Potassium Channels And Their Modulators From Synthesis To Clinical Experience

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Potassium Channels and their Modulators: From Synthesis to Clinical Experience

LEADING EDGE BOOKS IN PHARMACEUTICAL SCIENCES NEW AND FORTHCOMING TITLES 1995/1996 International Pharmaceutical Product Registration : Aspects of Quality, Safety and Efficacy (Cartwright & Matthews) 013474974 X Advanced Drug Design and Development: A Medicinal Chemistry Approach (Kourounakis & Rekka) 0133367932 Pharmaceutical Design and Development: A Molecular Biology Approach (Ramabhadran) 013 553884 X Reverse Transcriptase PCR (Larrick and Siebert) 013 123 118 9 Biopharmaceutics of Orally Administered Drugs (Macheras, Reppas and Dressman) 013 108093 8 Pharmaceutical Coating Technology (Cole, Hogan and Aulton) 013 662891 5 Dielectric Analysis of Pharmaceutical Systems (Craig) 013 210279 X Autonomic Pharmacology (Broadley) 074840 556 9 Photochemical Stability of Drugs and Drug Formulations (Tonnesen) 074840 449 X Potassium Channels and Their Modulators : From Synthesis to Clinical Experience Pharmacokinetic Profiles of Drugs (Labaune) 074840 559 3 (Evans et al) 074840 557 7 Flow Injection Analysis of Pharmaceuticals : Automation in the Laboratory (Martinez-Calatayud) 074840 445 7 Pharmaceutical Experimental Design and Interpretation second edition (Armstrong and James) 074840 436 8 Handbook of Drugs for Tropical Parasitic Infections second edition (Gustafsson, Beerman and Abdi) 07484 0167 9 hbk/ 07484 0168 7 pbk Biological Interactions of Sulfur Compounds (Mitchell) 0748402446 hbk / 07484 0245 4 pbk Paracetamol: A Critical Bibliographic Review Review (Prescott) 0748401369 Zinc Metalloproteases in Health and Disease (Hooper) 07484 442 2 Cytochromes P450 (Lewis) 074840 443 0

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Potassium Channels and their Modulators: From Synthesis to Clinical Experience Edited by J.M.EVANS T.C.HAMILTON S.D.LONGMAN & G.STEMP SmithKline Beecham Pharmaceuticals

UK Taylor & Francis Ltd, 1 Gunpowder Square, London EC4A 2DE This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” USA Taylor & Francis Inc., 1900 Frost Road, Suite 101, Bristol, PA 19007 This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © Taylor & Francis Ltd 1996 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, electrostatic, magnetic tape, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-30441-1 Master e-book ISBN

ISBN 0-203-34216-X (Adobe eReader Format) ISBN 07484 0557 7 (formerly 013 009283 5) Library of Congress Cataloguing Publication data are available Cover design by Jim Wilkie

Contents

Contributors Foreword Preface

viii x xiii

1

The Synthesis and Chemistry of Benzopyran Related Potassium Channel Activators D.G.SMITH

1

2

Structure-Activity Relationships of Benzopyran Based Potassium Channel Activators J.M.EVANS & G.STEMP

32

3

Syntheses and Structure-Activity Relationships of Pyridine Based Potassium Channel Activators M.N.PALFREYMAN

66

4

Conformational Analysis of Potassium Channel Activators C.M.EDGE

91

5

The Structure-Activity Relationships of Potassium Channel Blockers R.CROSSLEY & A.OPALKO

128

6

Potassium Channels: Diversity, Assembly and Differential Expression R.LATORRE & P.LABARCA

156

7

Potassium Channel Electrophysiology in Vascular Smooth Muscle Cells and the Site of Action of Potassium Channel Openers P.I.AARONSON & C.D.BENHAM

194

8

Effects of Potassium Channel Activators in Isolated Blood Vessels U.QUAST

214

9

In Vivo Vascular Effects of Potassium Channel Activators J.C.CLAPHAM

242

vii

10

Cardiac Potassium Channel Modulators: Potential for Antiarrhythmic Therapy M.C.SANGUINETTI & J.J.SALATA

273

11

Cardioprotective Properties of Potassium Channel Modulators G.J.GROSS

316

12

Potassium Channel Activators: Airway Pharmacology and Bronchial Asthma J.R.S.ARCH

336

13

Potassium Channels in Pancreatic β -Cells: Modulation, Pharmacology and their Role in the Regulation of Insulin Secretion M.J.DUNNE, J.H.JAGGAR, E.A.HARDING, C.KANE & P.E.SQUIRES

369

14

Potassium Channels and their Modulation in Urogenital Tract Smooth Muscles A.F.BRADING & W H.TURNER

406

15

Potassium Channel Modulators and the Central Nervous System H.HERDON

434

16

Potassium Channel Modulators: Clinical Experiences and Future Prospects T.J.COLATSKY & T.C.HAMILTON

460

Abbreviations

494

Index of Compounds

499

Index

503

Contributors

P.I.AARONSON, Department of Pharmacology and Medicine, UMDS Guy’s and St. Thomas’s Hospitals, Lambeth Palace Road, London SE1 7EH, UK. J.R.S.ARCH, Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. C.D.BENHAM, Department of Biophysical Sciences, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. A.F.BRADING, Department of Pharmacology, Oxford University, Mansfield Road, Oxford, OX1 3QT, UK. J.C.CLAPHAM, Department of Vascular Biology, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. T.J.COLATSKY, Division of Cardiovascular and Metabolic Diseases, WyethAyerst Research, CN 8000, Princeton, New Jersey 08543, USA. R.CROSSLEY, Wyeth Research UK, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 0PH, UK. M.J.DUNNE, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. C.M.EDGE, Computational Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. J.M.EVANS, Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. G.J.GROSS, Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA. T.C.HAMILTON, Department of Neurology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. E.A.HARDING, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.

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H.HERDON, Psychiatry Research Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. J.H.JAGGAR, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. C.KANE, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. P.LABARCA, Centro de Estudios Científicos de Santiago y Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 16443 Santiago 9, Chile. R.LATORRE, Centro de Estudios Científicos de Santiago y Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 16443 Santiago 9, Chile. A.OPALKO, Wyeth Research UK, Huntercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 0PH, UK. M.N.PALFREYMAN, Rhône-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex, RM10 7XS, UK. U.QUAST, Department of Pharmacology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany. J.J.SALATA, Department of Pharmacology, Merck Research Laboratories, West Point, PA, USA. M.C.SANGUINETTI, Division of Cardiology, University of Utah, Salt Lake City, UT, USA. D.G.SMITH, SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey, KT18 5XQ, UK. P.E.SQUIRES, Cell Biology Research Group, The Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, UK. G.STEMP, Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. W.H.TURNER, Department of Urology, Inselspital, Bern, Switzerland.

Foreword

Ion channels have come of age. Channels have been classified, sequenced and reconstituted and recognized as one of the major classes of biological effectors and Nobel prizes have been awarded. Ion channels have gone from being a molecular black box in the transduction machinery of the cell to being an increasingly well characterized and defined piece of molecular machinery. Ion channels are, in fact, a class of pharmacological receptor. As such we expect them to have the following general properties: 1 Possess binding sites for activator and antagonist drugs with defined structure-activity relationships. 2 These drug binding sites should be coupled to the functional machinery of the channel. 3 They may be linked to members of the G protein family. 4 They will be subject to up-and down-regulation by homologous and heterologous influences. 5 Their numbers and function will be altered in disease states, both experimental and clinical. As this book illustrates these expectations have been fulfilled for K channels. That ion channels are important to the health and welfare of the cell is scarcely surprising. One of the earliest decisions that the first-formed cells had to make was that of selective transport across the cell membrane. The cell membrane was the critical stage in the evolution of the cell as machine, but if all that it achieved was to keep the inside in and the outside out it would have functioned only in a reactionary manner. The issues of transmembrane transport by the cell were solved in several ways—passive diffusion, facilitated diffusion, active transport and ion channels. It is often convenient to consider these pathways as totally distinct, but they are not. They all have the same passengers, although the fare for transport may be paid in different currencies. The underlying molecular motifs of the proteins that comprise these transport systems have common components that contribute to the determination of ion selectivity and transport rates. Some classes of device, such as the cystic fibrosis

xi

transmembrane regulator, share functional and structural properties of both ion channels and ATP-dependent transporters. Ion channels may be conveniently classified by several distinct processes. They can be classified as ligand—and voltage—gated according to their primary mode of stimulus. They can be classified according to their electrophysiological properties-the kinetics of activation and inactivation, their conductance, large or small, and by their pharmacological sensitivity to toxins and synthetic drugs. Increasingly, they are classified according to their sequences—a process that has demonstrated that ion channels exist as ‘super—families’ with considerable structural homology between members, despite very different electrophysiological and pharmacological properties. Finally, of course, ion channels can be classified according to the ions to which they are selectively and remarkably permeable—Cl−, Na+, Ca2+ and K+. This book focuses on K channels. This class of ion—selective channels has been the subject of increasing electrophysiological, pharmacological, molecular biological and therapeutic attention. This is appropriate because K channels are particularly important regulators of cell responsiveness in both electrically excitable and non—excitable cells and they may represent ancestral ion channels. This importance was indicated in the early proposal by Bernstein in 1902 that cellular excitability arose from the selective permeability of cell membranes to K+ ions and their modulation. From the pharmacological and therapeutic perspectives progress in a field depends critically upon the type and quality of the investigational tools available. K channels were hampered, relative to Na and Ca channels, by the general unavailability of potent, selective small molecule ligands. Rather, the pharmacology of K channels was dominated by exotic toxins, frequently of limited and expensive availability and purity, and small molecule ligands of the quaternary ammonium ion class that were neither potent nor selective. This situation should be compared with that for Na and Ca channels where the availability of potent and selective small molecule synthetic ligands generated major therapeutic drug categories. This picture is now changing very rapidly for K channels, particularly those of the KATP class. The hypoglycemic sulfonylureas are already widely employed in the therapy of type II diabetes and the radioligand [3H] –glibenclamide is a popular molecular tool. The corresponding K channel activators, including the prototypical benzopyran cromakalim, offer wide therapeutic possibilities for cardiovascular, noncardiovascular and central nervous system disorders. They offer, in fact, the therapeutic possibility of being complementary to the available Ca channel antagonists. These possibilities are strengthened by the recent cloning of this channel. With the increasing molecular dissection of the ion channels contributing to the cardiac action potential there is continuing interest in both activators and antagonists of the K+ currents as antiarrhythmic agents. Given the continued widespread occurrence of ‘sudden cardiac death’, continued focus in this area can only be welcome. A major, although still potential, therapeutic

xii

target for K channel modulators is in the central nervous system in disorders including neurodegenerative diseases and ischemia. No single volume of manageable size could claim to offer a comprehensive treatment of K channels. This book is no exception. It does, however, manage to embrace a remarkably broad range of topics from chemistry to structure— activity relationships and molecular modelling to molecular cloning through smooth, cardiac and neuronal physiology and pharmacology. This broad coverage reflects, in fact, the current intellectual and practical excitement in the field of K channel modulators. Long may this excitement continue. David J.Triggle, School of Pharmacy, State University of New York at Buffalo.

Preface

When two of us published an article in Chemistry in Britain describing the discovery of cromakalim, the first compound to be shown to exert its pharmacological action purely by K channel activation, we received an invitation from Ellis Horwood OBE to use the article as a basis for a book. From this initial approach came the opportunity to produce the series of articles that comprise this book, covering the theme of K channels and their modulators. In compiling this work we have drawn on the pool of experience built up within SmithKline Beecham, the pharmaceutical company that pioneered K channel activators, and have augmented it with contributions from internationally known experts in the field of structure and properties of K channels and K channel modulators. It is clear that the K channel story did not start with the fundamental discovery of the mode of action of cromakalim by Hamilton, Weston and Weir in 1986, as nicorandil was already known to possess this mode of action in addition to its propensity to activate guanylate cyclase. However, it must be said that the discovery of cromakalim’s single mechanism of action in vascular smooth muscle acted as a spur to the generation of several series of benzopyran analogues and led to the discovery that many existing compounds, such as pinacidil and RP 49356, were forerunners of extensive families of K channel activators. In addition the availability of cromakalim initiated many novel biological studies involving K channels. Perhaps one of the most important discoveries was that by workers at Beecham, Rhône Poulenc and Sandoz who demonstrated that the channel modulated by cromakalim was blocked by glibenclamide (glyburide), a sulphonylurea drug known to block ATP-sensitive K channels in pancreatic β cells. This finding brought together the more mature field of K channel blockers, where drugs are already in the healthcare market, and the new and burgeoning field of K channel activators. Such discoveries served to confound the sceptics who thought little of the ideas underpinning the mechanism of the activators, and who merely dismissed the class as just another series of vasodilators. As K channels are found in virtually every body tissue, it was to be expected that compounds that are selective for the differing tissues would emerge. This

xiv

has happened to some extent in that K channel blockers are used clinically as antiarrhythmic and antidiabetic agents. As for the activators, we are only at the start of the search for selective agents to attenuate the disease states that are associated with different tissues. Besides hypertension, the (patent) literature indicates that activators are potentially useful in the treatment of angina, myocardial ischaemia, asthma, urinary incontinence and CNS disorders such as epilepsy. The search for K channel opening selectivity encompasses several approaches. First, there is the structural modification of known compound series, for example that of the cromakalim series has produced the in vivo airways selective compound BRL 55834, the cardioselective compound BMS-180448 and the CNS active benzamides. Second, the search for different structures that modulate ATPsensitive channels in specific tissues, for example the recent ZD-6169 that is selective for the bladder in vivo. Third, there is the search for novel structures that modulate K channels other than the ATP-sensitive K channel, exemplified by NS 004 that is reported to modulate the big calcium-activated K channel and to be of use in CNS disorders. Such investigations involve many different groups of scientists including medicinal chemists, molecular modellers, pharmacologists and electrophysiologists. Each group is dependent upon the contribution of the others for data on the K channel targets that they are seeking to modulate. The central chapters in this book attempt to show how such data are generated and using the ligands described by the chemists in the early chapters, the results that are obtained for individual tissues and how they are interpreted. The importance of molecular biology in the structural determination of the plethora of K channels illustrates the widening range of techniques that are being utilised in K channel studies. Finally, building on the results of such studies, there is the experience in the clinic of the marketed K channel blockers and the potentially clinically useful activators. In conclusion we must thank the contributors to this book for their dedication in not only producing succinct reports of the areas in which they have built up their expertise but also their major efforts in unravelling the K channel story. In the future much will depend on their present and future studies and those of the other investigators responsible for the work described herein. That may lead us to the discovery of tissue selective K channel modulator molecules with the exciting potential for attenuating a variety of disease states. The opportunity has been taken to keep the information in the text as up-todate as posssible by the inclusion of the most recent literature references where applicable.

1 The Synthesis and Chemistry of Benzopyran Related Potassium Channel Activators D.G.SMITH SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey, KT18 5XQ, UK. 1.1 Introduction Since the disclosure of antihypertensive activity in a series of 4-amido benzopyran-3-ols (Ashwood et al., 1986), and the emergence of cromakalim (CRK 1) as the prototype (Hamilton et al., 1986) of a new class of smooth muscle relaxant offering therapeutic potential (Longman and Hamilton, 1992), the literature in the potassium channel activator (KCA) area has increased dramatically.

Not only has the scientific literature grown over the last few years but from the patent literature it is evident that a large number of organisations have been, or are still, involved in the search for similar entities, and numerous compounds based on the CRK lead have undergone further development. Thus compounds where the aromatic ring has been variously substituted or replaced by other aromatic moieties (pyranopyridines, thienopyrans) have been investigated. Similarly benzothiapyran, indane, benzoxepine, tetralin (tetralone), tetrahydroquinoline and benzoxazine analogues of the benzopyran have been explored and have given rise to active derivatives. Positional substitution of the non-aromatic ring has shown that the C-2 and C-3 positions of CRK are relatively intolerant to structural change whereas C-4 is remarkably flexible in the type of substituent it can accommodate; the cyclic lactam being just one example of a multitude of both cyclic and acyclic amide moieties, or their equivalents, which confer KCA activity. Details of structure-activity

2 K CHANNELS AND THEIR MODULATORS

relationships (SAR) brought about by such changes have been reviewed recently (Evans et al., 1992; Buckle and Smith, 1993) and have been updated in Chapter 2 of this book. The intention of this chapter is to bring together the disparate compound types which can be collectively regarded as analogues of the benzopyran series of KCAs, and to review their preparation and salient features of their chemistry which have been developed over the last few years. The review covers the chemistry, medicinal chemistry and significant patent literature from the time of the first chemical paper relating to CRK (Ashwood et al., 1986) until the end of 1993. 1.2 General Synthetic Aspects—Racemic Derivatives The standard route to 4-amido-3, 4-dihydro-2H-1-benzopyran-3-ols 6 is shown in generalised form in Figure 1.1, regiospecific, trans orientation of amide and hydroxyl functionalities usually being assured through ring opening of the precursor epoxide 5 by amide nucleophiles at C-4 (Ashwood et al., 1986, 1990). The reaction is usually carried out on the epoxide itself or, in those cases where the epoxide is either relatively unstable or is difficult to form directly, in situ generation from the precursor bromohydrin 7 is favoured (Ashwood et al., 1986). With relatively high temperatures, long reaction times and/or excess base further reaction can occur resulting in dehydration to the corresponding benzopyran 8. This can also be formed by base-induced elimination of the mesylate 9 (Buckle et al., 1990). Direct formation of the epoxide from the benzopyran 4 is usually straightforward using a peracid, but in those cases where the epoxide is unstable to the acidic conditions successful isolation has been achieved by inclusion of a peracid-KF complex (Houge-Frydrych, C.S.V., personal communication).

THE SYNTHESIS AND CHEMISTRY OF BENZOPYRAN RELATED KCAS 3

Figure 1.1 General synthetic approaches to 4-amidobenzopyrans

The commonly used synthesis of benzopyrans 4 involves condensation of an appropriately substituted phenol 2 with 3-chloro-3-methylbutyne followed by Claisen rearrangement of the aryl propargyl ether 3, the reaction being facilitated by the presence of the gem dimethyl group (Harfenist and Thom, 1972). (Indeed attempted use of the reaction to prepare the benzopyran precursor to the C-2 nordimethyl analogue of CRK was not satisfactory (Buckle et al., 1991c).) The rearrangements are usually carried out in high boiling solvents such as 1, 2dichlorobenzene or N, N-diethylaniline at reflux and result in good yields of the desired benzopyran 4. The generally accepted mechanism for this reaction (Rhoads and Raulins, 1975) is a 1, 5 sigmatropic shift followed by electrocyclic rearrangement of the initially formed allene intermediate 10 and usually occurs without formation of benzofuran products. However, thermal cyclisation of the pyridyl acetylene 11 gave a concentration dependent mixture of the benzopyran 12 and benzofuran 13, higher concentration and base catalysis favouring formation of the benzofuran (Attwood et al., 1991b).


The Claisen rearrangement, although generally successful for the preparation of 6-substituted benzopyrans, is not particularly suitable for synthesis of the corresponding 7-substituted isomers. Thus for the 3-methoxy- (Anderson and LaVoie, 1973), 3-cyano-, 3-nitro- (Evans et al., 1983) and 3-trifluoromethyl (Buckle et al., 1990) phenylpropargyl ethers mixtures of 5- and 7-isomers are obtained with the 5-isomer 14 predominating. To overcome this problem a regioselective route to the 7-trifluoromethyl derivative 15 (R=CF3) was developed (Buckle et al., 1990) whereby 4-bromo-3-fluorobenzotrifluoride was regiospecifically coupled with 2-methyl-3-butyne-2-ol under Heck conditions and the derived acetylene partially hydrogenated to the cis olefin 16 which was cyclised to the desired derivative. This route has proved satisfactory for the preparation of 2, 2-trifluoromethylbenzopyrans whose synthesis is problematic via the standard route (Fenwick, 1993).

An alternative to the propargyl ether cyclisation which has been utilised frequently to provide benzopyran intermediates is the condensation of an acetophenone with a ketone (Kabbe, 1978). Thus reaction of a range of 2hydroxyacetophenones with acetone gave the corresponding benzopyran-4-ones 17 which, after reduction and dehydration, generated the required benzopyran 4 (Buckle et al., 1990; Bergmann and Gericke, 1990). Appropriate substitution of the acetophenone mitigates against isomeric mixtures and hence problems of regiochemistry are obviated.


The benzopyran-4-one intermediates to the C-2 nor-dimethyl 18 and monomethyl 19 analogues of CRK have been prepared in high yield by alkylation of 4-cyanophenol with the appropriate lactones, followed by cyclisation of the acid intermediates 20 (Buckle et al., 1991c). This would appear to offer a general and high yielding route to this type of compound but the regiochemical cyclisation of asymmetric substrates was not investigated. A high yielding route to CRK via the benzopyran involved condensation of 4cyanophenol with isoprene under acidic conditions to give the dihydrobenzopyran 21 Bromination at C-4 and dehydrohalogenation gave the required benzopyran 4 (R=6-CN) in 82% overall yield (Timer et al., 1991). The less potent racemic cis-isomer of CRK was originally prepared (Ashwood et al., 1986) by treatment of the epoxide 5 (R=6-CN) with HBr to give the trans-4-bromo-3-ol 22. The tetrahydropyranyl (THP) ether of 22, was subsequently reacted with NaN3, reduced and deprotected to give the cisaminoalcohol which, after acylation and cyclisation furnished the cis-isomer 23. A more facile method for the preparation of cis-isomers involves isomerisation of the trans-amidoalcohols using diethylaminosulfur trifluoride (DAST) (HougeFrydrych and Pinto, 1989; Burrell et al., 1990b; Buckle et al., 1991b). This reaction is further discussed in Section 1.6.3.

The racemic dihydrobenzopyran 25 is less active in vivo than CRK and was originally prepared by catalytic hydrogenation of the corresponding benzopyran


(Ashwood et al., 1986). The analogous pyridone 26 has also been reported, formed in low yield (4%) from the 4-bromodihydrobenzopyran 24 and pyridone anion (Bergmann and Gericke, 1990). The C-2 position of CRK would appear optimally substituted by a gemdimethyl (Ashwood et al., 1986; Buckle et al., 1991c) or methyl, ethyl (Attwood et al., 1991a) moiety, but the synthesis of potent gem-trifluoromethyl (Fenwick, 1993) and gem-monofluoromethyl (Koga et al., 1993d) analogues have also been described. Spiro derivatives (Bergmann and Gericke, 1990; Lang and Wenk, 1988) and compounds where one of the methyl moieties has been replaced by a ketal or thioketal substituent (Yoo et al., 1992b) are also known. Mono substitution at C-2 is complicated by the presence of a further chiral centre. The 6-nitro compound 27 was reported with unassigned stereochemistry (Ashwood et al., 1986) but more recently both isomers of the 6-cyano derivative 28 have been prepared and characterised (Buckle et al., 1991c; Attwood et al., 1991a). Dehydration of the latter com-pounds, and the corresponding 2, 2–unsubstituted derivative, gave mixtures of 2, 3 and 3, 4-enes or solely the 2, 3-derivative depending on conditions used (Buckle et al., 1991c). High potency is associated with a group of compounds substituted with methyl at C-3, particularly in conjunction with an O-linked heterocycle at C–4 e.g. 29. Preparation of the intermediate benzopyran-4-one 30 was achieved either by reaction of 4-cyano-2-hydroxypropiophenone with acetone, or via reduction of the β -keto methylene analogue 31, and elaboration to the epoxide (Gericke et al., 1991a). In contrast to the relative ease of epoxide ring opening shown by the C-3 nor-methyl derivatives, the epoxides derived from this series were more resilient to attack by amide nucleophiles, several days reflux being required for complete reaction. Syntheses of CRK analogues where the C-3 hydroxyl group is replaced by other moieties have also been described (Buckle et al., 1991b; Gericke et al., 1991d) and are reviewed in Section 1.6.4.

In a novel departure from KCAs of standard structural type the synthesis and activity of a series of potent compounds where C-3 and C-4 functionalities are transposed has been described (Cassidy et al., 1992). Thus treatment of the benzopyran 4 with t-butyl N, N,-dichlorocarbamate followed by reduction with sodium metabisulfite gave regio– and stereoselectively the trans-4-chloro-3carbamate 32 from which the aziridine 33 was formed after base hydrolysis (Orlek and Stemp, 1991). Acid hydrolysis of 33 followed by deprotection gave


the aminoalcohol 34, from which a range of amido derivatives was produced. Dehalogenation and deprotection of the initially formed adduct 32 gave amine 35, from which analogous derivatives were prepared. Ring opening of the aziridine 33 (R=CN) with other nucleophiles gave further C-4 variants.

Numerous benzopyran KCAs have been prepared to explore the effect of aromatic ring substitution. Since synthesis generally follows the routes outlined above or results from simple functional group transformation of intermediate compounds (Evans et al., 1992; McCaully, 1991 and references therein) their preparation need not be elaborated further. A range of C-6-heterocyclyl derivatives has been prepared via reaction of the appropriate substrate with CRK or its 6amino, carbamoyl or formyl analogues using standard heterocyclic methodology (Bergmann and Gericke, 1990). Largely because of its ready ease of modification, but also because it would appear to occupy a critical role in the KCA pharmacophore, the C-4 position has similarly been subject to considerable variation. Whilst most of the modifications have either retained the amide or an equivalent pharmacophore, and were prepared using the syntheses described above, other groupings which are not normally regarded as amide surrogates (e.g. pyridine N-oxide and sulfoxide) have been prepared by alternative methods. The preparation of amides and ureas is normally straightforward. However, in those cases where the epoxide appears particularly recalcitrant towards ring opening, reactions have been enhanced in the presence of Lewis acids such as MgBr2 (Buckle et al., 1992a) or BF3.Et2O (Gericke et al., 1991b) or by use of tetramethyl ethylenediamine (TMEDA) to increase the nucleophilicity of the anion in the case of C-4 pyrrole derivatives (Buckle et al., 1992b). For those amides which can react as ambident nucleophiles (e.g. 2-pyridones) then mixtures of O- and N-linked compounds are generally obtained. Results of Nversus O-alkylation in the reaction of a wide variety of heterocycles with benzopyran epoxides have been documented (Bergmann and Gericke, 1990; Bergmann et al., 1990). Pinacidil 36 and CRK are both openers of the ATP-sensitive K (KATP) channel (Cook et al., 1988). This, together with the known isosterism between N-


cyanoguanidine and thiourea (Durant et al., 1977), probably accounts for the number of publications describing compounds designed to exploit this equivalence at C-4. Representative preparations of generalised compound 37 were by reaction of epoxide 5 with a diamine (R2R3 linked) followed by treatment with dimethyl-N-cyanodithioiminocarbonate, or by treatment of the appropriate thiourea 38 with cyanamide (Shiokawa et al., 1989, 1990; Atwal, 1991, 1992b, Atwal et al., 1990, 1992a, 1992b; Stemp et al., 1990; Stenzel et al., 1990). The N-cyano group in these compounds has been replaced by moieties known to act as amide surrogates in the H2-antagonist area and were prepared by processes analogous to those already described (Burrell et al., 1993; Butera and Bagli, 1993). N-cyanoamidines have also been prepared via epoxide opening with the anion derived from the appropriate heterocycle to give e.g. 39 (Garcia et al., 1991), or by reaction of the amino alcohol 40 with an N-cyanoimidate to give 41 (Ohtuka et al., 1991).

A number of C-4 C-linked derivatives have been prepared which retain KCA activity, seemingly by virtue of an appropriately orientated carbonyl group. Thus reversed amides of type 42 and ester 43 have been prepared from opening of the epoxide with the appropriate lithium salt (Ashwood et al., 1991). The latter compound gave the propanone 44 after hydrolysis and decarboxylation (Ashwood et al., 1991). C-linked pyridones 45 have been similarly prepared via the anion of the N-benzyl derivative (Gericke et al., 1991c). A series of C-4 amides/thioamides has been described which was prepared by reaction of the enolate derived from the 3-ketone 46 with an isothiocyanate to give 47, followed by reduction to the diastereomeric alcohols 48. Dehydration of the trans-isomers via the mesylate gave the benzopyrans 49. Alternatively the dithioester 50, formed by reaction of 46 with CS2 and Mel was aminated (Arch et al., 1991). Similar syntheses were used by others for the preparation of such compounds (Koga et al., 1993a, 1993b, 1993c, 1993d; Ishizawa et al., 1993).


Spirocyclic benzopyran imidazolones 52 have been prepared as rigid analogues of CRK by treatment of the corresponding ethoxy derivatives 51 with amines, and many of the compounds were potent KCAs despite lacking the C-3 hydroxyl (Gadwood et al., 1993). In an alternative preparation of the C-4 pyridine N-oxide derivative 54, the enol triflate 53 was treated with 2-(tributyltin)pyridine under palladium catalysis followed by oxidation (Yoo et al., 1992a). The sulfoxide group has been investigated as an N-oxide mimic and 56 was found to be as potent as CRK, although about 30 times less potent than 54. The preparation of 56 was achieved by treatment of the benzopyran-4-one 17 (R=6NO2), with the lithium salt of the cyclic sulfoxide 57 followed by dehydration (NaI/ TFAA). This also resulted in reduction of the sulfoxide to 55 but selective reoxidation using oxone gave 56 (Yoo et al., 1993).


1.3 Preparation of Enantiomers Standard routes to benzopyranol KCAs provide the compounds in racemic form. For CRK there is a 100-fold potency difference in vitro between the eutomer (3S, 4R) and the distomer (3R,4S) (Hof et al., 1988; Attwood et al., 1992) and at least a 30-fold difference in vivo in the spontaneously hypertensive rat (Ashwood et al., 1986). Similar potency differences exist between enantiomers of other KCAs (Buckle et al., 1990, 1991a; Bergmann et al., 1990). Individual enantiomers have been prepared by ring opening of the racemic benzopyran epoxide with a suitably substituted chiral pyrrolidinone followed by separation of the diastereomers obtained. In this way benzopyrans bearing the 5’methyl pyrrolidinone (Englert et al., 1988), and more complex derivatives (Regnier et al., 1990; Pinto et al., 1993) at C-4 have been obtained. The C-3 hydroxyl group provides a handle by which to attach chiral auxiliaries, and enantiomers have been separated via the diastereomeric β -methylbenzyl carbamates (Ashwood et al., 1986; Buckle et al., 1990; Quagliato et al., 1991), camphorsulfonates (Bergmann et al., 1990) and camphanates (Bergmann et al., 1990). Some workers have relied upon separation at an earlier stage to provide chiral ester derivatives of the bromohydrin 7 which were separated, hydrolysed and cyclised to the (3S,4S) epoxide 58. Thus N-Boc-(L)-alanine (Shiokawa et al., 1989), (-) camphanic acid (Blarer, 1988), chiral alkylcarbonylthiobutyric acids (Setoguchi et al., 1990), and (L)-proline (Yamanaka, 1991) derivatives have been used. The (3S,4S) epoxide has also been synthesised from the chiral aminoalcohol 60, prepared by resolution of the tartrate salt or derivatives thereof (Attwood et al., 1992; Genain and Pinhas, 1990; Quagliato et al., 1991). Successful separation of the (3S,4S) epoxide by entrainment crystallisation has been reported (Devant and Gericke, 1991) and a similar procedure was used for the C-3 methyl analogue 59 (Gericke et al., 1991a). Microbial oxidation of the benzopyran 4 (R=6-CN) to the (3S,4S) epoxide 58 has been attempted using a variety of organisms but the highest enantiomeric excess (ee) reported was only 10%. By contrast a number of organisms showed high enantioselectivity (>96% ee) of epoxidation to the undesired (3R,4R) enantiomer, but only in low (100 μ M, suggesting that at therapeutically administered doses these compounds will relax smooth muscle cells without having effects upon the regulation of insulin secretion. The second series of ethers is exemplified by EMD 57283 57 and the closely related 2-oxo-4-pyridyl analogue 58 (Bergmann et al., 1990) that is of similar potency to the racemate of compound 57 in the SHR. Removal of the N-methyl groups does not result in any significant change in the potency that remains in the 6–24 μ g/kg dose range to elicit a 30 mm Hg fall in mean BP, five to tenfold less than required of CRK. Other isomers of both the pyridyl and pyridazinyl series showed markedly lower potencies, and served to define a very tight set of SARs in this series of KCAs. More recently certain diazabicycloalkene derivatives, exemplified by compound 59, have been disclosed (Mimura and Kubo, 1993) that are derived from the EMD 57283 57 series. These compounds, where it appears that the cyclopropyl group acts as a bioisosteric replacement for the double bond, possess ED50 values in the range 0.0023–0.40 mg/kg.


Three entirely different approaches to those described so far in this section have also been documented. In the first, a 2-pyridyl N-oxide has been linked directly to the chromene nucleus in Ro 31–6930 60 (Attwood et al., 1991), and this appears to be the first instance of pyridine N-oxide and lactam bioisosterism. Interestingly, there are significant differences reported between CRK and compound 60. Thus the latter was an order of magnitude more potent than CRK in rat models of hypertension, although unlike CRK it did not reduce renal vascular resistance in the anaesthetised dog (Paciorek et al., 1990). Both compounds reduced renal vascular resistance in the anaesthetised rat, but only CRK exerted a selective action on the renal vascular bed (Duty et al., 1990). The importance of the N-oxide is illustrated by its replacement by a phenolic hydroxy group as in compound 61, that resulted in an approximate 100-fold loss in potency (Attwood et al., 1988). It is also interesting to observe high potency in a benzopyran analogue where the substituent is attached by carbon to position 4, and one can speculate that unlike the carbon attached compounds 28 discussed above, the sp2 nature at the point of attachment is partly responsible for this high potency. The second approach is based on the observation that the lactam ring of CRK prefers to adopt an orthogonal relationship with respect to the benzopyran nucleus (Cassidy et al., 1989), a likely feature of other benzopyran KCAs. In this approach (Gadwood et al., 1993), the relationship is fixed in

conformationally rigid spirocyclic benzopyran imidazolones 62–67 (Table 2.4). Evaluation of the compounds was by a membrane potential assay, where hyperpolarisation—a typical effect of KCAs—was detected by a decrease in fluorescent intensity of a voltage-sensitive dye. The nature of the substituent at position 2 of the spirocyclic group affected the level of potency of the compounds, as alkoxy- 62 and alkylamino- 64–67 compounds were more potent than alkylthio compound 63. The length and branching of the alkylamino substituent exerted an influence on potency, leading to optimal potency in the n-propyl compound 66. Compound 66 was slightly more potent than CRK in this test and it lowered mean arterial blood pressure in normotensive rats by about 50 mm Hg at a cumulative dose of 0.04 mg/kg whereas CRK caused a fall of about 23 mm Hg under the same conditions. Overlap comparison of computer-generated

K CHANNELS AND THEIR MODULATORS 49

structures of CRK and the tautomeric representation used here of compound 66 indicated that the imidazolone C=N nitrogen, adjacent to the carbonyl group, occupies the same space relative to the benzopyran ring as does Table 2.4 Effect of spirocyclic 2-substituted-imidazolones on membrane potential

Membrane Potential Assaya Compound No

R

concn (μ M)

fluorescence decrease %

CRK 62 63 64 65

― EtO EtS MeNH EtNH

66

n-PrNH

1.0 1.0 1.0 1.0 1.0 0.1 1.0 0.1 1.0

22 20 6 19 25 8 27 21 10

67 aA10

i-PrNH

cell line derived from rat embryonic rat aorta. Gadwood et al., 1993.

the carbonyl group of CRK, and it was thought that this particular nitrogen atom serves as a hydrogen bond acceptor group. If this is indeed the case then these rigid analogues support the notion that CRK most likely interacts with its putative receptor in a close to orthogonal conformation. The final approach utilised an appropriately substituted pyrrole as the substituent attached via nitrogen to position 4 (Smith et al., 1992). An exemplar of the series is compound 68 that is about twice as potent as CRK in relaxing the spontaneous tone of guinea pig isolated trachea. The pyrrole ring is thought to adopt an orthogonal relationship with respect to the benzopyran nucleus, much in the same manner as that envisaged for CRK, and the potency is derived from the substituted enamine function acting as a bioisostere for the pyrrolidinone of CRK. It is also of note that a related structure 69 was also classified as a KCA, although it was slightly less potent than CRK, despite the rigidity imposed in the molecule that precludes the attainment of an orthogonal disposition. This observation contrasts sharply with that of the rigid orthogonal spirocyclic compounds described in the second approach to benzopyran KCA design above.


2.3.2 Position 3 The hydroxyl group at this position has been shown to be a necessary feature for good activity, as its replacement by a hydrogen atom diminished activity (Ashwood et al., 1986). However, its elimination to form chromene analogues can in some instances provide potent KCAs. The topic of chromene analogues is discussed later in this section. Esterification of the hydroxyl group to give formate, acetate and nitrate groups (Ashwood et al., 1986; Houge-Frydrych and Evans, 1989; Bergmann and Gericke, 1990), and oxidation to the ketone (Buckle et al., 1991c) furnished analogues that retained the potency of CRK. However, a range of other substituents such as carbonyl, hydroxymethyl, nitro, fluoromethyl and formyl attenuated activity (Buckle et al., 1991c). The influence of the hydroxyl group stereochemistry at position 3 on the activity of benzopyran and other KCAs was discussed in section 2.2. A methyl group has been inserted in addition to the hydroxyl group at this position in the conventional CRK series (Gericke et al., 1991) but this caused an attenuation in the potency of relaxation of acetylcholine-induced contractions in pig coronary artery. But in marked contrast, in O-linked KCAs such as compound 70, potency was enhanced, and it is notable that this methyl group is incorporated in the closely related series of compounds exemplified by compound 59 above. It is thought that the effect of this methyl group is to modify the orientation of the C(4) substituent, and this topic is enlarged upon in Chapter 4.


Although the replacement of the hydroxyl group by a hydrogen atom diminished activity, it can be eliminated together with the hydrogen atom at position 4, to give chromenes such as compound 71, that retain the potency of CRK (Ashwood et al., 1986). By selecting this structural type of KCA, the question of stereochemistry at positions 3 and 4, and the necessity of stereoselective synthesis or resolution, discussed in Chapter 1, can be obviated. Thus, several analogous chromenes such as Ro 31–6930 60, KC-399 31, bimakalim 12 and SR 47063 46 have been designated as development candidates. However, the advantage of choosing chromenes for development may not be entirely the result of ease of synthesis, as certain chromenes have been found to be more potent than their chromanol counterparts. Indeed the potencies of the chromenes and the chromanols do not always parallel each other, a difference first seen in a close analogue of CRK, the 4-acetamide chromene 72 (Ashwood et al., 1990) that was about tenfold less potent than the corresponding chromanol.

This difference has subsequently been observed in other series. Thus compound 12 lowers mean arterial BP in SHR by approximately 20% more than the racemate of emakalim 13, and this differential appears to be a general characteristic of the pyridone series of KCAs (Bergmann and Gericke, 1990). In contrast, in the series of O-linked KCAs, the chromanols such as the racemate of HMD 57283 57 have a similar potency level to the racemate of chromanol 13, whereas the corresponding chromene 73 did not alter significantly BP in the SHR (Bergmann et al., 1990). In the indane series (Buckle et al., 1991a) the differences were variable, but in the thieno[3,2-b]pyran series dehydration led to compounds that were markedly less potent than the thienopyranols (Sanfilippo et al., 1992). To further complicate matters, in the tetralone series the introduction of unsaturation tended to enhance potency of blood pressure lowering ability and relaxation of guinea pig isolated trachealis (Almansa et al., 1993). While limited by the conditions of comparison of results obtained from in vivo and in vitro models, discussed in the introduction, one can speculate on the reasons for the differing potencies of closely related chromenes and chromanols. Thus it might be postulated that the loss of one molecular characteristic namely the hydroxyl group, that possibly aids binding to the putative receptor, is


compensated in some instances, but not in others, by a closer fit of other characteristics such as the amide carbonyl group at position 4, or the dialkyl group at position 2, that may enhance its binding capability at the receptor. A most unusual development at this position has been the discovery of KCAs with transposed trans C(3) and C(4) substituents, that retain the potency of CRK in the SHR (Cassidy et al., 1992). Optimal potency in SHR was associated with the presence of a urea or thiourea group at position 3, substituted by a bulky alkyl terminal group, as in compound 74 (R=t-Bu). Some interesting differences between this series and C(4) urea substituted series have emerged. Thus the presence of a terminal methyl group, as in compound 74 (R=Me) that is normally associated with optimal activity in conventional benzopyran KCAs, leads to a decrease in potency. The parallel with pinacidil terminal group SAR, discussed in Chapter 3, is also notable. Also in contrast to the CRK series, the piperidinone 75 (R1/R2=– (CH2)4-), acetamide 75 (R1=H, R2=Me) and benzamide 75 (R1=H, R2 =para F–Ph) displayed low levels of potency in the SHR. These are substituents that confer high potency when incorporated at position 4 in benzopyrans (Ashwood et al., 1986, 1990).

2.3.3 Position 2 In the series of KCAs based on the benzopyran system, the presence of alkyl or substituted alkyl groups at position 2 has been found to be crucial in conferring activity. In a study of dihydro, mono-, or dimethyl substituted compounds, optimal activity was associated with the dimethyl group (Ashwood et al., 1986). The dihydro compound was virtually devoid of activity, and confirmation of this observation comes from the 1,1-dimethylindane series (Buckle et al., 199la), where absence of this group from compound 76 diminished its ability to inhibit spontaneous contractions in guinea-pig trachealis by at least an order of magnitude. One possibility is that conformational differences between the dimethyl- and more flexible dihydro compounds are responsible for the difference in potency, although binding to a hydrophobic site on the putative receptor may also be of importance (Evans et al., 1992). The presence of the single methyl group at this position enhanced potency over the dihydro compound, but not to the levels associated with the dimethyl group (Ashwood et


al., 1986). Subsequently, both isomeric monomethyl analogues 77 and 78 of CRK have been studied (Buckle et al., 1991b) by NMR and X-ray crystallographic techniques, and although differences in conformation were elicited, they were not correlated with biological activity. A second study (Attwood et al., 1991) described both isomers as relaxants of 20 mM KCl induced contractions in rat isolated portal vein with an IC50>30 μ M, compared with an IC50 for CRK of 0.24 μ M. The conformation of the pyran ring in these isomers, discussed in more detail in Chapter 4, is described as a distorted half-chair in the β -methyl isomer 77, and a half-chair in the β -methyl isomer 78.

Higher alkyl (Buckle and Smith, unpublished results) and cycloalkyl groups at position 2 (Bergmann and Gericke, 1990) in general reduce potency in benzopyran-3-ols. In tetralones (Almansa et al., 1993) the data are somewhat equivocal, but in the two chromene analogues described, the tendency was to a reduced potency. However there are two exceptions to this reduction in potency on homologation of the gem-dimethyl group. One is the racemic 2-ethyl-2methyl analogue 79 that has twice the potency of Ro 31–6930 60 in relaxing rat isolated portal vein (Attwood et al., 1989). The second exception in the benzopyran-4-carbothioamide series (Sato et al., 1993) was exemplified by a wide range of alkyl substituents in compounds 80–87 (Table 2.5) that were examined for their relaxant effects on rat isolated aorta precontracted with 30 mM KCl. Increase in size of the dimethyl group in this series enhanced the potency in compounds 81–84, but reduced potency in the methyl/n-butyl combination in compound 85. Optimal potency was observed in compounds containing the methyl/n-propyl combination 82 and the cyclobutyl 86 and cyclopentyl 87


groups that were about ten thousand times more potent than CRK. The presence of a single large alkyl group, the t-butyl group in compound 84, was also sufficient to produce good potency, but enlargement of the cycloalkyl group to six membered or higher attenuated potency. Functionalised alkyl groups, in particular fluoroalkyl groups, have been inserted in KCAs such as KC-399 31. This compound was shown to possess a longer duration of action than LCRK in the SHR, with reduced tachycardia and a slower onset of action (Koga et al., 1993c). This property of longer duration of action is also shared

Table 2.5 Vasorelaxant property of benzopyran-4-carbothioamides; effect of the group at position 2

Compound No

R1

R2

Rat aortaa pEC50

CRK 80 81 82 83 84 85 86 87

― ― 6.77 ±0.03 Me Me 8.87 ±0.05 Me Et 9.74±0.15 Me nPr 10.77 ±0.34 Et Et 9.43 ±0.25 H tBu 9.40 ±0.30 Me nBu 7.39±0.11 ― (CH ) ― 10.68 ±0.12 2 3 ― (CH ) ― 10.60 ±0.21 2 4 a Negative log [M] required to relax rat isolated aorta pre-contracted with 30 mM KC1 at 50% Intrinsic activity for each compound. Sato et al., 1993.

by the gem-trifluoromethyl substituted compound 88 that was prepared during studies aimed at airways selective KCAs (Fenwick, 1993). The propensity to prolong the antihypertensive effects of KCAs in the SHR has been correlated with the lipophilicity of the agents (Soll et al., 1991), and this may be an important factor in the prolonged action of compounds 31 and 88.


2.4 Replacement of the Benzopyran Nucleus The replacement of the benzopyran ring system by a variety of other ring systems discussed below, has led to several new series of KCAs. In general, by the judicious choice of substituents the potency of certain analogues in these new series has been enhanced over the standard compounds CRK and LCRK. It is also notable that although the SARs of these published series have much in common with the benzopyran KCAs, there are certain significant differences that will be discussed under the appropriate sections. 2.4.1 Replacement of the Pyran Ring When the benzopyran nucleus was replaced by benzothiopyran in a 6-methyl analogue 89 (X=S) of CRK, potency was maintained (Smith, 1990), but oxidation to sulphoxide and to sulphone 89 (X=SO and SO2, respectively) gave less potent compounds. Replacement by the tetrahydroquinoline 90, and tetrahydronaphthalene 91 nuclei caused a decline in potency of about tenfold in BP lowering in SHR, compared with the benzopyran series (Ashwood et al., 1991). Using the indane skeleton as a benzopyran substitute as in compound 92 produced a series of analogues only slightly less potent than the benzopyrans in relaxing guinea-pig isolated trachealis (Buckle et al., 1991a). Besides ring contraction, ring expansion to benzoxepines such as compound 93 have been described (Buckle et al., 1991b), but this modification significantly reduced potency in relaxation of spontaneous tone in guinea-pig isolated trachealis.


In a recent paper, the tetrahydronaphthalen-1-one nucleus has been used to provide a series of KCAs, exemplified by UR-8225 11 (see section 2.3.1), but this variation also caused overall potency to be slightly diminished compared to the benzopyran series. Modification of the carbonyl group by reduction or oxime formation only served to reduce potency further. Compound 11, having an IC50 of 0.6 μ M against noradrenaline-induced contraction in rat isolated portal vein, is about twice as potent as LCRK in lowering SHR systolic BP. This high potency is probably the result of, in part, the presence of the 4-pyridone substituent. One of the most potent compounds in a series of 1,4-benzoxazines is the pyridine N-oxide analogue 94, YM-934, that was described as about tenfold more potent than CRK in inhibiting spontaneous contractions in rat isolated portal vein (Matsumoto et al., 1991). Little SAR detail is reported for this series, but it seems likely that the pyridine N-oxide group, a key feature of compound 59 discussed previously, is probably partly responsible for the high potency of compound 94. 1,3-Benzoxazines such as compound 95 have also appeared in the patent literature (Baumgarth et al., 1990) but without pharmacological data.


2.4.2 Replacement of the Aromatic Segment A study of electrostatic potentials indicated that the pyridine group could be a useful cyanophenyl surrogate in benzopyran KCAs (Stemp and Evans, 1993). Of a series of the four possible isomeric pyranopyridines 96–99, the pyrano[3,2-c] pyridine isomer 96, with the nitrogen atom occupying the key 6-position (see section 2.5) was found to possess the highest potency in the SHR (Burrell et al., 1990b). The corresponding N-oxide had a similar potency. The pyrano[2,3-c] pyridine analogue 97 was at least threefold less potent than the pyrano[3,2-c] pyridine 96, while the pyrano[3,2-b]- 98 and -[2,3–b]pyridines 99 were at least 20-fold less potent than the parent compound 96.


Details have emerged of the SARs of thieno[3,2-b]pyrans containing cyclic amido substituents at the 7-position, (equivalent to the benzopyran 4-position). The series is exemplified by the 2-nitro analogue 100, RWJ 29009, that is reported to be tenfold more potent than CRK in the SHR, and has potential in the indication of angina and ischemia. The 2-nitro group is thought to mimic the 6cyano group of CRK, while the thiophene replaces the CRK aromatic ring. Despite the difficulty of comparing results derived from different dosing regimes, it is clear that comparison of CRK and the thiophene equivalent 101 of CRK (ED30 in SHR of 0.19 and 0.07 mg/kg administered orally, respectively), that there is a differential in potency of about threefold in favour of the thiophene compound. Other comparative data tend to support this observation, that represents the first instance where an alternative ring system has been proved to confer superior antihypertensive potency to the benzopyran ring system (Sanfilippo et al., 1992).

Thieno[3,4–b]pyrans and thieno[2,3-b]pyrans have subsequently been studied to see if this interesting differential extends to all the thienopyran regioisomers (Press et al., 1993). The thieno[3,4-b]pyran 102 and the thieno[2,3-b]pyran 103 had similar potencies to the thieno[3,2-b]pyran 104, the three isomers reducing mean arterial BP in SHR by 49, 43 and 53% respectively on administration of an oral dose of 20 mg/kg. Thus by extrapolation it appears that the three thienopyran ring systems confer higher potency than the benzopyran ring system. The impact of addition of aromatic substituents to these thienopyrans is considered in section 2.5.


2.5 Aromatic Substitution Studies of the aromatic substituents have indicated that their position and nature are of prime importance in influencing the level of potency of benzopyran KCAs (Ashwood et al., 1986; Burrell et al., 1990b; Buckle et al., 1990). The original work (Ashwood et al., 1986) in SHR revealed that BP lowering was optimal when a nitro substituent was sited at position 6. Relocation to position 7 lowered potency by about tenfold, and to position 8 virtually abolished activity. In subsequent studies similar observations were made (Buckle et al., 1990) while observing the relaxation of spontaneous tone in guinea pig isolated trachealis. In addition it was noted that a trifluoromethyl group conferred high potency at position 6 but when located at position 5 potency declined markedly. Taken together these data indicate a regioselective preference for position 6, followed by position 7. Since some of the most potent benzopyran KCAs possess substituents located at the 6- and 7-positions of the benzopyran nucleus (see later) it is interesting to note the introduction of the benzoxadiazole ring in compound 105, NIP 121, as a replacement for the cyanophenyl group. No SAR data are available for this series but the modification enhances the potency of compound 105 tenfold over CRK in the SHR (Arakawa et al., 1990).

Optimal potency was originally associated with the presence of strong electron withdrawing groups such as nitro, trifluoromethyl, nitrile, acetyl, methoxycarbonyl, formyl and chloro, in approximate order of descending potency (Ashwood et al., 1986; Burrell et al., 1990b) in SHR. To these can be added the phenylsulphonyl group incorporated in rilmakalim (HOE 234) 106 that confers greater potency than the nitrile of CRK (Klaus et al., 1990), the trifluoromethoxy group of compound 25 that confers approximately 20-fold higher potency than the nitrile group (Soll et al., 1991), and the pentafluoroethyl group of BRL 55834 107 that confers a tenfold increase in potency over the nitrile group in relaxing spontaneous tone in guinea pig isolated trachealis (Buckle et al., 1990). Compound 107 is the first airways-selective KCA (Buckle et al., 1992). A possible source of this selectivity is its ability to open both KATP and BKCa in bovine trachealis (Ward et al., 1992), a property not shared by LCRK. In view of this requirement for electron withdrawing capability it was surprising to find that alkyl groups could also be incorporated at position 6, and


still retain good potency, the 6-ethyl group conferring about one third of the potency of the nitrile group in SHR (Burrell et al., 1990b). Thus it appears that an electon withdrawing group is not mandatory at position 6. The dimensions of the group have some influence on the degree of potency, an approximate order of activity being ethyl, isopropyl, t-butyl >n-propyl, cyclopentyl >methyl >phenyl. The strong electron withdrawing group requirement for optimal potency appears to hold good for the other ring systems described in the last section. However, one interesting exception, is the thieno[3,4-b]pyran nucleus. Thus insertion of bromo or acetyl groups on either side of the ring sulphur in compound 102, either individually or in combination, lowered the potency of the parent compound (Press et al., 1993). The difference between this situation and the effect of 5- and 8-substituents in benzopyran compounds which are broadly neutral is quite striking. As the highest potency in benzopyran KCAs is associated with position 6 and to a lesser extent with position 7, it prompted limited investigations of compounds bearing substituents at both these positions. Incorporation of an acetylamino, or amino substituent at position 7 in a 6-nitro compound enhanced potency, whereas the reverse combination gave rise to a reduced potency (Ashwood et al., 1986). In contrast to the pairing of an electron withdrawing and electron donating group, the combination of two electron withdrawing groups, exemplified by the 6-bromo-7-nitro- and 6-cyano-7-nitro-benzopyrans, is the most potent substitution pattern (Ashwood et al., 1990). In support of these observations, the 6,7-dichloro combination of groups was found to confer higher potency than either the 6-cyano-or 6-chloro groups in the tetralone series (Almansa et al., 1993). 2.6 Conclusions This chapter has illustrated the huge reported increase in SAR studies in compounds containing benzopyran and related ring systems that belong almost exclusively to the class of ATP-sensitive KCAs. While novel compounds are still being discovered as agents with even higher potency as antihypertensives and smooth muscle relaxants, there are studies in progress searching for compounds with differing profiles of activity and selectivity for different channels and smooth muscle types for the amelioration of a variety of disorders (Longman and Hamilton, 1992). There are tantalising glimpses of profile change and selectivity, ranging from the longer acting antihypertensive compounds such as celikalim 25, and the prodrug Y 27152 27 (R1=PhCH2O, R2=Me) that lowers blood pressure with little associated tachycardia, to the potential anticonvulsant benzamide 32 (R=F) and the enhanced airway selective compound BRL 55834 107. Fundamental to these searches are the examination of SARs that may give the first insights into profile change and selectivity that until now, with the few


exceptions noted above, have not been easily attained in this class of KCAs. It is clear that further SARs will be forthcoming and it is to be hoped that the discovery of suitable selectivity will enable the potential of this class of compounds to be realised. References ALMANSA, C, GOMEZ, L.A. CAVALCANTI, F.L., RODRIGUEZ, R., CARCELLER, E., BARTROLI, J., GARCIA-RAFANELL, J. & FORM, J. (1993) J. Med. Chem., 36, 2121–2133. ARAKAWA, C., YUKINORI, M., YOKOYAMA, T., KAWAMURA, N. & TANAKA, S. (1990) Jap. J. Pharmacol., 52, (suppl. 1) 311P. ARCH, J.R.S., BUCKLE, D.R., CAREY, C., PARR-DOBRZANSKI, H., FALLER, A., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G. & TAYLOR, S.G. (1991) J. Med. Chem., 34, 2588–2594. ASHWOOD, V.A. & EVANS, J.M. (1985) European Patent Application 158 923 to Beecham. ASHWOOD, V.A., BUCKINGHAM, R.E., CASSIDY, F., EVANS, J.M., FARUK, E.A., HAMILTON, T.C., NASH, D.J., STEMP, G. & WILLCOCKS, K. (1986) J. Med. Chem., 29, 2194–2201. ASHWOOD, V.A., CASSIDY, F., COLDWELL, M.C., EVANS, J.M., HAMILTON, T.C., HOWLETT, D.R., SMITH, D.M. & STEMP, G. (1990) J. Med. Chem., 33, 2667–2671. ASHWOOD, V.A., CASSIDY, F., EVANS, J.M., GAGLIARDI, S. & STEMP, G. (1991) J. Med. Chem., 34, 3261–3267. ATTWOOD, M.R., JONES, P.S. & REDSHAW, S. (1989) European Patent Application 298 452 to Hoffman-La Roche. ATTWOOD, M.R., JONES, P.S., KAY, P.B., PACIOREK, P.M. & REDSHAW, S. (1991) Life Sci., 48, 803–810. ATTWOOD, M.R., BROWN, B.S., DUNDSDON, R.M., HURST, D.N., JONES, P.S. & KAY, P.B. (1992) BioMed. Chem. Lett., 2, 229–234. ARAKAWA, C, YUKINORI, M., YOKOYAMA, T., KAWAMURA, N. & TANAKA, S. (1990) Jap. J. Pharmacol., 52 (suppl. 1) 3IIP. ATWAL, K.S., MORELAND, S., MCCULLOGH, J.R., AHMED, S.Z. & NORMANDIN, D.E. (1992) BioMed. Chem. Lett., 2, 87–90. ATWAL, K.S., GROVER, G.J., AHMED, S.Z., FERRARA, F.N., HARPER, T.W., KIM, K.S., SLEPH, P.O., DZWONCZYK, S., RUSSELL, A.D., MORELAND, S., MCCULLOUGH, J.R. & NORMANDIN, D.E. (1993) J. Med. Chem., 36, 3971–3974. AUCHAMPACH, J.A., MARUYAMA, M., CAVERO, I. & GROSS, G.J. (1992) Circulation, 86, 311–319. BARTMANN, W. (1989) In: Trends in Medicinal Chemistry, van der Groot, H., Domany, G., Pallos, L. & Timmerman, H. (eds). Elsevier, Amsterdam, pp. 629–657. BAUMGARTH, M., GERICKE, R., BERGMANN, R., DE PAYER, J. & LUES, I. (1990) German Patent Application DE 4 010 488 to E Merck. BERGMANN, R. & GERICKE, R. (1990) J. Med. Chem., 33,492–504.


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EVANS, J.M., HADLEY, M.S. & STEMP, G. (1992) In: Potassium Channel Modulators: Pharmacological, Molecular & Clinical Aspects. Hamilton, T.C. & Weston, A.H. (eds). Blackwell Scientific, Oxford, pp. 341–368. FENWICK, A.E. (1993) Tetrahedron Lett., 34, 1815–1818. FOZARD, J.R., MENINGER, K., COOK, N.S., BLARER, S. & QUAST, U. (1990) Br. J. Pharmacol., 99, 7P. GADWOOD, R.C., KAMDAR, B.V., DUBRAY, L.A.C., WOLFE, M.L., SMITH, M.P., MlZSAK, S.A. & GROPPI, V.E. (1993) J. Med. Chem., 36, 1480–1487. GARCIA, G., DI MALTA, A. & SOUBRIE, P. (1990) European Patent Application 370 901 to Sanofi. GENAIN, G. & PINHAS, H. (1990) European Patent Application 377 966 to Syntex. GERICKE, R., HARTING, J., LUES, I. & SCHITTENHELM, C. (1991) J. Med. Chem., 34, 3074–3085. GROVER, G.J., MCCULLOUGH, J.R., HENRY, D.E., CONDER, M.L. & SLEPH, P.O. (1989) J. Pharmacol. Exp. Ther., 251, 98–104. HOUGE-FRYDRYCH, C.S.V. & EVANS, J.M. (1989) Patent Cooperation Treaty Application 89/05808 to Beecham. ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. KLAUS, E., LINZ, W., SCHÖLKENS, B. & ENGLERT, H.C. (1990) NaunynSchmiedeberg’s Arch. Parmacol., 342, (suppl.) R17. KLAUS, E., ENGLERT, K.E., HROPOT, M., MANIA, D., RAJAGOPALAN, R. & ZWERGEL, U. (1991) Naunyn-Schmiedeberg’s Arch. Pharmacol., 344, R35. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993a) BioMed. Chem. Lett., 3, 1115–1118. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993b) BioMed. Chem. Lett., 3, 625–631. KOGA, H., SATO, H., IMAGAWA, J., ISHIZAWA, T., YOSHIDA, S., SUGO, I., TAKA, N., TAKAHASHI, T. & NABATA, H. (1993c) BioMed. Chem. Lett., 3, 2005–2010. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., NABATA, H., IMAGAWA, J., YOSHIDA, S.& SUGO, I. (1993d) BioMed. Chem. Lett., 3, 1111–1114. LONGMAN, S.D. & HAMILTON, T.C. (1992) Med. Res. Rev., 12, 73–148. MANLEY, P.W. & QUAST, U. (1992) J. Med. Chem., 35, 2327–2340. MARTIN, C.A.E., NALINE, E. & ADVENIER, C. (1993) Drug Develop. Res., 29, 63–72. MATSUMOTO, Y., TSUZUKI, R., MATSUHISA, A., TAKAYAMA, K., YODEN, T. & UCHIDA, W. (1991) Abstr. Papers Am. Chem. Soc. 202 Meet. Pt 1, MEDI 99. MCCAULLY, J. (1991) Current Drugs-Potassium Channel Modulators KCMB 5–19. MIMURA, T. & KUBO, H. (1993) European Patent Application 571 822 to Daiichi. MURAOKA, K., NAGAO, H., HORI, T., SAKAYA, S., HOSHINO, T., MIYAO, Y., MURAI, T., EDANAGA, M. & NAKANISHI, M. (1991) Jap. J. Pharmacol., 55, (suppl. 1) 341P. NAKAJIMA, T., SHINOHARA, T., YAOKA, O., FUKUNARI, A., SHINAGAWA, K., AOKI, K., KATOH, A., YAMANAKA, T., SETOGUCHI, M. & TAKARA, T. (1992) J. Pharmacol. Exp. Ther., 261, 730–736.


OSHIRO, G.T. & COLATSKY, T.J. (1991) Current Drugs-Potassium Channel Modulators KCMB 20–28. PACIOREK, P.M., BURDEN, D.T., BURKE, Y.M. COWLRICK, I.S., PERKINS, R.S., TAYLOR, J.C. & WATERFALL, J.F. (1990) J. Cardiovasc. Pharmacol., 15, 188–197. PETERSEN, H.J., NIELSEN, C.K. & ARRIGONI-MARTELLI, E. (1978) J. Med. Chem., 21, 773–791. PRESS, J.B., MCNALLY, J.J., SANFILIPPO, P.J., ADDO, M.F., LOUGHEY, D., GIARDINO, E., KATZ, L.B., FALOTICO, R. & HAERTLEIN, B.J. (1993) BioMed. Chem., 1, 423–435. QUAGLIATO, D.A., HUMBER, L.G., JOSLYN, B.L., SOLL, R.M., BROWNE, E.N.C., SHAW, C. & VAN ENGEN, D. (1991) BioMed. Chem. Lett., 1, 39–42. QUAST, U. & VILLHAUER, E.B. (1993) Eur. J. Pharmacol.- Molecular Pharmacol. Secn., 245, 165–171. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FITZPATRICK, L.J., URBANSKI, M.J., KATZ, L.B., GIARDINO, E., FALOTICO, R., SALATA, J., MOORE, J.B. & MILLIER, W. (1992) J. Med. Chem., 35, 4425–4433. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FALOTICO, R., GIARDINO, E. & KATZ, L.B. (1993) BioMed. Chem. Lett., 3, 1385–1388. SATO, H., KOGA, H., ISHIZAWA, T., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 2627–2630. SHIOKAWA, Y., TAKIMOTO, K., TAKENAKA, K. & KATO, T. (1989) European Patent Application 344 747 to Fujisawa. SMITH, D.G. (1990) J. Chem Soc. Perkin Trans. 1, 3187–3191. SMITH, D.G., BUCKLE, D.R., FALLER, A. & PINTO, I.L. (1992) BioMed. Chem. Lett., 2, 1595–1598. SOLL, R.M., QUAGLIATO, D.A., DEININGER, D.D., DOLLINGS, P.J., JOSLYN, P.L., DOLAK, T.M., LEE, S.J., BOHAN, C, WOJDAN, A., MORIN, M.E. & OSHIRO, G. (1991) BioMed. Chem. Lett., 1, 591–594. STEMP, G. & BURRELL, G. (1992) US Patent 5 147 866 to Beecham. STEMP, G., EVANS, J.M. & BURRELL, G. (1991) European Patent Application 431 741 to SmithKline Beecham. STEMP, G. & EVANS, J.M. (1993) In: Medicinal Chemistry 2nd. Edition. Ganellin, C.R. & Roberts, S.M. (eds). Academic Press Ltd, London, pp. 141–162. TOOMBS, C.F., NORMAN, N.R., GROPPI, V.E., LEE, K.S., GADWOOD, R.C. & SHEBUSKI, R.J. (1992) J. Pharmacol. Exp. Ther., 263, 1261–1268. WARD, J.P.T., TAYLOR, S.G. & COLLIER, M.N. (1992) Br. J. Pharmacol., 107, 49P.

Recent Literature ATWAL, K., GROVER, G.J., FERRARA, F.N., AHMED, S.Z., SLEPH, P.G., DZWONCZYK, S. & NORMANDIN, D.E. (1995) Cardioselective Antiischaemic ATP-Sensitive Potassium Channel Openers. 2. Structure-Activity Studies on Benzopyranylcyanoguanidines: Modification of the Benzopyran Ring. J. Med. Chem., 38, 1966–1973.


BERGMANN, R. & GERICKE, R. (1994) The Influence of Substituents in 3-Position on the Activity of Chroman-Type Potassium Channel Openers. Arch. Pharm. (Weinheim), 327, 169–173. ISHIZAWA, T., KOGA, H., SATO, H., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1994) Substituent Effects of Benzopyran-4-(N-Cyano)carboxamidine Potassium Channel Openers for Selectivity to Guinea Pig Trachealis. BioMed. Chem. Lett., 4, 1995–1998. RUSSELL, K., BROWN, F.J., WARWICK, P., FORST, J., GRANT, T., HOWE, B., KAU, S.T., LI, J.H., MCLAREN, P.M., SHAPIRO, H.S. & TRIVEDI, S. (1993) A Highly Potent Series of Fluoroalkyl Benzoxazine Pyridine-N-oxide Potassium Channel Openers. BioMed. Chem. Lett., 3, 2727–2728. SATO, H., KOGA, H., ISHIZAWA, T., MAKINO, T., TAKA, N., TAKAHASHI, T. & NABATA, H. (1995) Vasorelaxant Activity of 2-Fluoroalkyl-6-nitro-2H-lbenzopyran-4-carbothioamide and Carboxamide K2+ Channel Openers. BioMed. Chem. Lett., 5, 233–236. SOLL, R.M., DOLLINGS, P.J., MCCAULLY, R.J., ARGENTIERI, T.M., LODGE, N., OSHIRO, G., COLATSKY, T., NORTON, N.W., ZEBICK, D., HAVENS, C. & HALAKA, N. (1994) N-Sulfonamides of Benzopyran-Related Potassium Channel Openers: Conversion of Glyburide Insensitive Smooth Muscle Relaxants to Potent Smooth Muscle Contractors. BioMed. Chem. Lett., 4, 769–773. TAKA, N., KOGA, H., SATO, H., ISHIZAWA, T., TAKAHASHI, T. & IMIGAWA, J-i. (1994) Vasorelaxant Activity of 2-FluoromethylBenzopyran K2+ Channel Openers. BioMed. Chem. Lett., 4, 2893–2898. TAKAHASHI, T., KOGA, H., SATO, H., ISHIZAWA, T., TAKA, N. & IMIGAWA, J-i. (1994) Synthesis and Vasorelaxant Activity of N-Imino-2-(Benzopyran-4-yl) Pyridine K2+ Channel Openers. BioMed. Chem. Lett., 4, 2899–2902.

3 Syntheses and Structure-Activity Relationships of Pyridine Based Potassium Channel Activators M.N. PALFREYMAN Rhône-Poulenc Rorer, Dagenham Research Centre, Rainham Road South, Dagenham, Essex, RM10 7XS, UK. 3.1 Introduction Potassium channels comprise the most diverse group of ion channels so far investigated. Amongst these, the ATP sensitive K channels (KATP channels) have been the most studied. During the last ten years compounds have been discovered which can activate or block these KATP channels. In particular K channel activators (KCAs) have been found, along with many other activities, to be smooth muscle relaxants with their main utility in hypertension and bronchodilatation. There are at least seven classes of activators (Edwards and Weston, 1990) of which the main four are shown in Figure 3.1. The benzopyrans, typified by cromakalim (CRK, shown as its (–) 3S, 4R-enantiomer levcromakalim (LCRK)), have been the class most thoroughly explored with more than thirty companies claiming patents. In comparison there have been few reports of structural modifications in the other classes of KCAs. This chapter covers the syntheses and structure-activity relationships (SARs) of the pyridine classes of KCAs: the thioformamides, cyanoguanidines and organic nitrates together with their hybrid molecules. A detailed description of SARs is complicated by the plethora of pharmacological models used to illustrate KCA-dependent potency. Where possible, potencies derived from isolated organ bath studies will be used supplemented by measurements of hypotensive effects in in vivo models of hypertension. The precise identity of the K channel responsible for the pharmacological effects of the compounds shown in Figure 3.1 is still unknown. It is therefore uncertain that all classes interact with the same site on a given channel or even with the same channel.

SYNTHESES AND STRUCTURE-ACTIVITY RELATIONSHIPS OF PYRIDINE BASED KCAS 67

3.2 Thioformamides 3.2.1 Aprikalim and Isomers The discovery of aprikalim The discovery of aprikalim 1 (RP 52891), initially as the racemate RP 49356, arose from the study of a series of 2-(2-pyridyl)tetrahydrothiophene-2thiocarboxamides

Figure 3.1 Main closes of KATP Channel Activators

(Aloup et al., 1987) which had antisecretory and antiulcer activity, possibly resulting from inhibition of K+ /H+ ATP-ase and which led to the selection of picartamide 2 for further evaluation. As part of this work, a number of structural modifications were carried out including the synthesis of the isomeric 2-(3pyridyl)tetrahydrothiophene 3 (n=1). Whereas 3 (n=1) was inactive as an antiulcer and antisecretory agent, it possessed a marked blood pressure lowering activity when given orally (5 mg/kg p.o.) to spontaneously hypertensive rats (SHR). Further analogues were then synthesised (Aloup et al., 1990) and the tetrahydrothiopyran analogue 3 (n=2) was found to be more active (0.5 mg/kg) in the SHR screen. The onset of the antihypertensive effect was slow and the activity was found to be due to the sulfoxide metabolite. Aprikalim was subsequently synthesised and found to be active in the SHR screen at 0.05 mg/kg.


At this time it was not realized that aprikalim was exerting its pharmacological actions by K channel activation.

The synthesis of racemic RP 49356 and its cis analogue 10 is outlined in Scheme 3.1 starting from 3-chloromethylpyridine hydrochloride 4. Formation of the isothiouronium salt of 4 followed by alkylation gave the chlorosulfide 5. Oxidation Chemical class benzopyran thioformamide cyanoguanidine organic nitrates

Typical member cromakalim aprikalim pinacidil nicorandil

Origin SmithKline Beecham Rhone-Poulenc Rorer Leo Chugai

Scheme 3.1 Synthesis of RP 49356 and its cis isomer 10 The depicted stereochemistry is relative Reagents : (i) NH2CSNH2, EtOH; (ii) aq. NaOH. Br(CH2)4Cl; (iii) MCPBA, CH2CI2; (rv) KOtBu, THF; (v) a) NaNH2; b) MeNCS; (vi) a) KOtBu, THF; b) CS2, Mel; (vii) MeNH2, EtOH: (viii) P2S5, CH2CI2; (ix) MCPBA, CH2CI2; (x) a) MeNH2, EtOH; b) chromatography.

followed by cyclisation with base gave a cis/trans mixture of the thiopyran oxides 6 which could be separated by column chromatography. Treatment of the mixture or separated isomers with strong base followed by methyl isothiocyanate


gave the thioamide 1 (RP 49356). The thioamide 1 could also be synthesised indirectly from 6 via the treatment of the carbanion with CS2/MeI to give the dithioester 7 followed by treatment with methylamine. Both direct and indirect routes gave exclusively the isomer in which the thioamide is trans to the sulfoxide. The cis isomer 10 was synthesised from the dithioester 7 by reduction to the sulfide 8, which upon reoxidation gave a mixture of cis and trans sulfoxides 9. The mixture was condensed with methylamine to give, after chromatographic separation, the cis thioamide 10. The active enantiomer of RP 49356 (aprikalim) was synthesised by the route outlined in Scheme 3.2 (Aloup et al., 1990). The acid 11 was resolved via the tBu-L-prolinate amides and the R (–) enantiomer 12 converted to the thioamide 13. Conversion of 13 to the dithioester 8 was followed by oxidation to give a mixture of diastereomeric sulfoxides, from which the desired sulfoxide 7 was obtained by column chromatography. Amination of 7 with methylamine alforded aprikalim. Aprikalim is the laevo rotatory enantiomer of RP 49356 with an absolute configuration (1R, 2R) as determined by X-ray diffraction. KCA potency of aprikalim and close analogues Later studies have shown that 1 acts by activating the KATP channel (Mondot et al., 1988; Escande and Thuringer, 1989; Brown et al., 1992). A variety of screens have been described in the literature for the evaluation of KCAs. In the thioformamide series the in vitro screen is based upon the concentration (IC90) of compound required to relax by 90% the contraction induced by 20 mMKCl on rat aortic strip (Brown et al., 1992). The smooth muscle relaxation was reversed by the addition of

Scheme 3.2 Synthesis of Aprikalim The depicted stereochemistry is absolute Reagents : (i) a) SOCl2; b) tBu-L-protinate; c) cHCI; (ii) a) SOCI2; b) MeNH2; c) P2S5; (iii) a) BuLi, Mel; b) pyridine HCI, H2S; (iv) MCPBA; (v)MeNH2

the KATP channel inhibitor glibenclamide or by high concentrations of KCl (60 mM).


The KCA in vitro potencies of aprikalim and close analogues are collected into Table 3.1. Whereas the parent sulfide 13 is inactive, aprikalim exhibits an activity similar to cromakalim and twice that of RP 49356. The other enantiomer (1S, 2S) of RP 49356 and the racemic cis analogue 10 are both inactive. Stereochemistry and molecular modeling of aprikalim The absolute configuration of aprikalim has been established by X-ray determination (Brown et al., 1992) and the trans relationship between the thioamide and sulfoxide

Table 3.1 In vitro KCA activities of aprikalim analogues (IC90)μ M 13 1

10 cromakalim

RP 49356 aprikalim (1S, 2S) cis (racemate)

>30 0.7 0.4 >30 >30 0.2

confirmed. The authors also described conformational analysis studies with full geometry optimization using the semiempirical molecular orbital programmes MOPAC and the AM 1 Hamiltonian. It was found that several low-energy conformations could exist within a 2 kcal/mol energy band of the lowest energy structure. The global minimum energy conformation of aprikalim is depicted in Plate 1 and corresponds to that determined by X-ray crystallography. The energy difference between this conformation with an axial thioamide in contrast to an equatorial group is small (― E 1.7 kcal/mol), some stabilization coming from an internal hydrogen bond (S― O…H—N) as shown in Plate 2. Similar conformational studies on the inactive cis sulfoxide showed that the lowest energy conformation of the molecule has the thioamide group axial (Plate 3), but twisted almost 180° relative to aprikalim, so as to possibly form an internal hydrogen bond to the now equatorial sulfoxide oxygen (S― O…H—N, 2. 1 Å from MOPAC/AM1 calculations). Conformations with the orientation of the thioamide and pyridyl groups corresponding to those in aprikalim, which could be considered as representing the ‘active’ conformation, have an energy greater than 5 kcal/mol above the lowest energy structure. 3.2.2 Structural Modification of Aprikalim The syntheses and SARs of a series of aprikalim analogues have been reported (Brown et al., 1992). For purposes of discussion the structural modifications may


be conveniently divided into changes to the pyridine, thioformamide and thiopyran ring. Modification of the pyridine ring The analogues in which the 3-pyridyl group of aprikalim has been modified synthesised by routes similar to that outlined in Scheme 3.1 for RP 49356, were starting from the appropriate chloromethylaryl analogue of 4. The efficiency the conversion of the sulfoxide into its carbanion and subsequent thioacylation of dependent upon the choice of base and reaction conditions. In all cases was compounds in which the thioamide is trans to the sulfoxide were formed. only The KCA potencies of the analogues are listed in Table 3.2. Linkage of pyridyl ring at the 2- or 4- rather than the 3-position reduces potency but potency the was maintained when a 6-chloro substituent was introduced into the 3-pyridyl analogue. Whilst the 3-quinolyl analogue exhibited a tenfold increase in potency over 1, other heterocyclic replacements showed only modest potency. In the series, the enhancement of potency by the presence of an electron withdrawing aryl group in the meta position was demonstrated by the Cl, CF3, CN and substituted analogues when compared to the unsubstituted phenyl compound. F This potency is reinforced by the presence of an extra para Cl substituent. parallel with the 3-quinolyl analogue, the 2-naphthyl analogue was more potent In than the phenyl analogue, indicating either an electron withdrawing hydrophobic effect. or a Modification of the thioformamide group Analogues of aprikalim with modified thioamide groups were synthesised (Brown et al., 1992) by the methods outlined in Scheme 3.1 from the intermediate

Table 3.2 Aryl and heterocyclyl thioformamides R

IC90(μ M)

3-pyridyl 2-pyridyl 4-pyridyl 6-Cl-pyrid-3-yl 3-quinolyl 2-Ph-thiazol-3-yl 5-isothiazolyl 2-benzthiazolyl phenyl 3,4-diCl-phenyl 3,5-diCl-phenyl 3-CF3-phenyl

0.7 >30 25 0.3 0.006 4 7 29 9.5 0.3 0.4 0.8


R

IC90(μ M)

3-CN-phenyl 3-F-phenyl 4-Cl-phenyl 2-naphthyl

1.6 7 2.8 2.0

dithioester 7 by reaction with the requisite amine; yields were generally low. The methylformamide was synthesized directly from 1 by treatment with nitronium tetrafluoroborate. The alcohol and thiol analogues were synthesised from the nitrile intermediate. SARs are collected into Table 3.3 and the requirement for a small alkyl group, preferably ethyl, is apparent indicating the presence of a small hydrophobic pocket. The total loss of activity of the amide is striking and the very precise requirements for high activity is reflected in the results for the dithioester, nitrile, alcohol and thiol analogues. Modification of thiopyran-1-oxide The activities of compounds with a modified thiopyran-1-oxide group are collected into Table 3.4. The essential requirement of a sulfoxide group, trans to the thioamide, is indicated by the loss in activity of the sulfone, thiane, 1,3dithiane, 1,3-oxathiane, tetrahydrothiophene and tetrahydrofuran analogues. The unsubstituted cyclohexane was unexpectedly active at 3 μ M. Full synthetic routes to these analogues have been described (Brown et al., 1992). Summary of SARs A summary of the SARs for optimal KCA potency in aprikalim analogues described here is represented in Figure 3.2. Optimal biological activity requires a 3-substituted

Table 3.3 Thioformamide analogues

R

IC90μ M

CSNHMe CSNHEt CSNHPr CSNHBu CSNHPh

0.7 0.3 0.6 8 >30


R

IC90μ M

CSNHNH2 CONHMe CN CH2OH CH2SH CSSMe

>30 >30 >30 >30 >30 >30

Table 3.4 Thiopyran-1-oxide analogues X

Y

IC90μ M

SO (trans) SO (cis) SO2 S S S S O CH2 CH2

CH2 CH2 CH2 CH2 S O db db CH2 db

0.7 >30 >30 >30 >30 >30 >30 >30 3 30

db=direct bond

pyridyl or quinolyl group and a thioformamide attached to the C-2 atom of a thiopyran-1-oxide. The absolute stereochemistry at the C-2 atom and a trans relationship of the thioformamide and sulfoxide groups are crucial for good activity. 3.2.2 Cyclohexanone Analogues The initial objective in modifying the sulfoxide of aprikalim was to synthesise the trans alcohol 18 (Cook et al., 1987); the route used is outlined in Scheme 3. 3. A


Figure 3.2 Summary of the SARs of aprikalim analogues

Wittig reaction on pyridine-3-carboxaldehyde gave the olefin 14, which, when treated with NBS, gave the bromohydrin 15. The rearrangement of 15 to the ketone 16 was accomplished with silver perchlorate and 16 was converted to the racemic thioformamide 17 by carbanion formation followed by thioacylation with methyl isothiocyanate. Potassium borohydride reduction of 17 furnished a mixture of alcohols (9:1 trans/cis) which were separated by column chromatography to give the trans alcohol 18. Whereas 18 had disappointing KCA potency (IC90 7.5 μ M), the intermediate ketone 17 was equipotent with RP 49356 (IC90 0.8 μ M). This unexpected discovery led to the syntheses and investigation of many cyclohexanone analogues. These changes have been briefly reviewed (Chapman, 1991, 1993).


Alkene and alkane analogues The first compounds to be synthesised (Scheme 3.4) were the alkene analogues (Brown et al., 1993) and the synthesis of the phenoxy analogue 24 is illustrative of the methods employed. Lithiation of 3-bromopyridine followed by reaction with 2-methoxycyclohexanone gave the intermediate alcohol 19, which when treated with c. H2SO4 resulted in a high yield conversion to the cyclohexanone 16. Reaction of the carbanion of 16 with CS2/MeI gave the dithioester 20 which was converted to the β , β -unsaturated nitrile by the Wadsworth-Emmons modification of the Wittig reaction. The nitrile was reduced to the aldehyde 22 with DiBAL, which upon further reduction with NaBH4 gave the alcohol 23. The alcohol was converted to the phenoxy thioamide 24 by a Mitsunobu reaction followed by treatment with ethanolic methylamine. The KCA potencies of the alkene and alkane analogues are shown in Table 3.5. The β , β -unsaturated nitrile 25, aldehyde 26 and alcohol 27 had reduced activities when compared to the ketone 17. Introduction of an aromatic nucleus by functionalisation of the alcohol as phenoxy 24 or thiophenyloxy 28 led to a 500-fold increase in activity, suggesting the occupation of an extra binding site. Changing the sp2 geometry of the unsaturated nitrile 25 to sp3 as in the alkane 29 (Table 3.5) gave a threefold increase in activity. Interestingly it was subsequently

Schema 3.4 Synthesis of alkene analogues


Table 3.5 Alkene and alkane derivatives of ketone 17

compd

R

17

IC90μ M

compd

R

IC90μ M

0.800

29

CN

0.3

25

CN

10

30

CH2OH

1.7

26

CHO

10

31

CH2OCOCH3

0.03

27

CH2OH

30

32

CH2OCOPh

0.03

24

CH2OPh

0.065

33

CH2OCOC6H4-p-F

0.1

28

CH2SPh

0.003

34

CH=NOCH2Ph

0.03

35

CH2NHPh

0.003

36

CH=CHPh

0.1

37

CH=CHC6H4-p-F

0.01

found that the cyanomethyl side chain in 29, obtained by LiAlH4 reduction of 38 (Scheme 3.5), was trans to the thioformamide group therefore paralleling the arrangement in aprikalim. A range of substituents were synthesised similar to the alkene analogues and the increase in activity was maintained. Further functionalisation with analogues containing an extra aromatic nucleus in the form of an ester 32–33, oxime ether 34, amine 35 or styrene 36–37 gave the expected increase in activity. A low energy conformation of the nitrile 29 can be superimposed over the Xray conformation of aprikalim (Figure 3.3) and a good overlay of the axial sulfoxide of aprikalim with the cyanomethyl side chain can be obtained (Brown et al., 1993). Sulfonamide analogues As an extension of the alkane chemistry, a series of sulfonamide analogues were synthesised and the SARs were found to follow a similar pattern culminating in the synthesis of 40 (RP 66784) with a KCA IC90 of 0.3 nM; the aryl sulfonamido group contributing to the increased activity. The synthesis of 40 is outlined in Scheme 3.5 starting from ketone 17. Reaction with diethylcyanomethyl phosphonate in a Wadsworth-Emmons reaction gave the β , β -unsaturated nitrile 38. Room temperature reduction of 38 with LiAlH4 gave exclusively the saturated trans nitrile 29. This selectivity can be understood by complexation of LiAlH4 with the thioformamide group followed by delivery of hydride from the same face. The


Figure 3.3 Stereoplot of cyanomethyl analogue 29 superimposed on aprikalim

trans geometry was unambiguously established (Brown et al., 1993) by X-ray analysis of an ester derivative. Further reduction of 29 with LiAlH4 at elevated temperature gave the ethylamine 39 which was acylated with PhSO2Cl to give racemic sulfonamide 40. Oxime, hydroxylamine and amine analogues Oximes 41 and derived hydroxylamines 42 were synthesised from 17 (Cook et al., 1987) by standard procedures. In all cases the oxime was isolated as a single compound and assumed to have the anti-configuration. As in the case of the alkyl amine 39, only the trans N-substituted hydroxylamine 42 was formed upon reduction of 41 with LiAlH4. SARs for both series parallel those of the alkane series with optimal activity where R=substituted phenyl.


As an extension of the oxime chemistry, two asymmetric syntheses of 17 have been developed (Hart et al., 1992). In the first route (Scheme 3.6) condensation of the ketone 16 with (S)-(-)-l-amino-2-(methoxymethyl)pyrrolidine (SAMP) using Enders methodology (Enders and Eichenauer, 1976) gave the hydrazone 43. Lithiation followed by treatment with methyl isothiocyanate produced a mixture containing, for the most part, the diastereomer 44 in 80% diastereomeric excess (d.e.) as measured by hplc. Acid hydrolysis of the crude reaction mixture gave the (S)-ketone thioformamide 17 which was easily purified to homochirality by recrystallisation. Because problems were encountered in recycling the chiral auxiliary, cheaper alternatives were investigated. This led to the use of (R)-(+)-β -methylbenzylamine (Pfau et al., 1985) which when condensed with 16 afforded the Schiffs base 45. Lithiation and thioacylation gave 46 in a d.e. of 80%. Hydrolysis led

to the chirally pure (S)-ketone 17. The diastereomer 46 was also reduced with NaCNBH3 to give the chirally pure trans benzylamine 47, which was found to be an extremely potent compound (IC90 0.03 nM). Ester analogues Given the increase in KCA potency imparted by the extra phenyl group, it was decided to synthesise ester analogues of the original trans hydroxy compound 18 (Scheme 3.3). Both chiral and nonchiral esters were made (Hart et al., 1989) and the most potent compounds are collected into Table 3.6. In the benzoate ester, the activity resides in the (1R, 2S) enantiomer.


Summary In the cyclohexanone series SARs also parallel those of the sulfoxides regarding the pyridyl group and thioformamide modifications. The chirality is an important feature as is the trans relationship of the thioformamide group with adjacent substituents, the absolute configuration of eutomers being the same as that in aprikalim. The enhanced potency associated with hydrophobic derivatives provides evidence for the existence of a lipophilic binding site at the receptor. The aromatic binding group can accommodate differing substituents and a variety of linking groups, both as sp2 (alkene, oxime) and sp3 (alkane, sulphonamide, amine and ester). The most potent analogues had subnanomolar KCA potency in the primary screen. 3.2.3 Other Syntheses and Hybrid Structures An alternative approach to the synthesis of aprikalim analogues via a DielsAlder reaction on the thioketoester 48 to the carboxamide 49 has been reported (Pinto et al., 1992). The conversion of the amide to aprikalim was not described Table 3.6 Ester analogues of 18

R

IC90nM

Me nBu Ph (1R 2S) Ph (IS 2R) 4-F-Ph 3-pyridyl

100 1 0.1 3000 0.3 0.1

presumably because of synthetic difficulties. Whereas no hybrid aprikalim analogues have been described, the pyrrolidinone group in CRK has been successfully replaced by thioformamide in structures of type 50 (Ishizawa et al., 1993). The SAR of such compounds is discussed in Chapter 2.


3.3 Cyanoguanidines 3.3.1 Pinacidil and Close Analogues Discovery and initial SAR studies Pinacidil was developed from a series of N-alkyl-N'-pyridyl thioureas, which were known to have hypotensive activity (Petersen et al., 1978) and, as was the case with aprikalim, the KCA properties of pinacidil were unknown. Pinacidil 53 was synthesised from the 4-pyridyl isothiocyanate 51 via the thiourea 52 (Scheme 3.7). The enantiomers of pinacidil were initially synthesised by kinetic resolution of the tartrates (Arrigoni-Martelli et al., 1980). More recently the enantiomers have been synthesised via chiral reduction (Manley and Quast, 1992). Reaction between pinacolone with (R)-β -methylbenzylamine (Scheme 3.7) gave the chiral imine 54. Reduction with diborane resulted in the addition of hydrogen to the azomethine double bond from the face opposite to that occupied by the bulky phenyl group to give the diastereomer 55. Catalytic hydrogenolysis afforded optically pure amine 56, which was converted through to the (R)enantiomer of pinacidil by the route used for 51–53. The corresponding (S)enantiomer was synthesised using the corresponding (S)- β -methylbenzylamine. The initial structure/activity data on pinacidil and analogues was based upon SHR hypotensive data (Petersen et al., 1978) and is summarized in Figure 3.4. In general 3-substituted pyridyl compounds were up to 20-fold more potent than the 4-pyridyl analogues; however pinacidil was an exception in being more potent than the 3-pyridyl isomer. Further ring substitution in the 3-series was usually detrimental. In the side chain, the thiourea was more active than the urea, but the cyanoguanidine


Figure 3.4 Hypotensive SARs of pinacidil analogues

was 200-fold more potent than the thiourea. Replacement of the cyano group with, for example, COOEt, OMe, OH and CONH2 led to a reduction in activity. A branched alkyl group containing 4–5 carbon atoms was optimal at the terminal position of the side chain. KCA activity of pinacidil analogues It is now known that pinacidil relaxes blood vessels by additional non K channeldependent mechanisms (Cook et al., 1989) making blood pressure (BP) data unreliable as an indicator of KCA potency. Consequently KCA potency data based upon the inhibition of spontaneous myogenic activity in rat portal vein was generated (Manley and Quast, 1992) and is collected into Table 3.7. The data demonstrate the higher activity of the NCN, CHNO2 and to a lesser extent S analogues over the ureas 57 and 61. The 3-pyridyl analogues were invariably more active than the corresponding 4-pyridyl counterparts e.g. 62 and 59, 63 and 53, 64

Table 3.7 KCA activities of pinacidil and analogues (rat portal vein) Pyr

57 58 59 53 60 61 62 63 64

Pyr

R

X

pIC50

4-pyridyl 4-pyridyl 4-pyridyl 4-pyridyl (pinacidil) 4-pyridyl 3-pyridyl 3-pyridyl 3-pyridyl 3-pyridyl

CH2CMe3 (R) CHMeCMe3 (S) CHMeCMe3 (±) CHMeCMe3

O S S NCN

5.5 6.3 6.5 7.2

(±) CHMeCMe3 CH2CMe3 (±) CHMeCMe3 (±) CHMeCMe3 (±) CHMeCMe3

(E)-CHNO2 O S NCN (E)-CHNO2

6.0 5.7 7.3 7.8 7.5


65 66

Pyr

R

X

pIC50

3-pyridyl 3-pyridyl

(R) CHMeCMe3 (S) CHMeCMe3

(E)-CHNO2 (E)-CHNO2

6.0 8.0

and 60. In the nitroethene series the higher activity resided in the (S)-enantiomer 66 rather than the (R)-enantiomer 65. Stereochemistry and molecular modeling of pinacidil analogues A receptor binding model (Figure 3.5) has been developed to explain the KCA potencies of pinacidil analogues (Manley and Quast, 1992). The relative inactivity of the ureas was explained on conformational grounds since only the cyanoguanidines, nitroethenediamines and thioureas can adopt similar relatively low energy staggered conformations of type 67–69, which could correspond to the active conformation at the receptor. Interestingly an X-ray determination of pinacidil (Pirotte et al., 1993a) has the unusual conformation 70. It was also suggested that the pyridine nitrogen was acting as a hydrogen bond acceptor with the interaction being less favourable for the 4-pyridyl compound. Evidence from pKa measurements suggested that the relative acidity of the pyridyl amino group determined the ease to which it could act as a hydrogen bond donor. The disparate geometry requirements for activity between enantiomers was explained by the receptor undergoing conformational changes in order to accommodate either the bulkier CHNO2 or NCN moieties. In essence three receptor binding elements are required as shown in Figure 3.5: a H-bond donating site (pyridyl NH), flanked by a H-bonding acceptor site (pyridine N atom) and a lipophilic site (CH(Me)CMe3 group).


3.3.2 Other Pinacidil Analogues Substituted phenyl analogues of pinacidil Paralleling the replacement of the pyridine in aprikalim with substituted aryl groups (Table 3.2), a series of aryl cyanoguanidines have been synthesised by methods

Figure 3.5 A minimum energy conformation of 66 showing proposed receptor binding interactions. Copyright ACS 1992.

similar to those used for pinacidil (Atwal et al., 1992a). SARs are collected into Table 3.8 and, as expected, the presence of an electron withdrawing group is optimal for activity, although there is no clear preference for the location of the substituent at either the meta or para positions. The bulky 1,2,2-trimethyl propyl group is optimal for activity. The analogues 71 (Nishimaru et al., 1990) and 72 (Lenfers et al., 1991) have been reported to have KCA potency. The potency of 72 is surprising in that one would predict an electron donating group to reduce potency. However 72 is reported to be equipotent with pinacidil.

Aminopyridine analogues The KCA potency of a series of aminopyridine thioureas has been reported (Takemoto et al., 1994). They were synthesised by the route similar to that used for pinacidil (Scheme 3.7) and their activities are collected into Table 3.9. The activity increased with increasing bulk of the terminal alkyl group, but altering


the position of the amino substituent on the pyridine ring did not have a profound effect upon activity. Maximal hypotensive activity was seen in the 6-amino compound, and all further changes had this group constant. Activity increased with a 1,2,2-trimethylpropyl group (the pinacidil substituent) and was optimal as a l-methyl-2-norbornyl group. Isosteric transformation of thiourea to cyanoguanidine gave an even more active compound (pEC100 7.5). 3.3.3 Hybrid Analogues The cyanoguanidine group has been incorporated into CRK with both aryl 73 (Atwal et al., 1993) and cycloalkyl 74 (Atwal et al., 1992b) derivatives. It is claimed that 73 possesses anti-ischaemic properties without significant vasodilatation. The

Table 3.8 Phenyl analogues of pinacidil Ar

IC50μ M(rat aorta)

Ph 3-CNphenyl 4-CNphenyl 2-NO2phenyl 3-NO2phenyl 4-NO2phenyl pinacidil

0.88 0.42 0.022 0.24 0.17 5.15 0.07

Table 3.9 Aminopyridine analogues of pinacidil R'

R

pEC100 (rat portal vein)

Me nBu cyclohexyl cyclohexyl cyclohexyl

4-NH2 4-NH2 4-NH2 2-NH2 5-NH2

IA 3.0 4.0 4.0 5.0


R'

R

pEC100 (rat portal vein)

cyclohexyl 1-Me-2-norborny1 l-Me-2-norbornyl (NCN) pinacidil

6-NH2 6-NH2 6-NH2

5.0 7.0 7.5 7.0

cycloalkyl analogue 74 was equiactive with cromakalim as a vasodilator. The diazoxide hybrid 75 has also been reported (Pirotte et al., 1993b) and found to be a powerful inhibitor of insulin release. Further discussion of certain of these compounds appears in Chapter 2.

3.4 Organic Nitrates 3.4.1 Nicorandil Nicorandil 77 (Furukawa et al., 1981) was the first organic nitrate to demonstrate KCA potency and was synthesised (Scheme 3.8) in two steps from methyl nicotinate via the alcohol 76. It has also been shown that nicorandil stimulates guanylate


Figure 3.6 Nicorandil analogues

cyclase in vascular smooth muscle (Holzmann, 1983) and has therefore a dual mode of action and the association of KCA and organic nitrate-like potency leads to powerful vasodilators. 3.4.2 Analogues of Nicorandil Several analogues of nicorandil 78–83 have been reported and are collected into Figure 3.6. The closest analogues all retain the nicorandil side chain together with a substituted pyridine 78 (Miura et al., 1990), a pyrazine 79 (Ito et al., 1991) or a thiazole 80 (Satake et al., 1992). The amide of nicorandil can be replaced by a cyanoamidine 81 (KRN 2391; Ishibashi et al., 1992). When the nitrate group of 81 was replaced with a phenyl group as in 82, a potent vasodilator was obtained which was found to act by K channel activation only (Okada et al., 1993). The cromakalim hybrid 83 has been reported (Evans and Frydrych, 1990). 3.5 Other Pyridine KCAs 3.5.1 Anilide Tertiary Carbinols During an investigation of anti-androgen compounds, the KCA activity of a series of anilide tertiary carbinols e.g. 84 (Russell et al., 1992) was unearthed and found to be about eightfold more potent than CRK on guinea pig detrusor (bladder) strip mildly depolarized with 15 mMKCl (Table 3.10). The compounds were synthesised by the reaction between the requisite aniline and acid chloride


in the presence of DMAP. Subsequently the activity of the (S)-pyridylsulfone 85 (Grant et al., 1993) and the phenylsulfone 86 (Russell et al., 1993) have been reported. The compounds are being pursued mainly in the area of urinary urge incontinence.

Table 3.10 Anilide tertiary carbinols IC50 μ M KCA potency (g.p. detrusor strip) 84 85 cromakalim

0.07 1.6 0.57

3.6 Conclusion A wide variety of structural types exist within the pyridine based KCAs. Combining these structures with CRK, common pharmacophoric features have been suggested (Atwal et al., 1992a, 1992b) and a common pharmacophoric model proposed (Koga et al., 1993). Although there are indeed common structural features between the benzopyrans, pyridyl thioformamides and cyanoguanidines, the evidence for a common pharmacophore is not compelling. Functional studies with ET-1 and different classes of KCAs suggest that the benzopyrans interact at a different site to pinacidil and aprikalim (Lawson et al., 1992). Binding studies using a tritiated pinacidil analogue (Manley et al., 1993) suggest that differing structural types of KCA probably interact with different receptor binding sites further weakening the evidence for a common pharmacophore. Exploration of pyridine based KCAs has led to some very potent compounds; some thioformamides have subnanomolar activity. Within the thioformamide series, a clear understanding of SARs has been obtained. The secondary thioformamide is crucial for good activity and must be in a trans relationship with a sulfoxide (aprikalim analogues) or an aryl side chain (cyclohexanone analogues). The enhanced potency associated with the hydrophobic side chain provides evidence for the existence of an extra lipophilic binding site at the receptor. More limited SARs are available for pinacidil and nicorandil analogues and they possess additional properties to that of K channel activation to account for their pharmacological profiles. Aprikalim has been selected for development for angina, and at nonhypotensive doses, may prove useful in the treatment of ischaemia. Pinacidil


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ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. ITO, Y., KATO, H., ETSUCHU, E., OGAWA, N., MITANI, K., IWASAKI, N. & HOKURIKU SEIYAKU (1991) Japanese Patent 89056667-A. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993) BioMed. Chem. Letters, 3, 625–631. LAWSON, K., BARRAS, M., ZAZZI-SUDRIEZ, E., MARTIN, D.J., ARMSTRONG, M. & HICKS, P.E. (1992) Br. J. Pharmacol., 107, 58–65. LENFERS, J.B., MUSCHALEK-LETINA, V., NIEMERS, E., JANIS, R.A. SCRIABANE, A. (1991) U.S. Patent 661720. MANLEY, P.W. & QUAST. U. (1992) J. Med. Chem., 35, 2327–2340. MANLEY, P.W., QUAST, U., ANDRES, H. & BRAY, K. (1993) J. Med. Chem., 36, 2004–2010. MIURA, K., KOYAMA, H., SUGAI, T., YAMADA, H., SAKURAI, E., HORIGOME, M. (1990) European Patent 385350. MONDOT, S., MESTRE, M., CAILLARD, C.G. & CAVERO, I. (1988) Br. J. Pharmacol., 95 (Supp), 813 P. NlSHIMARU, N., IWAMOTO, T., SUKAMOTO, T., YOSHIIZUMI, K., SEKO, N. & YOSHINO, K. (1990) Japanese Patent 01657. OKADA, Y., YOKOYAMA, T., JINNO, Y., KASHIWABARA, T., IZAWA, T., FUKUSHIMA, H. & OGAWA, N. (1993) Eur. J. Pharmacol., 241, 177–181. PETERSEN, H.J., NIELSEN, C.K. & ARRIGONI-MARTELLI, E. (1978) J. Med. Chem., 21, 773–781. PFAU, M., REVIAL, G., GUINGANT, A. & D’ANGELO, J. (1985) J. Am. Chem. Soc., 107, 273–274. PINTO, I.L., BUCKLE, D.R., RAMI, N.K. & SMITH, D.G. (1992) Tetrahedron Lett., 33, 7597–7600. PlROTTE, B., DUPONT, L., DE TULLIO, P., MASEREEL, B., SCHYNTS, M. & DELARGE, J. (1993a) Helv. Chim. Acta., 76, 1311–1318. PIROTTE, B., DE TULLIO, P., LEBRUN, P., ANTOINE, M-H., FONTAINE, J., MASEREEL, B. , SCHYNTS, M., DUPONT, L., HERCHUELZ, A. & DELARGE, J. (1993b) J. Med. Chem., 36, 3211–3213. RUSSELL, K., OHNMACHT, C.J. & GIBSON, K.H. (1992) European Patent 524781. RUSSELL, K., EMPFIELD, J.R., OHNMACHT, C.J. & GIBSON, K.H. (1993) WO 9323358. SATAKE, N., KIVOTO, S., ZHOU, Q., MATSUO, M. & SHIBATA, S. (1992) Faseb J., 6, Abs. 360. TAKEMOTO, T., EDA, M., OKADA, T., SAKASHITA, H., MATZNO, S., GOHDA, M., EBISU, H., NAKAMURA, N., FUKAYA, C., HIHARA, M., EIRAKU, M., YAMANOUCHI, K. & KAZUMASA, Y. (1994) J. Med. Chem., 37, 18–25.

Recent Literature EDA, M., TAKEMOTO, T., ONO, S., OKADA, T., KOSAKA, K., GOHDA, M., MATZNO, S., NAKAMURA, N. & FUKAYA, C. (1994) Novel Potassium Channel Openers: Preparation and Pharmacological Evaluation of Racemic and Optically


Active N-(6-Amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine Derivatives. J. Med. Chem., 37, 1983–1990. FRANK, C.A., FORST, J.M., GRANT, T., HARRIS, R.J., KAU, ST., NAKAJIMA, T., KASHIWABARA, T., IZAWA, T. & NAKAJIMA, S. (1994) StructureActivity Studies of N-Cyano-3-pyridinecarboxamides and their Amide and Thioamide Congeners. BioMed. Chem. Lett., 4, 2485–2488.

4 Conformational Analysis of Potassium Channel Activators C.M. EDGE SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

4.1 Introduction Potassium channel activators (KCAs) represent a varied structural class of molecules that interact with ATP-sensitive potassium channels (KATP). Their action on these ion channels results in smooth muscle relaxation and has led to KCAs being proposed as potential drugs for the treatment of asthma and hypertension, for example. A study of the conformations adopted by this class is of interest in the design of novel therapeutic agents and in the characterization of the KATP channel itself. This chapter therefore includes conformational analysis data on the three main classes of KCA—namely, cromakalim (CRK), aprikalim, pinacidil and some of their analogues. The majority of the calculations reported in this chapter are at the semi-empirical MNDO level of theory, using the AMI Hamiltonian. This seems to have been accepted as the de facto standard in this field, notwithstanding its limitations, such as low barrier heights and poor description of some sp2 nitrogens. Novel calculations made for this chapter have used this theoretical method for consistency with previously published work. 4.1 Conformational Analysis of Cromakalim 4.1.1 Structure The structure of CRK, 1, has been studied using the techniques of X-ray crystallography (Cassidy et al., 1990) and nuclear magnetic resonance (NMR) (Thomas and Whitcombe, 1990). These reports showed that the pyran ring adopts a half-chair conformation, in which the 4-pyrrolidinone is pseudoequatorial and the 3-hydroxyl is equatorial. The 3- and 4-substituents are trans to each other, with the more potent enantiomer having 3-(S), 4-(R) stereochemistry.


Plate 4 shows the X-ray crystal structure of this enantiomer, levcromakalim (LCRK).

4.1.2 Rotation of the 4-pyrrolidinone in Cromakalim The rotational preference of the 4-position of CRK has been the subject of some debate. NMR experiments conducted in deuteriochloroform at 270 MHz seemed to suggest that there was a single, rigid conformer, 2 (Cassidy et al., 1990). Semiempirical calculations (Cassidy et al., 1990), using the AMI Hamiltonian (Dewar et al., 1985) suggested that there was an energy difference of 2.4 kcal/mol between this minimum and a higher energy rotamer, 3, found upon rotation of the C4-N1' bond. Assuming a Boltzmann distribution, this corresponds to about a 98:2 prevalence of the lower energy form. The presence of 2% of the higher energy rotamer was not detected by the original NMR studies.

CONFORMATIONAL ANALYSIS OF KCAS 93

In contrast to the earlier studies, the NMR spectrum in deuteriomethanol showed the presence of two structural forms at -60°C (Thomas and Whitcombe, 1990). The major component was shown to be identical to that identified by Cassidy et al.—the rotamer in which the carbonyl oxygen is on the same side of the molecule as the 4-proton. (This is the lower energy structure, 2, found in the AM1 calculations.) The minor component at -60°C corresponded to the higher energy structure, 3, in which the sp2 oxygen and the 4-H are on opposite sides. The barrier to rotation was estimated to be between 11.5 and 13.5 kcal/mol. From this NMR study, the difference in energy between the two forms was calculated to be 2.2 kcal/mol, which accords with the AM1 calculation mentioned above. Figure 4.1 shows a graph of the energy of LCRK versus the C10-C4-N1'-C2' dihedral angle. The calculation, like most in this chapter, has been made using the semi-empirical AM1 Hamiltonian (Dewar et al., 1985). Since this particular method is well known for failing to maintain the planarity of sp2 amide nitrogens (Stewart, 1990), a constant improper dihedral of 180° was imposed in the Zmatrix definition of The following colour plates are referred to in Chapters 3 and 4. Plate 1 Stereoview of the X-ray conformation of aprikalim 1. Copyright ACS, 1992. Plate 2 Stereoview of an AM1 low energy conformer of 1 with equatorial thioamide and sulfoxide groups. The dashed line (orange/white) represents a possible hydrogen bond. Copyright ACS, 1992. Plate 3 Stereoview of the AM1 lowest energy conformer of the cis isomer 10. The dashed line (orange/white) indicates a hydrogen bond between the equatorial sulfoxide and axial thioamide groups. Copyright ACS, 1992. Plate 4 Orthogonal views of the X-ray crystal structure of LCRK. Plate 5 The half-chair and boat structures of LCRK, clockwise from top left 2, 6, 5, 7. Plate 6 Notional coordinate system for the flipping of the pyran ring of LCRK. Half-chair loci are in red; boat loci in blue. The Cremer and Pople β parameter runs from 0–180° from pole to pole. The β parameter runs from 0–360° longitudinally. Plate 7 Structures of the 3-(R),4-(S) enantiomer, 14, of LCRK and the 3-(R),4(R) enantiomer, 15, of cis CRK.


Plate 8 The proposed overlap of indanol (blue) and chromanol (red) structures. Plate 9 An overlap of benzoxepine (blue) and chromonol (red) structures. Plate 10 Electrostatic potential surface of thieno [2,3-b]pyran, thieno [3,2-b] pyran and thieno [3,4-b]pyran structures. Areas of negative potential are coloured red. Plate 11 Local minimum energy conformations of the trans diaxial and tram diequatorial structures of RP52891, reported in Table 4.4. Plate 12 Local minimum energy conformations of six twist boat structures of RP52891, reported in Table 4.4. Plate 13 The X-ray crystal structure of pinacidil. Plate 14 Contour map of AM1 energy for two driven torsion angles of pinacidil. The torsion angles are shown in yellow and pink, and the loci of the X-ray crystal structure are shown in yellow and the loci of the proposed overlap structure are shown in red.















Figure 4.1 Graph of AM1 heat of formation versus C10-C4-N1'-C2' dihedral angle for LCRK

the third atom connected to the pyrrolidinone nitrogen. The graph shows minima at -130° and at 70°, corresponding to structures 2 and 3 respectively. Full geometry optimization of these two structures (including the aforementioned dihedral angle) resulted in structures of energy -65.8 and -63.4 kcal/mol respectively. The dihedral angles C10-C4-N1'-C2' were -129° and 83° respectively. The difference in energy between the two minima is 2.4 kcal mol. The barrier to interconversion between the two minima can be estimated to be 8.2 kcal/mol from a consideration of the lower of the two maxima on the graph in Figure 4.1. The barrier to free rotation, based on the higher energy maximum, is 9.7 kcal/ mol. It must be emphasized that these are only estimates of the barrier, since the maxima are not true transition states on the reaction coordinate. Also, AM1 barriers are known to be underestimates of the true case by a factor of about 2 or 3 (Fabian, 1988; Gundertofte et al., 1991). The structure of the graph shows that the obstacle to rotation is the 5-position hydrogen on the benzopyran nucleus. This atom clashes with the 5'-hydrogens of the pyrrolidinone for the lower barrier and with the carbonyl oxygen for the higher one. The form of the barrier can also be reproduced by simple molecular mechanics forcefields, such as the one in the Sybyl molecular modelling program (Tripos Associates), also suggesting that it is governed by the steric constraints of the system. In contrast to the above, the carbonyl oxygen of the piperidinone of 4, a BRL55834 carbamate analogue, was shown to point away from the C4 proton by X-ray crystallography (Buckle et al., 1992).

4.1.3 Flipping the Pyran Ring In addition to the rotational preferences of the 4-substituent in LCRK, the conformational preferences of the pyran ring have also been investigated. The experimental evidence points toward one half-chair structure for the benzopyran


ring, 2. Other possible structures include the opposite half-chair, 5, and two boat structures, 6 and 7. These structures are shown in Plate 5. An inspection of the energy surface with respect to the pyran ring internal dihedrals was attempted using the AM1 Hamiltonian. The position of n ring atoms may be described using n-3 dihedral angles—3 angles in the case of LCRK. One of these angles, that of Ol-C9-C10-C4, can be assumed to be 0°, as the pyran ring is fused to a benzene ring. Thus we are left with two dihedral angles to describe the ring geometry, namely C9-C10-C4-C3 and C10-C9-O1-C2.

Plate 6 shows a diagram of LCRK on a notional rotational coordinate, using the ― and β values from the Cremer and Pople method (Cremer and Pople, 1975) of characterizing ring conformations. The Cremer and Pople method gives values of ― of 132° and 49° and values of β of 23° and 203° for the two minima 2 and 5 (a cyclohexane half-chair would give ― values of 129.2° or 50.8° and β values of 30, 90, 150, 210, 270 or 330°). These structures are shown as red points on the β , β surface. The difference in energy for these two structures is 3.5 kcal/mol. Thus we would expect to see a preponderance of the lower energy half-chair, 2. This is in accord with the NMR and X-ray studies mentioned in section 4.1.2. In addition to the two minima discussed above, two saddle points are discernible in Plate 6. These are shown as blue points on the β , β surface. The Cremer and Pople values for the pyran rings have been calculated. Structure 6 has the following values: β =93°, q3=0.0, q2=0.53, Q=0.53, β =116° and structure 7 has β =80°, q3=0.1, q2=0.52, Q=0.53, β =280°. The characteristic values for boat and twist-boat structures are β =90°, q3=0, q2=Q. Perfect boat structures have β values of 0, 60, 120, 180, 240 or 300° and perfect twist-boats have values of 30, 90, 150, 210, 270 or 330°. Thus, it can be seen that structure 6 is a true boat structure whereas structure 7 is a distorted form, between a boat and twist-


boat. These are stationary points on the LCRK energy surface, but are not minima. They are true transition structures, each having one calculated imaginary frequency. One can estimate the barrier to interconversion, in an analogous manner to the interconversion of rotamers above. The AM1 barrier is 4.8 kcal/mol between the lower energy half-chair, 2, and 7, the boat with the equatorial 3-OH. The barrier via the other boat, 6, with the axial 3-OH, is higher, at 6.5 kcal/mol. A full ‘pseudorotation’ of the ring would therefore require at least 6.5 kcal/mol. It is striking that the points on the β , β surface almost form a plane, suggesting that there may be a circular path, as shown in Plate 6. This is not necessarily the case and may reflect the nature of the dihedral sampling used to identify the stationary points. There are probably two other saddle points on the ‘pseudorotation’ path as there are minima close to the saddle points, but it has proven to be extremely difficult to find these. One of the main difficulties lies in the facility in finding trivial transition states due to rotation of the 2-methyl and 3-hydroxyl groups. It is not unusual to see two or three negative eigenvalues of the force constant matrix of a suspected transition state. The 2-monomethyl analogues of LCRK have been synthesized (Buckle et al., 1991a) and the structures of the two isomers were identified by NMR techniques. The 2β -monomethyl compound, 8, was shown to exist predominantly in one form. This was the 2-(R), 3-(S), 4-(R) form in which all three substituents were equatorial (or pseudo-equatorial), with the benzopyran ring in a half-chair conformation. This structure is the global energy minimum as calculated using the AM1 Hamiltonian (Table 4.1). The flipped half-chair structure, 9, is destabilized slightly, compared to the dimethyl analogue of LCRK. The energy difference between the two half-chairs is 3.9 kcal/mol rather than 3.5 kcal/mol found for LCRK. The 2β -monomethyl compound, 10, exhibited a more complicated set of nOe difference spectra, compared with that of compound 8. These were rationalized as being due to the presence of an equilibrium between two structures, 11 and 12. Semi-empirical molecular orbital calculations on these structures show that 11 is 2.4 kcal/ mol lower in energy than 12. This is a lower energy difference than that seen for LCRK, as the higher energy half-chair has an axial hydrogen at the 2position rather than a methyl group. This proton will have less of a steric repulsion from the pyrrolidinone ring than the methyl group. Interestingly, the calculations identify 13 as the global energy minimum for the 2β -monomethyl compound, rather than 11. This structure is broadly similar to 11, except that the pyrrolidinone carbonyl group is on the Table 4.1 The AM1 energies of 2-monomethyl analogues of LCRK, showing both minima upon rotation of C4-N1'. Structure

Energy (kcal/mol)

C10-C4-N1'C2' dihedral

8 9

-63.6 -59.7

-140.0° -116.5°


Structure

Energy (kcal/mol)

C10-C4-N1'C2' dihedral

8 9 11 12 11 12

-62.6 -59.5 -63.1 -60.7 -62.1 -64.0

91.0° 74.7° -138.9° -136.4° 90.7° 66.9°

opposite side of the ring from the 4-hydrogen. The carbonyl oxygen of 13 is 2.17 Å away from the axial 2-hydrogen, a suitable distance for an electrostatic interaction such as a hydrogen bond. However, this disagrees with the published solid state X-ray crystal structure, which is in the configuration represented by 12; it also does not accord with all the nOe data, since mutual nOes are observed between the 2-H and 5'-H protons. Although the calculations correctly predict that the 2-(S) compound, 10, is more likely to exist in a number of conformational states than the 2-(R) compound, 8, it seems that the predicted rank ordering of the minima is different from the X-ray structures. In part these results can be rationalized by considering that the AM1 calculations simulate the situation in the gas phase at zero Kelvin, which favours the formation of intramolecular hydrogen bonds. In contrast, the X-ray crystal structure contains an intermolecular hydrogen bond between the carbonyl of one molecule and the hydroxyl of another, thus determining the conformation adopted.


4.7.4 Stereochemistry LCRK, the more active enantiomer of CRK, has a trans 3-(S), 4-(R) stereochemistry. This structure has been examined above, in section 4.1.1. The other trans enantiomer, 14, is 100-fold less potent. Obviously, the relationship between the carbonyl group of the pyrrolidinone and the benzopyran ring is exactly reversed in the 3-(R), 4-(S) molecule. The dihedral angle across the C4N1’ bond, C10-C4-N1’-C2’, is 130°, rather than -130° found in LCRK for the global minimum structure. It is probable that this relationship between aromatic ring and hydrogen bond acceptor is not acceptable to the receptor associated with the KATP channel.


Both the enantiomers of cis CRK have been shown to possess potassium channel modulator properties (Quast and Villhauer, 1993). The 3-(R), 4-(R) enantiomer of cis CRK, 15, is at least twice as potent as the 3-(S), 4-(S) enantiomer, 16, and three times more potent than the 3-(R), 4-(S) enantiomer, 14 of CRK. Calculations show that the preferred structure of 15 is a half-chair similar to that of LCRK itself, but with the 3-hydroxyl group in an axial position. This interferes with the rotational characteristics of the 4-substituent, raising the energy of the opposite rotamer because of steric and electrostatic repulsions of the 2’-carbonyl oxygen and the 3-hydroxyl oxygen. The difference in AM1 energy of the two minima upon rotation of the 4-pyrrolidinone is 6.9 kcal/mol compared to 2.4 kcal/mol for LCRK. The higher energy rotamer is also pushed further round, away from the 3-position, making a dihedral angle of 47° for C10C4-N1’-C2’, rather than the more usual 70°. This is shown in Plate 7. The fact that the global minimum energy structures of LCRK and the cis 3-(R),4-(R)enantiomer of cis CRK, 15, differ only in the orientation of the 3-hydroxyl suggests that either the 3-hydroxyl is not important in KATP binding, or that a putative hydrogen bond donor (to the hydroxyl oxygen) or acceptor (from the hydroxyl proton) must be able to interact with both the axial and equatorial positions.


4.2 Studies on Benzopyran Ring Replacements 4.2.7 Replacements for the Pyran Ring The pyran ring of CRK has been replaced by a five-membered ring to give 3aminoindanol compounds, following molecular modelling studies (Buckle et al., 1991b). The compound, 17, was designed to have the same trans relationship between the 2-hydroxyl and the 3-amino substituent as between the 3- and 4positions of LCRK. Overlap studies showed that one could overlay these positions and keep the phenyl rings in the same plane by allowing the indane aromatic group to slide slightly toward the 1-position. This is shown in Plate 8.

The benzopyran ring system has also been replaced by a benzoxepine (Buckle et al., 1991b), to investigate the effect of increased bulk and conformational flexibility. In this series, the 2,3,4,5-tetrahydro-2,2-dimethyl-benzoxepine, 19, was of modest potency, but the di-nor-methyl compound, 18 was more potent than compound 19. Plate 9 shows a possible overlap of a chromanol and benzoxepine. The C2, C3 region of the benzoxepine, 18, occupies a region of space between the two methyl groups of LCRK and this accommodation may in part be responsible for the enhanced potency of compound 18. 4.2.2 Replacements for the Benzene Ring The electrostatic potential in the plane of the benzene ring of 6-cyanopyran was compared to that of a series of pyranopyridines (Stemp and Evans, 1993). On the basis of the similarity of the electrostatic potential maps, it was predicted that the [3,2-c]pyranopyridine ring system would most closely resemble the benzopyran. It was subsequently found that this pyranopyridine was a more potent KCA than [3,2b], [2,3-c] and [2,3-b]pyranopyridines. The benzene ring of CRK has been replaced by a thiophene ring by workers from R.W. Johnson (Sanfilippo et al., 1992, 1993; Press et al., 1993). Thieno[3,2-


b]-and thieno[2,3-b]pyran compounds, such as 20 and 21, with electronwithdrawing substituents have good activity that is comparable, or better than CRK. An unsubstituted thieno[3,4-b]pyran, 22, also has good activity, but the addition of the usual electron-withdrawing groups ortho to the sulphur atom reduces or abolishes activity. The authors suggest that not only is the electronwithdrawing function of the substituents important, but also the location is critical. They claim that the electrostatic potential maps calculated for typical thieno[2,3-b]pyran and thieno[3,2-b]pyran molecules have a similar structure, with what seems to be a large negative region extending over the middle of the thiophene ring and its substituent, directed away from the pyran. The thieno[3,4b]pyran series was claimed to exhibit a shift in the location of this negative region. The MNDO Hamiltonian (Dewar and Thiel, 1977a, 1977b, 1977c) was used to calculate these electrostatic potential maps. Plate 10 shows the electrostatic potential surrounding these structures. All three structures have a negative region of electrostatic potential above the carbons of the thiophene ring. This merges with the negative region due to the pyran oxygen in each case, but the cyano-substituted thieno[2,3-b]pyran and thieno[3,2–b]pyran structures have a more extended negative region, running towards the nitrile group.

4.3 Pyrrolidinone Replacements There have been many replacements for the pyrrolidinone ring of CRK reported in the literature. Chapter 2 contains an up-to-date review of these. The variety in the tolerated replacements allows us to sketch out some structural requirements for the benzopyran 4-position. The conformational properties of the 4substituents will be discussed in this section. The substituents have been arbitrarily divided into cyclic and acyclic replacements. A subsequent section will deal with attempts to build pharmacophore models, based on tolerated replacements.


4.3.7 Cyclic Replacements As discussed in detail above (section 4.1.2), the archetypal 4-substituent— pyrrolidinone—adopts an orthogonal orientation, relative to the benzopyran ring system. This is likely to be because of the steric repulsion suffered by the carbonyl and the 5’-hydrogens of the ring as they approach the peri-hydrogen at position 5, as discussed in section 4.1.2. Perhaps not surprisingly, many other rings of a similar size can adopt a similar orthogonal conformation. Many of these are five-or six-membered rings, with a nitrogen attached to the 4-carbon of the pyran ring. Table 4.2 lists some of the rings that have successfully replaced pyrrolidinone, along with the orientation of the internal N-C bonds of the rings, calculated using the AM1 Hamiltonian. One can see that each of these rings adopts a similar preferred conformation, with the ring approximately at right angles to the benzopyran. For all of these substituents, there are two possible minima found upon rotation of the C4-N1’ bond. The rotamer with the carbonyl oxygen on the same side of the ring as the 4-proton is the lower energy structure, usually by a few kcal/mol. Thus, all the ring structures in Table 4.2 can adopt similar orientations to pyrrolidinone, presenting a similar hydrogen bond accepting locus to the interaction site. The conformational behaviour of the piperidinone structure, 23, has been studied by NMR in a variety of solvents (Thomas and Whitcombe, 1990). A ratio of rotamers of the order of 88:12 was found in deuteriomethanol and deuteriodimethyl sulphoxide, and a ratio of 91:9 was found in D2O. These results may be compared to those for CRK mentioned in section 4.1.2.

Table 4.2 The orientation of various cyclic 4-substituents relative to the benzopyran template, derived from geometry optimized AM1 Hamiltonian calculations. R

Energy*

C10-C4N1’, -C2’, dihedral

Energy*


Energy* Difference

morpholinone

-102.1

-132.4

-98.4

70.5

3.7

oxazolidinone

-59.4

-119.1

-59.9

102.1

-0.5

piperazinone

-55.8

-131.7

-52.2

69.7

3.6

pyridone

-31.2

-142.5

-26.7

74.2

4.5

thiamorpholinone

-56.3

-133.6

-51.9

64.8

4.4

thiazolidinone

-28.9

-144.2

-28.2

99.9

0.7

* AM1 geometry optimized heat of formation

It has been reported that a 5’-methyl substitution on the pyrrolidinone enhances activity (Bartmann, 1989). This structure has a slightly modified energy profile


upon rotation of the C4-N1’ bond, because of the extra steric influence of the 5’ methyl group. The result of this steric influence is to rotate further the pyrrolidinone substituent in its lower energy form, giving a C10-C4-N1’ -C2’ dihedral angle of -66°, rather than -129° in the equivalent CRK structure. The difference in energy between the two minima on the rotation pathway is also reduced from the usual 2.4 kcal/mol to only 1.4 kcal/mol. The change in the preferred orientation of the 4-substituent suggests that the 5’-methyl group is destabilizing the lower energy rotamer, relative to the higher one, resulting in a lower energy difference between the minima. The fact that this compound and the piperidinone, 23, mentioned above, both have this smaller difference in energy and are both more potent than CRK, suggests that the higher energy rotamers may be closer to the required binding conformation.

Gadwood et al. have replaced the pyrrolidinone ring with conformationally restricted spirocyclic structures, such as the imidazolone, 24 (Gadwood et al., 1993). The structure contains a formal orthogonal relationship between imidazolone and benzopyran due to the strictures of the spiro connection. The authors found that there were two low energy forms of the benzopyran, differing in the pyran ring pucker. The higher energy structure of the two was used in a proposed overlap with LCRK, as this was only 1.2 kcal/mol higher in energy— presumably calculated with the AM1 Hamiltonian. Using the overlap as a basis, the authors suggest that the C=N nitrogen of the imidazolone is the likely hydrogen bond acceptor, rather than the oxygen atom of the adjacent carbonyl group. Evidence in support of this is observed in the crystal structure, where the nitrogen atom accepts an intermolecular hydrogen bond. This nitrogen atom can be overlaid easily on the C=O oxygen of LCRK. Furthermore, the activity of the imidazolones correlated with the degree of electron density on this nitrogen atom, since 2-ethoxy and 2-(alkylthio) imidazolones were generally less potent than 2(alkylamino) imidazolones. 4.3.2 Acyclic Replacements Many acyclic replacements of the pyrrolidinone ring have been investigated. Acyclic amides have been prepared (Ashwood et al., 1990). Whereas the


acetamide, 25, possessed reasonable activity, the formamide, 26, was less potent. Also, larger alkyl group substitutions flanking the carbonyl group did not retain the level of activity of 25. Table 4.3 shows the calculated energies of some of these amides. Inspection of Table 4.3 The orientation of various acyclic 4-substituents relative to the benzopyran template, derived from geometry optimized AM1 Hamiltonian calculations.

R

Energy*


Energy*


Energy* Difference

-75.4

-147.5

-70.6

72.6

4.8

-69.5

-148.2

-65.0

74.8

4.5

-67.7

-166.0

-66.4

55.7

1.3

-67.3

-139.4

-63.2

69.3

4.1

-64.1

-125.8

-63.2

60.3

3.8

* AM1 geometry optimiz ed heat of formation

the table shows that the acetamide has a similar preferred orientation of the C=O oxygen to LCRK, with a C10-C4-N1’ -C2’ dihedral angle of -147.5° and a difference in AM1 heat of formation of 4.8 kcal/mol. However, when the acetamide N-H hydrogen is replaced by a methyl group, the more stable structure is the (Z)-form 27, in which the N-Me and C=O are on the same side of the amide bond for the lower energy C4-N1’ rotamer. The higher energy rotamer has equal energy (Z) and (E)-isomers.


4.4 The Aprikalim Series Aprikalim was discovered via general screening of a putative antiulcer compound, 28, which possessed slow onset hypotensive activity. This activity was due to the formation of the potent sulphoxide metabolite (RP49356), 29 (Aloup et al., 1990). The (1R, 2R) (-)-enantiomer (RP52891), known as aprikalim, possesses typical KCA behaviour. The detailed SAR of molecules related to aprikalim can be found in Chapter 3.

Some molecular modelling work has been reported on this compound (Brown et al., 1992). Conformational analyses of the (-)-enantiomer and the cis-isomer of aprikalim have been performed. These will be discussed below, along with other calculations.


4.4.1 Structure of Aprikalim X-ray crystallography studies of aprikalim, 28, (Brown et al., 1992) show an axial position for the sulphoxide oxygen in the thiacyclohexane ring. Other crystal structures are known in which thiacyclohexane sulphoxides have an axial oxygen (Robert and Gauchotte, 1977; Miler-Srenger et al., 1981; Yuasa et al., 1990), but there is also evidence for equatorial forms (Robert, 1977; Miler-Srenger et al., 1981). It appears that the location of the oxygen atom is very sensitive to other substituents on the ring and will adopt either equatorial or axial positions to accommodate the preferences of other groups. The thioamide group is trans to this oxygen, in a trans diaxial arrangement, while the pyridine ring is obviously cis to the sulphoxide. The disubstitution at position 2 of the thiacyclohexane ring causes a steric crowding and thus interactions between the ring and the two substituents influence the conformation of the ring itself and the disposition of the substituents. The X-ray crystal structure of 28 corresponds to the AM1 global energy minimum reported by Brown et al., (1992). There is some conformational freedom, however, as a series of conformers exists within a few kcal/mol of this minimum. For instance, it is reported that the difference in energy between the axial thioamide structure and an equatorial form is of the order of 1.7 kcal/mol. Table 4.4 shows calculated energies of eight conformers of 28, two chair forms, corresponding to the trans diaxial and trans diequatorial structures mentioned above and six twist boat structures. Plates 11 and 12 show these conformations and Table 4.5 shows that the rings all have distinct pucker parameters. The structures were generated using the DGEOM program and were subsequently geometry optimized, using the AM1 Hamiltonian in MOPAC and SPARTAN. Sulphur parameters were incorporated into MOPAC from Dewar’s publication (Dewar and Yuan, 1990). One can clearly see Table 4.4 The heats of formation of various conformers of RP52891 29 and its cis isomer 30, using geometry optimized AM1 Hamiltonian calculations. Structure

AM1 Energy*

Energy relative to lower chair

Chair1trans Chair2trans Twist Boat1trans Twist Boat2trans Twist Boat3trans Twist Boat4trans Twist Boat5trans Twist Boat6trans

26.2 22.0 35.4 25.4 24.3 31.4 26.5 24.1

4.2 0.0 13.4 3.4 2.3 9.4 4.5 2.1


Structure

AM1 Energy*

Energy relative to lower chair

Chair1cis 28.0 Chair2cis 25.7 Twist Boat1cis 30.3 Twist Boat2cis 32.9 Twist Boat4cis 29.1 Twist Boat6cis 31.2 *AM 1 energies with zero-point correction for 298 K

2.3 0.0 4.6 7.2 3.4 5.5

Table 4.5 Cremer and Pople puckering coordinates for various conformers of RP52891 29 and its cis isomer 30, using geometry optimized AM1 Hamiltonian calculations. Structure

β

q2

Q

q3

β

Chair1trans Chair2trans Twist Boat1trans Twist Boat2trans Twist Boat3trans Twist Boat4trans Twist Boat5trans Twist Boat6trans Chair1cis Chair2cis Twist Boat1cis Twist Boat2cis Twist Boat4cis Twist Boat6cis

8 175 85

0.09 0.06 0.90

0.68 0.68 0.91

0.68 -0.68 0.08

57 200 32

87

0.89

0.89

0.04

90

95

0.83

0.84

-0.08

153

96

0.94

0.95

-0.10

208

93

0.91

0.91

-0.05

268

88

0.86

0.86

0.04

335

8 168 85

0.10 0.14 0.89

0.67 0.68 0.90

0.66 -0.67 0.08

50 223 37

85

0.90

0.91

0.08

84

98

0.85

0.86

-0.11

206

83

0.80

0.81

0.10

346

that the ring could easily adopt a range of conformations, as the energy differences between conformers are low. One interesting feature of some of the structures is a possible intramolecular ‘hydrogen bond’ from the thioamide NH to the sulphoxide O. This is noticeable in the AM1 calculations, but is probably more of a non-directional charge-charge


interaction, rather than a true hydrogen bond. The geometry is not that of a classical hydrogen bond, as the N-H-O angle is only 117°, but it has been suggested that this interaction helps to stabilize the trans equatorial structure (Brown et al., 1992). 4.4.2 Calculations on the Cis-isomer Calculations have been reported for the cis-isomer, 30, of aprikalim, which is inactive in vitro (Brown et al., 1992). The global energy minimum structure has a chair structure for the thiacyclohexane ring, but both the thioamide and the pyridine ring are rotated relative to the lowest energy trans form. This allows the thioamide NH to interact with the sulphoxide O in an analogous ‘hydrogen bond’ to that found in the opposite chair structure of 28 mentioned above. Table 4.4 shows calculated AM1 energies for various ring conformations of the (1S,2R) form. It was not possible to find minima for all six twist boats; two structures, corresponding to Cremer and Pople β values of 150° and 270°, could not be isolated. These ring conformations are probably unstable due to steric crowding caused by the substituents at position 2 of the ring. It is also apparent that the separation in energy between the twist boat and the chair forms is greater for 30, compared with 28. It is possible that this inflexibility of 30 does not allow the compound to adopt a conformation recognized by the receptor, thus accounting for its lack of potency.

4.5 Studies on Pinacidil Pinacidil, 31 and related cyanoguanidines were developed as bioisosteric replacements for hypotensive thioureas (Petersen et al., 1978) and were discovered subsequently to be KCAs. Chapter 3 contains data on the SAR of this class of compounds. Conformational aspects of the class are covered below.


4.5.1 Structure of Pinacidil The (R)-isomer of pinacidil, 31, is the more potent form. Although this is a 4pyridyl-substituted molecule, it is noteworthy that most of the analogues reported by Petersen et al. (1978), were more potent as 3-pyridyl compounds. The crystal structure of 31 has been reported (Pirotte et al., 1993) and this structure is shown in Plate 13. It is interesting to note that the cyano group is cis to the pyridyl amino group.

4.5.2 Rotation of the N- and N’-substituents of Pinacidil Theoretical studies on pinacidil have been performed, to investigate its Conformational preferences. Plate 14 shows the Ramachandran diagram (AM1 energy versus torsion angle) for rotation of the bonds connecting the pyridine ring to the cyanoguanidine group for the 3-pyridyl pinacidil analogue, LY222674, 32. The two bonds were rotated through 360°, with full relaxation of the remainder of the molecule. The figure shows that there is relatively free rotation possible about the pyridine-NH bond and restricted rotation possible about the NH-C(NCN) bond of the cyanoguanidine.

Plate 14 shows the location of the corresponding torsion angle values for the Xray crystal structure of (R)-pinacidil on the Ramachandran energy surface. It also shows the location of a structure which has been reported to overlap with cromakalim. It can be seen that there is a low energy pathway between these two conformations. This suggests that if a conformation corresponding to the overlap conformation were required at the receptor, this is attainable for a low energy cost.


4.6 Possible Pharmacophore Models There are many different structural classes of KCAs now extant. These pose several questions. Do these diverse structures act at the same receptor? If so, do they interact in a similar manner, with the same set of amino acids in that receptor? A reasonable pharmacophore model requires that each of these questions needs to be answered in the affirmative. There is evidence that KCAs act at the same receptor, in that they are blocked characteristically by glibenclamide. This may be taken as evidence that KCAs are acting in a similar manner, though this is certainly not conclusive proof. Further evidence that KCAs are acting at the same site has been provided (Manley et al., 1993). It has been shown that thirteen structurally distinct KCAs all displaced a tritiated pinacidil analogue from the receptor in radioligand binding studies. In general, the correlation between the pD2 for inhibition of rat portal vein spontaneous activity versus the pKi for inhibition of radioligand binding is good, (r=0.96, slope=0.88, n=13). However, although there is little difference between the concentration of compound needed to inhibit either radioligand binding or spontaneous activity for pinacidil analogues, the concentrations of benzopyrans needed to inhibit radioligand binding are higher (by about half a log unit) than those necessary for inhibiting activity. This is presented as evidence that the KCAs do not all bind at the same site. If this is the case, then attempts at constructing pharmacophores are doomed to failure. Despite this evidence—or before it was published—many workers have made and tested hybrid KCAs, based on pharmacophore models. The rest of this section will describe these models.

Work in finding new hybrid structures has shown that it is possible to mix some of the structural elements of cromakalim and pinacidil and obtain potent compounds (Atwal et al., 1992, Burrell et al., 1993). Atwal et al., (1992) synthesized cyclic and acyclic cyanoguanidines, such as 33 that have antihypertensive activity. It was suggested that these hybrids provided evidence that cyanoguanidine could mimic the pyrrolidinone amide of CRK. Additionally, the benzene ring of CRK was equivalent to the pyridine of pinacidil and both compounds had lipophilic groups, namely the alkyl sidechains, to give a three


point pharamacophore. Burrell et al., (1993) have reported that a series of urea analogues, including the cyanoguanidine, 34, have antihypertensive activity. However, the structure-activity relationships in this series do not correlate exactly with those of the pinacidil series. Thus, replacement of the cyanoguanidine group by the urea group leads to a loss of potency in pinacidil analogues, but not in benzopyrans. Replacement of the cyanoguanidine group by the nitroethene substituent retains potency in pinacidil analogues, but, in contrast, this change reduces potency in the benzopyran series. These observations suggest that the two series of KCAs act at different receptor sites. A series of publications has appeared where the authors claim to have designed compounds based on a pharmacophore model (Koga et al., 1993a, 1993b, 1993c, 1993d; Ishizawa et al., 1993). The pharmacophore consists of four elements: two regions of lipophilic interaction and two vectors representing interaction with hydrogen bond donors. The lipophilic regions correspond to the 2, 2-dimethyl group of CRK and the benzene ring. The hydrogen bond acceptors are defined as the carbonyl oxygen and at the cyano nitrogen of CRK. The authors claim to have designed compounds such as 35 based on this pharmacophore model. 4.7 Conclusion This chapter has presented some of the calculations and analyses that have been attempted on the ATP-sensitive potassium channel activators. Work has been published on conformational preferences, analysis of X-ray crystal structures and pharmacophoric overlaps. Novel work has also been presented here on ring flipping of some KCAs and on aprikalim conformations. It can be expected that studies on KCAs will continue to be based on their structural properties, until there is information available on the structure of their molecular targets. There are still many issues that require study and clarification in this area. For instance, the SAR of the 6-position of CRK is not satisfactorily resolved—why do certain alkyl groups retain potency, when most potent compounds have strong electron-withdrawing groups? The problems associated with the universal pharmacophore models mentioned in section 4.6 also need further study. It may prove necessary to classify KCAs into small groups to be confident of reasonable overlap at the receptor. Alternatively, it may be possible to use newer threedimensional techniques, such as DISCO and CoMFA to define a generic pharmacophore model. References ALOUP, J.C., FARGE, D., JAMES, C., MONDOT, S. & CAVERO, I. (1990) Drugs of the Future, 15, 1097–1108.


ASHWOOD, V.A., CASSIDY, F., COLDWELL, M.C., EVANS, J.M., HAMILTON, T.C., HOWLETT, D.R., SMITH, D.M. & STEMP, G. (1990) J. Med. Chem., 33, 2667–2672. ATWAL, K.S., MORELAND, S., MCCULLOUGH, J.R., AHMED, S.Z. & NORMANDIN, D.E. (1992) BioMed. Chem. Lett., 2, 87–90. BARTMANN, W. (1989) In: Trends in Medicinal Chemistry, van der Groot, H., Domany, G., Pallos, L. & Timmerman, H. (eds). Elsevier, Amsterdam, pp. 629–657. BROWN, T.J., CHAPMAN, R.F., COOK, D.C., HART, T.W., MCLAY, I.M., JORDAN, R., MASON, J.S., PALFREYMAN, M.N., WALSH, R.J.A., WITHNALL, M.T., ALOUP, J.-C., CAVERO, I., FARGE, D., JAMES, C. & MONDOT, S. (1992) J. Med. Chem., 35, 3613–3624. BUCKLE, D.R., EGGLESTON, D.S., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., READSHAW, S.A., SMITH, D.G. & WEBSTER, R.A.B. (1991a) J. Chem. Soc. Perkin Trans I, 2763–2771. BUCKLE, D.R., ARCH, J.R.S., EDGE, C., FOSTER, K.A., HOUGE-FRYDRYCH, C.S.V., PINTO, I.L., SMITH, D.G., TAYLOR, J.F., TAYLOR, S.G., TEDDER, J.M. & WEBSTER, R.A.B. (1991b) J. Med. Chem., 34, 919–926. BUCKLE, D.R., EGGLESTON, D.S., PINTO, I.L., SMITH, D.G. & TEDDER, J.M. (1992) BioMed. Chem. Lett., 2, 1161–1164. BURRELL, G., EVANS, J.M., HICKS, F. & STEMP, G. (1993) BioMed. Chem. Lett., 3, 999–1002. CASSIDY, F., EVANS, J.M., SMITH, D.M., STEMP, G., EDGE, c. & WILLIAMS, D.J. (1990) J. Chem. Soc., Chem. Commun., 377–378. CREMER, D. & POPLE, J.A. (1975) J. Amer. Chem. Soc., 97, 1354–1358. DEWAR, M.J.S. & THIEL, W. (1977a) J. Amer. Chem. Soc., 99, 4899–4907. (1977b) J. Amer. Chem. Soc., 99, 4907–4917. (1977c) Theor. Chim. Acta, 46, 89–104. DEWAR, M.J.S. & YUAN, Y.-C. (1990) Inorg. Chem., 29, 3881–3890. DEWAR, M.J.S., ZOEBISCH, E.G., HEALY, E.F. & STEWART, J.J.P. (1985) J. Amer. Chem. Soc., 107, 3902–3909. FABIAN, W.M.F. (1988) J. Comp. Chem., 9, 369–377. GADWOOD, R.C., KAMDAR, B.V., CIPKUS DUBRAY, L.A., WOLFE, M.L., SMITH, M.P., WATT, W., MISZAK, S.A. & GROPPI, V.E. (1993) J. Med. Chem., 36, 1480–1487. GUNDERTOFTE, K., PALM, J., PETTERSSON, I. & STAMVIK, A. (1991) J. Comp. Chem., 12, 200–208. ISHIZAWA, T., KOGA, H., OHTA, M., SATO, H., MAKINO, T., KUROMARU, K., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993) BioMed. Chem. Lett., 3, 1659–1662. KOGA, H., OHTA, M., SATO, H., ISHIZAWA, T. & NABATA, H. (1993a) BioMed. Chem. Lett., 3, 625–631. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., NABATA, H., IMAGAWA, J., YOSHIDA,S. & SUGO, I. (I993b) BioMed. Chem. Lett., 3, 1111–1114. KOGA, H., SATO, H., ISHIZAWA, T., KUROMARU, K., MAKINO, T., TAKA, N., TAKAHASHI, T., SATO, T. & NABATA, H. (1993c) BioMed. Chem. Lett., 3, 1115–1118. KOGA, H., SATO, H., IMAGAWA, J., ISHIZAWA, T., YOSHIDA, S., SUGO, L, TAKA, N., TAKAHASHI, T. & NABATA, H. (1993d) BioMed. Chem. Lett., 3, 2005–2010.


MANLEY, P.W., QUAST, U., ANDRES, H. & BRAY, K. (1993) J. Med. Chem., 36, 2004–2010. MILER-SRENGER, E., STORA, C. & HUGHES, N.A. (1981) Acta Crystallogr., Sect B, 37, 356–360. PETERSEN, H.J., NIELSEN, C.K. & ARRIGONI-MARTELLI, E. (1978) J. Med. Chem., 21,773–781. PIROTTE, B., DUPONT, L., DE TULLIO, P., MASEREEL, B., SCHYNTS, M. & DELARGE, J. (1993) Helv. Chim. Acta, 76, 1311–1318. PRESS, J.B., MCNALLY, J.J., SANFILIPPO, P.J., ADDO, M.F., LOUGHNEY, D., GIARDINO, E., KATZ, L.B., FALOTICO, R. & HAERTLEIN, B.J. (1993) BioMed. Chem., 1, 423–435. QUAST, U. & VILLHAUER, E.B. (1993) Eur. J. Pharmacol. Mol. Pharm., 245, 165–171. ROBERT, F. (1977) Acta Crystallogr., Sect B, 33, 3480–3484. ROBERT, F. & GAUCHOTTE, S. (1977) Acta Crystallogr., Sect B, 33, 3484–3487. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FITZPATRICK, L.J., URBANSKI, M.J., KATZ, L.B., GIARDINO, E., FALOTICO, R., SALATA, J., MOORE, J.B. & MILLER, W. (1992) J. Med. Chem., 35, 4425–4433. SANFILIPPO, P.J., MCNALLY, J.J., PRESS, J.B., FALOTICO, R., GIARDINO, E. & KATZ, L.B. (1993) BioMed. Chem. Lett., 3, 1385–1388. STEMP, G. & EVANS, J.M. (1993) In: Medicinal Chemistry, 2nd Edition. Ganellin, C.R. and Roberts, S.M. (eds). Academic Press Ltd., London, pp. 141–162. STEWART, J.J.P. (1990) J. Comp. Aided Mol. Des., 4, 1–105. THOMAS, W.A. & WHITCOMBE, I.W.A. (1990) J. Chem. Soc., Chem. Commun., 528–529. YUASA, H., TAKENAKA, A. & HASHIMOTO, H. (1990) Bull. Chem. Soc. Jpn., 63, 3473–3479.

Recent Literature OHTA, M., KOGA, H., SATO, H. & ISHIZAWA, T. (1994) Comparative Molecular Field Analysis of Benzopyran-4-carbothioamide Potassium Channel Openers. BioMed. Chem. Letts., 4, 2903–2906.

5 The Structure-Activity Relationships of Potassium Channel Blockers R.CROSSLEY & A.OPALKO Wyeth Research UK, Huntercombe Lane South, Taplow, Maidenhead, Berkshire 516 OPH, UK.

5.1 Introduction To date very few drugs have been developed as potassium channel blocker (KCBs). This may seem to be a strange introduction to a chapter concerned with the Structure-Activity Relationships (SAR) of such agents but it is nevertheless true. In the main, compounds in this chapter had originally been developed with various therapeutic goals in mind and were screened accordingly in an in vitro or an in vivo model which reflected this goal. Subsequently, the lead compounds in the various series were found to be KCBs and the responses of these lead compounds at various K channels were then examined in some detail. This distinction may seem to be rather arbitrary and inconsequential, but it means that the derived SAR of such compounds not only reflect their abilities as KCBs, but also other factors as well. The presence of lipophilic groups, for example may reflect the need for a drug to cross a membrane rather than to locate a hydrophobic pocket on a receptor and, for these compounds, it is not possible to determine a priori what the interactions at the receptor level actually are. In spite of this, the SAR discussed here will assume that it is concerned with activity at the channels. More recently, drugs are being developed from their inception with due consideration both to their primary K channel blocking effects and with their eventual use in mind. There are many different kinds of potassium channel blockers but it is not possible to deal meaningfully with certain classes of these. Some compounds, for example tetraethylammonium (TEA) and quinine, are quite promiscuous in their effects on K channel subtypes but may be used in general classification of channels. Others, for example charybdotoxin, apamin, mast cell degranulating peptide (MCDP), and dendrotoxin, are structurally complex and there is some speculation, but little hard evidence, as to which of their elements are involved in binding. A general review of these types of compound is to be found elsewhere (Cook and Quast, 1990).

THE STRUCTURE-ACTIVITY RELATIONSHIPS OF KCBS 129

5.2 ATP-Sensitive K Channel Blockers 5.2.1 Introduction The coupling of the metabolic state of a cell to its excitability is largely governed by the presence of K channels in the cell membrane which are inhibited by a rise in intracellular levels of the purine nucleotide ATP. These ATP-sensitive K channels (KATP) have been identified in cardiac (Fosset et al., 1988; Lederer and Nichols, 1989), pancreatic (Schmid-Antomarchi et al., 1987) and nerve (Bernardi et al., 1988; Mourre et al., 1989; Levesque and Greenfield, 1991) cells and in skeletal (Weik and Neumcke, 1989) and smooth muscle (Gopalakrishnan et al., 1991) cells. They thereby present significant therapeutic opportunities in all these areas, especially in the CNS (Miller, 1990), although only two of these have been extensively exploited to date (Gopalakrishnan et al., 1993). The subject of smooth muscle KATP channel activators has already been dealt with in previous chapters and an established use of pancreatic KATP channel blockers is in the control of blood sugar levels by mediating the release of insulin. In the pancreas, a rise in intracellular glucose leads to activation of mechanisms which produce an increase in ATP and this leads to inhibition of the KATP channel. This in turn alters the membrane potential to the threshold for voltage dependent Ca channels, leading to opening and influx of calcium, which in turn triggers insulin release. Drugs which have a similar effect to this in inhibiting the KATP channels in the pancreas form the main line of drug treatment for early stage diabetes. 5.2.2 Sulphonylureas and Related Molecules The hypoglycaemic sulphoriylureas have been used in the therapy for type II diabetes for many years with various speculations as to their mode of action (Rasmussen et al., 1981; Asmal and Marble, 1984; Gylfe et al., 1984), but it is only relatively recently that their effect in lowering blood glucose has been directly linked with blockade of KATP channels (Schmid-Antomarchi et al., 1987; Gaines et al., 1988; Henquin, 1988; Bernardi et al., 1989; Ashford, 1990). They were originally developed from the antibacterial sulphonamides, which were noted to produce hypoglycaemia when administered at high doses (Loubatieres, 1957). The original group of sulphonylureas are typified by the relatively simple first generation compounds such as tolbutamide, tolazamide, chlorpropamide, carbutamide, gliclazide and glibornuride (Figure 5.1). These compounds all have a simple aryl substituent and a lipophilic urea substituent. The second generation compounds (Figure 5.2) retain this arylsulphonylurea moiety but extend it significantly by the introduction of ethylaminoacylaromatic units which extend


the pharmacophore. This has led to the production of gliquidone, glisoxepide, glibenclamide (glyburide), glipizide and glisindamide (HOE 036). It is these second generation compounds which, because of their greater affinity, have also provided the radioligands [3H]-glyburide (Geisen et al., 1985; Bernardi et al., 1989), [125I]-glyburide (Robertson et al., 1990) and the 5-[125I]-2-hydroxy analogue of glyburide (Rajan et al., 1993). The availability of these ligands, along with a labelled pinacidil analogue (Manley et al., 1993) have led to the isolation and identification of the sulphonylurea receptor as a 150 kDa protein which has been considered to either

Figure 5.1 Structural formulae of first generation sulphonylureas


Figure 5.2 Formulae of second generation sulphonylureas

form the ion channel or is more likely closely associated with it (Bernardi et al., 1988; Aguilar-Bryan et al., 1990; Rajan et al., 1993; Manley et al., 1993). The use of these ligands has also indicated the presence of subtypes of these channels (Gopalakrishnan et al., 1991; Lazdunski et al., 1992; Rajan et al., 1993). The observed SAR of the sulphonylureas led to the initial proposal (Rufer et al., 1974) for a putative receptor site which covered all the first and second generation compounds and which can be extended to include other compounds active at this receptor (Figure 5.3). These include sulphonamides such as glymidine, thus providing a link with the antibacterial sulphonamides. The proposed model also extends (Biere et al., 1974; Rufer and Losert, 1979) to analogues with reversed amide functionality as well as to some chiral derivatives such as (-)-S-gliflumide and the less potent (-)-S-4-N-(l-(5-fluoro-2methoxyphenyl)ethyl)carbamoyl)methylbenzoic acid (1) both of which were considerably more potent than their enantiomers, with eudismic ratios of 40 and 18 respectively. There is some evidence that the positions of the aromatic and alkyl sites on the pharmacophore may exchange when the amide group is reversed and this presumably reflects a conformational change (Biere et al., 1974). Finally, some drugs such as meglitinide (HB-699) and AZ-DF 265


(Figure 5.3) dispense with the sulphonylurea functionality and in its place have a carboxylic acid. These compounds can also be incorporated into a pharmacophore model. The chiral AZ-DF 265 also demonstrates enantioselectivity with the (-)-enantiomer being the eutomer with a eudismic ratio of around 10 (Garrino and Henquin, 1988). The single pharmacophore model which results from combining all these compounds illustrates, amongst other things, the non-criticality of the sulphonylurea function (Figure 5.4). In this model a central aromatic moiety is extended to right and left to pick up ancillary binding sites. The various molecules in this class may occupy either the right hand end (the first generation compounds and sulphonamides), both left and right ends (the second generation compounds) or just the left hand end (benzoic acid derivatives). At the left is an aromatic ring, preferably electron rich, an alkyl group and an amide arranged as in the S configuration of gliflumide or, as in the case of


Figure 5.3 Formulae of benzoic acid and sulphonamide analogues of sulphonylureas

Figure 5.4 Pharmacophore model for sulphonylurea binding site

gliquidone (Figure 5.2), a heterocycle containing these elements. To the right there appears to be an acidic sulphonamide or sulphonylurea NH or carboxylic acid group and a lipophilic group which presumably picks up a hydrophobic interaction at the receptor site. The range and variety of the analogues which have been studied make the sulphonylurea binding site one of the best characterised there is. The proof that this forms part of the KATP receptor comes from the observation that the hyperglycaemic compound diazoxide (Figure 5.3), a cyclic sulphonamide, opens these channels (Zunkler et al., 1988), and from extensive correlations of the activities of several sulphonylureas with channel blocking activity (Schmid-


Antomarchi et al., 1987; Bernardi et al., 1988; De Weille et al., 1989; Amoroso et al., 1990). There is, for example, an excellent correlation, for a whole series of compounds, including most of those in Figures 5.1 and 5.2, between the inhibition of [3H]-glibenclamide binding and rubidium efflux in insulinoma cells and a similar correlation between binding in this preparation and in heart and brain microsomes. In addition, a correlation of the KATP channel inhibition by sulphonylureas and the release of GABA in the substantia nigra indicates that they may also modify the responses in the brain to diabetes, ischaemia and anoxia and hence present alternative therapeutic opportunities for these compounds (Amoroso et al., 1990). 5.2.3 Imidazolines and Related Molecules Imidazoline-containing noradrenergic β 2-receptor antagonists have been used to treat diabetes for some time and their mechanism of action was assumed to be a result of the activity at this receptor being indirectly responsible for insulin release (Mohrbacher et al., 1987). More recently, some of these compounds (Figure 5.5) have been shown to antagonise the vascular actions of cromakalim (CRK) in a concentration dependent manner with an order of potency alinidine = phentolamine > tramazoline = naphazoline (McPherson and Angus, 1989). The relative and absolute potencies of the compounds, coupled with the observation that the closely related tolazoline was inactive as were other standard β 2-receptor antagonists, is strongly suggestive that they have the additional property as KATP channel blockers in smooth and cardiac muscle. The effect on pancreatic channels is less clear as tolazoline does seem to potentiate insulin release along with phentolamine. This is still proposed to be a consequence of the imidazoline structure


Figure 5.5 Structural formulae of imidazoline derivatives

rather than β 2-receptor blockade per se as antazoline, which has only very little activity as an β 2-receptor antagonist, is also effective in promoting insulin release (Schulz and Hasselblatt, 1989). Apart from the observation that the imidazoline moiety is essential for activity (the related compound 1-benzylimidazole is inactive), there is as yet little SAR available which can be directly related to KATP channel blockade and it remains to be seen if is possible to design out the β 2-receptor properties and develop these compounds into therapeutically effective KATP channel blockers. 5.2.4 Miscellaneous Compounds KATP channels are also blocked by relatively non-specific (Cook and Quast, 1990) KCBs such as TEA, 4-aminopyridine, quinine and quinidine as well as by more specific compounds such as lidocaine and several barbiturates, although at relatively high concentrations (Ashcroft and Ashcroft, 1990). There are, however, some compounds, linogliride, ciclazindol and BRL 31660 (Figure 5.6), which may turn out to be the progenitors of relatively specific KATP channel blockers. Linogliride Linogliride is a member of a class of guanidine-based insulin secretogogues (Mohrbacher et al., 1987) and has some structural similarities with the imidazolines. It is unclear as yet if this similarity is of consequence in its interaction with a receptor or if linogliride has the same mode of action as the


sulphonylureas. Nevertheless, linogliride has been shown electrophysiologically to inhibit KATP channels in pancreatic β -cells, an effect which is sensitive to pretreatment with tolbutamide (Figure 5.1). It may, therefore, prove to be a lead to a new chemical class of KATP channel blockers. Other guanidine hypoglycaemics do not seen to have been examined as KATP channel blockers in any detail.

Figure 5.6 Structural formulae of miscellaneous KATP channel blockers

Ciclazindol The antidepressant drug ciclazindol, and the related compound mazindol (Figure 5.6), mediate their antidepressant effect mainly through inhibition of monoamine uptake (Oh et al., 1979) and, although there are differences between the compounds, both have some similarities to the imidazolines. Ciclazindol and mazindol do reduce blood glucose levels in a non-insulin-dependent manner, putatively by increasing uptake into human skeletal muscle (Kirby and Turner, 1977). Subsequently, ciclazindol has been shown to block the effects of K channel activators (KCAs) in rat portal vein and bladder, an effect which is associated with KATP channel blockade, but ciclazindol is not totally selective for these channels (Noack et al., 1992). Notably, there are no reports on the effects of mazindol. Because ciclazindol does not displace [3H]-glibenclamide from porcine brain, its effect is presumed to be a result of interaction at a site different from that of the sulphonylureas. This may, however, reflect the involvement of a different subtype of KATP channel. Interactions of ciclazindol with KATP channels in the ventromedial hypothalamic nucleus may be responsible for the anorectic effects of this drug (Noack et al., 1992) and this may lead to uses for KATP channel blockers by centrally mediated mechanisms.


BRL 31660 The novel combined Class I and Class IV antiarrhythmic agent BRL 31660 inhibits the effects of CRK and other KCAs in guinea pig trachea, (Taylor et al., 1989; Arch et al., 1991) an effect which is associated with inhibition of K+ efflux. The compound is a sulphonamide but it is not clear if it is mediating its effects through interaction with the sulphonylurea receptor site or elsewhere. It has some structural features, such as a basic centre and naphthalene ring, which are not commonly found in the sulphonylureas but are present in the imidazolines. It may also prove to be a useful tool to elucidate the mechanisms of action of KATP channels in different tissues (Taylor et al., 1989).

5.3 Class III Antiarrhythmic Agents 5.3.1 Introduction The treatment of arrhythmias by prolonging the repolarisation in the heart has been achieved by a number of synthetic agents through a variety of pharmacological actions. The majority of these mechanisms involve the modulation of voltage gated ion channels of one type or another and these modes of action have been classified into five distinct classes (Vaughan Williams, 1984) based on the pharmacological properties of the drug. Most early antiarrhythmic drugs were Class I agents, and these compounds block the fast sodium channels, which are responsible for depressing the rapid phase of depolarisation, and thereby stabilise cell membranes. Class II agents inhibit the activity of the sympathetic nervous system; the β -adrenoceptor (beta blockers) are examples of drugs which manifest their actions by this mechanism. Class III agents prolong the action potential duration by blockade of one or more types of K channels, whereas Class IV agents block Ca channels and Class V agents slow the rate of sinus depolarisation by modulating Cl channels. There are least eight different K channels found in the heart (Colatsky and Follmer, 1989; Colatsky, 1991) which are thought to influence the action potential under normal physiological conditions. Amongst those channels, suitable targets for drug intervention, are the delayed rectifier channel (IK), which is subdivided into the rapidly (IKr) and slowly activating (IKs) subtypes (Noble and Tsien, 1969; Sanguinetti and Jurkiewicz, 1990), a voltage dependent transient outward current (IA) (Giles and Ginneken, 1985; Kenyon and Sutko, 1987; Tseng and Hoffman, 1989) and the inward


Figure 5.7 Analogues of clofilium

rectifier (background) current (IK1) (Sakmann and Trube, 1984). A number of Class III antiarrhythmic drugs such as d-sotalol (Figure 5.8), E-4031 (Figure 5.10) and MS-551 (Figure 5.10) have been examined for their effects on two types of cloned rat K channels, RH1 (Kvl.2) and RH10 (Kvl.4) isolated from rat heart, but they had no effect (Yamagishi et al., 1993). Some Class III agents have been identified which are more selective for distinct K channel subtypes such as the delayed rectifier channel (IK) Agents which have been shown to be selective for (IK) are risotilide (Figure 5.8), E-4031 (Figure 5.10) and dofetalide (Figure 5.9). Drugs which are less specific are d-sotalol (Figure 5.8) which blocks (IK), (IK1) and (IA.) and amiodarone and clofilium (Figure 5.7) which block (IK) and (IK1). Also, some Class Ia drugs such as quinidine and disopyramide also block potassium currents such as (IK), (IK1) and (IA) and Class Ic drugs such as encainide and flecainide block (IK) as well as sodium currents (Colatsky et al., 1990). Although a number of Class III agents have shown selectivity for (IK). there have been no systematic structure-activity studies correlating antiarrhythmic efficacy with (IK) blocking ability. Accordingly this section will concentrate on reviewing how the current agents were developed and what the structural requirements for Class III activity are.


Figure 5.8 First generation analogues


Figure 5.9 Second generation phenoxyalkylamines

There have been a number of reviews of Class I and Class III drugs (Arrowsmith and Cross, 1990), Class III agents (Morgan Jr. and Sullivan, 1992), KCMs (Robertson and Steinberg, 1990; Atwal, 1992) and KCBs (Cimini and Gibson, 1992). As the biological aspects of antiarrhythmic therapy will be discussed later in this book (Chapter 10) the focus of this section will be on the chemistry of Class III agents. There are three main classes of chemical structures which have served as leads, these are clofilium, a quaternary compound (Figure 5.7), sotalol and sematilide (Figure 5.8). All the series were developed initially by virtue of their actions in vivo and subsequently the connection with K channel blocking activity has been established. 5.3.2 Quaternary Compounds Clofilium (Steinberg et al., 1984), a quaternary ammonium compound, was the starting point for a number of variations to its structure (2, Figure 5.7). The first of these imposed some rigidity into the alkylamine chain by the introduction of unsaturation and this gave compounds which were just as active as clofilium


(Morgan Jr. et al., 1986). This suggests that clofilium in its active conformation is in an extended form. The conformational flexibility at the quaternary nitrogen was also examined by constraining the quaternary ammonium moiety into 2- and 3-substituted phenylalkyl quinuclidines (Morgan Jr. et al., 1987) or an imidazolium group as in CK-1649

Figure 5.10 Miscellaneous second generation analogues

(Lis et al., 1987b) (Figure 5.7). Generally the 2-substituted analogues had both Class I and III activity whereas the 3-substituted derivatives had more Class III selectivity. Following the demonstration that the β -adrenoceptor antagonist (β -blocker) sotalol (Figure 5.8) also possessed Class III activity (Singh and Williams, 1970) some of its features, such as the sulphonamide and the ethanolamine chain were combined with these quaternary compounds to give a series of imidazolium derivatives (Lis et al., 1987b), for example CK-1649 (Figure 5.7). Quaternization of the amine group in various β -blocking drugs leads to greatly diminished β -blocking activity but maintains Class III antiarrhythmic activity (Paterson et al., 1980). A series of quaternary derivatives of the β -blocker propranolol, pranolium UM272 (Eller et al., 1983), UM301 (Gibson et al., 1986) and UM424 (Figure 5.7, Gibson et al.,. 1985), were developed and found to have respectable antiarrhythmic and antifibrillatory activity. Unfortunately, the oral bioavailability of such quaternary compounds is rather poor and so clinical development has not been possible.


First generation analogues Class I antiarrhythmic agents such as encainide, flecainide or moricizine (Morganroth and Bigger, 1990) used to be the most frequently used agents clinically, until results from the cardiac arrhythmia suppression trial (CAST) revealed that there was an increased risk of morbidity when patients were treated with these drugs. This resulted in a change in the focus of research interest to Class III agents, where the majority of effort has revolved around sulphonamide derivatives. The methylsulphanilide moiety features in most of the agents, for example sematilide (Figure 5.8), dofetalide (Figure 5.9) and E-4031 (Figure 5.10) which are in clinical trials and the d-enantiomer of sotalol (Figure 5.8). Procainamide is a Class 1A antiarrhythmic (Figure 5.8), which is readily acetylated in humans (Karlsson, 1978) to give the N-acetyl derivative, NAPA (Figure 5.8) and this metabolite has been shown to be primarily a Class III agent (Dangman and Hoffman, 1981). Replacement of the acetamide group with the more stable and also more acidic methylsulphonylamino group, present in the structurally related molecule sotalol, resulted in sematilide (Lumma Jr. et al., 1987). Sematilide, is fifty times more potent than NAPA, as a selective Class III compound. The same group found that this moiety could be replaced with an imidazol-1-yl function (3, Figure 5.8) (Morgan Jr. et al., 1990b) to give a compound which shows similar efficacy and potency to sematilide. Various modifications of the basic sematilide structure have resulted in compounds which have combined Class I and III activity (4, Figure 5.8) (Philips et al., 1990). It was found that substitution at R1, R2 or R3 in the general formula (4) with phenyl or substituted phenyl led to more active Class III compounds than sematilide, but also resulted in the introduction of Class I activity which was generally detected at higher concentrations. Combined Class II/III antiarrhythmic agents have been obtained by combining β -blocking (Class II) activity into the sematilide structure (Philips et al., 1992). An arylpiperazine moiety (5, Figure 5.8) provides the Class II or β -blocking pharmacophore1 and this was combined with features from sematilide. A variety of linkages X such as CH2O, CH2NH and CONH were examined, but the CONH moiety used in sematilide was found to be the most potent. This compound was found to prevent electrically stimulated arrhythmias in dogs (Class III activity) and to be effective against epinephrine-induced arrhythmias in halothane anaesthetised dogs (Class II activity). Risotilide (Figure 5.8), which is in preclinical studies, and is structurally similar to sematilide was derived from a series of benzene sulphonamides (Colatsky and Follmer, 1989). Electrophysiological studies on this compound indicate that it is a selective blocker of the delayed rectifier current without effect on IKI and IA, or on Na+ currents (Follmer et al., 1989). Sotalol (Figure 5.8) was originally developed as a β -blocking agent (Larsen and Uloth, 1969), for the treatment of hypertension, and in the early seventies it


was discovered to have Class III antiarrhythmic activity (Singh and Williams, 1970). Separation of the racemate into its enantiomers resulted in the discovery that the Class II effect resided in the l-isomer, whereas the Class III effect resided in the d-isomer (Hoffmeister et al., 1991). The d-enantiomer of sotalol was given FDA approval in the beginning of 1993. The amino function of sotalol can be constrained in a cyclic form such as the imidazole (6, Figure 5.8) analogue of the quaternary compound CK-1649 (Figure 5.7) (Lis et al., 1987a). This analogue has comparable activity to its quaternary analogue CK-1649 in vitro but was ineffective in vivo. Ibutilide (Figure 5.8) can be viewed as a modified sotalol analogue in which the secondary ethanolamine chain has been extended to a tertiary butanolamine and thereby removing the Class II activity (Hester et al., 1991). In addition, ibutilide has a novel mode of action compared with the other Class III antiarrhythmic agents. Besides its Class III activity, it activates a slow inward Na+ current at subnanomolar concentrations (Lee et al., 1990) and this dual action has the effect of prolonging the action potential duration at low concentrations and shortening it at high concentrations, so producing a bell-shaped dose response curve. All the analogues described so far, have a simple pharmacophore which comprises of a para substituted aromatic ring, normally substituted with a methanesulphonamide. This aromatic ring is linked to an secondary or tertiary amine by a three to four atom linked spacer. Second generation analogues A second generation of compounds has sought to extend the pharmacophore by the incorporation of other aryl and heterocyclic moieties, see Figures 5.9 and 5.10, but the majority of compounds still retain a sulphonamide group. In view of the Class III activity observed by including an ethanolamine β -blocking pharmacophore such as in sotalol (Figure 5.8), it is not surprising that the corresponding oxypropanolamine substituent also has activity. In a similar manner to the ethanolamines, the β -blocking effect can be removed by replacing the secondary amine with a tertiary amine. Such a modification has provided WAY-123,223 (Figure 5.9) (Butera et al., 1991) and removal of the alcohol to give the deshydroxy analogue also resulted in a very potent and selective Class III agent, WAY-125,971 (Figure 5.9). Another group deliberately set out to incorporate β 1-blocking activity with Class III activity, based on the hypothesis that combined modes of action would be potentially useful against re-entrant and catecholamine-dependent arrhythmias, at doses below those which cause β -blocker-mediated hypotension and cardiac depression. This has been achieved with both an aryl piperazine 5

1 This is not the usual pharmacophore associated with β -blocking activity, see Philips et al. (1992) for examples of β -blocking activity associated with this group.


(Figure 5.8) using a non-classical β -blocking pharmacophore and CK-3579 (Figure 5.9) using a ‘classical’ β -blocking pharmacophore. The latter retains a secondary amine and the S-enantiomer was found to be the eutomer (Lis et al., 1990; Morgan Jr. et al., 1990a; Connors et al., 1991). Replacement of the secondary amine to give cyclic amine derivatives, also results in selective molecules (7, Figure 5.9) (Connors et al., 1991). Another phenoxyalkylamine, dofetalide (Figure 5.9) (Cross et al., 1990) is in clinical trials. It prolongs cardiac action potential duration below 5 nM and is claimed to be one of the most potent Class III agents prepared to date (Gwilt et al., 1991). Modification of risotilide (Figure 5.8) by the further addition of a benzimidazolyl group (Figure 5.10) gave WAY-123,398 (Ellingboe et al., 1992). A number of heterocyclic groups such as quinolyl or pyrimidyl have also been examined, but the 2-aminobenzimidazole group was shown to be the most potent. WAY-123,398 was shown to have good oral bioavailability and to produce a 3-fold increase in ventricular fibrillation threshold in anaesthetised open-chest dogs. Two sulphonamide analogues (Figure 5.10) which incorporate cyclic amines are UK-66,914 and E-4301 (Oinuma et al., 1990). UK-66,914 has a pyridylpiperazino moiety in place of the isopropylamino found in sotalol, and this modification diminishes β -blocking activity. The second aromatic group probably locates an extra binding site and this makes it more potent than sotalol (Gwilt et al., 1988). UK-66,914 was withdrawn from clinical studies because of toxicity considerations. There are a number of other potential therapeutic agents, being evaluated in humans (Figure 5.10), which have different electron withdrawing groups in place of sulphonamide. MS-551 was originally derived from a β -blocking agent MS-3579 (Figure 5.10) (Katakami et al., 1992) but it is devoid of the Class II effects normally associated with the β -blocking pharmacophore. It has also been reported (Endoh et al., 1993) that MS-551 was able to rescue patients in cardiac arrest which had been induced by Class I antiarrhythmic agents. Almokalant (Figure 5.10) is a selective Class III agent, even though it contains a β -blocking pharmacophore. In rabbit ventricular cells, almokalant blocks the delayed rectifier K+ current in a time-and voltage-dependent manner and so lengthens the action potential duration (Carmeliet, 1993). Both MS-551 and almokalant have replaced the methyl sulphonamide with other electron withdrawing groups. It is still possible to find leads in this area, and the development of a novel series of spiro[benzopyran-2,4’-piperidines] led to L-691, 121 (Figure 5.11) which is essentially devoid of significant activity for the β 1 receptor, a problem found with some of the series (Elliot et al., 1992). It is a selective blocker for the rapidly activating component (IKr) of the delayed rectifier current in isolated guinea pig ventricular myocytes, with no effect on the slowly activating component (IKs) current, the inward rectifier (IK1) current and the L-type Ca2+ current (Claremon


Figure 5.11 Development of spiro[benzopyran-2,4’-piperidines]

et al., 1993). The series was derived from a 5-HT3 antagonist GR-38032, which prolongs the effective refractory period with a potency similar to sotalol. A variety of tricyclic ring systems were evaluated starting with the thienothiopyran (8) which led to thienothiopyranone spiropiperidine derivative (9), and eventually benzopyran spiropiperidine (10). Subsequent introduction of a methylsulphonamide produced L-691,121. A major metabolite of this compound is the N-dealkylated product and, in order to overcome this problem, a series of conformationally restrained derivatives were examined leading to L-702,958. Reduction of the ketone to the alcohol occurs metabolically and both enantiomers were found to be equally active in vitro, but the S-enantiomer L-706,000, is six times more potent in vivo. The two second generation compounds L-706,000 and L-702,958 have good oral bio-availablity and longer duration of action in animals, than the original compound L-691,121.


5.3.3 Combined Class III Pharmacophore Most of the Class III antiarrhythmic agents can be accommodated by one pharmacophore outlined in Figure 5.12. An aromatic ring is substituted with an electron withdrawing group, preferably methylsulphonamide in the para position, and this helps reduce liability to Class I effects (e.g. conversion of procainamide into sematilide). Further substitution of the nitrogen NH of the sulphonamide or extending the S-methyl group into the ethylsulphonamide leads to diminished activity (Lumma Jr. et al., 1987). The quaternary compounds outlined in Figure 5.7 also fit this pharmacophore. Apart from sulphonamide the substituent can be other electron withdrawing groups, such as halogen (clofilium, Figure 5.7), nitro (MS-551, Figure 5.10), nitrile (almokalant, Figure 5.10), acetamide (NAPA, Figure 5.8) and imidazol-1-yl (3, Figure 5.8). The aromatic ring is connected to a nitrogen atom, which is presumably charged at the receptor, by a 3 to 4 atom spacer. This spacer can be a (CH2)4 moiety in which one or two of the methylenes can be replaced with O, SO2N, NH or CO or substituted with an alcohol. It has also been possible to incorporate fused rings at this

Figure 5.12 Pharmacophore Model for Class III Binding Site

position (Figure 5.11). Substitution with an alcohol in combination with a secondary amine, can lead to a molecule with β -blocking activity (Class II), as found in sotalol (Figure 5.8), but converting the secondary amine into a tertiary amine tends to abolish this activity. The amine functionality can be a quaternary group, or a secondary or tertiary amine, and this can be acyclic, cyclic or heterocyclic. Second generation compounds are distinguished by the addition of another group to the amine, normally aromatic or heterocyclic, and this is separated by a one to three atom spacer. This has the effect of increasing potency, and this enhanced activity


suggests that an additional binding site has been located in this region. Owing to the lack of information it has not been possible to correlate K blocking activity with structure. 5.3.4 Miscellaneous Class III Agents Some compounds do not fit the pharmacophore just described in all respects, and this is reflected in their K channel blocking properties. Tedisamil (Figure 5.13) has a totally different structure to all the other class III antiarrhythmic agents and this difference in structure seems to be manifested in a different profile as a KCB. Not only is it a blocker of both the fast and slow components of the delayed rectifier current, but it also blocks the transient outward current in mammalian cells and at higher concentrations inhibits Na+ currents (Dukes and Morad, 1989; Dukes et al., 1990). Ambasilide, an analogue

Figure 5.13 Miscellaneous Class III antiarrhythmic agents

of tedisamil (a symmetrical dibasic molecule), is unsymmetrical with only one basic centre and it has been shown to block the delayed rectifier current in guinea


pig myocytes (Zhang et al., 1992) in a dose dependent manner. A blocking effect of Na + currents in canine Purkinje fibres and ventricular muscle was observed, which indicates that ambasilide also has some Class I activity (Takanaka et al., 1992). (-)-S-Terikalant (RP-62719) is the active enantiomer of RP-58866 and is 150 times more potent than the R-enantiomer (Escande et al., 1992) in blocking the inward rectifier K+ current. This is unusual because the usual Class III agents selectively block one or both components of the delayed rectifier current. Another unusual structure which displays selective Class III activity is NE-10064 (Figure 5.13). Information on the compound is scarce; it was reported that it blocked the slowly activating component of the delayed K+ rectifier current. Surprisingly the compound also blocked the mini K channel when expressed in Xenopus oocytes (Busch et al., 1993). The piperazinoguanadine RS-87337 (Figure 5.13) is a combined Class III/IA antiarrhythmic agent (Dumez et al., 1989). Another novel structure with combined Class I and III activity is a benzodiazepine (11, Figure 5.13) and the Renantiomer which is slightly more active than the S-enantiomer was selected for further biological and toxicological evaluation (Johnson et al., 1993). 5.4 Aminopyridines Prior to the discovery of the very potent scorpion and snake toxins the aminopyridines were a useful tool in helping dissect out various K+ currents. 4Aminopyridine (4-AP) is selective for the delayed rectifier type K+ current and is particularly active against the slowly inactivating outward current (IKS). As there are already some excellent reviews (Marshall, 1982; Glover, 1982; Cook and Quast, 1990) on the effects of aminopyridines on K channel types and sub-types it is not intended to cover this aspect here. 4-AP and its derivatives have been known to block K+ currents since the early seventies (Pelhate and Pichon, 1974; Gillespie and Hutter, 1975; Yeh et al., 1976; Meves and Pichon, 1977) and some years later there was a thorough review of the pharmacological properties and an attempt at studying the SARs of the aminopyridines was reported (Glover, 1982). There have also been more systematic studies on the SARs of aminopyridines and derivatives on axonal K+ conductance (Pelhate and Malecot, 1989; Marshall, 1982). The structure of 4-AP is an extremely simple one (Figure 5.14). Nevertheless, some manipulation of this structure has been attempted (Marshall, 1982). The amino position has been switched from position 4-to 2-(2-AP) and 3-(3-AP), but these are generally found to be less active. Diamino derivatives are also active, the most potent of which is 3,4-diaminopyridine (3,4-DAP). The order of potency for the various amine analogues is 3,4-DAP>4-AP>2,3-DAP>3-AP>2AP>2-amino-3-hydroxypyridine. Replacement of amino with hydroxy leads to less active compounds as does further substitution of amino compounds with


chloro, bromo, methyl, or ethoxy substituents (Pelhate and Malecot, 1989). Replacement of pyridine with other heterocyclic groups such as piperidine, pyrazine, pyrimidine, triazine and morpholine leads to totally inactive molecules (Pelhate and Malecot, 1989). Tacrine which has been used for the treatment of Alzheimer’s disease, by nature of its anticholinesterase activity, also has K+ blocking activity (Osterrieder, 1987;

Figure 5.14 4-Aminopyridine derivatives

Schauf and Sattin, 1987; Freeman et al., 1988) which is not too surprising, owing to its strong relationship to 4-AP. Attempts have been made to improve the K+ channel blocking abilities of aminopyridines and a series of 3-substituted-4aminopyridines were examined in the hippocampal slice (Waser et al., 1988), the most interesting of these compounds was 3-methoxy-4-aminopyridine (Figure 5.14) and this was evaluated in various preparations, such as guinea pig ileum, and for effects on botulism toxin paralysis (Berger et al., 1989a). The 14C labelled form was used to examine its distribution in mice and high levels were found in cholinergically innervated secretory organs, also in the adrenal medulla, the hippocampus, the thalmic nuclei and the cortex (Berger et al., 1989b). Another aminopyridine examined for its ability to release neurotransmitters such as noradrenaline (Foldes et al., 1988) and acetylcholine, was LF-14 (Potter et al., 1989). A number of related ureas were also examined (Ohta et al., 1982), but were found to be less active. LF-14 was evaluated in a variety of other preparations and found to be a peripherally acting compound, with less central action than 4-AP (Biessels et al., 1985). The aminopyridines block K channels in both the periphery and in the CNS. The central actions are potentially interesting because they cause the release of neurotransmitters in the brain and so could be useful in improving neurotransmission. They are also convulsant with a small therapeutic ratio which means that the aminopyridines are not suitable for use in the treatment of Alzheimer patients. They have however, been used in the treatment of botulism


in humans, particularly 3,4-DAP, which is less able to penetrate the blood-brain barrier, and therefore has less propensity to cause convulsions. 3,4-DAP has also been shown to be effective in multiple sclerosis (Bever et al., 1990) but two of ten patients did suffer seizures. It appears that the clinical potential of the aminopyridines is limited by their toxicity and lack of potency. 5.5 Miscellaneous Blocker 5.5.1 1-(4-methoxyphenyl)indole Neurosearch has filed a patent on this indole (Figure 5.15) for use as an antidepressant or for use in Alzheimer’s disease (Olsen et al., 1992). Antidepressant

Figure 5.15 1-(4-Methoxyphenyl)indole

activity was detected in a tail suspension assay at 10 to 100 mg/kg i.p. The compound which is claimed to block the BKCa in bovine aortic smooth muscle cells as well as in pancreatic β -cells has alsox been shown to have memory enhancing effects in a social recognition model in rats. 5.6 Conclusions The examples in this chapter serve to illustrate several points. First and foremost that KCBs are useful drugs which possess the same subtleties and range of interactions of any other class of drugs. This serves to dispel their sometimes public image of being mere bungs which prevent the passage of ions through pores in membranes. The fact that the existing channel blockers were developed from the serendipitous exploitation of their eventual functional effects and not by an attack on the molecular target itself opens up two futures. First, that other existing therapeutic agents will have their mode of action shown to be K channel blockade is a probability, and second, that blockers of so far untapped channels will become important drugs is a certainty. In fact with the wide availability of different K channel types, the emphasis will switch to identifying selective blockers (and enhancers) of these individual clones. Accordingly, the eventual


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6 Potassium Channels: Diversity, Assembly and Differential Expression R. LATORRE & P. LABARCA Centro de Estudios Cientfficos de Santiago y Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 16443, Santiago, Chile. 6.1 Introduction The wealth of evidence on K channel structure, function, diversity and differential distribution in animal tissues obtained in the past few years presents us with an amazing panorama that is only now beginning to be revealed. Yet, in spite of the formidable progress that has followed the cloning of a K+-selective channel (Baumann et al., 1987; Kamb et al., 1987; Tempel et al., 1987) our knowledge of the basic principles that govern K channel structure and function is still in its infancy. Such principles concern the most basic phenomena associated with K channel function: their ability to catalyze with high efficiency the passive flow of K+ across the cell membrane, their various selectivity and permeation profiles, and the mechanisms of interaction with the membrane electric field (reviewed in Jan and Jan, 1992; Pongs, 1992; Caterall, 1993; Hoshi and Zagotta, 1993; Bezanilla and Stefani, 1994). As a minimum, a basic understanding of K channel structure-function implies the development of a common set of rules that are sufficient to account for the occurrence of K channels exhibiting variable open conductances, different selectivity sequences and permeation mechanisms. Likewise, the development of the basic rules that give rise to K channel voltage dependence should suffice to account for the various gating properties of K channels. K channel diversity seems to have accompanied animal cells throughout evolution and some K channels are among the most conserved proteins in eukaryotes (Jan and Jan, 1990b, Rudy et al., 1991a; Salkoff et al., 1992). K channels became fundamental to animal cell physiology very early in evolution and became part of ancient transduction mechanisms which enabled eukaryotes, through K channel-mediated control of membrane potential, to couple the cell inner dynamics to the outer environment. A differential expression of K channel mRNAs is found to accompany the events of animal development and this also occurs in adult animal tissues. This differential expression of K channels is particularly noteworthy in the brain where it has been documented from

K CHANNELS: DIVERSITY, ASSEMBLY AND DIFFERENTIAL EXPRESSION 157

invertebrates to mammals. It is not too risky to predict that differential expression of K channels in the nervous system will be demonstrated to be relevant to and accompany neuronal differentiation, and be associated to plastic phenomena as well as neurological deficiencies. Although for the sake of clarity, we have divided this chapter into several sections, it contains two main topics. First, we describe the varieties of the K channels that have been cloned until now and their possible topologies in the lipid bilayer membrane. Second, since we posses a wealth of information regarding the S4-related potassium channel family (Jan and Jan, 1990a), we reviewed in some detail its diversity, subunit assembly, and differential expression. 6.2 Varieties of K Channels Before the application of molecular biology into the field of electrophysiology, K channels were placed arbitrarily in five different groups (e.g. Rudy, 1988): (1) Voltage-dependent K channels which in turn can be subdivided into channels showing fast inactivation (A-type channels) and delayed rectifiers of the Hodgkin and Huxley type (Hille, 1992); (2) Ca-activated K channels that are usually classified into two different classes of high (maxi, BKCa) and small (SKCa) conductance, although they can also be dissected pharmacologically according to their sensitivity to scorpion and bee toxins (Blatz and Magleby, 1987; Latorre et al., 1989; McManus, 1991; Latorre, 1994); (3) inward rectifiers that give rise to a current that activates with hyperpolarizing voltages in contrast to A-type and delayed rectifiers channels that activate with depolarizing voltages (e.g Rudy; 1988); (4) adenosine triphosphate-dependent channels (KATP; Noma, 1983); and (5) K channels coupled to receptors, as is the case of those coupled to muscarinic receptors in the heart muscle. With the exception of the apamin-sensitive SKCa, channels of all the classes described above have been cloned and expressed in oocytes or in cell lines in culture. Structurally, channel taxonomy has become simpler because the classes described above can be grouped into the following three large superfamilies. 6.2.1 K Channels Belonging to the S4 Channel Superfamily (Jan and Jan, 1990a) As determined from hydrophatic plots, these channels have six transmembrane segments (S1–S6; Figure 6.1). The fourth (S4) segment is characterized by having several, up to eight, positively charged amino acid residues (lysine or arginine) at every third position, with non-polar or hydrophobic residues in between. K channels in this family share the S4 segment with voltage-dependent Na and Ca channels (Figure 6.1B). For example, the S4 segment of the Shaker Atype K channel has about a 50% identity with the corresponding segment in


domain IV of the Na channel and more important these two domains contain a similar number of positive charges (7 in Shaker and 8 in domain IV of rat brain II Na channels). Because of its peculiar structure, S4 is postulated to be part of the voltage sensor (for reviews see Catterall, 1993; Bezanilla and Stefani, 1994). In the case of Na and Ca channels, Figure 6.1 A shows that the channel-forming protein is a polypeptide consisting of four domains or pseudosubunits (I–IV) each of which contains the S1 to S6 transmembrane segments. K channels belonging to this superfamily have a molecular weight that is about a quarter of that of Na or Ca channels. This suggests that K channels are tetramers.

Figure 6.1 Transmembrane folding models for voltage dependent Na and Ca channels (A), voltage-gated and calcium-activated K channels (B), inward rectifier channels (C) and min K channels (D). In some voltage-dependent K channels the N-terminal is an integral part of the inactivation gate and provides a recognition site for heteromultimeric channel assembly and prevents coassembly between subfamilies. The S1 segment is important in the coassembly of homo- and heteromultimeric channels. The S4 is supposed to be the channel voltage sensor and the S5–S6 linker (region P) forms part of the pore walls. (For detail and references see text)


In voltage-dependent and Ca-activated K channels (Atkinson et al., 1991; Adelman et al., 1992) an hydrophobia polypeptide segment with the shape of a hairpin located between transmembrane segments S5 and S6 forms the channel pore (H5 or P region). This region is extremely well conserved in all K channels belonging to this superfamily. The isolation of a cDNA clone from ether à go-go (eag) locus of Drosophila (Warmke et al., 1991) showed that it also encodes a polypeptide that shares a S4 transmembrane domain and its S5–S6 linker has a high degree of identity with voltage-gated K channels (Figure 6.1B). The sequence also contains a putative cyclic nucleotide binding site in the carboxy terminal region, making this channel a relative to both, voltage-dependent channels and cyclic nucleotide-activated channels (Guy et al., 1991). Expression of eag cRNA in oocytes induces a voltage–and cAMP-dependent K channel (Brüggemann et al., 1993). Although eag has been identified in mouse and human sharing 71 and 48% identity, respectively, with the Drosophila protein (Warmke and Ganetzki, 1993, 1994), the physiological importance of this channel in mammals is unclear at present. A K inward rectifier channel from the plant Arabidopsis thaliana also belongs to the S4 superfamily (Schachtman et al., 1992). Apparently, this channel has a different mechanism of activation than ‘classical’ inward rectifier channels since it activates at large hyperpolarizing voltages regardless of the value of the K+ equilibrium potential. The most salient feature of ‘classical’ inward rectifier channels is that they allow current to pass only below the K+ equilibrium potential, i.e. the position in the voltage axis of their voltage activation curves varies with external K+ concentration (Hagiwara and Takahashi, 1974). 6.2.2 Channels with Monomers Containing Two Membrane Spanning Regions Cloning of three different inward rectifier channels (Kubo et al., 1993a, 1993b; Ho et al., 1993; Dascal et al., 1993) has revealed a new superfamily of K channels. In this case the proteins only feature two putative membrane-spanning segments (M1 and M2, Figure 6.1C). However, the potential P region of these channels (with one notable exception; see Suzuki et al., 1994) has a high degree of similarity with the P region of the channels belonging to the S4 superfamily. The K channel coupled to muscarinic receptors in the heart [(5) above] belongs to this superfamily (Kubo et al., 1993b; Dascal et al., 1993). The number of inwardly rectifying channels is expanding very fast. So far five different channel genes have been identified (Kir1.0–Ki5.0), and the mRNAs of the subfamilies Kir1.0, Kir2.0 and Kir3.0 undergo alternative splicing (Doupnik et al., 1995). Inward rectification arises as a consequence of a Mg2+ and/or as a consequence of a polyamine (e.g., spermine, spermidine, putrescine) voltage-dependent block. However, intrinsic channel voltage dependence can also contribute to the inward rectification in these channels (reviewed in Doupnik et al., 1995). The ATP-


sensitive (inhibited) type of K channels from rat heart have been cloned, sequenced and expressed in Xenopus oocytes (Ashford et al., 1994). The cloned rcKATP is inhibited by ATP and activated by intracellular nucleotide diphosphates. The amino acid sequence revealed two putative transmembrane segments which flank a pore region with a high degree of identity with the Pregion of other K channels. Thus, the rcKATP channel belongs to the inward rectifier K channel family. 6.2.3 Channels Formed by Monomers Containing a Single Membrane Spanning Region A third family is represented by a gene putatively coding for a voltagedependent K channel with a hydropathy profile showing a single membrane spanning β -helix (Takumi et al., 1988; Figure 6.1D). The protein is a relatively small polypeptide of only 130 amino acids; the small size of this channelforming protein has prompted workers in the field to name this channel ‘minK’ (minimal K; Hausdorff et al., 1991). In oocytes the minK protein induces a slowly activating voltage-dependent K+ current that is inwardly rectifying (Blumenthal and Kaczmarek, 1993). The gene has been cloned from kidney (Takumi et al., 1988), heart (Folander et al., 1990), uterus (Murai et al., 1989), and T-lymphocytes (Attali et al., 1992), but its physiological role is still unclear. In the heart, Folander et al. (1990) have suggested that minK induces the slow component of the delayed rectifier currents. 6.3 Origin of Diversity in K Channels Belonging to the S4 Superfamily K channels are ubiquitously distributed in different cells and tissues and, therefore, K channel diversity is of great importance in determining the variety of electrical responses of cells when subjected to stimuli (e.g. Hille, 1992). Below we discuss three possible mechanisms able to originate K channel diversity: 1) alternative splicing; 2) multiple genes; and 3) formation of heteromultimeric channels (for reviews see Jan and Jan, 1990b; Rudy et al., 1991a; Pongs, 1992; Salkoff et al., 1992).


6.3.1 Shaker and Mammal Genes Encode More Than One K Channel Protein by Alternative Splicing Drosophila The molecular biology era of K channels started thanks to the existence of a Drosophila mutant named Shaker, characterized by its leg-shaking phenotype under ether anesthesia. Early studies, using intracellular recording, indicated that the neuromuscular transmission in two Shaker mutants was abnormal. The postsynaptic potentials in the mutant are abnormally large and prolonged and they are a consequence of a sustained Ca2+ current at the nerve terminals. It was later demonstrated that Shaker mutants showed prolonged action potential duration and also altered A-type K+ currents (Tanouye et al, 1981; Salkoff and Wyman, 1981). Several cDNA clones were isolated from the Drosophila Shaker locus. The sequence showed the characteristic primary structure of voltage-dependent channels: S1–S6 transmembrane segments with a positively charged S4 segment (Tempel et al., 1987; Papazian et al., 1987; Iverson et al., 1988; Baumann et al., 1987, 1988; Kamb et al., 1987, 1988). Subsequently, Shaker proved to be a family of different but closely related proteins encoded by alternatively spliced transcripts and this mechanism was proposed to be a convenient one to generate K channel diversity (Schwarz et al., 1988). It was found that all the spliced variants share the central region of the channel (i.e. S1–S6; Figure 6.1) and that they differ at the amino- and carboxy-terminal (two variants). There are five alternatively spliced amino–terminal variants of Shaker (Schwarz et al., 1988; Pongs et al., 1988; Kamb et al., 1988) and two carboxyl termini (Iverson et al., 1988; Kamb et al., 1988; Schwarz et al., 1988; Timpe et al., 1988b). Moreover, three variants (ShB, ShC, and ShD; Table 6.1) inactivate rapidly (Table 6.1; time constants (β ) of tens of milliseconds or less) whereas the other two (ShD2 and ShH37) inactivate with a β in the hundreds of milliseconds (Timpe et al., 1988a, 1988b; Zagotta et al., 1989; Aldrich et al., 1990; Iverson and Rudy, 1990). ShA and ShB (also called ShH4) have the same amino-terminus but they differ in their carboxyl terminal sequence. Kinetically, these two Shaker channels differ mainly Table 6.1 Nomenclature and kinetic properties of S4 superfamily-related K channels in invertebrates and mammals1 Subfamily of K Channels Shaker subfamily

Type of Current

Species

References

ShA2

Fast Inactivation

Drosophila

ShB

Fast Inactivation

Drosophila

Tempel et al. (1987) Pongs et al. (1988) Schwarz et al. (1988) Kamb et al. (1988)


Subfamily of K Channels Shaker subfamily

Type of Current

Species

ShC ShD

Fast Inactivation Fast Inactivation

Drosophila Drosophila

References

Schwarz et al. (1988) Schwarz et al. (1988) Pongs et al. (1988) 3 ShD 2 Drosophila Stocker et al. (1990) ShH37 Slow Inactivation Drosophila Iverson et al. (1988) AKv1.1a/AK01a Fast Inactivation Aplysia Pfaffinger et al (1991) rKvl. 1/RCK1/BK1/RBK1 Sustained Rat Baumann et al. (1988) McKinnon (1989) Christie et al. (1989) mKv1.1/MBK1 Mouse Tempel et al. (1988) rKv1.2/RCK5/BK2/ Sustained Rat Stühmer et al. (1989) NGK1/RH1/RK2 McKinnon (1989) Yokoyama et al. (1989) Ishii et al. (1992) Po et al. (1992) mKv1.2/MK2 Mouse Chandy et al. (1990) XKv1.2 Xenopus Ribera (1990) rKv1.3/RCK3/RGK5/KV3 Slow Inactivation Rat Stühmer et al. (1989) Douglass et al. (1990) Swanson et al. (1990) mKv1.3/MK3 Sustained Mouse Grissner et al. (1990) Chandy et al (1990) hKv1.3/HPCN/HLK3/ Slow Inactivation Human Philipson et al. (1991) HGK5 Attali et al. (1992) Yun–Cai et al. (1992) rKv1.4/RCK4/RK3/ Fast Inactivation Rat Stühmer et al. (1989) RHK1/RH10 Roberds and Tamkun (1991) Tseng–Crank et al. (1990) Meyerhoff et al. (1992) Okada et al. (1992) hKv1.4/HK1/hPCN2 Fast Inactivation Human Tamkun et al. (1991) Philipson et al. (1991) rKv1.5/RCK7/KV1/RK4 Betsholtz et al. (1990) Swanson et al. (1990) Roberds and Tamkun (1991) mKv1.5 Slow Inactivation Mouse Attali et al. (1993) mKv1.5 5’ Mouse Attali et al. (1993) hKv1.5/HK2/hPCN1 Slow Inactivation Human Tamkun et al. (1991) Philipson et al. (1991)


rKv1.6/RCK2/KV2

Sustained

Rat

hKv1.6/HBK2 mKv1.7/MK6/MK4 Shab subfamily AKv2.1/AShab rKv2.1/DRK1/cDRK1

Sustained

Human Mouse

mKv2.1 Kv2.1 Shaw subfamily Kv3.1a/NGK2/KShIIIB

Sustained Sustained

Mouse Human

Sustained

Rat, Mouse Yokoyama et al. (1989) Weiser et al. (1994)

Kv3.1b/Kv4/Raw2

Sustained

Rat, Mouse

rKv3.2a/RKShIIIA Raw 1 rKv3.2b/KShIIIA3

Sustained Sustained

Rat Rat Rat

Slow Inactivation Aplysia Sustained Rat

rKv3.2c/KShIIIA.2

Rat

rKv3.3a/KShIIID.1

Slow Inactivation Rat

rKv3.3b/KShIIID.2

Slow Inactivation Rat

rKv3.4a/Raw3 hKv3.4b/KShIIIC rKv3.4c

Fast Inactivation Fast Inactivation

Shal subfamily mKv4.1/mShal rKv4.1/RShal Kv4.2/RK5

Rat Human Rat

Sustained Mouse Slow Inactivation Rat Rat

Grupe et al. (1990) Swanson et al. (1990) Grupe et al. (1990) Grissmer et al. (1990) Quattrocki et al. (1994) Frech et al. (1989) Hwang et al. (1992) Pak et al. (1991a) Albrecht et al. (1993)

Luneau et al. (1991a) Grissmer et al. (1992) Rettig et al. (1992) McComack et al. (1990b) Rettig et al. (1992) Luneau et al. (1991b) Rudy et al. (1992) Luneau et al. (1991b) Rudy et al. (1992) Vega–Saenz de Miera et al. (1992) Vega–Saenz de Miera et al. (1992) Schröter et al. (1991) Rudy et al. (1991b) Vega–Saenz de Miera et al. (1994) Pak et al. (1991b) Baldwin et al. (1991) Roberds and Tamkun (1991)

in their recovery from inactivation. For ShA, recovery from inactivation has a β of 400 ms whereas the same process in ShB has a β of 20 ms (Wittka et al., 1991).


Thus, the Shaker subfamily consists of several K channels with different degrees of inactivation. A digression on inactivating mechanisms The fact that Shaker K channels with different amino-terminal domains have different inactivation rates strongly suggested that this region of the protein molecule was (or was part of) the inactivation gate. Hoshi et al. (1990) studied the effect of mutations of this domain in ShB. Deletion mutations allowed the identification of the first 20 amino acids of the amino–terminal as the inactivation gate. For example, a 41 amino acid deletion (6–46) produced a channel that, once expressed in oocytes, does not inactivate with a rapid time course. Restoration of inactivation in the Sh 6–46 mutant can be obtained if a peptide consisting of the first 20 amino acids of the ShB channels is added to the cytoplasmic side of the channel (Zagotta et al., 1990). These results indicate that fast inactivation in Atype channels is the result of the occlusion of the pore by the protein N-terminal domain. Structurally, the N-terminal is hydrophobic and is followed by a stretch of positively charged amino acids. Both hydrophobicity and the net charge of the inactivating peptide are crucial in determining the degree and the kinetics of the inactivation process (Murrel-Lagnado et al., 1993; Toro et al., 1994). This type of inactivation is called N-type inactivation as opposed to the C-type of inactivation that involves amino acid residues in the S5, P and S6 segments (Hoshi et al., 1991). In contrast to N-type, C-type of inactivation is modulated by external cations and most of the mutations that affect C-type inactivation also modified ion conduction through the pore (LopezBarneo et al., 1993). Increasing external K+ slows down C-type inactivation. Mammals In mammals, K channel diversity arises, at least in part, from the expression of alternatively spliced RNA (Luneau et al., 1991a, 1991b; McCormack et al.,

1

In the case mammalian K channels, in the first column the first denomination for a given channel corresponds to the nomenclature of Chandy (1991) and is followed by the other names given to the same channel in the literature. The second column refers to the channel kinetics. ‘Fast Inactivation’ means that the channel inactivates in the order of tens of milliseconds or less. ‘Slow inactivation’ refers to channels that show inactivation processes with time contents in the 100–1000 ms range. Potassium channels with time constants larger than 1 s are referred to as ‘Sustained’. 2 For other names of the Drosophila Shaker subfamily, the interested reader should consult, for example, Iverson and Rudy (1990). In particular, ShB is also called ShH4. This alternatively spliced variant has been used in numerous biophysical studies. 3 In the case of ShD2 there is a small component of fast current decay, but at all voltages the steady state component is large.


1990b; Rettig et al., 1992; Rudy et al., 1992; Vega-Saenz de Miera et al., 1992; Attali et al., 1993; Weiser et al., 1994). In rat it has been shown that a K channel (Kv4; see Table 6.1) structurally related to the Drosophila Shaw protein (see below), is originated by alternative splicing. In rat, NGK2, the first cloned K channel of this family (Yokoyama et al., 1989), arises from alternative exon usage at a locus that also encodes Kv4. NGK2 and Kv4 have quite different carboxyl-terminal domains. On the other hand, Schröter et al. (1991) and Rettig et al. (1992) have described a family, also related to the Shaw gene, composed of three alternatively spliced variants and denominated Raw1, Raw2, and Raw3 in the rat nervous system. In the Raw subfamily alternative splicing produces K channel proteins with different Ctermini. However, the alternative C-termini appear not to play an important role in ion channel characteristics, leaving open the question about the reason and the importance for the existence of Raw K channel variants. When expressed in oocytes Raw1 and Raw2 cRNAs induce non-inactivating currents; Raw3 cRNA, on the other hand, expresses A-type inactivating currents (Ruppersberg et al., 1991a; see below). Raw3 possesses an N-terminus which is 28 amino acids longer than the N-terminus of Raw1 and Raw2. Deletion of these extra amino acids completely eliminates fast inactivation in Raw3 (Rettig et al., 1992). Raw3 also shows a marked inward rectification that is a consequence of a voltage-dependent internal Mg2+ blockade (Rettig et al., 1992). Table 6.1 shows that the Kv3 subfamily is very rich in splice variants and Vega-Saenz de Miera et al. (1992) described in detail two alternatively spliced transcripts: KSHIIID.1 (Kv3.3a) and KSHDIIID.2 (Kv3. 3b). Kv3.3a has slow activation kinetics compared with Shaker A-type K channels, but like other A-type channels the current inactivates with a time constant of about 100 ms at 50 mV. The N-terminal of the Kv3.3a channel shares a stretch of amino acids with the Raw3 channel (ser-ser-val-cys-val). The cysteine in this sequence plays an important role in the modulation of inactivation by reducing agents such as glutathione. Oxidation of the cysteine residue inhibits N-type of inactivation (ball and chain mechanism) of Raw3 channels (Ruppersberg et al., 1991b). Table 6.1 shows that several mammalian Shaker K channels have been cloned and most of them appeared to be encoded by separate intronless genes (Stühmer et al., 1989; Beck and Pongs, 1990; Chandy et al, 1990). However, Attali et al. (1993) reported the existence of at least three mRNA isoforms able to encode the mouse cardiac delayed rectifier K channel (Kv1.5). Two alternatively-spliced variants encoded for a long (602 amino acid) protein and a short form (Kv1.5 ― 5’) in which the first 200 amino acids from the N-terminus have been deleted. A third clone codes for a non-functional protein with a truncated C-terminus (Kv1. 5 ― 3’). Kv1.5 ― 3’ mRNA co-injection with mRNA coding for the long form inhibited expression of the Kv1.5 channel.


6.3.2 Multiple Genes Inside a subfamily like Shaker different A-type channels can originate from alternative splicing of the Shaker gene. However, it is clear that this type of mechanism cannot account for the K channel diversity found in excitable tissues of Drosophila. It is important to remember here that the Shaker mutant still has K + currents. This fact strongly suggests the presence of a family of genes coding Shaker-related K channels allowing for the variety of K channels with different voltage dependencies, Ca2+ sensitivities, and pharmacological properties. Using a Shaker complementary DNA probe and low-stringency hybridization, Butler et al. (1989) were able to clone three new genes which they dubbed Shab, Shaw, and Shal each of which coded for proteins with a considerable degree of identity (― 38%) to the Shaker protein. The identity between members of this K channel family is considerable when the transmembrane segments are considered. For example, the identity between the S6 transmembrane segment of Shaker and Shaw is 86%. When expressed in Xenopus oocytes this family of voltagedependent K channels was shown to have different rates of activation and inactivation (Wei et al., 1990). Thus, whereas Shaker and Shal K channels displayed fast inactivation processes, Shab and Shaw are of the delayed rectifier type (Figure 6.2). Soon it became clear that a member of a subfamily in mammals was more similar to one of the four members of the Drosophila family than to a mammalian member of a different subfamily. For example, the Shaker protein shares about 70% identity with mammalian homologs (e.g. RCK1 (76%); Baumann et al., 1988), but has only

Figure 6.2 Ionic currents induced by expression in Xenopus oocytes of ShakerH37, Shal2, Shab11, and Shaw2 potassium channels. Depolarizing pulses were applied from a holding voltage of –90 mV in 10 mV steps in the range of –80 to 20 mV. Data are adapted from Wei et al. (1990).

38% identity with the Shab subfamily.1 Table 6.1 shows that Shaker genes are homologs of the Kv1 subfamily of mammalian genes; Shab are homologs of the Kv2 subfamily; Shaw are Kv3 homologs; and Shal are Kv4 homologs. The nomenclature for the mammalian genes is that of Chandy (1991) where K stands for potassium-channel gene, v for voltage-dependent, the first number corresponds to the subfamily and the second number is the number of the gene.


For instance the Kv1 subfamily is at present composed of seven different genes whereas only four genes have been found in the case of the Kv3 subfamily. Alternative nomenclatures can be found in the literature; for example ShI, ShII, ShIII, and ShIV for Kv1 Kv2, Kv3, and Kv4 respectively (e.g. Rudy et al., 1991a, 1991b; Weiser et al., 1994). It is notable that the genes of a given subfamily are extremely well conserved among invertebrates and vertebrates considering that they diverged about 570 million years ago. On the other hand, the fact that the same subfamilies of K channels are found in vertebrates and invertebrates suggest that the K channel genes diverged before eukaryotes diversified into vertebrates and invertebrates. Gene duplication and variation of these precursor genes may explain the origin of the various members of the subfamilies found in mammals (Rudy et al., 1991a). The largest subfamilies of K channels in vertebrates are Kv1 (Shaker-like) and Kv3 (Shaw-like). In contrast to the Shaker subfamily where diversity is a consequence of alternative splicing of a single gene, in the Kv1 subfamily, diversity is obtained through gene duplication. In the case of the mouse, Chandy et al. (1990) showed that K channel genes coding for MK1, MK2 and MK3 exist as a single uninterrupted exon in the mouse genoma. This prevents the generation of different K channels by alternative splicing. In the rat (RCK gene family; reviewed in Pongs, 1992) six members of the Kv1 subfamily have been characterized and each protein is encoded in a separate gene. The lack of alternative splicing in this subfamily has a notable exception in Kv1.5 (see section 6.3.1; Table 6.1; Attali et al., 1993). In contrast, the Kv3 subfamily consisting, at present, of four genes (Rudy et al., 1991b; Weiser et al., 1994) that show a large number of spliced variants (Rudi et al., 1991b; Luneau et al., 1991a, 1991b; Rettig et al., 1992; Rudy et al., 1992; Vega-Saenz de Miera et al., 1992; Weiser et al., 1994). The different K channel subfamilies generate currents that can be transient or of the delayed rectifier type. Figure 6.3 shows that the different genes belonging to the same (Kv3) subfamily are also able to express transient and sustained currents. Notice that Kv3.2a and Kv3.1a transcripts express delayed-rectifier type currents while Kv3.4b and Kv3.3a give rise to transient currents, although with different time courses (fast and slow inactivating currents). In this regard both Kv3.4 and Kv3.3 can be distinguished from Kv3.2 and Kv3.1 by the presence of N-terminal inserts likely to be the structures responsible for their inactivation process (Vega-Saenz de Miera et al., 1992). These currents activate relatively slowly and at voltages larger than –20 mV. Therefore, as discussed by Vega-

1 Percentage of identical amino acids in analogous positions between pairs of K channel proteins in other subfamilies are for example: Shab/Kv2.1 72%; Shaw/Kv3.2a 52%; Shal/ Kv4.1 82% [for more information see Rudy et al. (1991a, 1991b)]. Rudy et al. (1991a, 1991b) have pointed out that some of the Sh proteins appear to be among the most conserved proteins known. For example, the human Shaker Kv1.2 shows about 99% identity with the rat Kv1.2.


Saenz de Miera et al. (1992), they could not be involved in subthreshold phenomena (e.g. regulation of resting potential) and it is doubtful that they activate during a single fast action potential. However, these currents may play an important role during prolonged depolarizations or in neurons showing repetitive firing. Only a few K channels belonging to the Kv2 (Shab-like) and Kv4 (Shal-like) subfamilies have been cloned. The channels of the Kv2 subfamily (rKv2.1, Frech

Figure 6.3 Current time courses induced by alternatively spliced variants of the Kv3 subfamily of K channels. Notice that the current time courses for the different alternatively spliced variants, similarly to the current kinetics seen when channels from different subfamilies are expressed, are of the sustained type (A and B) or transient [fast (C) and slow (D) inactivation]. Data from Weiser et al. (1994).

et al., 1989; Hwang et al., 1992; mKv2.1, Pak et al., 1991a; hKv2.1, Albrecht et al., 1993) activate slowly and show delayed rectifier properties. In the case of mKv2.1 a very slow inactivation process with a time constant of several seconds has been reported (Pak et al., 1991a). In contrast, the Aplysia Kv2.1, although it activates slowly like the others members of the Kv2 subfamily, it inactivates within several hundred milliseconds (Quattrocki et al., 1994). Interestingly, the inactivation process of the Aplysia Kv2.1 channel remains intact after truncation of the N-terminal. This finding suggests that in this case inactivation is not


mediated by a mechanism of the ‘ball and chain’ type. Despite the fact that the rat, mouse, and human Kv2.1 channel proteins are highly conserved, their electrophysiological and pharmacological properties differ. For example, despite the fact that there is 100% identity in the S4 segment of these three channels, rKv2.1 is more voltage-dependent than the mouse or the human Kv2.1 (Albrecht et al., 1993). Pharmacological differences are also profound. The rat Kv2.1 is about 200-fold more sensitive to tetraethylammonium (TEA) than its mouse and human counterparts. In the case of the rat Kv2.1, it was found that deletions in either the N- or the C-terminal were able to promote major changes in channel kinetics (VanDongen et al., 1990). Surprisingly, removal of both N- and Cterminal produced a channel with almost identical characteristics to those of the wild-type Kv2.1 channel. Van Dongen et al. (1990) observations indicate that although N- and C-termini are not essential, they play an important modulatory effect on activation and inactivation kinetics. This may explain the differences in the electrophysiological properties between rat, mouse, and human Kv2.1 since the only differences in these channel proteins are present outside the membrane spanning domains. In general, the properties of these channels suggest an involvement in action potential repolarization and/or broadening. In the case of the Aplysia Kv2.1 it has been shown that the slow inactivation process contributes to action potential broadening that occurs in bag cell neurons at the onset of neuropeptide secretion. So far, two genes have been found in the Kv4 (Shal-like) subfamily (Baldwin et al., 1991; Pak et al., 1991b). The rat Kv4.1 shows a complex inactivation kinetics that cannot be described with a single-exponential decay (Baldwin et al., 1991). At least three exponentials are necessary to fit the inactivation time course of the rKv4.1 channel and both the fast (β =155 ms) and the intermediate (β =688 ms) are slowed down when positively charged amino acids (35–37) are deleted from the amino-terminal. On the other hand, deletion of amino acids 2–32 of the N-terminal of the mKv4.2 has only a small effect on the inactivation process in this channel (Pak et al., 1991b). Probably positively charged amino acids, like in the case of the ShB channel (Hoshi et al., 1990), are important in determining inactivation kinetics in Kv4.1, but whether or not a ball and chain model is appropriate in this case is not clear. Salkoff et al. (1992) pointed out that it is possible that, like in the mammalian Shab, the central core region may play an important role in determining channel gating kinetics. Although the electrophysiological properties of rKv4.1 and mKv4.1 are similar, there is only 77% identity between these two channels. In contrast, the different rat and mouse members of the Kv1 subfamily share a much higher degree of identity [almost 100% for rKvl.l (RCK1) and mKv1.1 (MBK1)]. Kv4.2 was cloned from rat heart (Roberds and Tamkun, 1991) and it has a very high degree of identity with the rat brain Kv4.1 at the N-terminal and S1–S6 region, but they diverge at the C-terminal region.


6.3.3 Formation of Heteromultimeric Channels is Fundamental in Subunit Composition and Diversity of K Channels The K channel proteins of the S4 superfamily (Figure 6.1) resemble one of the four internal repeats of Na or Ca voltage-dependent channels suggesting that functional K channels are multimers (tetramers). If K channels are formed by aggregation of subunits, heteromultimer formation may be another molecular mechanism to generate K channel diversity. Cotransfection of HeLa cells with cDNAs or coinjection of two different cRNAs into Xenopus oocytes corresponding to the RCK1 and RCK4 proteins were found to give rise to channels with mixed RCK1 and RCK4 channel properties (Ruppersberg et al., 1990). Heteromultimeric channels have propertie s distinct from those of the homomultimeric channels as if mixing of subunits is unrestrained. For example, RCK1 channels do not inactivate and are sensitive to dendrotoxin2 and TEA. In contrast, RCK4 channels show fast inactivation and are insensitive to dendrotoxin and TEA. The result of RCK1 and RCK4 cRNAs coinjection into Xenopus oocytes is a transient K channel sensitive to dendrotoxin and TEA (Ruppersberg et al., 1990). Coinjection of RBK1 and RGK5 cRNAS into Xenopus oocytes (Christie et al., 1990), coinjection of the RNAs coding ShA. and ShB containing a truncated N-terminal (inactivation removed) or coinjection with ShB and RCK1 cRNAs into Xenopus oocytes (Isacoff et al., 1990), or coinjection of two Sh cRNAs containing either different N-terminals or different C-terminals into Xenopus oocytes (McCormack et al., 1990a), all led to the formation of heteromultimeric channels. McCormack et al. (1990a) proposed some ‘mixing rules’ in the formation of heteromultimeric channels. First, when mixing channels with different N-terminals, the channel with the N-terminal that produces more inactivation dominates the kinetics. This can be understood if in N-type inactivating channels a single inactivation gate (one N-terminal) is enough to inactivate a channel. The coinjection of ShH4 and ShH4IR (inactivation removed; the channel has also a mutation that renders this channel toxin-insensitive to a scorpion toxin) cRNAs led to the conclusion that a single gate is sufficient for inactivation (MacKinnon et al., 1993). The reasoning is as follows: coinjection of ShH4 and ShH4IR channels into Xenopus oocytes induces the expression of channels containing 0, 1, …, 4 inactivation gates. With the proviso that a single wild-type subunit is enough to induce toxin sensitivity, if one inactivation gate is enough to inactivate a channel, all toxin sensitive channels (containing 1, …, 4 inactivation gates) will inactivate. The experimental outcome exactly matched this expectation. Measuring the inactivation time constant at increasing ratios ShH4IR cRNA: ShH4 cRNA demonstrates that channels with a single gate inactivate with an inactivation time constant that is a quarter that of channels with four gates. In contrast, the rate of recovery from inactivation is independent of the number of gates. This result suggests that the gates act independently and a gate closes a channel in a


mutually exclusive manner. Second, if the heteromultimeric channel is formed by mixing and matching proteins containing different C-terminals, the resulting heteromultimeric functional channel recovers from inactivation at a rate closer to the channel containing the C-terminal that recovers faster. It is remarkable that invertebrate (ShB) and mammal (RCK1) proteins can mix in the oocyte membrane originating functional heteromultimeric K channels (Isacoff et al., 1990). This finding indicates that, despite the evolutionary differences between these two species, the tertiary structure of the ShB and RCK1 proteins is similar enough to allow functional subunit interaction. Different subfamilies express independent K channel systems The reports described above demonstrate that closely related polypeptides (i.e. belonging to the same Kv1 subfamily) are able to form functional heteromultimeric channels. However, coinjection of cRNAs of two different subfamilies into Xenopus oocytes do not form heteromultimeric channels (McCormack et al., 1990a; Covarrubias et al., 1991). Indeed, the detailed study of Covarrubias et al. (1991) showed that Shaker, Shal, Shab, and Shaw express independent channel systems. Even when the cRNAs of all subfamilies are coinjected into a single oocyte the independence of channel systems is maintained. Mixing of subunits of different subfamilies is also hindered in vertebrates (McCormack et al., 1990a). 6.4 Structural Determinants for K Channel Assembly Covarrubia et al. (1991) hypothesized that the lack of heteropolimerization between K channels of different subfamilies was due to the existence of a ‘molecular barrier’. In the case of ShB channels such a molecular barrier was restricted to the N-terminal (Li et al., 1992). When ShB is coexpressed together with Kv2.1, the macroscopic currents are well fitted by the sum of two independent currents (e.g. Figure 6.4). However, if a chimeric cDNA is constructed such that it codes for a protein containing the ShB N-terminal and the hydrophobic core and C-terminal of Kv2.1, the resultant chimeric monomer (NShB ― 6–46-TmCDRKl) is able to form functional heteromultimeric channels when mixed with ShB. The results of Li et al. (1992) have been extended by Shen et al. (1993), Hopkins et al. (1994) and Lee et al. (1994). Lee et al. (1994) showed that heteromultimeric channels between hKv1.5 and hKv1.4 cannot be formed if a deletion (― 28–283) is introduced in the N-terminal of hKv1.4. However this deletion does not preclude the formation of homomultimeric

2

Dendrotoxin is a venom peptide from the mamba snake and blocks K channels with high affinity by binding to a site located in the external mouth of the channel.


channels. Moreover, deletion of the N-terminal of Kv2.1 allowed formation of hybrid channels with the ― 28–283hKv1.4 mutant. In other words, N-terminal deletions allow for the mixing of monomers of distant families of K channels. Contrasting with the results of Lee et al. (1994), deletion of large domains of the N-terminal of mKv1.1 prevents formation of functional homomultimers and of heteromultimers with mKvl.3 (Hopkins et al., 1994). This last result allows the possibility that N-terminal domains of different K channels belonging to the same family have more than one role in channel assembly. Shen et al. (1993) constructed progressive deletion clones of the Aplysia Kv1.1a (Pfaffinger et al., 1991) to investigate the minimal requirements for membrane insertion of the channel-forming protein. All deletion clones containing the S1 domain are able to incorporate into the membrane. That is, a clone containing nothing but the Nterminal linked to S1 is inserted into the lipid bilayer. Therefore, S1 provides a signal for membrane incorporation. Biochemical studies showed that once inserted, subunits are able to form stable multimeric structures.

Figure 6.4 Potassium channels of different subfamilies do not mix. Mixtures of cRNAs coding for Shaker and Shaw in a 3:1 ratio (upper-right) and Shal and Shab in 1:1 ratio (lower-right) were coinjected into Xenopus oocytes. Additive effect means digital addition of individual currents. Voltage pulses were 1 s in duration and ranged from –80 to 20 mV in 20 mV steps. Data Adapted from Covarrubias et al. 1991.

Homomultimeric structures are not formed if a domain comprising amino acids 66–194 from the N-terminal is deleted. Therefore, this region is essential for subunit coassembly in homomultimeric AKv1.1a K channels and was named T1


domain (first identified tetramerization domain; Shen et al., 1993). The results of Li et al. (1992) and Shen et al. (1993) (see also Lee et al., 1994 and Hopkins et al., 1994) provide strong evidence that the cytoplasmic N-terminal domain of the Shaker-like subfamily plays an important role in determining the assembly of subunits. However, coimmunoprecipitation experiments show that there is coassembly between T1(–) and T1(+) subunits to form tetramers. Furthermore, expression in oocytes of the amino-terminal truncated Kv1.5― 5’ cRNA produces currents similar to that of wild-type Kv1.5 albeit with lower efficiency (Attali et al., 1993). The coimmunoprecipitation experiments of cotranslated Kv1.1 and Kv1.2 of Babila et al. (1994) showed that the S1 segment plays a crucial role in the coassembly of homo–and heteromultimeric K channels. They proposed that the S1 domain is essential in homo–and heteromultimerization and in the stabilization of the interaction between subunits. On the other hand, the Nterminal confers selectivity and specificity to the K channel coassembly. 6.5 K Channels are Tetramers The fact that K channel proteins of different channels of the same subfamily can form heteromultimeric channels allowed MacKinnon (1991) to show that K channels of the S4 superfamily are tetramers. In this case the experimental strategy was to coinject the cRNAs of ShH4 and of ShH4 containing a mutation that makes the binding of a toxin about 1000-fold weaker (an aspartate in position 432 is mutated to asparagine). Assuming that monomer mixing is unrestrained, the probability (Pi) of having a channel with i mutated subunits is a function of the number of monomers forming a channel (n) and the fraction of wild-type and mutated subunits present (coinjected) initially. In this case a binomial distribution determines Pi. On the other hand, the fraction U of channels that are not blocked at a given concentration [T] of toxin is given by: U = ― (i=0.n)PiUi where Ui = Ki/(Ki +[T]) and Ki is the dissociation constant of the reaction of a channel containing i mutated subunits with the toxin. If one wild-type subunit in a channel is enough to confer tight toxin binding to a channel U ― fnmutUmut since U, Umut, and fmut can be experimentally determined, the channel stoichiometry, n, can be calculated. For two different fractions (f) of coinjected mutated cRNA values for n were 3.8 and 3.7, values in good agreement with the assumption that K channels are tetramers. K channel stoichiometry was also determined for Kvl.l (Liman et al., 1992). They mixed Kv1.1 cRNA and that of a Kv1.1 mutant that does not form functional channels (a tyrosine in position 379 was mutated to a lysine). Tyrosine 379 forms part of the S5-S6 linker. When these two cRNAs are coinjected into oocytes, the expressed channels have a much lower sensitivity to TEA than the wild-type Kv1.1 channel indicating that the mutant is able to mix with the wild-type protein. Coinjection of wild-type


Kv1.1 trimer cRNA with mutant Kv1.1 cRNA monomers also forms functional channels with low tetraethyl ammonium (TEA) affinity. However, coinjection of wild-type tetramer cRNA with mutant monomers produces only channels with a TEA affinity equal to that of Kv1.1. In other words, tetrameric wild-type constructions are unable to incorporate mutant monomers suggesting that functional K channel structures are tetrameric. More recently, Li et al. (1994) were able to image Shaker K channels using the electron microscope. The analysis of the Shaker protein revealed a structure of 8 nm × 8 nm × 6.7 nm with a fourfold symmetry, consistent with a tetrameric assembly of monomers. 6.6 Differential Expression of K Channels A counterpart to the wide variety of K selective channels found in animal cells is their differential distribution in animal tissues and brain regions. The electrophysiological studies provided an early indication that different K channels operate in different excitable cells. The wealth of information on K channel structure and function derived by applying DNA recombinant approaches to their study, as well as the powerful methods it provided, made it possible to start gathering direct evidence that a differential expression of K channels in animal tissues represents quite a general phenomena. At least, a differential distribution of K channels offers a fundamental mechanism underlying the diversity of electrical stereotypes exhibited by excitable cells. It follows that a differential pattern of K channel expression must accompany the events of tissue differentiation during development. The methods provided by molecular biology have demonstrated that K channel diversity is paralleled by specific patterns of channel mRNA expression and channel product in tissues and in different areas of the nervous system. However, in the nervous system, the evidence that differential mRNA’s expression implies functional specific K channels is scant. Ion channel functionality needs to be demonstrated since K channel localization patterns derived from immunohistochemical analysis yield details in channel product location which are not apparent for mRNAs analyzed by in situ hybridization. The above considerations are more relevant if we take into account the lack of information regarding the detailed electrophysiological properties of the variety of cells and cell processes that occur in the animal brain. Indeed, such information would help to strengthen the notion that differential K channel patterns, revealed by in situ hybridization, immunohistochemistry and biochemistry, correlate with relevant functional differences in the electrical properties of cellular components in different regions of the brain. Although the field of differential K channel expression is new, several questions concerning this fascinating problem that have been addressed are: (1) how general is the phenomenon of differential distribution of K channels in animal


tissues?; (2) are heteromultimeric K channels formed in vivo?; and (3) what factors modulate channel expression in cells and tissues? 6.6.1 Differential K Channel Expression is a General Phenomenon The molecular cloning of the renowned Shaker gene of Drosophila, and the realization that this gene encodes a family of K channel components pertaining to the A-class (sections 6.2 and 6.3), paved the way to begin addressing the question of differential ion channel distribution in animals. As discussed earlier, through alternative splicing, multiple transcripts arise from the Shaker gene yielding a variety of A-type channels exhibiting distinctive structural and kinetic properties. This knowledge was taken advantage of by Schwarz et al. (1990) to raise antibodies to three portions of the predicted sequences of Shaker channels. Immunoblots revealed a nonuniform distribution of products in the brain of the adult fly. Products were found to be prominently associated with the optic system and the mushroom bodies, a complex structure containing different cell types and inputs. Shaker products were detected both in the neuropils and in axons. Moreover, Schwarz and collaborators provided evidence that two splicing variants, ShA. and ShB, were present in only a subset of regions that express Shaker products, indicating that Shaker-type products do not have a fixed composition in all cells. On the other hand, the immunoblot assay was unable to show the presence of Shaker products in muscle cells, which are well known to contain A currents of the Shaker type. The immunohistochemical analysis carried out by Schwarz and coworkers was not accompanied by in situ hybridization studies which would have helped to establish how Shaker mRNA’s patterns compared to those of Shaker products revealed by the immunohistochemical assay. An electrophysiological complement supporting the observations of Schwarz et al. (1990) was provided by Baker and Salkoff (1990) who found that deletion of the Shaker gene abolished Shaker-type K+ currents only in a subset of neurons in late stage Drosophila pupae. An intriguing aside to the studies of Schwarz et al. (1990) concerns their observation that Shaker products are prominent in the mushroom bodies in the brain of Drosophila. The mushroom bodies receive inputs from olfactory systems and other sensory areas and Shaker alleles have been reported to be deficient in a conditioned odor-avoidance learning and retention protocol (Cowan and Siegel, 1986). Indeed, there is evidence that mushroom bodies are implicated in odor-avoidance associative learning and retention (Davis, 1993, 1995) and, recently, it has been documented that chemical ablation of mushroom bodies in flies causes deficiencies in this form of associative learning (De Belle and Heisenberg, 1994). The observations by Schwarz et al. (1990) raise the unexpected possibility that, owing to differential expression in well defined structures of the nervous system, K channel mutations might cause specific neurological deficiencies. In support of the above idea is the evidence that synaptic transmission and synaptic plasticity


is abnormal in the neuromuscular junction of Shaker mutants (Jan et al., 1977; Delgado et al., 1994). Differential expression of K channels has been documented convincingly also in the brain of higher animals. Thus, the distribution of members of the Shakerrelated RCK channel family (RCK1, RCK3, RCK4 and RCK5) has been investigated in the rat nervous system (Beck and Pongs, 1990). As discussed earlier in this chapter, the RCK gene family expresses a spectrum of voltagegated delayed rectifier and transient K+ currents. RNA blot hybridization revealed a differential distribution of RCK mRNAs in the adult rat brain, as well as a differential pattern of appearance during development. RCK1 and RCK5 mRNAs were found to predominate in the adult nervous system (postnatal days 30 (P30) and P90 animals). RCK3 and RCK4 were detected throughout all developmental stages. In adult rat central nervous system (CNS; P30 and P90), maximum levels of RCK1 were found in caudal regions of the brain, lower ones in the rostral regions and in the retina. Likewise, RCK5 mRNA levels were higher in the caudal regions of the CNS but the corpus striatum, midbrain and superior colliculus exhibited lower levels than the cerebral cortex. In the case of RCK3 mRNA levels were almost undetectable in cerebellum and high in the inferior colliculus, medula-pons and olfactory bulb. RCK4 mRNA levels were higher in the midbrain and forebrain areas of the CNS, including retina, with lower expression in cerebral cortex. RCK mRNAs were monitored also in the peripheral nervous system (PNS), leading to the conclusion that RCK1 and RCK5 mRNAs are coexpressed in the PNS, although at different levels, while faint RCK3 and RCK4 mRNAs signals could be assessed only for dorsal root ganglia. The above observations seem to indicate that both in the CNS and in the peripheral nervous system RCK1 and RCK5 are most abundant in the rat. The study of the developmental time course of RCK K channel mRNAs in the CNS of the rat by Beck and Pongs (1990) indicated the detection of RCK1 mRNA around embryonic day E15, with levels increasing during the second and third postnatal weeks in midbrain and hindbrain areas with only a moderate increase in forebrain areas. The developmental profile of RCK5 is somehow similar to that of RCK1. In contrast, RCK3 mRNA is present in all CNS regions throughout all developmental stages (E10 to P90), although RKC3 mRNA levels are low. RCK4 mRNA levels are substantial at E10 increasing slightly until the end of the first postnatal week. Thereafter, levels are maintained in the forebrain and midbrain areas but decrease in the hindbrain. Concerning the PNS, a similar developmental pattern to that of the CNS was observed for RCK1 mRNA in dorsal root ganglia (E18 to P60) suggesting that, perhaps, the developmental patterns of the other mRNAs for other RCK potassium channels might be similar in central and peripheral nervous system. The expression of RCK1, a K channel exhibiting sensitivity to the mast cell degranulating peptide (MCDP) and to dendrotoxin I (DTX1) (Stühmer et al., 1989) was investigated by Sequier et al. (1990) in rat brain. These agents are convulsant, induce neurotransmitter release, and can induce the β rhythm


associated with arousal. MCDP induces long term potentiation when applied to hippocampal slices (Bidard et al., 1987; Bidard et al., 1989; Cherubini et al., 1987). Transcript signals were prominent in granule cells of the hippocampal formation and pyramidal cells of Ammon’s horn, levels of expression being conspicuous in pyramidal CA3 cells compared to CA1 and CA2 cells. Interestingly, it is in the CA3 area of the hippocampus that the epileptogenic properties of MCDP have been traced (Cherubini et al., 1988). Other brain areas exhibiting a high RCK1 hybridization signal include the cerebellum, mainly at the level of the granular layer and in the Purkinje cell layers and in layers II, III, V and VI of the telencephalon. In contrast, levels of mRNA were low for the whole hypothalamus with the exception of the ventromedial nucleus. The distribution of mRNAs corresponding to Raw channels from the rat brain (Raw 1–3), namely a class of channels pertaining to Shaw-related K channels, was pursued by Rettig et al. (1992). With respect to tissue distribution of Raw mRNAs, it was reported that Raw1 and Raw2 mRNAs are expressed predominantly, if not exclusively, in the brain as compared to kidney, heart or skeletal muscle. Raw3 was detected both in the brain and in skeletal muscle, reflecting, perhaps, the existence of closely related Raw3 mRNAs or the occurrence of alternatively spliced variants. The distribution of Raw, investigated in the rat brain by in situ hybridization, revealed that different Raw mRNAs are expressed differentially in the nervous system. For example, each Raw mRNA exhibits a distinct pattern of distribution within the hippocampus. A high level of Raw3 RNA is found in the dentate gyrus and Raw1 RNA expresses in a gradient such that CA3 exhibits higher expression than CA1. Moreover, the granulated expression pattern of Raw1 and Raw2 would suggest that, in CA1 and CA3 hippocampal areas, they are confined to particular cells. Immunocytochemical studies revealed that, in the hippocampus, Raw3 and RCK4 mRNAs are expressed both in the same neuron and in different ones, raising the possibility that in the rat central nervous system the same cell can express multiple, independently assembled K channels and that unrestrained mixing of subunits does not occur. Weiser et al. (1994) carried out an intense scrutiny of the distribution in rat brain of the four known Shaw-related genes, or ShIII, (KV1, KV2, KV3 and KV4) using Northern blot analysis and in situ hybridization. Northern blot analysis indicated that Kv3.2 and 3.3 are expressed mainly in brain, with low levels of expression of Kv.3.1 in skeletal muscle. Kv3.4 transcripts were more abundant in skeletal muscle. In brain, ShIII transcript distribution was nonhomogeneous and the expression patterns of Kv3.1, Kv3.3 were similar. Kv3.4 mRNAs, which are less prominent in the CNS, were found mainly in areas in which Kv3.1 and/or Kv3.3 occurred in high levels. In many areas, Kv3.2 transcripts did not overlap with the products of other ShIII genes. Table 6.2 summarizes the distribution of transcripts for four ShIII genes in some regions of the CNS in the rat, as reported by Weiser et al. (1994). Higher-resolution studies of hybridization patterns, achieved by microscopic analysis of emulsion-dipped sections, revealed that


transcripts were present only in neuronal somas and not in glial cells. Many neuronal populations exhibiting Kv3.1 transcripts expressed also Kv3.3 mRNAs. Kv3.4 transcripts, at lower levels, were observed in some neuronal populations containing Kv3.1 and/or Kv3.3 mRNAs, indicating a potential Table 6.2 Distributions of transcript from four Shlll genes in some areas of the rat CNS.

Olfactory bulb Periglomerular cells Tufted cells Mitral cells Neocortex Interneurons Pyramidal cells Pyriform conex Hippocampus CA1 pyramidal cells Str. radiatum Str. oriens CA3 pyramidal Str. radiatum Str. oriens DG granule cells Borders with str. granulosum Hilus Thalamus (dorsal) Anterodorsal nu. Anteroventral nu. Anteromedial nu. Laterodorsal nu. Parataenial nu. Reunions nu. Mediodorsal nu.

KshIIIA (KV3.2)

KShIIIB (KV3.1)

KShIIIC (KV3.4)

KShIIID (KV3.3)

–

+

–

+

–

+ ++

– –

+ ++

– +

Layers II– IV>V–VI – ±

Layers II– IV>V–VII – +

Layers II– IV>V–VI –

+

+

–

+

– +++ ++ ++ ++ –

++ ++ +++ + + +++

– – – – – ++

+ + + + + ++

–

+

+++

±

–

–

++++

+

–

±

++++

±

–

–

++++

±

–

–

+++ ++++ +++ ++++

– – ± ±

– – –

± – –

Layers V–VI

±


KshIIIA (KV3.2)

KShIIIB (KV3.1)

KShIIIC (KV3.4)

KShIIID (KV3.3)

Intermediodors + + – – – al nu. Lateral +++ + – – posterior nu. Ventral ++++ ++ – – posterolateral nu. Ventrolateral ++++ ± – – nu. ++++ ++ – + Ventral postenomedial nu. Ventro medial ++++ ± – – nu. Central medial +++ ± – – nu. Posterior nu. ++++ ± – – Dorsal lateral ++++ ++ – + geniculate Medial ++++ – – – geniculate Thalamus (Ventral) Reticular ± ++++ ± ++ thalamic nu. Ventral lateral – + – + geniculate nu. Cerebellum Molecular cell – + + + layer Purkinje cells – + + +++ Granular cell – ++++ ± ++ layer Deep cerebellar + + +++ – +++ nu. Spinal cord Dorsal horn – +++ – +++ – + ++ +++ Ventral horn The symbols indicate signal intensity as follows: - (signals undistinguishable from background); ± (very weak signals but clearly above surrounding background);+(weak);+ + (moderate);+ + +(high);+ + + + (very high). (Modified from Weiser et al., 1994) Nu=nuclei; str=stratum.


for heteromultimer formation between these three gene products. Of great potential significance is the observation that coinjecting Xenopus oocytes with small amounts of Kv3.4 cRNA and an excess Kv3.1 or Kv3.3 cRNA yielded channel products with fast-inactivating properties resembling those of Kv3.4 channels. Thus, as discussed in section 6.3.3, it is probable that even a single Kv3.4 subunit is competent to confer fast-inactivating properties to ShIII channels. The higher-resolution analysis also showed that, although the expression patterns of Kv3.1 and Kv3.3 in the CNS are similar, they are not identical. Thus, Kv3.1 signals in some neurons were more intense than those of Kv3.3 while the opposite was true in other cases. In several brain areas, for example the cerebral cortex, hippocampus and caudate-putamen, individual ShIII probes labeled specifically a small number of neurons which otherwise could not be distinguished from neighboring neurons in the area. Kv3.3b, an alternatively spliced form of the Kv3.3 Shaw-type K channel identified by Goldman-Wohl et al. (1994), was found to be at a high level in mouse brain, particularly in the cerebellum. There, expression was confined to Purkinje cells and deep cerebellar nuclei. Expression of Kv3.3b mRNA appeared to be developmentally regulated in cerebellar Purkinje cells beginning between P8 and P10, to be present in virtually all Purkinje cells by P12, and continue through adulthood. P8–P10 corresponds to the period at which Purkinje cells establish synapses with granule cell parallel fibers. Since Purkinje cells exhibit aberrant morphology in agranular cerebellar cortex, the observations by Goldman-Wohl et al. (1994) suggest that, perhaps, expression of this gene is regulated by mechanisms that involve the interaction between Purkinje and granule cells. It is of interest to point out that, according to Goldman-Wohl et al.’s (1994) observations, cell type specificity and developmental regulation of this gene was maintained in dissociated primary culture of mouse cerebellum. In culture, Purkinje cells were identified by immunocytochemistry for calbindin and the time course of Kv3.3b expression was monitored by in situ hybridization. Kv3.3b mRNA was undetectable in E18 Purkinje cells cultured for one week but it was expressed in calbindin-positive cells after two to three weeks in culture and expression was not dependent on neurite extension. Hwang et al. (1992, 1993) investigated in the rat brain the localization of CDRK and DRK1 channels pertaining to the Shab subfamily by immunohistochemistry. They found that both products occur predominantly in


neurons, displaying regional variations with overall levels of expression in the various brain regions being similar for both channels. However, cellular distribution of the two channels differs greatly, differences that were particularly obvious in the cortex. For example, the DRK1 immunoreactivity was restricted mostly to pyramidal cells and dendritic processes. CDRK was not evident in pyramidal cells and was restricted to a population of smaller nonpyramidal elements resembling cortical interneurons. CDRK was detected also in the processes of such cells. Differential location of CDRK and DRK1 products was also apparent in the hippocampus where the dentate gyrus was enriched in CDRK immunoreactivity with respect to DRK1. On the other hand, although in the cerebellum there was coexpression of CDRK and DRK1, both in Purkinje and granule cells, their localization at the subcellular level differed with DRK1 localized in the somata and proximal dendrites and CDRK associated with the cell body. Rudy et al. (1992) investigated the differential expression of KShIIIA (Kv3.2a), a Shaw-like gene that encodes several alternatively spliced transcripts in the rat brain. In oocytes, KShIIIA transcripts yield voltage-dependent K+ currents exhibiting slow activation at voltages above –20 mV, Because of these properties, KShIIIA channels are not expected to be relevant to subthreshold events and they are much too slow to be active during a single, fast Na+ depolarizing phase (see Section 6.3.2). Northern blot analysis with a KShIIIA probe that does not distinguish spliced transcripts revealed that hybridization was stronger in thalamus-enriched RNA than in whole-brain RNA. It indicated the presence of two major bands at 7.5 and 6.5 kb, in addition to a diffuse one around 4 kb, and the band at 6.5 kb was not obtained with thalamus RNA. The several bands obtained could not be accounted for as arising from alternatively spliced transcripts since similar bands were obtained when probes specific for KShIIIA1, KShIIIA2 and KShIIIA3 were used. These results were interpreted as indicative of the existence of differentially processed species or of subtypes of each of the transcripts. In situ hybridization confirmed that, in rat brain, KShIIIA transcripts display a differential distribution, being most prominent in the nuclei of the dorsal thalamus and in the optic layer of the superior colliculus. Less prominent hybridization signals were found in the deep layers of all cortical areas of the neocortex, the piriform cortex, the red nucleus and the CA3 region of the hippocampus. Discrete to weak labeling was obtained in the cochlear nucleus and in the neocortex. A differential expression of alternatively spliced KShIIIA transcripts could not be documented in this study and the analysis did not address the possible subcellular distribution of the products. However, the distribution pattern of the transcripts suggested that they are expressed at highest levels in neurons. Bright field images showed that in the thalamic nuclei hybridization grains were concentrated over the soma of large neuronal cells and the density of hybridization in glia appeared similar to background. In the same study, Rudy et al. (1992) compared the hybridization patterns of KShIIIA transcripts to those of NGK2-KV4, a transcript that, in Xenopus oocytes, induces


currents with properties similar to those of KShIIIA. The analysis demonstrated that, if NGK2-Kv4 transcripts are concentrated in certain brain areas, their pattern of distribution differs to that of KShIIIA. For example, NGK2-Kv4 mRNAs were abundant in the cerebellar cortex where KShIIIA expression is minimal. In the hippocampus both KShIIIA and NGK2-Kv4 expressed, albeit in a different fashion. Thus, KShIIIA transcripts were more prominent in the CA3 field of Ammon’s horn while NGK2-Kv4 was found in the dentate gyrus as well as in CA3. Immunofluorescence assisted Freeman and Kass (1993) in showing that an antibody directed against the minK channel reacted with a membrane antigen on adult guinea pig ventricular myocytes and sinoatrial cells, suggesting that such a channel might actually be responsible for slow-rising cardiac delayed-rectifier K + currents. This evidence is of extreme importance considering the peculiar structure of this putative minimal K channel consisting only of 130 residues and a single transmembrane domain. In Xenopus oocytes minK cRNA yields slow voltage-gated, non-inactivating outward currents. Previous to the Freeman and Kass (1993) report, minK channel activity had not been detected in mammalian cells raising the concern that minK transcript injection could yield in oocytes a regulatory protein of endogenous K+ currents rather than a K channel. In addition to showing that cardiac cells are immunoreactive to minK antibodies these authors were able to achieve the expression of minK currents in a transfected mammalian cell line (HEK 293). The currents expressed exhibited properties similar to those recorded from guinea pig ventricular myocytes strengthening the idea that minK gives rise in mammalian cells to slow-activating, noninactivating voltage-dependent channels with delayed-rectifier properties. Although the experimental evidence indicates the existence of differential expression of K channels in animal tissues, this does not mean that expression of a given K channel type is confined only to a specific tissue. For example, the delayed rectifier (IKs) K channel has been cloned from neonatal heart and ovariectomized diethylstilbestrol-primed rat uterus and expressed in oocytes. Northern blot analysis failed to document channel expression in adult heart, nonprimed uterus or brain (Folander et al., 1990). However, an identical channel (IKs) was demonstrated earlier in rat kidney (Takumi et al., 1988). The studies by Folander et al. (1990) would indicate that, at variance with the claim of Takumi et al. (1988), expression of IKs channels would not be restricted to epithelial tissue in mammals. In the human heart, the studies of Tamkun et al. (1991), using Northern blot analysis, indicated that HK2 K channels, much more abundant in ventricle than atrium, bear a great degree of identity (86%) to Kv1, a rat brain K channel. In addition, expression of channels belonging to the RCK family (rat cortex K channels) have been reported by Betsholtz et al. (1990) in insulinproducing cells. Douglass et al. (1990) reported the cloning of RGK5, a K channel derived from rat thymus which was postulated to represent n-type currents in immature thymocytes and T lymphocytes. Northern blot analysis revealed that RGK5 transcripts are found both in thymus and rat brain.


6.6.2 Heteromultimeric Channels in vivo The occurrence of heteromultimeric K channels in the brain has also been addressed. The fact that different K channel subunits show overlapping expression patterns in the brain fulfills one of the prerequisites of in vivo heteromultimer channel products (Drewe et al., 1992). The studies by Sheng et al. (1992, 1993) indicated that Kv1.2 and Kv1.4 mRNA, two K channel subunits of the Shaker subfamily, exhibited distinct, but overlapping expression patterns in the rat brain and these authors provide convincing evidence that Kv1.2 and Kv1.4 form heteromultimers which are differentially distributed in the brain. It is worth mentioning here that Kv1.2/Kv1.4 channels combine properties of both parent subunits to form a novel A-type channel characterized by a fast inactivation, rapid recovery from inactivation and high sensitivity to the K channel blocker 4-aminopyridine. Monitoring of the distribution of Kv1.4 and Kv1.2 in rat brain, using immunohistochemistry, indicated that these channel subunits coassemble in the rat brain and that the heteromultimers are localized in axons and nerve terminals, consistent with a role in the control of neurotransmitter release. Kv1.2 immunoreactivity overlapped with Kv1.4, particularly in the neuropil of the cerebral cortex, axon tracts of the corpus callosum and in well defined terminal fields of the hippocampus. Evidence for multimer formation was supplemented by biochemical evidence indicating the cofractionation of Kv1.2 and Kve1.4 proteins. Following detergent solubilization and separation in an anion-exchange column, identical elution patterns for both products were obtained. Moreover, the use of gene-specific antibodies demonstrated that Kv1.2 and Kv1.4 polypeptides could be coimmunoprecipitated by antibodies specific for either gene. Thus, to the growing evidence for the differential expression of homomultimer K channel products in the brain, the differential expression of heteromultimeric K channels must be added. Support for heteromultimeric K channels in the brain has also been provided by Wang et al. (1993) for mKvl.l and mKvl.2 in the juxtaparanodal regions of nodes of Ranvier and in terminal fields of basket cells in mouse cerebellum. Coimmunoprecipitation of both products could be achieved with antibodies specific for either mKvl.l or mKvl.2. The studies by Wang et al. (1993), in addition to demonstrating heteromultimeric channels in vivo, showed that such heteromultimers occur in subcellular regions in the brain. In vitro translation in transfected mouse L-cells, combined with immune purification approaches, enabled Deal et al. (1994) to gather evidence for the cotranslational assembly of Kv1.1/ Kv1.4 heteromers. Glycosilation of Kv1.1 at the extracellular N207 site was not required for assembly and did not affect channel turnover or function.


6.6.3 Regulation of K Channel Expression Little is known currently of the rules governing the striking phenomenon of K channel differential expression in the brain and even less to its relation to neurological deficiencies in higher animals. The scant information available includes the work of Tsaur et al. (1992). In the rat brain, changes in the distribution of mRNAs from K channels. Kv1.1 and Kv1.2, encoding delayed rectifier channels of the Shaker-RCK subfamily, and Kv4.2, an A-type channel of the shal subfamily, were monitored following seizure induced by the convulsant pentylenetetrazol. Kv1.1 and Kv1.2 were expressed at higher levels in the hippocampus, thalamus, cerebral cortex and cerebellum. In turn, the highest levels of Kv4.2 expression were found in the cerebellum, intermediate levels in the hippocampus and media habenula, with lower levels in the cerebral cortex and thalamus. Following seizure, a reduction of Kv1.2 and Kv4.2 mRNAs was detected only in the dentate granule cell layer of the hippocampus, a brain region in which excitatory glutamatergic neurons predominate. No brain region exhibited changes in Kv1.1 mRNA levels. Importantly, the changes in mRNA levels could not be attributed simply to stress-related activation of the hypothalami-adrenocortical axis, since the mRNA levels of the three K channels were not altered after treatment with adrenocorticotropic hormone. Moreover, the changes in mRNA levels observed after seizure would seem to be related to neuronal activity, since administration of diazepam, prior to pentylenetrazol, both protected the animal from seizure and prevented reduction in Kv1.2 and Kv4.2 mRNA levels in the dentate granule. Takimoto et al. (1993) demonstrated that dexamethasone, a glucocorticoid agonist, induced rapid Kv1.5 channel mRNA transcription in clonal pituitary cells, an effect that could not be attributed to a change in Kv1.5 mRNA turnover. Moreover, the steroid was found to increase the expression of the Kv1.5 protein, as evidenced by immunoblots, without changing its half-life. Increases in a noninactivating component of the voltage-gated K+ currents accompanied the induction of the Kv1.5 protein, supporting the notion that hormones and neurotransmitters may affect, within hours, excitability by controlling K channel gene expression. In neuroblastoma cells, which exhibit neuronal characteristics, the effects of dimethylsulfoxide (DMSO) and retinoic acid (RA), agents which cause differentiation-like events in cell culture, were investigated by Smith-Maxwell et al. (1991) using the patchclamp technique. Under control conditions, the cell line used (N2AB-1) exhibited transient, voltage-dependent Na+ currents but little delayed rectifier outward K+ currents. Treatment with RA or DMSO, under low serum conditions, caused the expression of delayed rectifier K+ currents. It was observed, however, that there was not necessarily a correlation between K channel expression and inhibition of cell division, cellular hypertrophy or the elaboration of processes. In cultured mouse hippocampal neurons there is evidence for astroglial modulation of transient K+ current development (Wu and Barish, 1994) suggesting that


astroglial-induced plasticity could mediate long-term modulation of excitability in the hippocampus. The mechanisms by which astroglia influenced the appearance of outward currents of the A-type were most probably surface or extracellular matrix associated ones and living glial cells were required for modulation. Glial neurons treated with an inhibitor of RNA synthesis for 5–7 days had reduced A-current amplitudes and the action of the inhibitor was localized to the astroglia. The regulation of Kv1.5 by platelet-derived growth factor (PDGF) or fibroblast growth factor (FDG) was investigated by Timpe and Fanti (1994) in Xenopus oocytes by the simultaneous expression of Kv1.5 channels and either receptor. PDGF or FDG receptor activation in oocytes led to a slow decline in K channel amplitude with a half-life of about 20 minutes without any major change in channel voltage-dependence or kinetics. Depression of K+ currents was found to require activation of phospholipase C. The time course of current decline was too fast to be caused by altered gene transcription. Since K channels and PDGF and FGF receptors can occur in the same cell type, for example progenitor cells for oligodendrocytes and type 2 astrocytes, it is conceivable that the modulation of K channels investigated by Timpe and Fanti (1994) might also occur in other cell types. 6.7 Conclusion K channels can be now grouped according to their primary structure. Structurally K channels can be divided in three large superfamilies: K channels belonging to the S4 super family, K channels with subunits containing two membrane spanning regions (e.g., inward rectifiers), and K channels with a single membrane spanning region (minimal K channel). The large variety of K channels in the S4 superfamily, that include delayed rectifiers, A-type, and Caactivated K channels among many others, is a consequence of mRNA splicing, multiple genes, and formation of heteromultimeric channels. In this superfamily, K channels are tetrameres and some of the assembly rules of monomers into tetrameres have been elucidated. Inside a K channel subfamily mixing of monomers of different types of K channels is possible. However, monomers of K channels belonging to different subfamilies do not mix. The N-terminal and the S1 transmembrane segment of the K channel-forming proteins appear to be, at least in part, the structures regulating mixing and matching of monomers in this superfamily. Differential expression of K channels is a general phenomenon, although the factors that govern the expression of a given K channel in different cells and tissues are still unknown. Both homo- and heteromultimeric K channels show differential expression in vivo. Acknowledgements This work was supported by grant FNI-1940227, Human Frontier in Sciences Program, by a European Communities research contract, by institutional support to the Centre de Estudios Científicos de Santiago provided by SAREC (Sweden),


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RETTIG, J., HEINEMANN, S.H., WUNDER, F., PARCEJ, D.N., DOLLY, J.O. & PONGS, O. (1994) Inactivation Properties of Voltage Gated K+ Channels Altered by the Presence of Beta Subunit. Nature, 369, 289–294. SCOTT, V.S., MUNITZ, Z.M., SEWING, S., LICHTINGHAGEN, R., PARCEJ, D.N., PONGS, O. & DOLLY, J.O. (1994) Antibodies Specific for Distinct Kv Subunits Unveil a Heterooligomeric Basis for Subtypes of β -Dendrotoxin-sensitive K+ Channels in Bovine Brain. Biochemistry, 33, 1617–1623. TSENG-CRANK, J., FOSTER, C.D., KRAUSE, J.D., MERTZ, R., GODINOT, N., DICHIARA, T.J. & REINHARDT, P.H. (1994) Cloning, Expression, and Distribution of Functionally Distinct Ca2+ -activated K+ Channel Isoforms from Human Brain. Neuron, 13, 1315–1330. VIVIENNE, N. & PFAFFINGER, P.J. (1995) Molecular Recognition and Assembly Sequences Involved in the Subfamily-specific assembly of Voltage-gated K+ Channel Subunit Proteins. Neuron, 14, 625–633. WALLNER, M., MEERA, P., OTTOLIA, M., KACZOROWSKI, G., LATORRE, R., GARCIA, M., STEFANI, E. & TORO, L. (1995) Cloning, Expression of, and Modulation by a Subunit of a Maxi KCa Channel from Human Myometrium. Receptors Channels, 3, 185–199.

ADDENDUM Diversity of K Channels is More Rich than our Imagination News from Paramecium tetraaurelia The ciliate protozoan, Paramecuim, can do amazing things. It can swim forward, or backwards when it finds an obstacle to resume swimming forward some seconds later, or when bumped from the rear, it escapes very fast from the aggressor. All of these Paramecium movements are controlled by a highly efficient set of Ca2+ and various types of K channels. Paramecium tetraaurelia has a family of K channels (Pak1 and Pak2) with a very low degree of homology to the metazoan voltage-dependent K channels (Jegla and Salkoff, 1994). According to Jegla and Salkoff (1994) the uniqueness of these K channels may be a consequence of evolutionary pressures at play in single vs multicellular organisms. The Pak channels contain six transmembrane segments and a P region. Although the pore region has a high degree of identity with other K channels, the overall identity with, for example, the Shaker K channel is only 19 per cent. In particular, the S4 transmembrane segment in Pak channels contains very few positively charged residues compared with Shaker K channels. Some K channels appear to be dimers Ketchum et al. (1995), using as a hook the P domain from several K channels, fished from gene databases a novel K channel from Saccharomyces cerivisiae. This K channel, denominated TOK1, contains two pore domains in tandem within the same polypeptide. Eight potential transmembrane regions, S1–S8, were deduced from the primary sequence of the TOK1 channel, interrupted by a


P1 region located, as with the S4 superfamily, between S5 and S6 and a P2 region between S7 and S8. The S4 domain in TOK1 channels does not have positively charged amino acid residues. However, injection of TOK1 cRNA into Xenopus oocytes induces outwardly rectifying K+ currents. Interestingly, the position in the voltage axis of the conductance-voltage relationship varies with external K+ as in ‘classical’ inward rectifying channels. In TOK1 channels current is outward at voltages larger than the potassium equilibrium potential. Moreover, TOK1 channels are blocked by external Ca2+ in a voltage-dependent fashion and once this divalent cation is removed, channels can conduct ions inwardly. Thus, as in inward rectifier channels, the voltage dependence of TOK1 channels is not an intrinsic property of the channel protein but a consquence of a voltage-dependent blockade. Above we discussed that K channels of the S4 superfamily are tetramers requiring four P domains to form the walls of the pore (see section 6.5). The particular structure of TOK1 channels implies, thus, that their functional stoichiometry is that of dimers (for a review see Salkoff and Jegla, 1995). Additional References JEGLA, T., & SALKOFF, L. (1994) A Multigene Family of Novel K+ Channels from Paramecium tetraaurelia, Receptors Channels, 3, 51–60. KETCHUM, K.A., JOINER, W.J., SELLERS, A.J., KACCZMAREK, L.K. & GOLDSTEIN, S.A.N. (1995) A New Family of Outwardly Rectifying Potassium Channel Proteins with Two Pore Domains in Tandem. Nature, 376, 690–695. SALKOFF, L., & JEGLA, T. (1995) Surfing the DNA Databases for K+ Channels Nets yet more Diversity. Neuron, 15, 489–492.

7 Potassium Channel Electrophysiology in Vascular Smooth Muscle Cells and the Site of Action of Potassium Channel Openers P.I.AARONSON1 & C.D.BENHAM2 1

Department of Pharmacology and Medicine, UMDS Guy’s and St Thomas’s Hospitals, Lambeth Palace Rd, London, SE1 7EH, UK. 2

Department of Biophysical Sciences, SmithKline Beecham Pharmaceutical, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK. 7.1 Introduction The application of patch-clamp electrophysiology to single smooth muscle cell preparations is generating a bewildering wealth of information about K+currents in vascular muscle cell membranes as it is in many other excitable cells. Functional studies such as these are hampered by a lack of a comprehensive range of specific pharmacological probes to clearly identify K+ currents. Aside from specific agents such as charybdotoxin and the relatively selective glibenclamide, many studies attempt to draw conclusions from data using tetraethylammonium (TEA) and 4-aminopyridine (4-AP), compounds with a broad K channel blocking profile. The recent advances in the molecular biology of K channels now provides the opportunity to put the functional studies into a structural framework and hopefully reveal a little more order. In this chapter we will consider work using both approaches in an effort to develop an overall picture of these channels in vascular smooth muscle. This information will then be used to consider the likely sites of action of the K channel openers, a group of compounds that have attracted interest both because of their therapeutic potential and because of the insights they have provided on vascular smooth muscle function. 7.2 Structural Division of K Channels Deduced from Molecular Biology Molecular biology has defined three families of K channels (reviewed by Hoshi and Zagotta, 1993). The largest group has homology to the Shaker encoded channel in Drosophila. This gene encodes a voltage dependent, inactivating K channel that electrophysiologists had termed an A current. Four gene sub-families have been cloned from Drosophila and their mammalian homologues have been called Kvl.x, Kv2.x, Kv3.x and Kv4.x. These channels are all voltage dependent

K CHANNEL ELECTROPHYSIOLOGY IN VASCULAR SMOOTH MUSCLE CELLS 195

K channels, opening in response to membrane depolarization but showing varying rates of activation and inactivation. The classically described delayed rectifier and transient outward currents fall into this group (for review see Pongs, 1992). Recently, a Ca dependent K channel has been cloned with some homology to the Shaker group of channels. It seems likely that this group of voltage dependent channels are part of the larger Shaker superfamily. Thus, the great majority of voltage sensitive K channels that shape repolarization in excitable cells can be gathered into this one large group. The second group is unusual in being defined by a single gene, MinK, that codes for a voltage dependent K channel with very slow activation kinetics (for review see Blumenthal and Kazcmarek, 1992). This channel is distinguished by a subunit structure that contains only one putative transmembrane spanning region in the 130 amino acid subunit sequence. The number of subunits required to form a functional channel is unknown. This structure is in marked contrast to the Shaker family which share a common structural motif of six membrane spanning regions (S1–S6) in each subunit and have been shown to exist as tetramers in Xenopus oocyte membranes. The gene is expressed in a variety of glandular tissues (Takumi et al., 1988) but also in heart and smooth muscle. The first functional expression was in fact from mRNA purified from oestrogen-primed uterine smooth muscle (Boyle et al., 1987) which preceded the identification of the MinK gene. A third major family is now coming into focus. This includes K channels that can open close to the resting membrane potential, several of which behave as inward rectifiers passing inward current more readily than outward current. Also included are a group of channels that appear to be under metabolic control, regulated by the cytoplasmic concentration of ATP and other intermediary metabolites. This last group also appear to be the site of action of the K channel opening drugs such as cromakalim. So far, sequence data for only two members of this family are available. ROMK1 (Ho et al., 1993) and IRK1 (Kubo et al., 1993) conform to a putative two membrane spanning domain structural signature. ROMK1 cloned from rat kidney and expressed in Xenopus oocytes exhibits some of the biophysical properties of an ATP-sensitive K channel (KATP). However, millimolar concentrations of cytoplasmic ATP do not close the channel as seen for native channels although the channels require low ATP concentrations to prevent rundown. Thus it seems likely that these two genes code for channels related to the KATP channel and that KATP channels will turn out to have some homology to these channels but be new additions to this family. 7.2.1 Vascular Smooth Muscle Representatives of these Families As with other excitable cells, a number of the 50 or more identified voltage dependent K channels (Pongs, 1992) appear to be expressed in vascular smooth muscle. The most ubiquitous channel in excitable tissues (at least as defined by


mRNA expression), is that coded by the DRK1 gene. This is the mammalian homologue of the Drosophila shab gene and DRK1 is abundantly expressed in rat aorta (Roberds and Tamkun, 1991). The electrophysiological properties of this channel most closely resemble the classical delayed rectifier current described by Hodgkin and Huxley (Frech et al., 1989). Nine members of a highly homologous family of genes, corresponding to the Shaker family, have been expressed from rat, designated RCK1–9, that encode channels displaying a complete range of properties from rapidly inactivating A-type currents to noninactivating delayed rectifier currents (Pongs, 1992). Of these nine, representatives of at least four types are expressed in aorta (Roberds and Tamkun, 1991). Finally, a homologue of the Shal gene is expressed in rat aorta (Roberds and Tamkun, 1991) which might be expected to have an intermediate kinetic fingerprint (Pongs, 1992). The relative abundance of these expressed mRNAs suggests that, in aorta (Roberds and Tamkun, 1991) and probably in a number of other blood vessels, the most important K channels for conferring the overall electrical properties of the cells are the delayed rectifier DRK1 and the Shaker homologue RCK7 or Kv1. This latter channel shows relatively slow inactivation over hundreds of milliseconds (Swanson et al., 1990). Functionally, we would therefore expect to see a TEA-sensitive delayed rectifier current in vascular smooth muscle cells. In rabbit portal vein the IC50 for TEA on the delayed rectifier in this cell is about 5 mM (Beech and Bolton, 1989b). This is consistent with the properties of DRK1, IC50=6 mM (Pongs, 1992). Clearly, the complex (and no doubt incomplete picture) described above provides an obvious explanation for the widespread variation in the electrophysiological characteristics of voltage dependent K channels in different vascular cells. A large number of subtly different electrophysiological phenotypes can be defined by the palette of channels available. Further work on the molecular biology of K channels in different blood vessels will be important in unravelling this complexity. In particular the functional expression of particular channel types will allow the identification of specific antagonists that can then be used to probe the functional role of these different channels in the intact tissues. 7.3 K Channels in Vascular Smooth Muscle Cells: Electrophysiological Overview As with many other excitable tissues, characterization of K channels in vascular smooth muscle has been hampered by a relative lack of specific antagonists. However, over the last ten years the use of voltage-clamp techniques to investigate currents in single cell preparations has allowed significant progress to be made. Although K channels and K+ currents with a wide range of properties have been characterized in smooth muscle cells from many sites in the vasculature, most of these fall into one of three major classes. These include delayed rectifier-type


channels (IK), large-conductance Ca-activated (BKCa) channels, and KATP channels. While it is unlikely that a complete characterization of K channels in any one type of vascular smooth muscle cells (VSMCs) has yet been achieved, it is apparent that these three types of K channel co-exist in VSMCs from a number of blood vessels. Whether each type of channel therefore has a distinct function, and what that function is, remains unclear. In general, however, K channels are thought to be involved in setting the resting potential and suppressing or limiting membrane depolarization. 7.3.1 Ca-activated K Channels Most of the KCa channels which have been reported in VSMCs have a conductance of approximately 100 pS when the K+ gradient is set to a physiological level, this value is roughly doubled in symmetrical high K+ solutions (e.g. Benham et al., 1986; Gelband and Hume, 1992). The opening probability (P0) of these channels is sensitive both to increases in intracellular [Ca2+] and to depolarization. Single channel studies have indicated that at likely resting conditions of membrane potentials negative to –60 mV and intracellular Ca2+ of 100 nM, the probability of opening of the KCa channels will be very low (Benham et al., 1986). However, the large number of channels and their large unitary conductance means that even with a low Po, they could contribute a functionally important component of the total K + conductance under resting conditions. KCa channels appear to be virtually ubiquitous in the smooth muscle cells of the vasculature; their high conductance contributes a characteristic ‘noisy’ appearance to cell outward currents. In addition, large current oscillations, termed spontaneous transient outward currents (STOCs), are often observed at negative membrane potentials (Benham and Bolton, 1986). These are thought to be caused by stochastic Ca2+ release from the sarcoplasmic reticulum, resulting in the brief activation of a local group of channels. KCa channels are selectively blocked at submillimolar concentrations by TEA, and are also potently and selectively suppressed by charybdotoxin (Brayden and Nelson, 1992). These blockers are therefore useful in evaluating the role of KCa channels in controlling the membrane potential. Bath application of TEA causes depolarization and action potential generation in a variety of isolated blood vessels (reviewed by Bolton, 1979). In a recent study, Brayden and Nelson (1992) reported that the vasoconstriction of rabbit cerebral arteries induced by elevation of luminal pressure was potentiated by TEA and charybdotoxin; they therefore suggested that KCa channels act as a negative feedback pathway to control the myogenic response.


7.3.2 Delayed Rectifier-type K Channels Whole-cell membrane currents carried by voltage-gated delayed rectifier K channels are also widely distributed in VSMCs (Beech and Bolton, 1989a). The single channel conductances underlying IK are relatively small, and although extensive studies have not been carried out, appear diverse (Beech and Bolton, 1989a; Gelband and Hume, 1992). IK typically becomes apparent when cells are depolarized beyond –40 or –30 mV. The activation rate is voltage-dependent. The current subsequently inactivates, although the rate of this process varies in different VSMCs, again pointing to the existence of more than one type of IK channel in the vasculature. The contribution of IK to membrane potential regulation is not well understood; this may in part be the result of the problem that no selective blockers of IK have as yet been described. 4-AP in millimolar or submillimolar concentrations was shown to suppress IK to a much greater extent than KCa in several types of VSMCs (Beech and Bolton, 1989b), and also caused membrane depolarization in single cells and isolated blood vessels (Hara et al., 1980; Gelband et al., 1993; Smirnov et al., 1994). These results are, however, ambiguous, since 4-AP also may block KATP channels (Beech and Bolton, 1989b). Several aspects of the regulation of IK in different VSMCs have recently been reported. For example, acute hypoxia greatly attenuates a 4-AP-sensitive K+ current in cultured rat pulmonary, but not mesenteric, arterial cells (Yuan et al., 1993). A K+ current in canine pulmonary artery has also been shown to be reduced in amplitude by acute hypoxia, although the channels involved were of high conductance and were Ca2+ -sensitive (Post et al., 1992, 1993). It was suggested that this latter effect involved a reduction in cell redox state. Longterm exposure of rats to hypoxia causes a chronic depolarization of pulmonary artery, and also reduces the amplitude of IK in cells isolated from pulmonary arteries after prolonged hypoxia (Smirnov et al., 1994). IK is also inhibited by rises in intracellular Ca2+ and Mg2+. The molecular characterization of IK channels is likely to result in further and rapid advances in our understanding of the regulation and role of this current in vascular cells. 7.3.3 KATP Channels and Inward Rectifier Channels in Vascular Smooth Muscle These channels can be grouped together because they show the properties of being open near the resting membrane potential, showing little inactivation, and having ohmic current voltage relationships or a preference to passing current in an inward direction.


Inward rectifier currents An inward rectifier K+ current (a conductance preferentially passing inward current at negative membrane potentials) was first identified in intact segments of small arterioles (Edwards and Hirst, 1988). Attempts to further characterize this conductance in single cells have been thwarted until recently by an inability to identify such a current in single cell preparations. This could be due to a localization of these channels to small resistance vessels. Such a possibility is given further weight by the recent observation of this current in single cells isolated from small resistance sized cerebral arteries (Quayle et al., 1994). This conductance showed high selectivity for K+ ions and inward current through the channels was blocked by low concentrations of barium (IC50=2.2 μ M). It is of interest that increases in extracellular K+ in the 5–15 mM range hyperpolarize and dilate these vessels (McCarron and Halpern, 1990), which may be important as a mechanism of autoregulation in these vessels and as a mechanism for increasing cerebral blood flow in response to elevated neuronal function that will be signalled by elevated extracellular K+. The localization of the inward rectifier current in these cerebral vessels may underlie this unique functional response, since the activation of this current, which leads to hyperpolarization, is shifted to more positive potentials by an elevation of extracellular K+. Regulation by ATP of KATP channels Although KATP channels are defined by the inhibitory action of ATP upon their activity, it is well established that low concentrations of ATP also stimulate channel activity (reviewed by Ashcroft and Ashcroft, 1990). The presence of MgATP is necessary to prevent the disappearance of KATP activity in isolated membrane patches in a number of types of cell. Non-hydrolyzable ATP analogues are not effective in this respect, indicating that phosphorylation of the channel or an associated regulatory protein is needed to maintain its viability. Evidence for a potentiating effect of ATP in VSMCs has been presented by Noack et al. (1992) and by Pfründer et al. (1993), both of whom found that low concentrations of ATP in the presence of excess Mg2+ greatly enhanced the K+ current elicited by ATP depletion; channel run-down was however not slowed. Although persistent KATP activity has been shown in isolated patches or dialyzed cells under ATP-free conditions (Standen et al., 1989; Clapp and Gurney, 1992; Lorenz et al., 1992), this might reflect the presence of residual ATP, or the absence of cellular phosphatases. An inhibitory effect of ATP on K channels has been demonstrated in membrane patches from a number of types of VSMC, although the range of channel properties reported is diverse. Standen et al. (1989) reported that in isolated membrane patches from rat and rabbit mesenteric artery VSMCs, ATP inhibited the opening of a slightly voltage-dependent, K-selective channel with a conductance of 135 pS. In the presence of ATP, the opening of this channel was


increased by cromakalim (CRK), and then decreased by glibenclamide (glyburide). The inhibitory effect of glibenclamide on KATP channel activity has been described in a variety of cell types, and this compound is widely used to elucidate the role of these channels in cellular responses thought to be due to KATP activation (e.g. hyperpolarization and relaxation of VSMCs) under conditions where it is difficult or impossible to directly establish channel identity. In a subsequent report (Nelson et al., 1990) the glibenclamide-sensitive K channel in these cells was reported to have a much smaller conductance (20 pS between –20 and –50 mV). A large (258 pS) conductance ATP- and glibenclamide-sensitive (but Ca insensitive) K channel was also observed in VSMCs from rabbit kidney afferent arterioles (Lorenz et al., 1992). This channel also demonstrated a marked voltage-dependence, distinguishing it from KATP channels in other types of cells. An ATP- and glibenclamide-sensitive K channel with a conductance of 30 pS has also been described in porcine coronary artery (Miyoshi et al., 1992). This channel appears, however, to be atypical in that it is activated by extracellular Ca2+, and was only weakly inhibited by ATP unless the extracellular Ca2+ concentration was reduced to 0.1 μ M. K+ currents sensitive to intracellular ATP have also been recorded in VSMCs using the whole-cell patch-clamp technique. Clapp and Gurney (1992) compared membrane potential and K+ currents in rabbit pulmonary arterial cells dialyzed with either no, or 1 mM, ATP. In the absence of cellular ATP, cells were hyperpolarized and demonstrated a glibenclamide-sensitive non-inactivating K+ current near the resting potential. In the presence of ATP, cells were more depolarized and showed no non-inactivating current. Noack et al. (1992) demonstrated similar effects when comparing rat portal vein cells dialyzed with a solution either containing or lacking glucose and Krebs cycle intermediates. Silberberg and van Breemen (1992), using the perforated patch technique in order to preserve the intracellular milieu, found that metabolic inhibition of rabbit mesenteric artery cells with iodoacetic acid and dinitrophenol also elicited a timeand voltage-independent, glibenclamide-sensitive K+ current. Other intracellular modulators of KATP Measurements of the ATP-sensitivities of KATP channels in isolated patches from VSMCs and other tissues show that channel opening is half-maximally inhibited at [ATP]i 20–100 μ M (Nichols and Lederer, 1991). Since cellular ATP has been measured to be in the millimolar range, questions have been raised concerning whether the ATP concentration ever falls enough to allow significant channel opening (Ashcroft and Ashcroft, 1990), and additional or alternative regulators of the KATP channel have been sought. There is a wealth of information which indicates that ATP itself is not the only, or indeed the primary, physiological regulator of KATP channels in VSMCs and other cell types. Regulation of these channels by nucleotide diphosphates, pH, endogenous agonists, and second


messenger systems has been described (Ashcroft and Ashcroft, 1990; De Weille and Lazdunski, 1990). Nucleotide diphosphates (NDPs) such as ADP and GDP influence KATP channel activity in various types of cell, including VSMCs. A number of NDPs have been shown to promote the opening of KATP channels in pancreatic β -cells (Findlay, 1987) and cardiac ventricular cells (Lederer and Nichols, 1989); in both cases this effect required the presence of Mg2+. It has been proposed that this effect may involve competition at the ATP binding site (e.g. Lederer and Nichols, 1989), and also that ADP acts at a separate site (Tung and Kurachi, 1991) which may be located on a diffusable cytoplasmic component (Larsson et al., 1993). Kajioka et al. (1991) observed a 15 pS ATP-sensitive K channel in rabbit portal vein cells which opened in the presence of pinacidil in cell attached, but not isolated, membrane patches; this implied that a cytoplasmic factor was necessary for channel activation. Channel opening could be restored in isolated patches by GDP (>0.1 mM) applied to the internal surface of the patch; GTP had a similar but weaker effect, and GMP was ineffective. The channel was inhibited by ATP (IC50=29 μ M). Pfründer et al. (1993) found that 0.1 mM ADP in the absence of ATP elicited a glibenclamide-sensitive, non-inactivating conductance in guinea-pig portal vein cells, which was not present in the absence of both ATP and ADP. This current was not entirely suppressed even in the presence of 5 mM ATP, if 0.1 mM ADP was present. Beech et al. (1993a, 1993b) also found little glibenclamide-sensitive K+ current in rabbit portal vein cells dialyzed with an intracellular solution lacking any nucleotides. Glibenclamide-sensitive currents of similar magnitude were however evoked in the absence of ATP when any of several nucleotide diphosphates, including GDP, CDP, and UDP, were present. ADP also had a similar, but lesser, effect; nucleotide tri- and monophosphates were essentially ineffective. The channel underlying this current had a conductance of 24 pS. Figure 7.1 illustrates that the glibenclamide-sensitive K+ current in rat pulmonary arterial cells is also only negligibly activated in the absence of added intracellular ATP, unless a nucleotide diphosphate is also present. Panel A shows that in the absence of intracellular ATP only a minor effect of glibenclamide on the whole cell K+ current can be demonstrated. When cells are dialyzed with an ATP-free pipette solution containing 1 mM GDP however, a large glibenclamidesensitive K+ current is observed. These results were obtained in cells which were bathed in a high K+ solution in order to shift the K+ reversal potential to 0 mV. Under such conditions a large K driving force is present at the normal resting membrane potential and the lack of voltage-dependence of the glibenclamidesensitive current in the presence of GDP was clearly revealed. The potentiating effect of nucleotide diphosphates on KATP channels in rabbit portal vein cells appears to require Mg2+ (Beech et al., 1993a), as it does in ventricular myocytes and β -cells. The block of the KATP channel by ATP in portal vein cells was also relieved by Mg2+, indicating that ATP is more effective in blocking these channels than is MgATP. This characteristic has previously been


shown to apply to KATP channels in pancreatic β -cells, but not in ventricular myocytes. pH has also been shown to regulate KATP channel activity in a number of cell types, although both increases and decreases in channel opening have been reported,

Figure 7.1 Whole cell currents recorded from pulmonary artery VSMCs, where the patch pipette contained either no nucleotide (A) or 1 mM GDP (B). The K+ solution on both sides of the membrane was set at 135 mM. Cells were held at –60 mV and 100 ms voltage ramps from –100 to +90 mV were applied every 10s. The open circles show the resulting current under control conditions, and the solid circles represent the current measured in the presence of glibenclamide (10 μ M). Mean and S.E.M. values are from 3 cells in A and 5 cells in B.

depending on cell type (Misler et al, 1989; Davies, 1990). Koyano et al. (1993) have recently shown in guinea-pig ventricular cells that acidosis both decreases the sensitivity of the channel to ATP, and also reverses channel rundown. Similar studies in VSMCs are lacking, but it is worth noting that neither agonists, nor severe hypoxia (Aalkjaer and Lombard, 1994) have been shown to cause important changes in intracellular pH in VSMCs under physiological conditions. Hypoxia has been shown to cause dilation of guinea-pig coronary arteries (Daut et al., 1990) and inhibition of myogenic reactivity of renal afferent arteries (Loutzenhiser and Parker, 1994); both effects were glibenclamide sensitive,


implying a role for KATP channels. The mechanism by which hypoxia activates KATP channels is, however, unclear. One possibility, suggested by Daut et al. (1990), is that hypoxia leads to a fall in intracellular [ATP] or the [ATP]/[ADP] ratio sufficient to activate KATP channels. This concept, however, runs counter to a number of reports that cellular [ATP] and energy state are maintained at normal levels in the face of hypoxia, being depleted only during anoxia or severe metabolic inhibition (e.g. Buescher et al., 1991). It is also thought that the nearmembrane [ATP] is highly dependent upon glycolysis rather than oxidative phosphorylation (Weiss and Lamp, 1987). Loutzenhiser and Parker (1994) observed that the glibenclamide-sensitive inhibitory effect of hypoxia was not associated with increases in arterial NADH, implying that oxidative ATP production was not compromised. It is therefore possible that hypoxia may activate KATP channels via an ATP-independent pathway. 7.4 Are Vascular KATP Channels Open under Basal Conditions? Although it has been demonstrated that the membrane potential of isolated VSMCs can be altered by changing the intracellular ATP concentration, either directly or via metabolic inhibition (Clapp and Gurney, 1992; Silberburg and van Breemen, 1992), the extent to which KATP channels are normally contributing to the maintenance of the resting membrane potential under physiological conditions remains unclear. Quast and Cook (1989) found that the infusion of glibenclamide into rats, which would be expected to block any basal KATP channel activity and therefore lead to depolarization and vasoconstriction, caused only a transient rise in blood pressure, implying that the K+ conductance sensitive to this drug was unlikely to be exerting a significant control over the resting potential. Similarly, glibenclamide infusion had no effect upon blood pressure in dogs (Samaha et al., 1992; Imamura et al., 1992). Glibenclamide also has been shown not to increase vascular tone in a number of types of isolated blood vessels (Quast and Cook, 1989; Eltze, 1989; Yuan et al., 1990). Glibenclamide decreases basal tone in canine cerebral and basilar arteries (Zhang et al., 1991). On the other hand, glibenclamide was shown to increase resting tone in vivo in both the hamster cheek pouch and cremasler muscle microcirculalions (Jackson, 1993). In addition, infusion of high concentrations of glibenclamide into the coronary arteries of anaesthetized dogs led to significant coronary constriction under non-ischaemic conditions (Samaha et al., 1992; Imamura et al., 1992). It would therefore appear that KATP channels are open under basal conditions in some, but not all, vascular beds. Although this might reflect the influence of endogenous agonists (see below) which may be tonically activating these channels, particularly in vivo, it is also possible that the regulation and/or


structure of these channels may differ significantly between different sites in the vasculature. 7.4.1 KATP Channel-independent Effects of Glibenclamide An interpretation of the effects of glibenclamide on vascular tone is complicated by the fact that this drug has also been shown to suppress tension development induced by vasoconstricting prostanoids such as PGF2― (Zhang et al., 1991; Neilsen-Kudsk and Thirstrup, 1991) and U46619 (Cocks et al., 1990) in arteries from the rat and dog. This inhibitory effect is nol endothelium-dependent (Zhang et al., 1991), and appears to involve a competitive interaction al the prostaglandin receptor(s) involved (Cocks et al., 1990). The EC50 for this effect is typically in the micromolar range; similar or higher concentrations are widely used to block KATP channel activity in most in vitro studies. Glibenclamide has also been observed to suppress contractile responses to high concentrations of K+ solution in coronary arteries (Neilsen-Kudsk and Thirstrup, 1991), and to a number of different spasmogens in airways smoolh muscle (Neilsen-Kudsk and Thirstrup, 1993). Figure 7.2 illustrates that glibenclamide has a potent inhibitory effect on the PGF2― contracture in rat pulmonary artery. This effect is contrasted wilh that of



Figure 7.2 Effects of KATP channel blockers BRL31660 and glibenclamide on force development In rat pulmonary artery rings precontracted with a submaximal dose (2 μ M) of PGF2― . Vertical and horizontal scale bars represent 0.25 g and 10 min respectively.

BRL 31660, a novel antiarrhythmic compound which has also been shown to block KATP channels (Taylor et al., 1989). BRL 31660 caused a concentrationdependent increase in force development in the presence of PGF2― (Figure 7.2). BRL 31660, but not glibenclamide, also induced force development over a similar concentration range in the absence of any agonist (not shown). It therefore seems that glibenclamide is quite effective at relaxing responses to vasoconstricting prostanoids, and may also have additional, although less potent, inhibitory effects on responses to other types of stimulation. The contrasting effects of glibenclamide and BRL 31660 suggest that the widespread use of glibenclamide as a supposedly selective inhibitor of the KATP channel may have led to an underestimate of the role of this channel in regulating basal tone and membrane potential under physiological conditions. 7.4.2 Agonist Mediated Modulation Regulation of KATP channel activity by agonists, acting through G proteins and protein kinases A and C, is likely to play a major role in controlling VSMC membrane potential, and thus vasoconstriction. Effects of both vasoconstrictors and vasodilators on KATP channels have been demonstrated, both directly, and indirectly using glibenclamide. In cultured VSMCs from porcine coronary artery, endothelin (20 nM) caused a marked depolarization, which could be reversed by the KATP channel opener nicorandil. Openings of a KATP channel of 30 pS conductance were detected in cell attached and isolated membrane patches, and were potently and completely suppressed by endothelin (IC50 near 1 nM). Vasopressin had a similar effect on these channels at low concentrations (Miyoshi et al., 1992; Wakatsuki et al., 1992). This channel was somewhat atypical of KATP channels, however, in that it was regulated by the extracellular Ca2+ concentration, such that the channel was mostly open at physiological [Ca2+]0, even if intracellular [ATP] was raised to 1 mM. Vasoconstrictor effects were recorded at 0.1 mM [Ca2+]0, raising questions concerning their physiological relevance. It is becoming increasingly clear that activation of KATP channels contributes to the actions of several vasodilators. Standen et al. (1989), demonstrated that glibenclamide inhibited both the opening of KATP channels in isolated patches from rat and rabbit mesenteric artery VSMCs, and the hyperpolarizations induced in intact arteries by acetycholine and vasoactive intestinal peptide. A subsequent report showed that calcitonin gene-related peptide (CGRP), a potent vasodilator, caused hyperpolarization and the opening of K channels in rat mesenteric artery VSMCs (Nelson et al., 1990). Both effects were blocked by glibenclamide, although the CGRP-induced relaxation was partially insensitive


to this compound, suggesting a KATP independent mechanism was also present. Recent data provide good evidence that the CGRP mediated modulation of KATP activity is through activation of protein kinase A (Quayle et al., 1994). Evidence has also been presented that the vasodilation of guinea-pig coronary arteries by adenosine was mostly blocked by glibenclamide (von Beckerath et al., 1991), as was the inhibition of myogenic reactivity in rat kidney afferent arterioles caused by low concentrations of this autocoid (Loutzenhiser and Parker, 1994). Dart and Standen (1994) have recently demonstrated that adenosine, acting at A1 receptors, causes a glibenclamide-sensitive K+ current in porcine coronary artery cells. 7.5 K Channel Openers and their Site of Action Over the last several years a number of structurally diverse molecules have been identified with the common mechanism of hyperpolarizing vascular smooth muscle. The first compound with this selective action was CRK (Hamilton et al., 1986) and a comprehensive listing can be found in Edwards and Weston (1993). The key characteristics of the profile of these compounds, in addition to hyperpolarizing the membrane potential of smooth muscle cells, is that the relaxant effects can be overcome by elevation of external [K+] above 50 mM and can be blocked by sulphonylureas such as glibenclamide at submicromolar concentrations. This profile was interpreted to suggest that the compounds mechanism of relaxant action was as potassium channel openers (KCOs). Since these initial observations, a large body of experimental evidence has been accumulated to test this hypothesis and has been reviewed by Quast (1993). 7.5.1 The K Channel Opened by KCOs Following the description of the hyperpolarizing action of CRK (Hamilton et al., 1986), the next problem to resolve was the nature of the K channel that was opened. An early study by Beech and Bolton (1989b) examined the pharmacology of the CRK-activated current in VSMCs from portal vein and compared this current with the delayed rectifier and BKCa current in these cells. These studies showed a pharmacological profile similar, but not identical to, the delayed rectifier current although the KCO activated current showed no voltage dependence unlike the delayed rectifier. The whole-cell current activated by KCO also showed no apparent increase in current noise suggesting that the channels activated were not large conductance like BKCa channels. A subsequent study revealed no significant effect of levcromakalim (LCRK) on the Caactivated and delayed rectifier K+ currents in these cells (Russell et al., 1992). In the last five years a number of studies have built on these observations and indicate that the major conductance that is activated in whole cells by LCRK is a


KATP channel whose key properties are little voltage sensitivity, no inactivation and block by micromolar glibenclamide. Noack et al. (1992) showed that LCRK (1 μ M) activated a glibenclamide-sensitive current in rat portal vein and that this had very similar properties to a current Imet that developed in the same cells when intracellular ATP was depleted. Moreover, when Imet ran down after prolonged ATP depletion (characteristic of KATP channels) then LCRK was no longer able to activate the KCO current. The unitary conductance of this current was estimated at 10–20 pS from noise analysis of the whole-cell currents. Other KCOs such as aprikalim and P1060 also seem to activate the same conductance (Ibbotson et al., 1993). In VSMCs from rabbit pulmonary artery, a whole-cell K+ current with similar properties has been identified (Clapp and Gurney, 1992), and the unitary conductance of this current has also been estimated by noise analysis to be 16 pS (Langton et al., 1993). In addition to this enhancement of the KATP current, inhibition of the delayed rectifier has been reported in rat portal vein cells (Noack et al., 1992). This effect is likely to be functionally less relevant in VSMC than the activation of a conductance at or near the resting membrane potential of the cell that will dominate the response. However it does suggest that KCOs have more than one channel target. 7.5.2 Other Actions of KCOs The major experimental evidence that challenges the straightforward hypothesis that KCOs act by opening K channels is the ability of KCOs to relax tone induced by noradrenaline in tissues where it is known that Ca antagonists are unable to reverse noradrenaline-induced tone (Bray et al., 1991). These data suggest that KCOs must be inhibiting an intracellular mechanism as the noradrenaline-induced tone clearly does not depend on voltage-gated Ca2+ influx. This could be explained either by KCO induced hyperpolarization modulating other second messenger pathways, in which case multiple sites of action do not need to be invoked, or by direct intracellular effects of the KCOs themselves on these pathways. A link between membrane potential and phospholipase C activity would provide a membrane potential dependent pathway. Itoh et al. (1992) have demonstrated that in rabbit mesenteric artery, noradrenaline induced IP3 production is inhibited by pinacidil, and inhibition is relieved by elevation of external K+ and by K channel blockers (KCBs). Such an inhibitory profile would be expected if hyperpolarization inhibited IP3 production. This neatly explains the inhibitory effects of pinacidil on tension and intracellular Ca2+ that were measured in conditions of zero extracellular Ca2+. Direct evidence for modulation of IP3 production by membrane potential has now been provided. Ganitkevich and Isenberg (1993) showed that in guinea-pig coronary artery smooth muscle cells held under voltage clamp, hyperpolarization inhibits intracellular Ca2+ transients generated by muscarinic activation of phospholipase


C. The effect is independent of extracellular Ca2+ and not seen with caffeine induced intracellular Ca2+ transients. The potential sensitivity of phospholipase C might be explained if, as a membrane bound enzyme, it directly senses the membrane potential through an intrinsic voltage sensor. The complex effects of membrane potential on second messenger function fail to explain all the paradoxes of experimental data. In particular, experiments showing that KCBs can inhibit the effects of KCOs on membrane potential without blocking their relaxant actions clearly imply intracellular targets for KCOs (reviewed by Quast, 1993). The data seem most consistent with the idea that reuptake of Ca2+ into intracellular stores is inhibited by KCOs. Evidence to support this comes from studies on permeabilized airways smooth muscle cells (Chopra et al., 1992). Taking all this data together (Figure 7.3), it seems that the most important action of the KCOs is to open a K channel that has very similar properties to a KATP channel. Surprisingly, some of the relaxant properties of these compounds do not mimic the actions of voltage-gated Ca channel antagonists because of the recently identified effects of membrane potential on other signal transduction systems in smooth muscle. Finally, it is also possible to identify actions that must be completely independent of changes in membrane potential although, as yet, the functional significance of these effects is not clear.

Figure 7.3 Plan illustrating the identified mechanisms of action of KCOs and the functional consequences thereof.


7.6 Conclusions Much of this chapter has focussed on the properties of a K channel or channels that can open close to the resting membrane potential of vascular smooth muscle cells. This property makes them well suited to exert subtle control of the membrane potential and hence excitability of these cells, particularly as the voltage dependent entry of Ca2+ is likely to be mostly a graded trickle dependent on membrane potential rather than large quantal influxes associated with action potentials. The activity of these channels is modulated both by intracellular nucleotide levels and by G-proteins, indicating that both internal and external signal transduction systems couple to these channels to regulate membrane potential. In contrast, the large group of voltage dependent K channels might be predicted to have a relatively less important functional role, reflecting the smaller contribution of action potential discharges to the overall pattern of electrical activity of these tissues. We can look forward to clarifying these speculative conclusions as molecular approaches identify all the pieces in the K channel puzzle that make up the K conductance of each type of smooth muscle cell. Understanding the extent of the diversity of channels in different tissues will help to explain the tissue variation in experimental data. Selective functional expression of channels will facilitate the search for selective blockers. These agents will then in turn be used to clarify physiological function in the original vascular preparations. References AALKJAER, C. & LOMBARD, J. (1994) J. Vas. Res., 31 (Suppl 1), 1. ASHCROFT, S.J.H. & ASHCROFT, F.M. (1990) Cell Signall., 2, 197–214. BEECH, D.J. & BOLTON, T.B. (1989a) J. Physiol, 412, 397–414. (1989b) Br. J. Pharmacol., 98, 851–864. BEECH, D.J., ZHANG, H, NAKAO, K & BOLTON, T.B. (1993a) Br. J. Pharmacol., 110 (2), 573–582. (1993b) Br. J. Pharmacol., 110(2), 583–592. BENHAM, C.D. & BOLTON, T.B. (1986) J. Physiol., 381, 385–406. BENHAM, C.D., BOLTON, T.B., LANG, R.J., & TAKEWAKI, T. (1986) J. Physiol., 371, 45–67. BLUMENTHAL, E., & KACZMAREK, L.K. (1992) Neurochem. Res., 17, 869–876. BOLTON, T.B. (1979) Physiol. Reviews, 59, 606–718. BOYLE, M.B., AZHDERIAN, E.M., MACLUSKY, N.J., NAFTOLIN, F. & KACMAREK, L.K. (1987) Science, 235, 1221–1224. BRAY, K.M., WESTON, A.H., DUTY, S., NEWGREEN, D.T., LONGMORE, J., EDWARDS, G. & BROWN, T.J. (1991) Br.J. Pharmacol., 102, 337–344. BRAYDEN, J.E. & NELSON, M.T. (1992) Science, 256, 532–535. BUESCHER, P.C., PEARSE, D.B., PILLAI, R.P., LITT, M.C., MITCHELL, M.C. & SYLVESTER, J.T. (1991) J. App. Physiol., 70(4), 1874–1881.


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ZHANG, H., STOCKBRIDGE, N., WEIR, B., KRUEGER, C. & COOK, D. (1991) Eur. J. Pharmacol, 195, 27–35.

8 Effects of Potassium Channel Activators in Isolated Blood Vessels U.QUAST Department of Pharmacology, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany.

8.1 Introduction In most cells, the plasma membrane at rest is more permeable to K+ than to Na+, Cl- and Ca2+; therefore, K channels determine the resting membrane potential and, hence, the excitability of the cell. (A notable exception from this rule is skeletal muscle where the resting membrane potential is essentially governed by Cl-channels (Hille, 1992)). The opening of K channels shifts the membrane potential towards the K+ equilibrium potential which is around -90 mV. For smooth muscle, which generally has a resting membrane potential of ― -60mV, this means that opening of K channels hyperpolarizes the tissue; however, the value of -90 mV will only be reached asymptotically as the permeability to K+ becomes absolutely dominating over that of the other ions (Hille, 1992). In excitable cells endowed with depolarization-activated Ca channels (voltage-gated Ca channels, VOCs), hyperpolarization will prevent such channels from opening and, hence, Ca2+ entry via this pathway. In cells devoid of VOCs, e.g. endothelial cells, leukocytes and others, hyperpolarization will, by increasing the driving force for Ca2+ entry into the cell, promote Ca2+ influx via pathways which are active at such (hyperpolarized) membrane potentials. In view of the physiological importance of K channels it is no surprise that this class of ion channels is particularly heterogeneous (for details, see Chapters 6 and 7 of this book or Hille, 1992). Within the large group of voltage-gated K channels, the A channels (KA) open rapidly in response to depolarization and inactivate quite rapidly again if depolarization is sustained. Another, again heterogeneous, group responds to depolarization much more slowly (delayed rectifier K channels, KV); some of these KV channels do not inactivate at all. Both groups of channels let the K+ current pass only out of the cell and are both of great importance for repolarization of the cell; in addition, KA channels are involved in setting the firing frequency of pacemaker cells (Hille, 1992). Another large group of K channels, the inwardly rectifying K channels (KIR), let K+ currents pass much better into the cell than outwards. Such channels have

EFFECTS OF KCAS IN ISOLATED BLOOD VESSELS 215

been cloned recently from rat kidney (Ho et al., 1993), mouse macrophages (Kubo et al., 1993a) and rat heart (Kubo et al., 1993b). In many cells, KlR channels determine the resting membrane potential (Hille, 1992); this is particularly well documented for cardiac myocytes. The KIR channel cloned from rat heart was opened by muscarinic receptor activation and by activated Glproteins (Kubo et al., 1993b) and is abundantly expressed in the atrium. It therefore most likely represents the K channel which mediates the regulation of cardiac frequency by the vagus nerve. Regulation of K channels by G-proteins and thus by neurotransmission constitutes a very important signalling pathway (Brown, 1990). Two families of K channels have caught the particular interest of the pharmaceutical industry, e.g. the ATP-sensitive K channels (KATP) and, more recently, the large conductance Ca-dependent K channels (BKCa). BKCa channels have a conductance of 100–200 pS under quasi-physiological conditions and form a heterogeneous and almost ubiquitous class of K channels. They open at depolarized membrane potentials in response to elevated intracellular Ca2+ concentrations (for review see McManus, 1991; Hille, 1992); hence they provide an important coupling mechanism between the intracellular Ca2+ level and the membrane potential. BKCa channels are blocked with high affinity by the scorpion toxins, charybdotoxin and iberiotoxin (review: Garcia and Kaczorowski, 1992). Recently, BKCa channels have been cloned from Drosophila and from murine skeletal muscle; their core is structurally related to the voltage-gated (KA and Kv) K channels (Atkinson et al., 1991; Butler et al., 1993). The KATP channels constitute a heterogeneous class of K channels found not only in excitable tissues like pancreatic β -cells, heart, skeletal and smooth muscle, neurons (for reviews see Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993) and the rat adenohypophysis (Bernardi et al., 1993), but also in tissues like kidney epithelium (Wang et al., 1990) or the follicular cells surrounding the Xenopus oocyte (Honoré and Lazdunski, 1991, 1993). In general, KATP channels are closed by ATP binding to an intracellular binding site; they open when ATP dissociates from this site and/or when Mg salts of nucleoside diphosphates (NDP) like MgADP and MgGDP bind to an activatory site which is different from the inhibitory ATP site (Nichols and Lederer, 1991; de Weille, 1992; Edwards and Weston, 1993; in vascular smooth muscle: Pfründer et al., 1993; Beech et al., 1993a). Thus, the opening of KATP channels is regulated by the quotient of ATP/NDP; hence, these channels link the metabolic state of the cell to cellular excitability. The activity of KATP channels is also modulated by phosphorylation (reviews: Ashcroft and Ashcroft, 1990; Nichols and Lederer, 1991; Edwards and Weston, 1993). A characteristic feature of KATP channels is their inhibition by sulphonylureas like glibenclamide with widely varying affinities in different tissues (Quast and Cook, 1989a; Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). The primary structure of a typical KATP channel is still unknown; the K channel recently cloned from rat kidney


(Ho et al., 1993) has not been shown to be inhibited by intracellular ATP or the sulphonylureas; however, these properties could be conferred to the channel by an additional, yet unidentified subunit (Nichols, 1993). On the other hand, evidence has been presented to show that the KATP channel in rat portal vein is a dephosphorylated form of the delayed rectifier K channel, Kv (Edwards et al., 1993). In the pancreatic β -cell, KATP channels regulate insulin release in response to the plasma glucose level (which, in turn, determines the ATP concentration in the β -cell near the channel); in most other tissues, they are generally closed and open only when the tissue is metabolically compromised, i.e. the quotient of ATP/NDP falls. In some vascular beds these channels are opened by vasorelaxant neurotransmitters and hormones (see below). This article will focus on the effect of KATP channel activators in isolated vascular preparations. After a short presentation of natural and synthetic activators of vascular KATP channels, the available binding data and the recent progress made in elucidating the K channels opened by these compounds and their mode of activation will be reviewed. This is followed by an analysis of the vasorelaxant profile of the KATP channel activators (KCAs) and of the mechanisms leading to vasorelaxation. The chapter is closed with an overview of recently described activators of the BKCa channel. 8.2 Natural and Synthetic Activators of Vascular KATP Channels In several blood vessels, hormones and neurotransmitters produce a glibenclamide-sensitive hyperpolarization and/or relaxation, suggesting that these effects are mediated by opening of KATP channels, probably via the intermediate activation of a G-protein. Well documented examples are the relaxant effects of adenosine (Daut et al., 1990; Dart and Standen, 1993) and prostacyclin or its analogue iloprost (Jackson et al., 1993) in the coronary vasculature of isolated guinea-pig or rabbit heart preparations or in isolated coronary artery cells. Part of the calcitonin gene-related peptide (CGRP)-induced relaxation in rabbit mesenteric small arteries is due to the opening of KATP channels (Nelson et al., 1990). A detailed mechanistic study in this preparation has shown that CGRP activates adenylate cyclase; the subsequent phosphorylation of KATP channels by cAMP-dependent protein kinase (PKA) induces KATP channel opening (Quayle et al., 1994). The field of synthetic KCAs is described in detail in earlier chapters of this book. It started in 1981 with the discovery that the coronary vasodilator nicorandil, a pyridine with a nitro-group in its side chain, hyperpolarized vascular smooth muscle cells by increasing the K+ conductance of the cell membrane (Furukawa et al., 1981). Shortly thereafter it was found that nicorandil, in addition to its K channel opening action, also acted as an organic nitrate and increased intracellular cGMP (Holzman, 1983). This compound thus possesses two


independent vasodilator mechanisms and it depends on the vessel under study and on the stimulus as to which of them predominates (Borg et al., 1991; Magnon et al., 1994). The observation that cromakalim (CRK), a compound structurally unrelated to nicorandil and much more potent as a vasodilator, apparently acts solely by opening K channels (Hamilton et al., 1986), represented a major advance. Subsequently, other ‘directly acting’ vasodilators like pinacidil (Bray et al., 1987; Cook et al., 1988a), diazoxide (Quast and Cook, 1989b; Winquist et al., 1989) and minoxidil sulphate, the active metabolite of minoxidil (Meisheri et al., 1988; Winquist et al., 1989) were recognized to act by K channel opening. New KCAs, structurally unrelated to the compounds mentioned above, e.g. the thioformamide aprikalim, have been synthesized (reviews: Atwal, 1992; Evans et al., 1992; Robertson and Steinberg, 1990; Edwards and Weston, 1990a,b; Chapters 1–4 of this book). 8.3 Binding Studies with KATP Channel Activators in Vascular Smooth Muscle With the introduction of the tritiated KCA, 3H-P1075 (Manley et al., 1993), binding studies with the KCAs have become feasible in intact rings of rat aorta (Bray and Quasi, 1992a, Quast et al., 1993), in rat aortic smooth muscle cells in culture and in freshly dissociated canine cardiac myocytes (Dickinson et al., 1993). Using another tritiated KCA, 3H-BAY-X-9228, Hoffmann et al. (1993) found specific KCA binding in rat insulinoma cells. In the vascular preparations, 3H-P1075 binding was found to be of high affinity (K ― 3–6 nM) and relatively D low capacity [Bmax in aortic rings ― 20 fmol/mg wet weight (Bray and Quast, 1992a), in cells ― 5000 sites/cell (Dickinson et al., 1993)]; in cardiocytes, KD was 30 nM and Bmax ― 117 000 sites/cell (Dickinson et al., 1993). 3H-P1075 binding in rat aorta was diminished by metabolic poisoning in parallel with the intracellular ATP concentration [ATP]i. It was, however, not affected by depolarization with 55 mM KCl or by lowering pH from 7.4 to 6.0 (Quast et al., 1993) and hardly influenced by interventions which activate the cAMP signalling chain (Linde et al., 1994). 3H-P1075 binding was inhibited by representatives from all major groups of KCAs and by the sulphonylureas with regular inhibition curves which gave the same rank order of potencies as that obtained in functional assays (Bray and Quast, 1992a; Quast et al., 1993; Dickinson et al., 1993). The potencies of the KCAs determined in the binding assays essentially coincide with those determined in vasorelaxation assays; however, they are about 40 times higher than those determined in the 86Rb+ efflux assay (the latter is a qualitative measure of K channel opening, see below). It was concluded from these studies that the major KCAs bind to the same target, but possibly to different sites at this target, to elicit their effects (Bray and Quast, 1992a; Manley et al., 1993); the quantitative relationship between agonist binding and K channel opening may be complex (Quast et al., 1993). The


sulphonylurea glibenclamide binds to a site different from and negatively allosterically coupled to the 3H-P1075 binding site (Bray and Quast, 1992a). Since there has been no success in detecting binding of KCAs in broken cell preparations (Dickinson et al., 1993; Hoffmann et al., 1993; Quast et al., 1993), the biochemical characterization of the KCA receptor is still lacking and the question whether the drug receptor of the KCAs is the KATP channel itself or a different protein, remains open. Intriguing results were obtained by Meisheri and colleagues (1991, 1993a) with minoxidil sulphate (MxS). Using two radiolabelled MxS derivatives, 35SMxS and 3H-MxS, these workers found that the sulphate group of MxS remained in the tissue longer than the tritiated piperidine moiety and that MxS covalently sulphated a 116 kD protein in rings from rabbit mesenteric artery and in A7r5 cells, a cell line derived from rat aorta (Meisheri et al., 1991), and in hair follicles from human skin (Meisheri et al, 1993a). This sulphate acceptor appears to be a membrane-associated protein with (an) isoelectric point(s) of 5.2–5.7 (Meisheri et al., 1991; 1993a). The relationship between the sulphation of the 116 kD protein and the KATP channel opening effect of MxS needs further clarification. 8.4 K Channel Opening The electrophysiological effects of the KCAs are described in detail in Chapter 7 of this book; therefore, only a short account of the most recent developments is given here. 8.4.1 Recent Electrophysiological Studies Type of K Channel activated by the KCAs Studies at the single channel level have shown that the KCAs can open a variety of K channels in vascular smooth muscle cells. Several authors have reported that KCAs increased the open probability of BKCa channels in isolated patches derived from vascular smooth muscle cells (Klöckner et al., 1989; Gelband et al., 1989; Hu et al., 1990; Gelband and McCullough, 1993). However, the pharmacological profile of the KCA-induced vasorelaxation differs profoundly from that expected if it was mediated by BKCa channel activators; in particular, differences in the sensitivities to the K channel blockers charybdotoxin, glibenclamide and tetraethylammonium (Winquist et al., 1989; Quast and Cook, 1989b; Quast, 1987) suggest that the BKCa-activating effect of the KCAs is not important physiologically.


Recent patch-clamp studies converge to show that in vascular smooth muscle cells the KCAs open a K channel with a relatively small conductance (15–20 pS, Kajioka et al., 1990;. 1991; Beech et al., 1993b); this is in contrast with the activation of a large-conductance KATP channel (135 pS) described earlier (Standen et al., 1989). Noise analysis of the K+ current induced by various KCAs in vascular smooth muscle cells also points to a small conductance (10–20 pS) K channel (Beech and Bolton, 1989; Noack et al., 1992a; Ibbotson et al., 1993a; Langton et al., 1993). The pharmacological and biophysical properties of the K+ current elicited by the KCAs in vascular smooth muscle cells are very similar to those of the current induced by depletion of intracellular ATP in these cells (Clapp and Gurney, 1992; Noack et al., 1992b; Silberberg and van Breemen, 1992; Beech et al., 1993b). The finding that the KCAs act on precisely the K channel that opens under ischaemic and hypoxic conditions may explain the therapeutic benefit found with the KCAs in animal models of cardioprotection (Escande and Cavero, 1992) and intermittent claudication (Cook et al., 1993). Mechanisms of K channel opening The mechanism by which the KCAs open the channel in vascular smooth muscle is still a matter of debate. In the absence of KCAs, the channel opens when the concentration of Mg salts of nucleoside diphosphates increases and the ATP concentration falls (Pfründer et al., 1993; Beech et al., 1993a); these changes in intracellular nucleotide concentration occur when the cell is metabolically stressed, e.g. by ischaemia and/or hypoxia. In addition, and as mentioned above in section 8.2, stimulation of cAMP-dependent phosphorylation in intact vascular smooth muscle cells isolated from rabbit mesenteric artery induces a glibenclamide-sensitive K+ current, indicating that this intervention also opens KATP channels (Quayle et al., 1994); similar observations have been made on KATP channels in follicular cells surrounding the Xenopus oocyte (Honoré and Lazdunski, 1991; 1993). The fact that KCAs are able to open KATP channels in isolated patches from vascular smooth muscle cells if appropriate conditions are met (Standen et al., 1989; Kajioka et al., 1990; 1991; Beech et al., 1993b) does, however, not necessarily mean that the KCAs act on the channel directly; the isolated patch may contain proteins (be they integral membrane proteins or tightly associated to the membrane) which might be essential for activation of the channel, e.g. kinases or phosphatases. The KCAs could indeed act on such proteins to open the channel. In recent investigations into the mechanism of activation of KATP channels by the activators in rat portal vein cells, it was found that these compounds inhibited the K+ current (IKv) flowing through the delayed rectifier channel (Kv) and that they simultaneously induced a K+ current (IKATP) flowing through KATP channels such that the sum of the two currents remained approximately constant; both effects were inhibited by glibenclamide (Noack et al., 1992a; Ibbotson et al, 1993a). These effects of the KCAs were mimicked by several measures that


decrease the phosphorylation state of the cell, in particular by inhibition of protein kinase A (PKA) with a specific protein inhibitor (Edwards et al., 1993). The authors suggested that the KCAs, by a yet unknown mechanism (but not by inhibiting PKA), induce dephosphorylation of the delayed rectifier channel Kv, and thereby convert KV into the activated form of KATP (Edwards et al., 1993). The finding that in rat portal vein, inhibition of PKA induces KATP channel opening is difficult to reconcile with the observation in rabbit mesenteric artery, that stimulation of PKA activates the KATP Channel (Quayle et al., 1994). We have found that in rings of rat aorta dibutyryl-cAMP (Bt2-cAMP), a membranepermeant activator of PKA, enhances the 86Rb+ efflux stimulating effect of levcromakalim (LCRK) (Linde and Quast, 1995; note that 86Rb+ efflux is a qualitative measure of K channel opening, see below). In rat portal vein, the potentiating effect of Bt2-cAMP on CRK-stimulated 86Rb+ efflux was, however, weaker (Quast, 1987), indicating differences in the modulation of KATP channels by PKA in different blood vessels. The hypothesis that dephosphorylation converts Kv into KATP has to accommodate the fact that these channels show a number of differences in their biophysical and pharmacological properties, most notably in their gating properties: Kv are gated by depolarization, due to the voltage-sensing S4 segment in their structure (Jan and Jan, 1992) whereas KATP channels in vascular smooth muscle are not (Edwards and Weston, 1993). The long awaited cloning of the KATP channels will resolve this matter. 8.4.2 Electrophysiological and Tracer Efflux Studies The direct manifestation of K channel opening is a K+ current which, in vascular smooth muscle under physiological conditions, is directed outward, leading to hyperpolarization of the cell. This K+ efflux can also be observed in tracer efflux experiments using 42K+ or 86Rb+. It has been extensively documented that the KCAs induce all these effects in various vascular preparations (whole tissue or isolated cells; for reviews see e.g. Cook and Quast, 1990; Edwards and Weston, 1990b; Edwards et al., 1992; Longman and Hamilton, 1992; Quast et al., 1994; electrophysiological studies: Chapter 7 of this book); therefore only a few comments regarding tracer efflux studies will be made here. Recent experiments show that relatively low concentrations (0.1 μ M) of LCRK or pinacidil induce sizable K+ currents in isolated rat portal vein cells (Noack et al., 1992a) and hyperpolarizations of rabbit mesenteric artery (Itoh et al., 1992). This corresponds well to the detection limit established for these compounds in tracer efflux studies in rat aorta (Quast and Baumlin, 1988; Bray and Quast, 1992b) or rat portal vein (Quast, 1987), so that earlier discrepancies between the results obtained by the two methods have collapsed. Tracer efflux studies with KCAs suffer from several shortcomings. First, they reflect the total K+ efflux from reaction of the whole tissue instead of that from a specific cell. Second, membrane potential is not clamped during the application


of the KCA; hence, as the K+ channels open and hyperpolarization develops, the driving force for the radioisotope to leave the cell is decreased. Hence, the effect of the KCA will be quantitatively underestimated (Videbaek et al., 1990), a bias that is particularly significant at high opener concentrations which induce a substantial hyperpolarization. A further critical point is the use of 86Rb+, which is often preferred to 42K+ for its more convenient half-life and the less penetrating radiation. Since different K channels have a different permeability ratio of 86Rb+ to 42K+, this may lead to further distortions of the flux signal. There is general agreement that under basal conditions, the ratio of 86Rb+ to 42K+ efflux is about 70% in large (rat aorta: Smith et al., 1986) and small vascular preparations (rat mesenteric resistance vessels: Videbaek et al., 1988). Detailed comparison of the 42K+ and 86Rb+ fluxes elicited by CRK in rat portal vein and aorta (Quast and Baumlin, 1988; Bray and Quast, 1991a) have shown that this compound, at low concentrations, opens K channels which are more selective for K+ over Rb+ than those opened at higher concentrations. This seems, however, not to be the case for minoxidil sulphate in rat aorta, where a constant permeability ratio of K+ to Rb + was found over the whole concentration range of the compound (― 40%; Bray and Quast, 1991a); in contrast, other workers found that minoxidil sulphate opened a Rb impermeable channel in rat aorta (Newgreen et al., 1990). Despite the limitations mentioned above it is our experience that 86Rb+ efflux experiments provide useful information on the K channel opening properties of the KCAs, in particular when these compounds are used in the low concentration range. 8.4.3 Inhibitors of KATP Channel Opening Inhibitors of the KATP channel activation have been reviewed previously (Cook and Quast, 1990; see also Robertson and Steinberg, 1990; Atwal, 1992). Here, some quantitative aspects of the sulphonylurea blockers will be discussed and hitherto unpublished data presented; then recent developments in the field of nonsulphonylurea blockers will be discussed. Glibenclamide The long chain sulphonylurea, glibenclamide, is the standard inhibitor of the effects of the KCAs (Cavero et al., 1989; Quast and Cook, 1989b; Winquist et al, 1989; Wilson, 1989; for further references see Cook and Quast, 1990; Meisheri et al., 1993b). It is well established that glibenclamide blocks KATP channels in the pancreatic β -cell at nM concentrations (Zünkler et al., 1988); in vascular smooth muscle, however, concentrations β 100 nM are required to induce substantial inhibition of the vasorelaxant and 86Rb+ efflux stimulating effects of the KCAs (see e.g. Quast and Cook, 1989b and Figure 8.1 below). At higher concentrations, however, glibenclamide has numerous additional effects (e.g.


Villar et al., 1986; Quast and Cook, 1989b; Cocks et al., 1990; Yoshitake et al., 1991; Zhang et al., 1991; further references in Panten et al., 1989). Thus, care must be taken when it is inferred from the inhibition of a (vasorelaxant) effect of a compound by



Figure 8.1 (Continued) glibenclamide at high concentrations (e.g. ― μ M), that opening of KATP channels was involved and additional data should be provided in support of this contention. Increasing concentrations of glibenclamide induce a gradual rightward shift in the concentration- 86Rb+ efflux curves obtained with CRK in rat portal vein (Quast and Cook, 1989b). Schild analysis of this data (which showed quantitative deviations from a strictly competitive behaviour) gave a pA2 value of 7.0 for glibenclamide in this preparation. Figure 8.1 shows new data obtained in rat aorta. Panel A compares the inhibition by glibenclamide of P1075-induced 86Rb+ efflux obtained at different P1075 concentrations. With increasing concentration of the agonist, the inhibition curves were shifted rightward and became steeper; the latter would not be expected for a strictly competitive mechanism of inhibition. Cheng-Prusoff analysis (Cheng and Prusoff, 1973) of the IC50 values from these curves gave an approximate Ki value of 18±19 nM for glibenclamide (Figure 8.1A, inset). Qualitatively similar results were obtained for the inhibition of CRK (1 and 3 μ M)-induced 86Rb+ efflux (Figure 8.1B); here, the (tentative) Cheng-Prusoff analysis gave a Ki value of 40 nM for glibenclamide (Figure 8.1B, inset). Since these data show quantitative deviations from the competitive model, the Cheng-Prusoff analysis has only a tentative character. However, the Ki value of 20–40 nM determined here for glibenclamide blocking the open channel (at zero concentration of opener) agrees very well with the inhibition constants (IC50 values) for glibenclamide as an inhibitor of KCA-induced vasorelaxation in rat aorta (Bray and Quast, 1992b; note that the vasorelaxation assays were conducted at low opener concentration). They are, however, 10 times lower than the Ki value of glibenclamide determined in the 3H-P1075 binding assay in rat aorta (Bray and Quast, 1992a; see Quast et al., 1993, for possible explanations). Other sulphonylureas and related insulinotropes The insulinotropic sulphonylureas (and related compounds) are generally grouped into three classes. Class A, the first generation sulphonylureas like tolbutamide and Figure 8.1 Inhibition of KCA-induced 86Rb+ efflux from rat aortic rings by glibenclamide (GBC). A: 86Rb+ efflux was stimulated by superfusion of P1075, (― ), 20 nM for 20 min; (― ), 60 nM for 20 min; (― ), 180 nM for 10 min. The curves were analyzed according to the Hill equation giving the following parameters (from left to right): IC50 (nM) 27± 3, 97±4, 180 ±22; Hill coefficients were 1.22±0.12; 1.58±0.11, 1.77±0.30. The inset shows a plot of the IC50 values versus P1075 concentration (Cheng-Prusoff analysis): Linear regression analysis gave an ordinate intercept (i.e. the affinity of glibenclamide for the open KATP channel in the absence of activator) of 18±19 nM and a slope of 1.1±0.2. B: Cromakalim (CRK) was applied as the KCA: (― ), 0.1 μ M for 20 min; (― ), 1 μ M for 20 min; (― ), 3 μ M for 10 min. The parameters of the inhibition curves were: IC50 values (nM): 40, 76±3, 155±20; Hill coefficients 1.2±0.1, 1.0±0.1. Inset: Linear regression of the


IC50 values versus CRK concentration gives an ordinate intercept of 35±1 nM and a slope value of 0.04±0.0004. Each preparation was stimulated twice with the same concentration of agonist with a washout period of 80–100 min in between. Glibenclamide was applied 20 min before the second stimulation until the end of the experiment and the degree of inhibition was determined by comparing the areas under efflux rate constant versus time curve as described (Bray and Quast, 1991a); n=4–6.

glibornuride are relatively short molecules, class B molecules (second generation) like glibenclamide and glipizide are the long chain sulphonylureas with a greatly increased affinity to the pancreatic KATP channel, and class C compounds like meglitinide (HB 699; Garrino et al., 1985), AZ-DF 265 (Garrino and Henquin, 1988), and UL-DF 9 (Garrino et al., 1986) possess a carboxylic acid group instead of the sulphonylurea group (Rufer and Losert, 1979). Some of these compounds have been examined as inhibitors of the vasorelaxant effects of CRK (Wilson, 1989). We have examined the inhibition of CRK (3 μ M)-induced 86Rb+ efflux from rat aorta by 12 insulinotropic compounds, 4 of each class. The results are listed in Table 8.1 together with the affinity of these compounds to the KATP channel in a rat insulinoma cell line determined in a binding assay (Schmid-Antomarchi et al., 1987). Also listed is the effect of the insulinotropes on basal 86Rb+ efflux from rat aorta as a reflection of their interference with other K+ exchange systems of the cell. It is seen that all insulinotropics, also the non-sulphonylurea derivatives (group C), are able to inhibit cromakalim-induced 86Rb+ efflux, with the (S)-(-) enantiomer of AZ-DF 265 having the second highest potency (most potent: glibenclamide). Figure 8.2 compares the potency of the compounds for binding to the pancreatic KATP channel with that for blocking the channel in the aorta. Considering all compounds, a significant linear correlation (r=0.817) is Table 8.1 Effects of insulinotropes on cromakalim-stimulated and basal 86Rb+ efflux from rat aorta and inhibition of 3H-glibenclamide binding to microsomes from RINm5F cells. Substance

Classa

Inhibition CRKstimulated 86Rb+ effluxb pIC50

Increase in the 3H-GBCC rate pKi constant of basal 86Rb+ efflux, ― k (%) at –log concentr. (M)

(–) AZ-DF 265 (+) AZ-DF 265 glibenclamide glibornuride gliclazide glipizide

C C B A A B

6.17±0.06 5.43±0.09 6.80±0.03 4.90±0.05 4.40±0.08 5.50±0.05

20 at 5.5 5 at 5.0 0 at 5.5 0 at 4.0 0 at 4.0 13 at 4.5

7.8d 6.1d 9.5 7.0 6.2 9.1


Substance

Classa

Inhibition CRKstimulated 86Rb+ effluxb pIC50

Increase in the 3H-GBCC rate pKi constant of basal 86Rb+ efflux, ― k (%) at –log concentr. (M)

gliquidone B 5.20±0.10 6 at 4.2 9.0 glisoxepide B 5.00±0.05 0 at 4.0 7.9 glymidine A 3.30±0.20 30 at 3.0 5.2 meglitinide C 5.30±0.05 8 at 4.3 6.6d tolbutamide A 3.50±0.30 10 at 3.0 5.0 UL-DF 9 C 4.00±0.02 0 at 3.5 5.0d aClassification as A: short (first generation), B: long (second generation) sulphonylurea; C: carboxylic acid containing compound. b pIC = negative logarithm of the midpoint (IC50, M) of the inhibition curve of CRK(3 50 μ M)-stimulated 86Rb+ efflux from rat aorta. c pK = negative logarithm of the midpoint (IC , M) of the 3H-glibenclamide (3H-GBC) i 50 displacement curve in microsomes from RINm5F cells after correction for the concentration of the radiolabel (0.3 or 0.1 nM). Values from SchmidAntomarchi et al. (1987). d M. Fosset, personal communication.


Figure 8.2 Relationship between the inhibition by insulinotropes of CRK-stimulated 86Rb + efflux from rat aorta and of 3H-glibenclamide (3H-GBC) binding to RINm5F cells. The data are taken from Table 8.1; (― ), class A compounds; (O), class B; (― ), class C. Linear correlation analysis for all compounds together (—) and for the 3 subgroups separately (…) gave the following results (n=number of compounds, s=slope, r=correlation coefficient, SL=significance level of fit). Group

n

s

r

SL

all

12

0.52±0.12

0.81

0.001

A

4

0.79±0.12

0.98

0.02

B

4

0.90±0.54

0.76

0.23

C

4

0.74±0.17

0.95

0.03

found. The slope of 0.52 in the double logarithmic correlation indicates that the changes in molecular structure affect the affinity for the pancreatic channel more than that for the vascular channel, reflecting the fact that these compounds were optimized for the channel in the β -cell. When a subgroup analysis is made, a good correlation is found for the members of class A or C, whereas the


correlation is less good for the class B compounds. This data suggests that the sulphonylurea receptor in the aorta is related to, but different from its counterpart in the insulinoma cell. Other blockers Symmetrical tetra-n-alkylammonium ions These are well known K channel blockers (see e.g. French and Shoukimas, 1981; for tetraethylammonium: Stanfield, 1983). We have investigated such compounds as inhibitors of cromakalim-induced 86Rb+ efflux from rat aorta (Quast and Webster, 1989). The inhibition curves (Figure 8.3) show that the potency of these compounds increased with chain length from the tetramethylhomologue (IC50 ― 20mM) up to the n-pentyl-homologue (IC50 ― 0.1 μ M). Increasing chain length further decreased potency; in addition, the hexyl- and heptyl-homologues at higher concentrations and the octyl-compound at all concentrations tested increased basal 86Rb+ efflux from the organ, probably by depolarizing the preparation. In order to minimize this effect, these experiments were conducted in the presence of the dihydropyridine Ca entry blocker, isradipine (0.1 μ M). Even then, the octyl-homologue no longer gave any block (Figure 8.3). The data show that the tetrabutyl- and pentylammonium compounds are potent blockers of the vascular KATP channel without affecting basal K+ exchange mechanisms. The data suggest that the tetraalkylammonium site of the KATP channel has a strong preference for bulky, lipophilic compounds with a sharp cut-off at chain length ― 7 (n-heptylammonium). The increase in blocking potency with increasing chain length of tetra-n-alkylammonium ions has also been observed for the K+ conductance of squid axon (French and Shoukimas, 1981).


Figure 8.3 Symmetrical tetraalkylammonium ions (TAA) as inhibitors of CRK (3 μ Minduced 86Rb+ efflux from rat aorta. The inhibition curves are numbered according to chain length from 1 (tetramethylammonium) to 8 (tetraoctylammonium). The curves were analyzed according to the Hill equation yielding the parameters listed in the inset; plC50= -log(IC50, M) and nH=Hill coefficient. The data were obtained as described in Figure 8.1; from the hexyl- to the octyl-compound, experiments at concentrations ― 10 μ M were performed in the presence of isradipine (0.1 μ M) in order to minimize the increase in 86Rb + efflux induced by these compounds at high concentrations.

Imidazolines, guanidines and structurally related blockers Recently, a growing number of inhibitors of the KCAs has been described which contain an imidazoline or a guanidinium group, the most prominent of them being the β -adrenergic blockers, phentolamine and antazoline and the bradycardic agent, alinidine (McPherson and Angus, 1989). In the higher micromolar range, this group of compounds produces a non-competitive inhibition of the vasorelaxant effects of CRK (McPherson and Angus, 1989; Ibbotson et al., 1993b); similar concentrations (30 μ M) completely prevent the KATP channel opening in rat portal vein cells (Ibbotson et al., 1993b). Similar data were obtained for the guanidine, guanabenz (Ibbotson et al., 1993b) and the imidazoisoindole, ciclazindol (Noack et al., 1992c). The guanidinium-related compound U-37883A, an amantadine derivative containing a carboximidine moiety, also inhibits the vasorelaxant and 42K+ efflux stimulating effects of several KCAs in the μ M range (Meisheri et al., 1993c). Interestingly, this compound, at low concentrations that do not yet inhibit the KCA-induced vasorelaxation, augments the inhibitory potency of glibenclamide and vice versa. Hence, these two compounds display a functional synergy as inhibitors of the KCAs, suggesting that they interfere with different steps of the signal chain which mediates the KCA-induced vasorelaxation (Ohrnberger et al., 1993). Blockers that inhibit KCA-induced channel opening more than vasorelaxation Several chemically unrelated molecules have been found to inhibit the K channel opening effect of the KCAs in vascular tissue without much inhibition of vasorelaxation. This was first observed with the sparteine derivative, tedisamil (Bray and Quast, 1991b; 1992b), a K channel blocker (KCB) with bradycardic and class III antiarrhythmic actions (Beatch et al., 1991); a similar profile was found with the inorganic KCBs, Rb+ (Foster et al., 1992; Greenwood and Weston, 1993), and Ba2+ (Quast and Bray, 1991; Quast et al., 1995). [Note that Rb+, which is used in μ M concentrations as a convenient substitute for 42K+ in tracer flux experiments (see section 8.4.2, above), is a general KCB at mM concentrations (Greenwood and Weston, 1993)]. It appears from these studies


that the majority of KATP channels can be blocked by the inhibitor without substantially impairing the vasorelaxant potency and efficacy of the KCA; at higher degrees of channel block, however, the KCA-induced vasorelaxation becomes transient or even disappears (Foster et al., 1992; Greenwood and Weston, 1993; Quast et al., 1994). The preferential inhibition of the KCAinduced channel opening by these compounds is in sharp contrast with the behaviour of glibenclamide, which inhibits both the K channel opening and the vasorelaxant effects of the KCAs at similar concentrations (Quast and Cook, 1989b; note that due to the quasi-competitive inhibition pattern of glibenclamide shown in Figure 8.1, similar KCA-concentrations are used to measure channel opening and relaxation). Taken together, these data suggest that the relationship between the channel opening and vasorelaxant effects of the KCAs is not as simple as originally thought (see below, 8.6). 8.5 Vasorelaxant Properties of the KATP Channel Activators The KCAs are potent vasorelaxants with a quite characteristic profile (Longman and Hamilton, 1992; Cavero and Guillon, 1993; Cook and Quast, 1990; Edwards and Weston, 1990b; Quast, 1993). First, their vasorelaxant effect is abolished in media containing high (>50 mM) concentrations of K+ (Hamilton et al., 1986). This is a direct consequence of their mechanism of action since depolarization by a high K+ solution results in a shift in the membrane potential towards the K+ equilibrium potential (see e.g. Furukawa et al., 1981) and the opening of additional K channels by the KCA will not significantly affect membrane potential. Figure 8.4 examines the ability of LCRK to relax rat aortic rings precontracted by different concentrations of KCl in the bath. Considering only the data up to 10 μ M, it is seen that with increasing KCl the amount of tension that can be relaxed by LCRK diminishes and is almost 0 at 55 mM KCl; in addition there is a gradual rightward shift of the relaxation curve. Thus, KCl is a non-competitive inhibitor of the vasorelaxant effect of LCRK, which is entirely expected from the changes in membrane potential and of the K+ equilibrium potential with KCl concentration, and from the known voltage-dependence of the L-type Ca channels (Hille, 1992; Furukawa et al., 1981; Häusler, 1983). At high concentrations of LCRK (>10 μ M), there is another component of relaxation which we have not characterized further and which may be nonspecific (Figure 8.4). The KCAs also relax contractions to agonists which mobilize intracellular Ca2 + via the inositoltrisphosphate (IP ) pathway (Cook et al., 1988b; reviews: 3 Edwards et al., 1992; Quast 1993) with a potency approximately similar to that against low concentrations of KCl (for LCRK compare Table 8.2 and Figure 8.4). We have


Table 8.2 Comparison of the vasorelaxant potency of KATP channel activators under polarized and depolarized conditions in rat aortic ringsa Substance

Polarized EC50, (μ M)

Depolarized EC50, (μ M)

Selectivity ratio

levcromakalim 0.03 180 6000 (+)enantiomer 6.3 >1000 >160 SDZ PCO 400 0.04 16 400 (+)enantiomer 16 >300 >19 pinacidil (racemate) 0.06 22 367 (–)enantiomer 0.04 13 310 (+)enantiomer 0.32 39 122 P1075 0.008 >100 10000 diazoxide 7 >250 >36 RP 49356 0.2 >100 >500 a Rat aortic rings were contracted by noradrenaline (0.1 μ M) under polarized (5 mM KCl) or depolarized conditions (55 mM KC1, NaCl reduced from 120 to 70 mM, in the presence of the dihydropyridine Ca2+ blocker, isradipine (0.1 μ M) to inhibit KCl-induced tension (Cook et al., 1988b)). Concentration-relaxation curves were obtained cumulatively; maximum tension was similar under either condition (1.0 to 1.2 g). EC50=midpoint of concentration relaxation curve; Selectivity ratio=EC50 (depolarized)/EC50 (polarized).


Figure 8.4 Relaxation of KCI-induced contractions of rat aorta by LCRK. The concentration-relaxation curves were obtained cumulatively in a buffer where NaCl was reduced from 120 mM as KCI was increased above 5 mM. The data were subjected to Hill analysis (for two components where necessary). The maximum tension (Fmax) and the parameters of the high affinity component are listed in the following table (A, maximum relaxation in % of Fmax; EC50, midpoint of relaxation curve in nM; nH, Hill slope). KCI (mM)

Fmax (mg)

A (%)

EC50 (nM)

nH

20 700 ± 150 94 ± 2 14 ± 1 1.5 ± 0.1 25 1310 ± 100 96 ± 2 28 ± 2 1.5 ± 0.1 30 1600 ± 210 81± 1 70 ± 2 1.8 ± 0.1 35 1650 ± 106 36 ± 1 69 ± 1 1.9 ± 0.1 40 1750 ± 114 16 ± 1 123 ± 7 2.3 ± 0.3 55 1800 ± 200 6±1 130 ± 100 2±1 Analysis of the low affinity component at 35 and 55 mM KCI gave the following parameters: A (%) = 50 ± 1, 87 ± 2; EC50 (μ M) = 229 ± 2, 272 ± 8; nH = 2.0 ± 0.1, 1.7 ± 0.1.

compared the potency of some KCAs against noradrenaline under normal (5 mM KCI) and depolarized (55 mM KCI) conditions, the latter in the presence of the Ca antagonist, isradipine (0.1 μ M), in order to have comparable maximum


tension in the absence of KCA. The results are listed in Table 8.2 and allow the calculation of a selectivity ratio which indicates the concentration range over which the KCA acts solely by the K channel opening mechanism. This selectivity is lowest for pinacidil and its enantiomers which have repeatedly been shown to have vasorelaxant mechanisms in addition to KATP channel activation (Cook and Quast, 1990; Xiong et al., 1991; Itoh et al., 1991). A further characteristic trait is the inhibition of the KCA-induced vasorelaxation by the sulphonylureas, in particular glibenclamide (see above). The fact that glibenclamide acts with similar potency against structurally quite diverse KCAs (see e.g. Meisheri et al., 1993b) provides additional support for the conclusion from binding studies (Bray and Quast, 1992a) and electrophysiological investigations (Ibbotson et al., 1993a) that these compounds have a common pathway leading to KATP channel activation and vasorelaxation. 8.6 Mechanism of KATP Channel Activator-induced Vasorelaxation The mechanisms by which the KCAs induce vasorelaxation have been reviewed recently (Quast, 1993). The existing evidence suggests that these compounds relax vascular smooth muscle primarily by opening K channels. The ensuing hyperpolarization clamps the cell at sufficiently negative values to prevent depolarization-induced Ca2+ entry by voltage-sensitive Ca channels (Hamilton et al., 1986). In this respect the KCAs may be looked at as indirect Ca channel blockers. In addition, the KCAs have recently been shown to inhibit the agonistinduced accumulation of IP3 and thus to interfere with agonist-stimulated Ca2+ mobilization from intracellular stores (Ito et al., 1991; Itoh et al., 1992; Yamagishi et al., 1992); they also reduce the intracellular Ca2+ concentration in vascular smooth muscle at rest in both Ca2+ -containing and Ca2+ -free media (Ito et al., 1991; Itoh et al., 1992; Yamagishi et al., 1992) and decrease the Ca2+ -sensitivity of the contractile elements in strips of canine coronary artery (Okada et al., 1992; 1993). All these effects are abolished in depolarizing medium and reversed by glibenclamide suggesting that they are somehow linked to the ability of the KCAs to hyperpolarize the cell membrane by opening KATP channels in the plasmalemma. The precise mechanism by which hyperpolarization of the cell membrane regulates these phenomena remains to be established; however, in recent investigations in isolated cells from guinea-pig coronary artery it was clearly demonstrated that membrane potential modulates the acetylcholineinduced Ca2+ transient, and indirect but strong evidence suggested that this was due to a modulation of acetylcholine-stimulated IP3 liberation (Ganitkevitch and Isenberg, 1993). The ability of the KCA to inhibit both the agonist-induced mobilization of Ca2+ from intracellular stores and the increase in Ca2+ sensitivity of the contractile apparatus predicts that the vasodilator profile of these compounds will differ from that of the Ca2+ antagonists and explains earlier


findings that the KCAs relax contractions which do not depend on depolarizationinduced Ca2+ entry (Cook et al., 1988b; Quast and Baumlin, 1991, further references in Quast, 1993). The studies with the KCBs tedisamil Ba2+ and Rb+ have demonstrated that the large majority of the K channels opened by the KCAs can be blocked without much effect on vasodilatation (see section 8.4.3). Despite the fact that these compounds are relatively nonselective KCBs and have many additional actions (see references above), these studies strongly suggest that LCRK and other KCAs, but minoxidil sulphate to a lesser degree (Bray and Quast, 1992b; Greenwood and Weston, 1993) possess mechanisms of vasorelaxation, independent of plasmalemmal KATP channel opening. The characteristic profile of the KCA-induced vasorelaxation requires these mechanisms to be abolished by depolarization and to be sensitive to inhibition by glibenclamide. One such mechanism is the (partial) inhibition of intracellular Ca2+ store refilling, produced by LCRK in vascular tissues (Cowlrick et al., 1988; Bray et al., 1991; Greenwood and Weston, 1993). In this context it is important to remember that the mode of channel activation by the KCAs is not yet elucidated (see section 8.4.1 above). If the KCAs, by binding to their target, are able to interfere with phosphorylation phenomena (Edwards et al., 1993), this could open multiple pathways leading to vasorelaxation, the most important of them being the opening of KATP channels. Such an indirect mechanism of action could also resolve the paradoxical observation that the concentrations of LCRK which induce 50% vasorelaxation are generally three to five times lower than those required to detect the direct manifestations of K channel opening (see Quast, 1993, for references). 8.7 BKCa Channel Activators As mentioned in the Introduction, the BKCa channels provide an important negative feedback mechanism between membrane potential and [Ca2+]i in many excitable cells. The availability of high affinity toxins which block BKCa channels, e.g. charybdotoxin and iberiotoxin, has helped to recognize the fundamental role that these channels play in many tissues (review: Garcia and Kaczorowski, 1992); in arterioles, they are major regulators of tone (Brayden and Nelson, 1992). In addition to the gating of these channels by depolarization the activity of some BKCa channels is modulated by cAMP-dependent phosphorylation (Kume et al., 1989; Sadoshima et al., 1988), G-proteins (Gsβ ; Scornik et al., 1993), the guanine nucleotides GMP and cGMP (Williams et al., 1988) and other factors (review: Armstrong and White, 1992). In view of the physiological role of BKCa channels, tissue-selective openers of BKCa channels can be expected to be of great therapeutic interest. Recently, three classes of molecules have been described that act as activators of BKCa channels. The imidazopyrazine SCA40 has been shown to completely relax rings from


guinea-pig trachea (Laurent et al., 1993) and rat aorta (Michel et al., 1993) contracted by 20 mM KCl, but was a much weaker and less efficacious relaxant against contractions to 80 mM; the relaxant activity in guinea-pig trachealis against 1 μ M carbamylcholine was competitively inhibited by charybdotoxin (Laurent et al., 1993). This is indeed the pharmacological profile expected for a BKCa channel activator; however, this data can also be explained on the basis of functional antagonism (Huang et al., 1993; Cook et al., 1995) and definite proof of the proposed mechanism of action has to come from electrophysiological experiments. In guinea-pig trachea SCA40 caused full suppression of spontaneous tone without a change in membrane potential and it was concluded that K channel opening was not important in this tissue (Cook et al., 1995). The situation is the opposite with the benzimidazolones, NS004 (Olesen et al., 1993;1994a) and NS1619 (Olesen et al., 1994b). Electrophysiological data clearly show that these compounds, at micromolar concentrations, are effective activators of BKCa channels in a variety of cell types (bovine aorta and coronary artery; mouse cerebellar granule cell and cortical neuron; rat pancreatic β -cell). NS1619 activates the channel in bovine aorta by shifting the voltage-activation curve towards more negative values, i.e. it mimicks the effect of an increased [Ca2+]i, but it cannot activate the channel in the virtual absence of Ca2+ (Olesen et al., 1994b). However, in rat aorta, NS004 relaxed contractions to 110 mM KCl with a similar potency (― 10 μ M) as those to phenylephrine (0.3 μ M); the relaxation was not inhibited by iberiotoxin (50 and 200 nM) (Sargent et al., 1993). This rules out a significant contribution of BKCa channel activation to the observed vasorelaxation which may be due, at least in part to the inhibition of (Ltype-) Ca channels produced by NS004 at μ M concentrations (Sargent et al., 1993; Olesen et al., 1994a). However, the complete relaxation of phenylephrineinduced contractions of rat aorta (note that these depend largely on the mobilization of intracellular Ca2+) requires the presence of a vasorelaxant mechanism other than Ca channel inhibition; but as stated above, this mechanism cannot be an opening of K channels. Most interestingly, NS004, at submicromolar concentration, is able to activate the cystic fibrosis transmembrane transport regulator (CFTR), even in its mutated form (― F508; this mutation causes the most prevalent form of the disease) (Gribkoff et al., 1994). This effect, the mechanism of which needs further clarification and which is probably unrelated to the aforementioned properties of the molecule, offers exciting therapeutic possibilities (Gribkoff et al., 1994). A third class of exogenous compounds which have been shown to activate BKCa channels are the soyasaponins (McManus et al., 1993). These compounds, isolated from a medicinal herb which is used in Ghana as a spasmolytic in the treatment of asthma, activate the channel from the inside of the cell in the presence of Ca2+ and inhibit charybdotoxin binding by a negative allosteric interaction. Some of the compounds act at quite low concentrations (from 10 nM on); however, their poor membrane permeation limits their therapeutic use


(McManus et al., 1993) and obviously prevents a direct assessment of these compounds in functional tests. 8.8 Conclusion In recent years there has been considerable progress in the field of the KATP channel activators, in particular the long standing controversy as to which channel is the principal mediator of the effects of the KCAs in smooth muscle has been settled in favour of a KATP channel of ― 20 pS conductance. The KATP channel has eluded cloning and expression efforts up to now but, undoubtedly, this problem will be overcome. This will probably also solve the question of how the KCAs activate the channel, whether by a direct or an indirect mechanism. If the existing KCAs have not yet fully lived up to the original therapeutic expectations (often due to limited tissue specifity) they have been important tools in examining the effects of hyperpolarization on smooth muscle. The emerging field of BKCa channel activators which is again pioneered by the pharmaceutical industry deserves greatest attention. The activators already available leave ample space for improvement; hopefully compounds will be found in the area of the BKCa channel activators which serve both the clinician and the pharmacologist. Acknowledgement I am grateful to Dr Michel Fosset, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice-Sophia Antipolis, for determining the binding affinity of some sulphonylureas. Own unpublished work reported here was performed at Sandoz Pharma, Basel, Switzerland with the excellent technical assistance of Y.Baumlin and J.Dosogne. I thank C.Linde, C.Löffler and F.Metzger (Tübingen) for artwork and help with the manuscript. References ARMSTRONG, D.L. & WHITE, R.E. (1992) Trends Neurosci., 15, 403–408. ASHCROFT, S.J.H. & ASHCROFT., F.M. (1990) Cell. Signal., 2, 197–214. ATKINSON, N.S., ROBERTSON, G.A. & GANETZKY, B. (1991) Science, 253, 551–555. ATWAL, K.S. (1992) Med. Res. Rev., 12, 569–591. BEATCH, G.N., ABRAHAM, S., MCLEOD, B.A., WALKER, M.J.A. & YOSHIDA, N.R. (1991) Br. J. Pharmacol, 102, 13–18. BEECH, D.J. & BOLTON, T.B. (1989) Br. J. Pharmacol., 98, 851–864. BEECH, D.J., ZHANG, H., NAKAO, K. & BOLTON, T.B. (1993a) Br. J. Pharmacol., 110, 573–582. (1993b) Br.J. Pharmacol., 110, 583–590. BERNARDI, H., DE WEILLE, J.R., EPELBAUM, J., MOURRE, C., AMOROSO, S., SLAMA, A., FOSSET, M. & LAZDUNSKI, M. (1993) Proc. Natl. Acad. Sci. USA, 90, 1340–1344.


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9 In Vivo Vascular Effects of Potassium Channel Activators J.C.CLAPHAM Department of Vascular Biology, SmithKline Beecham Pharmaceutical, The Frythe, Welwyn, Hertfordshire, AL6 9AR, UK. 9.1 Introduction This review will address the vascular changes that occur in whole animals following activation of ATP-sensitive potassium channels KATP by the potassium channel activators (KCAs). The molecular mechanisms involved have already been discussed in Chapters 6 to 8. Indeed, there have already been many comprehensive reviews on the KCAs and some have included the activity of these agents in vivo (Richer et al., 1990b; Edwards et al., 1992; Longman and Hamilton, 1992). Whilst previous reviews have tended to cover the broader issues, I intend to restrain this chapter to the purely vascular aspects of KCAs in both anaesthetised and conscious animal models. 9.2 Acute Blood Pressure Studies Studies on the antihypertensive effects of KCAs have made extensive use of the spontaneously hypertensive rat (SHR; Table 9.1). These drugs were also very effective blood pressure (BP) lowering agents in other models of hypertension such as the deoxycorticosteroid (DOCA)/NaCl rat (Aloup et al., 1990; Morin et al., 1990), the Goldblatt (renal) rat (Aloup et al., 1990; Morin et al., 1990) and the Dahl salt-sensitive rat (Hirawa et al., 1989, 1992). Efficacy was also demonstrated in models of renal hypertension in non-rodent species such as the cat (Clapham et al., 1991a) and dog (Schliep et al, 1989; Clapham et al., 1991a; Nakajima et al., 1992). The ability of the KCAs to reduce arterial pressure in several species is now well established (Buckingham, 1989; Edwards et al., 1992; Longman and Hamilton, 1992) and accepted as being the principal measurable response in an animal. Despite this apparently universal BP lowering effect, differences in time course have emerged with the introduction of more compounds of this class (Table 9.1). In the SHR, aprikalim (RP49356, Aloup et al., 1990), levcromakalim

IN VIVO VASCULAR EFFECTS OF KCAS 243

(LCRK) (Clapham et al., 1991a), NIP-121 (Masuda et al., 1991) and HOE 234 (Linz et al., 1992), for example, elicit acute antihypertensive responses that are rapid in onset, their maximum effect Table 9.1 Oral antihypertensive effects of some KCAs in conscious SHR KCA

Dose-range mg.kg–1

HR

Time to max

Duration

Ref

HOE-234 0.1–1.0 20 min ~3h [1] bimikalim 0.0361 * * ~4h [2] levcromakalim 0.038–0.15 20 min ~4h [3] NIP–I21 0.013–0.05 1.5h ~4h [4] SDZ PCO400 0.1–0.3 5h [5] Ro 31–6930 0.01–0.1 8h [6] 1 celikalim 0.25 2–6 h ~24 h [7] Y–27152 0.1–0.3 7h ~24 h [8] aprikalim 0.1–1.0 * 5h [9] 1 Indicates dose calculated to produce a 30 mmHg reduction in arterial pressure. * data not given,

occurring 20 min following an oral dose. Thereafter, BP returns towards control levels with the effect lasting around 3 h. Ro 31–6930 (Paciorek et al., 1990) exhibits a similarly rapid onset of action, but the antihypertensive response persists for up to 8 h. However, at least two new KCAs have emerged with strikingly different profiles in the SHR. Celikalim (WAY–120,491; Morin et al., 1990) and Y–27152 (Nakajima et al., 1992) have a very slow onset of action, taking up to 7 h to reach a maximum fall in BP with a duration of action claimed to be 24 h. It appears, from one study at least, that the duration of action of the benzopyranrelated KCAs in SHR is related to the intrinsic lipophilicity of these compounds (Soll et al., 1991). These authors demonstrated that a long duration of action was associated with increased lipophilicity, regardless of potency, in a study comparing cromakalim (CRK), LCRK, EMD–52692, Ro 31–6930, SDZPCO 400, NIP–121, celikalim and a number of analogues of celikalim. However, the slow onset of action of Y–27152 was related to the pro-drug nature of this compound (Nakajima et al., 1992). Y–27152, which is inactive in vitro, is converted in vivo to the active desbenzyl form, Y–26763, by a cytochrome P450dependent mechanism (Nakajima et al., 1992). In contrast, the antihypertensive effects of LCRK are mediated by unchanged parent compound (Clapham et al., 1993).


In non-rodent models of hypertension the duration of action of the antihypertensive response to LCRK was slightly prolonged (~5 h) in the renal hypertensive cat and dog (Clapham et al., 1991a) such that the duration in these models is similar to that of Ro 31–6930 (Paciorek et al., 1990). In contrast, the difference in the duration of action between LCRK and Y–27152 is maintained in the conscious hypertensive dog (Nakajima et al., 1992). 9.3 Chronic Blood Pressure Studies The majority of the published haemodynamic data on KCAs report their acute actions. In addition there are a number of studies where antihypertensive effects have been monitored after repeat dosing. In the studies listed in Table 9.2 there were no signs of tolerance to the antihypertensive effects of these KCAs. Since the dosing regimen used in these studies was once daily dosing, lack of tolerance may not be altogether surprising. The short duration of action in SHR of some of the compounds could reflect the sum of a number of acute challenges. However, reproducible antihypertensive responses were obtained for the longer acting KCAs such as Ro 31–6930 (Paciorek et al., 1990) and Y–27152 (Nakajima et al., 1992) and in Dahl salt-sensitive rats with LCRK (Hirawa et al., 1989). In addition, an antihypertensive response was still evident in SHR exposed to dietary incorporation of LCRK (resulting in a drug intake of around 0.35 mg.kg– 1day–1) for up to 6 weeks (Hamilton et al., 1993). Thus, there is no evidence that tolerance develops to the antihypertensive activity induced by KATP -channel activation in animal models of hypertension. When administered to young SHR (4 weeks of age), the KCAs CRK and bimikalim (EMD52692 or SR44866), fail to prevent the development of genetic hypertension despite exhibiting favourable haemodynamic effects (Mulder et al., 1989). In the Dahl-salt sensitive rat, LCRK reduces arterial pressure concomitant with a reduction in aortic thromboxane A2 (TXA2) formation both in vivo and in vitro (Hirawa et al., 1992). Furthermore, since TXA2 may play a role in smooth muscle cell growth and proliferation (Ishimitsu et al., 1988), this would give a theoretically favourable effect with respect to vascular hypertrophy. However, studies in this area are relatively few and more will be required to establish a consensus as to whether KCAs have real potential in vascular remodelling. When the KCAs bimikalim and CRK (Mulder et al., 1989) and LCRK (Clapham et al., 1991b) are administered once daily to SHR, heart weight/body weight ratio remains unchanged indicating that the compounds do not reverse cardiac hypertrophy in the SHR. Furthermore, these data are in agreement with earlier work where pinacidil failed to reverse cardiac hypertrophy in the SHR despite an aggressive BP lowering regimen (Jespersen et al., 1986). On the other hand, there was no induction of further work-related cardiac hypertrophy.


Table 9.2 Repeat oral dose studies of some KCAs in conscious hypertensive animals KCA

Dose mg.kg-1 day-1

Species

Duration days

Tolerance

Ref

cromakalim 0.2 RHD 28 no [1] HOE-234 2.0 SHR 5 no [2] levcromakal 0.05 & 0.1 SHR 28 no [3] im levcromakal 0.5 DAHL 14 no [4] im levcromakal 6 ppm in SHR 42 no [5] im diet1 NIP– 121 0.025–0.1 SHR 15 no [6] SDZ 0.1 SHR 4 no [7] PCO400 Ro 31–6930 0.03 SHR 22 no [8] Y–27152 0.1 RHD 56 no [9] 1 Equivalent to approximately 0.35 mg.kg-l day-l , ppm = parts per million [1] Buckingham et al., 1986; [2] Linz et al., 1992; [3] Clapham et al., 1991b; [4] Hirawa et al., 1989; [5] Hamilton et al., 1993; [6] Masuda et al., 1991; [7] Fozard et al., 1990; [8] Paciorek et al., 1990; [9] Nakajima et al., 1992.

9.4 Heart Rate and Plasma Renin Activity KCA-induced reductions in arterial pressure were accompanied almost invariably by tachycardia, with the possible exception of Y–27152 (Nakajima et al., 1992) which has an extremely slow onset of action which may account for the lack of tachycardia (Nakajima et al., 1992). However, tachycardia induced by KCAs is sensitive to β -adrenoceptor blockade (Clapham et al., 1991a; Masuda et al., 1991) indicative of a reflex rise in sympathetic nervous system activity in response to a reduction in total peripheral resistance (TPR). In addition, when admixed with diet, where it is assumed that drug intake occurs over time (Hamilton et al., 1993), reflex elevations in heart rate following LCRK are attenuated. This lends support to the explanation of Nakajima et al. (1992) that a slow onset of action would lead to a reduced involvement of counter regulatory mechanisms. Reflex activation of the sympathetic nervous system may also give rise to an increase in plasma renin activity (PRA) and this was shown, for example, in the renal hypertensive cat, SHR and the conscious rhesus monkey (Fozard et al., 1990; Clapham et al., 1991a; Clapham et al., 1991b). As with heart rate, Y–


27152 did not increase plasma renin activity in the renal hypertensive dog following repeat administration (Nakajima et al., 1992). Suppression by propranolol of these counter regulatory responses to a KCAinduced reduction in arterial pressure increases the duration and magnitude of the antihypertensive response (Clapham et al., 1991a). Furthermore, in the Dahl saltsensitive rat, which has suppressed reactivity of the renin-angiotensin system, blood pressure lowering activity of LCRK was still evident 24 h post-dose (Hirawa et al., 1989). 9.5 The Venous System There are relatively few reports describing the potential venodilator effects of KCAs in whole animal models. In the anaesthetised rabbit or cat (Clapham and Buckingham, 1988; Hof et al., 1988; Clapham et al., 1991a), CRK or LCRK have no effect on, or increase, central venous pressure when TPR was reduced by these drugs. In the anaesthetised dog, cromakalim and pinacidil lower BP together with a concomitant increase in total venous return and pulmonary artery blood flow (Gotanda et al., 1989; Kaneta et al., 1993). Together these data imply that these drugs do not increase venous capacitance via a venodilator action, but act solely on the arterial side of the circulation. Indeed, direct observation of skeletal muscle microcirculation by intravital microscopy in SHR (StruykerBoudier et al., 1992) reveals that at BP lowering doses, LCRK dilated arterioles with very little effect on venules (see Figure 9.3 for example). By contrast, intravenous, but not intracoronary, administration of bimikalim to anaesthetised pigs results in a dose-related reduction in left ventricular end diastolic pressure that is independent of heart rate (Sassen et al., 1990). Although not measured directly, these data suggest that bimikalim possesses some venodilating property. KCAs possessing a nitrate moiety, however, might be expected to elicit a venodilator effect, since this is recognised as the primary mode of action of nitrovasodilators in vivo. Indeed, Kaneta et al. (1993) have shown that nicorandil and nitroglycerine elicit qualitatively similar effects on venous haemodynamics in the anaesthetised dog. These workers showed also that the nitrate containing KCA, KRN2391, appeared to have a bimodal effect on venous haemodynamics. First, KRN2391 increased venous return in a similar way to CRK. This was followed by a nitrate-like action where KRN2391 decreased venous return via an increase in venous capacitance. The sulphonylurea KATP channel blocker, glibenclamide, abolished the initial CRK-like effect on KATP channels unmasking a purely nitrate-like action of KRN2391 (Kaneta et al., 1993).


9.6 Regional Haemodynamics The mechanism by which KCAs lower BP in animal models is without doubt due to a reduction in total peripheral resistance resulting from arteriolar vasodilatation. The study of the contribution of individual vascular beds to the overall reduction in total peripheral resistance, however, has been carried out largely in anaesthetised animals using flow-probe or microsphere technology. Recently, data have begun to emerge from conscious animal studies that are beginning to strengthen our knowledge of the haemodynamic profile of KCAs (see Figure 9.1). Thus, there is a consensus view regarding the effects of KCAs on the coronary and splanchnic vascular beds but data from the renal vasculature remain equivocal. On the other hand, data on the pulmonary circulation are not so abundant and further evaluation, particularly in the area of hypoxic pulmonary constriction and pulmonary hypertension, is still required. 9.6.1 The Cerebral Circulation Intravenous administration of CRK, LCRK or bimikalim increases cerebral blood flow, when measured by radioactive microspheres or [14C]-iodoantipyrine, in normotensive rabbits, rats and pigs (Hof et al., 1988; Schliep et al., 1989; Sassen et al., 1990). However, there is no increase in radioactive microspheres in the brain following oral administration of CRK in SHR (Shoji et al., 1990). In a study where stroke-prone SHR and their normotensive counterpart, the Wistar Kyoto (WKY) rat, were compared, aprikalim increased basilar artery diameter in the WKY rat but not in the stroke-prone SHR (Kitazono et al., 1993). The effect in WKY rats was glibenclamide-sensitive and it is possible that KATP channel function/density is diminished in the cerebral vasculature of genetically hypertensive rats. In a study comparing non-diabetic and streptozotocin-diabetic rats, aprikalim dilated pial arterioles in a glibenclamide-sensitive manner in nondiabetic rats (Mayhan and Faraci, 1993). However, a slight vasoconstriction occurred in the pial arterioles in streptozotocin-diabetic rats following aprikalim, nitroglycerine was effective equally in both non-diabetic and streptozotocindiabetic rats. Thus, it appears that not only can KATP channel function in cerebral blood vessels be impaired by both hypertension and streptozotocin-diabetes, but that dilatation of cerebral blood vessels does not always occur in anaesthetised normotensive animals in response to KCAs. It is possible that KATP channels are not widely distributed throughout cerebral blood vessels. Indeed, it has been reported that rat brain microvessels of the cerebral cortex lack specific [3H]glibenclamide binding sites implying a lack of KATP channels in this vessel type (Sullivan and Harik, 1993).


Figure 9.1 Haemodynamic profile of levcromakalim (8.7 nmol.kg–1 iv) in conscious, instrumented dogs. (Reproduced with permission from Shen and Vatner, 1993).

Despite these findings however, KCAs may still have a potentially beneficial effect in cerebral ischaemia. For example intracerebroventricular administration


of LCRK, pinacidil and nicorandil (10 nmol.5 μ l–1) blocked ischaemia-induced expression of the immediate early genes, c-fos and c-jun and of the mRNAs for the 70 kDa heat shock protein and amyloid β -protein precursor genes in the rat (Heurteaux et al., 1993). In addition, these drugs protected against neuronal death and all of the anti-ischaemic events reported were sulphonylurea-sensitive indicating an involvement of KATP channels. 9.6.2 Coronary Circulation KCAs representing most chemical classes elicit marked increases in coronary blood flow (diazoxide, Rubin et al., 1962; nicorandil, Sakai et al., 1981; Sakai et al., 1983; pinacidil, Arrigoni-Martelli and Finucane, 1985; aprikalim, Aloup et al., 1990; LCRK, Clapham et al., 1991a; YM934, Uchida et al., 1994; RWJ29009, Damiano et al., 1994). Indeed, in those studies where coronary blood flow was measured in animals instrumented for full haemodynamic profiling, KCA-induced reductions in coronary vascular resistance (–52%) are much greater than reductions in resistances in other vascular beds (–17 to –28%) (Shen and Vatner, 1993; Figure 9.1). This exaggerated effect of KCAs on the coronary circulation may reflect a greater density of KATP channels in this tissue but also indicates that KATP channel mechanisms are probably of major importance in the coronary vasculature (Duncker et al., 1993). Increases in coronary blood flow in response to KCAs are not simply a consequence of metabolic vasodilatation due to the demands of increased heart rate. For example, myocardial oxygen consumption in response to coronary vasodilatation in anaesthetised dogs is unchanged by pinacidil (Dubé and Greenfield, 1991) and RWJ29009 (Damiano et al., 1994). Furthermore, coronary vasodilatation is still evident following pinacidil or LCRK when heart rate changes are suppressed by propranolol (Giudicelli et al., 1990; Drieu la Rochelle et al., 1992; Shen and Vatner, 1993) in anaesthetised or conscious dogs. Despite the apparently exaggerated response of the coronary vasculature to KCAs compared to other vascular beds (Shen and Vatner, 1993), the coronary vasodilator effects of KCAs are also glibenclamide-sensitive (Cavero et al., 1991) confirming that an action at KATP channels underlies this effect. Interestingly, the coronary vasodilator activities of nicorandil and KRN2391, that have additional nitrate-like activity producing stimulation of soluble guanylate cyclase, (Holzmann, 1983; Kashiwabara et al., 1991), were antagonised by glibenclamide, and not methylene blue (Cavero et al., 1991; Ogawa et al., 1992), suggesting that even with vasodilators of mixed character, expression of the KATP channel effects is the overriding mechanism dilating coronary resistance vessels. Furthermore, KRN2391, despite its nitrate moiety, did not induce acute tolerance, either to itself or to isosorbide dinitrate or nitroglycerine (Kaneta et al., 1992). Dilation of large coronary vessels by these compounds however is more complex (see below). The evidence that KATP


channel modulation and control of coronary blood flow are intimately linked is further strengthened by evidence that glibenclamide, per se, reduces basal coronary blood flow in the anaesthetised dog (Imamura et al., 1992). It has recently emerged that the effects of the KCAs, CRK and pinacidil, are not uniform throughout the coronary vascular bed. Both KCAs induced a dosedependent reduction in coronary vascular resistance and an increase in diameter of large coronary arteries in conscious dogs, a response unaffected by propranolol (Giudicelli et al., 1990). This contrasts with the effects of nitroglycerine, that is, dilatation of the large coronary arteries only. Furthermore, when blood flow through the large coronary artery is held constant, the coronary dilator effects of CRK and pinacidil, but not nitroglycerine, are markedly reduced (but not abolished). Thus, it appears that in the dog, KCAs may dilate the coronary vascular bed by both direct (resistance vessels) and indirect, flow-dependent, (large vessels) mechanisms. This flow-dependent coronary vasodilator response to CRK and pinacidil appears to be endothelium-dependent (Drieu la Rochelle et al., 1992). In contrast, however, the relaxant effects of CRK are not endotheliumdependent in rat isolated aorta (Taylor et al., 1988) and dog isolated coronary artery (Drieu la Rochelle et al., 1992). This discrepancy is attributed to a dilution effect in the large coronary artery in vivo thereby preventing CRK and pinacidil from reaching the required local concentration to elicit a pharmacological effect. Nicorandil, like CRK and pinacidil, dilated both large and small coronary arteries in the same model but, unlike CRK and pinacidil, nicorandil-induced coronary vasodilatation was not affected when flow was restricted (Berdeaux et al., 1992) and was therefore exhibiting both KCA- and nitrate-like actions. As discussed, KCA-induced increases in coronary blood flow, with concomitant reductions in coronary vascular resistance, are readily demonstrated by standard flow probe techniques. When blood flow distribution across the ventricular wall is measured by microspheres, interpretation of data is more equivocal. For example, Bache et al. (1990a) showed that low doses of pinacidil produced uniform increases in blood flow across the left ventricular wall thereby maintaining the subendocardial/ subepicardial blood flow ratio in normal hearts. When the dose of pinacidil was raised, a further increase in blood flow to the outer, subepicardial layer, with no change to the inner, subendocardial layer resulted in an apparent alteration in the ratio of blood flow distribution within the myocardium. However, absolute blood flow to both regions of the myocardium remained greater than control throughout the experiment such that there was no underperfusion. However, in order to model occlusive coronary artery disease, a severe coronary artery stenosis to restrict blood flow to ~30% above basal, was performed by Bache et al. (1990a) in the anaesthetised dog. They reported that those doses of pinacidil eliciting favourable effects in normal hearts, increased subepicardial perfusion at the expense of subendocardial perfusions; though when a moderate stenosis (flow restricted to ~60% above basal) was applied blood flow to all layers was greater than control after pinacidil. In another study by the same group (Bache et al., 1990b), in dogs with a chronically overloaded


hypertrophied left ventricle, pinacidil reduced the subendocardial/subepicardial blood flow ratio from 1.06 to ~0.8. This again implied that there was a diversion of blood away from the subendocardial layers. However, absolute values of blood flow to the subendocardium following pinacidil were substantially (139%) greater than control values and, as with the previous study (Bache et al., 1990a), pinacidil did not actually underperfuse the inner layers of the hypertrophied ventricle. Further studies are clearly required in this area to determine the effects of other KCAs on the distribution of blood flow across the ventricular wall. In addition, existing studies have tended to reflect the acute effects of KCAs. Thus, investigation of chronic administration of these drugs on blood flow distribution within the ventricular myocardium would be extremely useful. 9.6.3 Splanchnic Circulation In general, KCAs increase blood flow and reduce vascular resistance through the mesenteric artery. Thus, in the anaesthetised rat, aprikalim, CRK, Ro 31–6930 and SDZ PCO 400 reduce mesenteric vascular resistance (Cavero et al., 1989; Aloup et al., 1990; Duty et al., 1990; Fozard et al., 1990). Where measured, the reduction in mesenteric vascular resistance closely reflect reductions in systemic vascular resistance and arterial BP (Cavero et al., 1989; Aloup et al., 1990). These findings are also seen in conscious animals where LCRK elicits a doserelated increase in mesenteric blood flow in Long Evans rats (Gardiner et al., 1991). Similar results have also been shown for CRK, Ro 31–6930, KRN2391 and nicorandil in anaesthetised (Paciorek et al., 1990; Ogawa et al., 1993a) and conscious (Shen and Vatner, 1993) dogs. Where microspheres have been used to measure regional haemodynamics however, the data are not as clear. In the anaesthetised pig, bimikalim elicited doserelated increases in blood flow to the small intestine whereas stomach blood flow was increased only at the highest dose used (Sassen et al., 1990). In the anaesthetised rabbit, CRK and LCRK increase blood flow markedly to both the stomach and small intestine (Hof et al., 1988) while in conscious SHR (Shoji et al., 1990) CRK has no effect on small intestine blood flow whilst increasing stomach blood flow by 78%. These differences may reflect species differences, as suggested by Richer et al. (1990b). They may also reflect possible differences in time-course between responses of each vascular bed since the microsphere technique reflects a ‘snap-shot’ at a single time point. However, in conscious dogs (Shen and Vatner, 1993; Figure 9.1) and anaesthetised cats (Clapham and Gentry, unpublished data), LCRK increases coeliac and mesenteric blood flow implying that both stomach and small intestine receive an increased blood flow.


9.6.4 Renal Circulation When the haemodynamic effects of KCAs were reviewed previously (Buckingham, 1989; Richer et al., 1990b; Edwards et al., 1992) there was no real agreement expressed about the renal haemodynamic effects of these drugs. In our laboratory we have observed that CRK increases renal blood flow in both anaesthetised and conscious cats (Clapham and Buckingham, 1988; Clapham and Longman, 1989) and others have demonstrated increases in renal blood flow in anaesthetised dogs (Dumez et al., 1988). However, these effects are not seen in anaesthetised rats or rabbits (Hof et al., 1988; Shoji et al., 1990) and, in anaesthetised pigs, bimikalim increased slightly, then decreased, renal blood flow depending on the dose used (Sassen et al., 1990). The reduction in total renal blood flow observed in the pig comprises a decrease in cortical (predominant) blood flow whereas medullary blood flow is increased significantly, producing an uneven distribution of total renal blood flow (Sassen et al., 1990). In other studies using anaesthetised normotensive rats, reductions in renal vascular resistance, following bolus ascending doses of cromakalim, but not Ro 31–6930, have been reported though both drugs elicited the same absolute falls in arterial pressure (Duty et al., 1990). Thus different changes in renal haemodynamics can be observed even within the benzopyran family of KCAs. A definitive answer to this problem is an important goal since it was declared during the mid-eighties that, if new vasorelaxant drugs were to play a major role in the future treatment of hypertension, they must exhibit a salutary effect on the kidney (Struyker-Boudier et al., 1984; Hollenberg, 1987). The earlier and sometimes conflicting data for changes in renal haemodynamics has centred largely around CRK and pinacidil. Now, with the introduction of many more KCAs and published haemodynamic studies, a slightly clearer picture has emerged. More importantly, the increased number of studies in conscious animals (Clapham and Longman, 1989; Gardiner et al., 1991; Shen and Vatner, 1993; see Figure 9.1) should, theoretically, provide the strongest evidence, since the effects of anaesthesia are obviated. In animals, KCAs tend to elicit a reduction in renal vascular resistance which is occasionally accompanied by an overt increase in renal blood flow (Table 9. 3). Where increases in renal blood flow have been recorded, it is possible that increased basal tone of the renal vasculature could be responsible for this effect and this is worthy of further investigation. Hollenberg et al. (1978) have estimated that up to two thirds of patients with essential hypertension have a 20% reduction in renal perfusion associated with renal vasoconstriction. Therefore, KCAs that dilate the Table 9.3 Effects of some KCAs on renal haemodynamics in animals KCA Species Anaesthesia


Method RBF RVR Ref aprikalim rat Yes -NS[1] cromakalim rat Yes Doppler [2,3] dog Yes EM [4] cat Yes EM

[5] cat Yes EM [6] cat Conscious Doppler [7] cat Conscious Doppler

[8] diazoxide dog Yes EM [9]


bimikalim pig Yes MS [10] dog Yes -NS[11] KRN2391 dog Yes EM [12] levcromakalim rabbit Yes MS [13] rat Conscious Doppler [14] cat Conscious Doppler

[15] dog Conscious Doppler

[16] dog Yes EM

[17] nicorandil


dog Yes EM [12] cat Yes EM [6] pinacidil cat Conscious Doppler

[8] cat Yes EM [6] dog Yes EM [18] Ro 30–6930 rat Yes Doppler [2] dog Yes EM [4] YM934 dog Yes EM

[17] EM = electromagnetic flow probes, MS = microspheres, -NS- = not stated. RBF = renal blood flow, RVR = renal vascular resistance.


[1] Aloup et al., 1990; [2] Duty et at., 1990; [3] Cavero et al., 1989; [4] Paciorek et al., 1990; [5] Buckingham et al., 1986; [6] Longman et al., 1988; [7] Clapham and Buckingham, 1988; [8] Clapham and Longman, 1989; [9] Rubin et al., 1962; [10] Sassen et al., 1990; [11] Schliep et al., 1989; [12] Ogawa et al., 1993a; [13] Hof et al., 1988; [14] Gardiner et al., 1991; [15] Clapham et al., 1991a; [16] Shen and Vatner, 1993; [17] Uchida et al., 1994; [18] Olsen and Arrigoni-Martelli, 1983.

renal vasculature in addition to lowering BP, may beneficially affect a process involved in the progressive elevation of BP. However, as yet there are no reported effects of KCAs on renal haemodynamics in models of hypertension in larger animals or after chronic administration. When peripheral vascular resistances to a number of vascular beds have been measured simultaneously, the degrees of KCA-induced reduction in the various vascular resistances are not uniform. For example, the renal circulation dilates the least of all in response to KCAs (Cavero et al, 1989; Shen and Vatner, 1993; Uchida et al., 1994). However, this is not altogether surprising since the kidney can dramatically autoregulate its own blood flow over a wide perfusion pressure window, from about 80 to 180 mmHg (Cupples et al., 1990). Nevertheless, a reflex rise in sympathetic tone to the kidney may limit the degree of vasodilatation induced by KCAs in this bed compared to other regions. This possibility is supported by a recent study (Johns, 1993) where LCRK, given by close arterial infusion, did not affect reductions in renal blood flow elicited by β 1- and β 2-adrenoceptor agonists, or by electrical stimulation of the renal sympathetic nerves, in the anaesthetised rat. Adrenergic control of the renal vasculature would therefore have occurred normally in the presence of LCRK. In contrast, an earlier study performed in pithed rats (Richer et al., 1990a) showed that intravenous infusion of CRK or bimikalim inhibited renal vasoconstriction elicited by UK 14304 (β 2-adrenoceptor agonist) and spinal cord stimulation, but that the renal vasoconstrictor effects of cirazoline (β 1-adrenoceptor agonist) were unaffected by either KCA. The sympathoinhibitory effect observed was still apparent if BP was returned to pre-infusion level with vasopressin or prostaglandin F2― (PGF2― ). However, these studies are not directly comparable, since Johns’ (1993) study was conducted in anaesthetised rats and administration of agonists and sympathetic nerve stimulation primarily targeted the kidney itself. Vasoconstrictor responses in the former (Richer et al., 1990a) study were more generalised following intravenous administration.


9.6.5 Skeletal Muscle Circulation The measurement of hindquarters blood flow using periarterial flow probes (pulsed Doppler and electromagnetic) appears to be the favoured model used to determine the effects of KCAs on blood flow to skeletal muscle. Probes may be placed around femoral (Olsen and Arrigoni-Martelli, 1983; Buckingham et al., 1986; Paciorek et al., 1990; Ogawa et al., 1993a) or iliac (Duty et al., 1990; Shen and Vatner, 1993) arteries or, alternatively, around the lower abdominal aorta, thus encompassing both femoral and iliac blood flow (Aloup et al., 1990; Gardiner et al., 1991). In these systems however, particularly for lower abdominal flow, changes in flow to other structures such as the rectum, tail, testes and skin may have influenced the overall response. When femoral blood flow was measured in anaesthetised cats and dogs, pinacidil, CRK, NIP-121 and YM934 (Olsen and Arrigoni-Martelli, 1983; Buckingham et al., 1986; Ogawa et al., 1993a; Uchida et al., 1994) elicited only minor changes in femoral blood flow but CRK and Ro 31–6930 reduced femoral vascular resistance (Paciorek et al., 1990). This is in contrast to the Ca antagonist, nifedipine, which elicited marked increases in femoral artery blood flow in anaesthetised cats (Buckingham et al., 1986) and dogs (Ogawa et al., 1993a). Diazoxide caused a biphasic response, first decreasing then increasing femoral blood flow (Rubin et al., 1962). Nicorandil, whose effects were also biphasic on femoral blood flow in anaesthetised mini-pigs, exhibited the reverse pattern of diazoxide, namely an increase followed by a decrease (Sakai et al., 1983). In conscious dogs, LCRK increased iliac blood flow and reduced iliac vascular resistance (Shen and Vatner, 1993), whilst CRK and Ro 31–6930 reduced iliac vascular resistance in the anaesthetised rat (Duty et al., 1990). Hindquarters vascular resistance (mainly femoral and iliac) has been shown to be reduced by levels (45–50%) that are greater than the fall in systemic vascular resistance (35%) following administration of aprikalim or CRK (Cavero et al., 1989; Aloup et al., 1990). In the conscious Long Evans rat, LCRK increased hindquarters blood flow at low doses (1 μ g.kg–1 min–1); but at a higher dose (10 μ g.kg–1min–1) hindquarters blood flow remained unchanged (Gardiner et al., 1991). Using an alternative technique (radioactive microspheres), Hof et al. (1988) have shown that CRK had little or no effect on rabbit skeletal muscle blood flow whilst in anaesthetised pigs blood flow to the muscularis sternocleidomastoideus was doubled following intravenous administration of bimikalim (Sassen et al., 1990). In the latter study however, blood flow to a number of other muscle groups remained constant suggesting that there was a heterogeneous effect of KCAs in the skeletal muscle circulation.


In general therefore, KCAs have tended to reduce hind limb vascular resistance and in some, but not all, studies increases in muscle blood flow have been observed. An indication that the KCAs may be useful in the treatment of peripheral vascular disease occurred when Angersbach and Nicholson (1988) reported that the KCAs, CRK, pinacidil and nicorandil, but not the Ca antagonists, nifedipine, verapamil or diltiazem, markedly increased red cell flux and tissue oxygenation in chronically ischaemic (femoral artery ligation for 6 weeks) muscle of the rat hind limb. This finding gained further support when Hatton et al. (1991) communicated that CRK partially attenuated ischaemia-induced muscle fatigue in the rat hind limb. In other words, there is a functional correlate to the increased tissue perfusion and reoxygenation. However, the functional benefits of CRK have been disputed in a study using a rat model of acute hind limb ischaemia (Tresize et al., 1993). They found that CRK (10–100 μ g.kg–1 iv) and LCRK (15 μ g.kg–1 iv) reduced arterial pressure and hind limb vascular resistance concurrent with an increase in iliac artery blood flow but did not attenuate the acute ischaemic muscle fatigue induced by graded occlusion of the abdominal aorta. The reasons for the differences may be related to the different methodologies used in the two studies (Hatton et al., 1991; Tresize et al., 1993). Thus, Angersbach and Nicholson (1988) concluded that during chronic ischaemia, newly formed collateral blood vessels may be selectively dilated by the KCAs to account for the increased red cell flux and oxygenation. This would not occur in the acute situation and, as indicated by Tresize et al. (1993), comparable studies using chronically ischaemic hind limb are required to assess the contribution of newly formed collaterals using their methodology. 9.6.6 Pulmonary Circulation The pulmonary circulation differs from the systemic circulation in that it distributes output from the right ventricle to the gas exchanging surface of the alveoli. It is also a low pressure system with an average mean pulmonary artery pressure of about 15 mmHg. The phenomenon of hypoxic pulmonary vasoconstriction has been known for years and is thought to be a homeostatic mechanism by which pulmonary blood flow is diverted away from hypoxic areas to optimise gas exchange. However, the homeostatic event may deteriorate to become an undesirable event leading to pulmonary hypertension. The mechanisms for hypoxic pulmonary vasoconstriction are not yet fully understood but are likely to be multifactorial (McCormack et al., 1993). Data for KCAs on the pulmonary circulation have been relatively few but data from a number of studies are now emerging indicating increased interest in this area. In anaesthetised dogs, CRK reduces pulmonary artery pressure during hypoxic pulmonary vasoconstriction (Hicks et al., 1989; Martin et al., 1990) and both CRK and pinacidil have been shown to dilate the pulmonary vasculature, at


doses not affecting right ventricular contractile function (Minkes et al., 1991). The effects of CRK and pinacidil are glibenclamide-sensitive (Minkes et al., 1991) but there is no separation of pulmonary from systemic effects (Figure 9.2) (Hicks et al., 1989; Martin et al., 1990; Minkes et al., 1991). In another study in the anaesthetised cat,

Figure 9.2 Effect of cromakalim on hypoxic pulmonary vasoconstriction in the anaesthetised dog. Pulmonary resistance (― ), systemic resistance (― ) and cardiac output (― ). Hypoxia was induced using an air/N2 (2:1) mixture to reduce arterial pO2 from 92±4 mmHg to 48±8 mmHg. (Reproduced with permission from Hicks et al., 1989).

where pulmonary pressure was elevated by the thromboxane agonist U46619, LCRK elicited a dose-related reduction in intralobar pressure, when infused directly into the intralobar artery (Hood et al., 1991). This effect was also shown to be glibenclamide-sensitive (Hood et al., 1991; McMahon et al., 1992) and independent of the nitric oxide (NO) pathway (McMahon et al., 1992). Systemic vasodilatation was also evident. These data would suggest that although KCAs have clear actions on the pulmonary circulation, the effects in acute models


cannot be separated from systemic effects indicating that the populations of KATP channels on the respective vasculature beds are identical. When considering a role for the KCAs in pulmonary hypertension, it should be appreciated that in states of increased pulmonary vascular resistance, attempts to reverse the condition with vasodilators have been unsuccessful owing to a reduction in systemic vascular resistance (McCormack et al., 1993). In the models of acute hypoxia already discussed, the KCAs tested showed no dose separation between pulmonary and systemic dilatation. This lack of selectivity has also been observed in a chronic model of pulmonary hypertension (high altitude, chronically hypoxic rats; Oka et al., 1993). In these animals, the benzopyran based KCA, NIP-121, but not the Ca antagonist, nifedipine, reduced pulmonary artery pressure. However, as shown in acute studies with KCAs, pulmonary vascular resistance is reduced also at doses active on the systemic vasculature. In the study of Oka et al. (1993) however, the rats were acclimatised for two days under normoxic conditions, suggesting that the pulmonary dilatation was effective against a condition not associated with hypoxic vasoconstriction. The effects of NIP–121 were glibenclamide-sensitive and independent of NO release (Oka et al., 1993). Thus, the effects of KCAs in acute studies, and in one study in chronically hypoxic rats, appear to be qualitatively similar in vivo. In vitro studies would have predicted that the hypoxic pulmonary vasculature would have been more sensitive to the effects of KCAs since it is well known that the actions of KCAs can be augmented by hypoxia (Cook and Quast, 1990). In a study using rat isolated pulmonary artery precontracted with PGF2a, the potency of LCRK was enhanced significantly (IC50 0.52±0.05 μ M in normal versus 0.23±0.06 μ M in hypoxic tissue) in rats previously subjected to chronic hypoxia compared to their normoxic controls (Leach et al., 1992). This effect was not influenced by the absence or presence of endothelium. In a study of acute hypoxia, the activity of LCRK was augmented by hypoxia and inhibition of oxidative phosphorylation (Randall and Griffiths, 1993). The role of KATP channel modulation providing a means of selective pulmonary vasodilatation remains an attractive proposition (Peacock, 1993). 9.7 Microcirculation As mentioned previously, it is now widely accepted that KCAs reduce arterial pressure by a direct action on vascular smooth muscle to reduce systemic vascular resistance (Edwards et al., 1992; Longman and Hamilton, 1992). The studies discussed so far have, in the main, involved measurement of blood flow through large (accessible) conduit arteries. Together with systemic BP, the pressure/flow relationship can be calculated to give resistance to flow (or the reciprocal, conductance) as an indirect measure of the behaviour of the resistance vessels in that tissue. Resistance in this context, therefore, is the sum of all the


resistance vessels in the tissue to which flow was being measured. However, it is clear that the microcirculatory responses to vasodilators are not uniform throughout the arteriolar tree. In order to study the microcirculation in conscious rats, a lightweight thermoneutral chamber has been developed by Smith et al. (1985). This ‘dorsal microcirculatory chamber’ allows direct visualisation of skeletal muscle microcirculation by intravital microscopy. Using this technique, StruykerBoudier et al. (1992) have shown that LCRK (10–100 μ g.kg–1iv) elicits a doserelated reduction in arterial pressure in conscious SHR associated with preferential dilatation of the smallest (10–35 μ m diameter) arterioles. Larger (70–120 μ m diameter) vessels were significantly dilated only by the highest dose


Figure 9.3 Effects of levcromakalim on arteriole (upper panel) and venule (lower panel) diameters in conscious SHR measured by intravital microscopy. Values are mean ± S.E.M. Vessels are labelled in functional branching order with A1 or V1 vessels being the largest (70–125 μ m diameter), then A2/V2 (30–75 μ m diameter) and then the pre-capillary arterioles, A3/4 and post-capillary venules, V3/4 (10–30 μ m diameter). (Reproduced with permission from Struyker-Boudier et al., 1992).

tested (Figure 9.3). A similar profile was also found for the Ca antagonists nifedipine and verapamil (Messing et al, 1991). In contrast, the β 1-adrenoceptor antagonist, prazosin (Messing et al., 1990), dilated preferentially the larger arterioles. Thus, these important studies have provided direct evidence for heterogeneity in the response to vasodilator agents in the arteriolar tree in the conscious SHR. Intravital microscopy has shown also that, in the cutaneous microcirculation of conscious normotensive rats, bimikalim (6, 12 and 24 μ g.kg–1 iv) elicited a doserelated reduction in arterial pressure concurrent with an increase in erythrocyte flow velocity (Hertel, 1992). However, capillary diameter was increased following 6 and 12 μ g.kg–1 iv bimikalim only. At 24 μ g.kg–1 iv, vasoconstriction was observed. In summary, the data support the suggestion that the smallest blood vessels are under control of factors affecting membrane potential (Struyker-Boudier et al., 1992) and that KATP channels could be an important determinant of local tissue blood flow (Hertel, 1992). 9.8 In Vivo Mechanistic Studies 9.8.1 Glibenclamide Antagonism of the cardiovascular effects of KCAs by relatively high doses of the sulphonylurea, glibenclamide is now a hallmark for actions at KATP channels. In both conscious and anaesthetised rats (SHR and normotensive), glibenclamide (10–20 mg.kg–1 iv) has been shown to inhibit BP lowering effects of CRK and nicorandil (Cavero et al., 1989), SDZ PCO 400 (Quast et al., 1990), aprikalim (Aloup et al., 1990), LCRK (Clapham et al., 1991a), NIP–121 (Masuda et al., 1991) and Y–26763 (Nakajima et al., 1992). Glibenclamide is able to prevent the antihypertensive effects of KCAs and to reverse an already established antihypertensive response (Aloup et al., 1990; Clapham et al., 1991a). Importantly, glibenclamide appears to block selectively for hypotensive responses induced by KCAs. Thus, the vasodilator responses to dihydralazine, acetylcholine, vasoactive intestinal polypeptide and the Ca antagonists diltiazem and nifedipine, are unaffected by doses of glibenclamide that abolish the hypotensive effect of KCAs (Figure 9.4) (Cavero et al., 1989; Clapham et al., 1991a; Hood et al., 1991; Ogawa et al., 1993b).


Demonstration of the antagonism of the systemic effects of KCAs by glibenclamide has been extended to individual vascular beds in various species. Thus, glibenclamide antagonises the pulmonary (Hood et al., 1991; Minkes et al., 1991), mesenteric (Ogawa et al., 1993b) and coronary (Cavero et al., 1991; Ogawa et al., 1992) vasodilator responses to a variety of KCAs. These data provide in vivo evidence that glibenclamide is acting at the level of vascular smooth muscle. Conversely, antihypertensive doses of the KCAs, CRK or pinacidil, do not prevent nor reverse glibenclamide-induced hypoglycaemia in conscious SHR (Clapham et al., 1994). In contrast, diazoxide at equiantihypertensive doses did affect glibenclamide-induced hypoglycaemia. These data confirm, in vivo, the lack of effect of the newer KCAs on pancreatic KATP channels at BP lowering doses.


Figure 9.4 Interaction between intravenously administered glibenclamide on the blood pressure lowering activity of levcromakalim and nifedipine in anaesthetised SHR. a) glibenclamide (― ) or vehicle (― ) (first arrow) was administered 15 min prior to levcromakalim (second arrow), b) levcromakalim (first arrow) administered 15 min prior to glibenclamide (― ) or vehicle (― ) where indicated by second arrow, c) glibenclamide (― ) or vehicle (O) (first arrow) was administered 15 min prior to nifedipine (second arrow).


Values are mean ± S.E.M. * indicated Prenal>hind limb, with the effects of sympathetic nerve stimulation being more sensitive to inhibition than exogenous agonists (Richer et al., 1990a). Restoration of BP to


values observed in intact (as opposed to pithed) rats by PGF2― , had no effect on the inhibitory responses to CRK or EMD52692, whilst restoration of BP with arginine vasopressin (AVP) abolished the sympathoinhibitory effects of these KCAs (Buckingham 1988; Richer et al., 1990a). The discrepancy between the effects of PGF2― and AVP is not clear mechanistically but it is possible that AVP may act as a physiological antagonist through sensitisation of the vasoconstrictor actions to the stimuli used. The inhibitory responses of these drugs in this respect are therefore not dependent upon the prevailing level of BP. Richer et al. (1990a) postulated that the effects of CRK and bimikalim were pre-junctional since the effects of nerve stimulation were more sensitive than those of exogenously applied agonists to the inhibitory effects of these KCAs. However Johns (1993) found that LCRK had little or no effect on renal vasoconstrictor responses to β adrenoceptor agonists or sympathetic nerve stimulation. Furthermore, NIP–121 elicited a dose-related inhibition of pressor responses to nondrenaline, angiotensins I and II, AVP, PGF2― and the Ca agonist, BAY K 8644 (Masuda et al., 1991). In addition CRK inhibited pressor responses to endothelin I in pithed rats (Le Monnier de Gonville et al., 1990) indicating that a substantial postsynaptic involvement cannot be excluded. Tachycardia elicited by sympathetic-nerve stimulation was also unaffected by CRK or bimikalim (Richer et al., 1990a) confirming the results of an earlier study where CRK had no effect on sustained tachycardia evoked by stimulation of the spinal cord in the pithed rat (Clapham, 1988). Thus, if KCAs exert a prejunctional inhibitory effect on sympathetic nerves, the effect would appear to be specific for the vasculature. 9.9 Conclusion A feature that seems to have emerged from the haemodynamic studies described here, is the non-uniform effect of KCAs throughout the vascular tree which show a distinct rank order of vasodilator potential depending on the vascular bed and the size of arteriole. This may be explained partly by autoregulatory mechanisms in some vascular beds that would serve to offset the degree of vasodilatation possible. Although data are limited, it is also possible that there may be an uneven distribution of KCA-sensitive KATP channels throughout the vasculature. In certain blood vessels, such as cerebral arteries, expression of KATP function appears to be adversely affected by disease states such as hypertension and diabetes in animal models such as the SHR and streptozotocin-diabetic rat. Finally, it is clear from vascular studies in animals that KCAs have potential in the treatment of hypertension and angina. Possibilities also exist for the treatment of pulmonary hypertension where manipulation of KATP channels may yet provide a selective pulmonary vasodilator (Peacock, 1993) but further studies are clearly required. The KCAs seem also to be beneficial in a model of cerebral


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10 Cardiac Potassium Channel Modulators: Potential for Antiarrhythmic Therapy M.C. SANGUINETTI1 & J.J. SALATA2 1Division

of Cardiology, University of Utah, Salt Lake City, UT, USA,

2Department

of Pharmacology, Merck Research Laboratories, West Point, PA, USA. 10.1 Introduction

Cardiac arrhythmias arise from disturbances in the normal spread of cellular excitability and/or regional refractoriness, and can result from localized tissue damage (e.g., ischemia), anatomical anomalies or drug treatment. In the United States alone, sudden cardiac death is estimated to account for over half a million deaths yearly, and about 80% are believed to be caused by ventricular tachyarrhythmias (Morganroth and Bigger, 1990; Panidis and Morganroth, 1983). Modulation of K channel activity has been shown in numerous animal models and in some clinical studies to represent beneficial therapy in the treatment of arrhythmias that arise from such disturbances. The rationale for use of these agents is rather simplistic. Activation of K+ conductance reduces electrical excitability, an effect useful in arrhythmias arising from enhanced automaticity. Block of K+ conductance delays repolarization of action potentials, a useful mechanism to terminate some forms of reentrant-based tachyarrhythmias. The key to successful termination or prevention of an arrhythmia by either approach is to match the correct drug (and dosage) with an arrhythmia of defined origin, a difficult and often impossible clinical task. For example, lengthening of action potential duration (APD) with a K channel blocker (KCB) can prolong the refractory period sufficiently to slow or prevent a tachyarrhythmia based upon a reentrant circuit. However, excessive lengthening of action potential duration can result in the triggering of early afterdepolarizations (EADs), one probable cellular mechanism of torsades de pointes arrhythmias (Janse and Wit, 1989; Surawicz, 1989) (Figure 10.1). Druginduced torsades de pointes is a polymorphic ventricular tachycardia that is preceded by excessive QT lengthening and is characterized by a sinusoidal twisting of the QRS axis around the isoelectric line, with a cycle length that varies between 5–20 beats (Keren and Tzivoni, 1991; Surawicz, 1989). Several class 1A (e.g., quinidine, procainamide, disopyramide, aprindine) and class III (e.g., amiodarone, sotalol) antiarrhythmic drugs have been reported to induce


this arrhythmia. However, no useful correlation between plasma concentrations, or extent of QT prolongation by these drugs and their propensity to cause torsades de pointes has been established

Figure 10.1 Therapeutic and toxic effects of class III antiarrhythmic agents depend upon extent of APD prolongation.

(Surawicz, 1989). Treatment of torsades de pointes usually consists of cardiac pacing, or infusion of isoproterenol or magnesium sulfate (Keren and Tzivoni, 1991; Surawicz, 1989). In animal and tissue models, these drug-induced arrhythmias can also be terminated with an activator of ATP-sensitive K channels (KATP), e.g. cromakalim (CRK). Evidence from animal studies suggest that excessive doses of K channel openers are equally proarrhythmic, caused by an increased dispersion in refractoriness. Obviously, there is not a single mechanism that will terminate all types of arrhythmias and as discussed above, most K channel modulators (KCMs) can also induce arrhythmias when used inappropriately. This can easily be demonstrated in animal models. For example, in anesthetized rabbits infused with the β -agonist methoxamine, there is a striking correlation between doses of various class III agents required to prolong QTU interval by 20% and doses required to induce arrhythmias with features akin to torsades de pointes (Carlsson et al., 1990). Despite the obvious limitations of KCBs as antiarrhythmic agents there has been much recent effort directed towards their development. In large part this can be traced to the publishing of interim results of the Cardiac Arrhythmia Suppression Trial (CAST), where it was reported that encainide and flecainide (class IC agents)

CARDIAC K CHANNEL MODULATORS: POTENTIAL FOR ANTIARRHYTHMIC THERAPY 275

increased mortality relative to placebo (The Cardiac Arrhythmia Suppression Trial (CAST) Investigators, 1989). The CAST report obviously raised questions regarding the risk versus benefit of all then currently available antiarrhythmic agents, and provided great impetus to develop new drugs that acted via mechanisms other than slowed conduction. In this chapter we will review the rationale for using KCMs as antiarrhythmic drugs and discuss the underlying mechanisms of action for some of these agents. It should be stressed that other well established antiarrhythmic mechanisms exist (e.g., block of Na or Ca channels, or β -adrenergic receptors), but are not the subject of this chapter. Not all class III agents act by block of cardiac K channels. For example, ibutilide is reported to prolong action potentials by activating a slow inward Na+ current, and at high concentrations activates an unidentified instantaneous outward K+ current, thereby limiting the prolongation of APD mediated through Na channel activation (Lee, 1992). 10.2 Arrhythmogenic Mechanisms Susceptible to K Channel Modulators A broad spectrum of cellular mechanisms underly clinically important arrhythmias, many of which are poorly defined or have been studied only in animal models. Three major categories of arrhythmogenic mechanisms are usually recognized: enhanced or abnormal automaticity, triggered activity, and reentry (Task Force, 1991). Within each category exist several types of underlying mechanisms. For example, triggered activity can arise from either EADs or delayed afterdepolarizations (DADs). It is generally believed that arrhythmias arising from EAD-based, but not DAD-based triggered activity can be affected by KCMs. KCBs worsen, whereas K channel openers (KCOs) can terminate EAD-based arrhythmias. A very extensive review of the mechanisms underlying the complex ventricular arrhythmias that result from myocardial ischemia and infarction should be consulted for details on this subject (Janse and Wit, 1989). 10.2.1 Automaticity Agents that enhance outward K channel conductance can be useful in the treatment of some types of abnormal, or enhanced normal automaticity of atrial tissue. For example, opening of K channels that are activated by acetylcholine (IK (ACh)) slows phase 4 depolarization of atrial pacemaker cells and increases maximum diastolic potential of all atrial myocytes.


10.2.2 Triggered Activity Based on EADs Early afterdepolarizations (EADs) are slow response action potentials that are activated during the beginning of the terminal phase of repolarization (Figure 10.2). These mini-action potentials arise most readily when outward current is reduced and inward Ca2+ current is enhanced, such as may occur subsequent to treatment with certain KCBs. EADs can act to trigger tachyarrhythmias, and only occur at slow stimulation rates in vitro or slow heart rates in vivo. EADs can be suppressed by increased stimulation rate, or by enhancing outward current, for example by activation of the delayed rectifier K+ current (IK) or IK(ATP), or by block of Ca2+ current. 10.2.3 Reentry Reentry can be dependent upon Na channel or Ca channel function. Na channeldependent reentry is characterized by conduction of the excitable wavefront encroaching on refractory tissue (so-called short excitable gap). Figure 10.3 shows

Figure 10.2 Induction of early afterdepolarizations (EAD) by dofetilide. Superimposed action potential recordings from an isolated guinea pig ventricular myocyte during control at a stimulation frequency of 1 Hz and after 10 min of dofetilide (30 nM). (Unpublished observation)


Figure 10.3 Proposed mechanisms of reentry and its termination by class 1 agents (panel A) and class III agents (panels B and C). A: Area of impaired conduction and excitability is delineated by two small lines. B: Prolongation of refractoriness blocks reentrant wavefront. C: Fibrillation is caused by numerous reentrant pathways; prolongation of refractoriness terminates arrhythmias. From Task Force of the Working Croup on Arrhythmias of the European Society of Cardiology (1991) with permission.

schematics of reentrant circuits based upon long to very short excitable gaps and the changes in conduction or refractoriness that can theoretically terminate arrhythmias based upon these different reentrant circuits. Reentry that arises from long excitable gaps can be suppressed by slowing conduction with Na channel blockers, so-called class I antiarrhythmic agents. Reentry based upon short or very short excitable gaps are most effectively treated with agents that prolong refractoriness, so-called class III antiarrhythmic activity. Specific reentrant arrhythmias that are considered to be dependent on a short excitable gap include atrial flutter, atrial fibrillation, circus movement tachycardia in Wolfe-Parkinson-White syndrome, polymorphic and sustained monomorphic ventricular tachycardia, bundle branch reentry and ventricular fibrillation (Task Force, 1991). These arrhythmias can often be suppressed by prolonging APD, for example by blocking IK. 10.3 Cardiac K Channels The cellular basis for the long plateau phase of cardiac myocytes is complex, but results primarily from inward (‘anomalous’) rectification and/or very slow


activation of multiple types of K channels. Cardiac K channels can be grouped together into either voltage-dependent, cation-activated or ligand-activated channels. Voltage-dependent types conduct delayed rectifier (IK) inward rectifier (IK1) and transient outward K+ currents (IA). Cation-activated types conduct K+ currents activated by intracellular Na+ (IK(Na)) and intracellular Ca2+ (IK(Ca)). Ligand-activated K channels conduct currents activated by acetylcholine (IK (ACh)), arachidonic acid (IK{AA)) and phosphatidylcholine (IK(PC)), or blocked by intracellular ATP (IK(ATP)). Multiple types of IK and IA currents have been identified. Three distinct types of current have been identified in isolated cardiac cells based upon differences in rate of activation, rectification properties, and pharmacology using whole-cell voltage clamp techniques. These types are: 1) slowly activating, outwardly rectifying IK (IKs, Ix2); 2) rapidly activating, inwardly rectifying IK (IKr, Ix1), and 3) rapidly activating, outwardly rectifying IK (called IRAK in rats, IKur in human myocytes). Several of the channel proteins that conduct cardiac K+ currents have been cloned and sequenced. These include Kv4.2 and Kv1.4 (IA current), Kv1.1, Kv1. 2, Kv1.5 (ultrarapid delayed rectifiers, such as IRAK and HK2), IRK1 (IK1current), GIRK1 (IK(ACh) currennt), and IsK (IKs current). 10.4 K Channel Blockers Much effort has been directed in the past decade towards the discovery and development of drugs that have antiarrhythmic properties dependent on their ability to prolong cardiac refractoriness. These drug candidates have usually been discovered at pharmaceutical companies that screen their compound libraries, or a limited series of chemicals based upon unique structural similarities to known class III agents, for appropriate biological activity in simple assays. The usual assays rely on some measurement of cardiac refractoriness in isolated cardiac tissue preparations, most commonly isolated papillary muscles, Purkinje fibers or atria that are stimulated at a constant rate during which the effects of compounds on effective refractory period (ERP) or action potential duration (APD) are determined with a paired pulse protocol. Most often the APD is measured at the point of 90% repolarization, and is referred to as APD90. If microelectrodes are used to record action potentials, then the effect on maximum upstroke velocity (Vmax) and action potential configuration can also be determined. Limited information regarding possible mechanism of action can be determined by this method; e.g., Na channel blockers will decrease Vmax; IA blockers will reduce the size of the notch characteristic of phase 1 repolarization; Ca channel blockers will decrease the amplitude of the plateau phase; blockers of the inward rectifier K+ current will slow the rate of terminal (phase 3) repolarization (Martin and Chinn, 1992). Of course, the most likely scenario is that a specific compound will have multiple activities and therefore, change the configuration of the cardiac action potential in a complex and oftentimes


uninterpretable manner. An even simpler screening assay relies on only measuring the contractile force of papillary muscles or muscle strips using small force transducers. ERP can be determined in these preparations by observing the amplitude of contraction of the stimulated beat immediately after the paired pulse stimulation. Alternatively, compounds can be screened for their ability to displace radiolabeled drugs with known and desirable activity, such as [3H]dofetilide, a specific IKr blocker. Class III activity of some agents were determined after compounds were shown to have useful antiarrhythmic activity in standard arrhythmia models. The structures of several compounds with class III antiarrhythmic activity are shown in Figures 10.4 and 10.5. 10.4.1 Delayed Rectifier K Channel Blockers The majority of class III antiarrhythmic agents act at least in part by blocking the cardiac delayed rectifier K+ current, IK. Recent studies have shown that ‘IK’ is actually the composite of several distinct currents and that some class III drugs may be specific blockers of only one of these currents. Multiple types of IK channels The voltage-dependent outward K+ current activated during the plateau phase of the cardiac action potential, and recorded during depolarizing steps in whole-cell mode of voltage clamp is actually the composite of as many as three distinct K+ currents (IKs, IKr, IKur). The relative contribution of each of these distinct currents to net IK varies considerably among species and perhaps between different regions of the heart. For example, an IKr-like current is the largest IK component in rabbit and cat (Colatsky et al., 1990); IKs is the largest IK component in guinea pig (Sanguinetti and Jurkiewicz, 1990b); and IKur is by far the largest component of net IK in human atrial cells (Wang et al., 1993). Many recently developed class III agents block IKr at concentrations below that required to block other types of K+ currents (e.g., IKl, IA, IK(ATP)) or the other components of IK (IKs or IKur). A component of delayed outward current (‘ixl’) that displayed inward rectifying properties was originally described in Purkinje fibers by Noble and Tsien (1969). Subsequent studies (Attwell and Cohen, 1979; Cohen and Kline, 1982, Kline and


Figure 10.4 Methanesulfonanilide class III antiarrhythmic agents. Compounds are listed in decreasing order of potency for prolonging refractory perio in isolated ferret papillary muscle, ranging from 13 nM for (1) to 44 μ M for (9).


Figure 10.5 Class III antiarrhythmic agents not structurally related to sotalol. Compounds are listed in decreasing order of potency for prolonginj refractory period of isolated papillary muscle, ranging from 47 nM for (10) to 10 μ M for (15).

Cohen, 1984) documented experimental artifacts caused by inadequate space clamp and K+ accumulation between myocytes of this multicellular preparation, which left many investigators to question the original data (Jaeger and Gibbons, 1985). However, the original concept by Noble and Tsien that at least two components (ixl and ix2) of delayed rectifier current exist in mammalian cardiac myocytes appears to have been correct, as more recent studies have described two distinct, time-dependent components of IK in embryonic chick (Shrier and Clay, 1986), guinea pig (Balser et al., 1991; Balser and Roden, 1988; Sanguinetti and Jurkiewicz, 1990b, 1991), canine (Gintant, 1993) and human (Wang et al., 1993) cardiac myocytes. Similar to ‘ixl’, the property of IKr that distinguishes it from other cardiac delayed rectifier K+ currents (IKur and IKs) is a pronounced rectification at positive membrane potentials. IKr was originally defined in guinea pig cardiac myocytes as a component of IK that was specifically blocked by E– 4031 (Sanguinetti and Jurkiewicz, 1990b). Methanesulfonanilide class III antiarrhythmic agents such as E-4031 (4) and dofetilide (2) were shown to block net IK in a voltage-dependent manner, with block being greatest when assessed with short test pulses to potentials less than 0 mV (Carmeliet, 1992; Jurkiewicz and Sanguinetti, 1993; Sanguinetti and Jurkiewicz, 1990b). This effect of these drugs could either result from a voltage-dependent block of a single current, or from specific block of only one of two components of net IK. Several lines of evidence suggested that the latter interpretation was correct, including: (1)


differential modulation of the two components by β -adrenergic receptor agonists (only IKs is significantly activated by isoproterenol) (Sanguinetti et al., 1991); (2) removal of extracellular K+ increases IKs, but decreases IKr (Sanguinetti and Jurkiewicz, 1992); (3) absence of extracellular Ca2+ shifts voltage-dependence of IKs activation to more positive potentials, but IKr to more negative potentials (Sanguinetti and Jurkiewicz, 1992); (4) block of IKr but not IKs by 10 μ MLa3+ (Sanguinetti and Jurkiewicz, 1990a). As defined by sensitivity to block by 5 μ M E–4031 (Figure 10.6), IKr is half-activated at –22 mV, whereas IKs (current not blocked E–4031) is half-activated at +20 mV. The slope factor for IKr and IKs was determined to be 7.5 and 12.7 mV, respectively. In guinea pig ventricular myocytes IKs requires very long pulses to reach a pseudo steady-state level of outward current (time constants for activation: ― 1=400 msec, ― 2=2.4 sec at 0 mV), whereas IKr is fully activated in less than 500 msec (― =50 msec at 0 mV) (Sanguinetti and Jurkiewicz, 1994). Based on the kinetics of IK deactivation, Chinn (1993) also proposed that two distinct channel types contribute to net IK in guinea pig ventricular myocytes. Two distinct components of guinea pig atrial IK were also described at the single channel level, with single channel conductances of 1–3 pS (IKs?) and 10 pS (IKr?) (Horie et al., 1990). IKr and IKs contribute almost equally to net IK during the plateau phase of guinea pig ventricular myocytes (Courtney et al., 1992; Sanguinetti and Jurkiewicz, 1990b). When currents recorded during very long voltage clamp pulses are compared, IKs is about 11 times greater than IKr. Rabbit ventricular and nodal cells are reported to have only one type of Ik, with properties similar to IKr, including inward rectification and sensitivity to block by E-4031 (Shibasaki, 1987; Veldkamp et al., 1993). Shibasaki (1987) studied the properties of IK in rabbit atrial pacemaker myocytes using single channel recording techniques. The current characterized in the study by Shibasaki had several properties in common with IKr of guinea pig cardiac myocytes, including: (1) rectification at potentials >0 mV, (2) rapid activation, (3) halfmaximal activation at


Figure 10.6 Voltage-dependent block of delayed rectifier K+ current (IK) by dofetilide in isolated guinea pig ventricular myocytes. A and B: Currents recorded during 225 msec pulses to test potentials of –20, 0, +20 and +30 mV before and after exposure of cells to 1 μ M dofetilide. C and D: Magnitude of time-dependent lK (C) and lK tail current (D) before (o) and after 1 μ M dofetilide (― ). Dofetilide specifically blocks one component (lkr), having no effect on another component (lKs) of net lK. From Jurkiewicz and Sanguinetti (1993).

–25 mV and a slope factor of 7.4 mV, (4) little or no inactivation. It was not determined if IK of rabbit pacemaker cells was blocked by methanesulfonanilides, but IK of rabbit myocytes isolated from other regions of the heart are at least as sensitive to block by these agents as is IKr of guinea pig myocytes (Carmeliet, 1991, 1992, 1993). The mechanism of rectification of


rabbit nodal cell IK was studied by recording single channel current ensembles. Based upon the kinetics of deactivation of these currents, Shibasaki (1987) proposed that IK rectification results from an ultrarapid inactivation of channels that develops faster than activation at positive test potentials. The net result of these two opposing processes is a smaller current at positive test potentials than would be predicted solely upon the progressive increase in the electrochemical driving force for K+. Specific lKr blockers Several recently developed antiarrhythmic drugs that are still undergoing clinical evaluation were synthesized to mimic the main structural features of sotalol (9), a methanesulfonanilide (Figure 10.4). These new generation class III agents are extremely potent, blocking IKr and prolonging APD at concentrations as low as 1 nM, with half-maximal effects ranging from 13 nM [for L–691 121, (Lynch et al., 1993)] to 0.5 μ M [for UK–66 914 (6), (Baskin et al., 1991)]. Concentrations required to block IKr correlate well with those needed to prolong APD in vitro, or QTC in vivo. The term ‘specific blocker’ is a relative term at best. In the context used here it is meant to distinguish compounds that block IKr (or similar current) at concentrations at least 100–fold less than that required to affect other cardiac currents. For example, dofetilide blocks only IKr in cardiac myocytes, but also blocks a Ca-activated K channel in hippocampal CA1 neurons (McLarnon and Wang, 1991). IKr blockers lengthen cardiac refractoriness in a dose-dependent manner, with increases in QTC occurring at doses parallel to those that lengthen either effective or relative refractory periods of the ventricle. E–4031, UK–66 914, and dofetilide had no effect on mean arterial blood pressure, PR interval, QRS interval or ventricular conduction time, but did decrease heart rate, and increase maximum rate of left ventricular pressure development (LV+dP/dt) in anesthetized dogs (Wallace et al., 1991). D-sotalol had the same effects as these agents on all parameters except LV+dP/dt, which was unchanged. The increase in LV+dP/dt by these agents results from a positive inotropic effect secondary to prolonged APD (Baskin et al., 1991; Sanguinetti and Siegl, 1992). In human isolated ventricular strips, almokalant prolonged APD and contractile force in a concentration-dependent manner (Carlsson et al., 1991). At 1 μ M, almokalant (10) lengthened APD90 by 62%. Similar to studies in dogs, dofetilide (Sedgwick et al., 1991, 1992) and sotalol (Nademanee et al., 1985) produce QTC prolongation in humans without effects on conduction parameters (e.g., AH, HV, PR or QRS intervals). Dofetilide, UK–66 914, L–691121 (1), E–4031, sotalol, sematilide (8), almokalant, and MS–551 (13) exhibit clear suppression of ventricular tachyarrhythmias initiated by programmed electrical stimulation or an acute ischemic insult in dogs with previous myocardial infarcts (Lynch et al., 1992, 1993). In contrast to class I agents, E–4031, d-sotalol and L–691 121 do not


suppress the frequency of premature ventricular complexes (PVCs) early after myocardial infarction in dogs (Lynch et al., 1990, 1993). Many lethal arrhythmias develop several days after a myocardial infarction. An attempt to model this situation led to the development of a conscious (Patterson et al., 1982) and later an anesthetized canine model of sudden cardiac death. E–4031, L–691 121 and MK–499 (3) prevented death in response to a secondary acute myocardial infarction (induced by application of current to the lumen of the circumflex coronary artery) in the setting of chronic myocardial infarction in anesthetized dogs (Lynch et al., 1990, 1993, 1994). For inclusion in these studies, the dogs were required to have inducible ventricular arrhythmias in response to programmed ventricular stimulation 6 to 10 days after an anterior myocardial infarction. The initial infarction was produced by a 2 hour occlusion of the left anterior descending coronary artery followed by reperfusion. The survival of dogs administered either placebo or MK–499 for the 3 hour period after the onset of acute posterolateral myocardial ischemia is shown in Figure 10.7. IKr blockers prolong APD of isolated rabbit and cat myocytes to a greater extent than in isolated guinea pig ventricular myocytes. This reflects the fact that guinea pig myocytes have a large IKs, whereas the cat and rabbit have very little if any IKs. There is also significant variability in the extent of APD lengthening by IKr blockers in different regions of the heart. For example, as shown in Figure 10.8, dofetilide has a greater effect on dog Purkinje fibers than on ventricular muscle (Gwilt et al., 1991). Prominent age-related changes in the effect of some class III antiarrhythmic agents on cardiac APD suggest that the contribution of IKr to net repolarizing K+ current changes during development in the rat. Potent class III antiarrhythmic agents such as almokalant and dofetilide do not prolong APD of adult rat myocytes, although these compounds do block a very small outward K+ component in these cells (Abrahamsson et al., 1994). The component of current (presumedly IKr) blocked by these drugs probably has no significant effect on net outward current in these cells, and hence does not prolong action potentials. However, the action potential configuration of rat embryonic heart myocytes is more like guinea pig (prolonged plateau phase) than adult rat, due to the relative lack of a prominent IA in the fetal heart, and is markedly prolonged by these drugs. At high concentrations, these drugs induce EADs and rhythm abnormalities in the fetal heart, which may be the mechanism of the embryotoxic effects of these agents in rats (Abrahamsson et al., 1994).


Figure 10.7 Survival of MK–499 versus vehicle-treated postinfarction dogs with baseline inducible arrhythmias, expressed as a function of time after development of acute posterolateral myocardial ischemia. Adapted from Lynch et al. (1994).


Figure 10.8 Prolongation of action potentials recorded from canine ventricular muscle (A) and Purkinje fiber (B) by increasing concentrations of dofetilide. Adapted from Gwilt et al. (1991) with permission.

The effects of WAY–123 398 (5) on action potentials of isolated dog Purkinje fibers and membrane currents in cat ventricular myocytes have been compared with three other methanesulfonanilide compounds, dofetilide, E–4031 and dlsotalol (Spinelli et al., 1993). The concentrations required to prolong APD to –60 mV of the Purkinje fibers by 20% were 210 nM for WAY–123 398 and 6 nM, 30 nM, and 38 μ M for dofetilide, E–4031 and dl-sotalol, respectively. Unlike the other three drugs, the effects of WAY–123 398 on APD to –60 mV in muscles stimulated at 1 Hz reached a maximal value (from a control of 264 msec to 439 msec at 1 μ M). The mechanism for the plateau in effect is unknown, but is a useful property since it might limit the proarrhythmic potential of the drug. WAY–123 398 had no effect on Vmax of the canine action potential at 3 μ M, or


on L-type ICa, IA or IK1 in voltage clamped feline myocytes at 10 μ M. Consistent with APD prolongation and IK block in vitro, WAY–123 398 prolonged cardiac refractoriness without effects on conduction in anesthetized dogs (Spinelli et al., 1992). An interesting difference exists between the manner in which IK in guinea pig and cat myocytes is blocked by these drugs. In cat myocytes high concentrations of these drugs (e.g., 1 μ M WAY-123 398) completely block the deactivating tail current measured after a depolarizing pulse, whereas the timedependent current measured during the test pulse is only reduced by approximately 40% (Spinelli et al., 1993). In guinea pig myocytes, these drugs only block a fraction of the tail current, leaving a component that has an amplitude consistent with that expected for deactivation of the large IKs measured during the test pulse (Sanguinetti and Jurkiewicz, 1990b). Two possible explanations for this difference is that either these drugs block cat IK in a voltage-dependent manner (rapid block at less depolarized levels) or that the slowly activating outward current resistant to block by these drugs in cat myocytes is not a typical IKs. The efficacy of IKr blockers may also vary as a function of plasma levels of K+ in a manner not previously recognized. Torsades de pointes caused by class III antiarrhythmic drugs is often associated with hypokalemia (Janse and Wit, 1989; Keren and Tzivoni, 1991; Surawicz, 1989). The conductance of IKr is dependent upon [K+]e; low [K +]e reduces the magnitude of IKr in guinea pig ventricular myocytes (Sanguinetti and Jurkiewicz, 1992) and rabbit Purkinje fibers (Scamps and Carmeliet, 1989). It is possible that hypokalemia reduces the conductance of IKr channels in addition to its well-documented reduction in conductance of IK1. The resultant decrease in net outward K channel conductance, combined with IKr block produced by treatment with class III drugs may prolong action potentials beyond the point of therapeutic intent, resulting in torsade de pointes. The complex and interdependent effects of these factors on repolarization of cardiac action potentials are important considerations in the development of future antiarrhythmic drugs designed to specifically prolong refractory period at the onset of tachyarrhythmias. Nonspecific blockers of lK Several compounds that possess class III antiarrhythmic activity have many different, often poorly defined cellular mechanisms of action. Agents that were synthesized or developed for treatment of diseases other than arrhythmias have subsequently been discovered to block cardiac IK and have class III antiarrhythmic activity. Examples include ketanserin and ICS 205–930 (5–HT2 blockers), melperone (a neuroleptic), and tacrine (a cholinesterase inhibitor) (Morgan and Sullivan, 1992). The best example of a drug with multiple mechanisms of action is amiodarone; it is also arguably the most effective class III antiarrhythmic agent and is currently approved for clinical use. Amiodarone is a benzofuran derivative that was originally developed as a coronary vasodilator, but was subsequently discovered to have antiarrhythmic activity,


especially after chronic administration (Singh et al., 1989). It is one of only a few drugs (along with β -blockers) that has been reported to actually reduce mortality of patients following myocardial infarction (Ceremuzynski et al., 1992). The cellular mechanisms of action of amiodarone are quite complex, but includes block of IKs (Balser et al., 1991), INa, ICa, and β - and β -adrenergic receptors (Task Force, 1991; Singh et al., 1993). Agents with multiple activities (e.g., block of IKr channels and β -adrenergic receptors like sotalol) may have a wide spectrum of activity since arrhythmias that arise from diverse cellular mechanisms may be affected. For this reason, it was hypothesized that the complex electrophysiological actions of a compound such as amiodarone may be preferable to those that act specifically on a single channel type such as dofetilide or E–4031 (Singh et al., 1993). Racemic sotalol is both a β -adrenergic receptor blocker (β -blocker) and an IKr channel blocker. The l-isomer is a more potent β -blocker (about 50–times greater) than the d-isomer, but the two compounds lengthen APD equipotently (Kato et al., 1986). In isolated rabbit Purkinje fibers, 10 μ M sotalol lengthened APD by 41% and decreased IK by 50%, a concentration that was without significant effect on IK1, IA or INa (Carmeliet, 1985). In guinea pig ventricular myocytes sotalol blocks IKr but had no effect on IKs at a concentration of 100 μ M (Sanguinetti and Jurkiewicz, 1990b). Sotalol was shown to be more effective than six class I antiarrhythmic agents in preventing death and recurrence of ventricular arrhythmias in a multicenter study (Beta-blocker Heart Attack Trial Research Group, 1982). It is unclear whether the protection afforded by sotalol is related to block of β -adrenergic receptors, or to reduction of IKr, since β -blockers have also been demonstrated to reduce mortality hi patients with a prior myocardial infarction (Norwegian Multicenter Study group, 1981; Beta-blocker Heart Attack Trial Research Group, 1982). Quinidine (a class 1A agent) and MS–551 (a class III agent) have been reported to block IK in a time- and voltage-dependent manner. Quinidine (Roden et al., 1988) and MS–551 (Nakaya et al., 1993) delay the activation of IK in guinea pig and rabbit ventricular myocytes, respectively. This delay was quantified by measuring the amplitude of deactivating tail currents following pulses of increasing duration to a common test potential. The tail amplitude becomes progressively larger as the test pulse duration is increased, in proportion to the slowly activating current recorded during the test pulse. In the presence of quinidine or MS–551, the tail currents were decreased more following short (Rb+>Cs+>Na+>Li+) (Hadley and Hume, 1990; Hausdorff et al., 1991), and the reversal potential for IminK shifts approximately 58 mV per decade change in [K +]e, as predicted by the Nernst equation for a perfectly K+ -selective channel (Hausdorff et al., 1991). The permeability ratio of K+/Cs+ is 16.4 for IminK expressed in oocytes, but this ratio is reduced to 4.6 when a point mutation (F55T) is introduced into the minK protein (Goldstein and Miller, 1991). This rinding has been cited repeatedly as the best evidence that minK proteins form functional channels and are not simply activators of an endogenous channel. Both IKs and IminK are blocked by mM levels of external Ba2+ (Folander et al., 1990; Honore et al., 1991), and are enhanced by increases in [Ca2+]i or intracellular cAMP (Blumenthal and Kaczmarek, 1992; Busch et al., 1992; Honore et al., 1992). The single channel conductance of IKs in guinea pig ventricular myocytes (Walsh et al., 1991) and IminK in Xenopus oocytes (Hausdorff et al., 1991) is below detectable levels (less than 1 pS). The antiarrhythmic agents, clofilium and NE10064 block IminK and guinea pig IKs with approximately equal potencies (Folander et al., 1990; Varnum et al., 1993). The bulk of evidence is consistent with the hypothesis that minK protein is the primary subunit of functional cardiac IKs channels.


10.4.2 Inward Rectifier K Channel Blockers The conductance of inward rectifier K+ current (IK1) in atrial and ventricular cells is high at negative membrane potentials and as such ‘clamps’ the resting potential near the equilibrium potential for K+ (about–85mV). The inward rectifier K channel of cardiac and many other cell types exhibits pronounced rectification at potentials positive to EK, the equilibrium potential for K+. In a series of elegant studies, this was demonstrated to be caused by at least two distinct molecular mechanisms: a very rapid and voltage-dependent inactivation, and block of open channels by intracellular divalent cations, primarily Mg2+ (Ishihara et al., 1989; Matsuda, 1988, 1991; Matsuda et al., 1987; Vandenberg, 1987), but also Ca2+ (Delmar et al., 1991; Mazzanti and DeFelice, 1990; Mazzanti and DiFrancesco, 1989). Upon depolarization, IK1 channels close almost instantly. IK1 channels remain closed throughout the plateau phase and thus do not contribute to the repolarization process until later in the action potential cycle. Thus, it is the activation of other outward K channels during the plateau phase that initiate repolarization and return the membrane potential to a level (about –20 mV) where IK1 channels can again open and contribute to terminal (phase 3) repolarization. Block of IK1 channels is often viewed as an undesirable mechanism by which to prolong ventricular refractoriness, since excessive block of this conductance would increase membrane resistance during diastole which in turn could result in membrane depolarization. An expected consequence of membrane depolarization would be a graded decrease in conduction velocity due to a voltage-dependent inactivation of Na channels. Despite these theoretical limitations, at least one novel class III antiarrhythmic agent, terikalant (11) was recently developed that is reported to act by specific block of IK1 channels. The RP 58866 (racemate) and its active enantiomer, terikalant (the (S)(–)isomer, Figure 10.5) are benzopyran derivatives that were discovered in a screen using guinea pig papillary muscles. The mechanisms of action of these compounds and in vivo activities were described recently (Escande et al., 1992), and are summarized below. Terikalant is about 150 –times more potent in prolonging cardiac action potentials than the inactive enantiomer. Terikalant prolonged APD90 of guinea pig papillary muscles and right atria when paced at 1 Hz by 21–23% at a concentration of 0.3 μ M. Similar effects were observed in isolated human atrial tissue (37% increase in APD90 at 0.15 μ M). The compound also slowed the spontaneous firing rate of isolated guinea pig right atria by 36% at much higher concentrations (30 μ M). Resting membrane potential was unaffected by 30 μ M terikalant in an isolated guinea pig right atrium. The lack of effect on resting membrane potential by a compound that blocks IK1 by >50% suggests that only a fraction of the normal outward IK1 in this tissue is required to maintain the membrane potential near EK during diastole. Prolongation of action potential duration and effective refractory period was attributed to block of IK1. In guinea pig myocytes RP 58866 reduced IK1 measured at –40 mV by 31% and


51% when tested at 3 and 10 μ M, respectively. At concentrations up to 30μ M, RP 58866 had no effect on IKs, IKATP, IKACh, or T- and L-type Ca2+ currents. The effect of these compounds on IKr was not determined. In anesthetized dogs, terikalant dose-dependently (0.03–1 mg/kg) prolonged atrial, nodal, and ventricular refractory periods; with a maximal increase of 52, 57, and 21%, respectively. Thus, similar to the IKr blockers, a greater prolongation of ERP was observed in atrial than in ventricular tissue. QTC was increased by 57% at 0.1 mg/kg. At doses up to 1 mg/kg terikalant had no significant effect on AH, HV, or QRS intervals, nor did it alter tissue excitability. This ‘pure’ class III action was associated with antiarrhythmic activity in several models of reentrant ventricular tachycardia or fibrillation. For example, pretreatment with terikalant at 0.3 mg/kg protected 4 of 5 micropigs from ischemia-induced ventricular fibrillation resulting from a 20 min ligation of the left ventricular coronary artery. An interesting correlation was found between the concentration of terikalant and some other class III antiarrhythmic drugs (e.g., clofilium (12)) to lengthen action potential duration of isolated Purkinje fibers and that required to displace the binding of an ― 2 opioid receptor ligand ([3H]–3–PPP) from a rat cerebral cortex preparation (Jeanjean et al., 1993). All the compounds were about 10–100 times more potent at displacing the β 2 ligand compared to their class III activity. For example, the IC50 for inhibition of [3H]–3–PPP binding by RP5866 and clofilium were 6.1 nM and 40 nM, respectively, whereas the EC20 for prolongation of APD90 for the two drugs was 14nM and 3μ M, respectively. These studies suggest that the β 2 opioid receptor is a protein very similar to one or more cardiac K channels. The clinical significance of the binding of these class III antiarrhythmic agents with β 2 opioid receptor sites in the brain is unknown. 10.4.3 Transient Outward K Channel Blockers IA activates extremely rapidly upon membrane depolarization, then inactivates quickly with a voltage-independent time constant of about 50 msec. Thus, this current contributes to net repolarizing outward current predominantly during the initial phase of action potential repolarization. Before the channels can reopen during a subsequent action potential, the channel must undergo a process referred to as recovery from inactivation. When studied in rats and rabbits, IA is characterized by a rather slow recovery from inactivation. This slow recovery translates into a reduced current magnitude at high stimulation rates in vitro, or high heart rates in vivo. Because of this property, the contribution of IA to net repolarizing K+ current would diminish during a tachycardia. Block of this current would therefore seem not to represent a useful antiarrhythmic mechanism, since less prolongation of action potential duration would result during tachycardias, a frequency-dependent profile opposite to that most desired (see


section 10.5). However, unlike rat and rabbit, the rate of recovery from IA activation is rapid in human cardiac myocytes, such that the magnitude of IA is not diminished during rapid pacing (Fermini et al., 1992). The magnitude of IA is much greater in the epicardium than in the endocardium of some species such as the dog and cat (Furukawa et al., 1990). In the rabbit, IA is only slightly larger in epicardial than in endocardial myocytes isolated from the wall of the ventricle, however papillary muscles have a much reduced IA (Fedida and Giles, 1991). The variable expression of IA channels across the wall of the ventricle may have significant consequences with respect to the antiarrhythmic/proarrhythmic effect of compounds that block IA. This possibility needs to be explored in the future. IA channels may represent a useful antiarrhythmic target, but no specific and potent blocker of this current has been described. A few agents (e.g., tedisamil, clofilium) are relatively potent blockers of IA, but these compounds also block other K+ currents. Tedisamil (15) was developed as a bradycardic agent for treatment of angina pectoris in patients with impaired cardiac function (Grohs et al., 1989), but it also has antiarrhythmic activity. At 1–4 mg/kg, tedisamil reduced ventricular fibrillation induced by occlusion of the left anterior descending coronary artery in rats (Beatch et al., 1991). This compound prolongs APD in rat ventricular myocytes by increasing the rate of IA channel inactivation. This action was hypothesized to result from preferential block of channels in the open state, an effect that would mimic accelerated inactivation (Dukes et al., 1990). Tedisamil also prolongs APD of guinea pig ventricular myocytes. However, these cells do not express IA channels. In the guinea pig, tedisamil acts instead by blocking IK at concentrations similar to that required for block of IA in the rat (Dukes et al., 1990). Thus, this drug has class III activity in both species, but the underlying mechanisms of action are different. Other compounds such as clofilium (12) and quinidine also reduce rat IA by increasing the apparent rate of current inactivation (Castle, 1991; Imaizumi and Giles, 1987). The block of IA by clofilium is very sensitive to voltage and repetitive pulsing (‘frequency-dependent block’). The enhancement of block that occurs during repetitive stimulation (Figure 10.9A) is believed to result from a relatively slow rate of drug dissociation from the channel during the interpulse interval, whereas the voltage-dependence of block (Figure 10.9B) arises from a preferential block of open channels (Castle, 1991). The greater the depolarization, the greater the probability of open channels that become available for block. An unusual feature of clofilium is that exposure of cells for several hours is required before steady-state effects on IA magnitude are observed. Presumedly this ultraslow onset of activity by clofilium is related to its lipophilicity and quaternary ammonium group. The onset of IA block by LY97119, a des-ethyl analog of clofilium with a tertiary nitrogen group, is much more rapid than clofilium, requiring only a few minutes to reach steady-state. In addition, LY97119 also blocks IK1 with an IC50 of about 3μ M (Arena and Kass, 1988; Castle, 1991). Similar to tedisamil, clofilium also blocks IK of guinea pig


ventricular myocytes (Arena and Kass, 1988), albeit at concentrations 10–times that required for a comparable decrease of IA in rat myocytes. The class 1 antiarrhythmic agents, quinidine, flecainide and propafenone block IA (IC50=3.9, 3.7 and 3.3 μ M, respectively) in addition to INa and IK when studied in isolated rat ventricular myocytes (Slawsky and Castle, 1994). Flecainide and quinidine, but not propafenone exhibit significant frequency-dependent block of IA in this preparation.


Figure 10.9 Voltage- and use-dependent block of IA in isolated rat ventricular myocytes by clofilium. Top panel: Relative amplitude of current during repetitive pulsing at 1 Hz. Myocytes were preincubated with drug for 3–9 hours prior to applying the train of clamp pulses. Bottom panel: Voltage-dependence of use-dependent block of IA by clofilium. The


amplitude of IA measured during a test pulse to +40 mV was dependent upon the prepulse voltage during the preceeding train of 10 pulses. From Castle (1991) with permission.

10.4.4 lK(ACh) Channel Blockers Many class I antiarrhythmic agents antagonize the actions of acetylcholine in addition to blocking INa. Quinidine, disopyramide, procainamide, pilsicainide, flecainide and propafenone all decrease the effects of acetylcholine on IK(ACh) recorded in guinea pig ventricular myocytes (Inomata et al., 1993), although pilisicainide, procainamide and disopyramide do so by blocking ACh receptors (Inomata et al., 1993; Nakajima et al., 1989). Block of atrial IK(ACh) channels causes tachycardia in innervated hearts, shortens refractory period and accelerates A-V nodal conduction time. Activation of these channels greatly shortens APD and hyperpolarizes the resting potential of all atrial cells, a possible antiarrhythmic action under conditions in which atrial APD is excessively prolonged, such as could occur after overdosing with a ‘pure’ class III antiarrhythmic agent. 10.4.5 IK(Na) Blockers A large conductance K channel activated by high levels of intracellular Na+ was first described by Kameyama et al. (1984). Conditions that lead to marked increases in [Na+]i increase the open probability of these channels, in turn causing a shortening of APD. R56865 is a benzothiazolamine derivative that was developed as an antiischemic agent, but also has antiarrhythmic activity in several in vivo and in vitro models (Koch et al., 1990). R56865 was also reported to potently protect against ischemiaand reperfusion-induced arrhythmias in anesthetized rats (Garner et al., 1990). For example, ischemia-induced ventricular fibrillation was reduced from >50% to 8% in rats administered 2 mg/kg R56865. The cellular mechanism of action of these protective effects is not exactly clear, but may relate to its ability to block the transient inward current (TI) and IK(Na) (Leyssens and Carmeliet, 1991; Luk and Carmeliet, 1990), two currents that are normally activated after treatment of cardiac cells with toxic concentrations of cardiac glycosides. The reduction of TI by R56865 was not due to direct block of Na+/Ca2+ exchange current or the nonselective cation channel, the two components of TI . Instead, it appears that the drug may somehow reduce Ca2+ overload, perhaps via interferring with Ca2+ release from the sarcoplasmic reticulum (Leyssens and Carmeliet, 1991). As expected from block of outward current through IK(Na) channels, R56865 limits the extent of APD shortening normally caused by


ouabain intoxication (Vollmer et al., 1987). At concentrations up to 3 μ M, this compound had no effect on force of contraction or APD in isolated guinea pig papillary muscles, but markedly attenuated the APD shortening of ouabain (0.8 μ M) at concentrations as low as 1 nM. In these isolated papillary muscles, R56865 (10 nM) also prevented the extrasystoles that developed in the presence of ouabain when muscles were stimulated at a frequency of 1 Hz (Vollmer et al., 1987). In single guinea pig ventricular myocytes R56865 blocked INa, albeit at higher concentrations (IC50=1 μ M) than those required to antagonize the effects of ouabain, whereas other K+ currents (IK1, steady-state outward current) were unaffected at 10 μ M (Himmel et al., 1990). R56865 reduced IK(Na) activated by ouabain in guinea pig myocytes by 35–70% at 0.1 μ M and by 76–98% at 1 μ M. The importance of IK(Na) activation during ischemia or cardiac glycoside intoxication and the contribution of this current to net outward current during these conditions has not been quantified sufficiently to determine whether modulation of this current as an antiarrhythmic mechanism warrants further study. 10.5 Rate-dependent Effects of K Channel Blockers D-sotalol and more potent methanesulfonanilides such as dofetilide, almokalant (10), E–4031 and L–691 121 are characterized by a rate-dependent lengthening of cardiac APD or effective refractory period (ERP) in vitro (Baskin et al., 1991; Gwilt et al., 1991; Lynch et al., 1992) and of ERP or QTC interval in vivo (Lynch et al., 1992, 1993), such that diminished activity is observed as heart rate or stimulation frequency is increased. As noted above, these agents are specific blockers of a time-dependent, delayed rectifier K+ current (IKr) of cardiac myocytes (Carmeliet, 1985, 1991, 1992; Lynch et al., 1993; Sanguinetti and Jurkiewicz, 1990b). Theoretically, an ideal drug would prolong refractory period preferentially during tachyarrhythmias and thus, would block outward current (e.g., IKr) in a direct frequency-dependent manner (more block at high heart rates) (Carmeliet, 1993; Colatsky et al., 1990; Hondeghem and Snyders, 1990; Jurkiewicz and Sanguinetti, 1993). The rate-dependent efficacy of class III antiarrhythmic agents is further compromised in the presence of adrenergic stimulation. Isoproterenol functionally antagonizes the ability of E–4031 to prolong refractory period or APD in guinea pig papillary muscles or ventricular myocytes, respectively (Sanguinetti et al., 1991). The potential mechanisms for frequency-dependent drug action are many-fold, but only a few have actually been tested experimentally. Frequency-modulated actions of Ca or Na channel blocking agents have usually been attributed to either a channel-state-dependent or voltage-dependent block of channels. For example, class I antiarrhythmic drugs such as lidocaine decrease the upstroke velocity of cardiac action potential of single cells, decrease conduction velocity in multicellular cardiac preparations, and widen the QRS complex of the


electrocardiogram more at high, relative to low heart or stimulation rates. This augmented effect is a direct result of an increase in the number of Na channels that are blocked during more rapid, repetitive membrane depolarizations (Hondeghem and Katzung, 1984). By analogy, a logical explanation for the rate-dependent effect of class III agents would be the opposite phenomenon—a decrease in the number of K channels blocked by such agents as the frequency of repetitive depolarizations is increased (Hondeghem and Snyders, 1990). This potential mechanism has been evaluated and shown not to be the case for dofetilide (Carmeliet, 1992; Jurkiewicz and Sanguinetti, 1993), almokalant (=H234/09) (Carmeliet, 1991), or sematilide, d,l–sotalol and E–4031 (Krafte and Volberg, 1993). Dofetilide prolongs APD of isolated guinea pig ventricular myocytes more at 0.5 Hz than at 4 Hz when tested at a maximal effective concentration of 1 μ M. Whole-cell voltage clamp experiments revealed that 1 μ M dofetilide completely blocks IKr, regardless of stimulation frequency (Jurkiewicz and Sanguinetti, 1993). This observation rules out the possibility that the frequencydependent effects of these drugs on APD of cardiac cells results simply from a ‘use-dependent’ or voltage-dependent unblock of IKr. Even at submaximal concentrations, dofetilide and almokalant do not exhibit decreased blocking activity at elevated stimulation frequencies. In fact, Carmeliet has reported that these agents block IK of rabbit myocytes slightly more when voltage clamp pulses are applied at fast relative to slow rates (Carmeliet, 1991, 1992). Unlike guinea pigs, rabbit myocytes have a single type of IK, which is similar to IKr with respect to its rectification properties and sensitivity to methanesulfonanilides (Carmeliet, 1992; Shibasaki, 1987). The rate-dependent increase in block of rabbit IK by dofetilide and almokalant results from preferential block of IK channels in the open (as opposed to closed) state. Unblock of IK is extremely slow during interpulse intervals at ‘diastolic’ potentials (ADP>ADPβ S=AMP-PNP>2’deoxy-ATP>UTP>2’deoxyADP>GTP>GDP>UDP. Of course in vivo intracellular Mg2+ will always be available, but its concentration and subcellular localisation may be subjected to additional levels of regulation (Henquin et al., 1983). If this were the case, then


changes in the [Mg2+]i would have marked effects on the nucleotide-dependent gating of KATP channels. Pharmacology of KATP channels in β -cells Blockers of KATP channels Arguably the most important group of pharmacological agents capable of inhibiting KATP channels in the β -cells are the antidiabetic sulphonylureas. In this review we only have space to outline the effects of the sulphonylureas on ion channels and insulin secretion, and for a detailed review we recommend Ashcroft and Ashcroft (1992). Sulphonylureas, typified by the first generation compound tolbutamide, and the more potent second generation molecule glibenclamide, are a class of hypoglycaemia-inducing drugs that have been used for a number of years to treat type II or non-insulin dependent diabetes (reviewed by Hellman and Taljedal, 1975). These compounds promote insulin secretion from the pancreas by a mechanism associated with a decrease in the K+ permeability of the β -cell membrane (Henquin, 1980; Gylfe et al., 1984); which leads to a depolarisation of the cell, and voltage-gated Ca2+ influx (Gylfe et al., 1984; Henquin and Meissner, 1982). Patch-clamp studies have shown that these effects are mediated by the sulphonylureas selectively closing KATP channels (Sturgess et al., 1985; Dunne et al., 1987; Ohno-Shosaku et al., 1987; Zunkler et al., 1988b; Ashcroft et al., 1989; Dunne, 1990b). The potency of channel block falls over three orders of magnitude: tolbutamide KI=10–17 µM, meglitinide K1=2.1 µM, glipizide KI=6.4 nM and glibenclamide KI=4–20 nM (Zunkler et al., 1988a; Ashcroft et al., 1989; Sturgess et al., 1988). Indeed channel block by glibenclamide is more potent than that of either tetrodotoxin or saxitoxin for the voltage-gated Na channel, and this has successfully helped in the biochemical identification of sulphonylurea receptors. Receptor occupancy by the sulphonylureas was first shown to be closely associated to channel blockade by Schmid-Antomarchi and collaborators (1987). Once suitable corrections have been made for albumin binding to the drug (Aguilar-Bryan et al., 1990), the specificity and density of sulphonylurea binding sites correlates very well with the ability of the sulphonylureas to elicit secretion, and inhibit KATP channels (Niki et al., 1989; Schmid-Antomarchi et al., 1987). There is also a good correlation between the estimated number of sulphonylurea receptor binding sites and the number of KATP channels per cell (Dunne and Petersen, 1990; Rorsman and Trube, 1990). Because of this there has been a great deal of speculation as to whether the sulphonylurea binding protein is an integral part of the KATP channel or not. One way to address this is to provide insights into the mechanism(s) of action of the sulphonylureas. Gylfe and collaborators (1984) originally suggested that the drugs only act from the outside of the cell. However, patch-clamp experiments have shown that sulphonylureas are effective from both sides of the plasma membrane, closing channels in whole-cells as well as excised inside-out, outside-out and cell-

K CHANNELS IN PANCREATIC ― -CELLS 383

attached membrane patches (Trube et al., 1986). The cell-attached patch data are interesting in that direct access to channels from the bath solution is occluded by the patch-clamp recording pipette. These data suggest that the receptor must be reached from either the inside of the membrane or alternatively by lateral diffusion through the lipid domain of the membrane. A similar conclusion was also reached by Zunkler et al. (1989) who showed that channels are inhibited by the undissociated forms of tolbutamide and its related compounds. Inhibition of channels is not dependent upon the availability of Mg2+ (Dunne et al., 1987; Schwanstecher et al., 1994), although in the complete absence of internal Mg2+ the effectiveness of the sulphonylureas is reduced (Lee et al., 1994). There are however interactions between cytoplasmic nucleotides and the effects of tolbutamide. In particular sulphonylureas are more potent blockers in the presence of ATP, AMP-PNP, ADPβ S or ADP (Zunkler et al., 1988b; Schwanstecher et al., 1994); maybe indicating that the effects of the drugs are not directly onto the channel. The high affinity of glibenclamide for the KATP channel has meant that the drug can be used in the purification, and isolation of the putative channel protein. From porcine brain tissue, a protein of approximately 150 KDa has been isolated (Bernadi et al., 1988), whilst using a biologically active derivative of glibenclamide—5-iodo–2–hydroxyglyburide, a 140 KDa protein was subsequently isolated from the membranes of HIT insulinoma cells (AguilarBryan et al., 1990). This protein has been found to be an ADP-binding protein (Niki et al., 1990), but despite this, no sequence data have yet been reported, nor has the isolated protein been functionally reconstituted and shown to possess iontophoretic activity. This has fuelled speculation that the sulphonylurea receptor and the KATP channel are functionally and structurally distinct proteins, and aspects of patch-clamp data would tend to support this. First, in ventromedial hypothalamic neurones KATP channels are unaffected by tolbutamide in excised inside-out membrane patches but are blocked by the drug in cell-attached patches (Ashford et al., 1990); implying that the sulphonylurea receptor is not ‘tightly’ coupled to the KATP channel. Second, trypsinisation of the inside face of the cell membrane significantly reduces the effects of sulphonylureas on KATP channels in pancreatic β -cells (Proks and Ashcroft, 1993), and all but eliminates their action in cardiac myocytes (Nichols and Lopatin, 1993). In both the insulin-secreting cells and cardiac myocytes ATP-induced channel block is reduced but not eliminated by trypsin treatment, thereby indicating that proteolysis of the membrane uncouples the sulphonylurea- but not the ATP-dependent gating of the channel. Third, a subclone of the Cambridge insulinoma cell line CRI-G1 has recently been characterised—designated CRID11, and these cells have functional KATP channels, but unlike the CRI-G1 cells they are unresponsive to sulphonylureas in terms of ligand binding data, electrophysiology and insulin secretion (Ozanne et al., 1993). These cells provide another example of how the effects of sulphonylureas can be dissociated from nucleotide-dependent gating of KATP channels in insulin-secreting cells.


Finally, unpublished data from our laboratory suggest that although sulphonylureas can block KATP channels in patches immediately upon formation of an excised patch, many minutes later the effects are lost, but the channels can still be inhibited by ATP. This may be somewhat analogous to the situation in the central medial hypothalamic neurons, but occurring over much longer periods of time because of a relatively higher affinity of the sulphonylureas for KATP channels in the β -cell. Clinically the sulphonylureas are widely used in the treatment of type II diabetes but after several years of therapy many patients develop a resistance to their effects. Since it is still not clear why this should occur, alternative compounds have been developed in the hope of finding a more permanent and effective treatment. One of these compounds linogliride, is structurally distinct from the sulphonylureas, and will both lower blood glucose levels and promote insulin secretion from the pancreas (see Ronner et al., 1991a, 1991b). The mechanism that underlies the effect of linogliride on the pancreatic β -cell has been investigated, and as for the sulphonylureas its effects are mediated by closure of KATP channels (Ronner et al., 1991a). Overall linogliride is a much less effective blocker of KATP cannels (IC50 = approximately 20 µM) than glibenclamide, but the data do indicate how therapeutic intervention of these channels could lead to the development of more novel compounds that may be effective in the management of diabetes. There is also a great deal of interest in the effects of β -adrenoceptor antagonists as potential modulators of K channels in the insulin-secreting cell. For a number of years it has been recognised that certain compounds such as phentolamine (β 1 and β 2) and efaroxan (β 2) are able to initiate insulin secretion from β -cells by mechanisms not involving the β -adrenoceptor (Malaisse et al., 1967; Schultz and Hasselblatt, 1989; Chan and Morgan, 1990). Both compounds will also alleviate the effects of diazoxide on insulin-secreting cells, and we can explain these effects by the fact that both phentolamine and efaroxan are inhibitors of KATP channels (Henquin et al., 1982; Dunne et al., 1990c; Plant and Henquin, 1990; Chan et al., 1991; Jonas et al., 1992). Concentration-response relationships from excised patches suggest that 50% blockade of channels could be achieved by approximately 12 µM efaroxan and 0.7 µM phentolamine (Chan et al., 1991; Dunne, 1991). These compounds are therefore as effective at blocking KATP channels as tolbutamide, but not glibenclamide. ATP-regulated K channels can also be blocked by the β 2-adrenoceptor antagonists yohimbine (Plant and Henquin, 1990; Dunne, 1991), antazoline and tolazoline (Dunne, 1991; Jonas et al., 1992). With the exception of yohimbine, the common structural feature of these compounds is an imidazoline group. It is now believed that several tissues express specific and functional receptors for imidazolines that have been termed ‘imidazoline preferred binding sites’ (Michel and Insel, 1989; Michel and Ernsberger, 1992). These receptors are functionally distinct from β -adrenoceptors, and may comprise at least two subtypes—designated I1 and I2, which may differ in their tissue distribution, subcellular location and


pharmacological properties (Ernsberger, 1992; Atlas, 1991; Heible and Ruffolo, 1992; Kilpatrick et al., 1992). From electrophysiological studies we have the evidence that certain imidazolines will modulate KATP channels, but there is no indication from these types of studies that the effects are ‘receptor-mediated’. Indeed, the substituted imidazoline idazoxan (I2 receptor agonist), at low concentrations, has little effect upon insulin secretion in the absence of an adrenoceptor agonist (Ostenson et al., 1988; Chan and Morgan, 1990), but at higher concentrations (>100 µM) will partially reverse diazoxide-induced inhibition of insulin secretion (Chan and Morgan, 1990) and block KATP channels (Chan et al., 1991). If we compare the relative affinities of both efaroxan and idazoxan for the imidazoline receptor with the effects of the drugs on K channels and insulin secretion, we find that there is a very obvious negative correlation. Both efaroxan and phentolamine have a low affinity for the I2 receptor(s) (Langin and Lafontan, 1989) but are both very effective at closing KATP channels and initiating insulin secretion, whereas idazoxan has a very high affinity for the receptor but has relatively little effects upon K channels and secretion over the same concentration range. Therefore the relationship between imidazoline effects at the cellular level and inhibition of KATP channels may not be as straightforward as was originally thought. Recent experiments from our laboratory using human isolated pancreatic β -cells, indicate that imidazolines will elevate intracellular calcium levels ([Ca2+]i) through a depolarisation of the cell membrane caused by block of both the KATP channel and the large Ca- and voltage-gated Kmaxi (BKCa) channel (Dunne et al., 1994b). In summary, we believe that either the effects of imidazolines on K channels in the β -cell are not directly governed by receptor-operated events but involve direct binding with the ion channels, or that the β -cell possesses a novel subtype of imidazoline receptor that does not fall within the I1 (‘clonidine-preferring’) or I2 (‘idazoxan-preferring’) designated nomenclature (Chan et al., 1993). Concerning the mechanism (S) of channel block, we have evidence that the site of interaction on the KATP channel is distinct from that of the sulphonylureas. This is based primarily upon the fact that imidazolines block the channels after proteolytic treatment of the inside face of the cell membrane with trypsin (Dunne et al., 1994b); whereas the effects of the sulphonylureas under these conditions are markedly reduced (Proks and Ashcroft, 1993). Despite the uncertainty surrounding the nature of the imidazoline receptor expressed by the β -cell, and its relationship with the KATP channel, imidazolines may still provide an alternative therapy to the sulphonylureas in the treatment of type II diabetes (Kawazu et al., 1987). Finally, a large number of other compounds have also been found to block KATP channels in the β -cell: antiarrythmic agents such disopyramide (IC50=3.6 µM; Hayashi et al. 1993) and cibenzoline (IC50=1.5 µM; Kakei et al., 1993), the herbal extract ligustrazine (Peers et al., 1990), sparteine and the dopaminergic agonist amantadine (Ashcroft et al., 1992a), the protein kinase C inhibitor polymyxin B (Harding et al., 1994), TMB–8 (8(N,N-diethylamino)-octyl–3,4,5–


trimethoxybenzoate) (Szewczck et al., 1992), the local anaesthetics pentobarbitone, thiopentane, secobarbitone and phenobarbitone (Kozlowski and Ashford, 1991) and certain fluorescein derivatives which are capable of both channel inhibition and activation (De Weille et al., 1992). Finally, KATP channels are also closed by a number of more conventional K channel blockers (KCBs) such as; quinine (Findlay et al., 1985c) (Kd=40 µM), aminoacridine (100 µM) and 4– aminopyridine (100 µM) (Cook and Hales, 1984). Tetraethylammonium ions (TEA) at relatively low concentrations (100 µM, whilst the channels are more sensitive to AMP>ADP>ATP (Sturgess et al., 1987). Recent experiments undertaken in our laboratory however indicate that the channels may be far more sensitive to the adenosine nucleotides than previously thought—especially when more than one nucleotide is available. We find that these channels are present along with KATP channels in intact resting cells, and interestingly we see these channels more frequently in human than in either rodent or clonal β -cells. This may imply that the channel is able to contribute to the resting cell membrane potential or background K+ or Na + current. By analogy with other systems it may be involved in the generation of slow-waves of potential change, but as there are no known selective agonists or antagonists to these channels, it is difficult to assess their precise contribution to electrical events in the β -cell.


13.3.5 Transient Outward K (KA) Channels Only a short communication has described the presence of a 4–AP-sensitive K channel in mouse β -cells that is similar to one of the two transient outward K channels present in the myocardium (Smith et al., 1989). The inactivation profile of this channel in patch-clamp experiments indicates that it would be half maximally inactivated at normal resting membrane potentials, and completely inactive at membrane potentials experienced in the presence of glucose. This channel is not seen in all preparations of β -cells, and its role in the electrophysiology is unlikely to be significant. 13.3.6 Receptor-operated K (K1) Channels A K channel activated by hyperglycaemia-inducing hormones and neurotransmitters has recently been identified in mouse β -cells (Rorsman et al., 1991). This channel could only be examined through analysis of ‘noise’ associated with the receptor-mediated events, and may be the same channel as the low-conductance apamin-insensitive K channel (see above). So far this channel has been shown to be activated by adrenaline in a G-protein dependent manner, and by the adrenoceptor agonist clonidine. Activation of the K1 channel may therefore be responsible for the membrane hyperpolarisation evoked by adrenaline, and may possibly be involved in the effects of other inhibitory hormonal and neural influences on the β -cell. 13.3.7 ATP-activated K Channels A second type of ATP-regulated K channel has recently been described in the laboratory of Fran Ashcroft (Williams et al., 1993). This channel is functionally distinct from the KATP channel: it exhibits no inward current rectification, shows no sensitivity to the sulphonylureas, and is activated by internal ATP. Interestingly, this study was undertaken using β -cells isolated from a Type II diabetic human donor. ATP-activated K channels have not (so far) been reported in any other β -cell preparation, including human tissue. One important implication of these findings is that this type of channel could be a cause of (or indeed result from) the pathophysiological state of the β -cell. 13.4 Na Channels and Insulin-secreting Cells Na channels are perhaps the least well characterised of all β -cell ion channels. One possible reason for this may be that the contribution (s) of the Na+ current to


electrical events may vary significantly in β -cells from one species to the next. Tetrodotoxin, a specific inhibitor of voltage-gated Na channels has consistently been reported to inhibit glucose-induced secretion, and to be without effect (see Dunne et al., (1990d) for further discussion and references). In terms of the properties of Na channels, one study carried out upon mouse cells demonstrated that the Na + currents are inactivated at normal membrane potentials, and therefore could not contribute to the cell electrical activity (Plant, 1988). On the other hand, in the rat inactivation potentials are around–40 mV (Hiriart and Matteson, 1988), whilst in human and canine β -cells there is good evidence that Na channel activity contributes significantly to the action potential depolarisation (Pressel and Misler, 1991). In Table 13.1 Overview of ion channels in pancreatic β -cells and their possible physiological significance to cell electrophysiology β -Cell Ion Channel Calcium Channels ― L-type voltage-gated Ca2+ channel. ― T-type voltage-gated Ca2+ channel. Potassium Channels ― ATP-sensitive K channel. (KATP channel) ― ― ― ― ― ―

Calcium- and voltage KMaxi channel. Low-conductance KCa channel. Receptor-operated KI channel. Delayed rectifier KDR channel. Transient outward KA channel. Non-selective cation KNS channel.

― ATP-activated K channel. Sodium Channels ― Voltage-gated Na channel. –rodent β -cell –human, canine ß-cell –clonal ß-cell Chloride Channels ― Uncharacterised

Possible Physiological Role? Ca2+ influx during action potential Ca2+ influx during slow waves. Not established. Initiation of membrane depolarisation. Contribution to slow waves. Receptor-mediated events. Action potential repolarisation. Generation of slow waves. Receptor-mediated events. Action potential repolarisation. Not established. Contribution to initial depolarisation? Contribution to slow waves? Only seen in diabetic tissue.

Inactive at critical membrane potentials. Generation of action potential. Maintenance of electrical activity. Contribution to initial depolarisation? Contribution to slow waves?

clonal β -cells Na channels may have a more pronounced role since without external Na+ ions, nutrient-induced electrical events and the elevation of [Ca2+]i are abolished (Dunne et al., 1990d). In these cells phorbol esters will suppress


Na+ currents suggesting a possible role for protein kinase C in regulating their properties (Rorsman et al, 1986). 13.5 Summary With the approaches of conventional microelectrodes and patch-clamp techniques we now have a much better understanding of the sequences of events that are responsible for the complex changes in β -cell electrical activity elicited by glucose. It is obvious to relate the properties of voltage-gated Ca channels to the regulation of [Ca2+]i, but other ion channels particularly K channels are important in governing their operation. Therefore, the initiation, the maintenance and then finally the termination of glucose-induced electrical events in the β -cell involves the integrated control of several different types of ion channels (see Figure 13.1 and Table 13.1). We are also left with many more unanswered questions. For instance, what and how are cytoplasmic second messengers able to govern the opening of ion channels, and which ion channels are involved with the regulation of insulin secretion in response to the many hormones, neuropeptides and pharmacological agents that are able to influence the pancreatic β -cell. Moreover, the study of ion channel regulation in human isolated β -cells has only just begun, and whilst there is a great deal in common between rodent, clonal and human insulin-secreting cells there are some important differences in the properties of ion channels between the different species. Acknowledgements Work in our laboratory has been generously funded by project/equipment grants to MJD from The British Diabetic Association, The Wellcome Trust, The Yorkshire Cancer Research Campaign, The Medical Research Council, The British Heart Foundation, The Nuffield Foundation, The Royal Society, and the University of Sheffield. We are indebted to the continuing support of Roger James and Nick London at the University of Leicester, Department of Surgery for their collaboration on studies of ion channels and stimulus-secretion coupling mechanisms in human islets of Langerhans. References AGUILAR-BRYAN, L., NELSON, D.A., VU, Q.A., HUMPHREY, M.B., BOYD, A.E. III (1990) J. Biol. Chem., 265, 8218–8224. ÄMMÄLÄ, C., LARSSON, O., BERGGREN, P.-O., BOKVIST, K., JUNTTIBERGGREN, L., KINDMARK, H. & RORSMAN, P. (1991) Nature, 353, 849–852. ÄMMÄLÄ, C., BOKVIST, K., LARSSON, O., BERGGREN, P.-O. & RORSMAN, P. (1993) Pflugers Arch., 422, 443–448. ARKHAMMAR, P., HALLBERG, A., KINDMAK, H., NILSSON, T., RORSMAN, P. & BERGGREN, P.-O. (1990) Biochem. J., 265, 203–211.


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14 Potassium Channels and their Modulation in Urogenital Tract Smooth Muscles A.F.BRADING1 & W.H.TURNER2 1

Department of Pharmacology, Oxford University, Mansfield Road, Oxford, OX1 3QT, UK. 2

Department of Urology, Inselspital, Bern, Switzerland. 14.1 Introduction

Smooth muscles in the urogenital tract exemplify the amazing ability of these muscles to display quite different properties which fit them exquisitely to their separate functions. It is likely, for instance, that each muscle will have a unique combination of ion channels in its plasma membranes, which will govern its spontaneous electrical activity and its ability to respond to neurotransmitters and endogenous ligands. Amongst these channels will be the K channels, many types of which are expressed by smooth muscles. These channels will play an important role in determining the ‘resting’ membrane potential of the cells, the shapes and frequency of the spontaneous action potentials and the membrane response to receptor ligands. We do not yet know precisely which channels are present in which cells, but use of the increasing number of drugs and toxins that have been shown to modulate their activity, clearly demonstrates the importance of these channels. In this chapter, we will briefly describe the function and properties of urogenital smooth muscle, highlight the clinical problems, and discuss what is known about their K channels and the therapeutic potential of modifying K channel behaviour. 14.2 The Urinary Tract The smooth muscles of importance in the urinary tract are the ureters, the detrusor of the bladder and the urethral smooth muscle. 14.2.1 Ureters The ureters transmit urine from the kidneys to the bladder. This keeps renal pelvis pressure low, an essential function, since ureteric hold-up and consequent

K CHANNELS AND THEIR MODULATION IN UROGENITAL TRACT SMOOTH MUSCLES 407

raised renal pelvis pressure would soon stop filtration. Action potentials are initiated in the renal pelvis and sweep down the length of the ureter through the well-coupled smooth muscle cells, initiating a wave of contraction that pushes urine into the bladder. As in the heart, it is essential that tetanic contractions are avoided, since maintained tone would stop urine transport and allow build up of renal pelvis pressure. The action potentials thus have a long plateau, the contractile responses are very phasic in nature, and the contraction strength is related to the duration of the plateau (Shuba, 1981). Again like the heart, although there is a primary pacemaker, smooth muscle cells throughout the ureter can, under the right conditions, take over pacemaking activity (Golenhofen and Hannappel, 1973). Although there is little evidence for any direct sympathetic or parasympathetic innervation of the ureteric smooth muscles, activation of capsaicin sensitive sensory-motor nerves can modulate activity (Maggi and Giuliani, 1991), as can application of catecholamines and autacoids (Shuba, 1977). K channels in ureter Early experiments using a double sucrose-gap voltage clamp technique (Shuba, 1981) suggested that there were two components of voltage-sensitive outward currents in the ureter, one of which was blocked by tetraethylammonium ions (TEA). More recent work on enzymically dispersed single smooth muscle cells from guinea-pig ureter (Imaizumi et al., 1989; Lang, 1989) confirmed the presence of two main outward current components. The outward currents were a Ca-dependent K+ current, blocked by TEA, and a transient outward current blocked by 4-aminopyridine (4–AP). Unlike many other smooth muscles, there was little evidence of any voltage-dependent delayed rectifier channels. Single channel records (Imaizumi et al., 1990) demonstrate the frequent presence of large-conductance Ca-activated K channels (130 pS at physiological K+ gradients; BKCa or maxi-K), and a much less common small conductance channel (about 17 pS) similar to channels carrying the outward transient current (IA) in other preparations. It appears that the absence of the delayed rectifier channels allows the development of the long plateau. The role of the Ca-activated K channels may be in terminating the plateau due to a slow build-up of cellular Ca2 + during the contraction. The transient outward current plays no role during the plateau. It is probably partially active at the resting potential, and Imaizumi et al. (1990) have suggested that it would oppose the Ca2+ inward current responsible for the spike. It could thus help to ensure that spontaneous action potentials are not normally evoked in the main part of the ureter. The transient outward current is inactivated rapidly on depolarization and thus under local depolarizing conditions its braking action may be reduced, allowing local production of action potentials. It is possible that this mechanism would permit the ureter to try and overcome an obstruction.


The ureteric smooth muscle cells also seem to possess another K channel, since the K channel opening (KCO) drugs cromakalim (CRK), pinacidil, nicorandil and S 0121 have been shown to reduce the frequency and size of the K + -stimulated rhythmic activity in the rabbit and guinea-pig ureter (Klaus et al., 1990). CRK also produced a dose-dependent reduction in the size of the electrically evoked contractions of the ureter, completely abolishing the response at about 3 µM and it also suppressed the spontaneous activity of the renal pelvis. In the intact ureter in vivo, the KCO levcromakalim (LCRK) perfused through the ureter and decreased the frequency of the spontaneous contractions dosedependently (Kontani et al., 1993), but glibenclamide alone had no effect on the spontaneous activity. Microelectrode recordings (Klaus et al., 1990) and sucrose gap recordings (Maggi et al., 1994) demonstrated the ability of CRK to hyperpolarize the membrane. CRK also initially caused a shortening of the plateau before completely abolishing the action potential (Figure 14.1). These effects were reversed by application of glibenclamide, but TEA and 4AP were much less effective, and also less effective than they were on the Ca-dependent K + currents and the transient outward K+ current (Maggi et al., 1994). Although there is little evidence for excitatory neurotransmission in the ureter, it has recently been proposed that activation of sensory nerves can release calcitonin gene related peptide (CGRP) in the wall of the ureter, and that this peptide acts as an inhibitory neurotransmitter (Maggi and Giuliani, 1991). Electrophysiological recordings using the sucrose gap show that activation of intrinsic nerves, and application of CGRP cause hyperpolarization of the smooth muscle cells (Santicioli and Maggi, 1994). Interestingly, the effects of CGRP are abolished by glibenclamide, leading to the novel suggestion that this neurotransmitter may be activating the same channels that CRK can activate. Functionally this could be important in producing quiescence of the tissue when local damage and inflammation is enough to trigger the nociceptive sensory nerves and could be important in limiting retrograde transmission of action potentials and urine back to the kidney.


Figure 14.1 Sucrose gap recording of the effects of cromakalim (CRK) on electrically evoked activity of the guinea-pig ureter. The inset shows contractile responses (upper trace) and action potentials (lower trace) on a slow time base. The electrical recordings indicated by A (control) B and C (after application of 0.3 µM CRK) are shown expanded. Note the hyperpolarization and initial shortening of the action potential after addition of cromakalim. (From Maggi et al., 1994).

14.2.2 Detrusor The detrusor is the smooth muscle in the wall of the part of the bladder which lies above the trigone. It generates the contraction which empties the bladder during voiding. In the filling phase, the bladder wall does not behave as a floppy bag, but maintains the minimum surface area:volume relationship available to it anatomically, so that if micturition is initiated, the synchronous activation of the muscle can immediately raise intravesical pressure. Thus during filling the smooth muscle cells must lengthen but maintain enough tone to keep the bladder in shape. This is achieved through spontaneous electrical and mechanical activity. It is, however, essential that this activity does not prevent the bladder filling at low pressure, and evidence is available that synchronous activity is prevented by rather sparse electrical coupling between the cells. This poor coupling has been studied in some detail in the guinea-pig bladder, using measurement of input impedance (Brading et al., 1989) and more recently with the use of two


microelectrodes for recording potential and passing current (N. Bramich, Figure 14.2, personal communication). The latter experiments demonstrate that current flow at right angles to the cells’ longitudinal axis rarely occurs between cells separated by more than a few cell widths. However, the time course of the voltage response to current injection in a single cell suggests that each cell is probably coupled to its near neighbours. Activation of intrinsic motor nerves generates synchronous excitatory junction potentials and spikes in all cells in the strip, even if they are not electrically coupled, since the density of innervation is very high. In isolated tissue strips spontaneous activity occurs, and the pattern of spontaneous mechanical activity also probably reflects the poor coupling between cells. Spontaneous contractions are small in comparison to the maximum force a strip can develop in response to nerve stimulation, and they rise from and fall to the basal tension. Tetanic activity is normally never seen. The individual contractions often vary in size, and it seems likely that this is determined by the number of smooth muscle groups that happen to be firing action potentials at the same time. A characteristic of the strips is their response to sudden stretch, which results in a transient increase in activity which then decreases progressively with time, towards the basal tension. A major functional bladder disorder is detrusor instability, a condition in which during bladder filling the pressure does not remain uniformly low, but phasic increases occur, which cannot be suppressed voluntarily in man. This can lead to incontinence. Detrusor instability occurs in association with bladder outflow obstruction and with neuropathic disorders, but often there is no associated abnormality, so-called idiopathic detrusor instability. Artificial outflow obstruction in animal models can lead to conditions resembling detrusor instability, particularly in the pig (Jorgensen et al., 1983; Sibley, 1985; Speakman et al., 1987) and the rat (Malmgren, 1987; Saito et al., 1993). Instability in both humans and in the animal models is frequently associated with a reduction in motor innervation (see Brading and Turner, 1994) and smooth muscle strips from unstable bladders show changes to their properties which include a supersensitivity to agonist drugs, and in the pig and human, alteration in the spontaneous activity so that fused tetanic contractions are now seen (Brading and Turner, 1994). There is also evidence of increased electrical coupling between the cells, which may allow synchronous activity to


Figure 14.2 Simultaneous microelectrode recordings from two cells in strips of guineapig detrusor. The bathing solution contained 10−6 M nifedipine to prevent action


potentials. Excitatory junction potentials (EJPs) evoked by transmural stimulation. In A, note the simultaneous evoked EJPs in both cells, but non-synchronous spontaneous junction potentials (SEJPs). In B, the top pair of tracings show simultaneous evoked EJPs, and the bottom trace (from the same pair of cells) shows the lack of electrical coupling between the cells, since current injected into one cell did not affect the membrane potential in the second cell.

spread across the bladder wall leading to the unstable contraction in vivo (Fujii, 1988). K channels in the detrusor: contribution to shape of action potential Micro-electrode studies of action potentials in small mammal bladders show rising phases that are normally somewhat slower than the falling phase (rate of rise 4.2 V/s, rate of fall 5.4 V/s; Kurihara, 1975), with a pronounced but variable after-hyperpolarization. At present there are no published records of action potentials recorded with micro-electrodes from detrusor of larger mammals or humans. Action potentials have, however, been recorded under current-clamp conditions using the whole-cell recording mode from isolated human bladder myocytes (Montgomery and Fry, 1992), and show a similar shape, although without the after-hyperpolarization. The comparatively rapid falling phase suggests the opening of K channels, and whole-cell voltage clamp studies on single myocytes isolated from the bladder demonstrate the presence of voltage sensitive outward currents (guinea pig: Klöckner and Isenberg, 1985; human: Montgomery and Fry, 1992). The falling phase of the spike and the afterhyperpolarization can be differentially affected by various K channel blocking drugs (KCBs). In the guinea-pig, apamin (100 nM) and 4AP (5 mM) had no measurable effect on the rate of repolarization of the spikes, TEA (10 mM) and quinidine (100 µM) caused some prolongation of the falling phase (the time for half maximal repolarization increasing to 220% and 340% of control respectively), and procaine (5 mM) prolonged the falling phase by more than tenfold. At the above concentrations, procaine and apamin completely eliminated the after-hyperpolarization, 4AP and TEA had little effect and quinidine reduced it (Figure 14.3, Fujii et al., 1990). All of the KCBs so far tested, including charybdotoxin can enhance the spontaneous mechanical activity in the guineapig detrusor (Fujii et al., 1990; Zografos et al., 1992). These results strongly suggest that more than one type of K channel is present in guinea-pig detrusor. Voltage clamp studies (whole-cell and patch) have clearly demonstrated the presence of Ca- and voltage-activated maxi K channels (Markwardt and Isenberg, 1992; Suzuki et al., 1992) in the guinea-pig detrusor smooth muscles, and evidence strongly supports their presence in human bladder myocytes (Montgomery and Fry, 1992). In some conditions spontaneous transient outward currents (STOCS) can be seen, similar to those in other smooth muscles, in which they are thought to be caused by spontaneous release of stored Ca2+


triggering activation of maxi K channels (Bolton and Lim, 1989). The current through these channels is inhibited by cyclopiazonic acid, a specific inhibitor of the Ca2+ ATPase of the sarcoplasmic reticulum (Suzuki et al., 1992), by reduction in intracellular Ca2+ and by TEA. Markwardt and Isenberg have suggested that the rapidity of activation of these channels by intracellular Ca2+, would enable them to be responsible for the repolarization of the spike. Another possible role of the maxi-K channels is in the responses of the smooth muscles to stretch. Recently Wellner and Isenberg (1994) have demonstrated the presence of stretch activated non-selective cation channels in isolated myocytes from guinea-pig detrusor. Stretch generates net inward currents in the whole-cell mode, but this current declines spontaneously with time. Analysis shows that the stretch-activated non-selective cation channels do not themselves inactivate with time, but that the net current is reduced due to activation of the maxi-K channels,

Figure 14.3 The effect of KCBs on the action potentials from guinea-pig detrusor, recorded with microelectrodes. Action potentials on the left are controls, and on the right after addition of the blocking drug (from Fujii et al., 1990).


probably as a result of a local increase in intracellular Ca2+ close to the membrane, entering through the stretch activated channels. Further work by these authors has demonstrated that the rundown of the inward current is accelerated by cAMP analogues, which work largely through increase in the maxiK channel activity. This appears to be through a cAMP-dependent phosphorylation of the channels, increasing their sensitivity to Ca. Enhancement of maxi-K channel activity could thus be involved in the inhibitory effects of β receptor activation in the bladder, which can be evoked in response to circulating adrenaline. Effects of K-channel opening drugs on detrusor Although the role of K channels in determining the shape of the action potential and the physiological behaviour of the bladder is of interest, more attention has been focused on the effects of the KCOs on the properties of the detrusor. This work has recently been extensively reviewed (Andersson and Wyllie, 1992). The interest stemmed from the suggestion that drugs which reduced the excitability of the detrusor might be able to suppress the unstable contractions of the bladder which cause urinary incontinence in detrusor instability (Brading et al., 1986), and the obvious possibility that drugs that open K channels should be able to achieve this. The initial studies with CRK, the first of the benzopyran KCO drugs, showed it to be a powerful blocker of the spontaneous mechanical activity of smooth muscle strips dissected from the bladder of guinea pigs, pigs and humans (Foster et al., 1989a, 1989b). If the KCO drugs were to be of use clinically, it would be desirable for the bladder still to be able to respond to its normal parasympathetic input. High doses of CRK (10−5 M) sufficient to abolish completely spontaneous activity had no significant effect on the frequency response curves of bladder strips to intrinsic nerve stimulation in any of these species. This dose also had no effect on the dose-response curve of the guinea-pig detrusor to muscarinic agonists (Foster et al., 1989a), although it shifted the curves to the right and somewhat suppressed the maximum response in strips dissected from pig and human bladders (Foster et al 1989b; de Moura et al., 1993). Similar effects were seen with rat bladder, both with CRK and with pinacidil (Malmgren et al., 1990), although in this species the KCOs also caused a dose-dependent suppression of the maximum response to intrinsic nerve stimulation. The development of models of detrusor instability in the pig and rat (see above) provided smooth muscle from unstable bladders for in vitro testing, and also allowed the in vivo effects of the KCOs to be studied, and their potential as agents for the treatment of this disorder assessed. The effectiveness of CRK in abolishing unstable contractions was first demonstrated in the obstructed pig model (Speakman, 1988; Foster et al., 1989b), and then in the obstructed rat model (Malmgren et al., 1989), in which pinacidil was also effective. In both species substantial drops in blood pressure (BP) occurred. In the mini-pig model


however, extensive use of LCRK has shown that at medium doses of 0.02 mg/ kg, it does not invariably abolish unstable contractions (Turner, unpublished observations). So far published information on the clinical effectiveness of CRK and pinacidil comes from two preliminary trials, and the results have proved rather inconclusive (Restorick and Nurse, 1988; Nurse et al., 1991; Hedlund et al., 1991), although some improvement was noted in both trials (see Chapter 16). Experiments have also been carried out on detrusor strips from unstable bladders in rats, pigs and humans. Spontaneous activity in such strips often shows a different pattern than in normal bladders, with a larger degree of fused tetanic tone. Spontaneous activity in smooth muscle from unstable bladders is also abolished by the KCOs (Andersson et al., 1988; Restorick and Nurse, 1988; Foster et al., 1989b; Nurse et al., 1991). Mechanisms of action of the KCOs on detrusor Although it is the effects on contractile activity which will be important for the potential clinical effectiveness of this class of drugs, there has been considerable interest in bladder, as in other smooth muscles, about the mechanisms involved in these effects. The type of K channel involved and whether or not the effects are all ascribable to changes in the membrane K+ permeability, are of interest. Electrophysiological studies on guinea-pig detrusor demonstrated that the cessation of activity was accompanied by hyperpolarization of the membrane and a drop in membrane resistance, although the action potentials often stopped before significant hyperpolarization had occurred (Figure 14.4, Foster et al., 1989a). At


Figure 14.4 Effect of increasing concentrations of cromakalim (CRK; BRL 34915) on the membrane potential of guinea-pig detrusor smooth muscle. Note that the full sized action potentials stop before significant hyperpolarization of the cell occurs. The small deflections are probably caused by action potentials in neighbouring cells, and suggest that CRK may be reducing the coupling between the cells (from Foster et al., 1989a).

higher concentrations, CRK also caused an increase in radio labelled K+ fluxes across the cell membrane. In contrast to the channel opened in many other smooth muscles, those in the detrusor seem to be relatively impermeant to Rb+ (Foster et al., 1989a), and it was noticeable that spontaneous activity was abolished by concentrations of CRK some 50 times lower than needed to alter transmembrane fluxes. Initial studies of the effects of known KCBs on the activity of CRK on vascular smooth muscle cells led to the suggestion that the drug might be activating delayed rectifier K channels (Beech and Bolton, 1989). A similar suggestion was also made for guinea-pig bladder, judging from studies on the ability of various KCBs to attenuate the inhibitory effects of CRK on spontaneous electrical and mechanical activity (Figure 14.5, Foster et al., 1988; Fujii et al., 1990). An interesting observation was that CRK at increasing doses, was able to reverse the effects of quinidine on the action potential shape (Figure 14.6; Fujii, unpublished). However the demonstration that glibenclamide could antagonize the effects of CRK on smooth muscles, focused attention on ATP-dependent K channels. It has subsequently been found that glibenclamide can antagonize most of the effects of all the KCOs that have so far been tested on bladder smooth muscles (for examples see Andersson and Wyllie, 1992; de Moura et al, 1993; Ha et al., 1993). Recently an elegant study has been published in which the effects of LCRK on isolated smooth muscle cells from the guinea-pig bladder have been examined using whole-cell and single channel voltage clamp recording techniques (Bonev and Nelson, 1993a). To demonstrate the presence of ATP-dependent K channels in the cells, the bathing solution was a Iow-Ca2+ (100 µM) high-K+ (6 mM) solution, with the voltage sensitive Ca channels blocked with nimodipine, and the Ca-activated K channels blocked with TEA (1 mM) and iberiotoxin (100 nM). The cells were held


Figure 14.5 The effect of 5×10−6 M cromakalim on the electrical activity of guinea-pig detrusor in the presence of various K channel blocking drugs. Microelectrode recordings (from Fujii et al., 1990).

at −80 mV to prevent activation of delayed rectifier K channels. The high K+ pipette solution also contained EGTA (10 mM), GTP (1 mM) and ATP (0.1 mM), the latter concentration selected to allow some steady-state activation of the KATP channels if present. Under these conditions, activation of the KATP channels would produce inward current. The results clearly demonstrated that application of glibenclamide (10 µM) reduced the resting current, and LCRK (30 µM) increased it, an effect blocked by glibenclamide (Figure 14.7). If the pipette ATP concentration was increased to 3 mM, the glibenclamide sensitive currents were greatly reduced. Thus it appears that the detrusor smooth muscle cells do indeed contain typical KATP channels. Further experiments demonstrated that the


channels were voltage independent and more sensitive than the other K+ channels to Ba2 + (half block at −80 mV by 100 µM). Noise analysis of the whole-cell currents suggested that the mean single channel current under these conditions was 0.61±0.04 pA, and that the mean number of channels per cell was 425±74, giving

Figure 14.6 Interaction between cromakalim (BRL 34915) and quinidine on the electrical activity of the guinea-pig detrusor. Quinidine (10−4 M) increased the frequency of the action potentials, reduced the size of the after-hyperpolarization and prolonged the falling phase until a second spike was superimposed. Cromakalim reversed these effects. Marked action potentials from the continuous recording above are shown on an expanded time scale below.

a rather low density of about one in every 9 µm2. Using physiological K+ gradients at 0 mV, the mean single channel current was 0.29±0.04 pA. The authors also attempted single channel recordings from outside-out patches under these latter conditions (Figure 14.7B). They demonstrated the presence of a K channel with the same single channel current, and that LCRK (10 µM) increased the activity 6. 4–fold. The single channel conductance was 7.3 pS. In the absence of ATP on the internal surface, the channels had a high initial activity, which was abolished by 10 µM glibenclamide.


Bonev and Nelson (1993b) have also demonstrated that activation of muscarinic receptors can inhibit the KATP channels, and switch off the LCRK activated current in the whole-cell recording mode. The inhibitory mechanism appears to involve activation of protein kinase C (possibly via diacylglycerol, after its production through muscarinic receptor mediated stimulation of phospholipase C) since the inhibitory effect of carbachol was blocked by inhibitors of protein kinase C, and also by perfusion of the cells with a nonhydrolysable GDP analogue which prevents G-protein activation. Thus phosphorylation of the KATP channels may cause their closing. The authors suggest that muscarinic receptor stimulation could increase smooth muscle excitability by inhibition of KATP channels, leading to depolarization of the cell membrane.

Figure 14.7 The effects of levcromakalim (LCRK, LEM) and glibenclamide on K+ currents in guinea-pig bladder. A. Whole-cell currents recorded at a holding potential of −80 mV. External and internal K+ were 60 mM and 140 mM respectively, and the external medium contained 1 mM TEA and 100 mM iberiotoxin to block maxi-K channels. Glibenclamide (GLIB, 10 µM) reduces resting whole-cell current and prevents the effects of LCRK. Levcromakalim alone (30 µM) causes a large, glibenclamide sensitive increase in inward


current. B. Outside-out patch recording. External and internal K+ were 6 mM and 140 mM respectively, and the pipette medium contained 0.1 mM ATP. Holding potential 0 mV. Left hand panels: single channel records. Right hand panel: single channel current-voltage relationship. Means±SE. Arrows indicate closed channel level, dotted lines 0 current level (from Bonev and Nelson, 1993a).

Clinical potential The search is now on for KCOs that display selectivity between vascular smooth muscle and detrusor. Careful comparison between the effects of K channel modulators on vascular and detrusor smooth muscles suggest that there are differences which might be exploited (Zografos et al., 1992). Several drug companies are attempting to develop selective drugs, and there are compounds in existence that show in vitro selectivity between vascular and detrusor of the right order of magnitude, although the structures have not yet been published. There are also some interesting compounds thought to act as KATP channel openers which, although not showing in vitro selectivity, nevertheless in vivo reduce bladder activity in rats and dogs at concentrations that, in contrast to CRK, have no significant effect on BP or heart rate. One such compound (ZD6169), an anilide tertiary carbinol (see section 3.5.1) is in preclinical development. Wyeth Ayerst have examined the properties of 4–indazolinonyl derivatives of benzopyrans, and again found compounds which act in vitro through glibenclamide sensitive mechanisms (enhance outward currents in myocytes and relax vascular and detrusor smooth muscles strips) which in vivo can abolish unstable contractions in the obstructed rat model at concentrations which have little haemodynamic effect (Antane et al., 1994). It will be important to examine the effects of candidate drugs not only on the unstable detrusor contraction and the systemic BP, but also on the urethral pressure, since it would be preferable that a compound which reduces unstable contractions did not also lower urethral pressure. This could paradoxically reduce urethral closure pressure and thus might predispose to persistent incontinence.


14.2.3 Urethra Circular urethral smooth muscle is generally considered to contribute to urethral closure during filling, although the details of the mechanisms of urethral closure remain unclear (Torrens, 1987a). Measurement of the urethral pressure profile in women shows a high pressure zone of about 3 cm, some 2 cm distal to the bladder neck (Torrens, 1987b). This is also true in the female pig, and smooth muscle dissected from the pig’s high pressure zone, in great contrast to the detrusor and the ureter, shows continual spontaneous myogenic tone (Bridgewater et al., 1993). The muscle is innervated by excitatory and inhibitory nerves and it is likely that in vivo some of the urethral pressure is mediated by a tonic sympathetic activation of the smooth muscle. Sympathetic blockade however, typically only reduces maximal urethral pressure by 25–30% in the pig (Macneil et al., 1991) and in the human (Torrens, 1987a). The muscle in the pig is supplied by two types of inhibitory nerve, one nitrergic (Persson and Andersson, 1992; Bridgewater et al., 1993), mediating a rapid and transient relaxation, and the other, with an as yet unidentified transmitter, mediated a slower and prolonged inhibition (Bridgewater and Brading, 1993). In strips of human urethra precontracted with noradrenaline, field stimulation initiates relaxations (Andersson et al., 1988), as it does in rabbit urethra precontracted by exposures to prostaglandins (Ito and Kimoto, 1985). Nitrergic innervation mediating these relaxations occurs in the rabbit and probably human urethra (Andersson et al., 1992). There is little published information about electrical activity in urethral smooth muscle from humans or pigs, but in urethral smooth muscle from rabbits, dogs and wallabies (Callahan and Creed, 1985), spontaneous contractions are associated with action potentials, and in the rabbit, intrinsic nerve activation produced relaxations, associated with inhibitory junction potentials (Ito and Kimoto, 1985).


Figure 14.8 Whole-cell recordings from cells isolated from the female pig urethra, using a nystatin perforated patch. Cells were held at -50 mV and stepped for 2 sec to test potentials between −120 and +70 mV. Note transient and sustained outward currents at depolarized potentials, and the increased noise at more positive potentials. The control steps were repeated in the presence of 10−5 and then 10−4 M LCRK. Levcromakalim increases the outward current at depolarizing potentials, except that at +70 mV, 10−4 M LCRK actually suppresses the net outward current.

K channels in urethra Recently, in our laboratory, patch-clamp techniques have been used to examine the K channels present in smooth muscle cells isolated from the pig urethra (N. Teramoto, personal communication). Using a whole cell configuration with a nystatin perforated patch with a high K+ pipette solution, and a physiological


bathing solution (Figures 14.8 and 14.9), voltage dependent outward currents were

Figure 14.9 Current voltage relationships of the current at the end of a 200 msec test potential. A: net currents in the absence and presence of 10−5 and 10−4 M levcromakalim. B: Difference (levcromakalim sensitive) currents. Cells isolated from pig urethra, wholecell configuration, nystatin perforated patch, physiological ionic concentrations.


Figure 14.10 Effect of levcromakalim on single channel currents in a cell attached patch on isolated pig urethra smooth muscle myocyte. Symmetrical high K+ conditions. Holding potential −50 mV. A: continuous recording with a slow time base, showing the effects of adding 10−5 M levcromakalim to the bath solution. B: amplitude histogram before and during application of levcromakalim. The smooth lines are Gaussian curves.

elicited showing a transient and sustained component. The outward currents became very noisy at large depolarizations, suggesting activation of some large conductance channels. Addition of the KCO, LCRK caused a concentration dependent outward current at the holding potential of −50 mV. Between −120 and +20 mV, the LCRK sensitive current had a linear I-V relationship, reversing near EK, but with high concentrations of LCRK (100 µM) there was a surprising reduction in the net outward currents at potentials positive to +50 mV. In cell attached patches in symmetrical high K+, Ca2+ -free conditions, at least three types of channel could be seen. So far we have studied the maxi-K channels and


those activated by addition of LCRK. LCRK (10 µM) at a holding potential of −50 mV induced the opening of channels with an inward current (Figure 14.10). These channels were seen in about 25% of the patches, and showed evidence of clustering, since when found, more than one channel was normally present. The channels had a conductance of about 42 pS and a long mean open time (about 100–200 msec). The most common channel seen under these conditions, had the characteristics of the maxi-K channel in other smooth muscle cells (Figure 14.11). It was a Ca2+ and voltage sensitive channel with a conductance in symmetrical high K+ solutions of about 240 pS and a mean open time of a few msecs. This channel was found in virtually all patches studied. We have also studied the effects of KCOs in vivo on urethral pressure in pigs. In anaesthetised mini-pig, LCRK had no effect on maximal urethral pressure (Turner, unpublished observations). However, in these experiments, the maximum urethral pressure before LCRK was given, was 50–75% of the maximum achievable in these animals, and this may have masked any reduction due to LCRK. The normal variations in urethral pressure seen in the pig were however reduced by LCRK. 14.3 Female Genital Tract Smooth Muscles The smooth muscles in the non-pregnant female genital tract play a role in moving the released eggs into the uterus, in maintaining the uterine wall in the correct state for implantation and in helping the movement of sperm into the uterus. In the pregnant animal the smooth muscles must adapt to allow the foetus to remain in the uterus until the end of gestation, and then bring about dilation of the cervix and expulsion of the foetus at birth. The most important areas for pharmacological intervention are reducing uterine activity in dysmenorrhoea and in threatened miscarriage, and enhancing activity for the induction of labour or abortion. 14.3.1 Uterus The smooth muscle layers of the uterus, composed of an outer longitudinally arranged layer, a middle layer in which the muscle bundles run in all directions, and an inner layer which has both longitudinal and circularly arranged bundles, show spontaneous electrical and mechanical activity in both the pregnant and non-pregnant state. The activity is myogenic, the cells showing regular slow depolarizations with superimposed spike activity, that initiate contraction. The contractile activity is under hormonal control. The body of the uterus is innervated by cholinergic, adrenergic and peptidergic neurones, which innervate both the smooth muscle and the epithelial layers. Stimulation of the intrinsic nerves in strips of myometrial smooth muscle from


Figure 14.11 Effect of changing the holding potential on single channel currents (maxi-K +) in a cell attached patch on an isolated pig urethra smooth muscle myocyte. A: Current voltage relationship, B: single records at various potentials. Symmetrical high K+ conditions.

non-pregnant nonparous women excites the smooth muscle through muscarinic receptors (blocked by atropine) and β -adrenoceptors (blocked by phentolamine). There is no evidence of a nerve-mediated inhibition of activity. Exogenously applied VIP inhibits smooth muscle activity in the non-pregnant uterus, and causes vasodilatation. It may have a role in the mechanism of menstrual bleeding. During pregnancy there is an almost total loss of the innervation to the body of the uterus, although the smooth muscle continues to show myogenic activity. Waves of contraction pass along the uterus at regular intervals, but these do not elevate the intra-uterine pressure. The size and frequency of these waves change during pregnancy as the hormonal status changes. Towards the end of pregnancy


the smooth muscle becomes increasingly sensitive to oxytocin, which can powerfully enhance this myometrial activity. Just before delivery, there is a large increase in the number of gap junctions between the smooth muscle cells, which allows the electrical activity of the cells to be synchronized, thus initiating coordinated contractions of the smooth muscle which do elevate intra-uterine pressure, and are necessary to expel the foetus. K channels in rat uterus Most of the studies on the K channels present in the myometrial smooth muscle cells have been carried out on rat or on human cells. In the rat, work on nonpregnant myometrial cells has been reviewed thoroughly by Ludmir and Erulaker (1993), in which they point out that the hormonal status and developmental time may alter the expression of K channels. In studies on the pregnant rat uterus, Anwer et al., (1992) using whole-cell and cell attached voltage clamp of myometrial cells have demonstrated the presence of K channels which were activated by Ca2+ and by voltage, blocked by charybdotoxin and low concentrations of TEA (less than 1 mM) but not by 4AP. Single channel conductance was 143 pS in symmetrical high KCl. Inoue et al., (1993) also studying cells from pregnant rat uterus, used conditions in which the Caactivated K channels would not be activated. Whole-cell experiments demonstrated a voltage sensitive outward current with a threshold for activation of about −40 mV. This current could be dissected into a 4AP sensitive current (blocked by 2 mM 4AP) which had the characteristics of a transient outward current, and a TEA sensitive current which had the characteristics of a delayed rectifier K+ current (blocked by 5 mM TEA). Thus rat myometrium has at least three voltage activated K channels. Betaadrenoceptor agonists are known to inhibit contractions of the uterus associated with hyperpolarization of the membrane, thus implicating an effect on K channels. Anwer et al., (1992) demonstrated that the activity of the Ca-activated K channels in cell attached patches was greatly enhanced by activation of β adrenoceptors, suggesting that they may be involved in these effects. Inoue et al., (1993) demonstrated that the delayed-rectifier-like current was actually blocked by forskolin. This action, however, was independent of its ability to activate protein kinase A, since protein kinase A inhibitors did not block the forskolin effect and the addition of cAMP or non-hydrolysable GTP analogues did not affect the outward current. It seems most probable that the Ca-activated K channel is the dominant and most important K channel in the pregnant rat myometrium, and that it may be important in modulation of uterine activity. Interestingly, however, oxytocin, a potent stimulator of the uterus, did not have any effect on the K+ currents (Inoue et al., 1992). K channels in human uterus More recently results have been published of experiments on human myometrium. Erulaker and Ludmir (1993) have also demonstrated the presence of three types of voltage sensitive K+ currents, two non-inactivating and one fast


inactivating. A large conductance Ca-activated K channel has been characterized by incorporation of plasma membrane vesicles into lipid bilayers (Perez et al., 1993). It has all the properties of maxi-K channels seen in other smooth muscles (gated by voltage and intracellular Ca2+, blocked by charybdotoxin and low concentrations of TEA). The role of these channels in modulating contractile activity through regulation of membrane potential has been investigated by Anwer et al. (1993). Block of these channels with iberiotoxin depolarized the cells, and resulted in an increase in [Ca2+]i

Figure 14.12 Effects of levcromakalim on spontaneous mechanical activity of strips of myometrium from pregnant human, treated with oxytocin (from Morrison et al., 1993).


and initiated phasic contractions in myometrial strips. The β -adrenoceptor agonist hexoprenaline, which is used for tocolysis after the 26th week of pregnancy, was shown by Adelwoehrer and Mahnert (1993) to increase the open probability of the maxi-K channel, an effect that was independent of an increase in [Ca2+]i, and was also shared by CGRP. The CGRP effect appears to be mediated through direct coupling of a G protein to the K channels (Tritthart et al., 1992). K channel opening drugs Again, because of the clinical potential of drugs that can diminish uterine contractions, much attention has been given to the KCO drugs in the myometrium. Earlier studies were carried out on mechanical activity of the rat uterus by Hollingsworth and colleagues (Hollingsworth et al., 1987; Downing et al., 1989; Downing and Hollingsworth 1990) Micro-electrode studies showed that CRK (10 µM) caused a small hyperpolarization of these myometrial cells (Hollingsworth et al., 1987), although interestingly there were no effects on K+ or Rb+ tracer efflux from the tissues (Hollingsworth et al., 1987, 1989). The relaxant effects of the drugs were reversed by glibenclamide (Piper et al., 1990). These results have been reviewed recently (Andersson and Wyllie, 1992). Since this review, the effects of LCRK and pinacidil have been examined on mechanical activity of strips of human myometrium obtained from women during pregnancy and labour (Morrison et al., 1993), and the effects of aprikalim and LCRK have been studied on non-pregnant and pregnant human myometrial strips (Cheuk et al., 1993). As predicted, all these drugs were potent inhibitors of spontaneous activity, and activity induced by agonists such as oxytocin (Figure 14.12), phenylephrine and low (but not high) K+ concentrations. In contrast with rat uterus, a small increase in 42K efflux was seen on application of LCRK (Cheuk et al., 1993). Glibenclamide (but not tolbutamide) acted as if it were a competitive antagonist. Metabolic inhibition in the uterus, presumably resulting in a lowered ATP concentration, enhanced 86Rb efflux from rat myometrial strips (Heaton et al., 1993), an effect which was reversed by about 50% by glibenclamide, suggesting that KATP channels are involved, possibly implicating their activation in dystocia, a condition in which there is inadequate uterine activity during labour. 14.4 Male Genital Tract Smooth Muscles The smooth muscles of the male genital tract, unlike the female, show little spontaneous activity, and are under tight control of the autonomic nervous system. The smooth muscles are responsible for permitting erection, for movement of the sperm and the components of the seminal fluid into the urethra and for ejaculation. The clinical problem most likely to involve smooth muscle dysfunction is impotence.


So far, little attention has been paid to the K channels in the smooth muscles of the male genital tract. More attention has been paid to their innervation and the transmitters involved in controlling them. However, since one of the main events occurring in penile erection is the relaxation of the smooth muscles in the trabeculae of the corpora cavernosa, the effects of the KCOS on erectile tissue have been examined (monkeys: Giraldi and Wagner, 1990; rabbit and man: Holmquist et al., 1990a, 1990b; cat: Hellstrom et al., 1992). The results have been reviewed by Andersson and Wyllie (1992) and Andersson (1993). As expected, the KCOs relaxed isolated erectile tissue, and inhibited agonist evoked contraction. Intracavernous injection of KCOS in the monkey caused tumescence or erection (Giraldi and Wagner, 1990) and erection in the cat (Hellstrom et al., 1992). So far, however, little work has been carried out on the possibilities of these drugs in clinical treatment, largely because of the discovery of the importance of NO as a neural transmitter involved in the initiation of erection. Christ et al. (1993) have recently looked at the electrical properties of cultured corporeal smooth muscle cells, using patch-clamp analysis. In common with the other smooth muscles in the urogenital tract, these authors demonstrated a transient and sustained outward current in the whole-cell mode. The outward current was enhanced by the Ca-channel agonist, BAYK 8644, and by the KCO, pinacidil, suggesting the presence of Ca-activated K channels and KATP. Single channel analysis consistently showed maxi-K channels and a putative delayed rectifer channel. This diversity of K channels again suggests that they may play an important role in modulating corporeal smooth muscle tone. 14.5 Conclusions The diversity of K channels possessed by smooth muscles in general is reflected in the smooth muscles of the urogenital tract. Modulation of these channels can lead to increased or decrease mechanical activity in the smooth muscles, and the clinical implications of this are legion. Incontinence, impotence and premature labour are three immensely important clinical problems, none of which has adequate treatment. It can only be a matter of time and resources before selective drugs are developed. The clinical benefit from this could be enormous, with corresponding rewards for the drug company that develops specific drugs for treatment in these areas. References ADELWOEHRER, N.E. & MAHNERT, W. (1993) Arch. Gynecol. Obstet., 252, 179–184. ANDERSSON, K.-E. (1993) Pharmacol. Rev., 45, 253–308.


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RESTORICK, J. & NURSE, D. (1988) An in vitro and in vivo study. Neurourol. Urodyn., 7, 207–208. SAITO, M., WEIN, A.J. & LEVIN, R.M. (1993) Neurourol. Urodyn., 12, 573–583. SANTICIOLI, P. & MAGGI, C.A. (1994) Br. J. Pharmacol., 113, 588–592. SHUBA, M.F. (1977) J. Physiol., 264, 853–864. (1981). In: Smooth Muscle: an Assessment of Current Knowledge. Bülbring, E., Brading, A.F., Jones, A.W. and Tomita, T. (eds). Edward Arnold, London. SIBLEY, G.N.A. (1985) Br. J. Urol., 57, 292–298. SPEAKMAN, M.J. (1988) Studies on the physiology of the normal and obstructed bladder. M.S. Thesis, London. SPEAKMAN, M.J., BRADING, A.F., GILPIN, C.J., DIXON, J.S., GILPIN, S.A. & GOSLING, J. (1987) J. Urol., 138, 1461–1466. SUZUKI, M., MURAKI, K., IMAISUMI, Y. & WATANABE, M. (1992) Br. J. Pharmacol., 107, 134–140. TORRENS, M. (1987a) Chapter 9 In: The Physiology of the Lower Urinary Tract. Torrens, M. and Morrison, J. (eds) Springer-Verlag. Berlin. (1987b) Chapter 11 In: The Physiology of the Lower Urinary Tract. Torrens, M. and Morrison, J. (eds) Springer-Verlag, Berlin. TRITTHART, H.A., STARK, U., STARK, G., MAHNERT, W., STENDER, C.O. & SCHREIBMAYER, W. (1992) Ann. N. Y. Acad. Sci., 657, 216–227. WELLNER, M.C. & ISENBERG, G. (1994) J. Physiol., 480, 439–448. ZOGRAFOS, P., LI, J.L. & KAU, S.T. (1992) Pharmacology, 45, 216–230.

Recent Literature FRANK, C.A., FORST, J.M., GRANT, T., HARRIS, R.J., KAU, S.T., LI, J.H., OHNMACHT, C.J., SMITH, R.W., TRAINOR, D.A. & TRIVEDI, S. (1993) Dihydropyridine KATP Potassium Channel Openers. BioMed. Chem. Letts., 3, 2725–2726. GRANT, T., FRANK, C.A., KAU, S.T., LI, J.H., MCLAREN, P.M., OHNMACHT, C.J., RUSSELL, K., SHAPIRO, H.S. & TRIVEDI, S. (1993) Anilide Tertiary Carbinols: A New Structural Class of Potent Potassium Channel Openers. BioMed. Chem. Letts., 3, 2723–2724. HOME, B., HALTERMAN, T.J., YOCHIM, C.L., MY LINH DO, PETTINGER, S.J., STOW, R.B., OHNMACHT, C.J., RUSSELL, K., EMPFIELD, J.R., TRAINOR, D.A. et al. (1995) Zeneca ZD6169: A Novel KATP Channel Opener with in vivo Selectivity for Urinary Bladder. J. Pharmacol. Sup. Therap., 274, 884–890.

15 Potassium Channel Modulators and the Central Nervous System H.HERDON Psychiatry Research Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. 15.1 Introduction Potassium (K) channels are the most diverse and ubiquitous of all ion channels. They are present in virtually every eukaryotic cell, and exert major influences on cell function. In excitable cells, they play key roles in regulating action potentials, resting membrane potentials and cellular events such as learning and memory, whilst in non-excitable cells they regulate membrane transport processes and influence intracellular events. K channels have traditionally been identified and characterised on the basis of electrophysiological measurements of their kinetics, voltage-dependence and conductance. Their importance in excitable cell function was originally recognised using voltage-clamp techniques (Hodgkin and Huxley, 1952), and from the early 1980s onwards the development of patch clamp methodology (Hammill et al., 1981) has provided enormous advances in our knowledge of K channel properties. In recent years these methods have been complimented by the use of molecular biological techniques to clone, sequence and express ion channels. This has resulted in the identification of many new K channels, producing an exponential growth in information on the structural diversity of K channels and how this relates to their function. The combination of electrophysiological and molecular biological methods now provides a partial understanding of the molecular basis of some fundamental K channel properties (Pongs, 1992a). Many attempts have been made to produce a general classification of K channels, although none have so far proved totally satisfactory due to the remarkable diversity of these channels. In two recent complementary attempts, one system classified K channels into three overlapping categories: voltageregulated, voltage and ligand-regulated, and ligand-regulated (Quast and Weston, 1994), the other into four categories: voltage-regulated, Ca-activated, receptor-coupled and ‘others’ (Watson and Girdlestone, 1994). Such categories are not mutually exclusive; for example, certain K channels in the voltage-

KCMS AND THE CENTRAL NERVOUS SYSTEM 435

regulated category can be modulated by extracellular or intracellular ligands (see below). Such classifications are based largely on the electrophysiological and pharmacological properties of native channels, but many cloned β -subunits of channels corresponding to most of the main classes of voltage-regulated K channels have now been identified. These cloned channels can be divided into two ‘superfamilies’: Shaker-related (Shaker, Shab, Shaw Shal, Slo) channels, and a new family including ROMK1, IRK1 (members of the inward rectifier) and the recently discovered rcKATP1 (cloned cardiac KATP channel) (Ashford et al., 1994), which may correspond to the inner core of Shaker-related channels (Nichols, 1993; Pongs, 1993). However, other K channel families may also exist (e.g. Suzuki et al., 1994). Although such classifications are still incomplete and not wholly analogous, they serve as useful guides to this complex area until the IUPHAR recommendations on K channel nomenclature are finalised (Spedding and Vanhoutte, 1993). Most of the major types of K channels are present in the CNS. These include at least five different types of voltage-regulated channel, three types of Ca-activated channel and several ligand-gated/receptor-coupled channels (Halliwell, 1990; Aronson, 1992). A summary of the properties of some of these channels is given in Table 15.1. CNS K channels were originally characterised in terms of macroscopic currents, and subsequently by single channel analysis. Cloned K channel ― -subunits corresponding to several classes of voltage-regulated K channels have now been identified in the CNS. Some of these channels may be closely related; for example, certain KV-type delayed rectifier channels can be altered to display KA-type inactivating properties by association with a recentlydiscovered brain-specific β -subunit (Rettig et al., 1994). The K+ equilibrium potential is normally negative to the resting membrane potential, so the opening of most types of K channels generally produces hyperpolarisation and inhibition of neuronal excitability, whilst inhibition of K channels produces depolarisation. Such effects on neuronal excitability can occur as a result of not only direct effects on membrane potential but also indirect effects via changes in membrane conductance, which alter the influence other ionic currents have on membrane potential (Galvan, 1992). Some types of K channels (e.g. KV, KA) are activated mainly by depolarisation, and exert major influences on action potential characteristics. Other channels (e.g. KM) can remain open in the absence of depolarisation and influence resting membrane potentials. Still others (e.g. KIR) are Table 15.1 Summary of properties of some CNS K channels Symbol Channel type

Cloned family Modulators

KV

Delayed Rectifier

KA

A Channel

Shaker, Shaw, Blockers: TEA Shab Shaker, Shal Blockers: 4–AP, β -dendrotoxin, MCD peptide


Symbol Channel type

Cloned family Modulators

KD KIR KATP

Slowly Inactivating Inward Rectifier ATP-sensitive

? IRK, ROMK rcKATP (IRK, ROMK)

BKCa

Large-conductance Ca-activated

Slo

SKCa

Small-conductance ? Ca-activated Muscarinic-inactivated GIRK

KM

Blockers: 4–AP (potent) Blockers: TEA Blockers: Sulphonylureas (e.g. Glibenclamide) Openers: Cromakalim, Pinacidil, Diazoxide Blockers: Charybdotoxin, Iberiotoxin Openers: NS 004, NS 1619 Blockers: Apamin, Scyllatoxin ?

activated by hyperpolarisation and actually produce a small depolarisation, thus again regulating resting membrane potential. Many types of K channels can be regulated by neurotransmitters, second messengers etc. (see below) and produce effects independent of changes in membrane potential. It should also be borne in mind that K channels are present on glial cells as well as neurones (Bevan, 1990). Since glia have been shown to influence neuronal activity (Nedergaard, 1994), such glial K channels could produce indirect effects on neurones. These few examples serve to illustrate the diverse influences that K channels can have on neuronal function. 15.2 Endogenous Modulators of CNS K Channels Although the main subject of this chapter will be the actions of exogenous drugs and toxins which affect CNS K channels, it must be recognised that a wide variety of endogenous substances can also regulate the activity of these channels. In particular, many neurotransmitters and their receptors are known to affect K channels (Rudy, 1988; Nicoll et al., 1990; Storm, 1993). Amongst the best known is acetylcholine, which via muscarinic receptors inhibits the voltageregulated channel responsible for the M-current. This current can also be inhibited by other receptors coupled to the inositol phosphate/diacylglycerol effector system, including metabotropic glutamate, 5–HT and several peptide (e.g. bradykinin) receptors. Acetylcholine acting via muscarinic receptors can also inhibit the AHP current, which is probably carried by SKCa channels. This current is strongly inhibited by other neurotransmitters including monoamines, VIP and glutamate. Many of these effects may be mediated via actions on the cyclic AMP/adenylate cyclase effector system. Another major voltage-regulated K channel, KA, can be inhibited by both noradrenaline (via β -adrenoceptors) and acetylcholine (via muscarinic receptors); a similar K channel is activated by


GABAB receptors (Gage, 1992) and cannabinoid receptors (Deadwyler et al., 1993). Such examples serve to illustrate the concepts of ‘convergence’, where several different neurotransmitter receptors regulate the activity of a single type of K channel, and ‘divergence’, where a single type of receptor (e.g. muscarinic) can regulate the activity of several types of K channels (Nicoll, 1988). Dopamine has received a lot of attention as a possible modulator of K channels. Dopamine D2−type receptors can hyperpolarise neurones by increasing K+ conductance (Lacey et al., 1987), and non-selective potassium channel blockers (KCBs) can inhibit D2−autoreceptor function (Cass and Zahniser, 1991; Tanaka et al., 1992). Some D2 receptor effects on K channels may be mediated by Gi-type G-proteins (Lledo et al., 1992). Stimulation of D2−type receptors has been reported to open a K channel sensitive to quinine and sulphonylurea-type KATP channel blockers (Freedman and Weight, 1989; Roeper et al., 1990; Lin et al., 1993), although evidence against this has also been described (Hicks and Henderson, 1992). Thus, the type(s) of K channels affected by dopamine receptors remains unclear. Several peptides may also modulate ATP-sensitive K channels (KATP channels). There is indirect evidence that galanin opens CNS KATP channels (Ben-Ari, 1990), and opioid μ and β receptors may modulate this type of channel as well as KIR channels (North, 1989; Edwards and Weston, 1993). Endosulphine, a peptide extracted from brain, has been proposed as an endogenous inhibitor of KATP channels (Virsolvy-Vergine et al., 1992), though its location and function in the CNS remain unknown. Many of the effects of G-protein coupled neurotransmitter receptors on K channels are likely to involve actions via second messenger systems altering protein kinase activity and thus channel phosphorylation. However, some effects may be more direct and involve interactions of G-proteins themselves with the channels. Thus, Gi has been shown to activate both KACh and KATP channels (Birnbaumer, 1992), and several brain K channels activated by Go have also been described (VanDongen et al., 1988). 15.3 Toxin Modulators of CNS K Channels Amongst the first agents recognised to act as relatively selective modulators of K channels were a variety of peptide toxins. Toxins derived from snake, scorpion or bee venom have proved to be very valuable and versatile tools in studies on the properties of K channels and their effects on neuronal function. Such toxins have been used in electrophysiological and biochemical investigations, ligand binding assays and autoradiography, as well as functional studies both in vivo and in vitro. Although these experiments have resulted in the realisation that some toxins are not as specific for particular K channel types as was originally thought, these agents have still made important contributions to our understanding of K channels.


15.3.1 Scorpion Venom Toxins The most well-known scorpion venom toxin is charybdotoxin. This 37 amino acid peptide was originally classified as a specific blocker of BKCa channels, at which it is active at low nanomolar concentrations. However, recent investigations have shown that charybdotoxin can also block brain KA-type channels and probably other Ca-regulated K channels. The block of K channels by charybdotoxin is thought to involve physical occlusion of the channel pore (MacKinnon et al., 1990). Charybdotoxin has a high sequence homology to noxiustoxin, a 39 amino acid peptide, which can also block KA-type and BKCa channels. Iberiotoxin, a 37 amino acid peptide with substantial sequence homology to charybdotoxin, may be a more selective blocker of BKCa channels. In contrast scyllatoxin (leiurotoxin I), a 31 amino acid peptide, also has some sequence homology to charybdotoxin and noxiustoxin but can produce a selective block of SKCa channels. Several of these scorpion toxins have been shown to produce effects on brain function at a macroscopic level. Both charybdotoxin and noxiustoxin stimulate synaptosomal [3H]-GABA release, and charybdotoxin can increase intracellular Ca levels (see Brewster and Strong, 1992). Radioiodinated derivatives of charybdotoxin and scyllatoxin have been used to study the binding sites for these peptides in brain tissue; the scyllatoxin sites appear to be identical to those for the bee venom toxin apamin (see below) and have been taken as corresponding to SKCa channels. Autoradiographic mapping of charybdotoxin binding sites has revealed high densities in mesencephalic regions and white matter areas (Gehlert et al., 1992; Gehlert and Gackenheimer, 1993); these sites probably correspond to KA-type rather than BKCa channels (Vazquez et al., 1990). 15.3.2 Bee Venom Toxins Toxins from bee venom are generally smaller molecules than those from scorpion or snake venom. There are two of major importance: apamin and mast cell degranulating (MCD) peptide. The two are related in structure but have different pharmacological properties. Apamin, an 18 amino acid peptide, is a selective blocker of SKCa channels at low nanomolar concentrations, although some types of SKCa channel (e.g. in hippocampal neurones) seem resistant to apamin (Storm, 1993). Radioiodinated apamin has been used for autoradiographic mapping of brain binding sites thought to be related to SKCa channels. These sites are present in high densities in limbic and motor areas (Gehlert and Gackenheimer, 1993), and injection of apamin into the A10 dopaminergic region (which provides an input to the limbic forebrain) produces an increase in motor activity. Intraventricular injection of apamin can produce hyperactivity and convulsions; these symptoms can also be produced by


peripheral administration, though a much higher concentration of toxin is required (see Brewster and Strong, 1992). MCD peptide is a 22 amino acid molecule. In contrast to apamin, it appears to be a selective blocker of KA channels at low nanomolar concentrations. Autoradiographic studies with radioiodinated peptide show a high density of binding sites in cortex, hippocampus and cerebellum. The toxin can produce long-term potentiation in hippocampal slices (Cherubini et al., 1987), and in vivo central administration causes increased arousal followed by convulsions at higher doses. The channels blocked by MCD peptide seem closely related to those which are targets for the snake venom toxins, β -dendrotoxin and β bungarotoxin. 15.3.3 Snake Venom Toxins The most important snake venom toxins are β -bungarotoxin and the dendrotoxins. The latter consist of a group of 59 amino acid peptides. They are capable of blocking both Kv and KA-type channels, with the β and β dendrotoxins being more selective for KA (see Dolly et al., 1994). β bungarotoxin, a large 180 amino acid two chain peptide, is less selective as a KCB, since it also possesses phospholipase enzyme activity. However, it appears to block the same set of K channels as β -dendrotoxin. Allosterically interacting binding sites for β -bungarotoxin, β -dendrotoxin and mast cell degranulating peptide (MCDP) have been reported, and a family of brain proteins (‘DMB binding proteins’) have been identified to which all these toxins bind and the β subunits of which are related to the Shaker K channel family (Rehm and Tempel, 1991), though β -dendrotoxin-sensitive K channels also possess a β -subunit which affects channel properties but which appears to be unrelated to any known ion channel protein (Scott et al., 1994). Autoradiographic mapping of iodinated β dendrotoxin binding shows a wide distribution with highest levels in hippocampus and cerebellum. As with other K channel toxins, dendrotoxins can depolarise synaptosomes and stimulate both GABA and glutamate release, as well as causing severe convulsions after intraventricular administration (see Dreyer, 1990). 15.4 Drugs Modulating CNS K Channels The first synthetic compounds recognised as acting on K channels were simple molecules such as tetraethylammonium (TEA) and the aminopyridines. These agents are capable of blocking a variety of K channels, although they do exhibit some selectivity at lower concentrations. Thus, TEA is a much more potent blocker of Kv, Km and BKCa than of KA, SKCa or KATP. Conversely, 4– aminopyridine (4–AP) predominantly blocks KA and KD rather than Kv. Block of


K channels by TEA can occur at distinct extracellular and intracellular sites, and involves physical occlusion of the channel pore (Pongs, 1992b). Compounds such as quinine are also capable of blocking a range of K channels including Kv, BKCa SKCa and KATP. Excluding some of the toxins discussed above, compounds possessing highly selective direct actions have been discovered for very few types of K channels. Recent reports have described two novel benzimidazolones, the Neurosearch compounds NS 004 and NS 1619 (see Figure 15.1), which appear to be selective activators of BKCa channels in both smooth muscle cells and neurones (Olesen et al., 1994a, 1994b). A novel Bayer compound, Bay x9227 (Lenfers et al., 1993; see Figure 15.1), is reported (as the (–) enantiomer) to hyperpolarise neuronal cells with an EC50 of 3 pM (Hunnicutt et al., 1994). Although the mechanism of this effect of the (–) enantiomer is not disclosed, the (+) enantiomer of the compound is described as being a smooth muscle KATP channel opener. In addition, this (+) enantiomer is capable of blocking the effect of the (–) enantiomer (Lenfers et al., 1993). Therefore, K channels might also be involved in the mechanism of action of the (–) enantiomer. A series of benzoylamino benzopyrans related to cromakalim (CRK) have been disclosed in a SmithKline Beecham patent (Evans et al., 1992). Resolution of certain of these compounds into their 3S, 4R and 3R, 4S enantiomers (see Figure 15.1) has indicated that the 3S, 4R enantiomers possess antihypertensive (KATP channel opening?) activity, whereas the 3R, 4S enantiomers are minimally antihypertensive but do possess anticonvulsant activity (Blackburn et al., 1993). Whether this anticonvulsant activity is related to effects at K channels has not been reported. 15.4.1 KATP Channel Modulators Apart from the few novel compounds mentioned above, virtually all the agents which have been reported as direct and selective modulators of K channels belong to the class which have been described as either blockers/inhibitors or openers/ activators of peripheral ‘Type 1’ (Ashcroft and Ashcroft, 1990) KATP channels e.g. in pancreatic β -cells or smooth or cardiac muscle. The openers (KCOs) include agents such as diazoxide, pinacidil and CRK. The effectiveness of these compounds as antihypertensive agents, together with growing knowledge of their mechanisms of action, has led to the synthesis of a large number of molecules based mainly around the benzopyran, thioformamide or cyanoguanidine structures (for examples see Figure 15.2). Such compounds may differ greatly in their potency and


Figure 15.1 Novel compounds with possible K channel-modulating properties

pharmacokinetics, but are still classified as openers of KATP channels. However, recent reports of substantial selectivity between different smooth muscle tissues shown by certain KCOs (Weston and Edwards, 1992; Edwards and Weston, 1993) indicate that such agents can also differ in their pharmacodynamics, and may act preferentially on subtypes of KATP channels present in different tissues. Indeed, evidence has been presented recently that in certain smooth muscle cells levcromakalim (LCRK) can act on Kv channels, and that KATP channels may in fact represent a voltage-independent form of this channel (Edwards et al., 1993). However, this claim has not been supported by other studies (e.g. Evans et al., 1994), and the recent report of the cloning of a cardiac KATP channel (rcKATP1) indicates that such channels are members of the inward rectifier (IRK, ROMK) family rather than the Kv family (Ashford et al., 1994). It is also important to


remember that KCOs can show pharmacological activity independent of their KATP channel-opening abilities (Quast, 1993). Compounds classified as blockers/inhibitors of KATP channels are based mainly around the sulphonylurea structure and are typified by glibenclamide and tolbutamide

Figure 15.2 Examples of K channel opener and K channel blocker structures

(see Figure 15.2). These KCBs were developed as treatments for non-insulin dependent diabetes, and their mechanism of action in this case is thought to involve block of KATP on pancreatic β -cells. Sulphonylureas can also block smooth and cardiac muscle KATP channels, but with substantially lower potency. For example, glibenclamide is effective at concentrations of 1–10 nM on β -cells, but concentrations of 100–1000 nM are required in smooth muscle. Conversely, most KCOs (apart from diazoxide) show far greater potency on smooth muscle than on β -cells (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). As


with KCOs, sulphonylurea KCBs are not totally specific and can block other types of channel at micromolar concentrations (Ashcroft and Ashcroft, 1992). Although potassium channels sensitive to ATP have been identified in the CNS, it is not clear how their pharmacology relates to that of their peripheral counterparts. Unfortunately, most studies in CNS have used such high concentrations of KCOs and KCBs that they could not be considered as selective in terms of actions on peripheral-type KATP channels. For example, diazoxide (500 μ M) and tolbutamide (500 μ M) have been reported to modulate the activity of KATP channels in cortical neurones (Ohno-Shosaku and Yamamoto, 1992). Tolbutamide (100 μ M) also blocks KATP channels in ventromedial hypothalamic (VMH) neurones, but in this case the effect is actually reversed by 0.1 μ M glibenclamide (Ashford et al., 1990). This unexpected finding might be related to the fact that second generation sulphonylureas such as glibenclamide contain a benzoic acid as well as a sulphonylurea moiety, which could interact with a second site (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). These VMH KATP channels are likely to differ substantially from the well-characterised peripheral KATP channels, since they have different electrophysiological properties and are insensitive to a range of KCOs including CRK, pinacidil and diazoxide (Sellers et al., 1992). The exact relationship between the sites of action of KCOs/KCBs and the KATP channels that they modulate is also uncertain. In pancreatic β -cells and some smooth muscle cells, KCOs and KCBs can alter KATP channel activity in isolated membrane patches, suggesting that their site of action is closely related to the channel itself (Ashcroft and Ashcroft, 1990, 1992; but see Khan et al., 1993). However, the cloned cardiac KATP channel (rcKATPl) can be activated by pinacidil but is unaffected by glibenclamide, suggesting that the channel and the sulphonylurea binding site are separate entities (Ashford et al., 1994). The mRNA for this channel is also expressed in several brain areas, especially hypothalamus and preoptic area (Ashford et al., 1994), and in VMH neurones tolbutamide blocks KATP channels only in cell-attached not isolated patches, in agreement with the concept of channels and sulphonylurea binding sites as separate entities (Ashford et al, 1990). In cortical neurones, on the other hand, tolbutamide (at 500 μ M) is effective in isolated as well as cell-attached patches (Ohno-Shosaku and Yamamoto, 1992). Again, this discrepancy may be due to the existence of different types of CNS KATP channels. This uncertainty as to the site of action of KCBs in relation to the channels themselves also raises questions over the interpretation of radioligand binding studies using radiolabelled sulphonylureas. Although in pancreatic β -cells there is a good correlation between affinities of sulphonylureas in such binding assays and their effects on KATP channels, the same cannot be stated for CNS tissue. Radiolabelled glibenclamide binding is of similar affinity (ca. 0.5 nM) in brain as in β -cells, and the distribution of these binding sites in brain tissue has been taken as representing that of KATP channels (e.g. Mourre et al., 1990; Gehlert et al., 1991). However, functional effects of sulphonylureas on neurones generally


require concentrations several orders of magnitude higher than those effective at these high affinity binding sites (see next section). This has led to the suggestion that the lower affinity binding sites for radiolabelled glibenclamide (Kd 0.1–1 μ M) may also represent a site of action of sulphonylureas on neuronal KATP channels (Gopalakrishnan et al., 1991; Zini et al., 1993a). Neither high nor low affinity binding sites for radiolabelled glibenclamide are affected by KCOs except at very high (ca. 100 μ M) concentrations (Angel and Bidet, 1991; Gopalakrishnan et al., 1991; Schwanstecher et al., 1992; Zini et al., 1993a). This adds to the uncertainty over the relationship between the binding sites for KCBs, KCOs and the channels themselves. Numerous past studies have been unsuccessful in demonstrating specific binding sites for radiolabelled KCOs, but a pinacidil derivative (P1075) has now been used to label putative KATP channels in whole segments of rat aorta (Bray and Quast, 1992). Although this compound is not totally selective and can also act as a Cl channel blocker (Holevinsky et al., 1994), the pharmacology of both KCOs and sulphonylurea KCBs at this binding site correlates well with their functional effects (Quast et al., 1993). This technique has also provided evidence that the binding sites for sulphonylureas and KCOs may be allosterically coupled, at least in peripheral tissue (Bray and Quast, 1992). Unfortunately, the fact that this binding site could be detected only in intact tissue, but not in any membrane preparation (including brain), leaves the question of the functional significance of CNS sulphonylurea binding sites unresolved. Endosulfine, a peptide extracted from brain, can bind to the same sites as sulphonylureas (Virsolvy-Vergine et al., 1992). This peptide might represent an endogenous ligand for sulphonylurea binding sites (separate from KATP channels?), but its location and function in the brain remain unknown. 15.5 Effects of K Channel Modulating Drugs on CNS Activity Although there have been many studies on the effects of potassium channel modulators (KCMs) on a range of CNS activity models both in vivo and in vitro, very few have unequivocally identified the type of K channel involved. This is partly due to the use of non-selective modulators such as aminopyridines, but also to the use of ‘selective’ drugs at concentrations at which such selectivity becomes highly questionable. Setting these caveats aside, however, such studies still show the variety of actions which such drugs can produce. 15.5.1 Potassium Channel Blockers The effects of non-selective blockers such as TEA and especially aminopyridines have been widely studied. Both types of blocker have been shown to stimulate the release of several different neurotransmitters in vitro. Both basal and


electrically-evoked [3H]-noradrenaline release from hippocampal slices can be increased by TEA, 4–AP and 3,4–diaminopyridine (3,4–DAP) (Huang et al., 1989; Hu and Fredholm, 1991; Allgaier et al., 1993). Similarly, 4–AP and TEA have been reported to enhance acetylcholine release from both striatal (Drukarch et al., 1989; Dolezal and Wecker, 1991) and hippocampal (Fredholm, 1990) slices. Glutamate release from cortical synaptosomes (Nicholls, 1993) or hippocampal slices (Herdon, 1992) can be stimulated by 4–AP or 3,4–DAP respectively. The processes involved in the action of 4–AP on synaptosomal glutamate release have been studied in detail, and it has been suggested that blocking of nerve terminal K channels by 4–AP produces repetitive transient depolarisation of the terminals resulting in transmitter release (Tibbs et al., 1989). Investigations on the effects of 4–AP on electrical activity in hippocampal slices have shown that the compound can produce spontaneous repetitive depolarisations and increases in both EPSP and IPSP amplitude, generating long-lasting depolarisation and epileptiform activity (Perrault and Avoli, 1989, 1991). 4–AP induced epileptiform activity has also been observed in cortical slices (Mattia et al., 1993), and similar effects in human cortex have been reported (Avoli et al., 1992). In vivo studies, including those in humans, have shown that aminopyridines are powerful convulsant and epileptogenic agents (see Perrault and Avoli, 1989, 1991). The exact type of K channel involved in mediating these effects of aminopyridines is not known. However, the fact that the in vitro effects on transmitter release or electrical activity can be produced at relatively low aminopyridine concentrations (10–100 μ M) suggests that the slowlyinactivating D-current, which is particularly sensitive to block by 4–AP (Storm, 1993), may be of prime importance. 15.5.2 KATP Channel Modulators KATP channels have been of particular interest in studies on CNS K channel function. This is partly because of the wide range of pharmacological agents thought to act on these channels, but also because they provide a potential link between neuronal excitability and metabolism. However, evidence for the importance of such channels in neuronal function is still largely indirect, relying principally on studies using drugs which have been classified as modulators of smooth muscle or pancreatic KATP channels. The brain areas in which the actions of such drugs have been most investigated are the substantia nigra and hippocampus. Substantia Nigra The nigral region contains a high density of radiolabelled glibenclamide binding sites, making it a prime target for functional investigations. High concentrations


(500 μ M) of CRK or pinacidil, or intracellular ATP depletion, have been reported to hyperpolarise dopaminergic nigral neurones, with both effects being reversed by 10 μ M glibenclamide (Hausser et al., 1991). In contrast, another study has reported no effects of CRK (500 μ M), glibenclamide (30 μ M) or tolbutamide (300 μ M) on dopaminergic neurones (Hicks and Henderson, 1992). However, the same authors recently described presynaptic effects of tolbutamide (300 μ M) and ATP-depleting conditions on GABAB potentials in nigra (Hicks et al., 1994). A specific K channel has now been identified in nigral neurones which is activated by diazoxide (300 or conditions which deplete ATP, and inhibited by tolbutamide (100–1000 (Schwanstecher and Panten, 1993). Responses to cyanide or hypoxia have also been shown to be inhibited by tolbutamide (50–500 μ M) (Murphy and Greenfield, 1991, 1992). Neurochemical studies have also identified effects of KATP channel modulating drugs. KCOs including LCRK, CRK and pinacidil have been reported as potent (EC50 values 0.01, 0.3 and 0.4 μ M respectively) inhibitors of [3H]-GABA release and stimulators of 86Rb+ efflux from nigral slices (SchmidtAntomarchi et al., 1990). These effects are mimicked by conditions depleting ATP levels, and both types of action are reversed by a range of sulphonylureas, which in themselves stimulate [3H]-GABA release (Amoroso et al., 1990; Schmidt-Antomarchi et al., 1990). Certain of these results seem internally inconsistent, since LCRK is quoted as being 30–50 times more potent than CRK itself (Schmidt-Antomarchi et al., 1990). In addition, the low concentrations of KCOs required to produce these neurochemical effects (ca. 0.5 μ M for CRK or pinacidil) contrast with concentrations of these same drugs three orders of magnitude higher reported (by the same group) to produce electrophysiologically-measurable effects on nigral neurones (Hausser et al., 1991), raising questions as to whether the same mechanism of action is involved. Behavioural studies have indicated that tolbutamide and quinine can alter amphetamine-induced locomotor activity when injected directly into the substantia nigra (Levesque and Greenfield, 1991). Effects on locomotor activity have also been reported following injections of LCRK or glipizide into dorsal pallidum (Amalric et al., 1992). Effects of KATP channel modulators in key motor areas such as the nigra suggest that such drugs might be useful regulators of movement control. Hippocampus High concentrations (10–100 μ M) of CRK have been reported to hyperpolarise hippocampal neurones by increasing a voltage-dependent K+ current (Alzheimer et al., 1989; Politi et al., 1989). Conditions producing ATP depletion produced a similar effect to CRK, and both effects were blocked by 10 μ M. glibenclamide (Politi and Rogawski, 1991). In contrast, a K channel with different electrophysiological characteristics, which is opened by low concentrations (0.1–


1 μ M) of LCRK or hypoglycaemia and inhibited by 1 μ M glibenclamide, has also been described in hippocampal neurones (Tromba et al., 1992). Several studies have described hypoglycaemia and/or hypoxia-induced changes in hippocampal slices which can be modulated by KCOs/KCBs (see below for more detailed discussion). These include both inhibition of anoxiainduced hyperpolarisation by tolbutamide or glibenclamide (Grigg and Anderson, 1989; Godfraind and Krnjevic, 1993) and potentiation of anoxia-induced depolarisation by glibenclamide (Ben-Ari, 1990). However, in contrast to findings reported in substantia nigra, no effects of KCOs on either [3H]-GABA release or 86Rb+ efflux have been detected under either normal or hypoglycaemic/ hypoxic conditions (Nelson, 1989; Herdon et al., 1993). Although a potentiation of hypoglycaemia-induced [3H]-GABA release by high concentrations(10–100 μ M) of glibenclamide has been reported (Margaill et al., 1992), this may not involve KATP channels since it is unaffected by LCRK (Herdon et al., 1993). Such high concentrations of glibenclamide can also block other types of K channels (Crepel et al., 1992; Reeve et al., 1992), providing an alternative mechanism. Other brain areas In dorsal raphe, very high concentrations (100–1000 μ M) of glibenclamide inhibit cell firing; this effect can be reversed by LCRK (1–10 μ M) or by 50 μ M aprikalim (RP 52891). These effects are suggested to be indirectly mediated via actions of the drugs on GABAergic neurones (Haj-Dahmane et al., 1993). On the other hand, direct effects of such drugs are suggested in locus coeruleus, where cell firing rates are decreased by CRK (100 µM) or Ro 31–6930 (10 μ M), with tolbutamide (300 μ M) reversing this effect and increasing cell firing (Finta et al., 1993). In cerebral cortex slices, high concentrations (20–200 μ M) of diazoxide, CRK or pinacidil reduce [3H]-noradrenaline release, with some of these effects being reversed by 1 μ M glibenclamide (Takata et al., 1992). In the same study CRK (200 µM) was reported to increase 86Rb+ efflux, although this effect was not observed at lower drug concentrations in other reports (Nelson, 1989; Herdon et al., 1993). In conclusion, KCMs have certainly been shown by both in vitro and in vivo studies to have a wide range of effects on the CNS. However, a variety of limitations including the lack of selectivity and/or the high concentrations of some of the drugs used in such studies, together with questions over the pharmacological similarity between putative types of CNS K channels and their better-characterised peripheral counterparts, have meant that the exact identification of the type of K channel by which any drug acts to produce an effect on CNS function has not been made. Such identification will require both the further pharmacological characterisation of specific CNS K channels and the development of selective drugs for different types of such channels. Alternatively, the molecular cloning and characterisation of further CNS K


channels combined with the generation of antisense oligonucleotides for such channels (Wahlestedt, 1994) could allow another approach for examining their effects on CNS functions. Despite these limitations and unresolved questions, the pronounced effects of K channel modulating drugs in well-characterised areas such as the hippocampus and substantia nigra have led to suggestions that drugs of this type could be of use in treating CNS disorders associated with these areas such as stroke, epilepsy and movement disorders. Additionally, the overall concept of KCMs as having wide influences on neuronal excitability continues to promote their investigation in other CNS disorders. 15.6 Potential Uses of KCMs in CNS Disorders 15.6.1 Ischaemic Stroke Brain damage due to ischaemia involves a cascade of events. The initial loss of blood supply causes failure of energy-dependent processes such as ion pumps, resulting in depolarisation, massive neurotransmitter release, Na+ and Ca2+ influx etc.; this can produce rapid cell death. However, in areas in which blood supply is restored and normal homeostasis rapidly resumed, delayed neuronal death can still occur days or weeks after the initial ischaemia. This is thought to involve ‘excitotoxicity’, where the initial cell depolarisation and high levels of excitatory amino acids such as glutamate produce intracellular Ca overload, triggering a cascade of intracellular events including enzyme activation, free radical formation and gene expression. These events can enhance excitatory synaptic efficacy and weaken cells so that even normal glutamate levels cause further excitotoxicity, leading to a vicious circle of evolving neuronal damage even in areas which were unaffected by the initial ischaemia (Choi and Hartley, 1993; Hara et al., 1993). K channel opening can produce hyperpolarisation and inhibition of neuronal excitability, suggesting that drugs which act as KCOs could be of benefit in ischaemic stroke. Prophylactic administration of such drugs might limit the spread of the initial ischaemia-induced depolarisation, and administration even some considerable time after the initial ischaemia could reduce the evolving excitotoxicity and limit delayed neuronal death. This latter concept has been validated in animal models of stroke by late administration of drugs which limit excitotoxicity by other mechanisms e.g. glutamate antagonists or neuronal Ca channel blockers (Sheardown et al., 1993; Valentino et al., 1993). It should also be noted that KATP channels and their modulators have been proposed to play key roles in the protection of the heart from the effects of cardiac ischaemia (Escande and Cavero, 1992; Gopalakrishnan et al., 1993). Based on all these ideas and


findings, it could be predicted that KCOs should be neuroprotective in stroke. Indeed, there is now some experimental evidence for this. As summarised in the previous section, there is substantial evidence from in vitro studies that changes in neuronal activity caused by hypoxia and/or hypoglycaemia can be influenced by KCMs, especially those thought to act on KATP channels. In hippocampal slices, hypoxia induces a hyperpolarisation followed by a depolarisation in CA3 neurones. The hyperpolarisation has been reported to be inhibited by glibenclamide or tolbutamide (Grigg and Anderson, 1989; Mourre et al., 1989; Godfraind and Krnjevic, 1993). Conversely, the following depolarisation is increased by glibenclamide and inhibited by diazoxide or galanin (Ben-Ari, 1990; Ben-Ari et al., 1990). The hyperpolarisation is likely to represent a direct postsynaptic effect, whilst the depolarisation probably occurs as a result of presynaptic release of glutamate (Ben-Ari, 1990). In support of this idea, hypoxia/ hypoglycaemia-induced release of glutamate is partially inhibited by LCRK, RP 52891 or galanin, but increased by glibenclamide or gliquidone (Zini et al., 1993b). Additionally, in cultured hippocampal neurones excitotoxicity due to glutamate release and activation of NMDA receptors is inhibited by CRK or diazoxide, with the effects of these drugs being reversed by glibenclamide (Abele and Miller, 1990). However, a study on hypoxic/hypoglycaemic toxicity in cultured cortical neurones has not shown any protective effects of CRK or diazoxide (Koretz et al., 1994). Cyanideinduced neurotoxicity in hippocampal neurones can also be potentiated by glibenclamide, with this effect being reduced by diazoxide (Patel et al., 1992). As summarised above, neuronal damage due to ischaemic stroke is thought to involve glutamate-induced excitotoxicity. Therefore, these in vitro findings suggest that KCOs could be useful for the treatment of stroke. Unfortunately, the testing of such drugs using in vivo animal models of stroke is limited by their poor brain penetration, and interpretation of any neuronal effects observed after systemic administration is complicated by the potent cardiovascular actions of these drugs. In particular, such drugs can produce substantial hypothermia, which itself is strongly neuroprotective (Hara et al., 1993). However, it has been reported recently that i.c.v. injection of LCRK, pinacidil or nicorandil prevents ischaemia-induced gene expression and reduces delayed neuronal death in hippocampus in a rat global ischaemia model, with the actions of these drugs being reversed by glipizide (Heurteaux et al., 1993). This is a very encouraging finding, but the major systemic effects of such KCOs means that the development of neuronally-selective CNS-penetrating drugs will probably be required before this type of therapy is suitable for clinical development. 15.6.2 Epilepsy The basic causes of epilepsy are still uncertain, but some aspects of the pathology of the disease seem related to those of ischaemic-type delayed


neuronal damage. Thus lesions, tumours or other less clear factors can produce local neuronal hyperexcitability leading to epileptic seizures, and repeated brief seizures can produce excitotoxic-type neuronal damage and kindling phenomena in areas such as hippocampus and amygdala. These then lead to increased susceptibility to further seizures, resulting in a spreading cascade of damaging events (McNamara, 1992; Meldrum, 1993). As with ischaemic stroke, experimental seizures can be prevented by glutamate receptor antagonists (Rogawski, 1992), and many drugs used clinically for treatment of epilepsy are capable of modulating ion channel activity to reduce neuronal excitability, for example by blocking Na channels (e.g. phenytoin, carbamazepine) or potentiating GABA/C1 channels (e.g. benzodiazepines, phenobarbitone) (MacDonald and Kelly, 1993; Upton, 1994). Therefore, it is possible that KCMs might also represent a worthwhile treatment for epilepsy. In fact, the experimental evidence for the involvement of K channels in epileptic seizures is much stronger than it is for ischaemic stroke. As mentioned previously, non-selective KCBs such as 4–AP can produce epileptiform activity in brain slices and cause convulsions in vivo; relatively selective K channel blocking toxins such as apamin, MCDP and dendrotoxins are also powerful convulsant agents. Clinically-used anti-epileptic drugs such as carbamazepine, phenytoin, phenobarbitone and valproate have been reported to inhibit some aspects of 4–AP-induced epileptiform activity in hippocampal slices (Fueta and Avoli, 1992; Watts and Jeffreys, 1993), and to protect against dendrotoxininduced seizures in vivo (Coleman et al., 1992). These pro-convulsant effects of KCBs and the inhibition of their effects by clinically-effective anticonvulsant drugs suggest that agents having the opposite action, i.e. KCOs, should be beneficial in the treatment of epilepsy. Indeed, there is evidence that carbamazepine itself can enhance K+ currents at therapeuticallyrelevant concentrations (Zona et al., 1990). Therefore, a selection of KCOs, especially those classified as acting on KATP channels, have been studied in a range of in vivo and in vitro models of epileptic processes. In vitro studies in hippocampal slices have demonstrated that epileptiform activity induced by changes in ionic concentration or treatment with Ca antagonists can be inhibited by high concentrations (30–300 μ M) of CRK (Alzheimer and ten Bruggencate, 1988; Popoli et al., 1991). CRK (given i.c.v.) can also inhibit epileptic-type seizures induced by a variety of convulsant agents including pentylenetetrazole (Del Pozo et al., 1990), MCDP (Gandolfo et al., 1989a) and digoxin (Chugh et al., 1993), and decrease seizures in genetically epileptic rats (Gandolfo et al, 1989b). The actions of CRK on MCDP seizures could be mimicked by RP 49356, but neither drug could inhibit 4–AP or dendrotoxin-induced seizures (Gandolfo et al., 1989a). However, as with the potential treatment of ischaemic stroke, the poor brain penetration and major cardiovascular effects of present generation KATP channel openers limit greatly the further testing and possible clinical application of these drugs. Whether selective openers of other types of K channels might be effective as


anticonvulsant agents remains unknown. Certain compounds related to CRK have recently been described as being active as anticonvulsants but possessing minimal hypotensive activity (Blackburn et al., 1993), but whether this anticonvulsant activity is related to effects K channels has not been reported. 15.6.3 Pain As mentioned previously, μ - and β -opioid receptors are linked to CNS K channels (North, 1989). Therefore, KCMs might be able to mimic the actions of opiate drugs in producing analgesia. Non-selective KCBs such as 4–AP and quinine can inhibit behavioural and neurochemical effects of morphine (Pei et al., 1993), and the analgesic effects of morphine or the β -opiate peptide DPDPE can be antagonised by i.c.v. glibenclamide (Ocana et al., 1990; Wild et al., 1991; Narita et al., 1992). Sulphonylureas can also antagonise the antinociccptive effects of clonidine (Ocana and Baeyens, 1993). Conversely, i.c.v. CRK or pinacidil can potentiate morphine analgesia (Vergoni et al., 1992; Narita et al., 1993), and CRK alone can also produce antinociceptive effects (Narita et al., 1993; Kamai et al., 1994). In addition, i.c.v. CRK or diazoxide can inhibit some of the behavioural effects of morphine withdrawal (Robles et al., 1994). These findings suggest that brain-penetrant KATP channel opening drugs might have clinical utility as analgesic agents as well as for the treatment of opiate withdrawal. 15.6.4 Parkinson’s Disease As reviewed earlier, studies on the substantia nigra have shown electrophysiological, neurochemical and behavioural effects of KATP channel modulating drugs. Since the nigra is a major source of dopaminergic neurones, and nigro-striatal dopaminergic degeneration produces Parkinson’s disease, KATP channel modulators might be of benefit in this disease. Based on experiments with KATP channel inhibitors, it has been suggested that openers of these channels could both increase dopamine release and mimic the post-synaptic actions of dopamine, thus reducing Parkinsonian symptoms (Levesque and Greenfield, 1991; Murphy and Greenfield, 1991; see also Amalric et al., 1992). However, direct experimental evidence for this idea is lacking. 15.6.5 Alzheimer’s Disease Neuronal degeneration producing deficits in cholinergic transmission is thought to be a cause of some of the major symptoms such as memory impairment in Alzheimer’s disease. As summarised earlier, acetylcholine can alter the activity of several types of K channels; conversely, KCMs can affect acetylcholine


release. This reciprocal relationship suggests that KCMs could be of use in alleviating some of the effects of cholinergic deficit. In fact, non-selective KCBs such as TEA or aminopyridines have been reported to produce long-term potentiation in vitro (Aniksztejn and Ben-Ari, 1991) and to improve memory both in animal models and Alzheimer’s disease patients (see Lavretsky and Jarvik, 1992). This area is complicated by the fact that aminopyridines can also act as cholinesterase inhibitors; conversely, cholinesterase inhibitors such as tetrahydroaminoacridine (used clinically for treatment of Alzheimer’s disease) can block K channels. In fact, it has been suggested that agents with both cholinesterase-inhibiting and K channel-blocking properties would be suitable treatments for Alzheimer’s disease (Lavretsky and Jarvik, 1992). Another possible connection between K channels and Alzheimer’s disease has produced a lot of recent interest. It has been reported that a TEA-sensitive K channel present in normal fibroblasts is functionally absent in fibroblasts from Alzheimer’s disease patients (Etcheberrigaray et al., 1993). This finding obviously suggests a potential simple diagnostic test for the disease, but also raises the question of whether there is a similar defect in CNS K channel function. The fact that low concentrations of β -amyloid protein, a key factor in the CNS pathology of Alzheimer’s disease, can produce this same defect in normal human fibroblasts (Etcheberrigaray et al., 1994) certainly suggests that abnormalities in CNS K channels could be involved in producing some of the symptoms of Alzheimer’s disease. If this is the case, it could point to another role for KCMs in the treatment of this disease.

15.7 Summary and Conclusions A range of K channels are now recognised to be present in the CNS, and the electrophysiological and molecular biological properties of several types have been well characterised. In addition, the actions of neurotransmitters, toxins and nonselective blocking drugs on certain specific types of K channels have been studied in detail; such studies have helped to elucidate some of the mechanisms of regulation of K channels at molecular or single channel level. On the other hand, the CNS effects of a variety of drugs and toxins of differing selectivities have been studied at a macroscopic level on diverse functions such as cell firing, transmitter release or behaviour. However, it has proved difficult to link the two sets of findings together i.e. to determine the specific K channels on which any drug acts to produce a macroscopic functional effect. This problem has been largely a result of the lack of synthetic drugs which are highly selective for specific CNS K channels, and it is to be hoped that further efforts by the pharmaceutical industry together with an increased knowledge of CNS K channel properties will help to overcome this.


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16 Potassium Channel Modulators: Clinical Experiences and Future Prospects T.J.COLATSKY1 & T.C.HAMILTON2 1 Division of Cardiovascular and Metabolic Diseases, Wyeth-Ayerst

Research, CN 8000, Princeton, NJ 08543, USA. 2

Department of Neurology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK.

In this chapter we have reviewed, in separate sections for potassium channel activators (KCAs) (sections 16.1 to 16.4) and blockers (KCBs) (sections 16.5 to 16.16), clinical experiences and future prospects for their use in therapeutics. This approach seems logical to us in view of the disparate applications for these two drug classes. 16.1 KCAs Extensive pre-clinical evaluation of KCAs in a diverse range of animal models representative of a number of diseases (see Chapters 9, 11, 12, 14 and 15), has led to optimistic predictions of the likely uses of this class of drug in the clinic (Hammond et al., 1991; Andersson, 1992; Atwal, 1992; Gopalakrishnan et al., 1993; Poyser and Hamilton, 1994). This optimism is reflected by the intense efforts of a large number of pharmaceutical companies in the synthesis of many structurally novel KCAs (see Chapters 1, 2 and 3). However, whilst the extensive patent literature (see Current Drugs Database) and publications in pharmacology journals, give credence to the direction of drug discovery in this field, a relatively small number of papers describe the actions of these drugs in man (see Williams, 1992). It is our goal in this chapter to examine briefly the clinical potential of KCAs, then to review the available clinical findings for benzopyran KCAs (derived from cromakalim, CRK), for other chemical classes developed subsequently (such as thioformamides like aprikalim) and in the case of the cyanoguanidine, pinacidil, clinical data appearing since the review by Friedel and Brogden (1990). Finally, our opinions regarding future prospects for KCAs are discussed.

KCMS: CLINICAL EXPERIENCES AND FUTURE PROSPECTS 461

16.2 Therapeutic Potential Table 16.1 summarises a number of possible clinical uses of KCAs based upon the premise that such drugs, by opening membrane K channels, should raise membrane Table 16.1 Perspective of some potential therapeutic uses of KCAs

Cardiovascular System Hypertension Pulmonary Hypertension Peripheral Vascular Disease Angina Cardioprotection Congestive Heart Failure Respiratory System Chronic Obstructive Airways Disease including Asthma Inflammatory Airways Other Smooth Muscle Bladder instability Impotence Irritable Bowel Syndrome Uterine Disorders Secretory Diarrhoea Nervous System Epilepsy Pain Ischaemia (stroke) Others Hyperlipidaemia Hypotrichosis + in vivo data + (in vitro data) – no data

Pre-Clinical Findings

Clinical Findings

+ + + + + +

+ – + (+) (+) (+)

+ +

+ –

+ +(in vitro) – + +(in vitro)

+ – – – –

+ + +

– – –

– +

+ +

(+) data for nicorandil

potential (hyperpolarise) and thereby reduce tissue excitability. Support for this hypothesis has come from extensive pre-clinical data in in vivo studies using drugs such as CRK, aprikalim and pinacidil. In broad terms these potential uses may be categorised according to effects of KCAs upon body systems and tissues


(Table 16.1). Clearly, utility in many indications depends upon inhibition of smooth muscle contractility. For some others reduction in neuronal excitability is important whilst in hyperlipidaemia and hypotrichosis, the underlying mechanism (s) are less clear. Table 16.1 also reveals that the number of clinical indications in which the efficacy of KCAs has been demonstrated is confined to a few. Moreover, as described later, the available clinical data are derived from a limited number of studies using two or three drugs. Various speculative reasons could be proffered for the scarcity of clinical data but it is suffice to state here that, with one or two known exceptions, the biological profile as exhibited by CRK and pinacidil has been difficult to modify. Thus, for example, KCAs selective for one particular smooth muscle, or for neuronal K channels, have not yet emerged. This theme will be addressed later. 16.3 Clinical Experiences 16.3.1 Hypertension KCAs are directly acting smooth muscle relaxants and thereby produce vasodilation and reduce peripheral vascular resistance (see Chapter 9). Thus, these drugs have the ability to modify the primary haemodynamic abnormality present in essential hypertension, namely elevated total peripheral vascular resistance. Evidence has been obtained in man for KCA-mediated changes in the vasculature. In healthy volunteers, orally (0.5 to 2 mg) or i.v. (0.01 to 10 μ g/min) administered CRK increased forearm blood flow (Webb et al., 1989; Fox et al., 1991) and oral levcromakalim (LCRK) (0.5 and 0.75 mg) increased vessel diameter in the eye (Eckl et al., 1992). Oral CRK did not affect forearm venous capacitance (Fox et al., 1991) or, when infused i.v. (1 μ g/min), dorsal hand vein diameter (Haynes and Webb, 1991). Also, Thomas et al., (1990) reported that i.v. CRK (15 μ g/kg) reduced systemic, and pulmonary, vascular resistance by 29 and 24% respectively, in patients with ischaemic heart disease (IHD). Thus these KCAs are arterio-selective vasodilators in man. The ability of CRK to modify vasoconstrictor responses has been examined in volunteers. Nguyen et al. (1991) found that oral CRK (1 and 2 mg) blunted the vasoconstrictor responses to noradrenaline and angiotensin II. In contrast, CRK did not affect noradrenaline-induced venoconstriction (Webb et al., 1989) but did reduce similar responses due to endothelin (Haynes and Webb, 1991). In hypertensive patients, Lebel et al. (1991) found that CRK (1.5 mg daily for 3 days) reduced blood pressure (BP) while glomerular filtration rate was unchanged and effective renal plasma flow (ERPF) was slightly elevated. In


contrast, in hypertensive patients also receiving the β -adrenoceptor blocker, atenolol, CRK (1 mg orally) did not affect hepatic blood flow or ERPF (Donnelly et al., 1990). In the latter study the minor effect on BP of CRK, given alone or in combination with atenolol, suggested that the dose and/or plasma t1/2 (ca. 24 hours; Gill et al., 1988) for the parent drug were insufficient to produce an adequate lowering of BP of prolonged duration. However this conclusion is contrary to that from other studies with CRK (Vandenburg et al., 1986, 1987) and LCRK (Jain et al., 1991; see Hamilton et al., 1993) which showed that once daily oral administration (in the range 0.5 to 3 mg for LCRK) lowered supine DBP for at least 24 hours. In particular, in the double-blind, randomised, placebo controlled, parallel group study performed with LCRK, single daily doses of 0.5, 0.75, 1 and 1.5 mg were given daily for 8 weeks and produced significant decreases from baseline in trough supine DBP, without change in heart rate (HR) in all groups (see Hamilton et al., 1993) (Figure 16.1). Thus the pharmacodynamic and pharmacokinetic properties of this drug appear to be adequate for once daily treatment in mild to moderate hypertensive patients. Interestingly, the only other benzopyran, celikalim, tested so far in hypertension, has an exceptionally long plasma half-life (about 15 days) (Lasseter et al., 1992). As may be predicted from a direct vasodilator action, the KCAs cause reflexly mediated increases in HR, cardiac output and stroke volume (Thomas et al., 1990;

Figure 16.1 Mean supine diastolic blood pressure (mmHg) at baseline (

Fox et al., 1991; Senior et al., 1993). Indeed studies with oral bimakalim in volunteers, and i.v. CRK in IHD patients, suggested that KCAs may have


beneficial effects on cardiac function in patients with compromised left ventricular function or IHD. However, in the treatment of hypertension, an increase in HR is undesirable at BP lowering doses. Experiences with antihypertensive doses of CRK and LCRK in patients indicated that increases in HR are modest after single doses, and not an issue in chronic studies (Vandenburg et al., 1987; Erwteman et al., 1991). The long term effects of LCRK on cardiac mass remain to be determined but, in the case of pinacidil (Steensgaard-Hansen and Carlsen, 1988), left ventricular mass was reduced (−23%) in hypertensive patients receiving concomitant bendrofluazide treatment. Thus good prospects exist that KCAs will not exacerbate left ventricular hypertrophy. Another possible consequence of the use of directly-acting arterio-vasodilators such as LCRK and pinacidil, is reflex counter-regulatory stimulation of the reninangiotension-aldosterone system with the attendant risks of elevated plasma renin activity (PRA) and angiotensin II levels leading to peripheral oedema and, if severe, to increases in body weight due to Na retention. Although raised PRA has been reported following.CRK in volunteers (Ferrier et al., 1989; Lijnen et al., 1989a,b; Singer et al., 1989), this parameter has not been extensively studied in hypertensive patients receiving this KCA. Singer et al. (1989) found that single oral doses of CRK raised PRA, without altering plasma aldosterone, in both volunteers and hypertensive patients, but Lebel et al. (1988) found no change in PRA in a short-term daily dosing study in patients. In longer term studies, peripheral oedema has been reported after CRK and LCRK but without increased body weight (Vandenburg et al., 1987; Ertwemann et al., 1991; see Hamilton et al., 1993). Support for these findings is provided by lack of an effect of CRK on renal haemodynamics in short-term studies in patients (Singer et al., 1989; Lebel et al., 1991). However these results now need to be confirmed in longer term studies with LCRK. Most interestingly, CRK, LCRK and pinacidil have a beneficial effect on plasma lipid profile in hypertensive patients. First reported for pinacidil (see Friedel and Brogden, 1990) and CRK (Lacourciere et al., 1989), LCRK has also been shown to lower plasma triglycerides and total cholesterol, and to raise HDL cholesterol, when given in single daily doses, in the range of 0.5 to 1.5 mg, for 8 weeks (see Hamilton et al., 1993). Changes in high density lipoprotein (HDL) cholesterol and triglycerides were greater in a sub-set of patients with elevated baseline cholesterol (― 6.5 mmol/1) (Figure 16.2). Similarly, in Japanese hypertensive patients, LCRK (0.5 or 1 mg daily for 12 weeks) significantly lowered total cholesterol, triglycerides and apoproteins (Sasaki et al., 1994b). In a double-blind, comparator 12 week study, Japanese hypertensive patients were randomised to receive either LCRK (0.5, 1.0 mg) or the β 1 blocker, doxazosin (0. 5, 1.0, 2.0, 4.0 mg) (Sasaki et al., 1994a). The dose was titrated such that both drugs reduced DBP from 101 to 85 mmHg. Baseline total cholesterol levels were >250 mg/dl in both groups. Doxazosin had no significant effects on serum lipids whereas LCRK decreased total, VLDL, LDL and HDL3 cholesterol and triglycerides and increased HDL2 cholesterol. Between group differences were


detected with increases in HDL2 cholesterol and decrease in LDL/HDL. The beneficial changes in lipid profile produced by LCRK and pinacidil, distinguishes KCAs from most other classes of anti-hypertensive drugs (see Table 16.2) and, in the long term, may suggest a modifying effect on the development of atherosclerosis. Thus KCAs have the potential to modify two cardiovascular risk factors. However, in view of the prevalence of the use of combination therapy in the treatment of hypertension, it will be important to demonstrate that the changes in lipid profile produced by KCAs persist in the presence of concomitant low-dose diuretic and/or β -blocker treatment. In contrast to the benzopyran KCAs, the pharmacodynamic properties of pinacidil in the treatment of mild to moderate hypertension have been studied extensively (see Goldberg, 1988; Friedel and Brogden, 1990; Longman and Hamilton, 1992). The consensus view has emerged that this KCA is efficacious and exhibits both the pharmacodynamic and adverse event profile typical of a vasodilator antihypertensive agent. Overall pinacidil is associated with a high incidence of oedema, leading to increased body weight, and these effects have led to the recommendation from the FDA that pinacidil should be co-prescribed with a diuretic. Pinacidil is also associated with an incidence of hypertrichosis, possibly related to its mechanism of action as a KCA. Minoxidil sulphate, another KCA, is already marketed as a local application to treat hypotrichosis (see section 16.3.5). However, neither CRK nor LCRK have been associated with a single case of hypertrichosis. The short plasma half-life (2–3 hours) of pinacidil led to the development of a sustained release preparation, but twice daily dosing is still required (in contrast to once daily with LCRK). The new formulation of pinacidil may also have the advantage, by allowing the slower development of the maximum plasma levels of parent drug, of minimising reflexly-mediated counter-regulatory haemodynamic effects. Despite earlier reports of tolerability problems with pinacidil, a recent ‘Quality of Life’ study (Fletcher et al., 1992) presented a favourable outcome for pinacidil in



Figure 16.2 Effects of levcromakalim on plasma lipids following 8 weeks administration with single daily doses of 1.0 and 1.5 mg. Changes are % from the baseline established before administration and are shown for all patients and for a subset of hypercholesterolemic patients (total cholesterol >6.5 mmol/before treatment). (Reproduced from Poyser and Hamilton (1994) with permission). Table 16.2 Anti-hypertensive agents: changes in plasma lipid profile Class

Triglycerides

Total Chol.

LDL-Chol.

HDL-Chol.

HDL:Total

KCA

――

4

―

――

――

CCB

o

o

o

o

o

ACE-I

o

o

o

―

o

β 1-blocker

―

―

―

―

―

β -blocker

―

o

o

―

―

Diuretic

―

―

―

―

―

KCA=potassium channel activator; CCB=calcium channel blocker; ACE-I = angiotension converting enzyme inhibitor Chol.=Cholesterol; LDL=Low density lipoprotein; HDL=High density lipoprotein ― Decrease ― No change ― Increase

comparison with the dihydopyridine Ca channel blocker, nifedipine, in hypertensive patients also receiving a thiazide diuretic. Both drugs were given as sustained release formulations but unfortunately no placebo group was included in this study. Both drugs caused similar falls in supine DBP at 6 weeks, the target level being achieved in 57% and 63% of patients taking pinacidil and nifedipine respectively. Of the few differences noted between the drugs in this study, pinacidil increased body hair growth and nifedipine caused facial flushing in some patients. Recently, Buoninconti et al. (1993) found, in a 10 week study, comparable effectiveness (Figure 16.3) and tolerability for pinacidil and the ACE-inhibitor, captopril, whether given as monotherapy or combined with hydro-chlorthiazide, to achieve the target BP. Patients maintained on pinacidil alone for 6 months did not show increases in body weight. The mechanism of action of KCAs has inevitably led to questions regarding their-possible inhibitory effects on insulin secretion since this glucose-stimulated event is mediated via K channel closure in pancreatic β -cells. However, both in vitro and in vivo animal studies do not indicate a potential problem in the clinic with regard to either insulin release per se or interference with the ability of the sulphonylurea drugs (such as glibenclamide) to evoke insulin release (see Chapter 13). Recent clinical data obtained with pinacidil (up to 25 mg daily) has shown that, in healthy volunteers (Nielsen-Kudsk et al., 1990) and hypertensive patients (Ligtenberg et al., 1993) (Figure 16.4), the increases in fasting blood


insulin and glucose during oral and i.v. glucose tolerance tests were unaffected by the KCA. These findings therefore support those from animals and other evidence (see Chapter 13), that K channels in the vasculature and pancreatic β -cells are not identical. 16.3.2 Asthma The pharmacological properties of KCAs extend to relaxation of airways smooth muscle and to inhibition of bronchoconstriction produced by stimulation of nonadrenergic, non-cholinergic excitatory nerves to guinea-pig lungs (see Chapter 12). The latter nerves may be involved in releasing inflammatory mediators in airways. Limited clinical data exist to support the potential use of KCAs as bronchodilators but none exist so far for their anti-inflammatory activity in airways.


Figure 16.3 Supine blood pressure and heart rate (HR) during 10 weeks of treatment with pinacidil or with captopril, in monotherapy or in combination with hydrochloro-thiazide. (Values are expressed as mean±SEM [I].) *p

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