Nitric Oxide and the Cardiovascular System (Contemporary Cardiology) (Contemporary Cardiology)

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CONTEMPORARY

CARDIOLOGY

Nitric Oxide and the Cardiovascular System Edited by

Joseph Loscalzo, MD, PhD Joseph A. Vita, MD

HUMANA PRESS

NITRIC OXIDE AND THE CARDIOVASCULAR SYSTEM

CONTEMPORARY 9 CARDIOLOGY Christopher P. Cannon, Series Editor Management of Acute Coronary Syndromes, edited by Christopher P. Cannon, 1999 Minimally Invasive Cardiac Surgery, edited by Mehmet C. Oz and Daniel J. Goldstein, 1999 Annotated Atlas of Electrocardiography: A Guide to Confident Interpretation edited by Thomas M. Blake, 1999 Platelet Glycoprotein IIb/IIIa Inhibitors in Cardiovascular Disease, edited by A. Michael Lincoff and Eric J. Topol, 1999 Nitric Oxide and the Cardiovascular System, edited by Joseph Loscalzo and Joseph A. Vita, 2000

NITRIC OXIDE AND THE CARDIOVASCULAR SYSTEM Edited by

JOSEPH LOSCALZO, MD, PHD JOSEPH A. VITA, MD Boston University Medical Center, Boston, MA

HUMANA PRESS TOTOWA, NEW JERSEY

© 2000 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341, E-mail: [email protected]; or visit our Website: http://humanapr.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. All articles, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients. Further it is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication.

Cover design by Patricia F. Cleary. This publication is printed on acid-free paper. ' ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-620-0/00 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data

PREFACE The field of nitric oxide biology has expanded considerably over the past decade with a growing appreciation of its many roles in a variety of cell and organ systems. Nitric oxide was first discovered in the cardiovascular system, and the importance of this discovery led to the award of the 1998 Nobel Prize in Physiology or Medicine to Robert Furchgott, Louis Ignarro, and Fred Murad, well-known cardiovascular investigators. With this history, it should come as no surprise that our understanding of the role of nitric oxide in biology and pathobiology is, perhaps, best developed as it relates to cardiovascular biology and disease. For this reason, we felt it would be both timely and relevant to review in detail the role of nitric oxide in cardiovascular biomedicine. To this end, we assembled a group of contributing authors with expertise in areas that include the chemistry of nitric oixide, the biochemistry of its synthesis, the molecular biology of nitric oxide synthases, the pharmacology of nitrovasodilators, and the role of nitric oxide in vascular diseases. With the recent expansion of the field in directions that range from the development of novel nitric oxide donors for the treatment of myocardial ischemia and thrombosis to the development of gene therapy approaches for the restoration of endothelial function in atherosclerosis, the application of nitric oxide biology to investigative and clinical arenas in cardiovascular medicine is, indeed, rapidly evolving. This comprehensive overview should prove useful for basic and clinical investigators alike, as well as practicing clinicians in the fields of cardiology, hematology, and vascular medicine. With a balanced presentation of basic and clinically relevant subject matter, this text will provide a compendium of information that may guide the reader through the foundations of the most recent developments in this rich and exciting field.

ACKNOWLEDGMENT We thank Stephanie Tribuna for her assistance throughout the many phases of the development of this text, and Jalna Ross for her assistance in reference verification. Joseph Loscalzo, MD, PHD Joseph A. Vita, MD

v

To Anita, Julia, Alex, Gina, Olivia, and Sam

CONTENTS Preface ............................................................................................................................ v Contributors ................................................................................................................... ix

PART I BIOLOGY OF NITRIC OXIDE .................................................. 1 1 2 3 4 5 6 7 8 9 10 11 12

The Biological Chemistry of Nitric Oxide ....................................... 3 Joseph Loscalzo Cell and Molecular Biology of Nitric Oxide Synthases ................ 11 Olivier Feron and Thomas Michel Cellular Signal Transduction and Nitric Oxide .............................. 33 Stanley Heydrick Regulation of Gene Expression by Nitric Oxide ........................... 51 Ami A. Deora and Harry M. Lander Cytotoxicity, Apoptosis, and Nitric Oxide ..................................... 69 Stefanie Dimmeler and Andreas M. Zeiher Nitric Oxide and Ion Channels ....................................................... 85 Victoria M. Bolotina Role of Nitric Oxide in Vasomotor Regulation ........................... 105 Richard A. Cohen Nitric Oxide and Platelet-Mediated Hemostasis .......................... 123 Elisabeth M. Battinelli and Joseph Loscalzo Nitric Oxide Modulates Leukocyte–Endothelial Cell Adhesion . 139 Wolfgang Cerwinka and D. Neil Granger Nitric Oxide and Cardiomyocyte Function .................................. 153 Jean-Luc Balligand and Paul J. Cannon The Fibroblast and Nitric Oxide ................................................... 177 Peter Brecher Nitric Oxide in Cardiac Electrophysiology .................................. 191 Lü Fei and Douglas P. Zipes

PART II CARDIOVASCULAR PATHOPHYSIOLOGY OF NITRIC OXIDE ..... 205 13

Nitric Oxide and Endothelial Dysfunction ................................... 207 Gerard A. Dillon and Joseph A. Vita

14

Nitric Oxide and Hypertension ..................................................... 227 M. Audrey Rudd, María R. Trolliet, and Joseph Loscalzo

15

Nitric Oxide and Pulmonary Hypertension .................................. 247 John J. Leopore and Kenneth D. Bloch

16

Nitric Oxide in Atherosclerosis .................................................... 273 Robert T. Eberhardt and Joseph Loscalzo vii

viii

Contents

17

Thrombotic Disorders and Nitric Oxide Insufficiency ................ 297 Jane E. Freedman and Joseph Loscalzo

18

Myocardial Nitric Oxide in Heart Failure .................................... 309 Douglas B. Sawyer and Wilson S. Colucci

19

Shock States and Nitric Oxide ...................................................... 321 Hartmut Ruetten and Christoph Thiemermann

20

Stroke and Nitric Oxide ................................................................ 343 Nancy E. Stagliano and Paul L. Huang

21

The Role of Nitric Oxide in Ischemia-Reperfusion ..................... 357 Allan M. Lefer and Reid Hayward

PART III NITRIC OXIDE IN CARDIOVASCULAR THERAPEUTICS ...... 381 22

Nitrovasodilators ........................................................................... 383 John D. Horowitz

23

S-Nitrosothiols .............................................................................. 411 Jane A. Leopold and Joseph Loscalzo

24

Diazeniumdiolates (Formerly NONOates) in Cardiovascular Research and Potential Clinical Applications .......................... 431 Joseph E. Saavedra, Anthony L. Fitzhugh, and Larry K. Keefer

25

Inhaled Nitric Oxide Therapy for Acute Respiratory Failure ...... 447 William Hurford and Warren M. Zapol

26

Antioxidants and Endothelium-Derived Nitric Oxide Action ..... 473 Annong Huang and John F. Keane`y, Jr.

27

Coating Arterial and Blood-Contacting Surfaces • with NO -Donating Compounds .............................................. 503 John D. Folts and Joseph Loscalzo

28

Gene Therapy and Nitric Oxide ................................................... 515 Heiko E. von der Leyen

29

Nitric Oxide and Tissue Preservation in Transplantation ............ 529 David J. Pinsky and David M. Stern

30

L-Arginine: Its Role in Cardiovascular Therapy ......................... 547 Andrew J. Maxwell and John P. Cooke

Index ........................................................................................................................... 587

CONTRIBUTORS JEAN-LUC BALLIGAND, MD, PHD • Department of Medicine, Pharmacology Unit, University of Louvain Medical School, Brussels, Belgium ELIZABETH M. BATTINELLI, MSC • Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA KENNETH D. BLOCH, MD • Cardiology Division and the Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA VICTORIA BOLOTINA, PHD • Vascular Biology Unit, Evans Department of Medicine, Boston University School of Medicine, Boston, MA PETER BRECHER, PHD • Boston University School, of Medicine, Whitaker Cardiovascular Institute, Boston, MA PAUL J. CANNON , MD• Department of Medicine, Division of Cardiology, Columbia University College of Physicians and Surgeons, New York, NY WOLFGANG CERWINKA, MD • Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA RICHARD A. COHEN, MD • Vascular Biology Unit, Evans Department of Medicine, Boston University School of Medicine, Boston, MA WILSON S. COLUCCI, MD, FACC • Myocardial Biology Unit, Boston University School of Medicine, and Cardiovascular Division, Department of Medicine, Boston University Medical Center, Boston, MA JOHN P. COOKE, MD, PHD • Section of Vascular Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA AMI A. DEORA, PHD • Department of Biochemistry, Cornell University Medical College, New York, NY GERARD DILLON, MD • Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA STEFANIE DIMMELER, PHD • Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Germany ROBERT T. EBERHARDT, MD • Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA LÜ FEI, MD, PHD • Krannert Institute of Cardiology, Department of Medicine, Indiana University School of Medicine, and the Roudebush Veterans Administration Medical Center, Indianapolis, IN OLIVIER FERON, PHD • Pharmacology and Therapeutic Unit, Department of Medicine, University of Louvain Medical School, Brussels, Belgium ANTHONY L. FITZHUGH, MD • Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD JOHN D. FOLTS, PHD, FACC • Coronary Thrombosis Research Laboratory, Department of Cardiology, University of Wisconsin Medical School, Madison, WI JANE E. FREEDMAN, MD • Department of Clinical Pharmacology, Georgetown University, Washington, DC ix

x

Contributors

D. NEIL GRANGER, PHD • Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA REID HAYWARD, PHD • Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA STANLEY HEYDRICK, PHD • Boston University School of Medicine, Boston, MA JOHN D. HOROWITZ, MBBS, PHD • Cardiology Unit, The Queen Elizabeth Hospital, Woodville, Australia ANNONG HUANG, PHD • Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA PAUL L. HUANG, MD, PHD • Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA WILLIAM E. HURFORD, MD • Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA JOHN F. KEANEY, JR., MD • Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA LARRY K. KEEFER, PHD • Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD HARRY M. LANDER, PHD • Department of Biochemistry, Cornell University Medical College, New York, NY ALLAN M. LEFER, PHD • Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA JANE A. LEOPOLD, MD • Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA JOHN J. LEPORE, MD • Cardiology Division and the Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA JOSEPH LOSCALZO, MD, PHD • Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA ANDREW J. MAXWELL, MD • Cooke Pharma, Belmont, CA THOMAS MICHEL, MD, PHD • Cardiology Division, West Roxbury VA Medical Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA DAVID J. PINSKY, MD • Divisions of Cardiology and Circulatory Physiology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY M. AUDREY RUDD, PHD • Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA HARTMUT RUETTEN, MD • The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK JOESPH E. SAAVEDRA, PHD • Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD DOUGLAS B. SAWYER, MD • Myocardial Biology Unit, Boston University School of Medicine, and Cardiovascular Division, Department of Medicine, Boston University Medical Center, Boston, MA

Contributors

xi

NANCY E. STAGLIANO, PHD • Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA DAVID M. STERN, MD • Divisions of Cardiology and Circulatory Physiology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY CHRISTOPH THIEMERMANN, MD • The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK MARÍA R. TROLLIET, PHD • Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA JOSEPH A. VITA, MD • Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA HEIKO E. VON DER LEYEN, MD, PHD • Cardiogene AG, Erkrath, Germany, and Department Innere Medizin, Medizinische Hochschule, Hannover, Germany WARREN M. ZAPOL, MD • Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA ANDREAS M. ZEIHER, MD • Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Germany DOUGLAS P. ZIPES, MD • Krannert Institute of Cardiology, Department of Medicine, Indiana University School of Medicine, and the Roudebush Veterans Administration Medical Center, Indianapolis, IN

Chapter 1 / Chemistry of Nitric Oxide

I

BIOLOGY OF NITRIC OXIDE

1

2

Part I / Biology of Nitric Oxide


1

3

The Biological Chemistry of Nitric Oxide Joseph Loscalzo

INTRODUCTION Nitric oxide (NO•) is a heterodiatomic free radical that can participate in a wide range of biochemically relevant reactions to evoke a panoply of biological responses. In order to understand the biochemistry of NO•, we must first consider its chemistry. This introductory chapter provides an overview of the relevant chemistry of NO• and its derivative biochemical reactions, both with respect to normal biological actions and pathophysiological effects.

CHEMICAL PROPERTIES OF NO• In contrast to other biological free radicals, NO• is a free radical of limited reactivity. As one measure of this important property, NO• can diffuse over distances as great as several microns in aqueous solvent before engaging in collision-dependent reactions; superoxide anion radicals and hydroxyl radicals, in comparison, are much more reactive, diffusing over very much shorter reaction distances. The relative stability of NO• is a consequence of its unpaired electron being localized to a 2p-π-antibonding orbital. The bond order of NO• is 2.5 (defined by three bonds gained from the filled σx, πx, and πy molecular orbitals minus half a bond from the partially filled π* antibonding orbital), and this order does not change appreciably on dimerization (approx 5), again supporting the relatively low reactivity of the molecule. At room temperature, NO• is a colorless gas (boiling point, −151.7oC at 1 atm). The solubility of NO• is 1.9 mM/atm in aqueous solution (1,2), which is comparable to that of molecular oxygen. Because of its somewhat nonpolar nature and relative stability, NO• can diffuse at a rate of approx 50 µm/s (14) in aqueous solution; its half-life in vivo is approximately 10 s (4). The broad range of reactions in which NO• can participate is largely attributable to the variety of nitrogen oxide (NOx) species found in aqueous systems. NO• can undergo oneelectron oxidation (to nitrosonium, NO+) or reduction (to nitroxyl anion, NO−) with the following estimated half-cell potentials (5): NO• → NO+ + e−

E1/2 = −1.2 V

(1)

NO• + e− → NO−

E1/2 = −0.33 V

(2)

These reactions are analogous to oxidation and reduction reactions of superoxide radical to molecular oxygen and to hydroxyl radical/hydrogen peroxide, respectively. Yet the analogy is imperfect, as nitrosonium is isoelectronic to carbon monoxide, whereas nitroxyl anion is isoelectronic to molecular oxygen. The susceptibility of NO• to oxidation is consonant with From: Contemporary Cardiology, vol. 4: Nitric Oxide and the Cardiovascular System Edited by: J. Loscalzo and J. A. Vita © Humana Press Inc., Totowa, NJ

3

4


the fact that the highest occupied molecular orbital is an antibonding orbital. In support of this view, the ionization potential of NO• is low (9.25 eV) compared with that of N2 (15.56 eV), and the N–O bond length decreases by only 0.09 Å when NO• is oxidized to nitrosonium (6). NO• is central to the overall redox scheme for nitrogen oxides. This scheme begins with ammonia and ends with nitrate by way of a series of intermediates as shown in Eq. 3 with the nitrogen valence states indicated beneath each species: NH3 ' NH2OH ammonia hydroxyl amine −3 −1

HNO ' NO• ' hydrogen nitric oxide nitride (nitroxyl) +1 +2

'

NO2− nitrite

'

+3

NO3− (3) nitrate +5

REACTIONS WITH OXYGEN NO• reacts readily with oxygen, largely as a consequence of the diradical nature of the triplet species of molecular oxygen (with two unpaired electrons occupying degenerate π* antibonding molecular orbitals, formally given as 3O2). The product of this reaction is nitrogen dioxide (NO2), and the kinetics of its formation are second order in NO• and first order in O2, consistent with the third-order equation: d[NO•]/dt = k[NO•]2[O2]

(4)

where k = 7 × 106 M−2 • s−1 (7–10). Interestingly, this reaction is virtually unaffected by normal physiological ranges of pH and temperature. The importance of this rate process rests on its second-order dependence on NO•, indicating that the oxidative decomposition of the molecule is a strict function of its concentration: the higher the concentration, the more rapid its autooxidation. This property of NO• indicates that the further NO• diffuses from its source of synthesis, the more likely it is to be available to exert its biological effects. In aqueous environments, the terminal oxidation product of NO• is nitrite (NO2−), into which NO2 stoichiometrically decomposes by the following mechanism: 2NO• + O2 → 2NO2

(5)

NO• + NO2 → N2O3

(6)

N2O3 + H2O → 2NO2− + 2H+

(7)

REACTIONS WITH OXYGEN-DERIVED RADICALS Nitric oxide can react readily with superoxide anion at a rate that is essentially diffusion controlled (k = 6.7 × 109 M−1/s−1) (11) to form the oxidant peroxynitrite (−OONO): NO• + O2−• → −OONO

(8)

Importantly, this reaction is approximately fivefold faster than the dismutation of superoxide by superoxide dismutase. Peroxynitrite has a pKa = 6.8 and is a relatively stable species under alkaline conditions, slowly undergoing dismutation spontaneously to nitrite and molecular oxygen (12). Protonation destabilizes the molecule and facilitates anion isomerization to NO3−: −OONO

+ H+ ' HOONO → [•OH + NO2] → NO3− + 2H+

(9)

The formation of −OONO has two important consequences in biological systems: loss of bioactive NO• and oxidation of a wide variety of biological molecules. One of the principal stable oxidation products results from the reaction of −OONO with the amino acid tyrosine:


5

(10)

The precise nature of the oxidative intermediate derived from −OONO has not been well characterized to date. In addition, recent data from Pryor’s group (14) suggest that in carbonate-enriched buffer systems, CO2 reacts with −OONO rapidly to form the nitrosoperoxycarbonate anion adduct O = N − OOCO2−, which then rearranges to yield a nitrocarbonate anion O2N − OCO2−. The latter species may, in turn, serve as an oxidant in biological systems (by one- or two-electron oxidation reactions), engage in electrophilic nitration reactions, such as nitrotyrosine formation,

(11)

or undergo hydrolysis to yield nitrate and bicarbonate (13,14): O2N − OCO2− + H2O → NO3− + HCO3− + H+

(12)

Peroxynitrite also reacts with NO• to form nitrogen dioxide radical (NO2− •) and nitrite in an exothermic reaction. An analogous reaction between −OONO and H2O2 leads to the energetically favorable formation of nitrite and water; however, the physiological relevance of this reaction has yet to be demonstrated. There has also been some debate in the literature as to the possibility that HOONO undergoes homolytic cleavage to generate a hydroxyl radical; however, Koppenol and colleagues (12) have argued that the rate constant for homolysis is too slow to compete meaningfully with the rate of isomerization. In addition to superoxide, other peroxyl radicals react readily with NO• to form lipid peroxynitrite compounds (LOONO) (15): NO• + LOO• → LOONO

(13)

This reaction is, again, near diffusion-limited (k = 2 × 109 M−1 • s−1), and likely explains the effect of NO• on lipid peroxidation in cellular systems (16). Recent data suggest that NO• can react with H2O2 to form •OH (17). The mechanism for this reaction is as yet unknown, but may involve either a direct reaction between NO• and H2O2 or the formation of an intermediate that undergoes homolysis to yield •OH. Nitric oxide reacts with •OH at a diffusion-controlled rate (k = 1 × 1010 M−1 • s−1) (18) in aqueous solution by the following reaction: NO• + •OH → HNO2

(14)

In addition, NO• reacts with NO2 to form the nitrosating species N2O3 (written as δ−OONNO δ+) with a rate constant that is near diffusion-limited (k = 1 × 109 M−1 • s−1), and this species

6


can undergo hydrolysis to nitrite as shown in reactions (6) and (7). The rate constant for the latter reaction is only 1000 s−1, and this slow rate becomes important when we consider the nitrosation of thiols under physiological conditions (vide infra).

REACTIONS WITH THIOLS Although NO• does not directly react with the functional groups of biological molecules, oxidized derivatives of NO• with nitrosating capacity, such as N2O3, react with thiols to form S-nitrosothiols (19): N2O3 + RSH → RSNO + HNO2

(15)

This reaction competes favorably with reaction (7) in the intracellular milieu where glutathione concentrations in the millimolar range react 5–10-fold faster with N2O3 than does water (20). The formation of S-nitrosothiols occurs under normal physiological conditions (21,22), and the parent thiol can either be a low-molecular-weight species or a cysteinyl side chain of a protein. S-Nitrosation of proteins can modify protein function, and this form of posttranslational modification has been shown to have wide-ranging biological effects. The S-nitrosation of serum albumin’s single free cysteinyl group (cys 83) represents a special situation that yields a stable pool of −NO• equivalents. S-Nitroso-albumin is the most abundant extracellular − NO• pool (21), serving as a buffer for −NO• and stabilizing it from further oxidation in the comparatively oxidative extracellular space. S-Nitrosothiols also engage in trans-S-nitrosation reactions (23), which are thermodynamically and mechanistically equivalent to thiol–disulfide exchange reactions: RS − NO• + R'SH ' RSH + R'SNO

(16)

The transfer of −NO• between low-molecular-weight thiols and serum albumin is supported by this chemistry (24). Under anaerobic conditions, NO• reacts with low-molecular-weight thiols to produce the corresponding disulfide: 2RSH + 2NO• → RSSR + N2O + H2O

(17)

At pHs near or above the pKa of the thiol, this reaction is accelerated (24). Cysteinyl side chains of proteins, in contrast, can undergo oxidation to sulfenic acids when reacted with NO• under anerobic conditions (25); however, the relevance of this reaction to physiological conditions is questionable. S-Nitrosation of the cysteinyl side chains of proteins may lead to disulfide formation even under aerobic conditions when two thiols are vicinal, or in close proximity. It has been argued that the “activity” of the thiols, or their effective local molarity, sustains this reaction, and, in part, depends on the relative pKa of the two thiols. Thiols with anomalously low pKas can attack the sulfur involved in the −S–H bond by a nucleophilic mechanism:

(18)

NO•-mediated

inactivation of This type of mechanism has been postulated as the basis for the N-methyl-D-aspartate receptor in neurons (26) and for the NO•-mediated inactivation of Ca2+-dependent potassium channels (27).


7

Importantly, S-nitrosation reactions are quenched by O2− •, and oxidation of thiols by O2− • to the disulfide form mediated by peroxynitrite is quenched by excess flux of either NO• or O2− •. Thus, nitrosation and oxidation of thiols by O2− •/−OONO and NOx are dependent on their relative flux rates with peak oxidation occurring when fluxes are equivalent (28). Recently, a novel mechanism for the formation of S-nitrosothiols was identified that may have relevance to their formation in vivo. This mechanism involves the direct reaction of NO• with the thiol to produce the radical intermediate, RSN• − OH, which, in turn, reacts with an electrophile to yield the S-nitrosothiol; under aerobic conditions, molecular oxygen is the electron acceptor and is reduced to O2− • in the process (29). As yet another potential Snitrosating mechanism of physiological relevance, dinitrosyl–iron complexes (vide infra), which have been detected in tissues, can serve as direct nitrosating species that react with the thiolate functionality of serum albumin (30).

REACTIONS WITH OTHER NUCLEOPHILIC CENTERS In addition to thiols, biological systems contain a variety of other nucleophilic centers that are potentially susceptible to nitrosative attack, including amines, amides, carboxyl groups, and hydroxyl groups. Primary amine deamination and N-nitrosation of secondary or tertiary amines may occur in acidic environments and the latter species have been studied as mediators of carcinogenesis (31,32). In contrast, the biological relevance of the reaction of NO• with amides, carboxyl groups, and hydroxyl groups is speculative (33). Recently, we demonstrated that the indole group of tryptophan can undergo N-nitrosation, and that this derivative can engage in transnitrosation reactions that evoke biological responses (34). Aromatic rings also undergo nitrosation in a process that involves the formation of charge-transfer complexes between NO+ and aromatic electron donors (35): Ar − NO+ ' Ar• + − NO•

(19)

This type of one-electron transfer may provide a mechanism by which to interconvert the redox-related forms of NO• (36).

REACTIONS WITH METALS NO• reacts both with heme iron as well as nonheme iron. In contradistinction to other heme iron ligands, including O2 and CO, NO• can bind to both ferric [Fe(III)] and ferrous [Fe(II)] iron. The binding of NO• to Fe(III) leads to the formation of a charge-transfer complex that can be represented either as Fe(III) − NO• or Fe(II) − +NO (37). This latter complex can serve as a nitrosating species since the complex renders the nitrosyl moiety susceptible to nucleophilic attack: [Fe(III) − NO ↔ Fe(II) − +NO] + Nucleophile → Fe(II) + Nucleophile − NO

(20)

In addition, NO• can react readily with Fe(II) to form a strong ligand complex through donation of electrons from NO• to the metal as well as donation of electrons from the metal to NO• by a backbonding interaction between the d-orbitals of the metal and the antibonding orbitals of NO• (38). Recent X-ray crystallographic studies of the ferrous nitric oxide form of sperm whale myoglobin shows that the nitric oxide ligand is bent with respect to the heme plane with an Fe(II)N-O angle of 112o. This angle appears to be influenced by the both the proximal bond strength and hydrogen-bonding interactions between the distal histidine and the bound nitrosyl moiety. The ring nitrogen atom of histidine 64 is located 2.8 Å from the −NO• group’s nitrogen atom, suggesting that electrostatic interactions stabilize the Fe(II) − NO• complex (39), as shown in structure (21).

8


(21)

In the heme-binding site of guanylyl cyclase, the ligation of NO• to heme iron liberates the transaxial ligand, histidine, which leads to enzyme activation (40). Other heme ligands, such as O2 and CO, do not render the transaxial histidine ligand labile, and thus do not activate guanylyl cyclase. In the heme-binding site of oxyhemoglobin or oxymyoglobin, NO• binding leads to the formation of methemoglobin or metmyoglobin, respectively, by the following reaction scheme that involves the resonance forms of Fe(II) − O2 and Fe(III) − O2− (41): [Fe(II) − O2 ↔ Fe(III) − O2−] + NO• → Fe(III) + NO3−

(22)

This reaction is quite rapid and can serve as the basis of a spectroscopic assay for NO•. NO• also forms charge–transfer complexes with nonheme transition metals. In particular, NO• can complex with iron–sulfur centers in proteins or within cell membranes (42,43), often to form iron-dinitrosyl-dithiolate complexes of the following form, as shown in scheme (23). ON

SR Fe

ON

(23)

SR

Many of these nitrosyl complexes are effective electrophilic nitrosating species, having the general formula, Me − +NO, whereas others behave principally as nucleophilic nitroxylating species, having the general formula, Me − NO-. The unique chemistry of these metal–nitrosyl charge–transfer complexes offers another mechanism by which the redox states of NO• can be modulated (36).


9

CONCLUSIONS The chemistry of NO• is quite varied in physiological environments, and accounts for a rich and complex array of reactions. This chemistry serves as the basis for an equally broad range of biological effects, and these are discussed in turn throughout the first section of this book.

REFERENCES 1. Dean JA. Table 10-17: molecular elevation of the boiling point (ebullioscopic constants). In: Dean JA, ed. Lange’s Handbook of Chemistry (13th ed.). McGraw-Hill, New York, 1985, pp. 10–73. 2. Armor JN. Influence of pH and ionic strength upon solubility of nitric oxide in aqueous solution. J Chem Eng Data 1974;19:82–84. 3. Gally JA, Montague PR, Reeke GN Jr, Edelman GM. The NO hypothesis: possible effects of a shortlived, rapidly diffusible signal in the development and function of the nervous system. Proc Natl Acad Sci USA 1990;87:3547–3551. 4. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharm Rev 1991;43:109–142. 5. Stanbury DM. Reduction potentials involving inorganic free radicals in aqueous solution. Adv Org Chem 1989;33:69–138. 6. Cotton FA, Wilkinson G. The chemistry of the main group elements. In: Advanced Organic Chemistry, 5th ed. Wiley, New York, 1988, pp. 585–597. 7. Ford PC, Wink DA, Stanbury DM. Autoxidation kinetics of aqueous nitric oxide. FEBS Lett 1993; 326:1–3. 8. Wink DA, Darbyshire JF, Nims RW, Saavedra JE, Ford PC. Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: determination of the kinetics for oxidation and nitrosation by intermediates generated in the NO/O2 reaction. Chem Res Toxicol 1993;6:23–27. 9. Kharitonov VG, Sundquist AR, Sharma VS. Kinetics of nitric oxide autoxidation in aqueous solution. J Biol Chem 1994;269:5881–5883. 10. Lewis RS, Deen WM. Kinetics of the reaction of nitric oxide with oxygen in aqueous solutions. Chem Res Toxicol 1994;7:568–574. 11. Huie RE, Padmaja S. The reaction of NO with superoxide. Free Rad Res Commun 1993;18:195–199. 12. Koppenol WH, Moreno JJ, Pryor WA, Ischiropoulos H, Beckman JS. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol 1992;5:834–842. 13. Uppu RM, Squadrito GL, Pryor WA. Acceleration of peroxynitrite oxidations by carbon dioxide. Arch Biochem Biophys 1996;327:335–343. 14. Pryor WA, Lemercier JN, Zhang H, Uppu RM, Squadrito GL. The catalytic role of carbon dioxide in the decomposition of peroxynitrite. Free Rad Biol Med 1997;23:331–338. 15. Padmaja S, Huie RE. The reaction of nitric oxide with organic peroxyl radicals. Biochem Biophys Res Commun 1993;195:539–544. 16. Rubbo H, Radi R, Trujillo M, Telleri R, Kalyanaraman B, Barnes S, Kirk M, Freeman BA. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogencontaining oxidized lipid derivatives. J Biol Chem 1994;269:26,066–26,075. 17. Nappi AJ, Vass E. Hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide. Biochim Biophys Acta 1998;1380:55–63. 18. Buxton BF, Greenstock CL, Helman WP, Ross AB. Critical review of rate constants for reactions of hydrated electrons. Hydrogen atoms and hydroxyl radicals in aqueous solution. J Phys Chem Ref Data 1988;17:513–886. 19. Wink DA, Ford PC. Nitric oxide reactions important to biological systems: a survey of some kinetics investigations. Methods 1995;7:14–20. 20. Kharitonov VG, Sundquist AR, Sharma VS. Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J Biol Chem 1995;270:28,158–28,164. 21. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ, Loscalzo J. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci USA 1992;89:444–448. 22. Stamler JS, Jaraki O, Osborne JA, Simon DI, Keaney JF Jr, Vita JA, Singel DJ, Valeri CR, Loscalzo J. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc Natl Acad Sci USA 1992;89:7674–7677.

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23. Scharfstein JS, Keaney JF Jr, Slivka A, Welch GN, Vita JA, Stamler JS, Loscalzo J. In vivo transfer of nitric oxide between a plasma protein-bound reservoir and low molecular weight thiols. J Clin Invest 1994;94:1432–1439. 24. Pryor WA, Church DF, Govindan CK, Crank G. Oxidation of thiols by nitric oxide and nitrogen dioxide: synthetic utility and toxicological implications. J Org Chem 1982;47:156–159. 25. DeMaster EG, Quast BJ, Redfern B, Nagasawa HT. Reaction of nitric oxide with the free sulfhydryl group of human serum albumin yields a sulfenic acid and nitrous oxide. Biochemistry 1995;34: 11,494–11,499. 26. Lei SZ, Pan ZH, Aggarwal SK, Chen HS, Hartman J, Sucher NJ, Lipton SA. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 1992;8: 1087–1099. 27. Bolotina VM, Najibi S, Palacino JJ, Pagano PJ, Cohen RA. Nitric oxide directly activates calciumdependent potassium channels in vascular smooth muscle. Nature 1994;368:850–853. 28. Wink DA, Cook JA, Kim SY, Vodovotz Y, Pacelli R, Krishna MC, Russo A, Mitchell JB, Jourd'heuil D, Miles AM, Grisham MB. Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide-derived reactive intermediates. Chemical aspects involved in the balance between oxidative and nitrosative stress. J Biol Chem 1997;272:11,147–11,151. 29. Gow AJ, Buerk DG, Ischiropoulos H. A novel reaction mechanism for the formation of S-nitrosothiol in vivo. J Biol Chem 1997;272:2841–2845. 30. Boese M, Mordvintcev PI, Vanin AF, Busse R, Mulsch A. S-nitrosation of serum albumin by dinitrosyliron complex. J Biol Chem 1995;270:29,244–29,249. 31. Mirvish SS. Formation of N-nitroso compounds: chemistry, kinetics, and in vivo occurrence. Toxicol Appl Pharm 1975;31:325–351. 32. Challis BC, Fernandes MH, Glover BR, Latif F. Formation of diazopeptides by nitrogen oxides. IARC Sci Pub 1987;84:308–314. 33. Ridd JH. Diffusion control and pre-association of nitrosation, nitration and halogenation. Adv Phys Org Chem 1978;16:1–49. 34. Zhang YY, Xu AM, Nomen M, Walsh M, Keaney JF Jr, Loscalzo J. Nitrosation of tryptophan residue(s) in serum albumin and model dipeptides. Biochemical characterization and bioactivity. J Biol Chem 1996;271:14,271–14,279. 35. Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 1992;258:1898–1902. 36. Wayland BB, Olson LW. Spectroscopic studies and bonding model for nitric oxide complexes of iron porphyrins. J Am Chem Soc 1974;96:6037–6041. 37. Jameson GB, Ibers JA. Biological and synthetic dioxygen carriers. In: Bertini I, Gray HB, Lippard SJ, Valentine JS, eds. Bioinorganic Chemistry. University Science Books, Mill Valley, CA, 1994, pp. 167–252. 38. Traylor TG, Sharma VS. Why NO? Biochemistry 1992;31:2847–2849. 39. Brucker EA, Olson JS, Ikeda-Saito M, Phillips GN Jr. Nitric oxide myoglobin: crystal structure and analysis of ligand geometry. Proteins 1998;30:352–356. 40. Doyle MP, Hoekstra JW. Oxidation of nitrogen oxides by bound dioxygen in hemoproteins. J Inorg Chem 1981;14:351–358. 41. Henry Y, Ducrocq C, Drapier JC, Servent D, Pellat C, Guissani A. Nitric oxide, a biological effector. Electron paramagnetic resonance detection of nitrosyl-iron-protein complexes in whole cells. Eur Biophys J 1991;20:1–15. 42. Vanin AF. [EMR identification of ferro-cysteine complexes in biological systems.] Identifikatsiia metodom EPR kompleksov dvukhvalentnogo zheleza s tsisteinom v biologischeskihk sistemakh. Biokhimiia 1967;32:277–282. 43. Drapier JC. Interplay between NO and [Fe-S] clusters: relevance to biological systems. Methods 1997; 11:319–329.

Chapter 2 / Nitric Oxide Synthases

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Cell and Molecular Biology of Nitric Oxide Synthases Olivier Feron and Thomas Michel THE MAMMALIAN NO SYNTHASE ISOFORMS

This chapter focuses on the factors influencing cellular and molecular regulation of the three known mammalian nitric oxide synthase (NOS) isoforms in the cardiovascular system. It is now known that the different NOS isoforms can be found in numerous different human tissues, including diverse locales within the cardiovascular system. The overall amino acid sequence identity for the three human NOS isoforms is approx 50–55%, with particularly strong sequence conservation in regions of the proteins involved in catalysis (1). Alignment of the amino acid sequences of the different NOS isoforms reveals two domains of amino acid sequence similarity along the length of the proteins. The NOS C-terminal domain, comprising nearly half the molecule, has been termed the “reductase domain,” as it bears striking sequence similarity to the mammalian cytochrome P450 reductase, and even shows significant sequence similarities to archetypal reductases from plants and bacteria. The N-terminal domain, variably termed the heme or oxygenase domain, shows significant sequence similarities only among the three members of the NOS family, and contains the site for binding of the enzymes’ heme prosthetic group. These striking similarities in the proteins’ primary structure are likely to be reflected in homologies in their tertiary structure, but three-dimensional structural data currently exist only for the iNOS isoform (2,3). The three NOS enzyme isoforms are commonly denoted by prefixes that reflect the tissues of origin for the original isolation of their protein and cDNA: the nomenclature of nNOS, iNOS, and eNOS enzymes reflect their initial characterizations in neuronal tissue, immunoactivated macrophages, and endothelial cells, respectively (4). The official nomenclature of the corresponding human NOS genes reflects instead the order of isolation and characterization of human genomic clones: the human genes encoding nNOS, iNOS, and eNOS are thus termed NOS1, NOS2, and NOS3, respectively. The three NOS genes share many features in their overall genomic structure, with striking similarity in the size of the exons and the location of the splice junctions, suggesting that the three NOS isoforms derive from a common ancestral gene. However, as might be anticipated from their distinct modes of transcriptional regulation and tissue-specific expression, there are significant divergences in the putative promoter regions among members of the NOS gene family. The different NOS isoforms share a similar overall catalytic scheme, in which the homodimeric enzyme catalyzes the formation of NO• plus L-citrulline by oxidizing one of the two guanido nitrogens of the amino acid L-arginine. NOS catalysis involves the reduced form of nicotimamide adenine dinucleotide phosphate (NADPH) and molecular oxygen as cosubstrates, with the flavins adenine dinucleotide (FAD) and mononucleotide (FMN) representing key cofactors in promoting electron transfer to the NOS heme moiety; tetrahydrobiopterin From: Contemporary Cardiology, vol. 4: Nitric Oxide and the Cardiovascular System Edited by: J. Loscalzo and J. A. Vita © Humana Press Inc., Totowa, NJ

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represents another key cofactor, but the role of this compound in NOS catalysis remains less well understood. For all three mammalian NOS isoforms, binding of the ubiquitous Ca2+binding regulatory protein calmodulin (CaM) appears to be required for efficient electron transfer between the reductase and oxygenase domain of NOSs (5,6). The dependence of the different NOS isoforms on Ca2+/CaM constitutes, however, a major difference in the regulatory mechanisms of NO• production. Both nNOS and eNOS bind CaM in a reversible and Ca2+-dependent manner, but iNOS avidly binds CaM even at the low ambient intracellular Ca2+ concentration characteristic of resting cells. iNOS activity in the cell is therefore largely independent of changes in intracellular Ca2+ (7), whereas the temporal pattern of nNOS or eNOS activation is closely regulated by transient changes in intracellular Ca2+. The level of cellular iNOS activity appears to be closely related to the amount of iNOS protein, which, as a first approximation, is determined by mRNA abundance, which is in turn governed by the rate of iNOS gene transcription and by the stability of its mRNA. The principal form of iNOS regulation does appear to be at the level of genetic induction, hence its common appellation as an “inducible” enzyme. However, iNOS may be constitutively expressed under physiological conditions in some tissues, including pulmonary and bladder epithelia, and renal medulla (8,9). The eNOS and nNOS enzymes are often found expressed at stable levels in their characteristic tissues, and were originally denoted as “constitutive” enzymes. However, it has become clear that the expression of eNOS and nNOS genes can be regulated under different physiological and pathophysiological conditions (e.g., hemodynamic shear stress, nerve injury). In addition, posttranscriptional and posttranslational modifications importantly modulate the structure and function of all three NOS isoforms, as we discuss in detail in the following sections.

Neuronal NOS GENOMIC STRUCTURE AND MRNA PROCESSING The gene encoding nNOS (NOS1) includes 29 exons scattered over a region of 200 kb located on the human chromosome 12 (10,11). The full-length open reading frame of NOS1 encodes a protein of 1434 amino acids with a predicted molecular mass of 160 kDa (12,13). Two major transcriptional clusters, denoted as neuronal- and testis-specific, have been identified for human NOS1 (11,14); the identification of three potential polyadenylation sites adds to the complexity of NOS1 posttranscriptional processing (14). In the neuronal transcriptional cluster of the NOS1 gene, distinct first exons of nNOS appear to splice to a common exon 2, which contains the initiator ATG codon. These different mRNA species, therefore, each encode the full-length nNOS protein, but the existence of distinct transcription initiation sites in different first exons may reflect the existence of tissuespecific or developmentally regulated NOS1 promoters. By contrast to the neuronal transcription cluster, the site for transcription initiation from the testis-specific cluster is located between exons 3 and 4, and the predicted site for initiation of translation lies within exon 5. Translation at this site would yield a truncated protein of 125 kDa, which has been termed TnNOS. The existence of the TnNOS protein is inferred from the isolation of its cognate transcript by highly sensitive reverse transcriptase–polymerase chain reaction techniques (RT-PCR), and its corresponding cDNA can be expressed in heterologous cell systems, but the naturally occurring TnNOS protein has not itself been identified (15). The truncated TnNOS would lack key sequences at the protein’s extreme N-terminal (PDZ domain) thought to be important for nNOS targeting and protein–protein interactions in the full-length nNOS isoform. In the mouse, additional alternatively spliced nNOS transcripts have been identified: NOSβ and NOSγ appear to be analogous to the human TnNOS in that they lack exon 2 and would encode truncated nNOS without the N-terminal PDZ domain (16). Indeed, NOSγ and β were shown to account for the residual nNOS activity in nNOS knockout mice in which gene targeting inactivated the first coding exon but still allowed processing of


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transcripts starting downstream from the site of NOS1 gene disruption (17). Other exon deletions (exons 9/10) and insertions (between exon 16 and 17) have also been documented (11,18,19). Although it remains to be demonstrated that the exon 9/10 deleted transcript is actually translated in vivo, some of the nNOS splice variants appear to be differentially regulated in cellular models of morphine tolerance (20). Another alternatively processed nNOS transcript has been termed µNOS, which is formed by the insertion of 102 bp between exons 16 and 17. Expression of the µNOS protein has been established in rat and human penis as well as in rat skeletal muscle and heart (18,19), and µNOS represents the only alternatively spliced nNOS transcript that has clearly been shown to correspond to a naturally occurring novel nNOS protein. The functional role of µNOS remains less well understood, but the presence of a distinct nNOS enzyme generated by differential mRNA processing identifies another potential point of regulation.

CELLULAR EXPRESSION AND (POST-)TRANSCRIPTIONAL REGULATION To date, nNOS expression has been identified in diverse neurons throughout the central and peripheral nervous systems (20), and nNOS has also been found in numerous nonneuronal tissues including skeletal muscle (13,18), lung (21), and the genitourinary tract (15,19). Increases in the abundance of nNOS mRNA have been noted following physical and mechanical stresses, including spinal cord and nerve injuries (22), ischemia (23), hypoxia (21), as well as changes in plasma osmolarity (24). Neurotransmitters and hormones also influence the spatial and temporal pattern of NOS1 expression: upregulation has been reported in various tissues during pregnancy (25) and in cerebellar granule cells following inhibition of glutaminergic transmission (26); a development switch to µNOS (see above) was recently shown to occur in skeletal muscle in the process of myotube fusion (18). Downregulation of NOS1 expression is documented in rat brain following corticosterone treatment (27) and is also generally observed following bacterial lipopolysaccharide (LPS) and interferon-γ (28,29). Functional characterization of the NOS1 gene transcriptional regulatory regions has yet to be established despite the identification of several cis-acting and cis-regulatory elements in the neuronal and testis transcriptional clusters (11,14). Consensus sequences located in the putative NOS1 promoter region suggest a possible role for diverse regulatory elements, including AP-1, AP-2, Sp-1, transcriptional enhancer factor-1/M-CAT binding factor, CREB, ATF, cFos, Ets, NF-1, NF-κB motifs, GATA sites, p53 half-element, myocyte-specific enhancer factor 2 motif, and an insulin-responsive element. The presence of these putative regulatory motifs does not, in itself, establish their biological relevance, and the role, if any, of these diverse sequences for NOS1 gene regulation remains to be defined.

“Inducible” Nitric Oxide Synthase GENOMIC STRUCTURE AND MRNA PROCESSING The iNOS gene (NOS2) is located on the human chromosome 17 and contains 26 exons spanning a region of 37 kb (30). The full-length open reading frame encodes a protein of 1153 amino acids with a predicted molecular mass of 130 kDa. Alternatively spliced iNOS transcripts have been identified using RT-PCR chain reaction techniques (31). Although they were using recombinant iNOS mutants, these authors showed that alternative splicing of exons 8 and 9 is critical for the enzyme dimerization, the endogenous expression of novel iNOS proteins derived from these transcripts has not yet been detected in tissues or cells. CELLULAR EXPRESSION AND (POST-)TRANSCRIPTIONAL REGULATION Since the prototypical iNOS enzyme was first characterized expressed in murine macrophages, numerous studies have documented that the NOS2 gene can be induced in many different cell types. In human tissues, the initial report of iNOS expression in human hepatocytes (32) was followed by a plethora of studies showing iNOS expression following immunoactivation

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of macrophages, monocytes, myocytes, epithelial and endothelial cells, astrocytes, fibroblasts, keratinocytes, osteoblasts, and neutrophils/eosinophils (for review see ref. 33). Although iNOS induction may generally reflect a pathophysiological cellular response to immunoactivation, it has been recently found that iNOS may be constitutively expressed in some tissues without any known antecedent exposure to pathological immunoactivating stimuli (8,9), suggesting that iNOS may be subserving a physiological role in some tissues. Most commonly, however, transcriptional induction of iNOS appears to be a consequence of immunoactivation, and numerous reports in a wide variety of cell types have explored iNOS induction in response to bacterial endotoxin or following stimulation by cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-1 (IL-1). It should be emphasized that even though a large number of different cell types have been found to express iNOS under different conditions of gene induction, one must use caution in extrapolating the transcriptional regulatory mechanisms characterized for one cellular system to another. For example, in some cell types the NOS2 gene may be induced in response to cyclic adenosine monophosphate (cAMP)-elevating agents, by activation of protein kinase C (PKC), or by the action of various growth factors including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) (33). Comparison of the rodent and human NOS2 gene promoter indicates that although only 1 kb of the proximal 5' flanking region of the murine promoter is required to confer endotoxin and cytokine inducibility, other critical cytokine-responsive elements are present between 3.8 and 16 kb upstream the initiation codon of the human gene (34–36). Three γ-IRE as well as NF-κB, NF-IL6, and TNF-RE sites have however been identified in the very proximal 5'-flanking region of the human NOS2 gene (37–40). Among these elements, a functional role in transcriptional regulation of NOS2 gene has been demonstrated for γ-IRE and NF-κB sites, and these elements appear to be involved in IFN-γ- and endotoxin-stimulated gene expression, respectively (37,38). Shortly after the characterization of NOS2 gene induction in macrophages and hepatocytes, several groups provided evidence for a similar glucocorticoid- and TGF-β-sensitive induction of iNOS by cytokine and lipopolysaccharide (LPS) in cardiac myocytes and microvascular endothelial cells (41). As reported in other cell types, induction of the NOS2 gene by IL-1β and IFN-γ in cardiac myocytes appears to be preceded by activation of MAPK (ERK1/ERK2) and STAT1α phosphorylation, a finding compatible with the presence of AP-1 and GAS sequences in the NOS2 promoter (42). Downregulation of NOS2 gene induction is characteristically observed in various cell types following treatment with glucocorticoids or TGF-β (33). The mechanisms involved in the deactivation of iNOS are less well understood: depending on the cell type, plausible regulatory pathways might include the repression of NOS2 transcription, alterations in iNOS mRNA stability and/or processing, as well posttranslational mechanisms.

Endothelial Nitric Oxide Synthase GENOMIC STRUCTURE AND MRNA PROCESSING The gene encoding for eNOS (NOS3) is located on human chromosome 7 and contains 26 exons spanned on a region of 22 kb (43–45). The full-length open reading frame codes for a protein of 1205 residues with a predicted molecular mass of 135 kDa. Alternative polyadenylation sites have been identified in the 3' untranslated region of the eNOS mRNA (K. Sase and T. Michel, unpublished observations; see ref. 46), and may influence the differential stability or subcellular targeting of eNOS transcripts. Alternatively spliced transcripts encoding novel eNOS proteins have not been described. CELLULAR EXPRESSION AND (POST-)TRANSCRIPTIONAL REGULATION Immunohistochemical studies have located eNOS in various types of venous and arterial endothelial cells (47), and significant endogenous expression was also reported in myocytes (48), in neuronal cells (49), in platelets (50), and in various other tissues (see ref. 51).


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Present in the putative TATA-less promoter region of the eNOS gene are consensus sequences that potentially may serve as sites of binding for transcription/nuclear factors such as AP-1, AP-2, NF-1, IL6, as well as several putative NF-κB sites and several half-palindromic estrogen response elements (43–45). Recently, two tightly clustered cis-regulatory regions were identified in the proximal enhancer of the human eNOS promoter using deletion analysis and linker-scanning mutagenesis (52): positive regulatory domains I (−104/−95 relative to transcription initiation) and II (−144/−115). Analysis of trans-factor binding and functional expression studies revealed a surprising degree of cooperativity and complexity. The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional domain studies in Drosophila Schneider cells and endothelial cells revealed examples of positive and negative protein–protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ (52). Therefore, multiprotein complexes are formed on the activator recognition sites within this 50-bp region of the human eNOS promoter in vascular endothelium. Hemodynamic shear-stress and chronic exercise are among the stimuli that are associated with an increased abundance of the eNOS transcript (53,54). Consensus sequences present in the eNOS 5' flanking region may represent cis-regulatory elements responsive to shear stress, and the presence of several half-palindromic estrogen-responsive elements may provide the means for transcriptional regulation of the eNOS gene in response to physiological or pathophysiological perturbations. Hypoxia appears to influence eNOS abundance in cultured endothelial cells, causing a decrease in steady-state eNOS transcript levels associated with a decrease in eNOS mRNA stability (55). However, in animal models, chronic hypoxia appears to be associated with an increase in eNOS mRNA abundance (21). Cell proliferation also profoundly influences the expression of eNOS in cultured endothelial cells: eNOS transcript and protein abundance diminishes significantly when cells reach confluence (56). Thus, any factors that influence endothelial cell growth rate may confound the interpretation of primary effects on eNOS gene expression. The nature, magnitude, and physiological relevance of studies showing altered eNOS transcript abundance analyzed in cultured endothelial cell model systems continue to be actively investigated. Among many other perturbations, sex steroids (57,58) or lipoproteins (59–61) may also influence the abundance of the eNOS transcript; findings in different experimental models have yielded mutually contradictory results and must be interpreted with caution. Cytokines such as TNF-α are associated with a decrease in eNOS message and protein abundance in aortic endothelial cells, whereas paradoxically, NO synthesis increases (likely due to influences of the cytokine on NOS cofactor levels) (62–64). The TNF-α-induced decrease in the abundance of the eNOS mRNA in bovine aortic endothelial cells appears to be due to a decrease in eNOS mRNA stability (65); in contrast, transforming growth factor-β appears to increase the abundance of the eNOS transcript (66).

POLYMORPHISMS IN THE NOS3 GENE The central role of NO in blood pressure homeostasis has led to studies exploring whether polymorphisms in any of the three NOS genes, or abnormalities in NOS cellular regulation, may be associated with hypertension. To date, there have been compelling experimental or population-based studies in support of a relationship between human NOS1 polymorphism and hypertension. Although the NOS2 gene maps to a region in the rat genome linked to hypertension in Dahl salt-sensitive rats (67), disease association, and/or the relevance of this finding to human disease has yet to be established. Numerous studies have failed to link polymorphisms in introns of the human NOS3 gene with cardiovascular diseases (68); yet recent studies have documented the association of the missense variant Glu298Asp in exon 7 of the eNOS gene with essential hypertension and acute myocardial infarction in two independent populations of Japanese patients (69,70). Interestingly, the same missense variant Glu298Asp

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was shown to be an independent risk factor for vasospastic angina pectosis (71) in Japanese populations. In contrast, other authors failed to find a relationship between the Glu298Asp polymorphism and ischemic cerebral disease in Caucasian populations (72). Taken together, these results seem to indicate that, at least in Japanese populations, the missense variant Glu298Asp polymorphism is associated with, and could predispose to, various cardiovascular diseases. However, the functional consequences of the conservative amino acid change encoded by this eNOS genetic polymorphism have yet to be established.

Cardiovascular Phenotype of NOS Gene Knockout Mice The characterization of mice with targeted disruption of NOS genes (NOS “knockout” mice) has provided important insights into the physiological and pathophysiological roles of the individual NOS isoforms in the cardiovascular system. Despite the ubiquitous expression of the NOS and their striking evolutionary conservation (and contrary to some expectations), mice with homozygous deletions of individual NOS isoforms are entirely viable. The roles of NO in diverse physiological processes can apparently be largely supplanted or compensated by other regulatory pathways in the cardiovascular system and elsewhere. For example, the nNOS knockout mice do not show any gross neuroanatomic abnormalities or even alteration of long-term potentiation (LTP) processes (73), but do exhibit pyloric stenosis, and have been reported to display abnormal sexual and aggressive behavior (74). iNOS knockout mice are grossly normal, but show increased susceptibility to infection (75,76). From the standpoint of cardiovascular homeostasis, it is the eNOS knockout mice that document a most dramatic phenotype: eNOS−/− mice are hypertensive and show a mean arterial blood pressure approx 30% higher than wild-type littermates (77). This is a key finding: despite the wide array of compensatory mechanisms controlling vascular tone, the genetic abrogation of eNOS cannot be overcome and leads to hypertension. However, treatment of eNOS null mice with pharmacological NOS inhibitors led to a paradoxical decrease in blood pressure (77), suggesting perhaps a role for nNOS in the maintenance of blood pressure (note that nNOS −/− mice present a tendency toward hypotension under anesthesia [78]). Actually, the critical role of NO• produced by eNOS in the regulation of vascular tone was recently specifically addressed by the generation of transgenic mice overexpressing eNOS in the vascular wall (using murine preproendothelin-1 promoter) (79). These authors reported that in agreement with the observed increase in basal NO• release and cGMP levels in transgenic aorta, blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates. The central role of NOS isoforms becomes much more evident when NOS knockout mice are subjected to pathophysiological perturbations. For example, a murine model of operatively induced hindlimb ischemia was recently used to investigate the impact of targeted disruption of the eNOS gene on angiogenesis (80). Laser Doppler flow analysis and capillary measurements revealed that angiogenesis was impaired and was not improved by vascular endothelial growth factor (VEGF) administration in eNOS −/− mice versus wild-type. Moreover, in cerebral ischemia, NO• is known to rise dramatically associated with tissue damage, but the cellular source of NO• and its biological role were less well understood. Analyses of nNOS −/− mice with experimental cerebral infarcts documented reduced infarct size when compared with age-matched wild-type animals; these changes occur independently of alternations in blood flow (81). The nNOS −/− mice are also resistant to ischemic injuries (82), indicating that nNOS is importantly involved in neurotoxic damages. Interestingly, nonspecific NOS inhibitors appear to attenuate the reduced infarct size found in nNOS −/− mice, plausibly by inhibiting the eNOS-mediated relaxation of pial vessels. Indeed, transgenic mice that lack eNOS show, after middle cerebral artery (MCA) occlusion, increased infarct volume and reduced cerebral blood flow (83). Furthermore, nonspecific NOS inhibitors decrease the infarct size in eNOS −/− mice but not in wild-type animals, confirming both that eNOS plays a protective role by maintaining regional cerebral blood flow in the setting of


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ischemia and that nNOS contributes to neurotoxicity. iNOS also appears to be involved in late neuronal injury following experimental stroke: iNOS null mice show reduced infarct volumes when compared with wild-type animals (84).

COVALENT MODIFICATIONS OF NOS ISOFORMS Phosphorylation The three NOS isoforms can be phosphorylated in vitro by purified protein kinases and can be isolated as phosphoproteins in cultured cell systems. However, to date, the roles of specific protein kinases and phosphatases have not been specifically delineated any of the isoforms and the role and regulation of NOS phosphorylation remain incompletely understood. For example, although nNOS has been shown to serve as a substrate for a variety of protein kinases in vitro (49,85,86), phosphorylation of nNOS in neurons has not been definitively demonstrated. In addition, different studies have observed variable effects of nNOS phosphorylation on enzymatic activity, and these in vitro analyses have not yet been clearly correlated with the enzyme’s phosphorylation in native cells (85,86). Phosphorylation of iNOS has been even less extensively characterized (7), although a recent report suggests that tyrosine phosphorylation of the enzyme may serve to increase its activity (87). Serine phosphorylation of eNOS was shown to occur in endothelial cells subsequent to agonist-induced translocation of the enzyme to the cytosol (88), suggesting that eNOS deactivation may be regulated by phosphorylation. Treatment of endothelial cells with phorbol esters has also been shown to diminish NO• production, suggesting that phosphorylation may be associated with inhibition of eNOS activity (89). Possible clinical significance for these findings was suggested by studies reported by Craven et al. (90), who correlated increased PKC activity with decreased NO• production in glomeruli isolated from diabetic rats. These investigators further demonstrated that inhibition of PKC restored normal NO• production, suggesting that changes in eNOS phosphorylation may modulate the alterations in NO• signaling observed in diabetic vascular disease. Additionally, several groups have reported that the activation of eNOS by hemodynamic shear stress in cultured endothelial cells is influenced by reagents that modulate protein tyrosine kinase activity (91–93). Tyrosine phosphorylation of eNOS has been explored in several studies with contradictory results: several investigators failed to document any tyrosine phosphorylation of eNOS (92,94) whereas others reported either tyrosine phosphorylation (95) or dephosphorylation (93) of eNOS following incubation of endothelial cells with high concentrations of protein tyrosine phosphatase inhibitors. To date, no physiological agonists promoting the tyrosine phosphorylation of eNOS have been identified, although many agonists that modulate eNOS clearly affect tyrosine phosphorylation pathways, as well.

Acylation MYRISTOYLATION AND PALMITOYLATION eNOS is unique among the NOS isoforms in its being dually acylated by myristate and palmitate 14- and 16-carbon saturated fatty acids, respectively (for review see ref. 50), and importantly, both modifications are required for an efficient targeting of the enzyme to plasmalemmal caveolae (96). Myristoylation occurs cotranslationally on an N-terminal glycine residue within a specific consensus sequence (MGXXXS) (97) and is essentially irreversible, precluding its dynamic regulation by agonists or other stimuli (98). Moreover, the stable membrane association of myristoylated proteins often requires hydrophobic or electrostatic interactions in addition to those between myristate and membrane lipids; for eNOS, several lines of evidence (99,100) suggest that this membrane-targeting role is subserved by palmitoylation.

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Palmitoylation of eNOS takes place on two cysteine residues near the protein’s N-terminus (Cys-15 and Cys-26) that define a novel motif for protein palmitoylation (99,101). No general consensus sequence for protein palmitoylation has been identified (98,102), although some dually acylated G protein α subunits and members of the Src family of tyrosine kinases are palmitoylated at a cysteine residue within a conserved N-terminal sequence not found in the NOS isoforms. The two palmitoylated cysteines in eNOS flank an unusual Gly–Leu repeat [(Gly–Leu)5] not otherwise described in the protein sequence database. Mutagenesis of the palmitoylation site cysteine residues (to serine) markedly attenuates the association of eNOS with the particulate subcellular fraction, documenting a key role for this posttranslational modification in eNOS targeting (99). The myristoylation-deficient mutant eNOS also fails to undergo palmitoylation (100), plausibly because the mutant is not targeted to the plasma membrane, the presumed site for protein palmitoylation (98). The myristoylationdeficient eNOS is thus de facto an acylation-deficient enzyme, undergoing neither of the fatty acid modifications characteristic of the wild-type eNOS; this acylation-deficient enzyme is entirely cytosolic. The palmitoylation-deficient mutant eNOS still undergoes myristoylation, and, as noted earlier, its membrane targeting is reduced but not completely abrogated. Dual acylation of eNOS is thus required for efficient membrane localization, with cotranslational Nmyristoylation and posttranslational thiopalmitoylation playing key roles in enzyme targeting.

DYNAMIC REGULATION OF PALMITOYLATION Palmitoylation is a reversible posttranslational modification that has been shown to modulate the interaction of signaling proteins with the membrane (103). For example, agonist regulation of protein palmitoylation has previously been described for G protein-coupled receptors, such as the β-adrenergic receptor (104). For some peripheral membrane proteins, the loss of palmitate may correlate with protein redistribution to the cytosolic subcellular fraction. Agonists activating G protein αs appear to stimulate αs palmitate turnover, specifically accelerating depalmitoylation (102,105). There are striking parallels for eNOS: pulsechase experiments in endothelial cells biosynthetically labeled with [3H]palmitate showed that bradykinin treatment may promote eNOS depalmitoylation (100). However, it must be noted that another study (101) failed to document agonist modulation of eNOS palmitoylation; interpretation of the negative results of this latter study are confounded by the challenges of studying intracellular modulation of biosynthetically labeled proteins under non-steadystate conditions. This controversy aside, reversible palmitoylation of eNOS represents a plausible mechanism for modulating the binding of a large hydrophilic protein such as eNOS to membranes. Depalmitoylation could therefore be part of a cellular mechanism for the release of (myristoylated) eNOS from the plasma membrane and translocation to other cellular structures in response to agonist stimulation. Conversely, repalmitoylation could then facilitate the process of retargeting to specialized compartments such as caveolae (see following). The palmitoylation of several signaling proteins has been shown to influence their signaling activities as well as their subcellular localization (98,102). However, important differences have been noted in the regulatory roles of palmitoylation, even among closely related proteins. The receptor-mediated processes that regulate reversible palmitoylation of signaling proteins are not well understood, and few enzymes involved in the formation or hydrolysis of palmitoyl–protein thioesters have been extensively characterized. A protein palmitoylthioesterase was recently isolated and cloned from bovine brain (106). This palmitoylthioesterase is expressed in diverse cell types, including vascular endothelial cells (107), but its regulatory characteristics are not fully defined, and its relationship to eNOS palmitoylation is completely unknown. Another palmitoylthioesterase has been isolated that appears to be involved in the depalmitoylation of the G protein alphas (108), but the role of this enzyme in the regulation of eNOS depalmitoylation remains to be established. Another possibility is that eNOS activation itself could influence depalmitoylation, with NO production itself playing a role. It has recently been shown that NO reduces [3H]palmitate labeling of two nerve growth


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cone-associated proteins (109). NO might also regulate palmitoyl thioesterase activity or directly influence eNOS palmitoylation via nitrosothiol formation at the site(s) of palmitoylation. An understanding of the regulation of eNOS palmitoylation/depalmitoylation cycles has been facilitated by the development of heterologous cellular expression systems that permit the reconstitution of endogenous signaling pathways by cotransfecting cDNAs encoding wild-type and acylation-deficient eNOS along with constructs encoding specific cell surface receptors. Using this approach, it was shown that muscarinic cholinergic agonist stimulation rapidly induced the reversible dissociation of eNOS from caveolin. These studies revealed further that enzyme repalmitoylation markedly accelerated eNOS retargeting to caveolae following prolonged agonist stimulation (110).

FUNCTIONAL ROLE OF ENOS TARGETING The importance of eNOS myristoylation was recently illustrated in an elegant study examining the implication of eNOS in a form of synaptic plasticity termed long-term potentiation (LTP). Kantor and colleagues (111) showed that LTP can be attenuated in brain slices by pretreating them with the myristoylation inhibitor, hydroxymyristic acid (HMA). To rule out the possibility than a myristoylated protein other than eNOS was involved, they infected brain slices with a recombinant adenovirus vector encoding a chimeric eNOS protein wherein the N-terminal glycine required for myristoylation is replaced by the extracellular and transmembrane domain of CD8. Using this fusion protein, they showed that the chimeric eNOS could be targeted to plasma membrane by the CD8 transmembrane sequence (independently of eNOS acylation), and importantly, that the HMA-induced inhibition of LTP was fully rescued by the CD8–eNOS construct. The plasma membrane targeting of eNOS therefore appears necessary for NO to fulfill its proposed role of retrograde messenger, probably by promoting its release into the extrasynaptic region and also by facilitating the enzyme activation by plasmalemmal Ca2+ influx. eNOS acylation also appears to play a vital role in coupling the muscarinic cholinergic NO-mediated regulation of heart rate (112). Cardiac myocytes isolated from mice lacking a functional eNOS gene have proven valuable for the study of eNOS in myocyte function. Cardiac myocytes isolated from these eNOS gene-targeted mice lack the muscarinic cholinergic attenuation in beating rate seen in wild-type mice. The muscarinic cholinergic response can be reconstituted in these cells by the transfection of cDNA constructs encoding wild-type eNOS, but not by plasmids encoding the myristoylation-deficient eNOS mutant. In transfected cardiac myocytes expressing wild-type eNOS, the muscarinic cholinergic agonist carbachol completely abrogated the spontaneous beating rate and induced a fourfold elevation of the cyclic guanosine monophosphate (cGMP) level. By contrast, in the myr− eNOS myocytes, carbachol failed to exert its negative chronotropic effect and to increase cGMP levels. These data document an obligatory role for acylated eNOS, which is endogenously expressed in cardiac myocytes, and suggest an important role for eNOS in the modulation of heart rate control. Although nNOS is not acylated, nNOS directly interacts with two palmitoylated proteins, i.e., PSD-95 in neurons (113) and caveolin-3 in skeletal muscle (114). In both cases, the protein–protein association was shown to account for the targeting of the enzyme in these specialized cell compartments (see following) and it can, therefore, be postulated that any change in the palmitoylation of nNOS partners could have, as for the eNOS protein, dramatic effects on the location and functional activity of the enzyme.

SUBCELLULAR LOCALIZATION OF NOSs Criteria Almost every conceivable intracellular organelle has been postulated as a possible site for NO synthesis, from the plasma membrane to the cell nucleus. There is considerable

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controversy and confusion in this area, serving as a reminder that rigorous criteria must be applied to the study of the subcellular targeting of proteins. For example, the interpretation of immunohistochemical approaches must be substantiated by subcellular fractionation experiments utilizing an inclusive (rather than selective) range of organelle-specific markers. Moreover, although quantitative approaches, such as coimmunoprecipitation methodologies, are informative for the study of NOS interactions with organelle-specific proteins, other more inherently qualitative techniques are more difficult to interpret objectively and rigorously. This applies particularly to the NADPH diaphorase assay, a commonly used tissue staining technique, which exploits the NADPH-dependent reduction of nitroblue tetrazolium as a surrogate for NOS activity.

NOS in Plasma Membrane and Plasmalemmal Caveolae For several years, determining the specific particulate subcellular fraction to which the eNOS is targeted was an elusive goal; mutually contradictory reports have only been recently resolved by the study of Shaul and colleagues (96), documenting the localization of eNOS in plasmalemmal caveolae. The Latin term caveola intracellularis (115) had been introduced more than 40 years ago to describe plasma membrane invaginations identified by electron microscopy in a wide variety of cell types including epithelial and endothelial cells, as well as adipocytes and myocytes. Functionally, these 50–100 nm plasmalemmal vesicles were first shown to participate in the transcellular transport of macromolecules (transcytosis) and in the uptake of small molecules (pinocytosis). More recently, however, the discovery of a biochemical marker protein of these unusual organelles, termed caveolin, has provided the impetus for a new wave of studies suggesting that caveolae also participate in signal transduction by ensuring the compartmentalization of signaling molecules such as growth-factor and hormonal receptors, G proteins, protein kinases, as well as eNOS (for review, see ref. 116). Such a concentration of eNOS with other signaling proteins may facilitate, or improve the efficacy of, the coupling between the agonist stimulation and eNOS activation. We and others have, for instance, reported that G-protein-coupled receptors known to stimulate NO• production, such as the muscarinic and bradykinin receptors, are targeted to caveolae upon agonist stimulation (117,118). More recently, McDonald and colleagues (119) have reported the existence of a caveolar complex between the arginine transporter CAT1 and eNOS, thereby providing a mechanism for a highly efficient delivery of substrate to eNOS. In addition, the targeting of eNOS in plasmalemmal caveolae probably facilitates paracrine effects of NO•. In endothelial caveolae, this last statement is certainly verified, as the NO• produced finds most of its targets in the proximal myocyte layers or circulating blood cells such as platelets and red cells (120). The close association between plasmalemmal caveolae and the cytoskeleton could also reflect their importance in the vascular mechanotransduction mediated by NO•. The targeting of NOS to caveolae, which was first thought to be restricted to eNOS, is also likely to also occur for nNOS, which was recently shown to specifically interact with the muscle-specific caveolin isoform in skeletal muscle (114). In addition, caveolae were very recently identified in neuronal cells, and could therefore lead to the compartmentalization of nNOS in these specialized microdomains of the plasma membrane (121). As for iNOS, because the binding of caveolin and calmodulin are mutually exclusive, it seems unlikely that iNOS, which binds calmodulin avidly, is regulated by interactions with caveolin or is targeted to caveolae.

NOS in the Cell Nucleus and the Endoplasmic Reticulum Although it is now clearly established that NO• can cause G:C–A:T transitions and mediate DNA strand breaks (122), there is no definitive evidence that any of the NOS isoforms are localized to the cell nucleus. The nucleus being a cellular target for NO• may by itself explains the absence of constitutive NOS, considering the high risk of inducing genotoxicity


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(123). Although inconclusive, a recent immunohistochemical study has, however, provided suggestive data for the intranuclear localization of iNOS (124). Again, this could correspond to a pathological situation leading to cell death following DNA damage. A large proportion of nNOS immunoreactivity in neurons is associated with rough endoplasmic reticulum (125), but the functional implication of this compartmentation is still unknown. More generally, although synthesis of the NOS proteins clearly involves the nucleus and the endoplasmic reticulum, it is less clear that these locales constitute ultimate targets for the NOS isoforms.

NOS in Mitochondria In two successive papers, Giulivi and colleagues have documented the existence of a functionally active mitochondrial NOS (126,127). These reports succeeds to several studies providing interesting but inconclusive immunohistochemical data demonstrating mitochondrial staining for all three NOS isoforms. Giulivi and colleagues (127) have demonstrated, by using two different spectroscopic techniques, that rat liver mitochondria produce NO•: most of the enzymatic activity appears localized in the mitochondrial inner membrane and is sensitive to NOS inhibitors. In addition, Tatoyan and Giulivi (126) also reports the purification to homogeneity of the rat liver mitochondrial isoform (mtNOS). Monoclonal antibodies against iNOS are the only to show a crossreactivity with the mitochondrial NOS isoform. Furthermore, despite an apparent constitutive expression in the mitochondrial membranes, mtNOS exhibits kinetic parameters, molecular weight, and the requirement of a tightly bound calmodulin similar to the inducible NOS isoform. The authors, however, mention a distinctive proteolytic pattern of mtNOS, which suggests the existence of a splice isoform containing mitochondrial-targeting sequences. Thus, the exact identity of mtNOS remains to be established, but the production of NO• directly by the mitochondria opens new perspectives in the understanding of regulatory processes modulating oxidative phosphorylation in different biological systems. In this connection, a recent report by Clementi and colleagues (128) suggests that, beside the physiological regulation of the mitochondrial respiratory chain by NO• via its action on complex IV, long-term exposure to NO leads to persistent inhibition of mitochondrial complex I and may be of pathophysiological importance.

NOS in the Golgi Apparatus There have been numerous reports identifying eNOS in Golgi (129,130). The interpretation of many of these studies is confounded by the experimental challenges involved in the assignment of a (recombinant) protein to a specific subcellular organelle. Using green fluorescence protein (GFP) technology, Sessa and colleagues (129) reported that in more than 90–95% of transfected NIH 3T3 cells, GFP-eNOS was absent from the plasma membrane borders and exclusively located in the Golgi (130). As acknowledged by these authors, this observation is completely different when using native microvascular endothelial cells for expressing the GFP-eNOS, as 80% of these endothelial cells are positive for eNOS in plasmalemmal caveolae. These experimental differences aside, the dynamic nature of protein trafficking is very likely to account for differences in eNOS location noted between different cell types and experimental conditions. Prabhakar and colleagues (131) recently used a quantitative approach exploiting immunofluorescence microscopy to show that bradykinin stimulation of aortic endothelial cells promotes the translocation of eNOS from the cell membrane to intracellular structures, suggesting that transport of eNOS from plasmalemmal to intracellular structures is part of a physiological cycle highly sensitive to the state of cell activation (see following). Thus, even though it appears plausible that eNOS biosynthesis and/or recycling may involve the Golgi, the relevance of this organelle for the critical step of NOS activation and NOS release remains to be rigorously established.

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NOS in the Cytoskeleton There are suggestive studies that indicate eNOS may associate with cytoskeletal proteins (93,114). Intuitively, the targeting or association of eNOS with the cytoskeleton may provide a mechanism for mechanochemical coupling of changes in cell shape (e.g., with hemodynamic shear stress or cardiac myocyte contraction) to regulation of the enzyme. However, whether eNOS undergoes direct interactions with cytoskeletal proteins remains to be established by rigorous methodology. By contrast, the cytoskeleton association is much more clearly established for nNOS by virtue of its association with the cytoskeletal dystrophin complex in skeletal muscle (16,132) (see following). Furthermore, members of the postsynaptic-density 95 family of cytoskeletal proteins, which are known to interact with nNOS, have been shown to mediate receptor clustering at excitatory synapses in the brain (133).

NOS in Specialized Intracellular Organelles In primary macrophages, subcellular fractionation and immunohistochemical approaches have established the presence of iNOS in intracellular vesicles (phagosomes?), possibly reflecting a locale for NO•-dependent killing of opsonized intracellular microorganisms (134). The molecular mechanisms whereby iNOS is targeted to these macrophage vesicles is not clear, and it remains to be established whether iNOS is similarly targeted in other cells.

REGULATION OF NOS BY PROTEIN-PROTEIN ASSOCIATIONS Calmodulin: The First NOS-Associated Protein The requirement for calmodulin in nitric oxide synthesis is an essential characteristic of all three NOS isoforms, although this ubiquitous Ca2+ regulatory protein demonstrates important isoform-specific differences in its role as an allosteric activator. Indeed, nNOS and eNOS are low-output NOS whose activity is regulated by Ca2+/calmodulin, increasing as Ca2+ rises and decreasing as Ca2+ falls. By contrast, iNOS is a high-output NOS whose activity is essentially Ca2+-independent, as calmodulin is very tightly bound to the enzyme. Moreover, in the case of eNOS, and probably for nNOS in skeletal muscle, calmodulin activation of the enzyme involves not only the binding to the calmodulin binding motif within the NOS sequence but also the allosteric displacement of caveolin from NOS, thereby reversing the inhibitory effect of the scaffolding protein.

nNOS, PSD-93/95, and the Dystrophin Complex The sarcolemma of skeletal muscle contains a family of intracellular and transmembrane glycoproteins associated with dystrophin, linking the extracellular matrix with the actin-based cytoskeleton. The N-terminus of nNOS interacts with α1-syntrophin, a binding partner of dystrophin through a PDZ/GLGF protein motif of approx 100 amino acids present in both proteins (132). The PDZ-containing domain of nNOS also binds to PDZ repeats in postsynaptic density 95 (PSD-95). In certain nonneuronal cells, including developing chromaffin cells of the adrenal gland and secretory cells of salivary gland, nNOS is coexpressed with the related protein PSD-93 (16). Binding interaction between PDZ domains are selective. Though nNOS binds to the PDZ motif in α1-syntrophin and to the second PDZ motif of PSD-95, nNOS does not associate with the first and third PDZ motifs in PSD-95 (16). Certain PDZ domains are capable of binding to the extreme C-terminus of a family of receptors and ion channels, including N-methyl-D-aspartate (NMDA) receptors, Shaker-type K+ channels, and FAS. Brenman and colleagues found that nNOS and NMDA receptors compete for common or nearby binding sites within the second PDZ repeat of a PSD-95 subunit. The PDZ consensus sequence is present in a diverse family of enzymes and structural proteins. Many of


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these proteins are found concentrated at specialized cell–cell junctions, such as neuronal synapses, epithelial zona occludens, and septate junctions. PDZ domains may therefore be important elements of interactions required for signal transduction at the membrane. Furthermore, the finding that PDZ/GLGF is present in a heterogeneous family of enzymes has motivated suggestions that the Gly-Leu-Gly-Phe (GLGF) domain may regulate enzyme activities. However, Bredt and colleagues (12) found that deletion of the GLGF domain of nNOS does not alter NOS catalytic activity in transfected cells.

PIN and CAPON By means of yeast two-hybrid system screening, a 10-kDa protein was identified that physically interacts with and specifically inhibits the activity of nNOS (135). These authors named this protein PIN (for protein inhibitor of neuronal NOS) and presented evidence that the PIN/nNOS interaction leads to the destabilization of the nNOS dimer. Contradictory results (136; I. Rodriguez-Crespo, personal communication) were recently reported according which PIN inhibits all isoforms of NOS and has no effect on nNOS dimerization; stoichiometric analysis also shows that an approx 300-fold molar excess of PIN are required to inhibit nNOS by 50%. Moreover, PIN was simultaneously discovered as a light chain (LC) of dynein and myosin, with a highly conserved sequence over a wide spectrum of different organisms (137), and the role of the NOS inhibitor therefore appears as secondary to its other functions in myosin and dynein complexes. Clearly, the relevance of the PIN/nNOS remains to be rigorously established and considering that the stoichiometry represents a limiting factor, it may be most interesting to explore physiological or pathological conditions where high levels of PIN/dynein LC expression are observed. In this perspective, Gillardon and colleagues (138) recently reported that following global ischemia, mRNA expression of PIN/dynein LC was rapidly induced in pyramidal neurons of the hippocampal CA3 region and granule cell of the dentate gyrus which are resistant to ischemic damage. In vulnerable CA1 pyramidal neurons however, PIN/dynein LC remained at basal level after global ischemia and was associated to neuronal cell death. CAPON (for carboxyl-terminal PDZ ligand of nNOS) is a recently identified cytosolic protein highly enriched in brain that competes with particulate PSD95 for interaction with nNOS (139). The interaction CAPON/nNOS is highly specific, and although CAPON does not inhibit nNOS activity by itself, CAPON is thought to reduce the accessibility of nNOS to NMDA receptor-mediated calcium influx, thus diminishing the capacity of nNOS to acutely produce NO•. Although phosphorylation of nNOS by various kinases failed to alter the CAPON/nNOS interaction, phosphorylation of CAPON in its C-terminal region could be involved through disruption of its specific β-sheet conformation sequence in the regulation of the interaction (139). Clearly, more needs to be learned and studies are, no doubt, underway to examine the native CAPON/nNOS association in its physiological environment and to address specific questions such as the stoichiometry of the protein–protein interaction.

ENAP-1 or Hsp90 Stimulation of aortic endothelial cells with bradykinin produces cycles of tyrosine phosphorylation/dephosphorylation of a 90-kDa protein, termed ENAP-1 (for eNOS-associated protein-1) by Venema and colleagues (94). This protein, recently identified as the heat-shock protein 90 (Hsp90), appears to facilitate eNOS activation in endothelial cells by forming a heterocomplex with the enzyme following stimulation with a Ca2+-mobilizing agonist or shear stress (140). In the specific case of shear stress, the eNOS/Hsp90 association appears to be somewhat delayed relative to that seen following agonist stimulation (94) and may correspond with the slower change in detergent solubility of eNOS seen in endothelial cells following exposure to shear stress (93).

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Caveolin-1, Caveolin-3 and Their Regulatory Cycles Following the original observation of the caveolar localization of eNOS in plasmalemmal caveolae (96), several lines of evidence in endothelial cells and cardiac myocytes revealed that eNOS is quantitatively associated with caveolin, the structural protein within caveolae (141). Further experiments revealed that this association leads to the inhibition of the enzyme activity, and that a stable protein–protein interaction takes place between both proteins (142– 146). Consensus sequences were identified within both proteins: the scaffolding domain of caveolin, a juxtamembrane region of 20 amino acids in the C-terminal moiety of caveolin (143,145), and a putative caveolin-binding motif, a peptide sequence rich in aromatic residues localized in the oxygenase domain of eNOS (114,144,146); more recently, sites for caveolin inhibition of eNOS have been identified within the reductase domain of the enzyme (147) and within the caveolin N-terminal region (145). Like other modular protein domains, the scaffolding domain of caveolin appears to function by providing frameworks for the assembly of preassembled oligomeric signaling proteins, but, in addition, these structures maintain these diverse signaling proteins in their “off” state. The caveolin/eNOS interaction constitutes a new biological framework within which to understand the regulation of eNOS, yet many details still remain to be addressed to attribute to each interaction level the corresponding effect on the catalytic properties and/or targeting of eNOS. In order for eNOS to be fully activated, caveolin must dissociate from the enzyme. The ubiquitous Ca2+ regulatory protein calmodulin (CaM) disrupts the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion (142): caveolin serves as a competitive inhibitor of CaM-dependent eNOS activation (143). The CaM binding consensus sequence is located at the border of the NOS reductase and heme domains, and CaM binding to this site activates NO synthesis by enabling the reductase domain to transfer electrons to the heme domain. Caveolin appears to attenuate this electron transfer, and CaM apparently rescues the caveolin-inhibited eNOS at this level, probably by binding to sequences in the eNOS reductase domain (147). This close control of enzyme activity may be particularly important for eNOS in caveolae, where CaM is also largely enriched (96) and could thus lead to undesired enzyme activation if the interaction of caveolin with eNOS was not keeping the system in check. The relevance of the caveolin/CaM reciprocal regulation of eNOS was recently demonstrated in intact cells wherein transient increase in [Ca2+]i consequent to agonist activation was shown to promote the dissociation of eNOS from caveolin, associated with translocation of eNOS from caveolae (110,144). Such agonist-induced disruption of the caveolin/eNOS heterocomplex promotes the dissociation of the enzyme from proximity to the transporter of arginine (119) and thus, may serve as a feedback mechanism for eNOS activation (see earlier). An obvious question raised by the findings of the counterbalancing modulation of eNOS by caveolin and CaM is related to the cellular regulation of eNOS/caveolin interaction in the context of enzyme acylation. We have observed that the myr− and palm− eNOS mutants may both interact with caveolin in the cytosol (110,148); this association also leads to a marked inhibition of enzyme activity, which is completely reversed by addition of CaM (144). The regulatory caveolin/eNOS association therefore appears independent of the state of eNOS acylation, indicating that agonist-evoked Ca2+/CaM-dependent disruption of the caveolin– eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin’s tonic inhibition of enzyme activity. Thus, we propose that caveolin may serve as an eNOS chaperone regulating NO• production independently of the enzyme’s residence within caveolae or its state of acylation (110,148). These data suggest a dynamic cycle of eNOS–caveolin interactions initiated by agonistpromoted increases in [Ca2+]i that disrupt the caveolin–eNOS complex, leading to enzyme activation. Following more prolonged agonist stimulation, eNOS is likely to be depalmitoylated, and is no longer selectively sequestered in caveolae. The translocated enzyme then


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partitions into noncaveolar plasma membrane and also in the perinuclear region of the cell (131), the precise identity of which has not yet been established. Furthermore, several lines of evidence indicate that subsequently to the enzyme’s translocation, and following the decline in [Ca2+]i to basal levels, eNOS may once again interact with caveolin and is then retargeted to caveolae, a process accelerated (or stabilized) by enzyme palmitoylation (110, 144). The reassociation of eNOS with caveolin could occur either at the plasma or perinuclear membrane levels or even in the cytosol through which caveolin complexes may shuttle between caveolae and an internalized caveolar vesicle/trans-Golgi network.

G-Protein-Coupled Receptors A direct interaction between eNOS and seven-transmembrane-segment receptors has recently been described by Ju and colleagues (149): the bradykinin B2 receptor appears to be able to physically associate, through its fourth intracellular domain, with eNOS. Similar results were obtained using peptides derived from the angiotensin II receptor AT-1. This intriguing signaling paradigm was explored principally by studying purified proteins and synthetic peptides, and the role of these protein–peptide interactions remains to be established in intact cell systems. Although a small fraction of cellular eNOS appears to be physically associated with the B2 receptor (3 h) of endothelial cells to homocysteine results in impaired NO• production; however, if the exposure is brief (300 µM). In three in vivo studies, an L-arginine infusion (30 g over 30 min) decreased adenosine-diphosphate (ADP)-stimulated platelet aggregation, to 32–50% of control value within 15 min (126,216,217). There was no effect on collagen-stimulated platelet aggregation, which is consistent with the findings of Vallance and colleagues who showed no effect of EDNO on collagen-stimulated platelet aggregation in healthy humans ex vivo (218). The antiplatelet effect of L-arginine appears to be mediated by enhanced platelet cGMP production and inhibition of thromboxane B2 formation (219). Although the endothelial

Chapter 30 / L-Arginine

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isoform of NOS is present in platelets in small amounts, it is probably not completely responsible for the L-arginine-induced increase in platelet cGMP (220). Endotheliumderived NO• in the microvasculature may play a greater role in modulating platelet cGMP and reducing platelet reactivity. Platelets passing through the coronary microvasculature of the isolated perfused heart manifest an increase in intraplatelet cGMP. This effect is markedly enhanced by coadministration of ACh, which stimulates endothelial (but not platelet) NOS activity (221). These effects are abrogated by NOS inhibitors. In vitro administration of supraphysiologic concentrations of L-arginine to platelet-rich plasma enhances platelet cGMP levels and causes a reduction in platelet thromboxane formation. The effect on platelet aggregation in one clinical study cited earlier was associated with a 43% increase in platelet cGMP (216). The effect on cGMP levels is reversed by preincubation with NOS inhibitors demonstrating NO• dependency. It is interesting to note that polyamine metabolic products of L-arginine (putrescine, spermidine, and spermine) also have antiplatelet effects similar to L-arginine in normal and diabetic rats (222). These findings suggest that there may also be an NO•-independent antiplatelet effect of L-arginine in antithrombosis. L-Arginine has also been shown to inhibit the processes leading to fibrin formation and platelet-fibrin interaction and accelerate fibrin degradation in humans (223). Recently, L-arginine has been shown in vitro to accelerate tPA-induced plasmin generation and thus augment fibrinogenolysis (224). L-Arginine binds to plasminogen (probably at kringle 5) stimulating its conversion to plasmin with subsequent enhancement of fibrinogenolysis. This effect resulted in an acceleration of the early stages of clot lysis, although eventually clot lysis was equal in control groups as well. This finding is consistent with the findings in vivo in patients with peripheral arterial disease discussed shortly (225). Additionally, when L-arginine is given in high doses (30 g iv), it lowers blood viscosity (126). This effect is likely mediated by insulin because simultaneous administration of octreotide to inhibit endogenous insulin release prevents this effect. Furthermore, in diabetic patients, L-arginine infusion lowered blood viscosity and this effect was significantly amplified after an 8-wk treatment with metformin, which increases the peripheral sensitivity to insulin (226). These studies indicate L-arginine may also act as an antithrombotic agent beyond its antiplatelet effects.

PLAQUE FORMATION AND R EGRESSION By virtue of its ability to reduce superoxide activity, inhibit monocyte adhesion and reduce thrombosis, chronic supplementation of L-arginine inhibits the progression of aortic and coronary artery atherosclerotic plaque formation ([41,43,82,150–152,206,227,228], Fig. 4). In one particular study, the effects of L-arginine were compared to those of lovastatin (42). In that study, L-arginine restored EDNO activity, prevented an increase in superoxide free radical production and slowed the progression of intimal lesion formation. In contrast, the cholesterollowering therapy lovastatin also reduced intimal proliferation but not to the extent of L-arginine. Furthermore, lovastatin failed to increase EDNO activity, enhanced the activity of superoxide radical production, and failed to restore endothelium-dependent vasodilator dysfunction. In one study, this beneficial effect of dietary L-arginine was observed in male, but not female, hypercholesterolemic rabbits (152). However, this gender difference was not found in a murine model of hypercholesterolemia in which the LDL receptor was disrupted by homologous recombination. Regardless of gender, dietary L-arginine prevented intimal lesions and xanthoma formation that otherwise occurs when these mice are fed a high-fat diet (41). The reduction in atherosclerotic burden appears to be due to a reduction in both monocyte accumulation and in myointimal cell proliferation (201). Immunohistochemical detection of 5-bromo2'deoxyuridine (BrdU) incorporation demonstrated a 33% reduction in the proliferation rate of myointimal cells, whereas antibody staining demonstrated a 85% reduction in monocyte accumulation in L-arginine-treated animals. In this study, intimal/medial ratio of the L-arginine group was reduced about 63%.

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Part III / Nitric Oxide in Cardiovascular Therapeutics

Fig. 4. Microphotograph of thoracic aortae from hypercholesterolemic rabbits. New Zealand White rabbits were fed a 1.0% cholesterol diet for 10 wk. Half of the animals also received oral supplementation of L-arginine. After 10 wk the aortae were harvested for histomorphometry. Those animals receiving vehicle (left) demonstrated impaired endothelium-dependent vasodilation and lesions that involved 75% of the surface area of the thoracic aorta. By contrast (right), hypercholesterolemic rabbits receiving L-arginine supplementation exhibited normalized endothelium-dependent vasodilation, and reduced lesion surface area (about 25%). L-Arginine may have a beneficial effect on pre-existing lesions as well. New Zealand White rabbits fed a high-cholesterol diet for 10 wk develop atherosclerotic lesions that cover about 30% of the surface area of their aorta. If they continue on this diet for an additional 13 wk, the lesions progress to involve 57% of the surface area. By contrast, progression of the lesions is halted in rabbits fed a high-cholesterol diet that is supplemented with dietary L-arginine. Indeed, in a subset of these animals in which L-arginine persistently improved endothelial vasodilator dysfunction, an impressive regression of lesions was observed, with a final lesion surface area of only 5% (43).

R ESTENOSIS Because NO• inhibits vascular smooth muscle proliferation and monocyte recruitment, enhancement of vascular NO• activity may be beneficial in the prevention of restenosis following balloon angioplasty. Intravenous L-arginine (0.5 g/kg/d) initiated 2 d prior to and continued 2 wk following balloon injury to the thoracic aorta of normocholesterolemic rabbits reduced intimal proliferation by 39% (46). In a similar model, 4 wk of oral L-arginine therapy (2.25% in the drinking water) improved endothelium-dependent vasodilation to ACh and reduced intimal lesion area from 0.43 ± 0.08 to 0.24 ± 0.02 mm2 (229). In the hypercholesterolemic rabbit model of balloon injury, oral L-arginine improved endothelium-dependent vasodilation and reduced neointimal lesion area from 0.69–0.34 mm2) (230). This was associated with a significant reduction in monocyte recruitment to the area of balloon injury. In a recent study in heritable hyperlipidemic rabbits, L-arginine significantly reduced the extent of neointimal lesion formation following balloon injury, however, L-arginine reduced the endothelium-dependent inhibition of platelet aggregation and attenuated the vasodilator function of the injured vessel (45). The authors of this study suggest that the detrimental effect


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on endothelial vasodilator function may be related to the ongoing hyperlipidemia and the presence of lipids in the vascular wall in their model. This effect may also be caused by an effect of oxidized LDL to inhibit L-arginine uptake and NOS expression in platelets (110). More recently, Schwarzacher and colleagues have found that local delivery of L-arginine to the vessel wall could similarly reduce restenosis (44). Using a drug-delivery balloon, local administration of L-arginine was observed to increase NO• production in the rabbit iliac artery for up to 1 wk following the injury, and reduced intimal thickening at 4 wk following balloon angioplasty (44). This study suggests that a single intramural delivery of L-arginine is sufficient to enhance EDNO production for a sufficient amount of time to inhibit restenosis. Arginine supplementation has also been shown to inhibit intimal proliferation in vein grafts. Intimal proliferation is responsible for early stenoses in more than 30% of peripheral vein grafts within a year of placement. In the experimental model, L-arginine inhibits intimal thickness by 47% in vein grafts implanted in the position of the carotid artery (231). This effect of L-arginine was somewhat reduced if the rabbits were made hypercholesterolemic (24% reduction in intimal thickness) (151). BrdU incorporation experiments demonstrated less SMC proliferation in the L-arginine treated animals (232). Likewise, the vasorelaxation of venous rings to ACh remained intact in the supplemented animals. The mechanism by which L-arginine reduces intimal hyperplasia may be by inhibiting the expression of hyaluronan synthase-1 (233). Normally, within 7 d of implantation there is about a 20-fold rise in the expression of this enzyme, which is responsible for making hyaluronan, a key component of the extracellular matrix. L-Arginine supplementation reduces the expression to less than a fivefold increase. To date, there have been no studies demonstrating an inhibition or regression of plaque formation, an effect on venous engraftments, nor on the extent of restenosis following angioplasty in humans using supplemental L-arginine. However, investigators have demonstrated an effect on the key processes of human atherosclerosis as discussed previously.

A NGIOGENESIS There is a significant heterogeneity between patients in their angiogenic response to obstructive vascular disease. Some individuals develop an exuberant network of collateral vessels, proving them with a “biologic bypass” that reduces symptomatology. Other individuals are less fortunate. Recent insights into the regulation of angiogenesis have permitted pioneering efforts to induce therapeutic angiogenesis in patients with peripheral arterial disease (PAD) or coronary artery disease (CAD). Preliminary studies indicate that administration of angiogenic factors (e.g. vascular endothelial growth factor [VEGF] or basic fibroblast growth factor [FGF]) may improve collateral circulation in atherosclerotic obstructive disease. NO• may play an important role in this process (234). In endothelial cell culture, FGF upregulates the expression of eNOS and increases the elaboration of NO• while concurrently inducing tubule formation. FGF-induced tubule formation is mimicked by NO• donors and blocked by NOS antagonists (235). In other studies, Substance P, transforming growth factor β1 and VEGF all have been shown to increase the expression of eNOS and NO• elaboration, as well as to induce tubule formation (in vitro) or angiogenesis (in vivo) (236). The angiogenic effects of these factors are antagonized or abolished by NOS antagonists. Indices of capillary formation and blood flow are enhanced by L-arginine in animal models of hind-limb ischemia, whereas the angiogenic response to ischemia is markedly inhibited in the eNOS knockout mouse (237).

Coronary Artery Disease By virtue of its ability to restore vasodilation and slow the progression of atherosclerosis, L-arginine may be of particular therapeutic value in patients with CAD. These patients often have a paradoxical vasoconstrictor response to ACh (238). Systemic infusion of L-arginine

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reverses this vasoconstrictor response (239,240). Furthermore, Drexler and colleagues have shown that in patients with angiographically demonstrated coronary artery disease undergoing coronary artery catheterization, a single intravenous infusion of L-arginine (30 g) normalizes coronary blood flow response to the endothelium-dependent vasodilator, ACh (1). In patients with angina and normal coronary arteriograms (syndrome X), there is an endothelial vasodilator dysfunction of the microvasculature (241), as evidenced by reduced coronary blood flow response to ACh (242). This abnormality is reversed by intravenous infusion of L-arginine. However, when CAD is associated with hypertension, the endothelial vasodilator dysfunction does not appear to be reversible with L-arginine (239) consistent with other studies in hypertensive patients. In young men with documented CAD, an endothelial vasodilator dysfunction of the brachial artery also exists. L-Arginine improves brachial artery flowmediated vasodilation and, in addition, inhibits monocyte adhesion to endothelial cells in this population (20). These beneficial effects of L-arginine on coronary vasculature have functional consequences. Concomitant with the reversal of endothelial vasodilator dysfunction in patients with coronary artery disease, L-arginine (30 g) modestly lowers mean arterial pressure (approx 5 mmHg), reduces total peripheral resistance (by approx 16%), increases cardiac output (by 11%) and plasma cGMP (by 12%) (4). Recently, Lerman and colleagues demonstrated an impressive effect of dietary L-arginine on coronary vasomotor function in patients with significant CAD (243). Oral L-arginine (9 g/d for 6 mo) improved coronary blood flow response to intracoronary ACh by 150%. This was associated with a decrease in plasma endothelin levels and an improvement in patient’s symptoms scores. Ceremuzynski and colleagues have recently demonstrated an increase in exercise capacity following oral L-arginine in patients with stable angina pectoris (244,245). Patients included in this study all had documented CAD and a history of transmural myocardial infarction. Following 3 d of oral L-arginine (2 g t.i.d.), exercise time during a standardized treadmill protocol (Modified Bruce) was significantly increased (531 ± 195 to 700 ± 173 s, p < 0.0002). Along with the increased walking time was an increase in maximum work load (6.4 ± 2 to 7.4 ± 3 metabolic equivalents [METS], p < 0.006). Furthermore, the degree of maximum ST segment depression (summed for all leads) was less than before treatment. This study demonstrated that even moderate supplementation with oral L-arginine can reduce myocardial ischemia and improve cardiovascular activity in patients with stable angina.

Peripheral Arterial Disease L-Arginine

may have therapeutic utility in peripheral arterial disease (PAD) as well. In patients with severe PAD, intravenous L-arginine has been shown to improve limb blood flow (23). A single intravenous dose of L-arginine (30 g over 60 min) enhanced femoral artery blood flow (42% increase). The increase in limb blood flow was associated with an elevation of urinary cGMP and nitrate excretion rate consistent with an L-arginine-induced enhancement of EDNO synthesis. The effect of L-arginine infusion on limb blood flow was equal to that of intravenous prostaglandin E1 (29% increase, p < 0.05) in the same study. Furthermore, studies from the same group of investigators in Hannover, Germany, indicate that daily intravenous administration of arginine for two weeks enhances walking distance in patients with intermittent claudication (246). L-Arginine has also been shown to improve symptoms in patients with less severe PAD. Daily infusions of L-arginine (12.6 g) for 7 d increased calf blood flow and transcutaneous oxygen saturation, improved walking distance, shortened the time period to recovery from pain, and reduced platelet aggregation and clot lysis time (225,246). The enhancement in blood flow has been extended to the microvascular circulation as well. Thirty grams of intravenous L-arginine was able to enhance calf muscle blood flow from 1.7 ± 0.1 to 2.2 ± 0.2 mL/ min/100 g at 80 min following infusion (22). There was no effect from 8 g of iv L-arginine


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in this time period, however. Preliminary evidence from our clinical laboratory suggests that administration of an L-arginine-enriched food bar can increase walking distance and quality of life in these patients (Cooke and colleagues, unpublished observations). Patients with PAD have hyperaggregable platelets (212,213). Consistent with observations in other patient populations, daily infusions of L-arginine (60 mmol) reduced ADPinduced platelet aggregation and shortened euglobin clot lysis time in subjects with PAD (225). These antithrombotic effects of L-arginine may add to its vasodilatory effect to enhance overall walking ability and quality of life in this population.

Systemic Hypertension By virtue of its ability to enhance vasodilation, L-arginine can lower blood pressure. The major mechanism is likely via the enhancement of EDNO (247–249). In addition, L-arginine reduces serum angiotensin-converting enzyme activity and serum angiotensin II without affecting plasma renin activity, and these effects may contribute to the hypotensive effect of L-arginine (141). In normotensive individuals, intravenous L-arginine causes modest effects on blood pressure. Giugliano and colleagues conducted a dose response study and found that blood pressure changes were linearly correlated with dose of L-arginine up to 3 g (250). Multiple studies have shown that high doses (approx 30 g) of intravenous L-arginine cause a similar decrease in blood pressure to that of 3 g (approx 5–10 mmHg) (126,215,247,249,251–257). Because it has a modest effect on blood pressure, L-arginine may be adjunctive therapy in certain types of hypertensive disorders. In various animal models of systemic hypertension, oral L-arginine reduced or normalized blood pressure (258–261). Specifically, oral Larginine prevented salt-sensitive (258,259) and mineralocorticoid–salt (DOCA) hypertension (261) in rats. In several studies in spontaneously hypertensive rats however, oral L-arginine failed to lower blood pressure (165,259,262), although it did prevent hypertension-induced cardiac hypertrophy in these animals (262). In contrast, in animals with salt-sensitive hypertension, chronic oral supplementation with L-arginine prevented the salt-induced increase in blood pressure and drop in renal blood flow (263). Oral L-arginine also prevented adrenocorticotropic hormone (ACTH)-induced hypertension and did so partly by lowering serum cortisone levels (260). In contrast, dexamethasone-induced hypertension was not altered with L-arginine treatment (264), suggesting that ACTH-induced hypertension is not simply a glucocorticoid-mediated process. Intravenous L-arginine lowered blood pressure in the rat model of pre-eclampsia (265) and surgical coarctation (266). In another model of hypertension induced by lead, EDNO activity has been shown to be reduced and free radical activity increased (267). Infusion of L-arginine causes a rise in urinary NOx levels and a fall in blood pressure to normal. Interestingly, there is some evidence that part of the blood pressure effects of L-arginine are mediated by NO•-modulation of the baroreceptor reflex (268). Cervical and aortic sympathetic nerve activity decreased along with mean arterial pressure following L-arginine infusion in anesthetized rabbits. This effect was not seen with D-arginine. Similar to the animal models, in the clinical setting, L-arginine reduces blood pressure in only some forms of hypertension. Dietary L-arginine for 1–7 d in individuals with longstanding essential hypertension did not have any measured effects on hemodynamics (167). However, oral L-arginine (2 g t.i.d.) was able to lower blood pressure in newly diagnosed borderline hypertensive individuals within 1 wk of therapy (168). In responsive individuals, intravenous L-arginine (20–30 g infused over 30 min), causes a slight reduction in arterial pressure (5–10 mmHg), and reduces renovascular resistance (269), although this is diminished in salt-sensitive hypertension after salt loading (11). These findings were corroborated in a recent study on hypertensive African-Americans (13). L-Arginine infusion (500 mg/kg) caused a 12-mmHg drop in mean arterial pressure in salt-sensitive individuals but only a 4 mmHg drop in insensitive individuals. However, the increase in effective renal plasma flow observed in normotensive controls was blunted in salt-resistant individuals and even more

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so in salt-sensitive individuals. Part of the reason for the diminished effect may be due to an increased production of ADMA following salt loading (270). Concomitant with the reduction in hypertension is an improvement in ventricular ejection fraction (271) and increased cardiac output, urine output, and natriuresis (249,272). The effect of intravenous L-arginine on blood pressure has led to its study during repair of coarctation (257). Three adults undergoing coarctation repair received 10–20 g L-arginine during surgery, which resulted in desired reductions in mean arterial pressure of 5–20 mmHg during and immediately following surgery.

PULMONARY HYPERTENSION Just as in systemic hypertension, patients with pulmonary hypertension manifest an endothelial vasodilator dysfunction that may contribute to the abnormal pulmonary vascular reactivity and structural changes (169). In animal models of pulmonary hypertension, intravenous L-arginine reduces pulmonary pressure and reduced mortality (170,173,273). Specifically, in a rat model of pulmonary hypertension induced by chronic hypoxia or by injection of monocrotaline, concomitant daily injections of L-arginine nearly abolished the development of pulmonary hypertension and the associated structural changes of the pulmonary arteries and right ventricle (274). Just as in systemic hypertension, the clinical setting of the pulmonary hypertension and the mechanism of the defect in the vasodilation are a major factors determining the effectiveness of L-arginine as a therapy. Adults with primary pulmonary hypertension or pulmonary hypertension secondary to myocardial disease demonstrate a reduced expression of NOS (275) as opposed to newborns with persistent pulmonary hypertension, which demonstrates a relative arginine deficiency (276,277). Acute administration of intravenous L-arginine to adults with primary or secondary pulmonary hypertension reduces pulmonary pressure (15). However, only patients with certain forms of secondary pulmonary hypertension exhibit substantial pulmonary vasodilation in response to intravenous L-arginine that is comparable to the response observed with the therapeutic use of prostacyclin (278). In patients with primary pulmonary hypertension or pulmonary hypertension secondary to systemic sclerosis, intravenous L-arginine reduces systemic vascular resistance but, at best, transiently and minimally reduces pulmonary vascular resistance (4,16,278,279). L-Arginine may have greater therapeutic effects in these settings if started before pulmonary hypertension progresses to significant pulmonary vascular disease. L-Arginine has been used to treat persistent pulmonary hypertension of the newborn. McCaffrey and colleagues treated five newborns with 500 mg/kg (14). Ninety minutes after infusion there was a rise in PaO2 from 37–84 mmHg. There was a reduction in the oxygenation index in four of five of the newborns that persisted over 5 h and there were no adverse effects of therapy. Whereas short-term studies in humans with pulmonary hypertension have had disappointing results, these findings may have profound implications for the long-term use of L-arginine to prevent structural changes in such conditions as primary pulmonary hypertension and congenital heart disease.

TRANSPLANT VASCULOPATHY In heart transplant patients, there is an endothelial vasodilator dysfunction in allograft coronary arteries (29). These individuals have a coronary blood flow response to ACh that is about 50% of normal. The cause is probably multifactorial and includes hyperlipidemia, immune mediators, reperfusion injury, infection, and even cyclosporin A. An intravenous infusion of L-arginine (30 g) normalizes endothelium-dependent vasodilation in these patients (29). When intracoronary L-arginine is given in combination with the endothelial agonist, substance P, there is an associated increase in left ventricular contractile performance (30). These investigators were also interested in the physiologic significance of elevated iNOS content within the myocardium. Despite increased iNOS found on biopsy, there was no effect


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of direct L-arginine infusion on contractile performance in the absence of agonist. Furthermore, dietary L-arginine has been shown to reduce myointimal hyperplasia without affecting T-lymphocyte and macrophage infiltration in allograft coronary arteries in a rabbit cardiac transplant model (227). Dietary L-arginine has also been shown to reverse coronary and pulmonary endothelial dysfunction caused by cyclosporin A as described previously (194,196).

HEART FAILURE In patients with congestive heart failure (CHF), there appears to be a generalized reduction in endothelium-dependent vasodilation and these patients have reduced peripheral blood flow at rest and during exercise (280–282). Intravenous administration of L-arginine improves endothelial vasodilator function in this setting as evidenced by an increase in forearm blood flow response to ACh and reactive hyperemia (23–26,281). Intravenous L-arginine also decreased systemic vascular resistance and mean arterial pressure while concomitantly increasing ventricular stroke volume and cardiac output in patients with heart failure secondary to CAD (283). However, in one study in this population, oral administration of L-arginine (20 g/d for 28 d) did not improve vasodilator function (28). Despite this report, another study in patients with heart failure showed that oral L-arginine (5.6–12.6 g/d) taken for 6 wk increased forearm blood flow in response to exercise (27). In addition, these patients demonstrated an improved functional status with an increased walking distance and improved scores on the Living With Heart Failure questionnaire. There are no data presented on the difference in response of these two doses. Oral L-arginine also improved arterial compliance and reduced circulating endothelin levels. Interestingly, in patients progressing to cor pulmonale secondary to chronic obstructive pulmonary disease with renal vasoconstriction, L-arginine (20 g iv) had no effect at increasing intrarenal blood flow, suggesting a disturbance in the NOS pathway that is different from those seen in other forms of heart failure (284).

ISCHEMIA/REPERFUSION There is growing evidence that L-arginine may play a therapeutic role in tissue preservation during reperfusion following a period of hypoxia. Animal studies of cerebral, myocardial, hepatic, and other tissue ischemia/reperfusion generally show a benefit from administration of L-arginine (either iv or po) but not D-arginine. With respect to cerebral ischemia/reperfusion, L-arginine increased regional blood flow and reduced infarct size in areas of cerebral artery ligation in the rat model of focal cerebral ischemia (285). If ischemia persisted to 20 min, L-arginine had no effect unless the animals were pretreated with N G-nitro-L-arginine (L-NNA) (51). This finding suggests that NO• may play a cytotoxic role in the ischemic or early reperfusion phase of ischemia/reperfusion. A similar finding occurred in hypertensive rats made globally ischemic (286). In the rat model of traumatic brain injury, cerebral blood flow is significantly reduced within moments of injury. Administration of L-arginine (100 mg/kg) alone or in combination with SOD 5 minutes after injury, however, prevented a reduction in cerebral blood flow (287). In a rat model of thrombotic occlusion, however, L-arginine failed to improve regional blood flow (288). Similarly, in the cat model of global ischemia (temporary ligation of subclavian and brachiocephalic arteries with hemorrhagic hypotension), L-arginine failed to reduce neurological deficit scores and injury area (289). Finally, in the gerbil model of cerebral ischemia, L-arginine (at doses of 1, 10, and 100 mg/kg before and after occlusion) failed to lessen hippocampal neuronal cell death and, in fact, L-NNA prevented cell death, suggesting an adverse role of NO• in hippocampal ischemia (290). Nevertheless, the eNOS-deficient mouse manifests greater ischemic brain injury than normal animals, indicating that eNOS plays a protective role in ischemic brain injury (291,292). With respect to cardiac ischemia/reperfusion, such as that occurring with coronary occlusion, there is a ventricular dysfunction with an associated endothelial vasodilator dysfunction.

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In this setting, intravenous L-arginine increases NOS activity (50,293,294) and reduces levels of soluble adhesion molecules, lipid peroxidation, and neutrophil accumulation (48, 295). Increased NO• production during reperfusion appears to inhibit endothelin-1 (ET-1) release (296). ET-1 release caused cell necrosis and a 25-fold extracellular release of L-arginine. These combined actions of supplemental L-arginine are likely responsible for the observed reduction in infarct size (55,297,298) and improved recovery of cardiac function (50,295). One study in the isolated rabbit heart, however, showed a negative effect of exogenously applied L-arginine (299). In dogs with acute coronary occlusion, intrapericardial administration of L-arginine was able to reduce the severity of ventricular arrhythmias (300). Because of its ability to reduce the effective refractory period of the cardiac cell, the effect of L-arginine on cardiac rhythm appears to be via NO• modulation of sympathetic input. Skeletal muscle may also benefit from L-arginine during periods of ischemia. L-Arginine infusion (4 mg/kg/min) during and after 6 min of hind-limb ischemia prevented a decline in EDNO levels to undetectable levels and attenuated a rise in superoxide release following reperfusion (120). Consistent with the hypothesis that NOS loses its affinity for L-arginine under unfavorable conditions, the increase in superoxide in untreated animals appeared to be secondary to calcium-dependent eNOS activity. L-Arginine has been studied in liver ischemia/reperfusion as well (52,301,302). L-Arginine (540 mg/kg iv) given 1 h before and again during and after clamping of the hepatic hilum attenuated the rise in malondialdehyde content, aspartate transaminase and lactate dehydrogenase levels (303,304). In addition, hepatic cellular integrity, glycogen content, and the rate of apoptosis was significantly better in the treated animals. Finally, overall survival was better at 7 d following ischemia. These beneficial effects of L-arginine on ischemia/reperfusion have important implications in cardioplegic arrest, organ transplantation, and other surgeries involving periods of ischemia. Just as in cardiac ischemia/reperfusion secondary to coronary occlusion, cardiopulmonary bypass is associated with a ventricular dysfunction and endothelial vasodilator dysfunction despite the use of cardioplegia and hypothermia (305,306). In the isolated working rat heart model of cardioplegic arrest, exogenously applied L-arginine considerably improved the recovery of cardiac mechanical function and coronary flow rate when given in the reperfusate after hypothermic ischemia (54,307). Hiramatsu and colleagues investigated the effect of L-arginine added to cardioplegic solution in neonatal lambs (308,309). In these experiments, they examined the role of EDNO by adding either 10 mmol/L of D-arginine or 10 mmol/L of L-arginine to cardioplegia in isolated, blood-perfused neonatal lamb hearts having 2 h of cold cardioplegic ischemia. At 30 min of reperfusion, the L-arginine group showed a significantly improved recovery in left ventricular systolic function, diastolic function, coronary blood flow, endothelial function, and myocardial oxygen consumption compared with the control groups as those groups with D-arginine added to cardioplegia solution. This protective effect extends to the rat model of transplantation (310). L-Arginine given during reperfusion improves coronary endothelium-dependent vasodilation with recovery of systolic and diastolic function during early reperfusion. Similarly, in the rabbit model of lung transplantation, the addition of L-arginine or pentoxyfylline during reperfusion prevented pulmonary endothelial dysfunction (311). These studies have also been extended to the setting of liver transplantation (53). In the same way, L-arginine may beneficially impact skin flap survival as well. Exogenous administration of L-arginine to experimental tissue flaps increased the blood flow to the periphery of the flap (312). Despite demonstrating evidence of oxidative tissue damage, the L-arginine-treated flaps had a greater length of survival. In a similar study, L-arginine administration decreased neutrophil infiltration and flap necrosis (313). L-Arginine in combination with iloprost also reduced distal necrosis following reperfusion (314). Other combinations that demonstrated increased survival are L-arginine with TGF-β and L-arginine with growth hormone (315).


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ERECTILE DYSFUNCTION With the emerging body of evidence indicating the involvement of the NO• pathway in multiple aspects of the penile erectile response, the use of L-arginine supplementation is a promising therapy in erectile dysfunction. In animals and humans it appears that erectile function is dependent on intact systems of NO• generation within the cavernosum endothelium, the nonadrenergic, noncholinergic (NANC) nerves terminating within the penis, and centrally within the paraventricular nucleus (PVN) of the hypothalamus, which may be a center of arousal (316). This concept is supported by the finding that the NOS inhibitor, L-nitroarginine methyl ester (L-NAME), ablates erectile response (317). In addition, several risk factors for erectile dysfunction, including age (317), smoking (318), diabetes (319), hypertension, atherosclerosis, and hypogonadism are associated with reduced EDNO production, further supporting its important role in erectile dysfunction. In particular, age has been shown in the rat to be associated with inhibition of penile NOS activity and a reduction in the content of penile NOS isoforms (317). To examine the role of EDNO in the normal penile erection, Kim and colleagues isolated corpus cavernosus strips and mounted these in an organ bath (320). After precontraction with norepinephrine, electrical field stimulation (EFS) was performed. In the presence of L-nitroarginine, relaxation to EFS was attenuated. L-Arginine was capable of reversing the attenuated response to EFS unless the strips were deendothelialized. These results support the involvement of the EDNO in corpus cavernosus relaxation. Local neuronal NO• also plays an important role. Several risk factors for erectile dysfunction have been shown to decrease nitrergic nerve activity. nNOS enzyme content (measured by Western blot) is reduced to 35% of controls in the rat penis following castration/adrenalectomy (321). Along with the fall in nNOS level is a 55% reduction in NOS activity and a significant loss of erectile function following EFS. Similar to the studies in rats, human penile erectile function is mediated by NO• generated in response to NANC neurotransmission (322). Strips of human corpus cavernosus mounted in an organ bath relaxed to EFS. This effect was blocked by NOS inhibitors and methylene blue, an inhibitor of guanylate cyclase. The relaxation was not influenced by atropine, however, suggesting that the source of NO• was not endothelial. Although this study demonstrated the presence of NANC innervation of the human penis, there is no data demonstrating the relative involvement of NANC-derived NO• vs. EDNO in erectile function nor the level at which dysfunction occurs in functional abnormalities in humans. Simonsen et al. found that NANC innervation of small penile arteries (200–700 µm diameter) within the cavernosa is responsible for vasorelaxation (323). These nerve terminals contain NO• as well as another factor, the production of which is resistant to NOS inhibition. Central NO• activity is also important for control of penile erection. Injections of dopamine receptor agonists, oxytocin, and other substances into the lateral ventricles or the PVN caused both erections and yawning (316). These substances all increase NO• production within the PVN. Furthermore, these behaviors were abolished by injections of NOS inhibitors into the lateral ventricles, whereas NO• donors reproduced the behavior. The central role of NO• is cGMP-independent, as it could not be prevented by inhibitors of guanylate cyclase. Interestingly, NOS mRNA expression within the PVN of impotent rats is about half that of sexually potent rats (324). Therapy to restore NOS activity and erectile function in the animal model has involved L-arginine supplementation as well as gene therapy. Oral administration of L-arginine (2.25% in drinking water) for 8 wk to aged rats resulted in an increase in penile tissue levels of L-arginine, an increase in NOS activity and a complete reversal of erectile dysfunction (37). In one small clinical study of 20 impotent men involving a placebo controlled, crossover design, L-arginine in small doses (2.8 g/d for 2 wk) subjectively restored erectile function as

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well as vaginal penetration ability in 6 of 15 patients completing the study (36). This effect was not reported during the placebo phase.

RENAL INSUFFICIENCY Dietary intervention with L-arginine has resulted in improved renal hemodynamics in a number of experimental renovascular diseases, such as those caused by subtotal nephrectomy, diabetic nephropathy, cyclosporin A administration, salt-sensitive hypertension, ureteral obstruction, puromycin amino-nucleoside nephrosis, kidney hypertrophy resulting from high-protein feeding, glomerular thrombosis resulting from administration of lipopolysaccharide, thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, and even loss of function secondary to the normal aging process (67,325,326). With respect to uremia, L-arginine was as effective as captopril at ameliorating glomerular capillary hypertension (327), and normalizing creatinine clearance (328). At the doses of L-arginine required to lower blood pressure, renal blood flow was improved and the associated renal dysfunction was reversed or prevented (166,258,261,265). When renal failure is myoglobin-induced, L-arginine (150 mg/kg/min) can restore renal blood flow and creatine clearance (329). Oral L-arginine (1.6 g/kg/d) has also been shown to decrease preglomerular resistance and restore glomerular hemodynamic response to glycine infusion (NO•-mediated renal vasodilation) (195,330). At least part of the effects of L-arginine on renal blood flow are mediated through sympathetic neuronal mechanisms, as the effect of L-arginine to enhance renal blood flow was abolished in sympathetically denervated rats (331). In the rat model of subtotal nephrectomy, a high-protein diet leads to loss of NO• activity, renal hypertrophy, and glomerulosclerosis. Much of the loss of NO• results from a loss of inducible NOS (iNOS) content (332). When supplemented with L-arginine (1% in drinking water) renal hypertrophy was decreased (333). In the rat model of cyclosporin nephrotoxicity, tubulointerstitial injury, macrophage infiltration, and progressive interstitial fibrosis occurs secondary to accelerated apoptosis and ischemia (334). In a similar model, in addition to the aforementioned findings, arteriopathy and plasma renin levels were significantly less in the treated animals (335). L-Arginine supplementation in this model reduces tubulointerstitial apoptosis and a reduction in interstitial fibrosis. The effect on L-arginine on renal function was greater than that of allopurinol given to reduce the formation of oxygen radicals (336). L-Arginine has been administered to patients with end-stage renal disease during dialysis. These individuals demonstrate an endothelial dysfunction that is likely caused by the accumulation of plasma levels of ADMA (111,337). Hand and colleagues have recently demonstrated that hemodialysis reverses the endothelial dysfunction associated with renal failure (33). Similarly, administration of intravenous L-arginine, but not D-arginine, reversed the vasodilator dysfunction without the need for dialysis. L-Arginine has been used in other settings involving compromised kidney function. L-Arginine has been used to reverse the antinatiuretic effect of cyclosporin in renal transplant patients. In this study, L-arginine infusion (50–150 mg/kg/h) increased renal plasma flow, glomerular filtration rate, naturesis, and kaliureisis (338).

RAYNAUD’S PHENOMENON Raynaud’s phenomenon associated with occlusive arterial disease is due to normal fluctuations in vessel tone superimposed on fixed lesions (339,340). Raynaud’s phenomenon may also be secondary to changes in rheologic properties of blood. Cold agglutinins, cryoproteins, or hyperviscosity due to other disorders of serum proteins may induce intermittent ischemia of the digits in the absence of vasospasm. When Raynaud’s phenomenon occurs in the absence of organic arterial disease, it is thought to be due to digital artery vasospasm. This entity is known as Raynaud’s disease (339,340).


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That at least some cases of Raynaud’s disease may have a mechanism common to that of coronary vasospasm is underscored by the observation that patients with variant angina have a higher incidence of Raynaud’s disease (341). There are differences, however, between coronary and digital arteries in the determinants of vasomotion. Unlike the coronary arteries, α-adrenergic contraction predominates over β-adrenergic relaxation in limb arteries. The limb vessels are richly innervated by sympathetic fibers and are highly responsive to changes in sympathetic tone. Both α1- and α2-adrenergic subtypes are present on the postjunctional smooth muscle and initiate contraction when stimulated (342). Although aberrant sympathetic activity contributes greatly to the pathophysiology of Raynaud’s phenomenon and disease, the actions of platelets are also important. Aggregating platelets contract human digital arteries in vitro by releasing serotonin and thromboxane A2 (343). Each of these vasoconstrictors makes approximately equal contributions to the vasoconstriction induced by platelet aggregation in these vessels. Serotonin may contribute to the vasoconstriction induced by environmental cooling, as the latter is partly reversed with blockade of serotonergic receptors (344). The effect of L-arginine to inhibit platelet aggregation makes it a potential therapy in patients with these maladies. In reality the utility of L-arginine in patients with Raynaud’s phenomenon is mixed. In one study, patients with Raynaud’s phenomenon secondary to systemic sclerosis demonstrated enhanced cutaneous vasodilation (34). In this study, 12 patients were given oral L-arginine (8 g/d) for 1 mo. Following therapy there was an increase in digital vasodilation following warming and an increase in plasma levels of tissue-type plasminogen activator. In a similar study in patients with primary Raynaud’s phenomenon, 8 g/d of oral L-arginine did not restore the abnormal vasodilator response to ACh (35). This study underscores the minor contribution of abnormal EDNO production in the pathophysiology of Raynaud’s. Neither study examined the effect of L-arginine on platelet activity in this disease.

CONCLUSION Most of the known cardiovascular effects of L-arginine are exerted via its conversion to NO• by NOS. There are, however, NO•-independent actions of L-arginine that contribute to its beneficial action on the cardiovascular system. Accumulating evidence indicates that supplemental administration of L-arginine is sufficient to restore EDNO production in many disorders in which EDNO activity is reduced. A greater understanding of the pathophysiology of these disorders has helped to provide hypotheses to explain the paradox of supplemental L-arginine. Regardless of the mechanism, L-arginine may become a useful therapeutic approach to many cardiovascular disorders. The development of methods of practical L-arginine delivery in the quantities required will likely accelerate its acceptance in routine clinical practice.

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Contemporary Cardiology ™ Christopher P. Cannon,

MD,

Series Editor

Nitric Oxide and the Cardiovascular System Joseph Loscalzo,

Edited by MD, PhD and

Joseph A. Vita, MD

Boston University Medical Center, Boston, MA

Interest in nitric oxide biology has greatly intensified over the past decade in concert with our increasing appreciation of its many roles in the cardiovascular system. In Nitric Oxide and the Cardiovascular System, world leaders in the field comprehensively review the chemistry, biochemistry, molecular biology, physiology, and pathophysiology of nitric oxide in cardiovascular health and disease. These experts particularly illuminate nitric oxide biology, its cardiovascular pathophysiology, and its role in cardiovascular therapeutics. Topics also included are the development of nitric oxide donors for the treatment of myocardial ischemia and thrombosis, the development of gene therapeutic restoration of endothelial function in atherosclerosis, and the application of nitric oxide biology to investigative arenas in cardiovascular medicine. With its balanced presentation of basic and clinically relevant information, Nitric Oxide and the Cardiovascular System provides a comprehensive, authoritative guide for all those cardiovascular biologists, cardiologists, and cardiovascular surgeons engaged in today’s clinical or experimental research. Features • Comprehensive review of all aspects of nitric oxide in cardiovascular health and disease • Unique marriage of up-to-date experimental and clinical studies

• Comprehensive, timely, relevant, and thoughtful • Useful to the cardiovascular investigator, as well as the clinical scientist and clinician

Contents Part I. Biology of Nitric Oxide. The Biological Chemistry of Nitric Oxide. Cell and Molecular Biology of Nitric Oxide Synthases. Cellular Signal Transduction and Nitric Oxide. Regulation of Gene Expression by Nitric Oxide. Cytotoxicity, Apoptosis, and Nitric Oxide. Nitric Oxide and Ion Channels. Role of Nitric Oxide in Vasomotor Regulation. Nitric Oxide and Platelet-Mediated Hemostasis. Nitric Oxide Modulates Leukocyte–Endothelial Cell Adhesion. Nitric Oxide and Cardiomyocyte Function. The Fibroblast and Nitric Oxide. Nitric Oxide in Cardiac Electrophysiology. Part II. Cardiovascular Pathophysiology of Nitric Oxide. Nitric Oxide and Endothelial Dysfunction. Nitric Oxide and Hypertension. Nitric Oxide and Pulmonary Hypertension. Nitric Oxide in Atherosclerosis.

Thrombotic Disorders and Nitric Oxide Insufficiency. Myocardial Nitric Oxide in Heart Failure. Shock States and Nitric Oxide. Stroke and Nitric Oxide. The Role of Nitric Oxide in Ischemia-Reperfusion. Part III. Nitric Oxide in Cardiovascular Therapeutics. Nitrovasodilators. S-Nitrosothiols. Diazeniumdiolates (Formerly NONOates) in Cardiovascular Research and Potential Clinical Applications. Inhaled Nitric Oxide Therapy for Acute Respiratory Failure. Antioxidants and EndotheliumDerived Nitric Oxide Action. Coating Arterial and BloodContacting Surfaces with NO•-Donating Compounds. Gene Therapy and Nitric Oxide. Nitric Oxide and Tissue Preservation in Transplantation. L-Arginine: Its Role in Cardiovascular Therapy. Index.

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Contemporary Cardiology™ NITRIC OXIDE AND THE CARDIOVASCULAR SYSTEM ISBN: 0-89603-620-0

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Cardiovascular MRI: 150 Multiple-Choice Questions and Answers (Contemporary Cardiology)

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Preventive Cardiology: Insights Into the Prevention and Treatment of Cardiovascular Disease (Contemporary Cardiology)

Antithrombotic Drug Therapy in Cardiovascular Disease (Contemporary Cardiology)

Platelet Glycoprotein IIbIIIa Inhibitors in Cardiovascular Disease. Contemporary Cardiology

Angiogenesis and Direct Myocardial Revascularization (Contemporary Cardiology)

Platelet Glycoprotein IIb IIIa Inhibitors in Cardiovascular Disease (Contemporary Cardiology)

Platelet Glycoprotein IIb IIIa Inhibitors in Cardiovascular Disease (Contemporary Cardiology)

Cardiology

Cardiac Drug Therapy 7th ed (Contemporary Cardiology)

Management of Acute Coronary Syndromes (Contemporary Cardiology)

Management of Acute Coronary Syndromes. Contemporary Cardiology

Nitric Oxide and the Cardiovascular System (Contemporary Cardiology) (Contemporary Cardiology)