Current Topics in Membranes and Transport, Volume 05

Current Topics in Membranes and Transport Volume 5

Advisory Board

Robert W . Berliner Peter F. Curran I . S. Edelrnan I . M . Glynn Francois Morel Aser Rothstein Philip Siekevitz Torsten Teorell Daniel C. Tosteson Hans H . Ussing

Contributors

Karlheinz Altendorf Winfried Boos W i l l i a m A . Brodsky Emilio Carbone Peter F. Curran Franklin M . Harold Erich Heinz Theodore P. Schilb Stanley G . Schultz Ichiji Tasaki

Current Topics in Membranes and Transport

VOLUME 5

Edited by Felix Bronner Department of Oral Biology University o j Connecticut Health Center Farmingtm, Connecticut and Arnost Kleinzeller

Graduate Dillision of Medicine Universii y of Pennsylvania Philadelphia, Pennsylvania

1974

Academic Press

New York

San Francisco

London

A Subsidiary of Hareourt Brace Jovanovich, Publishers

COPYRIGHT 0 1974, BY ACADEMIC PRESS, [NC. ALL RIGHTS RESERVED. N O PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, w m o u r PERMISSION IN WRlTING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

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United Kingdom Edition published b y ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London N W l

LIBRARYO F

CONGRESS CATALOG CARD

NUMBER:70-1 17091

ISBN 0- 12- 15 3305 -0 PRINTED IN THE UNITED STATES OF AMERICA

List of Contributors, vii Preface, ix Contents of Previous Volumes, xi Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENIIORF I. 11. 111. IV. V. VI.

Introduction, 2 The Ion Balance of Bacterial Cells, 2 Ionophores, 9 Membrane Potential and Proton Transport, 14 Transport of K + and Na+, 26 Cation Transport and Cell Functions, 40 References, 45

Pro and Contra Carrier Proteins; Sugar Transport via the Periplasmic Galactose-Binding Protein WINFRIED BOOS

I. Introduction, 52 11. Properties of the MeGal Transport System, 57 111. Properties of the Galactose-Binding Protein, 95 IV. Evidence for the Essential Funrtion of the GalactoseBinding Protein in the Transport Mechanism of the MeGal Transport System, 116 V. The Involvement of the Galactose-Binding Protein in Chemotaxis, 122 VI. Regulation of the MeGal System and of the GalactoseBinding Protein Synthesis by Events Owurring during the Bacterial Cell Cycle, 123 VII. Pro arid Contra Carrier Funrtion of Periplasmic Binding Proteins, 126 References, 128

Coupling and Energy Transfer in Active Amino Acid Transport ERICH HEINZ

I. Introduction, 137 11. The Quasi-Chemical Notation of Irreversible Thermodynamics, 140 111. The Coupling of Amino Acid Transport to 1011 Flows, 144 References. 158 V

vi

CONTENTS

The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney WILLIAM A. BRODSKY AND THEODORE P. SCHILB Preface, 162 I. General Considerations, 162 TI. Acidification of the Luminal Fluid by the Turtle Bladder, 193 111. The Renal Mechanism of Acidification, 205 References, 223

Sodium and Chloride Transport across Isolated Rabbit Ileum STANLEY G. SCHULTZ AND PETER F. CURRAN

I. Introduction, 226 11. A Working Model of the Small Intestinal Epithelium, 227 111. The Shunt Pathway in Isolated Rabbit Ileum, 231 IV. Transepithelial Transport of Na and CI across in Vivo and in Vitro Preparations of Ileal Mucosa, 239 V. Influxes of Na and C1 across the Brush Border, 244 VI. Solute-Coupled Transport, 262 VII. Ion Transport and the Electrophysiology of Rabbit Ileum, 269 VIII. Concluding Remarks, 276 References, 278 A Macromolecular Approach to Nerve Excitation ICHIJI TASAKI AND EMILIO CARBONE

I. Introduction, 284 11. Abrupt Depolarization, 286 111. Instability and Excitability of the Axon Membrane, 294 IV. Interactions between Membrane Macromolecules and Small Ions, 299 V. Optical Studies of Excitable Membranes, 307 VI. Fluorescence Studies, 313 VII. Summary, 321 References, 322 Subject Index, 327

List of Contributors Numbers in parentheses indicate the pages on whirh the authors’ contributions begin. Division of Resenrch, National Jewish Hospital and Research Center, Ilenver, Colorado, and Ilepartment of Mirrotiiology, Llniversity of Colorado Medical Center, l)enver, Colorado* (1) Winfried Boor, Department of Biological Chemistry, Harvard Medical School, and the Biocheniicnl Research Lalmatory, Massachusetts General Hospital, h s t o n , Massachusetts (51) William A. Brodrky, llepartment of Physiology and Biophysics, Mount Sinai School of Medicine, The City University of New York. Nerv York, New York (161) Emilio Carbone, Laboratory of Neiirobiology, National Institute of Mental Health, Bethesda, Maryland ( 2 8 3 ) Peter F. Curran, Department of Physiology, Yale University School of Medicine, New Haven, Connecticut ( 2 2 5 ) Franklin M. Harold, Division of Research, National Jewish Hospital and Research Center, Ilenver, Colorado, and Ilepartment of Microbiology, University of Colorado Medical Center, I)enver, Colorado (1) Erich Heins, Ilepartment of Physical Biovhemistry, Gustav-Embden-Zentrum d. biol. Cheniie, J. W. Goethe-Universitat, Frankfurt-am-Main, Germany (137) Theodore P. Schilb, Department of Physiology and Biophysics, Mount Sinai School of Medicine, The City University of New York, New York. New York (161) Stanley 0. Schultz, Department of Physiology, Iiniversity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (225) Ichiii Taraki, Laboratory of Neurobiology, National Institute of Mental Health, Bethesda, Maryland (283) Karlheinz Altendorf,

* Present address: Institut fiir Biologie I1 der Universitat Tiibingen, Tubingen, Auf der Morgenstelle, West Germany. vii

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The fifth volume of Current Topics in Membranes and Transport presents chapters dealing with the interaction of solutes and membrane proteins, as exemplified by galactoside transport (Boos), and its energetic aspects in amino acid transport (Heinz). Three chapters treat electrolyte transport, in bacteria (Harold and Altendorf), in renal and bladder cells (Brodsky and Schilb), and in the intestine (Schultz and Curran). The final chapter, by Tasaki and Carbone, reviews the experimental basis for the macromolecular hypothesis of nerve excitation. We believe these reviews conform to our editorial policy of not shunning controversy. We therefore hope they will contribute to our understanding of the molecular basis of biological transport. FELIX BRONNER ARNOST KLEINZELLER

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Contents of Previous Volumes Volume 1

Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEYAND LLOYDBARR The Transport of Sugars acrofjs Isolated Bacterial Membranes H. R. KABACX Galactoside Permeasc of Escherichia coli ADAMKEPES Sulfhydryl Groups in Mrmbranc Structure and Function ASER ROTHSTEIN Molecular Architccturr of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index Volume 2

Thr Molecular Basis of Simple Diff usim within Biological Mrmbranes W. R. LIEBAND W. D. STEIN The Transport of Water in Erythrocytes ROBERT E. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCE A N D M. MONTAL Structure and Biosynthesis of the Mcmbrarle Adriiosine Triphosphatase of Mitochondria TZAGOLOFF ALEXANDER Mitochondria1 Compartmrnts: A Comparison of Two Models HENRYTEDESCHI Author Index-Subject Index xi

Xii

CONTENTS OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWAFZTZ, GEORGEE. LINDENMAYER, AND JULIUS C. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONY MARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W. J. ADELMAN,JR. AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODR~GUEZ DE LORESARNAIZ AND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: In Vitro Studies J. D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia RICHARD M. HAYS Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm WILLIAMR. HARVEY AND KARLZERAHN Author Index-Subject Index Volume 4

The Genetic Control of Membrane Transplant CAROLYN W. SLAYMAN Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMARJAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGAN AND CAROL F. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN Author Index-Subject Index The Editors wish to thank Mark Solomon for his photograph of Aharon Katzir-Katchalsky which appeared in Volume 4.

Cation Transport in Bacteria: K'. Na+. and H' FRANKLIN M . HAROLD and K A R L H E I N Z ALTENDORF* Division of Research. National Jewish Hospital and Research Center. Denver. Colorado. and Department of Microbiology. University of Colorado Medical Center. Denver. Colorado

I . Introduction . . . . . . . . . . . . . . . I1. The Ion Balance of Bacterial Cells . . . . . . . . . A. Cations and Anions . . . . . . . . . . . . B . Physical State of Cytoplasmic Cations . . . . . . C . Selectivity: Contributions of Membrane and Binding Sites . I11. Ionophores . . . . . . . . . . . . . . . . A . Membrane Composition and Ion Permeability . . . . B . Potassium Ionophores: Valinomycin and Monactin . . . C . Proton Conductors . . . . . . . . . . . . D . Nigericin and Monensin . . . . . . . . . . . E . Gramicidins . . . . . . . . . . . . . . IV . Membrane Potential and Proton Transport . . . . . . A. The Chemiosmotic Hypothesis . . . . . . . . . B . Measuring the Membrane Potential . . . . . . . C . Proton Transport in Bacteria . . . . . . . . . V. Transport of K+ and Na+ . . . . . . . . . . . .

VI .

. . . . . . . .

. . . . . . . .

. . . . . .

. . . . . .

.

.

. .

.

.

. . .

. . .

A . Streptococcus faecalis . . . . . . . . . . . . . . B . Escherichia coli . . . . . . . . . . . . . . . C. Other Bacteria . . . . . . . . . . . . . . . . Cation Transport and Cell Functions . . . . . . . . . . A . Alkali Metals m Metabolic Cofactors . . . . . . . . . B. Regulation of the Internal pH . . . . . . . . . . . C . Anion Transport . . . . . . . . . . . . . . . D . Osmotic Adaptation . . . . . . . . . . . . . . E Ion Gradients in Active Transport and Other Energy Transductions References . . . . . . . . . . . . . . . . . .

.

. . . . . . . .

. . . .

. .

. . .

.

2 2 3 5

7 9 9 10 11 12 13 14 15 18 21 26 27 34 40 40 41 42 42 43 44 45

* Present address: Institut fur Biologie I1 der Universitat Tubingen. Tubingen. Auf der Morgenstelle. West Germany

.

1

2

FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF

1. INTRODUCTION

The internal ionic milieu of bacteria generally differs radically from that of the medium. As a rule, K+ is by far the most abundant cytoplasmic cation, even though Na+ may predominate in the environment. Even when this generalization must be qualified, as is the case for halophilic bacteria, it remains true that a high internal concentration of Iclcd galactose, the counts-per-minutc valuc extrapolated to time zero for both the wild type and the mutant EH303.5 is reduced to about 530 cpni (2.75% of the total radioactivity present per filtered aliquot). This valuc is identical nit h that obt,ained in the presence or abscncr of additional unlabeletl galactose n ith anot,lier mutant, EH3027, negative in the MeGal transport system a s well as galactose-binding protein, and represents galact,ose remaining on the filtcr and in thr intcrcellular space. The diffcrencc bctwccn the two numbcrs must therefore rcprcscnt binding of galactose in the pcriplasmic space by the galitctosc4inding protein. In accordance u i t h the propwties of the protein in zlitra, the in U L U O binding of galactose is fast, independent of temperature between 5" and 35", and independent of energy uncouplcrs. From the binding plot of thc purified binding protein in iitro (Boos et nl., 1972) and the number of cclls present per milliliter in the transport assay, it can hc c.stimatec1 that each cell posse about 50,000 to 80,000 galactose-binding protein molecules outside its osmotic barrirr. Figurr 2 also demonstrates the specificity of galactose uptake at 0.5 df initial conccntration as a rricasure for the activity of the MeGal transport systcm; m g l and niglf strains cfin indeed he distinguished by their initial rate of uptake. Thc necessity of using a low galactose concentration in the transport assay, together with the large number of binding sites established by the

62

WINFRIED BOOS

- 5000

-4000

~

6

8 \

-3000

E

3 3h

kl -2000

c,

P 4

- iooo

0

I

I

10

20

TiME ( seconds

3

10 30

I

FIQ.2. The effect of the binding activity of the galactose-binding protein on the transport assay of the MeGal transport, system. Cells prepared for the transport assay were incubated a t 15" for 5 minutes. [1-~4C]galactosewas added a t time 0 to a final concentration of 0.5 p M . Aliquots (0.5 ml) were removed as fast as possible and filtered through Millipore filters (0.65-p pore size) without washing, and the filters were counted in a liquid scintillation counter. The total number of counts per minute per 0.5-ml aliquot was 20,000. The results are given in nanomoles of galactose accumulated per 2.5 X lo8 cells or 80 pg dry weight of cells. +, Wild type W3092cy-; A, strain EH3035 (negative in mgl but possessing a binding-active galactose-binding protein) ; 0 , strain EH3027 (negative in mgl and negative in galactose-binding protein synthesis) ; half-open symbols, transport assay in the additional presence of 30 mM sodium azide; closed symbols, transport assay in the additional presence of 1 mM unlabeled galactose. (Taken from Parnes and BOOS,1973.)

galactose-binding protein in the periplasmic space, make it impossible to study the rate of entry of galactose into the cytoplasm under conditions of energy uncoupling, i.e., equilibration of inside and outside galactose concentration. The amount of galactose simply bound on the surface of the cell by the galactose-binding protein (950 cpm in the above experiment), in addition to the amount of galactose trapped on the Millipore filter and in the intercelIular space (500-600 cpm) , is two orders of magnitude higher than the calculated amount of galactose in the cytoplasm (10 cpm). It is therefore impossible a t the present time to determine directly whether or not the MeGal transport system can in fact exert facilitated

63

PRO A N D C O N T R A CARRIER PROTEINS

diffusion, or a t least facilitated entry, under conditions of energy uncoupling. 2. TEMPERATURE DEPENDENCE OF ENTRY A N D EXIT Figure 3A shows the ability of wild-type cclls (strain W3092cy-) to take up galactose (initial concentration 0.5 p M ) at different temperatures, as well as the resulting decrease in thc outside concentration. The initial rate of uptake is 45 times higher a t 35" than a t 5", while the equilibrium level of accumulation reaches about the same value a t all temperatures. Similar temperature dependence can he shown for the exit process (Fig. 3B). The equilibrium level of accumulation is characterized by the constancy of the ratio of inside and outside concentrations, which is maintained over a period of at least 30 minutes. Under the given conditions of cell density and an initial [1-14C]galactose concentration of 0.5 pM a t 25", the steady-state outside concentration of galactose has a value of 50 nM. Assuming a cell volume of lo-'* ml (Winkler and Wilson, 1966), thr steady-state internal concentration of galactosr can be calculated to be 1 mM. A concentration ratio of 2 X lo4 is therefore maintained by the MeGal transport system under the above-stated condition of steady state of accumulation. [A value of 1 X lo5 has recently been reported by VoFiBek and Kepes (1972) .] The establishment of this considerable concentration gradient characterizes the MeGal transport system as an "active transport system" dependent upon metabolic energy. This can also be demonst,rated by the effrct of energy uncouplers on the transport system. In Figs. 2 and 4, it can be seen that net uptake of galactose is strongly reduced in the presence of sodium azide, an uncoupler of oxidative phosphorylation. It can be shown that the steady state of accumulation is characterized by simultaneous entry and exit of galactose; by adding trace amounts of [3H]galactose of high sprcific radioactivity, which does not significantly alter the equilibrium outside concentration, one can measure the initial rate of uptake of [ 3 H ] g a l a ~ tand ~ ~ ~calculate the rat(. of the steady-state flux. A value of 0.027 pmole per minute per 2.5 X lo8 cells at 15" was calculated, in good agreement with the initial rate of uptake at the same external substrate concentration but in the absence of intcrnal galactose (Parnes and BOOS,1973). 3. TRANSMEVBRANAL EFFECTSFOR EXITBUT DIFFUSION BARRIER

NOT

ENTRY;EXTERNAL

Exit of ['*C]galactose from preloaded cells can be observed by adding unlabeled glucose or galactose to the external niedium to prevent recapture

64

WINFRED BOOS

. . A

A

FIG.3. Entry and exit of galactose a t different temperatures. (A) Cells prepared for the transport assay were incubated for 5 minutes a t the desired temperature. Transport =say was performed as described in legend for Fig. 2. Aliquots of each filtrate were collected to determine the external [1-14C]galactose concentration. A, 5"; 0, 15"; 0, 25"; 0,35"; closed symbols stand far galactose taken up by the cells; open symbols stand for galactose concentration remaining in the external medium. (B) Cells prepared for the transport assay were incubated a t room temperature with [1-Wlgalactose for 5 minutes and then transferred for 2 minutes to a water bath of the desired temperature prior to the addition of 1 mM unlabeled galactose. Aliquots (0.5 ml) were withdrawn and filtered through Millipore filters (0.65-ppore size). Each filtrate (0.2ml) was measured for radioactive galactose in a scintillation counter. The results are given in nanomoles released from 2.5 X lo8 cells. Symbols as in (A).

65

P R O A N D C O N T R A CARRIER P R O T E I N S

A OZor

3 * oO

2

riME ( m i n u t e s )

-

0

0

I0

20

30

40

TIME f seconds)

FIG.4. Entry imd exit of gal:tctose i i i prelowtletl :ind poisotied cells. (A) Cells prepared for (.he 1r:insport. assay were iiiciibated at 23@for .i iiiiniites w i t h ~irilsbeledgal:icatose, 0.5 p M final concentration. T h e cult;iire w:is tlirir tr:irisferred 1 0 15" :tnd iricubuted for 5 ininiites. [l-"C](::tl:tc~ose, 0.5 pcrM firi:tl coric~eiitr:ttic~ii, was added :trid the priicediire continued :ts described i n the legend t o Fig. 2. Trmper:tt lire was 15". A, C'oritrol assay i n the :thserice of iitilaheled galactose; 0, assny :ifter preinciit):itioii with iinlnbeled g:il:tctose; closrd syiirhls, addition of 3 0 niM (fin:tl coriceiitriLtioii) s o d i i i m mide together with [1-'C]g:il:ictose. (€3) Cells prepared for thr lwiisport assay were incwl-)ntcd :it roo111 t~emper:itiire with [1-1T]galarti)se f o r 5 minutes and then tr:tnsferred for 2 miriiitcs I(, 1.;" prior l o t.he addition o f various coinpoiiiids: A, sodiitiii azide (30 m M ) ; 0, iitil:tl,eled galactose ( 1 m&fi; 0 , 30 niM sodiitin :uide together with 1 mM gnluctose; 0, gliicose (0.2 niM); 30 m M sodiiim axide together with 0.2 m M gluccise. Th e :issay w:ts performed as dewrihed in the legend for Fig. 3B. (T:tken f r u m Parries mid Ihtis, 1973.)

+,

of [14C]galactosr. This nicasiirenicnt o f rsit is not ncccssaril> identical with tlhc steady-stat(>flux undcr conditions of cquilibriuni accumulntion, since transnicnibran:il cffccts might occiir. Ind(wl, thc ratw of cxit arc 0.208 and O.O5,3 ninolc galactose p(>rminutc 1wr 2.5 x 1 0 8 cclls :It 15' at a saturating external concentration of glucosc :md g$:~I:tci osc, rcsprctivdy, in comparison to 0.027 pniolc p c b r minutc for t h r cquilibriun~ flux. Thus galactose and, to a groatcr extent, glucohc stimiilatr rxit trltnsmeinbranally. The half-maximal stimulation occurs \iith both sugars at 10 p M . Interestingly cnough, this valuc corresponds to thc scxond K,,,,, of the galactosrbinding protein with galactosr as ligmd (Boos et al., 1972). Thr involvement of the galactose-binding protein in transmcmbranal stimulation of exit is also indicated by thc observation that cxit in a n i u t m t with a structurally defect ivr galactose-binding protein is only slightly stiniulatcd

66

WINFRIED BOOS

by glucose and galactose. At this point, it is not clear whether the transmembranal stimulation of exit is caused by the entry process per se, or merely by binding on the transmembranal site. That transmembranal effects can indeed occur without actual countertransport has been observed in yeast by transmembranal inhibition with internal substrate, the exit of which cannot be observed (Kotyk and RihovA, 1972; Crabeel and Grenson, 1970). From the strong transmembranal stimulation of exit, one would expect that entry of external substrate also could be stimulated by internal substrate. The stimulation of both entry and exit by the transmembranal substrate has been demonstrated for the lactose system (Wong and Wilson, 1970). However, as seen in Fig. 4, preloading the cell with unlabeled galactose does not alter the initial rate of [14C]galactose entry. Therefore transmembranal stimulation of entry does not occur in the MeGal transport system. From the same experiment, it can also be concluded that the system does not show the phenomenon of an external diffusion barrier. This phenomenon has been observed in the lactose transport system of E . coli, and refers to the preferential recapture of substrate leaving the cytoplasmic membrane (Robbie and Wilson, 1969). Such a recapture seems plausible, since one can assume that the substrate concentration in the periplasmic space will be higher than that in the external medium under conditions in which internal substrate leaves the cell. The rate of uptake of labeled external substrate should therefore be reduced when the cell has been preloaded with unlabeled substrate. Considering the in vivo location of the galactose-binding protein in the periplasmic space, one is inclined to think that the MeGal transport system would exhibit the phenomenon of an external diffusion barrier. Indeed, the function of the transport system has been interpreted as a recapture or scavenger system (Wu et al., 1969). However, the lack of dependence of initial rate of uptake with preloading clearly demonstrates that such an external diffusion barrier does not exist for the MeGal transport system (Parnes and BOOS,1973).

4. THE GALACTOSE-BINDING PROTEIN MEDIATESENTRY BUT EXIT OF GALACTOSE

NOT

From the similarity of uptake specificity in vivo and the binding specificity of the galactose-binding protein in vitro, it is clear that the binding protein must be involved in the entry process. To determine whether the exit process is also mediated by the galactose-binding protein, we attempted to measure a K , value for exit of galactose stimulated by glucose. Figure 5 shows the dependence of the rate of exit upon the inside concentration of [14C]galactose. The ratte of exit does not reach saturation even

67

PRO A N D C O N T R A CARRIER PROTEINS

0

I

I

I

I

5

I0

I5

20

INTERNAL GALACTOSE CONCFNTRATION fmM1

FIG.5 . The initial rate of galactose exit a t different internal galactose concentrations. The cell suspension prepared for the transport assay was incubated with different [l-W]galactose concentrations (from 10 mM to 0.1 mM) at 23". After the equilibrium state of accumulation had been reached, the cells were transferred to 15" for 2 minutes and unlabeled glucose, 1 mM final concentration, was added. Aliquots (0.5 ml) were filtered as fast as possible through Millipore filters (0.65-p pore size). Each filtrate (0.2 ml) was counted in a scintillation counter. The rate of exit was calculated by interpolation of the first three time points. The results are given in nanomoles per minute per 2.5 X 108 cells. A, Rate of exit with untreated cells; 0, rate of exit in the additional presence of 30 mM sodium aside. (Taken from Parnes and BOOS,1973.)

a t an internal concentration of 20 mM. Therefore no K , of exit can be measured, and it is very unlikely that the galactose-binding protein (estimated Kdiss values 0.1 and 10 M M ) (Boos et al., 1972) establishes the recognition site for exit on the inside of the cytoplasmic membrane. This conclusion is corroborated by measuring entry and exit in an mgl mutant possessing a structurally defective galactose-binding protein (Boos, 1972). Under the standard transport assay conditions, this mutant exhibits only 6% of the initial rate of uptake in comparison to the wild type, while the rate of exit from preloaded cells is identical with that found in the wild type (Parnes and BOOS,1973). Presently, it cannot be determined whether galactose exit is mediated by an entirely different transport system or by components of the MeGal transport system other than the galactose-binding protein. The former possibility was indeed suggested by earlier studies on galactose transport in E . coli (Horecker et al., 1960) a t a tinic when periplasmic binding proteins had not yet been discovered. Also, the unilateral transmembranal stimu-

68

WINFRIED BOOS

lation of exit indicates that the carrier for exit is different from that for en try. Transmembranal stimulation (exchange diffusion) should be observed for both entry and exit on a carrier catalyzing both reactions (Heina and Geck, 1972). The failure to demonstrate transstimulation of entry by the “exit carrier” would then be explained by too low an external substrate concentration which we routinely use to specifically measure the MeGal transport system. However, there is also evidence for the possibility that galactose exit is mediated by components of the MeGal system: (1) entry and exit are inhibited by p-hydroxymercuribenzoate (PHMB) in a time-dependent reaction for which the half time of inactivation is identical (Parnes and Boos, 1973). This indicates a common component exists for entry and exit, or possibly for the energy coupling of both fluxes; (2) transmembranal stimulation of exit occurs only with substrates of the MeGal system, although not with all of them, and it is dependent on a functional galactosebinding protein. Similar observation of unidirectional transport activity in a substratebinding protein-mediated transport system has been made by Halpcrn et al. (1973a). Unidirectional transport activity has also been reported in yeast (Kotyk and Rihovd, 1972; Crabeel and Grenson, 1970). In this connection it might be worthwhile to refer to an observation made on the asymmetric effect on entry and exit of a variety of solutes in E. coli caused by phage-ghost adsorption (Duckworth and Winkler, 1972). From the asymmetric location of the galactose-binding protein in the periplasmic space of gram-negative bacteria, one is tempted to predict an asymmetric transport activity for all substrate-binding protein-mediated transport systems. To obtain more easily interpreted kinetic data, at least in the galactose system, it will be necessary to construct a mutant in which all transport systems for galactose, with the exception of the MeGal system, have been removed genetically. THE LEVELOF ENTRYA N D NOT EXITOF GALACTOSE; LACKOF COUNTERFLOW IN POISONED CELLS Reports on the lactose transport system show that energy uncouplers stimulate exit rather than inhibit entry of substrate (Winkler and Wilson, 1966; Koch, 1964; Wilson et al., 1972). [This conclusion has recently been questioned by Koch (1971). He found that further energy depletion of poisoned cells by energy consumption via an operating PTS results in a strongly reduced downhill entry of o-nitrophenyl-0-D-galactopyranoside (ONPGal) via the lactose transport system. However, the argument that energy is needed even for downhill transport via the lactose system has to be evaluated critically in view of Roseman’s observation that an operating

5. ENERGY COUPLING OCCURSON

69

PRO A N D C O N T R A CARRIER PROTEINS

PTS inhibits indirectly the activity of other unrelated transport systems (Roseman, 1972b).] The same uncouplers that affect the lactose system interfere severely with the not accumulation of galactose via the MeGal transport system (Figs. 2 and 4).It was therefore of interest to see whether energy coupling affects the same process in a system mediated by a periplasmic binding protein. As discussed earlier, the galactose-binding protein functions in entry but not exit of galactose. The failure to significantly accumulate galactose in the presence of uncouplers could be caused by a strong stimulation of exit via components of the MeGal transport system othcr than the galactose-binding protrin or via a different sugar transport system specific for galactose. In earlier studies the effect of the uncoupler dinitrophenol on galactose entry and exit in E . coli M L had been interpreted in a somewhat contradictory manner. Dinitrophenol was thought to interfere with entry (Horecker et al., 1960) or with exit (Kotman and Guzman, 1961), or, depending on its concentration, with both (Osborn et al., 1961). Recently VofiBek and Kepcs (1972) reported the influence of several energy uncouplers on the net accumulation of galactose in E. coli K12, without distinguishing between effects on entry and c.xit. Our experinients with the MeGal transport system, summarized in Table I1 show that the uncoupler sodium azide strongly inhibits galactose TABLE I1 1 mM [l-"C]GiLi\CTOSE DIFFERENTCONDITIONS FROM WILD TYPE MUTLNT OF MeGal T R ~ N S P OSYSTEM^ RT

I N I T I \L h T E O F E X I T OF P R E L O i D E D IT

13"

UNDER

ANI)

Addi tiori None Galactose 1 mM Glucose 0.2 mM h i d e 30 mkf Galactose 1 mM plus axide 30 mM Glucose 0.2 m M plus axide 30 m M None Sodium axide

I,

Wild type Mutant W3092cyEH3039 (nmoles/min/ (nmoles/min/ 2.5 x 10s 2.5 x 108 cells) cells) 0.027b 0.066 0.172 0.046 0.103 0.184 0.078c

0.011c

0.026 0.041 0.072 0.072 0.069 0.070 0.005~ -

From Parnes and Boos (1973). Rate of entry under conditions of steady-state accumulation. Iiiitial rate of entry of 0.5 p M [l-14C]galactose.

70

WINFRIED BOOS

entry and causes a weak stimulation of galactose exit. Energy coupling must therefore occur on the level of entry. The molecular mechanism of energy uncouplers is, of course, still a matter of speculation. They might dissipate a high-energy intermediate of oxidative phosphorylation (conformational state of the membrane) [for review, see Boyer and Klein, 19721 or collapse a proton gradient, or electrochemical potential, suggested to be the driving force for active transport (Mitchell, 1970, 1972; Greville, 1969; Skulachev, 1971 ; Harold, 1972). Energy uncouplers have different effects on quite different transport systems. For instance, inhibition of entry has been reported for glutamate transport in E. coli (Halpern el al., 1973a), a-aminobutyric acid uptake in yeast (Kotyk and kihovh, 1972) , and for hexose entry in Chlorella vulgaris (Komor et al., 1972), as well as Riaodotorula gracilis (Hofer, 1971), while inhibition of entry and exit of magnesium has been observed in E. coli (Lusk and Kennedy, 1969). On the other hand, energy uncouplers stimulate exit of substrate via the lactose transport system (Winkler and Wilson, 1966; Wilson, T . H., et al., 1972) and stimulate group translocation (entry) via PTS (Roseman, 1969; Winkler, 1971). Energy uncouplers have also been used to demonstrate counterflow in the bacterial active transport system for lactose. The term counterflow refers to the flux of solute against the concentration gradient due to the selective inhibition of a unidirectional flux from the trans side of the membrane. This was first demonstrated in the facilitated diffusion system of glucose transport in red blood cells (Rosenberg and Wilbrandt, 1957). I n the lactose transport system of E. coli, counterflow was demonstrated after rendering the active system into a facilitated diffusion system with energy uncouplers (Winkler and Wilson, 1966; Wilson et al., 1972). Recently, mutants for the lactose system have been isolated, which have acquired the properties of a facilitated diffusion system by mutation (Wong et al., 1970; T. H. Wilson et al., 1970). A similar mutation for alanine uptake has been isolated from Pseudomonas jluorescens (Hechtman and Scriver, 1970) . Also, galactoside transport in Streptococcus lactis can easily be converted from an active transport system to a facilitated exchange system in the absence of an energy source. The latter conditions allow the demonstration of counterflow (Kashket and Wilson, 197213). In the classic bacterial counterflow experiment for the lactose system, poisoned cells were preloaded to high internal substrate (unlabeled) concentrations and then exposed to low external concentrations of labeled substrate. The labeled substrate entered the cell and was prevented from exiting by the high concentration of unlabeled substrate which competed for the exit sites. Therefore it appeared as if labeled substrate “accumulated” temporarily against the concentration gradient. Figure 4 shows the result of a

PRO A N D C O N T R A CARRIER PROTEINS

71

similar experiment performed in the MeGal transport system. In the presence of sodium azide and internal unlabeled galactose, the initial rate of entry of [l-14C]galactose was identical with the rate observed in the control experiment. The lack of a transient accumulation of the labeled galactose shows that counterflow cannot be demonstrated by this technique in the MeGal transport system (Parnes and Boos, 1973). This failure to demonstrate counterflow for the entry process must be the result of the inhibition of the entry process b y energy poisons. C. Energy Coupling of Active Transport in Bacteria

The problem of energy coupling of bacterial transport has recently received considerable attention. One of the most intensively studied systems is that of the lactose transport system in E. coli. Several different proposals have been made in the past for this system. Scarborough et al. (1968) reported that ATP, under conditions in which the cells were made permeable to this compound, can increase the rate of downhill transport of ONPGal, indicating that energy could bc derived aerobically by oxidative phosphorylation or anaerobically by glycolysis. Recently, Schairer and Haddock (1972) studied the same transport system in mutants defective in the membrane-bound Mg2+,Ca2+-stimulated ATPase and showed that not ATP itself but rather an intormediate of oxidative phosphorylation is necessary for energy coupling of 0-galactoside transport. The intermediate can be derived either by oxidation of a suitable substrate via the respiratory chain, or by ATP hydrolysis via the membrane-bound ATPase. Studying amino acid transport in mutants defective in electron transport and the Mg2+,Ca2+-stimulated ATPase, Simoni and Shallenberger ( 1972) came to the same conclusion. Klein and Boyer's (1972) inhibitor studies of sugar and amino acid transport in E . coli also indicated that either phosphate-bond or oxidative energy could be used to drive active transport. Pavlasova and Harold (1969) found that uncouplers of oxidative phosphorylation inhibited TMG accumulation under anaerobic conditions, while the steady-state level of ATP remained constant. This observation led them to suggest that the energy source for the lactose system might be linked to a proton or ion gradient across the cell membrane, in accordance with the chemiosmotic hypothesis of energy coupling originally proposed by Mitchell (1970, 1972). '

1. THECHEMIOSMOTIC THEORY OF ENERGY COUPLING TO ACTIVE

TRANSPORT The chemiosmotic theory w a proposed ~ by Mitchell and developed as a n explanation of oxidative phosphorylation in mitochondria. It is outside

72

WINFRED BOOS

the scope of this article to discuss in detail the different arguments for and against the chemiosmotic hypothesis, and the reader must be referred to recent comprehrnsive revicws (Mitchell, 1970, 1972; Greville, 1969; Skulachev, 1971; Harold, 1972). Briefly, in analogy to the situation in mitochondria, proton gradicnts across the bacterial membrane are thought to serve as energy coupler for various activities: ATP synthesis in oxidative phosphorylation, transhydrogenation, reversed electron flow, and transport of solute against the concentration gradient. The necessary p H gradient can be established in two ways: (1) The respiratory chain is thought to be arranged across the membrane, and oxidation of substrate causes rxtrusion of protons and results essentially in the electrophorrtic separation of H+ and OH- across the membrane (alkaline insidc) . (2) Hydrolysis of ATP by the membrane-hound Ca2+-,Mg2+-stimulated ATPase also results in the extrusion of protons, leaving the cytoplasm alkaline. Thus respiration and glycolysis funnel in the same form of energy. Extrusion of protons results in both the chemical potential of the pH gradient, as well as the formation of an electrical potential, inside negative. Thus active transport of solute is thought to be coupled to the sum of both effects, the proton motive force: A P = A 9 zApH (A*, electrical potential; zApH, chemical potential expressed in electrical dimensions equal to

+

- ( R T / F ) (In CH+outsrdel/CH+,nslde)) Transport of solutes against their concentration gradients should then occur as an H+ symport or OH- antiport to make use of either the chemical potential of the pH gradient or the electrical potential, while uptake of cations could be driven directly by the membrane potential. Moreovcr, by postulating thc additional presence of transmembranal Na+/H+ exchange reactions (Harold and Papineau, 1972) , transport of solute could be linked to Na+ gradients, and solute “carriers” would play the role of Na+ symporters. The dependence of solute transport on ion gradient, particularly Na+ symport and K+ antiport, has been developed quite profoundly in studies of the accumulation of amino acids and other nutrients in animal cells (Schultz and Curran, 1970). There the necessary gradients of Na+ and K+ are maintained by the membrane-bound Na+, K+-dependent ATPase (Skou, 1971, 1973). One of the strong arguments for the unique role of proton gradients as energy-conserving mechanisms is the effect of energy uncouplers which are thought to function as lipid-soluble proton conductors (Mitchell, 1970; Harold, 1972). Indeed, it was shown that two compartments separated by an artificial bilayer and of different pH increase their transmembranal conductivity after the addition of uncoupler dinitrophenol (DNP), carbonylcyanide p-trifluoromethoxyphenylhydrazone (CF-CCP) , or

PRO A N D CONTRA CARRIER PROTEINS

73

carbonylcyanide m-chlorophenylhydrazone ( CCCP) under the simultaneous development of a transrnembranal potential (Hopfer et al., 1968). Also, one would predict the transport-inhibiting action of proton- conducting uncouplers is pH dependent, :t phenomenon t,hat indeed has been observed for the uptake of amino acids in yeast (Hunter and Segel, 1973). For opposing views on the mechanism of energy uncoupler, see (Wilson et al., 1971; Ting et nl., 1970; Hanstcin and Halefi; 1974). The exprrimcntal approach to prove the unifying schcmc of cncrgy coupling by a proton motive force cannot be discussed hcrc in dctail, but should be mentioned briefly. a. I o n Dependence of Solute Uptake. Several transport systems h a w been shown to be dependent on ions (Scarborough et al., 1968; Stock and Roseman, 1971; Thompson and MacLrod, 1971; Niven et al., 1973; Gale and Llew-ellin, 1971; Hslpern et a / . , 1973b; Frank and Hopkins, 1969; Shiijo and Miyajima, 1972; Willecke et al., 1973; Eagon and Wilkerson, 1972). I n the case of TMG uptak(1 via the rnelibiose transport system, evrn cotransport with Naf has been shown (Stock and Roseman, 1971). b, T h e Demonstration of p H Gradients (Interior A l k a l i n e ) and Membrane Potentials (Interior Negative). Extensive studies have been made with S. faecalis (Harold et al., 1970a,b; Harold and Papineau, 1972; Harold, 1972). This organism was found to lack oxidative phosphorylation, and active transport depends entirrly on glycolysis. Glycolyzing cells crrate a pH gradient (as measured by the distribution of the lipid-soluble weak acid dimethyloxazolidinedione) , which is abolished by energy uncouplers thought to act as proton conductors, as well as by dicyclohexylcarbodiimide (DCCD) inhibition of the membrane-bound ATPase (Harold et al., 1970a). Kf uptake in glycolyzing crlls occurs in response to an electrical potential interior nrgative created by the extrusion of protons as \wll as Na+ ions (Harold e.! al., 1970b). The formation of an electrical potential in glycolyzing cells of S, faecalis has been detnonstratcd by uptake of the lipid-soluble cation diniethyldibcnzvlaminonium (Harold and Papineau, 1972). In contrast, the demonstration of an electrical potential (interior negative) in respiring E. coli membranc vesicles is still debated (Lonibardi et al., 1973; Hirata et al., 1973). Proton extrusion from respiring bacteria has also been demonstrated for Micrococcus denitrijicans (Scholes and Mitchell, 1970), for E. coli (West and Mitchell, 1972), and in tnembrane vesicles of E. coli (Reeves, 1971). c. Cotransport of Protons with Solute Uptake. Studies by West, Mitchell, and Wilson have established that transport of TMG, and lactose via the E. coli lactose transport system, occurs with the concomitant translocation of protons with a 1: 1 stoichiometry (West, 1970; West and Mitchell, 1972, 1973; West and Wilson, 1973). Moreover, it has been shown in the

74

WINFRIED BOOS

anaerobe S. lactis not only that TMG uptake is concomitant with uptake of protons, but also that acidification of the medium can temporarily drive TMG uptake (Kashket and Wilson, 1973). Cotransport of protons with the accumulation of glutamate and aspartate has been observed in Staphylococcus aureus (Gale and Llewellin, 1971), as well as in amino acid uptake in yeast (Eddy and Nowacki, 1971). d . The Electrical Potential as Driving Force. I n S. lactis (Kashket and Wilson, 1972a, 1973), as well as in membrane vesicles of E. coli (Hirata et al., 1973), it has been demonstrated that the generation of a diffusion potential, interior negative, is able to drive solute uptake against the concentration gradient. 2. ENERGY COUPLING TO RESPIRATION Based on comprehensive studies on the lactose transport system, as well as on transport of amino acids, sugars, and Rb+, in membrane vesicles in E. coli, Kaback and his collaborators developed a model that intimately involves the respiratory chain in energy coupling of solute transport against the concentration gradient [for recent reviews see (Kaback, 1972; Hong and Kaback, 1973)l. According to this model, the carrier proteins are pictured as intermediates of the respiratory chain participating in the redox reactions through the reversible oxidation and reduction of certain S-H groups. Because of the reversible oxidoreduction, the carrier proteins exist in an equilibrium of two conformational states exhibiting different affinities for the transported solute. In the oxidized state the carrier has a high-affinity site for the solute, which is accessible on the outside of the cytoplasmic membrane. With the conformational change in the carrier due to reduction of S-S to S-H groups, the affinity of the carrier for its ligand is markedly reduced and the solute is released into the interior phase of the membrane. The reduced form of the carrier can also “vibrate” and catalyze a low-affinity, carrier-mediated, non-energy-dependent transport of ligand across the membrane. Although the primary electron donor for respiratory chain-linked transport in E. coli is D-lactate dehydrogenase (Kaback and Milner, 1970; Barnes and Kaback, 1971 ; Kaback and Barnes, 1971; Kerwar et al., 1972; Reeves et al., 1972; Lombardi and Kabaek, 1972; Walsh et al., 1972; Dietz, 1972) , a-glycerophosphate dehydrogenase can serve after induction (Barnes and Kaback, 1971; Heppel et al., 1972). In mutants lacking &lactate dehydrogenase, succinic dehydrogenase can replace the function of the defective enzyme (Hong and Kaback, 1972). D-Lactate dehydrogenase is also the primary electron donor for proline

75

PRO A N D CONTRA CARRIER PROTElNS

uptake in Mycobacteraurn phlei (Hirata et al., 1971). The primary electron donor is a-glycerophosphate dehydrogcnasc in S. aureus (Short et al., 1972a,b), NADH drhydrogenase in Bacallus subtzllis (Konings and Frcesc, 1972) and Bacillus licheniformzs (MacLcod et al., 1973), and malate dehydrogrnase in Azotobacter vinelandrz (Barnes, 1972) and Pseudomonas aerugznosa (Stinnet et al., 1973). Howwcr, the carrier is not couplcd in an obligatory way to any dehydrogenase, since artificial electron donors such as ascorbatr-PMS (Konings et al., 1971) can directly transfer electrons to reduce the carrier (Barnes, 1973) evvn under conditions in which lactate dehydrogcnase (E. colz) has becn inactivated with 2-hydroxy-3-butynoatc (Walsh et al., 1972). Moreover, the isolation of etc mutants (Hong and Kaback, 1972), ithich arc able to oxidize D-laCtatc normally but arc' unable to couple oxidative energy to thc accumulation of solute, has led to modification of the original model so that thc transport, carriers are now considered to represent shunts off the main rcspiratory chain rather than obligatory intcrniediates of respiration. Itccent experiments using the fluorescent galactosidc 2-(N-dans~I)aniinocth~I-~-r~-thiogalactosidc (a con+ petitive inhibitor of lactose transport hut not a substrate) have shown that l c ~ in binding of the. galactoenrrgization of E. cola nicmbrane v ~ ~ s i crcwilts side concomitant with its t ransfrr in a hydrophobic mvironmc~nt (Rccvcs et a1 , 1973a). Several points that have been prescntcd to prove or disprove the respiratory modcl of energy coupling arc worthy of discussion : 1. The ability of respiratory chain substratw to stirnulate transport is not parallclcd by their ability to bc oxidized. Moreovcr, inhibitors of n-lactate dehydrogenase (oxamate, 2-hydroxy-i,-but,ynoate) sprcifically inhibit transport in E. coli without affccting the oxidation of other substratm. Thcse and other observations have heen used (Kaback, 1972; Hong and Iiaback, 1973) to negate the possibility of a pH gradient as the transport driving force, sinoc this force should bc rcflcctcd in magnitude by the ratc of oxidation of the particiilar substrate used to generat,e it. This argumcn t is not necessarily valid. l<xpcriments to mcasurc dircctly proton extrusion upon oxidation of substrate have becn done only with n-lactate and NADII as substrates (Rcevcs, 1971). It, was obswvrd that ]>-lactate oxidation results indeed in acidification of the mrdium. The results with NADH w r r obscured hy alkttlinixation of tht. medium, probably rcsulting from the uptake of protons during thr oxidation of NADH on the outside of the mcxnbranc (NADH H+ $02 ---t NAD+ H&). Such an oxidation could not contribute to formation of the pH gradient that would be rcquird for transport, i.e., alkaline inside rclative

+

+

+

76

WINFRIED BOOS

to the medium. Thus not the rate of oxidation of substrate but the ability to form a pH gradient (or extrude protons) should be compared with the ability of a particular respiratory substrate to stimulate transport. 2. The D-lactate-dependent uptake of K+ in the presence of valinomycin is strongly reduced in mutants defective in K+ retention (Bhattacharyya et al., 1971). This cannot be explained by the Mitchell hypothesis in which simple diffusion of the valinomycin-K+ complex should occur with concomitant distribution of K+ according to the electrochemical potential. 3. The most severe criticism of a direct coupling of respiration to transport comes from the observation that uncouplers of oxidative phosphorylation such as DNP, CCCP, and others, severely inhibit the accumulation of solute but do not inhibit lactate oxidation via the respiratory chain. Nonspecific interference of these inhibitors with membrane structure or induction of nonspecific membrane leaks is highly unlikely. These uncouplers have been used extensively in the kinetic analysis of the lactose and other transport systems, and have been demonstrated not to affect transport significantly per se or to cause an increase in nonspecific membrane leakage (Wilson et al., 1972). 4. The argument of the physiological significance of D-lactate as a specific energy source for transport has been raised (Simoni and Shallenberger, 1972). It is clear that cells of E . coli grown under anaerobic conditions do actively transport lactose and other solutes (Pavlasova and Harold, 1969). If such transport is energized by means of a link to the respiratory chain, then one must make the additional assumption of electron acceptors other than oxygen. Indeed, it has recently been shown that transport of lactose in anaerobic membrane vesicles can be driven by either a-glycerophosphate plus fumarate, or formate plus nitrate, after appropriate induction under anaerobic growth conditions (Konings and Kaback, 1973). I n the framework of this article it is not possible to discuss and evaluate in dctail all the controversial arguments concerning the respiratory model and the chemiosmotic hypothesis (for reviews, see Kaback, 1972; Boyer and Klein, 1972; Harold, 1972). However, it might be fruitful to refer to a similar development in the study of energy coupling to active transport in cells of higher organisms. It is generally agreed that Na+, K+ ATPase (Skou, 1971, 1973) maintains inward K+ and outward Naf transmembranal gradients. Accumulation of solute is then explained by a cotransport of Na+ or antitransport of K+ or both (for review, see Schultz and Curran, 1970). However, the sole dependence of active transport on the respective potential gradients has recently been questioned by several observations. Ehrlich ascites cells poisoned with cyanide retained only 30% of their

PRO A N D C O N T R A CARRIER PROTEINS

77

glycino accumulation, w e n though thc cblcct,rolyte gradients \vcrc normal (Eddy, 1968) ; rcvcJrsal of thr Na+ or I 10-2

19.6 x 10-4 74.2 x 10-4

> 10-2 > 10-2

> 10-2 > 10-2 ~~

2 . 8 x 10-4 5.5 X 4 . 4 X lo-'

x 10-4 > 10-2

11.0

> 10-2 > 10-2

~

Taken from Brown and Hogg (1972).

contains an arabinose-binding protein, distinguishable from the galactosebinding protein (Schleif, 1969). It is not entirely clear whether or not the arabinose-binding proteins from B/r and K12 are identical with respect to their specificity, regulation, and mode of action, even though a high similarity of both proteins is rather likely. The binding affinity for L-arabinose and galactose seems to be higher in the K12 protein (Schleif, 1969; Hogg and Englesberg, 1969).] The system is induced by arabinose, galactose, and fucose (R. G. Parson and R. W. Hogg, unpublished data), and is genetically under the control of the araC gene of the arabinose operon, even though the inducer specificity is not identical with that of the arabinose operon. (Galactose is not an inducer, and fucose normally acts as an antiinducer for the enzymes of the arabinose operon). Moreover, the gene locus of the arabinose-binding protein is close to that of the galactose-binding protein (R. G. Parson and R. W. Hogg, unpublished data). The araE gene earlier described as the gene locus for the arabinose permease is not related to the arabinose-bindipg protcin-mediated system (Hogg and Englesbrrg, 1969) and controls a differrnt transport system for arabinose (Brown and Hogg, 1972), probably identical with the system still operating in isolated membrane vesicles of E. coli [Kerwar and Kaback, unpublished observation as referred to in Kaback (1972)]. Structural analysis of the arabinose-binding protein by circular dichroism (CD) and optical rotary

PRO A N D CONTRA CARRIER PROTEINS

93

dispersion (ORD) spectroscopy is similar to that obtained for the galactosebinding protein. This is also true of fluorescence measurements. The close relationship of both proteins is even c.xpressed in the similarities of the fingerprints of the tryptic peptides of both proteins and their weak crossreactivity (R. G. Parson and R. W. Hoggj. [Using goat antiserum against purified galactose-binding protein from E . coli K12, we could not observe cross-reactivity with arabinose-binding protein from E . coli B/r, yet there was full cross-reactivi ty against the galactose-binding protein from B/r. Also, rabbit antiserum against B/r galactose-binding protein (kindly supplied by Dr. Hogg) cross-reacted in our experiments only with the galactose-binding proteins from both strains hut, in contradiction to the observation by Parson and Hogg, not against arabinose-binding protein. Antiserum against arabinose-binding protein (kindly provided by Dr. Hogg) cross-reacted with all three binding proteins, in confirmation of results reported by Parson and Hogg.] Despite these similar properties of the arabinose- and galactose-binding protein-mediated systems, both can easily be distinguished by their affinity for glucose and by their mode of regulation, as well as by mutants defective in either system. The MeGal system recognizes glucose as substrate, while the arabinose system does not. The MeGal system is under the control of the mglR gene (Lengeler et al., 1971), while the arabinose system is under the control of the araC gene even though the inducer specificities of the two systems are similar (Brown and Hogg, 1972; R. G. Parson and R. W. Hogg). The arabinose system needs generally higher inducer concentrations. This is probably the reason for the observation that ga2K strains endogenously induced for the MeGal system by internal galactose are not induced for the arabinose system. In addition, mgl mutants that lack the galactose-binding protein are still normal in the arabinosebinding protein-mediated system. The relevance of the close similarity, and under certain conditions the simultaneous occurrence, of the MeGal and the high-affinity arabinose transport system is a t present only subject to speculation. F. Requirement for Unsaturated lipids of the MeGal Transport System

A variety of membrane-associated enzymic activities has been shown to be essentially dependent on the presence of phospholipids (Rothfield and Romeo, 1971; Cronan and Vagelos, 1972; Machtiger and Fox, 1973). It is therefore obvious that a typical membrane-associated activity such as transport must in some way be influenced by the composition of the membrane’s phospholipids. The kinetic paranietws of several bacterial transport

94

WINFRIED BOOS

systems (particularly the Arrhenius plot of solute uptake) have been shown to be dependent on the unsaturated fatty acid composition of the membrane phospholipids (G. Wilson et al., 1970; Overath et al., 1970; Holden and Bunch, 1972; Fox, 1972). Similar dependence of transport kinetics on the fatty acid composition of the lipids (ratio of cis to trans unsaturated fatty acids) has been observed in transport systems mediated by periplasmic binding proteins (Rosen and Hackette, 1972). In contrast to these reports, which seem to indicate that alterations in the phospholipid composition affect different transport systems in a similar way, Holden et al. (1973) showed recently that mutants that either overproduce fatty acids or excrete phospholipids display heterogeneous changes in the transport rates of different systems. For the MeGal transport system it has been shown that inhibition of unsaturated fatty acid biosynthesis by 3-decynoyl-N-acetylcysteamine affects transport activity without interfering with galactose-binding protein synthesis (Robbins and Rotman, 1972). Thus the assembly of the functional transport system seems to be inhibited upon cessation of unsaturated fitty acid biosynthesis. The latter conclusion has also been made regarding the lactose transport system by Fox et al. (Fox, 1969; Hsu and Fox, 1970), while Overath and his colleagues (1971) and Nunn and Cronan (1974) have demonstrated that phospholipid biosynthesis is not required for the functional assembly of the M protein. The same observation has been made for the lactose transport system of S. aureus (Mindich, 1971) and the citrate transport system in B. subtilis (Willecke and Mindich, 1971). However, the conclusion that the assembly of newly synthesized transport components can occur in the absence of phospholipid biosynthesis seems to be contradicted by a recent report that DNA, RNA, and protein biosynthesis are strictly dependent on phospholipid biosynthesis (Glaser et al., 1973). 111. PROPERTIES OF THE GALACTOSE-BINDING PROTEIN A. Location in the Cell Envelope of €. coli

The galactose-binding protein is a so-called periplasmic protein because, as the name suggests, it seems to be located in the periplasm (Mitchell, 1961), between the cell wall and the cytoplasmic membrane of gramnegative bacteria. The name of this location is purely operational, and its existence is indicated by several findings : 1. A mild cold osmotic shock after plasmolysis in hypertonic sucrosetris-EDTA solution a t room temperature releases the periplasmic pro-

P R O A N D C O N T R A CARRIER PROTEINS

95

tcins. The same group of proteins is rcmoved from the cell surfacc during sphcroplast formation by EDTA-lysozjmic (with thr cxception of ribonuclcasc) . The osmotic shock trcatnwnt shows indred an astonishing discrimination between pcriplasmic and cytoplasmic proteins. Thus 5'nuclcotidase, cyclic phosphodiestcrasc, and acid phosphatase are released to thc cxtrnt of 98, 105, and 767,, rcspectivcly, while only 0.8% of the cytoplasmic p-galactosidase is rrlcascd (Nru and Hcppcl, 1965). The extrnt of this rclease of thc prriplasmic proteins is somewhat dq)endrnt on the lipid composition of the ccll cnvelopc (Rosen and Hackrttr, 1972). Also, different pcriplasmic protcins arc rclrased by thr osmotic shock proccdure to a varying degrrrh whcn thc shock conditions arc changecl (Nossal and Hcppcl, 1966). Similarly, mutants have bcen isolated that appear to lrak pcriplasmic proteins into the growth medium, yct not all periplasmic proteins are lost to the sanic cxtcnt (Lopes ct al., 1972; Heppcl et al., 1972). Thesc mutants seem to I)c diffcrent from strains that have recently bcen found to excrrtc cytoplasmic p-galactosidase (Olden and Wilson, 1972). 2. Alkaline phosphatasc and other prriplasmic proteins can be assayed in uizm without penetration of thc substrate through thc cytoplasmic membrane (Brockman and Hcppcl, 1968). Similarly, the galactose-binding protein can be measured in UZ'Wby binding of galactJoseto cells trratcd with PHMB or uncouplcrs of oxidativr phosphorylation, undcr conditions in which translocation through thc cytoplasmic membrane docs not occur (Fig. 2). 3. The application of a dye, diazo-7-amino-l , 3-naphthalene disulfonate, which is not able to penetratc the cytoplasmic membrane, rracts with and inactivates a pcriplasmic sulfat,c-binding protcin but not the cytoplasmic B-galactosidase (Pardee and Wstanabe, 1968) . Thcsc and othcr rxperinicnls (as rcvicwxl, by Hcppcl, 1969, 1971; Costerou ~t al., 1974) arguc that pcriplasmic proteins arc indeed locatcd outside the cytoplasmic mcmbrsne. Anothrr question of considerable importance with rcgard to the mechanism of transport, but even morc important with respcct to chcmotaxis, is thr location of thr galactosc-binding protcin molcculcs within tlic cell cnvelopc of the sensing bacteria. Are thcy uniformly distributed ovcr the surface of the cntirr ccll? Or arrx thcy niorc concrntratcd a t oppositc cnds of the rod-shaprd ccll, i.c., in the polar caps? Or are they w e n concentrated preferentially a t one cnd of thr cell? Wctzel et a2. (1970) demonstrated by reaction product staining of alkaline phosphatase that the polar caps of the cells exhibit high density in the electron microscope, indicating a preferential concentration of alkaline phosphatasc. This suggests by extcn-

96

WINFRIED BOOS

sion that other periplasmic proteins may also concentrate at the opposite ends of the cell. However, this line of argument has been challenged by MacAllister et al. (1972). By using reaction product staining, as well as ferritin-coupled antibody precipitation, these investigators concluded that an even distribution of alkaline phosphatase along the cell envelope is a more likely picture. Recently, we demonstrated that the synthesis or in vivo assembly of the galactose-binding protein occurs only immediately after cell division and not during elongation of the cell. Furthermore, this synthesis does not occur a t the nonpermissive temperature in a temperature-sensitive cell division mutant. These observations suggest that synthesis of the galactose. binding protein is closely associated with septum formation (Shen and Boos, 1973). Possibly, this synthesis occurs in a compartment which becomes extrarnembranal after the septum has formed. Depending on the mobility of the protein in the periplasmic space, the cell would obtain, for a certain length of time, an asymmetric distribution of galactose-binding protein molecules at its respective ends. Furthermore, if the periplasmic proteins are more or less immobilized at their location of assembly, one should find the galactose-binding protein preferentially on both polar caps of the cell, and not uniformly distributed over the entire cell envelope. [A review on the topology of membrane growth was published by Kepes and Autissier (1972) .] B. Amino Acid Composition and Stability

The amino acid composition of the galactose-binding protein, as reported by Anraku (1968), comprises 53% nonpolar amino acids, and this does not seem to be abnormally high (an extremely lipophylic enzyme, the membrane-bound phosphokinase of S. aweus has 60.4%), but is rather comparable with typical membrane proteins which range from 47 to 55% nonpolar amino acid residues (Guidotti, 1972). Since typical soluble enzymes also show amino acid compositions of a similarly high percentage of nonpolar amino acids, it is not possible to demonstrate any preferential affinity of the galactose-binding protein for the cytoplasmic membrane of E. coli. Cysteine or cystine appears to be absent in the galactose-binding protein (McGowan, unpublished observation), even though its presence has been reported in arabinose- (R. G. Parson and R. W. Hogg), leucine- (Anraku, 1968), ribose- (Aksamit and Koshland, 1972), and cystine- (Berger and Heppel, 1972) binding proteins. The presence or absence of cysteine in binding proteins is of interest, since s-H groups may play a crucial role in the energy coupling of active transport. The function of S-H groups in the

PRO A N D CONTRA CARRIER PROTEINS

97

energy coupling of transport has recently been proposed for a variety of membrane-bound sugar and amino acid “carriers” in E . coli membrane vesicles (Kaback, 1972). Indcrd, membrane-bound binding proteins sohbilized by the nonionic detergent Brij 36-T have been shown to be sensitive to sulfhydryl reagents such as N-cthylmaleirnide and PHMB (Gordon et al., 1972). I n contrast, the binding activity of the soluble pcriplasmic binding proteins is not inhibited in uztro by these reagents, even though they strongly interfere with the in vioo transport activity of thc respective transport systems [for the galactosc-binding protein (Anraku, 1968; Parnes and BOOS,1973; Vofigek and Kepes, 1972)l. A general phenomenon observed with pcriplasmic binding proteins, including the galactose-binding protein, is considerable heat stability. Incubation for 10 minutes at 80” reduced the binding activity for galactose by only 10% (Anraku, 1968). Possibly connected with this phenomenon is the high percentage of saturation of airimoniuiri sulfate ( > 607,) required to precipitate the galactose-binding protcin, as well as other pcriplasmic binding proteins. The galactose-binding protein is quit(. insensitive to changes in pH. From pH 5 to 9 the binding activity changes less than 15%, (Anraku, 1968). This insensitivity to pH changes can also be seen by ultraviolet difference spectroscopy in the presence of substrate. Retween pH 5.3 and 9.3 the spectrum does not change. The reduction in thc difference spectrum that occurs below pH 5 seems to be due to protein denaturation. Howcver, a t a pH higher than 9.5 one can observe a qualitative change in the difference spectrum prior to denaturation. This sccnis to suggest that a t a pH near 10 substrate binding still occurs, but it occurs in a different manner (McGowan et al., 1974).

C.

Molecular Weight

The recent discovery that tho galactose-binding protein can be separated into two forms (Boos and Gordon, 1971) made a thorough study of thc molecular weight imperative. Early experimrrits by Anraku ( 1968) using sedimentation velocitjy in an analytical ultracentrifuge gave a molecular weight of 35,000, assuming a partial specific volume of 0.73 of the protein. Experiments in our laboratory with an analytical ultracentrifuge using sedimentation equilibrium and molecular sieve chromatography through Biogel P-150 gave molecular weights of 34,000 and 36,000, respectively, for the native protein (Boos et al., 1972). These values did not change upon addition of galactose. A single band corresponding t o a molecular weight of 36,500 was obtained by analytical polyacrylamide gel electro-

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WINFRED BOOS

phoresis in the presence of sodium dodecyl sulfate a method that gives the molecular weight of the denatured polypeptide chain (Weber and Osborn, 1969). D. Structural Features Little is known about the actual structure and conformation of the protein. The ORD and CD spectra, which are frequently used to interpret protein conformation (Beychok, 1968) are shown in Fig. 9. The salient features are: (1) an ORD trough near 230 nm and a crossover below 220 nm, and (2) a CD band a t 219 nm, a shoulder near 208 nm, and a crossover a t 203 nm. These spectral properties are indicative of a rather unusually high content of p structure. Since no typical a-helix band can be observed, the content of a helix is predicted to be less than 10%. The high content of p structure can also be demonstrated by infrared spectroscopy (Boos et al., 1972), which shows a strong band a t 1635 cm-l which has been attributed to p conformation (Susi et al., 1967). Similar features have

FIG.9. ORD and CD of the galactose-binding protein. The spectra were measured a t room temperature with a Cary 60 spectropolarimeter with a CD attachment. (A) CD M spectrum a t a protein concentration of 0.55 mg/ml in the presence and absence of galactose (the two curves are superimposible). Path length of the cell was 1 mm. (B) ORD a t a protein concentration of 0.4 mg/ml in 0.01 M tris-HC1 (pH 7.3)in the presence and absence of 10-4 M galactose (the two curves are superimposible). Path length wm 5 mm. Mean residue weight of the galactose-binding protein was calculated according to the amino acid composition to be 126. (Taken from Boos et al., 1972.)

PRO A N D C O N T R A CARRIER PROTEINS

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been found for the Ieucine- (Penrose et al., 1970) and the sulfate-binding proteins. I n the latter case crystallographic data indicate a highly ellipsoid shape with an axial ratio of 4 : I (Langridge et al., 1970). E. Measurement of Activity

The galactose-binding protein has been implicated in two cellular functions: galactose transport and cheniotaxis toward galactose. Neither phenomenon can be measured in an i n iiitro system using the isolated and purified protein. Thus measurements of activity arc restricted either t o assays using the binding affinity of the protein tonard galactose, or to assays of the intact structurc! of the protcin. Three differcnt kinds of assays are commonly in use: (1) the direct binding assay using radioactively labeled ligand; (2) measurements of spectral changes that occur in response to binding of ligand; ( 3 ) mrasurements based on the immunological properties of the protein. 1. BINDINGASSAY

a. Equilibrium Dialysis. T h r classic test for binding activity of any macromolecule is equilibrium dialysis. Prcscntly, apparatus is available that contains 24 double chambers of a minimal volume of 100 pliters to be operated under temperature control. Since the plastic material of the dialysis chambcrs adsorbs protein unspccifically, thc binding protc’in ‘ concentration has to be sufficiently high ( > 0 . 2 mg/ml), or a nonbinding active protein has to be added in cxccss to saturate the unspecific “binding sites” of the plastic material. In a typical exprriment using 0.4 mg per milliliter galactose-binding protein ( 11 p M , based on a molecular weight of 36,000), onr obtains cxcellent values by using 10 nM to 1 pM initial free galactose concentration. In this range the counts in thc cnzymc chainbrr are about 10 times thv amount found in thc ligand chamber. At ligand concentrations higher than 1 p M (initial value), this ratio decreascs and reaches 2 a t 10 p M free ligand concentration. Measurements at ligand concentrations higher than 10 therrforc rrflect increasing error due to the high background. A typical result of the above-mentioned experiment, plotted according to Lineweaver-Burk and to Scatchard (1949) are shown in Figs. 10 and 11, and are discussed in Scction 111, F. b. UltraJiltration. This binding assay is throretically frec of the limitations of equilibrium dialysis when ligand is used in concentrations higher than the dissociation constant of the binding protein and higher than the protein concentration. Thc binding protcin in the prrsence of the radioactive ligand is filtered through membranes impermeable t o the binding

100

I

WINFRIED BOOS

0

0.2

0.1

3 2

1

0

1

I

2

I

I

I

3 4 5 l / f r c e galactose (IIM) x lo7

1

I

I

I

6

7

8

9

FIG.10. Galactose-binding activity of the galactose-binding protein m a function of galactose concentration measured by equilibrium dialysis. Double chambers of 100-pliter volume separated by dialysis tubing were filled with 90 pliters of galactose-binding protein (0.4 mg/ml) in 0.01 M tris-HCI (pH 7.3) and 90 pliters of [l-WIgaIactose or [1-3H]galactose in the same buffer. The dialysis was performed at 4"for a t least 12 hours. Fifty microliters from each chamber were counted for radioactivity. (Taken from Boos et al., 1972.)

protein but not to the ligand (Paulus, 1969). The filter is subsequently measured for radioactivity. By using this method 10 pg galactose-binding protein can be easily detected (Boos and Gordon, 1971). Yet, quantitative binding assays for the galactose-binding protein with this method are not possible. During the filtration assay the protein solution is necessarily being concentrated, and independent studies by equilibrium dialysis have shown that the binding affinity is dependent on the protein concentration. This also appears to be true for the arabinose-binding protein of E. coli B/r (R. G. Parson and R. W. Hogg, unpublished data). However, the ultrafiltration assay is a convenient and sensitive method to detect qualitatively galactose-binding protein in different fractions during the purification of the protein. c. Preparative Polyacrylamide Gel Electrophoresis. Another excellent method to detect qualitatively galactose-binding protein in crude extracts of bacterial "shock fluid" is preparative polyacrylamide gel electrophoresis through gels polymerized in the presence of radioactive ligand (Boos, 1969). This method is based on a particular elution technique in which

101

PRO AND CONTRA CARRIER PROTEINS

2o

t

14

c 0

Pa

12

; .. 1 0

-t 0

\

0.8

0 U

L

06 01

U

5

0.4

0

n

0.2

0.0 I

1

1 1 I I I 1 1 I I I I I 7 8 9 10 11 12 13 3 4 5 6 2 bound gdlactoseltotal pr tein X tree galactose concentration (XI0 )

P

FIG.11. Scatchard plot of the binding data of Fig. 10. (Taken from Boos et ul., 1972.)

the eluting buffer docs not flow through the gel, but removes all the compounds that have migrated through the gel forced by the electrical potential, and which appear on the anodic end of the gel. The uncharged ligand galactose does not migrate in the gel, and radioactivity appears only together with the macromolccular binding protein. A potential hazard of this method is that the peak of the galactose-binding protein does not necessarily coincide with the peak of radioactivity. This can be seen when pure galactose-binding protein is subjected to electrophoresis under these conditions. The leading edge of the migrating protein may bind all the galactose, depleting the trailing edge of ligand. Therefore the lower the concentration of ligand in the gel, the larger the distance between protein and radioactivity peak. d. Precipitation with Ammonium Sulfate tn the Presence of Ligand. Once ligand is bound to the galactosc-binding protein, it is not removed during precipitation with 80% saturated ammonium sulfate solution. This phenomenon has been used as a test for galactose-binding activity (Kepes and Richarme, 1972). The ammonium sulfate precipitation is done in the

102

WINFRIED BOOS

presence of labeled galactose. The precipitate is filtered through a Millipore filter and measured for radioactivity. Even though the amount of galactose bound to the protein under these conditions is only about one-half the amount found with the same protein preparation by equilibrium dialysis, the assay represents a reproducible and fast qualitative binding assay, most useful to determine relative amounts of galactose-binding protein in crude protein preparations. 2. SPECTRAL CHANGES IN RESPONSE TO BINDING OF LIGAND

As discussed in Section 111, F, interaction of substrate and the galactosebinding protein results in the alteration of a variety of parameters. A convenient and very sensitive parameter to follow is tryptophan fluorescence (Boos et al., 1972). Figure 12 shows the excitation and emission spectra of pure galactose-binding protein in the presence and absence of galactose and glucose. The excitation spectrum (Fig. 12A) shows a peak a t 288 nm, which is increased in intensity by galactose and glucose, whereas

'"14

B A

90

260

280

300

320 300

320

340

360

380

400

Wavelength (nm)

FIG.12. Fluorescence spectra (uncorrected) of galactose-binding protein in the presence and absence of 10-4 M galactose and glucose. Galactose-binding protein (16.7 Ig/ml) in 0.01 M tris-HC1 (pH 7.3); excitation slit, 10 nm; emission slit, 8 nm. (A) excitation spectra. Emission wavelength was 330 nni. (B) emission spectra. Excitation wavelength was 290 nm. Temperature was 24". (Taken from Boos et al., 1972.)

103

PRO A N D C O N T R A CARRIER PROTEINS

the peak position remains unchanged. A t morr narrow excitation slits, thr excitation peak is observed to split into a peak a t 290 nm and a shoulder at 285 nm. Thest>presumably correspond either to two differrnt tryptophan residues or to tryptophan and tyrosinc, respectively. The emission spectrum remains unchanged in shape when protein is excited a t 280 or 295 nm, showing that the emission is due only to tryptophan (Udenfriend, 1962). The emission spectrum (Fig. 12B) shows a broad maximum a t 340 nni. Galactose increases the intensity and gives a 2-nm blue shift in thr emission maximum. The maximum percentage increase for galactose is observed a t 330 nm. Glucose also increases the intensity, but gives no shift in the emission maximum. This indicates, as discussed in detail in Section 111, F, that the active site contains a t lcast one tryptophanyl residue. The increase in the emission spectrum can conveniently be used as an activity test for the pure protein. Figure 13 shows the percent increase in the emission a t 330 nm a t an excitation of 290 nm upon the addition of varying concentrations of galactose and glucose. Both sugars result in a half-maximal increase a t a concentration of I p M , while the maximum percent increase is larger with galactose than with glucose. This method to detrrmine the binding affinity of the galactose-binding protein is free of the limitations

-2

10-2

I

'I1I1I1l

10-3

1 1 1 1 1 1 1 1 io-4

1 1 1 1 I

10-5

CONCENTRATION

1

lllllil I

10-6

,

I,. , , I !

10-7

1

10-8

, molar

FIG.13. Percent increase in fluorescence of galactose-binding protein versus total sugar concentration. 0 , Galactose; glucose. Excitation, 290 nm; emission, 330 nm. Excitation slit was 4 nm; emission slit was 10 nm. Protein concentration was lF.7 pglml. Temperature was 24". (Taken from Boos et al., 1972.)

m,

104

WINFRIED BOOS

of the equilibrium dialysis method when high substrate concentrations have t o be used. The method has proved useful to demonstrate the low binding affinity of a mutant galactose-binding protein for which no binding affinity could be detected with either equilibrium dialysis or ultrafiltration (Boos, 1972). Unfortunately, only preparations of somewhat purified galactose-binding protein ( >50% protein impurities with no affinity for galactose) can be used in this technique. Even though measuring the fluorescence increase seems to be an attractive replacement for the direct binding assay, an extrapolation to the exact binding constants of the protein for different ligands is not justified, since this method does not allow for determination of the number of ligands bound to the protein. It also would not account for the binding of substrate to a second site not containing tryptophan. However, measurement of the increase in fluorescence provides an excellent tool to compare the relative ability of different sugars to act as a substrate for the galactose-binding protein. This then enables one to compare the observed specificity with the known substrate specificity of the MeGal transport system. Table I shows such a comparison (Parnes and Boos, 1973). The sugars listed in Table I, for which the galactose-binding protein shows affinity, exhibit either a “glucose” or a “galactose” emission spectrum, depending on the configuration of the OH group in the 4-position of the glycon ring. All active galactosides tested so far have a p-glycosidic linkage to the aglycon. It was therefore of interest to see whether the fluorescence increase with galactose was due only to the p form of the sugar. When crystalline a-D-galactopyranose is equilibrated in 0.01 M tris-HC1 (p H 7.3), an equilibrium is reached in which 70% of the sugar has mutarotated to the p form. The addition of such an equilibrated solution to the galactose-binding protein results in an immediate change in fluorescence with no further subsequent increase. However, when a-D-galactopyranose solutions were prepared and immediately added to the protein, there was an initial fast increase followed by a slower increase which reached the value of the equilibrated galactose after about 30 minutes (Boos et al., 1972). The time course of this change is of the same order as the mutarotation required to reach equilibration of the a and p forms. Thus the fluorescence increase either is caused preferentially by the p form of galactose, or is caused by both, with the a form being less effective. Subsequent studies using ultraviolet difference spectroscopy (Section 111, F) indicate the latter possibility. Fluorescence changes in response to substrate binding have also been observed with sulfate- (Langridge el al., 1970), glutamine- (Weiner and Heppel, 1971), and arabinose-binding proteins (R. G. Parson and R. W. Hogg), but not with the leucine-binding protein (Berman and Boyer, 1972).

P R O A N D C O N T R A CARRIER PROTEINS

105

3. ASSAYSRASEDON IWWJNOLOGICAL PROPERTIES Purified galactosc-binding protein is an excellent antigen, and specific antibodicxs have been isolated from rabbits and goats. Qualitative tests for the presence, absence, or inducibility of the galactose-binding protein in mutants of the MeGal transport system or of galactose chcmotaxis have been made by thc Ouchterlony immunotliffusion method (Boos and Sarvas, 1970; Lengeler et al., 1971). This technique was also useful for following the prescmcc of a mutant galactosc-binding protein during purification. This protein exhibits a strongly reduced binding affinity, so that the usual binding assays could not be applied (Boos, 1972). A more direct immunological binding trst based on the precipitation of galactose-binding protein, even in crude extracts, with specific antibodies in the presence of radioactively labeled substrate has been reported (Rotman and Ellis, 1972). The antiserum has to he dialyzed extensively to remove glucose which otherwise would strongly interfere with galactose binding. The complex of antibody, galactose-binding protein, and substrate is filtcred through Millipore filttw, and the radioactivity remaining on the filter can be taken as a measure of binding activity. Yet, as discussed in the next paragraph, the conip1c.x formation appears t o change the binding affinity of the galactosc-binding protein. Therefore the binding properties of the native protein cannot be measured quantitatively by this technique. F. Conformational Change

1. CHANGEI N ELECTROPHORETIC MOBILITYUPON BINDINGWITH LIGAND

We observed recently that highly purified galactose-binding protein shows two bands on polyacrylamide gels at pH 8.4 when the protein was incubated with 20% sucrose prior to clectrophoresis (Fig. 14). (Sucrose was added to increase the density of the protein solution in order to facilitate the application on the gel.) Electrophoresis with gels equilibrated with 0.1 pM C3H]galactose denionstrated that both components were capable of binding galactose. Also, hot h components consisted of the same material, since reelectrophoresis of either component resulted again in the appearancc of the same two bands (Boos and Gordon, 1971). Subsequently, wc discovered (Boos et al., 1972) that, the simultaneous appearance of the two bands on these gels was caused by the presence of sucrosc in the incubation mixture prior to the electrophoresis. Replacing the sucrose by 20% nonpolyrnerized acrylamide, we obtaincd only one band. Yet, the position of the band changed when substrate was present in the gel as well as in

106

WINFRIED BOOS

FIG.14. Polyacrylamide gel electrophoresis of purified galactose-binding protein in the presence and absence of urea. The gel slab (4 X 120 X 160 mm) contained 7.5% acrylamide, 0.2% bisacrylamide, 0.1 M tris-borate (pH 8.4), and 0.002 M EDTA. Left: Untreated sample of galactose-binding protein. Middle: Sample incubated with 8 M urea in 0.01 M tris-HC1 (pH 7.4) for 1 hour. Right: Sample incubated with 8 M urea in 0.01 M tris-HC1 (pH 7.3) for 1 hour and subsequently passed through Sephadex G-100. The electrophoresis was run for 4 hours a t 300 V and 50 mA. (Taken from Boos and Gordon, 1971.)

PRO AND CONTRA CARRIER PROTEINS

107

the electrode buffers. The positions of the band in the presence and absence of substrate corresponded to the position of the two bands of the protein when it was run after incubation with sucrose. The change in electrophoretic mobility is highly specific; it occurs with galactose, glucose, and glycerol galactoside, but not with lactose, IPTG, and other 0-galactosides which are not substrates of the galactose-binding protein. (The substratedependent increase in the electrophoretic mobility on polyacrylamide gels a t pH 8.4 has been observed only with substrates of Kdlas< 2 X 10-6 M . Poorly bound substrates such as MeGal and D-fucose do not elicit this effect even when applied in concentrations higher than their respective KdiR8.The discrepancy in comparison to binding or fluorescence data is presently not understood.) The concentration of galactose to give the half-maximal effect (confluence of the two bands) is in the vicinity of 1 p M . This value represents the initial frce galactose concentration in the acrylamide gel. The actual free galactose concentration on the band position, however, might be considerably lower, since part of the galactose is removed by binding. A plausible explanation for the cause of the substrate-dependent change in thc migration through acrylamide gels was that the molecular weight of the galactose-binding protein changes and that the two forms represent monomer and dimer of the same polypeptide chain. However, subsequent detailed study of the molecular weight by different methods in the presence and absence of the substrate galactosc, as discussed above, showed that the protein exhibits a molecular weight of about 36,000 under all conditions. Therefore this interpretation seems unlikely. The occurrence of the two bands might be explained in several other ways: 1. Substrate binding alters the protein conformation, thus changing its retardation coefficient (Banker and Cotman, 1972). 2. Substrate binding is acconipanird by an alteration of surface charge. 3. A combination of both of the above. Evidencc indicating that the two forms differed in charge was obtained by the separation during electrofucusing of two species with pI values of 5.3 and 5.4 [T. Silhavy and W. BOOS,unpublished observation as referred to in Boos (1972)l. The more negatively charged form, i.e., the form with higher electrophoretic mobility tit pH 8.4 during electrophoresis, represents the form complexed with galactose. Assuming that the change in electrophoretic mobility is due to a change in surface charge, and that electrophoretic mobility increases linearly with the increase in negative charges, one can estimate that the surface charge a t pH 8.4 increases by 12-15%. That would mean that binding of galactose uncovers glutamic or aspartic acid residues, or buries the corresponding number of positively charged

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WINFRIED BOOS

amino acid residues. One would expect to observe such a change in the arrangement of amino acid residues by the classic methods of CD, ORD, and infrared spectroscopy. Yet, as discussed before, none of these methods showed any change in the spectrum of the galactose-binding protein in response to the presence of galactose a t the wavelengths where chromophores of the polypeptide backbone absorb. The change in electrophoretic mobility does not occur in a mutant galactose-binding protein (isolated from strain EH3039) , even a t galactose concentrations above the protein’s dissociation constant for galactose ( > 1 mM) (Boos, 1972). However, electrofocusing still reveals two species with different PI values. Therefore the mutant protein must still be able to occur in two different forms, and the difference in electrophoretic mobility a t pH 8.4 of the wild type is not the cause of the difference in PI of the two forms. 2. INCREASE IN FLUORESCENCE UPON BINDING WITH LIGAND

As discussed before, the tryptophan fluorescence of the galactose-binding protein (Figs. 12 and 13) increases up to 13.5% at 350 nm when excited a t 290 nm (Boos et al., 1972). This increase in fluorescence can be interpreted in severaI ways : 1. One or more tryptophanyl residues are part of the active site, and the microenvironment of this tryptophanyl residue (s) is changed upon the interaction with the substrate itself. 2. The tryptophanyl residue in question is not part of the active site itself, but changes its microenvironment because of a conformational change induced by the binding of galactose. 3. Both effects might occur a t the same time. The fluorescence spectra of the galactose-binding protein in the presence of glucose and galactose are not identical. This seems to indicate that the change in fluorescence is caused by the direct interaction of the carbohydrate with a tryptophanyl residue a t the active site, rather than by a change in microenvironment of an outside tryptophanyl residue due to a conformational change. The tryptophan fluorescence can be quenched in the presence of potassium iodide. The effect of quenching in the wild-type protein with this agent in the presence and absence of galactose is shown in Fig. 15. As can be seen, galactose protects a t all concentrations of iodide tested. Other substrates, such as glucose or glycerol galactoside have the same effect. In contrast, the quenching effect of potassium iodide in a mutant galactosebinding protein (EH3039) is unaffected by galactose concentrations above its Kdiss of binding (> 10 mM). As discussed in the following sections, the bulk solvent does not have access to the tryptophanyl residue of the active

109

PRO A N D C O N T R A CARRIER PROTEINS

site. Therefore it is w r y likely that the iodide anion also has no access to this tryptophanyl residue. Thus we conclude that one of the tryptophanyl residues affected by iodide is located on the outside of the protein in closc proximity to a charged group which greatly influences the quenching effect (McGowan et al., 1974). Binding of galactose, because of a small conformational change, alters the spatial arrangement of this charged group with this particular tryptophanyl residue without changing its accessibility to bulk solvent. It therefore appears as if galactose “protects” against quenching. In agreement with this cxplxnation is the observation that galactose does not protect against quenching in the mutant binding protein that also does not show the difference in electrophoretic mobility a t pH 8.4 in the presence and absence of galactose so characteristic of the wildtype protein. The data obtained \\ ith fluoresccncc quenching by potassium iodide, however, do not distinguish b e t w e n thfh two possibilities : a positive charge enhancing the quenching in the absence of galactose or a negative charge reducing quenching in the presence of galactose, since opposite

K I

CONCENTRAT/ON ( M .

FIG.15. Quenching of protein fluorescence emission by potassium iodide. T o wild-type galactose-binding protein (0.5 p M in 0.01 M tris-HC1, p H 7.3) in i,he presence or absence of 10 p M galactose, increasing amounts of G M potassium iodide were added. Mutant protein was treated identically, except that, the galactose concentrat,ion was 10 mM. The initial fluorescence Fois divided by the fluorescence F at, a given potassium iodide concentration. The emission wavelength monitored was 330 nm for the wild-type protein, and 336 for the mutant protein. 0, Wild-type protein; A, mutant, protein. Closed symbols, quenching in t,he absence of galactose; open symbols, quenching in the presence of galactose. (Modified from McGowan el d.,1974; copyright by the American Chemical Society.)

110

WINFRIED BOOS

charges also have opposite effects on the ability of potassium iodide to quench tryptophan fluorescence (Lehrer, 1971).

3. CHANGEIN LIGAND

THE

ULTRAVIOLET SPECTRUM UPON BINDINGWITH

The addition of galactose to wild-type and mutant galactose-binding proteins results in the production of an ultraviolet difference spectrum that closely resembles the solvent perturbation difference spectrum of N-acetyltryptophanyl ethyl ester (a model compound that resembles tryptophanyl residues in a polypeptide chain). The difference spectra are shown in Fig. 16. As can be seen, the mutant galactose-binding protein exhibits a spectrum slightly different in form than that of the wild type. The addition of glucose to both proteins results in a similar but not identical difference spectrum. These results correlate well with the previously discussed fluorescence studies, and are subject to the same three possible explanations. The addition of freshly prepared a-galactose to the wild-type galactosebinding protein results in a difference spectrum with a time-dependent increase in two absorption maxima; this time dependence is caused by the mutarotation of the LY form to an equilibrium mixture of the a and B forms. 002

A

260

270

280

290

300

31C

WAVELENGTH [ n t d

FIQ.16. Ultraviolet difference spectra of galactose-binding protein caused by galactose. A 1-ml protein solution (16 p M in 0.01 M tris-HC1, pH 7.3) was placed in two matched quartz cuvets; a baseline was obtained from 320 to 260 nm; 10 pliters of galactose, final concentration 10 mM, were added to the sample cell, and 10 pliters of water were added to the reference cell, and the resultant spectrum was recorded. (A) Wild-type protein. (B) Mutant protein. (Modified from McGowan et al., 1974; copyright by the American Chemical Society.)

PRO AND CONTRA CARRIER PROTEINS

111

The third peak of the difference spectrum is independcnt of mutarotation. This shows that both a- and @-galactoseare in fact suhstrates. The observation that glucose and galactose, as well as a- and @-galactose, give rise to similar but not ident,ical ultraviolet, difference spectra strongly suggests that a t least part of thr difference spectra must be caused by the direct interaction of substrate with a tryptophanyl residue a t the active site of the galactose-binding protein (McGowan et al., 1974). 4. SOLVENT

PERTURBATION I N THE P R E S E N C E A N D

ABSENCEO F LIGAND

One way of investigating the relative exposurc of chromophoric groups such as tryptophanyl or tyrosyl residues is k)y solvent perturbation (Herskovits, 1967). If a perturbant such as diincthyl sulfoxidc, ethylene glycol, or methanol is added to a protein solution, chromophores exposed to this agent will experience an alteration in their microenvironment. This alteration in environment is reflected by an altered ultraviolet absorption spectrum. By comparing solvent perturbation difference spectra with available model compound data (Herskovits and Sorensen, 1968), one can calculate the number of exposed chromophores present in a given protein. Studies with the galactose-binding protein and dimethyl sulfoxide, methanol, and ethylene glycol as perturbants indicated that two tryptophanyl and four to five tyrosyl residues are accessible. The smaller solvent DzO resulted in the perturbation of two additional tryptophanyl residues. If galactose produces a conformational change, then it may well alter the number of exposed chromophoric residues. Indeed, the addition of galactose to galactose-binding protein did result in an altered difference spectrum, but again this alteration is not uniquely conclusive for a conformational change. As discussed before, we concluded that the substrate must interact with a tryptophanyl residue at the active site. If in addition the interaction of the galactose-binding protein changes the nicroenvironment of the chromophoric groups outside the active site, then the solvent perturbation spectrum in the presence of galactose should be different from that in the absence of galactose. When t,his was investigated, both spectra were found to be identical. This indicates that the ultraviolet difference spectrum observed in the presence of substrate is due solely to an interaction between substrate and tryptophanyl residue a t the active site, rather than to a change in the environment of an outside tryptophanyl residue due to a conformational change. It should be noted, however, that thesc results do not exclude a conformational change in the protein. The identity of the solvent perturbation spectra in the presence and absence of galactose suggests that the tryptophanyl residue a t the active

112

WINFRIED BOOS

site is never accessible to the perturbing solvent. This must be the case, since it is exceedingly unlikely that the tryptophanyl residue a t the active site would be perturbed by any solvent in an identical way, regardless of the presence or absence of substrate (McGowan et al., 1974). It is interesting that the solvent perturbation difference spectra of the mutant galactose-binding protein (strain EH3039) in the presence or absence of substrate are not identical. The difference between these two spectra are slight but reproducible. This might indicate that perturbing solvents have some access to the active-site tryptophan in the mutant protein. 5. CHANGEIN BINDINGAFFINITYUPON BINDINGOF LIGAND

Examination of the binding activity a t different free galactose concentrations by equilibrium dialysis showed that the binding behavior is biphasic (Boos et al., 1972). Figure 10 gives the results plotted according to Lineweaver-Burk. Extrapolation of the points obtained between 0.3 p M and 10 nM free galactose concentration yielded an apparent dissociation constant of 0.1 p M , whereas extrapolation of the values obtained between 0.3 and 10 pM yielded an apparent Kdiasof 10 p M . As discussed above, the experimental data are still meaningful up to a concentration of 10 pM (protein concentration was 11 p M ) , When the data were plotted according to Scatchard (1949), the heterogeneous behavior was even more pronounced (Fig. 11). Surprisingly, the extrapolation of the curve to high free galactose concentrations indicated that 2 moles of galactose were bound per mole of galactose-binding protein of 36,000 molecular weight. All periplasmic binding proteins studied so far, including the galactosebinding protein (Anraku, 1968), have been reported as having only one binding site per polypeptide chain; however, the existence of a second binding site with strongly reduced binding affinity might have been overlooked because of the increasing error in equilibrium dialysis measurements a t high substrate concentrations. The differential ability of various substrates to protect against the inhibition by N-ethylmaleimide of the M protein, the membrane-bound transport protein of the lactose transport system in E. coli, suggests two binding sites per polypeptide chain (Carter el al., 1968; Kennedy et al., 1974). Thus the existence of two different binding sites in the fully saturated galactose-binding protein is not a completely isolated phenomenon. Our interpretation of the binding behavior at the time was that binding of galactose to the protein of the high-affinity form induces a conformational change resulting in a conformation with one additional binding site, but lower affinity. However, extrapolation of the number of binding sites of the high-affinity form

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(present a t low galactose concentrations) gave a t the most only 0.1 moles of galactose per mole binding protein (Fig. 11), an observation explainable by the continuous conversion to the low-affinity form with increasing galactose concentration. A better interpretation of the observed binding plot would be to postulate the existence of two different forms of the binding protein at any one time: form I, with low binding affinity, or completely binding inactive; and form 11, with both a high- ( K d I s s = 0.1 p M ) and a low- (Kd,84 = 10 H M ) affinity site. Both forms would be in equilibrium, form I being favored in the absence of galactose. The contribution of form I1 a t equilibrium would be too small to be detected beside form I during polyacrylamide gel electrophoresis, but would be large enough to exhibit high-affinity binding activity during equilibrium dialysis (one can extrapolate a contribution of 5-10% of form I1 in the absence of galactose). Binding of galactose would stabilize form I1 and shift the equilibrium in favor of form 11. Therefore only one form (11) can be detected a t high galactose concentration during polyacrylamide gel electrophoresis. G. A Working Model

Models of a reaction sequence in any one system usually function to conceal lack of knowledge about the actual mechanism. The model presented in Fig. 17 does not claim any resemblance to reality. Instead, it represents our current opinion extracted from a biased interpretation of available results (McGowan et a!., 1974). The galactose-binding protein occurs in two different conformational

FIG.17. Model of the conformational change occurring upon addition of galactose to tdheprotein. A, High-affinity binding site, active only in state 11; B, low-affinity binding site; TI, tryptophan in the active site; Tz, T , external tryptophan residues accessible to all perturbants (Ts is located close to ttn e l e c t r i d charge, which is different in state I and 11);T, and T , tryptophan residues not accessible to dimethyl sulfoxide, ethylene glycol, or methanol. but accessible to D20. (Taken from McGowan et ul., 1974; copyright by the American Chemical Society.)

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states, I and 11, which are in equilibrium with each other. In the abscncc of substrate, state I1 comprises less than 10% of the total protein. The protein has two binding sites, A and B. A has different affinities in states I and 11: Kdlnsvery high in I, and 0.1 p M in I1 with galactose as substrate. B has a Kdlssof 10 pM in 11, and may or may not be present in I with the same affinity. As a result of the presence of the highly active binding site in the absence of galactose, the presence of B in state I cannot be demonstrated experimentally. The only evidence for its presence in state I1 is the presence of two binding sites observed a t high galactose concentrations. Binding of substrate to A in I1 shifts the equilibrium to the right. I and I1 differ in their electrophoretic mobility in acrylamide gels, while thr fluorescence increase and the ultraviolet difference spectra are caused by interaction of the substrate with a tryptophanyl residue a t active site A in state 11. The charge difference in the two forms is the reason for thc protection by galactose against potassium iodide quenching. The position of this charge must be in the vicinity of a tryptophanyl residue on the outside of the protein not related to the active site. Increase in “binding activity” has been reported when specific antibodies were present in the binding assay (Rotman and Ellis, 1972). The order of additions was important for this increase. Galactose had to be added first to react with the galactose-binding protein before addition of thc antibodies. An explanation, ot,her than a trivial trapping of hound galactose by formation of the antigen-antibody precipitate can be provided by the model. Thc antibodies precipitate either form I or form 11, depending on whether or not substrate was present during the precipitation. The antibody interaction holds the galactowbinding protein in its rcspective state, and therefore in different binding affinities. From the results obtained from the ultraviolet difference spectra caused by glucose and galactose, it is clear that a t least one tryptophanyl residue occupies the active center. From the solvent perturbation studies with ethylene glycol, methanol, and dimethyl sulfoxide, one can conclude that two out of five tryptophanyl residues are accessible to the bulk solvent whether galactose is present or not. With D20 as perturbing solvent four tryptophanyl residues are accessible whether or not galactose is present. Therefore none of the perturbing solvents can have access t o the tryptophanyl residue a t the active site. The mutant protein does not change its electrophoretic mobility, even a t very high galactose concentrations sufficient to increasc its fluorescence maximally and to give rise to its ultraviolet difference spectrum. However, by electrofocusing one can still detect two different forms of the protein. We therefore conclude that the mutant binding protein can still occur in two states, even though the mutation has abolished the charge differcnces

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a t pH 8.4 characteristic of the wild-type protein. Incidentally, under these conditions the mutant protein migrates identically with the wild-type protein in the absence of substrate, the form with the higher positive charge. As in the wild-type, two tryptophanyl residues out of a total of five are accessible to bulk solvent in the mutlant, but the solvent perturbation difference spectrum is not entirely independent of the substrate. I n addition, the effect of potassium iodide in quenching the tryptophan fluorescence is not counteracted by the substrate. In terms of the model one could explain the mutant protein by saying that the mutation has led to a “loosening” of site A. The positive charge is not buried or the negative charge is not revealed any more when the protein is in state 11; hence galactose does not protect fluorescence against quenching by potassium iodide. I n addition the “loosening” of the active site, which results in a 7000-fold reduction in the binding affinity, allows limited access of bulk solvent to the tryptophanyl residue in the active site in the presence of galactose. The mutation also might have affected the equilibrium of the two forms and must have abolished binding site B. Despite the restriction implicated by the inaccuracy of the method of solvent perturbation, the tentative distribution of the different tryptophanyl residues (T,) would be: TI is part of the active site and is not accessible to the bulk solvent when the protein is in state I. When the protein is in state 11, TI interacts with galactose. Two tryptophanyl residues (Te, T3) are located on the outside of the protein unrelated to the active site. At least one of these residues must be located close to the positive charge of state I or close to the negative charge in state 11. The remaining two residurs (T4, T5) are half buried, i t . , they are accessible to D20but not to larger solvents such as dimethyl sulfoxide. These tryptophanyl residues also have no relation to the active site. The substrate-dependent equilibrium of two conformational states is not a gencral feature of periplasmic binding proteins, or a t least has not been recognized as such. Studies with the lcucinc-binding protein of E. coli (Penrose et al., 1970; Berman and Boyer, 1972) have shown that this protein does not exhibit substrate-dependcnt alteration of thc studied parameters, even though revcrsihility of denaturation was interpreted as an integral ability of the protrin to undergo conformational changes easily. Howevcr, substrate-dependent fluorescence changes have been reported for glutamine- (Weiner and IIeppel, 1971) , sulfate- (Langridge et al., 1970), and arahinose-binding proteins (R. G. Parson and R. W. Hogg). Also, it has h e w noted that the elution profile of a phenylalaninebinding protein of Coinamonas changes in the presence of substrate (Kuzuya et al., 1971). Moreover, it has bcen observed that cystine-binding protein

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(E. coli) can elute from a DEAE column a t two different positions (Berger and Heppel, 1972), as can arabinose-binding protein ( E . co2i) on polyacrylamide gel electrophoresis (R. G. Parson and R. W. Hogg). These latter observations might indicate that the occurrence of two conformational states in periplasmic binding proteins is not an unrelated peculiarity of the galactose-binding protein. IV. EVIDENCE FOR THE ESSENTIAL FUNCTION OF THE GALACTOSEBINDING PROTEIN IN THE TRANSPORT MECHANISM OF THE MeGal TRANSPORT SYSTEM A. Reduction in Transport Activity in Cells Treated with the Cold-Osmotic Shock Procedure of Neu and Heppel

As discussed briefly above, gram-negative bacteria subjected to a simultaneous osmotic and temperature shock after treatment with tris-HC1 (pH 7.3) containing EDTA release a variety of proteins (besides nucleotides, lipopolysaccharides, and other components) which are believed to have their in vivo location between the cytoplasmic membrane and the cell wall (Heppel, 1969, 1971; Heppel and Rosen, 1973; Costerou et al., 1974). So far, all transport-related substrate-binding proteins have been found in the class of proteins released by the cold osmotic shock procedure. One of the first evidences for participation of the binding proteins in transport was the observation that the transport activity for the respective substrate in shocked cells is more-or-less severely reduced in contrast to the transport activity of substrates for which no soluble binding protein could be detected. In fact, the reduction of transport activity in shocked cells has often been used as a criterion to postulate the existence of a shockable binding protein. In most cases the reduction of transport activity is not complete. This is generally for two reasons: (1) The release of the respective binding protein is not in all cases quantitative, Growth conditions and lipid composition of the outer membrane (Rosen and Hackette, 1972) are important factors for the yield in periplasmic proteins (Heppel, 1971). (2) Generally, the bacterium contains additional transport systems for the particular substrate, which are entirely bound to the cytoplasmic membrane (lactose type) and which are still operating in shocked cells, as well as in isolated membrane vesicles. Usually, the periplasmic binding protein-mediated system has the lowest transport K , for the substrate in question. Thus the use of substrate concentrations in the vicinity of the transport system’s K, improves the conditions for measuring loss of transport activity. Figure 18 shows the effect of the shock procedure of Neu and Heppel (1965), as

117

PRO AND CONTRA CARRIER PROTEINS 020r

FIG.18. Effect of a combined treatment of temperature and osmotic shock on the uptake of galactose. Cells were treated as described under “Materials and Methods” in Parnes and Boos (1973). Transport assay was performed a t 23” but otherwise as described in the legend for Fig. 1. @, No additions; 0 , addition of 20 mM D-lactate 2 minutes prior to the addition of [1-14C]galactose; +, addition of 20 mM ATP 2 minutes prior to the addition of [1-14C]gala~tose.Cells treated according to Neu and Heppel were assayed a t 23”. 0, No additions; 20 m M D-lactate added to shocked cells 2 minutes prior to the addition of [1-14Clgalactos.e;0 ,control, nonshocked cells. (Legend and figure taken from Parnes and Boos, 1973.)

a,

well as other shock procedures, on the transport activity of galactose a t an initial substrate concentration of 0.5 p M , the K , of the MeGal system. The Neu and Heppel treatment reduced transport to less than 5% of the activity of untreated cells. A t the same time no galactose-binding protein can be detected any longer by cross-reactivj t y against specific antibodies, while the cells are more than 85% viable. The addition of an energy source such as D-lactate does not restore transport activity in cells shocked by the Neu and Heppel method, in contrast to another shock procedure that depletes the cell of small molecules without removing the galactose-binding protein. The close connection of binding-protein release and reduction in transport activity strongly suggests the participation of these binding proteins in the transport mechanism, particularly since it is observed in all substratebinding protein-related systems. Of course this corelation is only indirect, and several other explanations for the reduction in transport activity could be given without invoking a necessary participation of substratebinding proteins in transport. To obtain direct proof for such a participation, experiments were performed in several laboratories to restore the

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transport activity in shocked cells by the addition of the respective substrate-binding protein. Successful restoration was reported in few cases (Anraku, 1968; Wilson and Holden, 1969; Iwashima et al., 1971 ;Medveczky and Rosenberg, 1970). In the case of the galactose-binding protein, Anraku (1968) reported that besides the purified binding protein an additional factor present in crude ‘[shockfluid’’ was necessary for maximal restoration, while in the case of the phosphate-binding protein a modified shock procedure (simple dilution with distilled water) was necessary to yield positive results. However, these reports could not be reproduced in all cases (H. Rosenberg, personal communication), and have since been questioned (Rosen, 1973a; Heppel et al., 1972; Pardee, 1970). Difficulties in the interpretation of restoration of transport activity might arise from the incomplete removal of binding proteins by the shock procedure and restoration of energy supply after incubation of shocked cells in the transport buffer, rather than restoration by exogenous binding protein. In fact, we demonstrated that radioactively labeled and purified galactose-binding protein did not bind to shocked cells or membrane vesicles (W. Boos, A. S. Gordon, and H. R. Kaback, unpublished research) under conditions in which restoration of galactose transport was reported. Restoration experiments with exogenous binding protein should bc carried out in mutants defective in transport as the result of a mutation in the respective binding protein (Medveczky and Rosenberg, 1970). It remains the goal of any biochemical approach to study the molecular mechanism of a particular transport system by restoration of active transport in an appropriate in vitro system using the purified components. Recently, the successful restoration of several quite different transport systems or transport-related functions has been reported. Storelli et al. (1972) found that the addition of purified sucrase-isomaltase (Cogoli et al., 1972) to black lipid membranes greatly increased the transport of sucrose through this artificial membrane while being hydrolyzed to glucose and fructose, a mechanism proposed to occur in the membrane of small intestinal brush borders (Semenza, 1972). Also, it has recently been shown that arginine-binding proteins from yeast are able to increase the permeability of arginine through a film of lipids extracted from the same yeast (Stuart and DeBusk, 1973). The generation of a membrane potential by the addition to mitochondria1 phospholipids of either cytochrome c plus cytochrome oxidase or ATPase (Jashftis et al., 1972) gives strong support for Mitchell’s chemiosmotic hypothesis for energy coupling of transport; Racker ( 1972) reported the successful reconstitution of calcium uptake by the addition of purified Ca2+-ATPase to vesicles of soybean phospholipids; The reversal of this reaction, i.e., the generation of ATP from ADP and P , during Ca2+ efflux from these vesicles, was demonstrated

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earlier by Makinosc and Hasselbach (1971) ; Reeves ~t al. demonstrated the reconstitution of n-lactatt.-depcritleIit stimulation of transport in vesicles of a lactate dchydrogenasc-negative niutant of E. coli by addition of solubilizcd enzyme from a wild-typr htrain (Kceves et al., 1973b). Restoration of active transport by the addition of galactose-binding protein t o membranc vesicles or cvvn shockcd cells remains to be seen. An estiniation of the concentration of galactose-binding protein of fully induced cells in the pcriplasniic space yiclds an astonishingly high value of 2 mM, corresponding to a protein concrntration of 70 mg per milliliter. Since thr cytoplasmic membrane does not seem to bind galactose-binding prott4n particularly well, one would havr to conclude that an exogenous protein concentration on the order of 2 n d i would b(1 necessary to exert active transport of galactose through t h r nirmhrane, conditions not feasible cxperimcritally. B. Comparison of Binding Specificity in Vitro and Transport Specificity in Vivo

Onc of the arguments frequently used for the close rdationship of substrate-binding proteins in the transport mechanism is the similarity of binding and transport specificity (Oxender, 1972a; Hcppel, 1969; Pardee, 1968; Baback, 1970). Such a similarity can also be observed in the case of the galactose-binding protein and the McGal system. Table I compares the binding specificity (expressed by the ability to increase the tryptophan fluorescence of the galactose-binding protein) with the transport specificity (expressed by the ability of the different sugars to inhibit galactose uptake via the MeGal system). The similarity of both assays is apparent. The comparison of transport data with direct binding data (Boos, 1969) or with another property of thc galactose-binding protein, its substratedependent increase in electrophoretic mobility on acrylamide gels (Boos et al., 1972), shows in general thp same pattrrn. The similarity of binding and transport specificities clearly indicates that binding proteins function at least as the specific recognition sites for the entry process in transport. That binding proteins are involvrd in rrgulation of transport rather than in transport itself (S.Roseman, personal objection) is unlikely a t lcmt for thc galactosc-binding protein for two reasons: ( I ) The inducer specificity of the MeGal system is different from the binding specificit!, of galactosr-binding protein; and (2) regulation of the biosynthesis of a structurally defective galactose-binding protein (7000fold reduced binding affinity) is unimpaired by the mutation. An indirect relationship of binding proteins to regulatory phenomena is indicated in some cases. The R2a protein classified by Garcn and Otsuji (1964) as a

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regulatory protein behaves like a typical periplasmic binding protein recognizing phosphate (Pardee, 1968). Mutations in R2a render the cell constitutive for alkaline phosphatase (Garen and Otsuji, 1964). However, as recently shown by Willsky et al. (1963), it appears that the R2a protein is involved in phosphate transport and represses the synthesis of alkaline phosphatase only indirectly, i.e., by accumulating phosphate inside the cell, which in turn represses synthesis of alkaline phosphatase. A related phenomenon can be observed with the galactose-binding protein in the MeGal transport system. Endogenous induction of the gal operon requires a functional MeGal system to prevent leakage of the internal inducer galactose (Wu, 1967; Wu et al., 1969). Thus binding proteins might be involved in regulation by controlling internal inducer or corepressor concentrations, rather than by being involved on the level of transcription or translation. C. Coregulation of Binding Protein Synthesis and Transport Activity

If the substrate-binding protein-mediated process establishes the ratelimiting step in translocation of the substrate through the membrane, one would predict that the rate of transport is reflected by the amount of binding protein present. Thus binding protein synthesis and transport activity should be coregulated. Indeed, with few exceptions (Guroff and Bromwell, 1970) this observation has been made in several systems (Penrose et al., 1968; Berger and Heppel, 1972; Hogg and Englesberg, 1969; Ames and Lever, 1970; Ohta et al., 1971; Pardee et al., 1966). Moreover, mutants that showed a simultaneous increase or decrease in both binding protein synthesis and transport activity (initial rate of uptake) have been isolated in several binding protein-mediated systems (Ames and Lever, 1970; Ohta et al., 1971; Pardee et al., 1966; Krajewska-Grynkiewicz et al., 1971). For the MeGal system it also has been observed that transport activity is a function of the amount of binding protein found per cell. This correlation is true for a variety of different inducible and constitutive mutants (Lengeler et al., 1971). This coregulation, even though necessary for essential participation of the galactose-binding protein in the translocation step, certainly does not prove such a correlation. A trivial explanation for the observed coregulation would be that the gene loci for the galactose-binding protein and the MeGal system occupy the same operon without being involved in a common function. By the same reasoning the finding of a close linkage between a gene necessary for galactose-binding protein synthesis and the mgl gene would be a necessary requirement for, but does not prove the function of, the galactose-binding protein in the MeGal system (Boos and Sarvas, 1970).

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D. Combination of Genetic and Biochemical Evidence

The strongest evidence for participation of the galactose-binding protein in the MeGal transport system comes from a combined genetic and biochemical approach. From a wild-type strain, positive in transport and galactose-binding protein, an m g l mutant was isolated (screening for the inability to accumulate low concentrations of external galactose) with a

SUGAR CONCENTffATION,

"0

1

2

3

4

5

5

molar

7

8

9

3

t / SUGAR CONCENTffATION, [ i / M ] . 10'

FIO.19. Percentage of increase in fluorescence of the mutant galactose-binding protein of strain EH3039 versus total sugar concentration. Protein concentration was 16 pg/ml in 10 mM tris-HC1 (pH 7.3). Excitation wavelength was 290 nm a t a slit width of 4 nm; emission wavelength was 336 nm for the protein of strain EH3039, and 330 nm for the protein of the wild type, W3092cy-, and the revertant, LA39. The emission slit was in all Galactose, protein of mutant strain EH3039; cases 10 nm. Temperature was 26". 0, 0 , glucose, protein of mutant strain EH3039; A, galactose, protein of revertant strain LA39; - - -, galactose, protein of wild-type strain W3092cy-. (A) Plot of percentage of increase of fluorescence versus sugar concentration. (B) Plot of inverse percentage of increase of fluorescence versus inverse sugar concentration. (Taken from Boos, 1972.)

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defect in the primary structure of the binding protein. This protein shows the same binding specificity as the wild type but has a 7000-fold reduced binding affinity. From the ultraviolet difference spectrum in the presence and absence of galactose, the alteration in the active site can be directly demonstrated (Fig. 16). A fingerprint of the tryptic digest, when compared with the fingerprint of the wild-type protein, shows that the mutation has indeed caused an alteration in the polypeptide chain. Reversion of the transport-negative mutant by selecting for a transport-positive phenotype yielded a galactose-binding protein with nearly wild-type properties. A comparison of the MeGal system activity and the binding affinity as measured by fluorescence increase of the purified proteins of all three strains is shown in Fig. 19. The correlation of both phenomena is obvious. Therefore the galactose-binding protein must be an essential component of the MeGal system (Boos, 1972). With a similar approach the essential function of the J protein (a specific L-histidine-binding protein) in a highaffinity histidine transport system of s. typhimurium has been demonstrated (Ames and Lever, 1972).

V. THE INVOLVEMENT OF THE GALACTOSE-BINDING PROTEIN IN CHEMOTAXIS

Kalckar (1971) first suggested that the galactose-binding protein might be involved in another phenomenon related to transport, namely, chemotaxis. Indeed, close examination by Hazelbauer and Adler (1971) of the specificity of several independent chemoreceptors showed a striking similarity of the galactose chemoreceptor to the galactose-binding protein and the MeGal system. Moreover, mutants lacking the galactose-binding protein also lack chemotaxis toward substrates recognized by the galactose chemoreceptor. Reversion of these mutants to a chemotaxis-positive phenotype shows simultaneously the appearance of a normal galactose-binding protein. Also, the examination of galactose chemotaxis in the three strains used t o prove the function of the galactose-binding protein in the MeGal system yielded similar results (J. Adler and co-workers, unpublished data). Thus the mutational alteration of the galactose-binding protein is accompanied by a simultaneous change in galactose chemotaxis. However, the first assumption, that galactose chemotaxis would be dependent on a functional MeGal system, turned out to be incorrect. Some mgl strains possessing an apparently normal galactose-binding protein were still fully active in chemotaxis. However, Hazelbauer and Adler (1971) isolated a mutant, AW551, with normal binding protein and MeGal system but

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defective in chemotaxis toward galactose. The galactose-binding protein was then interpreted as a necessary coniponent for both phenomena, acting as the recognition site for galactose chemotaxis as well as galactose transport. The niolecular mechanism by which the galactose-binding protein transfers the recognition event to the chcmotactic response is a t the present time largcly unknown, even though a response based on a memory mechanism (Macnab and Koshland, 1972) or spatial orientation (Berg and Brown, 1972) has been proposed for other chemoreceptors. The correlation of pcriplasmic binding proteins with chemotaxis does not swni to be a gcnchral one. A search for the possiblc role of other periplasrnic binding proteins in chemotaxis has shown that only one other periplasinic binding protein, a ribostl-binding protein of S. typhiinunum (Aksamit and Iioshland, 1972), might br involved in the chemotaxis toward ribosc. In contrast, the serinc and aspartate chemoreceptors of E. coli have no counterparts in soluble binding proteins and, vice versa, no chcniotaxis has been observed with histidine and lysine as attractants (Mesibov and Adlcr, 1972), even though binding proteins for these amino acids have been isolated (Lever, 1972; Kosen, 1971a). Also, an exclusive function of periplasniic binding proteins as the chemoreceptors is not warrantcd. It was recently obscrvrd that certain components of the PTS (EII’s) arc likely to be involved in the chemotaxis toward their respective substrates (Adler et n l . , 1973).

VI. REGULATION OF THE MeCSal SYSTEM AND OF THE GALACTOSEBINDING PROTEIN SYNTHESIS BY EVENTS OCCURRING DURING THE BACTERIAL CELL CYCLE

Studios by J. Lengrler, K. 0. Hcrman, and H. J. Unsold (unpublished) on the MeGal system have indicated that specific transport activity (active transport of galactose) exhibitc,d large fluctuations during logarithmic growth of the bacterial culturc. Several possible explanations of this phenomenon were proposed : (1) periodic alteration of the physiological state of the bacterium, affccting its ability to supply the energy to the rather 16 expensive” active transport systrm, or pcriodic alterations in feedback regulation of transport activity; (2) periodic synthesis or degradation of the transport system components during the bacterium’s cycle; (3) periodic assembly of these coniponents during thr bacterium’s cell cycle. With a sensitive inimunoassay we demonstrated that the alteration of transport activity mas due to changes in the level of the galactose-binding protein during the cell cycle of the bacterium. By pulse-labeling with

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125

radioactive amino acids and measuring specific incorporation of radioactivity into the galactose-binding protein in synchronized cells we demonstrated (Fig. 20) that binding-protein synthesis occurs only during or shortly after cell division but not while thr cell elongates without dividing. Furthermore, galactose-binding protein synthesis is shut off in a temperature-sensitive cell division mutant when the growth temperature is shifted from the permissive to the nonpermissive temperature. Shifting from the nonpermissive to the permissive temperature allows galactose-binding protein synthesis to proceed after cell division has occurred. In the wildtype, as well as the temperature-sc.nsitivc cell division mutant, the MeGal systcm activity follows closely alteration in the binding-protein synthesis (Shen and BOOS,1973). These observations indicate that, in addition to its regulation by the mglR gene with fucose and galactosc as inducers, thc MeGal system is under the control of events occurring during thc cell cycle of the bacterium. Alterations in L-a-glycerol phosphate uptake and cytochrome b l levels in synchronized cells of E. coli have been interpreted in the same way (Ohki, 1972). Also, from their studies with synchronized and synchronously growing cells, Kubitschek et al. (1971) have concluded that the number of carrier or binding sites for several transport systems per cell stays constant. Doubling of this number occurs a t a certain point in the bacterium's cycle, which can to sonic extent be manipulated by experimental conditions. Possibly, these observations have a common origin indicating a different regulatory mechanism for the synthesis or the in vivo assembly of a certain class of membrane or surface-related proteins.

FIG.20. Net transport activity and galactose-binding protein synthesis in synchronized cells of W3092cy-. Cells were grown in minimal medium A containing 0.4% glycerol a t 37" after inoculation froni a late stationary phase culture into warmed fresh medium. (A) Growth was followed by measurement of Klett units. (B) Cell number was measured by plating on nutrient broth agar plates. (C) For the transport assay [1-'4C]galactose, 0.5 p M final concentration, was added directly to 2 ml of the culture a t 35' and 0.5-ml aliquots were filtered without washing. Transport activity is given as initial rate of galactose uptake per 0.5-ml aliquot of the culture. (U) To 5-ml aliquots of the culture, 5 X 1 0 5 cpm of [U-l4C]leucine and CIJ-'4C]alanine, final concentrations 0.2 p M , were added for 5 minutes a t 37" before addition of 1 mM unlabeled amino acids. The culture was spun and resuspended in 50 pliters of 10 mM tris-HCI (pH 7.3);5 pliters of purified galactose-hinding protein (1.0 nig/ml) and 5 pliters of toluene-chloroform (1:1, v, v ) were added. The mixture was incubated for 1 hour a t 37" and applied directly on the diffusion plate. Staining was performed as descrilied. (E) The stained plate was dried (the agar attains the consistency of a thin foil) and exposed to x-ray film (Kodak, RP/ R.54) for 8 days. (Taken from Shen :mil Boos, 1973.)

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VII. PRO AND CONTRA CARRIER FUNCTION OF PERIPLASMIC BINDING PROTEINS

From the foregoing discussions of the properties of the galactose-binding protein and of the evidence of its function in the MeGal transport system, it is obvious that a t the present time it cannot be determined whether or not the galactose-binding protein or any other periplasmic binding protein acts as a carrier in the classic sense, i.e., whether or not it facilitates translocation of the substrate through the cytoplasmic membrane. All available results give information which (with some effort) could be interpreted in both ways. The easy release of binding proteins from the cell surface by osmotic shock has always been held to be a strong argument that these binding proteins could not carry out a ‘(membrane function.” Yet, the definition of membrane-bound protein is almost a matter of opinion (Guidotti, 1972), and the fact that these proteins are released by osmotic shock does not prove that they have no interaction with the cytoplasmic membrane in vivo. One interpretation of the function of binding proteins often heard is that they might facilitate transport of substrate through the periplasmic space and deliver it to the “permease proper” located in the cytoplasmic membrane. Yet, after removal of binding proteins for substrates transported by a kinetically homogeneous system (glutamine, arginine, diaminopimelic acid), no binding for these substrates in isolated membranes could be found, nor could transport be demonstrated. This observation argues that there is indeed no “second transport system,” or a t least no membranebound recognition site that takes over after accumulation of the substrate in the periplasmic space. Restrictions on the functions of periplasmic binding proteins to operate as reversibly translocating carriers come from the observation that the galactose- and the glutamate-binding proteins may function only in the entry but not the exit process. Other observations indicate that periplasmic binding protein-mediated transport systems are, unlike the lactose transport system, composed of more than one component. In almost all systems mutants have been isolated that apparently contain normal binding protein, even though they are defective in transport; also, transport is inhibited by PHMB, even though the binding activity of the purified binding protein is not. Restoration of transport activity by addition of purified binding proteins to isolated membranes or shocked cells, which would be strong support for a carrier function of binding protein, has never been demonstrated in an unambiguous way. But again, this does not prove the contrary.

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PRO AND CONTRA CARRIER PROTEINS A

I

Galactose molecules

Cell interior

0

FIG.21. Model for translocation of galactose across the cell membrane. GBP, Galactose-binding protein with two binding sites, h, arid b?; 15, high-mr)lecular-weight effector. See text for details of this model. (Taken from Rotman and Ellis, 1972.)

To conforni to Harold’s opinion of the behavior of a certain class of scientists connected with transport studirs (Harold, 1972), we will not end this chapter without presenting a transport model. Thus Fig. 21 shows a model recently proposed by Hotnian and Ellis (1972), which conies closest to what we do not know about periplasmic binding protein-mediated transport. A high-molecular-weight rffrctor u hich is membrane bound recognizes not free substrate but rathrr the binding protein-substrate complex. The membrane-bound effector then translocates the binding site and reduces its affinity for substrates. In our terms, the effector binds state I1 of t he galactose-binding protein complexed with galactose and converts it to state I (the rnergy-requiring step). Subscqucntly, galactose is liberated into the cytoplasm and the state-I galactose-binding protein into the periplasni, where it attains an equilibriuni between states I and 11, dcpending on the substrate concentration. To account for the lack of galactose-binding protein-mediated r.xit, onc would h a w to postulate that state I does not hind galactose nt all, an implication that is indicated by solvent perturbation studies (McGowan et d ,1974). In summary, one might say that it has been established, a t least for some systems, that binding proteins are integral parts of bacterial transport systems, even though their actual function in the translocation step remains to be elucidated.

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Hirata, H., Altendorf, K. H., and Harold, F. (1973). Proc. Nut. Acad. Sci. U.S. 70, 1804-1 808. Hochstadt-Ozer, J. (1972). J . Biol. Chem. 247, 2419-2426. Hochstadt-Ozer, J., and Stadtman, E. R. (1971). J . Biol. Chem. 246, 5304-5311. Hofer, M. (1971). Arch. Mikrobiol. 80, 50-61. Hogg, R. W. (1971). J . Bacteriol. 105, 604-608. Hogg, R. W., and Englesberg, E. (1969). J . Bacteriol. 100, 423432. Holden, J. T., and Bunch, J. M. (1972). Biochern. Biophys. Res. Commun. 46, 437442. Holden, J. T., Utech, N. M., Hegeman, G. I)., and Kenyon, C. M. (1973). Riochem. Biophys. Res. Commun. 50, 266-272. Hong, J. S., and Kaback, H. R. (1972). Proc. Nut. Acad. Sci. U . S . 69, 3336-3340. Hong, J. S., and Kaback, H. It. (1973). I n “Chemical Rubber Company Critical Reviews in Microbiology” ( A . L. Laskin and H . Lechavalier, eds.), Vol. 2, pp. 333-376. Chem. Rubber Publ. Co., Cleveland, Ohio. Hopfer, U., Lehninger, A. L., and Thompson, T. E. (1968). Proc. Nut. Acad. Sci. U S . 59, 484-490.

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Kaback, H. R . (1971). In “Enzyme Purificalion and Related Techniques” (W. B. Jakoby, ed.), Met,hods in Enzymology, Vol. 22, pp. 99-120. Academic Press, New York. Kaback, H. It. (1972). Biochim. Riophys. Acta 265, 367416. Kaback, H. It., and Barnes, E. M., Jr. (1971). J . B i d . Chem. 246, 5523-5531. Kaback, H. It., arid Milner, L. S. (1970). Proc. N a f . Acad. Sci. U S . 66, 1008-1015. Kalckar, H . M. (1971). Science 174, 557-565. Kamogawa, A., arid Kurahashi, K. (1967). J . Biochem. (Tokyo) 61, 220-230. Kashkct,, F:. It., and Wilson, T. H. (1972a). Hiochem. Biophys. Res. Commnn. 49, 615620. Kashkct, E. It., and Wilson, T. H. (1972b). J . Bacleriol. 109, 784-789. Kashket,, E. R . , arid Wilson, T. H. (1873). Proc. Nut. Acad. Sci. U.S. 7 0 , 2866-2869. Kennedy, R. P. (1970). In “The Lactose Operon” ( J . R . Beckwith and I). Zipser, eds.), pp. 49-92. Cold Spring Harbor I,&., Cold Spring Harbor, New York. Kennedy, E. P., liumley, M. K., arid Armstrong, .J. B. (1974). J . Biol. Chem. 249, 33-37. Kepes, A. (1970). Curr. T o p . Membranes Tm nsp. 1, 101-134. Kcpcs, A. (1971). J . Menahrane B i d . 4, 87-1 12. Kepes, A., and Autissier, F. (1972). Niochim. Biophys. Acla 265, 443-469. Kepes, A., arid Richarme, G . (1972). Fed. E u r . Riochem. Soc., Proc. 28, 327-338. Kerwar, (2. K., Gordon, A. S., and Kaback, H. R . (1972). J . Rid. Chent. 247, 291-297. Kimmich, G., Tucker, A . M., Barrel,, E,, and Ir,lt. (1972). FEBS (Fed. Eur. Biochenz. Soc.), Lett. 19, 31 1-314. Koch, A. L. (1964). Bi ochi ~tBiophys. . A d a 79, 177-200. Koch, A. L. (1971). J . hid. R i d . 59, 447-459. Komor, E., Haass, D., arid Tanner, W. (1972). Biochini. Biophys. Acta 266, 6494360. Konings, W. N., arid Frewe, E. (1972). J . Hiol. Chem. 247, 2408-2418. Konings, W. N., arid Kaback, Iisequilibriuin Method . . . . . C. Limitations of COPGradient Method . . . . . . . . 1) . COPProfiles . . . . . . . . . . . . . . . E . Acidification Coupling . . . . . . . . . . . . F . Leakiness to H and HCOI . . . . . . . . . . . G . Steady States of Minimal Lurninal pH . . . . . . . . H . COz Balance and Permeability . . . . . . . . . . I1. Acidification of the Lumirial Fluid by the 'I'urtle Bladder . . . . A. HCO, Transpnrt (Pro and Con) . . . . . . . . . . B . Ilependence of Acidificatioii on the Presence of Luininal HCO, . C. Acidification Rate as a Function of Lurninal [HCO,]. . . . 1). Conflicting Data . . . . . . . . . . . . . . E . Summary . . . . . . . . . . . . . . . . 111. The Renal Mechanism of Acidification . . . . . . . . . A . Background . . . . . . . . . . . . . . . R . The in-Milu Concentration of Luniinal COZ . . . . . . C. Apparent Reartion Disequilibrium . . . . . . . . . 1) . Distal Tubular hcidification . . . . . . . . . . . E . Reviewers' Bias on Renal Ac.idifiration . . . . . . . . References . . . . . . . . . . . . . . . . .

* Recipient

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. . . .

. . .

. .

. . . . .

. . . . .

162 . 182 . 164 . 165 . 167 . 17'2 . 176 . 183 . 186 . 190 . 193 . 193 . 197 . 201 . 204 . 205 . 205 . 205 . 207 . 212 . 215 . 221 . 223

of NIH Career Uevelo~mentAward K 04.GM.42431 . 161

162

WILLIAM A. BRODSKY AND THEODORE P. SCHllB

PREFACE

The present review is restricted to those articles wherein a serious attempt has been made to identify an acidification process as active transport of H into the fluid undergoing acidification, or as active transport of HC03 out of the fluid undergoing acidification. For a general review of the field of renal acidification, the reader is referred to the reviews by Pitts (1968), Berliner and Orloff (1956), Brodsky and Carrasquer (1961, 1962), Rector (1971), and Malnic and Giebisch (1972). For a general review on gastric acidification, the reader should consult the reviews of Heinz and Obrink (1954), Forte (1971), and Rehm (1972). For recent information on acidification in the stomach, intestine, pancreas, bladder, and kidney, the reader is referred to the symposium edited by Sachs et al. (1972). Special information on the role of carbonic anhydrase in several acidifying tissues (kidney, stomach, pancreas, ciliary body, and so on), appears in the review of hlaren (1967). The term active acidification, as used hereafter in this article, is defined as a luminal acidification mechanism that decreases the concentration of HCO3 in the luminal fluid to a level less than that in the interstitial fluid. Our definition is deliberately looser than the classic one of Rosenberg (1948) in that a decrease in luminal [HCO,] solely on account of the electronegativity of the luminal fluid is considered active in the context of this report, but not by the Rosenberg criterion. This is because effective net acidification of the luminal fluid takes place as long as the chemical concentration of luminal HCO, is reduced-even if the transmembrane electrical potential is the only force driving the HC03 ions out of the luminal fluid. When the concentration of luminal HC03 remains fixed or increases during reabsorption, factors such as bulk flow and sieving complicate the interpretation of methods currently used to identify the acidification mechanism. 1. GENERAL CONSIDERATIONS

Although it has become almost axiomatic to equate the accumulation of acid in secretory fluids with the transport of H ions from the cell, hard evidence for the existence of such proton transport is still lacking. As a matter of fact, evidence has been reported for the existence of an alternative mechanism of acid accumulation, namely, the transport of HCOI ions as such from the luminal fluid to the interstitial fluid in the urinary bladder of freshwater turtules (Schilb and Brodsky, 1966, 1972; Brodsky and Schilb, 1967, 1972).

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Similarities. Both mechanisms, H secretion and HCO3 reabsorption, result in the accumulation of H ions in the luminal fluid together with the delivery of HCO3 ions into the interstitial fluid. Therefore they are stoichiometrically identical with respect to certain parameters of acid-base balance (e.g., pH, rate of accumulation of titratable acid) which are the strictly quantitative indices of any type of acidification process. Both processes require active transport mechanisms in order to drive either the H or the HC03 ion against its transcellular gradient of electrochemical potential energy-as observed in systems such as the turtle bladder and the mammalian kidney and stomach. Therefore the acidification rate ought to be retarded if the transcellular gradient of electrochemical potential (of H and/or HC03) is oriented against the direction of flow of the transported ion, or accelerated if the gradient is oriented along the direction of flow of the transported ion. The passive permeability (leak parameters) of the membrane to the transported species ought to be less than that of the membrane to COZ. I n common with any active mechanism of ion transport, an active acidification mechanism is expected to be: ( 1 ) metabolically dependent; (2) located in a spatially oriented manner within one of the plasma membranes; and, consequently, (3) regulated by metabolically reactive hormones, extra- and intracellular electrolytes, drugs, and changrs in the activity of membrane-bound, transport-related enzymes. Therefore none of the properties associated with acid-base stoickiometry, active transport, metabolism, or membrane pcrmeability can be used to distinguish active transport of H in onc direction from active transport of HCO, ions in the opposite direction. In addition, the electrophysiological parameters (orientation of the transepithelial potential and short-circuiting current) are the same for both mechanisms. Diflerences. A fundamental diff erencc between the two mechanisms is the site of origin of the protons that accumulate in the luminal fluid during its acidification. In HCO3 reabsorption the protons originate in thc luminal fluid from the HzO molecules which react with COz (diffusing into the lumen from the cell) to form H&03, the dissociation of which (into H and HCO, ions) is initiated and maintained by the pump-induced removal of luminal HCO3. I n H secretion, the protons originate in the cell fluid from which they are made available to the pump mechanism-either in the form of free protons or in the form of H atoms which can be converted to free protons prior to secretion via the pump mechanism. This article deals with the theoretical and experimental methods used to distinguish one type of acidification mechanism from another-first, in a general sense; second, in the particular case of the reptilian bladder; and third, in the particular case of thc mammalian renal tubule.

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WILLIAM A. BRODSKY AND THEODORE P. SCHILB

A. Net Direction of luminal Reaction

The net accumulation of acid in any luminal fluid containing COZ and HC03 must start wifh a shift from the equilibrium position of the COzH&03 reaction in that luminal fluid. A secretory transport of H ions into the fluid induces an off-equilibrium state of the form H

+ HCOI S HzCO, ZHzO + COz

where the larger of the paired arrows denotes the direction of the luminal reaction. In contrast, a rcabsorptive transport of HC03 ions from the lumen induces an off-equilibrium state of the form HCOI

+H

HzCOs C HzO

+ COz

whcre thc larger of thc paired arrows denotes the direction of the HC03induced reaction which is opposite that of the H-induced reaction. In principle, if one knows the direction of displacement from equilibrium of the HzC03-C02reaction in the luminal fluid, one can identify the acidification mechanism as one of H ion secretion or as one of HC03 ion reabsorption (Brodsky, 1955; Brodsky et al., 1958; Kaim and Brodsky, 1959; Schilb and Brodsky, 1966; Brodsky and Schilb, 1967). Two experimental methods h a w been developed to determine thc direction of displacement of the C02-HzC03reaction from its equilibrium position: (1) the disequilibrium pH method (Walser and Mudge, 1960; Rector et al., 1965), and (2) the COz gradient method (Brodsky, 1955, 1958; Schilb and Brodsky, 1966). The disequilibrium pH method depends on the change in luminal pH from its in situ off-equilibrium level to its equilibrium level reached when the acidified fluid is isolated from the acidifying process. This method is applicable when the acidification rate is fast enough to exceed the rate a t which HzC03 reaches equilibrium with COz, and has been used extensively in studies on the renal tubule. The COZ gradient method depends upon the direction of change in the [COJ of the luminal fluid during its acidification, and consequently on the direction of the lumen-to-interstitial gradient of [COJ which develops. The method is applicable when the rate at which the luminal COZ-HzC03 reaction approaches equilibrium is faster than the rate of acidification. This is the case in the turtle bladder in which the rate of acidification is slow relative to that of the uncatalyzed rates of the COZ-HzC03 reaction (Brodsky and Schilb, 1972; Schilb and Brodsky, 1972), and possibly in the rat proximal tubule in which carbonic anhydrase may be available for the luminal C02-HzC03 reaction (Walser and Mudge, 1960; Rector et al.,

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165

1965)-which would ensure that the rate of transport is slow relative to that of the CO2-HzCO3reaction. 6. limitations of the pH Disequilibrium Method

The existence of an off-equilibrium state of the C02-H2COa reaction in the luminal fluid requires that the rate of acidification be greater than the rate of COZevolution in the case of H secretion, or greater than the rate of COZhydration in the case of HCO, reabsorption. The determination of such a state can be made from the direction of change in the pH of the luminal fluid after its isolation from the acidification mechanism. A proper isolation technique would include a sealed, gas-tight system for transfrrring the luminal fluid and for measuring its pH after the COz-H2C03 reaction in the sealed system has gone from an off-equilibrium to an equilibrium state. The direction of the p H change in going from the off-equilibrium level in the lumen to the equilibrium level in the isolated closed system is determined b y the nature of the in-situ acidification process as follows. 1. HCO:, PUMP

The rate of HCO, reabsorption must be fast enough to force the conccntration of H&OS to a value simificantly lower than that required to be in equilibrium with the prevailing COz concentration in the luminal fluid. Now, if the luminal fluid were suddenly transferred from the surrounding epithelial cell layer to a completely closed, perfectly impermeable container, the hydration of COz and the dissociation of H2C03 into hydrogen and bicarbonate would proceed until the constituents reach n. state of reaction equilibrium in the closed container. In this equlibrium state, the p H of the isolated luminal fluid system would be lcss than that of the same sample of luminn.1fluid while it had been in contact with the epithelial cell. 2. H-PUIIP

The rate of H secretion must be fast enough to force the concentration of H&O3 to a value significantly greatrr than that required to be in equilibrium with the luminal [CO,]. In this case, after transfer of the luminal fluid into thc gas-tight container, thv reaction proceeds to a final equililibrium while the p H of the isolated fluid increases with respect to its initial in situ level. In the published work concerning the existence and direction of a n offequilibrium pH in the renal tubule, the technique of the sealed system has not been used. Instead, the in situ p H is first determined. Then the luminal

166

WILLIAM A. BRODSKY AND THEODORE P. SCHllB

fluid is withdrawn, equilibrated under oil with 5% COZ,and the in vitro pH determined to provide a measure of the luminal HCO, concentration. However, the in situ concentration of the luminal COZis assumed, not measured. Therefore, the calculation of the equilibrium pH pressed in the form

which depends on the validity of the assumption that [CO& (assumed) = [CO,] (arterial plasma)

-an assumption made under any or all conditions in those parts of the renal tubule which are accessible to micropuncture from the cortical surface of the in situ kidney (Rector et al., 1965; Vieira and Malnic, 1968). With this assumption taken for granted, a calculated equilibrium pH has been compared with the measured intraluminal pH ( ~ H L )The . usual ~ interpretation is based on three possibilities: (1) PHL < p H e q , (calc.), which would be interpreted as H secretion; (2) pHL > ~H,,,L (calc.), which would be interpreted as HCO3 reabsorption or as COz diffusion from cell to lumen; and (3) ~ H =L pHeq,L(calc.) , which would be interpreted as an equilibrium level of the luminal pH, in which case the method could not be used to determine the direction of the intraluminal COz H2CO3 reaction. The limitations of the oil immersion technique become apparent [on the basis of Eq. (1)] when the concentration of COz is not really equal to, but is assumed equal to, that in the arterial plasma. The in situ luminal p H can be expressed in the general form PHL (meas.)

~ H , , , L(true) f ApH

(3) where the pH terms are self-explanatory and f A p H is the degree of the off-equilibrium state of the pH. By definition, pHeq,L (true)

=

=

pK,’

(meas.) + log [HCOJL [C OZ]L (true)

(4)

.eq

Substituting the defined value of p H e q , (true) ~ into the right side of Eq. (3), and then subtracting Eq. (1) from Eq. (3) yields the expression ~ H (meas.) L

- ~ H L , , (calc.) ,

= log

[CO,] (assumed) [COz]~,eq (true)

f

APH

(5)

which is the analytical form of what micropuncture physiologists have

HYDROGEN SECRETION VERSUS BICARBONATE REABSORPTION

167

actually measured, and which explicitly demonstrates the relation between the commonly used assumption on luminal [COJ and the degree of the off-equilibrium state of the pH. If there were no pH disequilibrium in the luminal fluid, the term ApH would vanish, and the measured ~ H would L be the true equilibrium pHL. But the pH difference on the left sidr of Eq. ( 5 ) may or may not vanish, depending on the choice of the assumed value for [CO,]. For example, in the p H disequilibrium produced by an active HC03 reabsorption, ~ H (measured) L must be greater than ~ H L , , ,(true), whence ApH in Eq. ( 5 ) is a positive number. However, if [CO& (assumed) were less than [COJ (true), the pH difference on the left side of Eq. ( 5 ) could vanish which would suggest that an equilibrium state prevailed in the luminal fluid when in reality there was a disequilibrium state due to a HCO, pump. In conclusion, the p H disequilibrium method, as now practiced, has little or no value for establishing the existence of an off-equilibrium state-let alone its direction. C. limitations of CO2 Gradient Method

The direction of the shift from equilibrium of the HzC03-C02 reaction in the lumen corresponds uniquely to t h r nature of the acidification mechanism, which implies that the luminal [CO,] ought to increase with H secretion, or decrease with HCO, reabsorption. However, this is not always the case, except in a CO2-impermeable system (Brodsky and Schilb, 1967; Schilb and Brodsky, 1972). In a COz-permeable system the directional change in luminal [COJ can sometimes be opposite that being induced by a particular acidification mechanism. For example, the luminal [CO,] can decrease during H secretion into a HC03-poor luminal fluid, or increase during HCO, reabsorption from a HC03-rich luminal fluid. As a matter of fact, the luminal [CO,] could increase in a system with no active acidification mechanism a t all. Such anomalous directional changes are occasioned by: (1) comparing the level of C 0 2 in the luminal fluid with that in the interstitial fluid rather than with that in the cellular fluid; and (2) setting the initial level of luminal COz equal to that in the interstitial fluid-as is conventionally done in experiments on the turtle bladder (Schilb and Brodsky, 1966, 1972; Green et al., 1970), or as happens naturally in the proximal renal tubule a t the site of entry of the glomerular filtrate. If it were possible to set the initial lcvrl of COz in the lumen equal to that in the cell, the luminal COz would remain unchanged in a nonacidifying

168

WILLIAM A. BRODSKY AND THEODORE P. SCHILB

system, would increase to a level greater than that in the cell during H secretion, and would decrease to a level less than that in the cell during HCO, reabsorption. But since it is not always possible to know the level of COzin the cellular fluid, the change in luminal [COZ] a t a given time is usually compared to the initial level of luminal COz, which in turn is set equal to that in the interstitial fluid. If it is assumed that the level of COZ in the cell is greater than the initial level in the luminal or interstitial fluid, the subsequent changes in luminal [GOz] can be predicted in a system with no acidification mechanism, in a system with active secretion of H ion, and in a system with active reabsorption of HCO, ion. I . No ACIDIFICATIONMECHANISM

I n an epithelial system with no active acidification mechanism and with few or no passive leaks of H or HCOI between the cellular and luminal fluids, the concentration of COz in the cellular fluid [COZlcis greater than that in the interstitial fluid [COZ],, and consequently greater than the initial level in the luminal fluid [COZlL,or

[coz]c.o > [COZ]L.O

=

[COZl8,"

(6)

where the subscripts c, L, or s denote the fluid compartment and the subscript 0 the initial time ( t = 0 ) , and where the volume of interstitial fluid (s) is considered to be that of an infinitely large, well-mixed reservoir, while that of the luminal (L) or cellular fluid (c) is considered to be small. Therefore [ C 0 J s is constant at all times, whiIe [COZlL varies with time. Under the initial conditions ( t = 0) described by Eq. ( 6 ) , the metabolic COZforming a t a constant rate Jg?: in the cell diffuses into both the luminal and the interstitial fluid, or

JZ::

=

J::'

+ J:"

(7)

where and JEo2 denote the rates of diffusion from cell to lumen and from cell to interstitial fluid, respectively, and where steady-state conditions prevail in the cell but not in the system as a whole because [C0zlc is assumed constant while [COZlLvaries with time. The portion of the COZ that diffuses into the interstitial fluid is washed away as soon as it enters thk large, well-mixed reservoir, while the portion that enters the luminal fluid accumulates therein until the concentration in the luminal fluid approximates that in the cell. When the concentration of Cot in the lumen fluid becomes equal to that in the cell ( t = m ) , steady-state conditions will prevail for the systnm as a whole, and thus [COZlc,w 2 [CO21L,m > [COZ18,W (8)

H Y D R O G E N SECRETION VERSUS BICARBONATE REABSORPTION

169

where [ C O Z ] ~ = , ~[COZ]~,~, and [C02]a,m = [COZ],,O;and where the steadystate level of C02 in the lumen is a good measure of that in the cell (see Fig. 5 ) . Therefore, under steady-state conditions for the system as a whole, and which means that all the metabolically produced COZ diffuses into the interstitial fluid under the steady-state conditions described by Eq. (8). Since [CO&,,m> [C02]L,o, and sincc there is no change in the luminal [HCO,] in a system with no acidification mechanism, the pH of the luminal fluid inevitably decreases between 2 = 0 and t = 00 to become less than that of the serosal fluid. In short,, the luminal fluid is acidified, even though there is no mechanism for either H secretion or HCO3 reabsorption. Moreover, the combination of a decrease in ~ H and L a n increase in [COJL, which occurs in a system with no active acidification mechanism, also occurs in a system with H secretion. A distinction between the two systems can be made by the change (or lack of change) in [HCO,]L, which decreases in a system with an active acidification mechanism but which undergoes no change or increases in a system with no active acidification mechanism. 2. H SECRETION

Whenever the concentration of luminal HCO3 is less than that of H during acidification of the luminal fluid, a concomitant decrease in the concentration of COz is just as compatible with H secretion as it is with HC03 reabsorption. Figure 1 shows how the secretion of H into a HCOe-poor luminal fluid is followed by a decrease (rather than an increase) in the initial or presecretory concentration of luminal C02. When the number of H ions secreted ( n ) exceeds the total amount of luminal HC03, the amount of luminal COz generated must be less than the equivalent number of OH ions ( n ) left behind in the cell as a consequener of the H secretion. The OH ions so accumulated in the cell react with CO,, lowering its concentration in the cell. When the cellular [CO,] decreases to a level less than that of the initial luminal [CO,], a lumen-to-cell diffusion of C02is initiated. The result is a lowering of the luminal [CO,]. However, lowering of the luminal [C02] is also a consequence of HCO3 reabsorption. Therefore the lowering of [CO,] during the acidification of a HC0,-poor luminal fluid can be a consequence of either H secretion or HC03 reabsorption, so that data from thc COz gradient method become

170

WILLIAM A. BRODSKY AND THEODORE P. SCHILB

ISF

FIG.1. H secretion. Events in the lumen and in an epithelial cell which actively secretes n H ions into the lumen and leaves an equal number of OH ions in the cell. The extra COZgenerated in the lumen diffuses into the cell. The n OH ions accumulating in the cell react with n COZ molecules to form n HCOI ions which diffuse from the cell into the interstitial fluid. In a HCOI-free luminal fluid, the H secretion process induces a decrease in the initial level of luminal COZ (see text).

equivocal with respect to identification of the acidification mechanism under these conditions (i.e., [HCO~IL [COZ],,) . Since a positively oriented COz gradient would be the inevitable result of luminal acidification in a bladder bathed by COz-free serosal fluid, Schilb and Brodsky (1972) performed experiments on bladders bathed by high (2.0 mM) and low (1.3 mM) levels of serosal CO,. In both groups of

HYDROGEN SECRETION VERSUS BICARBONATE REABSORPTION

195

bladders, the luminal levels of pH and HC03decreased to levels well below those in the serosal fluid. In both groups of bladders the final level of luminal COZ decreased to 1.47 mM. Thus the final transmural gradient of COz was negatively oriented (luminal < serosal) in the high serosal COz group, but was not significantly different from zero (luminal = serosal) in the low serosal COZ group. This finding resolved part of the difference in COz gradient measurements made by a pCOz electrode on the one hand (Green et al., 1970), and by the Henderson-Hasselbalch calculation on the other (Schilb and Brodsky, 1966, 1972; Brodsky and Schilb, 1967, 1972). The remainder of the apparent discrepancy in the data on luminal C02 was ascribed to the fact that one group (Green et al., 1970) had observed the acidification of alkaline luminal fluids (e.g., initial levels: pHL = 7.68; [HCOI]L = 23.6 mM), while the other (Schilb and Brodsky, 1972) had observed the acidification of acidic luminal fluid (e.g., initial luminal levels: PHL = 6.49; [HC031L = 4.28 mM). Testing the effect of these initial conditions, Schilb and Brodsky ( 1972) found that positively oriented transmural gradients of C02 could be reproduced a t will during acidification of HC03-rich luminal fluids. Under such conditions the magnitude of the maximal positively oriented gradient reported by Schilb and Brodsky (1972) was ca. 0.75 mM, and that reported by Green et al. (1970) was ca. 0.15 mM. In addition to the factor of the initial alkalinity of the luminal fluid, Schilb and Brodsky (1972) found that, the lower the serosal [COZ] and the lower the HCO3 transport ratc, the more positive the gradient of COP between the acidified luminal fluid and the serosal fluid. Low transport rates were achieved by incubating the sacs in Na-free media, and low transport clearances were found during acidification of HC03-rich, alkaline luminal fluids by bladders incubated in Na-rich media. The physical basis for the occurrence of such positive gradients in the presence of HC03 pumping has been discussed in Section I, E. The fact that acidification persists in Na-free luminal fluid has been found in turtle bladder (Gonzalez et al., 1967; Steinmetz et al., 1967) and rat proximal tubule (Solomon, 1966; Malnic and Giebisch, 1972), suggesting that there is little or no Na-H exchange involved in the acidification process. Figures 12 and 13, taken from the data of Schilb and Brodsky (1972) on turtle bladder sacs, show the relationship between the magnitude and orientation of the final transepithelial COz gradient and the transport rate expressed in terms of a normalized luminal clearance of HCO3. Both figures show that the gradients were positively oriented (luminal > serosal) a t

196

WILLIAM A. BRODSKY AND THEODORE P. SCHILB

FIG.12. Final transmural difference in COz concentration (mucosal (CO,] minus serosal [CO,]) versus normalized mucosal HCOI clearance (cubic centimeters per hour per 100 mg dry tissue) for 18 bladder sacs after acidification of mucosal fluid, while incubated in serosal fluid with a high concentration of free CO,.

low levels of HC03 clearance, occasioned by incubating the bladders in Na-free media with high initial levels (15 mM) of luminal HCO3. With higher levels of HC03 clearance and with lower initial levels of luminal HC03 (6 mM), COZ gradients became negatively oriented (luminal < serosal) during the luminal acidification. Comparing the two figures reveals that the lower the serosal [COZ] the greater the HCO3 clearance required for production of a negatively oriented gradient during acidification. Given that the COz gradients shown in Figs. 12 and 13 are valid, the development of negatively oriented COZgradients during luminal acidification constitutes unequivocal evidence for the active reabsorption of HCOI ions as such. Parenthetically, the negatively oriented COz gradients in Figs. 12 and 13 could not have been caused by active H secretion, because the concentration of HCOBwas always substantially greater than that of H in the acidified luminal fluid (see Section I, C).

FIG. 13. Final transmural difference in COZ Concentration (mucosal [COz] minus serosal [CO,]) versus normalized HCOa clearance (cubic centimeters per hour per 100 mg dry tissue) for 18 bladder sacs after acidification of mucosal fluid, while incubated in a serosal fluid with a low concentration of free Cot.

H Y D R O G E N SECRETION VERSUS BICARBONATE REABSORPTION

197

2. ESTIMATE OF CELLULAR [CO,]

A t the vanishingly low rates of HCOa reabsorption in some turtle bladder sacs (Schilb and Brodsky, 1972), the intraluminal [COZ] approximates a level 0.5 m M greater than that in the serosal fluid. This suggests that the luminal [COZ] increased to approximate the cellular [COzI-a confirmation of the previous prediction for a system with little or no active acidification (see Section I, D). Parenthetically, the readiness with which high levels of C 0 2 could be detected in acidified luminal fluids suggests that the low levels (detected under other conditions) are not spuriously induced by the loss of free COz during the handling of the samples for analyses. B. Dependence of Acidiflcation on the Presence of Luminal HCO,

The acidification of HCO,-free luminal fluid would constitute strong evidence against the HC03 reabsorption theory. With this in mind, Steinmetz (1967) and his colleagucs (Stcinmctz et al., 1967) observed the accumulation of H ions in a luminal fluid inztzally devoid of HC03 and COz. They showed that the net rate of arcumulation of luminal H ion (measured chemically) was equal to thc short-circuiting current density in turtle bladders bathed on both surfaces by Na-free Ringer’s solution in the absence of exogenously added COz and HCO,. Although substantial amounts of metabolic COZ were produced, thrse investigators claimed that little or no HCOs accumulated in the luminal fluid, and therefore concluded that the bladder is a H ion secret,or. However, it is inevitable that some HCO3 must form in a luminal fluid, which is statted a t a pH of 7.1 by the continuous addition of OH, and which is exposed to a continuous influx of metabolic COz. The extent of thc HCO, accumulation in such a luminal fluid can be estimated as follows (Brodsky and Schilb, 1972). Consider that the turtle bladder, mounted in an Ussing-Rchm chamber, is bathed on both surfaces by identical Ringer’s solutions, initially devoid of COZ and HCO, and gassed continuously with 100% 0 2 or with any suitable C02-free gas mixture. In the steady state the tissue produces q moles per hour of COZ ( $ 3 0 2 ) which are carried out of the tissue fluid system by the perfusing 0, bubbles. The serial path that must be taken by the metabolic CO2 in order for it to escape from the system includes the following sequence: the cell membranes, the unstirred boundary layers betwccn each o€ the cell membranes and the bulk solutions, the well-mixed, rapidly circulated bulk solutions through which the COz makes contact with the gas bubbles, the liquid boundary film surrounding O2 bubbles, the gas phase of the bubble, and

198

WILLIAM A. BRODSKY AND THEODORE P. SCHILB

FIG.14. Concentration of free COZversus distance z from the mucosal membrane interface at z = 0 to the bursting film of the gas bubble a t z = 6 4 and terminating in a C02-free(100% 02) ambient gas at z > 64. The cellular source of COz accounts for the boundary concentration at z = 0. The area between zero a.nd 61 is the unstirred boundary layer fluid adjacent to the mucosal membrane, that between ti1and 62 is the well-stirred bulk fluid, and that between 6 2 and 6s is the stationary liquid film around the ( c 0 . A ~ ) containing gas bubble. The break in the vertical lines indicates a change in the scale of the ordinate.

finally the liquid-ambient air interface where the C02-Oz-containing bubble bursts and the COz-02 gas mixture escapes into the ambient environment. Figure 14 is a schematic plot of the [GO,] profile in the steady state of GO2 diffusion from its site of production in the tissue to its point of exit from the system. The assumed values for the parameters shown in the figure are as follows. 1. The volume rate of wet gas flow V g bubbling into each hemichamber is 2.5-5.0 liters per hour; and consequently the mole rate of gas flow M g is 1.07 X lo-' to 2.14 X lo-' moles per hour. 2. The rate of production of metabolic COZ is ca. 4 x 10-6 moles per hour (LeFevre et al., 1970; Schilb and Brodsky, 1972) , of which 7.4 X lo-' moles per hour @On diffuse into and escape from the luminal fluid (Schwartz and Steinmetz, 1971). 3. The area of the exposed mucosal surface At is 8 cm2 on the basis of the system employed by Steinmetz (1967) and Gonzalez and Schilb (1969). 4. The thickness of the unstirred mucosal boundary film ( z = 61) is 10-3-10-2 cm on the basis of the estimates of Brodsky and Schilb (1965) in the case of osmotic flow of water across turtle bladder sacs. Unstirred layers in the boundary phase of solutions bathing cell surfaces have been estimated to vary from ca. 30 to 600 1.1 in a wide range of tissues including toad bladder (Hays and Franki, 1970), frog skin (Dainty and House, 1966), frog gastric mucosa (Harris and Edelman, 1964; Spangler

199

HYDROGEN SECRETION VERSUS BICARBONATE REABSORPTION

and Rehm, 1968; Kidder, 1970; Hersey and High, 1972), plant cells (Dainty and Hope, 1959) , and black lipid membranes (Andreoli and Trautman, 1971). The above thickness estimates were derived from data on several different parameters including vasopressin responsiveness, K fluxes, potential transients following step changes in the concentrations of K and C1 in the bathing fluids, and time requirements for reoxygenation of tissue cytochromes in situ. 5. The thickness of the stationary film surrounding each gas bubble (z = 63 - &) is taken as 5 X lop3cm. This estimate is based on the direct measurements of Ledig and Weaver (1924) on the absorption of gases from bubbles moving in liquids, and on those of Roughton (1941) on the influence of diffusion through liquid films in reactions involving COz and carbonic anhydrase. The principles on which these investigators estimated film thickness were those of Nernst on static liquid films and of Langmuir on liquid films moving tangentially to the bounding surface, as reviewed by Levich (1962) in his discussions on convective diffusion in liquids and on the motion of drops and bubbles in fluid media, and as reviewed by Helfferich (1962) in his text on ion exchange. 6. The radius of each gas bubble r b is 0.25 cm; whence the total area of 20 such bubbles (the number observed at any instant in the Ussing-Rehm apparatus) is Ab = 20(4?rrbe)= 15 cm2. I n the steady state the part of the metabolic COz that diffuses into the luminal fluid qCOzis equal to the rate of diffusion through the luminal fluid J c o ~where , Jcon denotes the rate of diffusion of COz (in moles per hour) normal to the tissue surface and parallel to the path denoted by the 2 axis of Fig. 14. The COz moves by diffusion across the boundary layers (61 - 0) and (63 - 62) where the decreases in concentration occur, and by convection across the well-mixed bulk of the bathing fluids (A2 - Sl) where no change in concentration exists. The steady-state rate of gas flow (in moles per hour) escaping from the bathing solution is M g (out) = 11;1g (in) 4COz (70)

+

The mole fraction of COZ XcoPin the gas bubbles is

XCO*= QCOz/Mg (out)

=

pCOz/P

=

pcoz

(71)

where Xcon= 3 X 1 e 6 to 6 X lop6atmospheres Applying Henry’s law to the equilibrium between the partial pressure of C02 in the gas phase and the molar concentration of COZ in the inner liquid boundary of the bubble film leads to the proportionality

[ c o 2 1 = a ( p ~ ~ =z 1)

x

10-7 to 2

x 10-7 M

=

0.1-0.2 r~

(72)

200

WILLIAM A. BRODSKY AND THEODORE P. SCHILB

where u = 3.61 x 10-2 moles per liter per atmosphere a t 22" (Harned and Davis, 1943; Harned and Bonncr, 1945). The concentration of 0.1-0.2 pM for free COZ at pH 7.1 would lead to a HCO3 concentration of 1.0-2.0 pM a t this gas-liquid interface; and Schwartz et al. (1971, 1974) have calculated a HC03 concentration of 1.28 pM a t this interface. However, they have assumed that the equilibrium concentrations of C02 and HCOI a t the gas-liquid or internal interface of the stationary film surrounding the bubble are the same as those in the bulk solution at the outer boundary of the liquid bubble film (i.e., a t the liquid-liquid interface between the stationary film and the bulk solution), and even the same as those in the boundary layer fluid bathing the mucosal membrane. Their assumption does not take into account the diffusion barrier presented by the stationary liquid films surrounding the gas bubbles, which means that these authors have ignored one of the two major factors governing the steady-state levels of C02 and HCOI in the bulk fluid and in the unstirred layer next to the tissue. The remainder of this analysis will demonstrate: that all of the C 0 2 diffusion from the tissue to the room air is driven by the concentration gradients across the two boundary fluids in the path of the COz escape; and that although the concentration of C02 in the escaping gas phasc is negligible in magnitude, the concentration in the bulk phase of the luminal fluid (and a t the tissue surface) is enough to violate the COz-free, HC03free requirements of the system. From the first form of Pick's law on the steady state rate of diffusion of C02 across the total surface of (A,,) of the bubble film of thickness, 8 3 8 2 = 50 p, where Jc0, = 7.4 x 10-7 moles per hour, it can be shown that

The maximal range of the value for [HC03]bulk, 10-70 p M , depends upon (1) the pH level a t which the mucosal fluid is statted, and (2) upon the assumed thickness of the bubble film, which has been found to vary from 10 to 400 p (Ledig and Weaver, 1924; Roughton, 1941). The value of 50 p used herein was chosen on the basis of Ledig's data on various-sized bubbles moving up a column of liquid in a system analogous to that of the air lift in the modified Ussing-Rehm chamber described previously (Gonzales et al., 1967). Similarly, a value of 30 p was chosen as the thickness of the boundary fluid phase between the luminal membrane and the bulk phase of the luminal fluid.

HYDROGEN SECRETION VERSUS BICARBONATE REABSORPTION

20 1

By analogous reasoning, the concentration of COz in the unstirred boundary layer a t the interface between thc cell membrane and the unstirred boundary fluid is [C0,lZ=o = 8 pM (75) and since [HCOI] in the unstirred fluid layer is minimal a t the tissueliquid interface ( z = 0) where the pump is located, there must be a finite decrement in the [HCO,] in going from z = 61 to z = 0. Now, if the rate of acidification is 1 . 1 pEq/hour in the steady state (Schwartz and Steinmetz, 1971), i t is readily shown that [HCO,],=o

7

36 p M

(76)

in order to satisfy Fick’s law; and consequently the pH (at z = 0) = 6.45, which is well within the expected physiological rangc for this rate of acidification. The maximal range of values for [HCO,lPa is 39 to 28 pM for film thicknesses of 10 to 100 p, respectively. Therefore, decreasing the thickness of the unstirred layer a t the mucosal cell surface should increase the boundary concentration of HCO, and consequently the rate of acidification by either HC03 reabsorption or H secretion. In this connection, Schwartz et al. (1974) have found such an increased rate of acidification after increasing the rate of bubbling of the luminal fluid. Conclusions from this analysis are: ( a ) The concentration of HCO3 reaches steady levels of 40-50 pM in the bulk phase of the luminal fluid (pH statted a t 7.1) and 36-46 pM in the tissue boundary fluid while both bathing fluids are being gassed with 100% 02.This concentration of HCOs in the bulk phase is a t about five times greater than the minimal [HCO,] attainable (8 p M ) in the luminal fluids of actively acidifying bladder sacs (Schilb and Brodsky, 1966; Brodsky and Schilb, 1972). (b) I n view of these bicarbonate levels in the bulk phase, the observed acidification of fluid initially devoid of bicarbonate constitutes no evidence against bicarbonate ion absorption. C. Acidification Rate as a Function of luminal [HC03]

If an active mechanism of HCO3 reabsorption were located in the luminal membrane, t,he rate of acidification ought to be a function of the luminal concentration of HCO, a t the luminal-facing surface of the membrane. However, if the mechanism were one of H secretion, the rate of acidification would not be expected to be a function of the luminal HCO3 concentration. With this in mind, Gonzalez and Schilb (1969) found that the short-

202

WILLIAM A. BRODSKY AND THEODORE P. SCHllB

circuiting current density is a Michaelis-like function of the isohydric (pH fixed a t 7.4) concentration of the luminal HCO3 in turtle bladders bathed on both surfaces by Na-free, C1-free media. Since HCO3 was the only transportable ion in both bathing media, these investigators inferred that the short-circuiting current would be completely accounted for by the active transport of HCO3 from lumen to serosa. What follows is a discussion of the limitations of these and other related data on electrical parameters with respect to the identification of a HCO3 or a H pump mechanism. 1. IDENTIFICATION OF THE ACIDIFICATION CURRENT The electrophysiological phenomena associated with the active transport of cation (e.g., H) across a membrane from one boundary fluid to the opposite one are indistinguishable from those associated with the corresponding transport of an anion (e.g., HC03) in the opposite direction. In the case of a three-compartment, two-membrane system such as an epithelial cell, transmembrane ionic gradients are always present. These gradients are symmetrically distributed and oppositely oriented across each of the two membranes when the cell is bathed on both surfaces by identical media, and are asymmetrically distributed across the two membranes when the bathing solution on one cell surface differs from that on the opposite surface. In the absence of a transcellular electric field, the short-circuiting current density must equal the algebraic sum of all net transcellular ion transfers: those driven by transcellular gradients in the case of passively transported ions, and those driven by active transport mechanisms in the case of actively transported species. In the experiments of Gonzalez and Schilb (1969), the short-circuiting current density (Iec)was measured across a bladder bathed initially by Nafree, C1-free, HCOI-free luminal fluid (choline sulfate) and HCO3-rich, Na-free, C1-free serosal fluid (Fig. 11). The observed I,, may be approximated in the form

- I H C O 8 leak, am in the presence of active HC03 reabsorption, or in the form I80

= IHCO8

pump

(77)

IHpump - I H C O s leak, ern (78) in the presence of active H secretion. The subscripts denoting active 1.c

=

(pump) and passive (leak) components of the ion transfers are self-explanatory. Gonzalez and Schilb next isohydrically increased the level of luminal [HC03] until it reached that of the serosal [HCO3] where the for either HCOa HCO3 leak current was zero and, consequently, I,, = Ipump reabsorption or H secretion.

HYDROGEN SECRETION VERSUS BICARBONATE REABSORPTION

203

If the pump component were constant and independent of the luminal HCO3, the changes in I,, would be restricted to those induced by the stepwise decreases in the HCO3 leak component [see Eqs. (77) and (78)]. If this variation in I,, were caused by passive diffusion driven by the transmural HCO, gradient, the function of I,, in Eqs. (77) and (78) should have been linear in accord with the Fick principle, rather than hyperbolic in accord with a Michaelis-like kinetic pattern. However, the HCO, leak may be negligible, and/or the “leak path” across the membrane may be restricted to a passive anion-exchange mechanism through which the net charge transfer is zero, causing no contribution to the I.,. [In connection with Cl-HC03 exchanges, Gonzalez et al. (1967) showed that neither the mucosa-to-serosa nor the net C1 flux in the presence of mucosal HC0 3 (no transepithelial gradients) was any different from the corresponding C1 fluxes in the absence of mucosal H C 0 3 (maximal serosa-to-mucosa gradient of HCO3). This suggests that a passive anion exchange mechanism for C1 and HCO3 plays little if any part in the transport of either anion.] If this were the case, the pump ( H or HCO3) would have to be sensitive to the luniinal [HCO,] a t constant pH in order to account for the pattern of I., versus [HCOSlL found by Conzalez and Schilb (1969) and illustrated in Fig. 15. Figure 15 shows the pattern of I,, versus luminal [HCOJ in a set of control bladders and in a parallel set of acetazolamide-treated turtle bladders. Acetazolamide, a t levels as high as 1.0 m M had no significant effect on the transmural resistance, even though the PD and I,, were reduced by 80-90%. This implies that ionic leaks, including that of HCO3, are unaffected by acetazolamide. 111111 (contrd)

0 0

5

10

I5 20 25 30

[HCO3]

: (mM)

FIG. 15. Short-circuiting current I., expressed as the normalized values (left ordinate) and as the ritw values (right ordinate) versus lriniinal [HCO,] for cwntrol and for acetazolamide-treated Idaciders. Each point plotted represents the mean value obtained from data of five experiments. Aoetazolnmide was added to interstitial fluid (final concentration, 2 x 10-4 M ) . Composition of iiiterstitial fluid was choline sulfate Ringer’s containing [HC03\,20 r ~ i M ;and thxt of the lumen fluid was initially choline sulfate. The isohydric change in luniinal [HCO,] wns achieved by the st,epnise addition of choline HCO,, together with the required increases in the pCOn of the luminal gas mixture.

204

WILLIAM A. BRODSKY AND THEODORE

P. SCHILB

Therefore subtraction of the value of the acetazolamide-inhibited current

(IBc,inhib) a t any luminal [HCO,] shown in Fig. 15 from the corresponding value of the control current ( I s c c, o n t r o ~ ) would yield the acetazolamideacst. Given that EHcoS,d i f f is the ion-specific sensitive current (Iac, ~ ion-specific permeability transepithelial diffusion potential and G H C Othe of the HCO, leak path, it can be stated that

- GHCO~EHCO~, diff ( I p u m p - A I p u m p ) - GHCOJHCO~, diff

I a c , control = I p u m p

I a c , inbib

=

and consequently that, I a c , m e t . sen,

=

AIpump

provided that G H c o ~ (at any given E H c O s , d i f f ) is not changed by acetazolamide. Since Fig. 15 shows that the acetazolamide-sensitive short-circuiting current is a Michaelis-like rather than a linear function of the luminal EHCO,], it could not be due to a diffusional leak of HC03. Instead, the acetazolamide-sensitive I,, is due to a pump mechanism which is sensitive to [HC03] on its luminal surface. It is reasonable to assume that such a pump mechanism is an active HCO3 transport simply because active transport mechanisms are generally sensitive to the concentration of the transported species bathing the membrane surface facing the side from which the net flux originates. Parenthetically, the finding of carbonic anhydrase activity in the turtle bladder epithelium (Scott et al., 1970; Schwartz et al., 1972) is not evidence for discriminating between H secretion or HCO, reabsorption. In this connection, Gonzalez (1969) found that acetazolamide inhibited the net chloride flux (from mucosa to serosa) as well as the HCO3 flux in the same direction. Therefore the intraepithelial carbonic anhydrase is involved in the active transport of chloride, an anion that has no direct relationship to acidification processes, especially under the short-circuiting conditions employed by Gonzalez (1969). D. Conflicting Data

Using the pC02 electrode technique, Schwartz et al. (1972, 1974), have reported that the [COJL decreases when the electrode is transferred from an acidified luminal fluid to the serosal bathing fluid. This means that [CO& > [CO2]i,f, and is in apparent conflict with the finding that [COZ]L < [C02lBobtained under the same conditions from measurements of pH and HC09 using the glass electrode and the Van Slyke apparatus (Schilb and Brodsky, 1966, 1972). These conflicting data need t o be resolved in terms of: (1) the magnitude

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205

of the experimental error of the technique relative to that of the observed physiological change, and/or (2) the likelihood of obtaining a precise measure of a spurious change induced by the measuring instrument. With regard to ( l ) , the magnitude of the maximal error in combined measurements of pH and HCO3 has been shown to bc but a small fraction ( < 15%) of the observed magnitude of the AICOz] between acidified luminal fluid and serosal fluid (Schilb and Brodsky, 1966, 1972). However, determinations of the corresponding error in measurements by the pCOz electrode are not yet available. With regard to ( 2 ) , a loss of luminal COz might be encountered during the transfer of the luminal fluid from the bladder sac to the Van Slyke apparatus. However, this has been tested for directly with the transfer technique used without detecting any significant loss of COZ ( I b i m and Brodsky, 1959) ; and increases of luniinal [CO,] when present have been readily detectable in both turtle bladder and frog stomach (Schilb and Brodsky, 1966). With respect to the pCOz electrodes, a spurious error could be obtained if the Teflon membrane were leaky to NaHC03 because of an imperfect seal and/or because its NaHCO~rc~lated reflection coefficient (UN,,HCO~) may be less than unity. The transfer of such an electrode from a HCOs-poor luminal fluid to a HC03-rich serosal fluid would result in a spuriously low value in the electrically estimated pCOn of the serosal fluid. E. Summary

The weight of currently avsilablr evidcncc, taken a t face value, supports the concept that an active IICO, transport is the mechanism of luminal acidification in the turtle bladder. This mcchanism is the unique explanation for the data on decreasing luminal [C'O,] during acidification, and is a fully adrquatci explanation for: increasing luminal [CO,] during acidification; H-accumulation in a luminal fluid initially devoid of HCO, and CO,; and the HC03-sensitive short-circuiting current. 111. THE RENAL MECHANISM OF ACIDIFICATION A. Background

1. LOCALIZATION

Pitts and Alexander (194.5) showed that the accumulation of hydrogen ions in the urine of intact dogs is a tubular rather than a glomerular process. Later, on the basis of micropuncture pH data from single nephron segments in rats, Cottschalk et al. (1960) showed that the process of

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WILLIAM A. BRODSKY AND THEODORE P. SCHILB

luminal acidification begins in the proximal tubule in the mammal, and not in the distal tubule as had been thought on the basis of earlier micropuncture data showing an unchanging pH in single proximal tubules of amphibia (Montgomery and Pierce, 1937; Pierce and Montgomery, 1935). According to Malnic and Giebisch (1972) 7045% of the filtered bicarbonate is reabsorbed in the proximal convoluted tubule, and the remaining 15-25%, in the distal tubule and loop under conditions of normal plasma pH, metabolic acidosis, and respiratory acidosis. The proximal reabsorption decreases to 55-60Oj, of the filtered loads under conditions of metabolic alkalosis, hypokalemic alkalosis, and respiratory alkalosis; and to 42% after the administration of Diamox. The minimal luminal pH in all of these tubular sites is 6.2-6.3 in both free-flow and stopped-flow experiments; while a minimal pH of 4, the minimal level in the final urine, has been found in the terminal 1-6 mm of single collecting ducts in the microcatheterization experiments of Ullrich et al. (1958). Thus, the site and the extent of the luminal acidification have been well established. However, the identification of the renal tubular acidification mechanism has not yet been achieved. 2. IDENTIFICATION OF THE MECHANISM Earlier physiologists had been well aware of the problem of distinguishing the reabsorption of HC03 or C03 or OH from the secretion of H or acid in the process of urinary acidification (Sendroy et al., 1934; Smith, 1937; Menaker, 1948; Kennedy et al., 1952). The only clearly defined principle for making such a distinction, first proposed by Brodsky (1955, Brodsky et al., 1958) is that of determining the direction of the shift away from the initial equilibrium state (or poise) of the COz-HzCO3 reaction in the fluid undergoing acidification (Section I, A), Expioiting this principle, two methods have been developed: the COZ gradient method, based on the assumed near-equilibrium poise of the COz-H2C03system (Brodsky et al., 1958; Kaim and Brodsky, 1959; Schilb and Brodsky, 1966, 1972; Brodsky and Schilb, 1967, 1972) ; and the pH disequilibrium (or off-equilibrium) method based on the assumed off-equilibrium state of the COz-HzCO3 reaction (Ochwadt and Pitts, 1956; Walser and Mudge, 1960; Rector et al., 1965). The details of the principle of the two methods are discussed in Section 11, A-C. It is important to reemphasize that the presence of either an off-equilibrium or an equilibrium state of the luminal COz-H$2Oa reaction has not been clearly established in the case of renal tubular acidification (Section I, B). Because of this uncertainty, it remains necessary to analyze data concerned with the identification of the renal acidification mechanism in terms of an assumed equilibrium state, as well as in terms of an assumed offequilibrium state of the luminal reaction constituents. With respect to the

207

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latter assumption, it has been pointed out that the measured parameters in the currently used p H disequilibrium method are most precisely expressed in the form of Eq. 5 or - PHI,,,, (calc.) ~ H (meas.) L

=

log

[cO~]~ ,e(assumed) q [cO,]~,eq (true)

f APH,

where it is critical to determine the quantity [COz]L,eq (assumed) in order to establish the presence or absence of an equilibrium state. What follows is a review of recent attempts to determine the intraluminal [CO,] (Section 111,B) and to analyze some of the other current micropuncture data in terms of assumed equilibrium as well as of assumed off-equilibrium conditions in the luminal fluid. B. The in-Situ Concentration of Luminal COS 1. C02 DIFFUSION METHOD

The principle of this method is to determine the level of intraluminal COz a t which there is no diffusion of COZ into or out of the renal tubular lumen-other than that due to the acidification mechanism. Malnic and Mello-Aires ( 1971) exploited this principle by injecting HCO, solutions of varying [CO,] into single proximal tubules. Their reasoning was as follows. When the concentration of C o nin the injectate exceeds that associated with the luminal acidification mechanism, a fast diffusional transient (of sharply increasing luminal pH levels) is superimposed on the time-dependent acidification function; and when the concentration of COz in the injectate is less than that associated with the luminal acidification, a diffusional transient in the reverse direction is obtained. However, when the COZ of the injectate equals that which would he produced by the acidification mechanism, the diffusional transients vanish; hence the intraluminal level of COZ is determined in the presence of the functioning acidification process. Their technique was first to inject a column of perfusion fluid and then a column of oil through a double-barrelled micropipete inserted into the lumen of a single renal tubule. An antimony micro-pH electrode had been inserted into the same tubule a little further downstream. They reinjected a column of perfusion fluid so that it split the oil droplet, thus becoming isolated from the rest of the lumen fluid, and moved downstream to make contact with the antimony electrode from which the pattern of luminal p H versus time was recorded continuously. In one set of studies, a 25 or a 100 m M injection fluid was preequilibrated with 15% COz; in another set the same fluid was equilibrated with air; and in a third set, the same fluid was equilibrated with 5% COz. In each case the pattern of p H versus time was obtained after the injection fluid had made contact with the intratubular p H electrode. The time-dependent

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WILLIAM A. BRODSKY AND THEODORE P. SCHllB

pattern of the change in luminal pH could bc represented as the sum of a fast and a slow exponential function. The half-time of the fast function, ascribable to transmural diffusion of COz, was ca. 1 second, while that of the slow function, ascribable to HC03 reabsorption was ca. 3 seconds. The crucial experiment, the injection of 25 or 100 m M HCO3 preequilibrated with COz at 34.5 mm of Hg (equivalent to [CO,] = 1mM) yielded a single exponential function ascribable to HC03 reabsorption alone on the basis of the half-time evaluation and on the basis of the absence of any detectable diffusion transients. Hence Malnic and Ifello-Aires (1971) concluded that the intraluminal [COZ] is ca. 1.0 mM under control conditions of proximal acidification. The kinetics of HCO3 reabsorption was fairly accurate insofar as the evaluation of the zero intercept ( t = 0) of the slow exponential usually led to a HCO3 concentration approximating that in the injected fluid, and insofar as the calculated steady-state level of HC03 reached was similar to that directly determined by Bank and Aynedjian (1967). However, the intraluminal [COJ of ca. 1.0 m M in the stopped-flow experiments of Malnic and Mello-Aires, (1971) should be reconciled with an intraluminal [COJ of ca. 2.0 mM in the free-flow experiments of Iiarlmark (1972) (see Section I, E ) , because the assumption of intraluminal equilibrium, and the experimental conditions (e.g., control) and the tubular locus (proximal) are the same in both experiments. These are two possible reasons for this apparent discrepancy. 1. The chemical composition of a small volume of loculated luminal fluid would be changed more drastically by any tubular transport mechanism than would that of a free-flowing larger volume of luminal fluid by the same tubular transport mechanism. This effect was experimentally amplified in the work of Malnic and Mello-Aires (1971) by minimizing the reabsorption of water through the addition of the nonpenetrant solute, raffinose, to the injectate in the stopped-flow experiments. 2. The luminal [COJ decreased from 3.0 to ca. 1.0 mM in going from the early to the distal segment of single proximal tubules (Karlmark, 1972). This means that the luminal [COJ decreases as a function of distance during free flow, and predicts that the luminal [CO,] should decrease as a function of time a t any given tubular locus during stopped flow. Parenthetically, the relationship between decreasing [CO,] and HCO3 reabsorption has been discussed in detail in Sections I and 11. 2. KINETICMETHOD

On the assumption that active H secretion is operative, an accumulation of [CO,] in the lumen to a level greater than that in the arterial plasma is the

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209

predicted result of the change in luminal [C02] calculated from the kinetic paremeters of Malnic and his colleagues (1972; Malnic and hlello-Aires, 1971; Malnic and Giebisch, 1972) for COz diffusion and HCO, reabsorption in the acidification process. The fact that the half-time of HC03 disappearance is two to three times longer than that of lumen-to-cell C02 diffusion means that two to three half-time’s worth of the evolved C 0 2 disappears for each half-time’s worth of HCO3 consumed; and consequently the amount of extra C02 accumulated in the lumen would be cqual to 1/22 to l/z3 of the concomitant decrement in luminal [HCO,). For example, let the luminal concentration of HC 0 3 be reduced from 24 m M to 12 mM (one half-time’s worth of HCO3) ; then 12 mmoles of COZ is evolved (for each liter of luminal fluid). Of the 12 mmoles evolved; 6 3 or 6 3 1.5 mmoles (two to three half-time’s worth of C02) diffuse out of the lumen, while 1.5-3 inmoles (over and above that already present) remain in the lumen to increase the concentration of the luminal c02. Thus C02 is apt to accumulate in the lumen over and above the initial luminal C02 when the kinetic coefficient of the diffusion process is but two to three times greater than that of the acidification process-an effect that was evidently discounted or overlooked by Rlalnic and colleagues (1972; Malnic and Mello-Aires, 1971 ; Malnic and Giebisch, 1972). On the assumption that an active Hco3 pump is operative in the proximal tubule, the injection of a C02-free, HC0 3 solution under stopped-flow conditions would be followed by the onset of a fast cell-to-lumen diffusion of C02 and a relatively slower HCO3 ion reabsorption; whence two timedependent functions of pH (or “apparent” [HC03]) would also be generated. The fast exponential would represent the cell-to-lumen diffusion and intraluniinal accumulation of C02, and the slow exponential would represent the active HC03 reabsorption. The fast exponential would also be eliminated in the case of a HCOs pump if the pC02 in the injected fluid were the same as that in the tubular cell fluid. Consequently, the kinetic method can be used to estimate changes in luminal [CO,] produced by either acidification mechanism, but neither this analysis nor the actual data on the time-dependent exponential functions are sufficient to distinguish H secretion from H C 0 3 reabsorption.

+

+ +

3. KARLMARK APPROACH

Assuming with Rector et al. (1965; Rector, 1971) and Malnic and Giebisch (1972) that the CO2-H2CO3 reaction in the proximal tubular fluid is a t or close to equilibrium under control conditions, Karlmark (1972) applied the Astrup technique to determine the [CO,] in samples of luminal

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WILLIAM A. BRODSKY AND THEODORE P. SCHlLB

fluid. His method was (1) to measure the in situ pH of the luminal fluid at a site in the proximal tubule, (2) to remove a sample of this fluid during free flow, and (3) to equilibrate the isolated luminal fluid under oil with various levels of pC02 and thus to determine the equilibrium level of pH as a function of the pCOz. The pCOz that returns the pH of the in vitro sample to its original in situ level is taken to be the pCOz of the in situ fluid, provided that the original in situ pH was at an equilibrium level. Karlmark found that the luminal [CO,] along the proximal tubule varied from 3.03 mM in the early proximal to 0.8 mM in the late proximal tubule, while the plasma [CO,] was 1.0 mM. Despite a substantial scatter in the data, the mean [COZ] in the middle half of the tubule was 2 mM and the luminal [COJ appeared to decrease as a function of increasing (Tubular Fluid/ Plasma) inulin ratios, (TF/Pinulin). Karlmark’s data show that the luminal [COJ is greater than the arterial [COJ which contradicts the conventional assumption that luminal [COJ equals arterial [COJ (Rector et at., 1965; Vieira and Malnic, 1968). This conflict can be resolved by assuming that there was no disequilibrium pH in the proximal tubular fluid (ApH = 0) in the work of Rector et at. (1965) and Vieira and Malnic (1968), that consequently the recalculated equilibrium level of [CO,] in the in situ luminal fluid of the proximal tubule is really greater than that in the arterial plasma. VIA THE EQUILIBRIUM ASSUMPTION If one assumes that the C02-H2C03system in the proximal tubule is always in reaction equilibrium, one can estimate the luminal [COJ from the in situ levels of proximal tubular pH and HCO3 for each one of the several conditions imposed in the free-flow experiments (e.g., control conditions, metabolic alkalosis, and acidosis (Vieira and Malnic, 1968; Malnic et at., 1972). Table I, taken from the data of Tables 8 and 9 in the report of Malnic et al. (1972), shows the mean values for the in situ determinations of pH and HCO3 concentration in the luminal fluid of single proximal tubules and in the arterial plasma under control conditions, metabolic alkalosis, and acidosis in the free-flow experiments of Vieira and Malnic (1968) and of Malnic et al. (1972). The values of luminal [CO,] are those calculated by us from the Henderson-Hasselbalch equation in order to satisfy an assumed state of reaction equilibrium among the luminal constituents (H, HCO3, and COZ) at the observed levels of intraluminal pH and [HCOI]. The equilibrium levels of luminal [COJ are about twice that of arterial CC0.J during metabolic acidosis or during control conditions, and about the same as that of arterial [CO,] during metabolic alkalosis. Inasmuch as

4. CALCULATIONS OF LUMINAL [COJ

21 1

H Y D R O G E N SECRETION VERSUS BICARBONATE REABSORPTION

TABLE I

PROXIMAL TUBULE METABOLIC STATES" Control

____

Parameter

PH HCOI (mM) COZeq ( m M )

Plasma 7.36 25.7 1.27

Metabolic alkalosis

Metabolic acidosis

_____

-

Lumen

Plasma

Lumen

Plasma

6.71 8.40 2.03*

7.61 55.5 1.76

7.44 36.4 1.82b

7.12 9.55 0.95

Lumen 6.43 3.73 1. 87b

a Mean values for pH and for concentration of HCOB and Cot in the plasma, for in siiu pH and the HC03 concentration of the proximal luminal fluid, and for the luminal [CO,] required for reaction equilibrium with the in silu luminal pH. Data taken from Vieira and Malnic (1968) and Malnic el 01. (1972). * Values calculated by Brodsky and Schilb.

these values of luminal [CO,] are uniquely required to satisfy the assumed equilibrium state, they are iiot in accord with the conventional assumption that luminal [COJ equals arterial [CO,], except under the conditions of metabolic alkalosis. If the conventional assumption (on equality of luminal and arterial [CO,]) were correct, there would have to be a nonequilibrium state among the i n situ luminal constituents (H, HCOB,and COz); and the so-called in vitro or calculated level of pH (pH,,) would have to be greater than the in situ or measured level of p H ( ~ H L. This ) is because pH,, as it is usually determined, is the level of luminal pH that satisfies the particular equilibrium conditions wherein [HCOS]~is equal to that determined and wherein [CO,], is equal to that in the arterial plasma (e.g., 1.27, 1.76, and 0.95 mM in the data of Table I ) . The mran values of pH,, - PHL calculated from the data in Table I amount to 0.2, 0.01, and 0.3 units, while those determined from all the experimental data in Table 10 of the report by Malnic et al. (1972) amount to 0.14, 0.07, and 0.16 units, respectively. The mean ApH of Rector et al. (1965) for metabolic alkalosis and control conditions amounts to 0.05 units. Both AZalnic et al. (1972) and Rector et al. (1965) emphasized that none of these pH differences was significant. n'evertheless a pH difference of 0.15 units corresponds to a discrepancy of 40% in the assumed level of luminal [CO,] (see the analysis in Section I, B). In short, neither these data (summarized in Table I ) , nor those on the ApH (equilibrium less nonequilibrium value) are sufficient to determine the presence or absence of a state of reaction equilibrium in the luminal fluid of the proximal tubule, which means that the in situ level of luminal [CO,] assumed could well be underestimated by a t least 40%.

212

WILLIAM A. BRODSKY AND THEODORE P. SCHllB

Unless a nonequilibrium state in the proper direction (pHea > ~ H Lis) definitely established in the proximal tubular fluid, a high level of luminal [CO,] per se cannot be taken as evidence in support of H secretion or against HCOa reabsorption. Alternatively, if the proximal luminal constituents are a t or near equilibrium a t all times, as postulated by Rector et al. (1965), Malnic et al. (1972), and Karlmark (1972), the high level of luminal [CO,] (especially in a luminal fluid of pH 6.4-7.4) is just as consistent with HCO, reabsorption as it is with H secretion (see Section I, C and E) . The possibility remains that a significant part of the proximal tubular HCO, was reabsorbed by bulk flow, which means that the active moiety (defined as that which produced dilution of the luminal [HCO,]) was operating a t a rate slower than that of the hydration-dehydration reaction. Finally, it should be noted that the luminal [COZ] of ca. 2 mM may well be in the neighborhood of the cellular [COJ, which in turn is probably sigficantly greater than the arterial [COz]. C. Apparent Reaction Disequilibrium

1. RESPIRATORY MANEUVERS

Table I1 shows the proximal tubular data of Malnic et al. (1972) under conditions of respiratory acidosis and alkalosis during free-flow micro-

TABLE I1

PROXIMAL TUHULE, RESPIRATORY STATES" Respiratory acidosis Parameter

. pH H C 0 3( m M ) COZ(mM) ApHdis

Plasma 7.00 27.4 3.5 -

Lumen (i.49 15.1 6. 14b 0.31

Respiratory alkalosis Plasma 7.64 18.0 0 . 52h -

Lumen 7.25 16.5 1.45h 0.24

a Mean values for pH and for concentrations of HCO3 and COZ in plasma, for in silu pH and HCO3 concentration of the luminal fluid, for the luminnl [COZ] required for reaction equilibrium with the in silu luminal pH, and for the apparent disequilibrium pH (ApHdJ. Data taken from Maliiic et (11. (1972).

* Values calculated by Brodsky and Schilb.

HYDROGEN SECRETION VERSUS BICARBONATE REABSORPTION

213

puncture experiments. On the assumption that the luminal [CO,] equals the arterial [CO,], these investigators reported significant disequilibrium pH values during respiratory acidosis and alkalosis. One of the problems noted by them is that of explaining the presence of a disequilibrium p H despite the presumed presence of carbonic snhydrase in the proximal tubular lumen. It is well known that the conditions prevailing in the luminal fluid during these respiratory maneuvers would not inhibit carbonic anhydrase in vitro. Moreover, the rates of HCO, reabsorption do not exceed those of HCOI reabsorption in control experiments in which there is no apparent disequilibrium pH. Assuming that CAH is present in the lumen, these investigators were forced to invoke the postulate that raising or lowering the cellular pCOz somehow causes a disappearance of the luminal carbonic anhydrase activity. If, however, the pCOz of the luminal fluid and the tubular cells were much higher than that assumed by these workers, the practice of equilibrating the luniinal samples with a pCOz equal to that in the arterial plasma would lead to a change in pH that would mimic a disequilibrium p H resulting from H secretion. Alternatively, on the assumption that luminal reaction equilibrium persists under these respiratory conditions, then the luminal [CO,] is greater than the plasma [CO,]; and, surprisingly, during respiratory acidosis, the luminal [CO,] would have to bc as high as 6.0 m M or nearly twice that of the plasma [CO,]. A luminal [CO,] of 6 mM is consistent with H secretion and an accumulation of luminal COz, the extent of which is close to that predicted by the data of Malnic and Mello-Aires (1971) on the kinetics of COZdiffusion and acidification (see Section 111, B, 1 ) . However, such a high luminal [CO,] is equally consistent with HCOl reabsorption in the presence of a cellular [CO,] that must be greater than 6.0 mM in respiratory acidosis and greater than 1.45 mM in respiratory alkalosis. The associated lumen-to-plasma gradients of [GO,], 2.6 m M and 0.9 for respiratory acidosis and alkalosis, respectively, are similar to some of the lumen-to-plasma gradients of [COJ found by Karlmark (1972) in the proximal tubule of rats under control conditions. 2. EFFECTOF CARBONIC ANHYDRASE INHIBITION

Since Rector et al. (1965) were unable to detect a disequilibrium p H in the proximal tubule, they inferred that carbonic anhydrase activity was available to the luminal reactants. Accordingly, these investigators, and subsequently Vieira and Malnic (1968), reasoned that inhibition of this

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WILLIAM A. BRODSKY AND THEODORE P. SCHILB

activity should elicit a disequilibrium pH, and indeed reported one in the direction of H secretion in the presence of C1-11,366 and/or acetazolamide. Although these data are consistent with active H secretion, they are equally consistent with active HC03 reabsorption or with no active acidification a t all. This is because after the introduction of acetazolamide or C1-11,366 the level of intraluminal [HCO3] in the proximal tubule remained the same as or increased to as much as three times that in the plasma (Clapp et al., 1963; Rector et al., 1965; Vieira and Malnic, 1968) during free-flow micropuncture experiments. This suggested to Clapp et al. (1963) that the drug may have completely inhibited HCOX reabsorption, although these investigators did not report any concomitant inulin data. In our opinion, the pattern of proximal HC03 reabsorption after carbonic anhydrase inhibition could be ascribed in part to a bulk reabsorptive effect (in which [HCOS] of the lumen remained equal to that of the plasma), and in part to a sieving effect in which the luminal [HCO,] increased, presumably out of proportion to the volume decrease in the tubule.

3. STOPPED FLOW Surprisingly, the proximal tubular data of Malnic and Mello-Aires (1971) from stopped-flow experiments after the introduction of acetazolamide appeared to contradict those of Vieira and Malnic (1968) obtained in the free-flow experiments. In the stopped-flow experiments, a calculated parameter called the apparent HCO3 concentration decreased to 5.83 mM, while the pH decreased to 6.71 in the luminal fluid. The apparent [HCOJ was calculated on the assumption that the luminal [CO,] was equal to the concomitant arterial [COZ]. It is noteworthy that (1) the actual luminal [HCO,] was not measured, and (2) the intraluminal pH in the stoppedflow experiments was about the same as that in the free-flow experiments after carbonic anhydrase inhibition. These data do not permit the inference that the luminal [HCOJ decreased after acetaxolamide treatment in the stopped-flow experiments. In fact, the level of the measured luminal pH, the only measured parameter, suggests that the luminal [HCOJ in the stopped-flow experiments, like that in the free-flow experiments, remained the same as or increased to levels greater than those in the plasma. If one assumes the existence of a reaction equilibrium in the luminal C02-HzC03 system even after acetazolamide treatment, the required luminal [COZ] in the free-flow experiments would have to be 7.1 mM (on the basis of the measured values of luminal pH as 6.67, and of [HCO,] as 26.6 mM), while the measured level of plasma COZwas 2.2 mM (Vieira and Malnic, 1968).

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215

I n addition, if one assumes that acetazolamide inhibits HCO, reabsorption under stopped-flow condit,ions in the same manner as it does under free-flow conditions, then the intraluminal [HCO,] would be ca. 26.6 mM. The measured pH was 6.73, and consequently the luminal [COz] would be 6.5 mM, which is in harmony with the COz level of 7.1 mM in the free-flow data determined on the basis of the equilibrium assumption. Extending this equilibrium assumption one step further leads to the conclusion that there exists a 5 niM difference between the [CO,] in the lumen and that in the arterial plasma after carbonic anhydrase inhibition. Whereas gradients of this magnitude have not been considered thus far, the calculated data of Tables I and I1 and those of Karlmark (1972) suggest that the luminal [COz] is 0.75-2.0 m M greater than plasma [COZ] under normal conditions, and 2.5 niM greater than plasma [CO,] under the conditions of respiratory acidosis reported by Malnic et al. (1972). I n this connrction, in turtle bladders with low rates of HCO, reabsorption, the luminal [COz] was as much as 0.6 niM greater than the serosal [CO, J as reported by Green et aE. ( 1970) and by Schilb and Rrodsky ( 1972) (see Fig. 13). Although t b s transepithelial gradient is smaller than those estimated in the mammalian nephron, the turtle bladders had been isolated from the animal and incubated a t 25" (instead of 38") in a synthetic interstitial fluid. In short, a 2.5 mM transepithelial gradient of COz can be rationalized in the rat nephron in the absence of acetazolamide (e.g., respiratory acidosis). A 5 m M gradient can be rationalizrd in the presence of acetazolamide, which according to Malnic and Giebisch (1972) decreases the COZ permeability of the nephron by a factor of 2 to 2.5. If such a reduction in COZ permeability were out of proportion to any drug-induced decrease in the metabolic rate, a cellulwr respiratory acidosis would ensue; and cellular [COJ could reach levels of 7 mM, with which the luminal [COZ] could equilibrate in the absence of an active acidification mechanism (see Section

I, D). D. Distal Tubular Acidiflcation

It is currently thought, (Rector, 1971; Malnic and Giebisch, 1972) that the acidiJicatiori inechanzs?n in the distal tubule is basically the same as that in the proximal tubule, but that the nczdiJication process is different insofar as carbonic anhydrase is not available to catalyze the intraluniinal COZH&O3 reaction in the distal tubule. According to Rector et al. (1965), the evidence for this difference is twofold: ( I ) the presence of an off-equilibrium pH (in the direction of H secretion) in the distal but not in the proximal

216

WILLIAM A. BRODSKY AND THEODORE P. SCHILB

tubular fluid; and (2) the elimination of the off-equilibrium pH in the distal tubule by the intravenous injection of the enzyme carbonic anhydrase. Findings similar to those of Rector have been reported by Malnic et al. (1972) in rats under conditions of metabolic and respiratory alkalosis, and acetazolamide treatment, but not in rats under conditions of respiratory or metabolic acidosis. 1. RESPIRATORY ACIDOSIS

The findings of Malnic and his colleagues (1972) in rats under conditions of respiratory acidosis merit special consideration because the mean value for the off-equilibrium pH (-0.13 units), was discounted solely on statistical grounds. In our opinion, this statistical conclusion was not warranted on account of the following considerations. In six out of the eight distal micropuncture samples in which all the required parameters were measured (see Malnic et al., 1972, the last eight experiments in Table 3 ) , the mean difference between the calculated equilibrium pH and the in situ pH in the lumen amounted to -0.32 units (total range, -0.08 to -0.46 pH units). In the other two distal samples, the corresponding differences in the off-equilibrium pH values, oriented in the opposite direction, amount to +0.53 and +0.27 pH units, which strongly suggests a frequency distribution characteristic of two distinctly different populations of data. Table I11 (Malnic et al., 1972, constructed from the data shown for the last eight experiments in Table 3) shows that there were two different sample populations in the distal micropuncture data during respiratory acidosis. This contention is supported by statistical evaluation of the parameters on disequilibrium p H (or equilibrium luminal [CO,]) , inulin TF/P ratio, and luminal [HCOs]. The one inulin TF/P ratio measurement made in the two aberrant samples was 3.01, while those in the main subgroup of six samples ranged from 4.08 to 4.63. The luminal [HC03] values in the two aberrant samples were 7.5 and 12.1 mM, while the total range of luminal [HCOs] values in the main group was 2.643.2 mM. According to Table 9 of the same article, the mean value (plus or minus standard error) for proximal tubular [HCOJ is 15.1 f 1.69 mM ( n = 13), and the mean value (plus or minus standard error) for the distal tubular [HCO,] is 5.76 f 1.07 mM ( n = 8). On the assumption of equilibrium of the C02-HzC03 reaction in the distal tubular fluid, the values of luminal [COJ were 8.41 and 5.45 m M in the two aberrant samples, while the entire range of [COZ] values in the main group of samples was 1.75-2.39 mM.

TABLE I11 DISTAL

6 v, m

TUBULE, RESPIRATORY !LCIDOSI~''

c

Groups

APHdu

Inulin (TF/P)*

HCO, (mM)

Cot, (mM)c

u,

C O ~ (mM) L

0

z

n

( a ) Major, n = 6 (b) Aberrant, n = 2 Total, n = 8 pa-b = 0

-0.32 f 0.055 +O. 27 + O . 53 - 0 . 1 4 f 0.127

Z 0

n

(h)

I

-2 1

3

45

5

2

38

-I

31

g

(i) ~~

B

rn W

>

(S) (R) norepi. ~

g

5

15

9

6

6

11 ~

~

5 ~

1 ~

0 ~

Z

_

v

W

(a) Schultz and Zalusky (1964a); (b) Schulta et al. (1964); (c) Clarkson (1967); (d) Clarkson and Toole (1964); (e) Al-Awqati et al. (1973); (f) Field et d.(1971); (g) Field (1971a); (h) Field et al. (1973); t i ) Field and McColl (1973). b (U), unstripped preparation; (S), stripped preparation; (R), rabbit; (H), human; (r), rat; chol, cholera toxin; theo, theophylline; CAMP, cyclic 3'5'-adenosinemonophosphate; glu, glucose (1-5 mM); norepi, norepinephrine. Numerical superscripts: 1, low HCO, or HCOa-free; 2, 25 mM HC03; 3, 1-5 mM glucose present in mucosal solution; 4, glucose present only in serosal solution (7.5-10 mM). Bracketed results represent data obtained on the same segment of tissue during a control period and experimental period. c All fluxes, the short-circuit current ( I a c ) ,and residual current (IR)are expressed in rmoles/cm2hr. A negative net flux designates net movement from serosa to niucosa. The transepithelial resistance R is expressed in ohm-cm2. All values have been rounded off without sacrificing statistical significance. a

_

_

242

STANLEY G. SCHULTZ AND PETER F. CURRAN

across short-circuited preparations of rabbit, rat, and human ileum, and Schultz et al. and Clarkson et al. found reasonable agreement between the short-circuit current I,, and the rate of active Na transport from mucosa to serosa. The data for rabbit and rat ileum were obtained using tissues that were not stripped of the underlying muscle layers and submucosal tissues, and Schultz and his collaborators and Clarkson and Toole employed low-HC03 or HC03-free bathing solutions. In contrast, Field and his collaborators (Field et al., 1971, 1972; Field, 1971a) employed astripped, short-circuited preparation of rabbit ileum and observed active Na absorption, small but statistically significant active C1 absorption (Table 111), and a “residual” current IR which appears to be attributable to active HCO, secretion (Dietz and Field, 1973). The presence of a significant component of active C1 absorption across stripped rabbit ileum has been confirmed by one of us (S.G.S.), but has not been observed by the other (P.F.C.) ; a more detailed presentation of these data and possible explanations for these apparent discrepancies are discussed in Section V, E. Finally, as shown in Table 111, active C1 absorption by short-circuited stripped rabbit ileum is not a consistent finding. One difficulty encountered using stripped preparations is that the bidirectional ion fluxes are large and the net flux, if any, represents a small difference between two large numbers; since J”,’, and J f A are determined on different tissues (in contrast to double-label experiments on a single segment of tissue), the experimental difficulties of demonstrating active C1 absorption are compounded. Thus, although active Na absorption by short-circuited preparations of rabbit ileum (as well as rat and human ileum) has been repeatedly demonstrated, active C1 absorption has been an inconsistent finding. Frizzell et al. (1974) demonstrated that stripped preparations of rabbit ileal mucosa consume more oxygen per unit area than unstripped preparations, in agreement with the earlier findings of Bronk and Parsons (1965) on rat small intestine. Thus the failure to observe active C1 absorption and HCO, secretion in unstripped preparations of rabbit and rat ileum may be related to inadequate oxygenation. This explanation is supported by the apparently close link between C1 absorption and H C 0 3 secretion. For example, Hubel (1967, 1969) has presented convincing evidence that HCO, secretion by rat ileum in vivo is inhibited when the lumen is perfused with a C1-free solution, and similar findings have been reported for rat colonic mucosa (Phillips and Schmalz, 1970). If, as postulated by Parsons (1956), these findings reflect the operation of a C1-HCO, exchange process, reduced oxidative metabolism by the tissue might be expected to impair C1 absorption. However, additional possible explanations for the elusive nature of active C1 absorption by in nitro preparations of mammalian ileum emerge

243

SODIUM AND CHLORIDE TRANSPORT

from the recent findings of Field and his collaborators on the effects of cyclic 3‘-5‘-adenosine monophosphate (CAMP) (Field, 1971a,b) and adrenergic agents (Field and McColl, 1973) on transepithelial movements of Na, C1, and HCO, across short-circuited, stripped rabbit ileum. As shown in Table 111, maximally effective doses of theophylline (10 mM) abolish active Na absorption and elicit active C1 secretion; similar effects are observed in the presence of cAMP (Field, 1971a), cholera toxin (Field et al., 1972), and prostaglandin E l (Kimberg et al., 1971) [the last-mentioned agents increase intracellular cAMP in rabbit (Kimberg et al., 1971; Sharp and Hynie, 1971), guinea pig (Kimberg et al., 1971), and canine (Schafer et al., 1970) ileal mucosa]. In contrast, adrenergic stimuli appear to enhance active Na and CI absorption and to abolish HCO, secretion by short-circuited rabbit ileum (Field and Dietz, 1973; Field and McColl, 1973) (Table 111). Other agents that have been shown to stimulate C1 secretion by mammalian small intestine in vivo include substituted phenols and cholinergic agents (Tidball, 1961a,b). Thus Na, C1, and perhaps HCO, transport by rabbit ileum appears to be influmced by opposing humoral and/or intracellular factors whose regulation under so-called control conditions is as yet poorly understood. Therefore, previously overlooked or “slighted” parameters such as eating patterns, seasonal and diurnal factors, level of activity on the part of the aniinal prior to sacrifice, method of sacrifice, presence of pathogenic bacteria, and so on, may have a more important effect on transport activity observed in vitro than previously appreciated, and may be largely responsible for the variability observed under apparently identical experimental conditions. Finally, it should be noted that in vitro preparations of rabbit ileum are capable of sustaining active transport processes for a t least 2 hours in the absence of exogenous substrate. Indeed, studies on O2 consumption and COz and HCO, production by niucosal strips of this tissue indicate that, in the absence of exogenous substrate, 0, consumption is maintained a t a level of approximately 75 pliters’cm-2.hr-l for a t least 2 hours; the metabolism of endogenous fatty acids is tho principal source of energy. In the presence of metabolizable exogenous substrates, 0 2 consumption is not markedly enhanced, but tho rate of total CO, elaboration is increased, thcrchy shifting the RQ toward the value that would be expected if the tissue were utilizing, a t least in part, the exogenous substrate in preference to the endogenous substrate(s). For example, in the presence of 10 m M glucose, O2 consumption averages 85 pliters cnr2.hr-’ and does not greatly exceed that observed in the absence of exogenous substrate, but the RQ averages 0.55. The rates of 0, consumption and COz and HCOs production by paired inucosal strips of rabbit ileuni from the same animal in the presence of a variety of substrates are summarized in Table IV. (More

-

244

STANLEY G. SCHULTZ AND PETER F. CURRAN

TABLE IV

METABOLISM OF MUCOSAL STRIPSOF RABBITILEUM' ~~~

Substrateb

~~

~

Qo2

HC03

Total

RQ

QCO~

{g:se,

10 mM

85 97

32 40

67 86

0.75 0.85

{!:zte,

10 mM

83 91

34 47

66 85

0.75 0.90

90 89

35 35

74 66

0.78 0.72

82 95

32 35

59 76

0.73 0.80

76 109

29 73

57 109

0.73

{PNgrionate {!En,, {m :; nie,

10 mM

10 mM

1 .oo

All values are expressed in pliters-cm-2. hr-1. Bracketed groups represent data obtained on paired mucosal strips from the same animals. (Data from Frizzell et al., 1974.) a b

properly the figure for HCO, production represents bound C 0 2 released by acidification. Nonetheless, it is fair to assume that bound C 0 2 largely represents HC03.) Of the substrates tested only glutamine markedly enhanced the rates of 0 2 consumption and C 0 2 and HC03 production. These results closely parallel those reported by Neptune (1965), and suggest that during the 2-hour duration of most in vitro experiments endogenous energy sources are present in sufficient quantity so that active transport processes are not likely to be energy-limited. However, the extent to which differences in the rate of C 0 2 and HCOa production may influence these transport processes is unclear. It is of interest to note that the rate of HCO, production by mucosal strips is equivalent to approximately 1.5-2 pmoles cm-2-hr-1, in good agreement with the value reported by Dietz and Field (1973) for the rate of HCO, secretion by rabbit ileal mucosa and with the residual current reported by Field and his collaborators (see Table 111) and Binder et al. (1973). V. INFLUXES OF Na AND CI ACROSS THE BRUSH BORDER

Having briefly summarized some of the overall features of Na, C1, and HC03 transport across in vivo and in vitro ileum, we now consider some

245

SODIUM AND CHLORIDE TRANSPORT

properties of the mucosal membrane with respect to the movement of Na and C1, and attempt to relate these findings to the central problem of transepi t helial transport . The routes of Na and C1 transport across the mucosal and basolateral (serosal) membranes of isolated rabbit ileum, in the absence of sugars or amino acids, are summarized in Fig. 2. At least three processes are involved in the influxes of Na and C1, one of which (pathway b ) is shared by both ions. A. Overall Na Influx (Pathways b and c)

The apparent intracellular concentration of Na in mucosal strips of rabbit ileum bathed by a solution containing 140 mM Na is approximately 40-50 m M (Schultz et al., 1966; Koopman and Schultz, 1969), and the cell interior is approximately 36 mV negative with respect to the mucosal solution (Rose and Schultz, 1971). Although many uncertainties becloud the interpretation of intracellular ion concentrations, in the case of Na there is abundant evidence suggesting that the overall volume-average Na concentration in a variety of cells is considerably larger than the cytoplasmic thermodynamic activity of Na due to binding and/or sequestration within intracellular organelles. In particular, Lee and Armstrong ( 1972) recently demonstrated, by means of cation-sensitive microelectrodes, that the activity coefficient of cell Na in bullfrog small intestine is approximately 0.5, whereas that of K is close to unity. Thus it appears safe to conclude that the entry of Na from th r mucosal solution into the cell is directed down a steep electrochemical potential gradient. This conclusion is corroborated by the observations that Na influx is not dependent upon metabolic energy, nor is it affected by ouabain (Chez et al., 1967). Further, MUCOSAL SOLUTION

CELL

FIG.2. Routes of Na and C1 transport across the mucosal and basolateral membranes. Bold arrows designate steps that appear to be dependent upon metabolic energy. (From Nellans el al., 1973.)

246

STANLEY G. SCHULTZ AND PETER F. CURRAN

Na influx does not appear to be subject to transconcentration effects, since it is unaffected by preincubation of the tissue in Na-free or C1-free media (Schultz et al., 196713; Nellans et al., 1973). These features are therefore characteristic of both processes b and c. However, Na influx does not appear to be due to simple ionic diffusion, since this process can be inhibited by K, Li, or guanidium in the mucosal solution (Frizzell and Schultz, 1972; Schultz et al., 196713). At least 50%, and perhaps as much as 85%, of the total Na influx is subject to inhibition by Li and, as discussed in Section V, E, Li appears to interact with both routes b and c. Finally, Na influx is inhibited by exposure of the mucosal surface of the tissue to conjugated bile salts (Frizzell and Schultz, 1970a), and by pretreatment of the rabbit with cyclohexamide (Frizzell et aZ., 1973b). Both of these procedures inhibit a variety of carrier-mediated influx processes, but have either no effect or a slight stimulatory effect on the influx of mannitol, which appears to be attributable to simple diffusion. Thus a large fraction of Na entry into rabbit ileal cells is a consequence of one or more carrier-mediated processes, a situation similar to that in other epithelia in which Na entry takes place down a n electrochemical potential gradient. Extrusion of Na from the cell across the basolateral membranes (d in Fig. 2) is an active transport process which is dependent upon a supply of metabolic energy and appears to be mediated by a Na, K-dependent, ouabain-sensitive ATPase similar to that identified in a wide variety of animal cells. This ATPase activity has been localized primarily if not entirely to the basolateral membranes by means of both assay of enzyme activity in fragmented cells (Quigley and Gotterer, 1969; Fujita et aZ., 1971, 1972) and autoradiographia techniques (Stirling, 1972). This localization is entirely consistent with the finding that active transepithelial Na transport is inhibited by the presence of ouabain in the serosal solution alone but is unaffected when this glycoside is present in the mucosal solution alone (Schultz and Zalusky, 1964a). Current evidence suggests that this ATPase is predominantly, if not exclusively, responsible for the maintenance of the low intracellular Na concentration, the high intracellular K concentration (ca. 140 mM), and active transepithelial Na transport. Although ethacrynic acid also abolishes active Na transport and results in an increase in intracellular Na concentration and an equivalent decrease in intracellular K, its effects mimic those of ouabain and appear to be attributable to an inhibition of the ATPase activity and perhaps energy-yielding metabolic processes as well (Chez et al., 1969). This notion is supported by the findings of Cassidy (1970) that, 1 mM ethacrynic acid is approximately as effective in inhibiting the Na, K-ATPase in rat jejunum as are the digitalis glycosides. Currently, there is no evidence

SODIUM AND CHLORIDE TRANSPORT

247

in rabbit ileum for the dual transport processcs a t thr basolateral membrancs postulated by Whitternbury and Proverbio (1970) and Whittembury (1971) for guinea pig kidncy slices, namely, an ethacrynic acid-sensitive NaCl extrusion mcchanisni and a ouabain-scnsitive Na-I< exchangc process, both of which are prcsuniably involved in active N a absorption. However, further study is certainly needed to define the actions of ethacrynic acid on rabbit ilcum completely. For example, AlAwqati, Field, and Greenough (personal communication) denionstrated that low concentrations of ethacrynic acid (0.1 m M ) do not affect active Na absorption by normal rabbit ileum but, in the prescnce of theophylline, inhibit active C1 secretion and restorr active Na absorption. As discussed in Section V, E, it seems likely that CI secretion in rcsponse to theophylline is mediated by a neutral NaCl rfflux mcchanisni at tlic brush bordcr, and, indred, all the observations of Al-Awqati et al. are consistent with the notion that ethacrynic acid a t low concentrations inhibits this brush border process. Thc important point is that in contrast to the model for proximal renal tubule suggested by Whittembury (1971) , in which there is a rheogenic, ouabain-insensitive Na extrusion mechanism located a t the basolateral membranes, active transepithelial Na transport by rabbit ileum is completely abolished by ouabain. [The term rhcogenic or currentgencrating is employcd instcad of the more common but ambiguous term electrogcnic. The reason for this choice of terminology, proposed by Schwartz (1971), is that neutral carrier mechanisms that bring about an equivalent exchange of cations or the cotransport of anions and cations can establish ionic asymmetries which may in turn affect thc electrical potential difference across a tissue. Thcsr ncutral but electrogeriic processes should be distinguished from carrier mrchanisms that do not bring about an equivalent transfer of anionr; or cations but aKe instead current-generating. The difference between thcsc two classes of carrier-mediated processes i s obscured by a “black-box” analysis of cpithelial tissues, but becomes of paramount importance in the attempt to dissect the “black box” and definc the processes responsiblo for ion transport across the t N o limiting membranes.] Finally, two additional points should bc noted : 1. As discussed in Section VII, C, the results of electrophysiological studics suggest that the active Na extrusion mechanism docs not result in a one-for-one exchange of Na for (prtmimahly) I< h u t , as in some other tissues (Kernan, 1970), is rheogcnic and is primarily responsible for the transepithelial PD. 2. Examination of the data in Tables I and I11 indicates that for unstripped rabbit ileum OdJEr(or OJ.Nm&) does not differ significantly from

240

STANLEY G. SCHULTZ AND PETER F. CURRAN

J,N," under short-circuit conditions. These observations suggest that the unidirectional flux of Na from serosa to mucosa may be attributed to ionic diffusion through the shunt pathway. This notion is further supported by the finding of Schultz and Zalusky (1964a), on unstripped rabbit ileum, conforms to Eq. (6) over the range of f50 mV and is not sigthat nificantly affected by ouabain or metabolic inhibitors which abolish active Na absorption solely by reducing JZE. Conformity of Jp,B with Eq. (6) has been confirmed by Nellans et al. (1974) for stripped rabbit ileum. However, preliminary findings by Desjeux and Curran (unpublished observations) suggest that OdJEis less than J g when stripped rabbit ileum is bathed in Ringer's solution containing 25 mM HCOa and 10 mM glucose; their data suggest that a portion of the serosa-to-mucosa flux of Na is not restricted to the shunt and proceeds via the transcellular pathway. An examination of the expression for JBNm&resulting from combining Eqs. (3) and (6) suggests a possible explanation for this apparent discrepancy. Thus,

JE

A linear relation between JBNm&and exp (5\Em,/2RT)that extrapolates to the origin meam that either the first term on the right of this equation is a multiple of exp ( W m , / 2 R T )or that it is equal to zero. However, JC","must be, a t least in part, the result of the active Na extrusion mechanism at the basolateral membranes and, as will be discussed below, J:: must be, a t least in part, mediated by a neutral Na-anion eflux mechanism a t the mucosal membranes. It is highly unlikely that these carrier movements would conform to the laws of strict ionic diffusion. Thus the finding of a linear relation between J Z and exp ( W m , / 2 R T )that extrapolates to the origin (Schultz and Zalusky, 1964a; Nellans et al., 1974) is most readily reconcilable with the conclusion that J F = 0 , implying that the basolateral membranes are virtually impermeable to Na and that the active Na extrusion mechanism is completely rectified. However, in the experiments of Desjeux and Curran, a plot of J,"m"versus exp ( W m , / 2 R T ) yielded a straight line with a positive intercept on the ordinate; the slope of this line corresponds to odJ2, whereas the intercept must reflect the transcellular contribution to J,",". Thus, under the conditions employed by Desjeux and Curran, J,"C"# 0. Recent studies by Garrahan and Glynn (1967) offer a possible explanation for this difference. These investigators showed that the active Na extrusion mechanism in the erythrocyte is capable of mediating a Na-K exchange and a Na-Na exchange, and that both intracellular and extracellular factors influence the relative magnitudes of these two exchange processes. The media employed in the ex-

SODIUM AND CHLORIDE TRANSPORT

249

periments by Nellans el al. (1974) and Desjeux and Curran on stripped ileum differed with respect to HCOl and K concentration, pH, and the presence or absence of glucose. All these differences might affect the relative magnitudes of the Na-Na exchange and Na-K exchange a t the basolateral membranes. In particular, Garrahan and Glynn showed that the ouabain-sensitive Na-Na exchange is abolished in the presence of extracellular K concentrations between 5 and 10 mM. I n the experiments by Nellans et al., the extracellular K concentration was 12 mM, whereas in those by Desjeux and Curran it was 5 m M . Thus it is quite possible that in the former experiments the active Na extrusion mechanism was completely rectified so that J,"C" = 0, whereas in the experiments carried out a t a lower K concentration a significant Na-Na exchange was present so that J E # 0. Needless to say, this explanation is a t best speculative, and further investigation is needed to clarify this issue. Nevertheless, a t present it is safe to conclude that the shunt pathway accounts for a t least approximately 7 5 4 0 % of JBNm&,and that the magnitude of this flux is largely determined by the total tissue conductance. B. Overall CI Influx (Pathways a and b)

The intracellular C1 concentration in mucosal strips of rabbit ileum bathed by a solution containing 145 mM C1 averages 66 m M and comprises a t least two compartments (Frizzell et al., 1973s). The first compartment exchanges very rapidly with extracellular 3Tl and amounts to approximately 58 mM; the second appears to be very slowly exchangeable and comprises the remaining 7-9 mM. Further, the slowly exchangeable compartment appears to be quite constant in size and is independent of the extracellular C1 concentration. Compartmentalization of cell C1 has been reported for several tissues, but the precise underlying mechanism (e.g., binding to cell proteins, sequestration) is unclear. Nevertheless, the concentration of the rapidly exchanging compartment appears to exceed significantly that which would be expected if C1 were distributed a t electrochemical equilibrium (38 m M ) , suggesting that the entry of C1 into the cell from the luminal solution is directed against an electrochemical potential difference. This suggestion is corroborated by the fact that C1 influx is a carrier-mediated process that conforms to saturation kinetics and is inhibited by metabolic inhibitors (unlike Na influx). Further, C1 influx is competitively inhibited by a variety of other monovalent anions. The sequence of inhibition is SCN > NOa > I > Br, which corresponds to the free-solution mobilities of these anions and represents Eisenman's (1965) sequence I, characteristic of an interaction with a

250

STANLEY G. SCHULTZ AND PETER

F. CURRAN

weak cationic field, Although the mechanism(s) responsible for C1 efflux (pathway e, Fig. 2) from the cell across the basolateral membranes is unclear, if our line of thought is correct, simple diffusion down an electrochemical potential difference might suffice. Finally, examination of the data in Tables I and 111 indicates that oJ2; (or is significantly less than Jf; under short-circuit conditions. Thus most of the J:A across rabbit ileum traverses the transcellular route, so that J:' # 0 and J:A # 0. The finding that J:' # 0 is of course compatible with an exit process attributable to simple ionic diffusion, but cannot be construed as proof of this notion.

,&A)

C. Coupled Na-CI Influx (Pathway b)

Prompted by the finding that theophylline brings about a simultaneous reduction in J:: and J:: (Table 111), the effects of theophylline on Na and C1 influxes in the presence and absence of the other ion have been examined (Nellans et al., 1973). As shown in Fig. 3, approximately 20% of C1 influx is inhibited by theophylline or by replacement of Na with choline, and approximately 20% of the Na influx is inhibited by theophylline or by replacement of C1 with SO4. Further, theophylline has no effect on either Na or C1 influxes in the absence of one or the other of these ions. Finally, the absolute inhibitions of Na and C1 influxes observed in the presence of theophylline or resulting from the removal of the other ion do not differ significantly. These findings cannot be attributed to changes in the P D across the brush border, inasmuch as ( 1 ) replacement of C1 with SO4has no effect on this P D (Rose and Schulta, 1971), and (2) any effect of theophylline on the P D would be expected to have opposite effects on the movements of Na and C1. These observations are the basis for the introduction of pathway b which represents a neutral NaCl influx process which is in.hibited by theophylline; this inhibition is restricted to pathway b. Further examination of this pathway has disclosed the following properties (Nellans et al., 1973): 1. NaCl influx via pathway b is an obligatory, neutral, one-for-one process. 2. Whereas choline and isethionate or SO4 are ineffective substitutes for Na and C1, respectively, Li can substitute for Na and I can substitute for C1 in a t least partially restoring the activity of this influx process. Thus this process is not highly selective with respect to cation and anion specificity. 3. The effect of lowering either the concentrations of C1 or Na in the

25 1

SODIUM AND CHLORIDE TRANSPORT

25 0

20 0 I

L

r N-

E

190 \ u)

-0

z

2 100 V

E

3

5 0

NO INFLUX

C I INFLUX

FIG.3. Effects of ion replacement and/or 10 m M theophylline 011 Na and C1 influxes across the brush border o f rabbit ileum. (From Nellans ct al., 1873.)

mucosal solution on the coupled NaCl influx is restricted to an inhibition of the maximal influx. The kinetics of this process are consistent with a carrier model (Fig. 4) featuring a random sequence of combination of Na and C1 with a membrane component to form a ternary complex with the provision that only the free (unloaded) carrier (X) or the ternary complex (XNaCl) can translocate across the mcmbrane. This model is essentially identical to that proposed by Goldner et al. (1969) to account for the kinetics of the coupled Na-sugar influx across the brush border of rabbit ileum. 4. The effect of theophylline on the kinetics of C1 influx is also restricted to the inhibition of maximal influx (Frizzcll et al., 1973a). Thus the direct or indirect effect of an elevation in intracellular CAMPmay be to decrease the ability of this influx process to bind either Na or C1 (or both). However, this possibility cannot he distinguished from possible effects of CAMP on other parameters that determine maximal influx, such as the

252

STANLEY G. SCHULTZ AND PETER F. CURRAN MUCOSAL SOLUTION

BRUSH BORDER

Not

x

C I t

XNa

CELL INTERIOR

t No'

-P-x'

ZK3 XNaCI-P-XNa

No t

XCI

CI t

IK2 X

X Na

t CI'

1

CI'

z

x CI'

t No'

X'

+CI'

FIG.4. Kinetic model for coupled NaCl influx across the brush border of rabbit ileum. (From Nellans et aZ., 1973.)

total carrier concentration or the rate constant for carrier translocation across the mucosal membrane. 5. Although the overall C1 influx is inhibited by metabolic inhibitors, this effect appears to be restricted to process a ; the coupled NaCl influx process does not appear to be directly dependent upon metabolic energy. Nonetheless, inhibition of the coupled NaCl influx process with theophylline or CAMPresults in a significant reduction in intracellular C1 concentration (Frizzell et al., 1973a). Thus the coupling of C1 influx to the downhill movement of Na may result in the movement of C1 against an electrochemical potential difference in a manner analogous to the role of the Na gradient in the active transport of sugars and amino acids (Schultz and Curran, 1970). The finding of a coupled NaCl influx process in the mucosal membranes of rabbit ileum is consistent with observations reported by other investigators suggesting an interaction between transepithelial movements of Na and C1 across small intestine. Thus Quay and Armstrong (1969) have reported that replacement of Na with choline inhibited C1 absorption and replacement of C1 with sulfate inhibited Na absorption by short-circuited bullfrog small intestine. These investigators suggested alternate mechanisms which result either in the coupled movements of Na and C1 across the mucosal membrane or across the basolateral membranes; however, their studies could not distinguish between these two alternatives. The model illustrated in Fig. 2 is virtually identical to one of the two alternatives suggested by Quay and Armstrong (1969, Fig. lob). Turnberg et al. (1970) also reported that replacement of C1 in the fluid perfusing normal

SODIUM AND CHLORIDE TRANSPORT

253

human ileum results in a simultaneous decrease in Na and C1 absorption, which cannot be attributed to a change in the transepithelial PD. Field et al. (1971) noted a significant correlation between Na and C1 transport across stripped short-circuited rabbit ileum, and speculated that this correlation may be indicative of a coupled interaction between the movements of these two ions. Further, evidence has been presented that Na and C1 transport across fish (Diamond, 1962) and rabbit gallbladder (Wheeler, 1963) are mediated by a neutral, coupled NaCl transport process, although the location of this mechanism was not specified. Unpublished observations by Frizzell and Schultz indicate the presence of a coupled NaCl influx process a t the brush border of rabbit gallbladder, which in many respects resembles that identified in rabbit ileum. In addition to the role of a neutral, coupled NaCl process in absorption, Taylor et al. (1968) and Munck (1972) have reported evidence suggesting the presence of a neutral NaCl secretory proccss in rat jejunum which can mediate active C1 secretion, and Powell et al. (1972) have suggested that the spontaneous secretory state observed in guinea pig ileum may be the result of a neutral Na anion pump mechanism that may be coupled to either HCO, or C1 with a greater affinity for the former. Norris et al. (1969) and Powell et al. (1973) have suggested that the secretion of NaCl and NaHC0, elicited by cholera toxin is mediated by a neutral Na anion secretory process, and additional evidence for the participation of pathway b in theophylline-induced C1 secretion is presented in Section V, E. Finally, Turnberg et al. (1970) have suggested that NaCl absorption by normal human ileum is mtldiated by a neutral exchange of Na for H and C1 for HCO,. It should be noted that thc model illustrated in Fig. 4 is potentially capable of NaCl or NaHC0, secretion, as well as an exchange of Na and C1 for H and HCO,; however, additional study of this process is necessary before further speculation regarding its rwersibility and selectivity is warranted. Suffice it to say that it is potentially capable of accounting for many of the neutral absorptivc, secretory, and exchange processes that have been described or postulated for small intestine from a variety of species. Finally, the effects of theophylline, CAMP, cholera toxin, and prostaglandin El on ion movements across short-circuited, stripped preparations of rabbit and human ileum appear to be largely restricted to (1) a reduction of J,”: to or below the level of JBNm&so that Na absorption is abolished or secretion is elicited, and (2) a decrease in JEL to the extent that this unidirectional transepithelial flux is now exceeded by J:L, resulting in active CI secretion. These agents apparently do not affect J,”,”and, although a small increase in JFA has been noted, t,his finding is inconsistent. AS indicated by Eqs. (2) and ( 3 ) ) the principal effect of a decrease in PH,on

254

STANLEY G. SCHULTZ AND PETER F. CURRAN

transepithelial unidirectional fluxes would be a decrease in PiB, so that the effects of intracellular cAMP (or agents such as theophylline, cholera toxin, and prostaglandin El that increase cell CAMP) on Na and C1 transport across in vitro rabbit ileum may be attributable at least in part to primary inhibition of the coupled NaCl influx process, a subsequent decline in cell C1 (Frizzell el al., 1973a) and Na concentrations, and in turn a decline in Jc"," as well as JZ', Currently, it is unclear whether cAMP by virtue of inhibiting influx simply unmasks preexisting processes that result in active C1 (and perhaps Na) secretion, or whether in addition to inhibiting influx cAMP stimulates de nouo active secretory processes. D. The Residual Influxes of CI (Pathway a) and Na (Pathway c)

In the presence of 140 m M Na and 145 m M C1, the unidirectional influxes of Na and C1 via pathway b average 4-5 pmoles.cm-2.hr-1. The unidirectional influx of C1 via pathway a is 8-10 pmoles-cm-2-hr-1, and the unidirectional influx of Na via pathway c is 10 pmoles.cm-2.hr-1; we refer to the influxes via pathways a and c as residual influxes, since they appear to contribute minimally t o transepithelial transport and vastly exceed the rates of net Na and C1 absorption by short-circuited rabbit ileum given in Table 111. Three possible explanations for these large residual influxes are: ( I ) they are the results of "exchange diffusion"; (2) they are artifacts resulting from the method employed to determine unidirectional influxes; and/or (3) they represent influxes into cells or compartments not involved in transepithelial transport. The possibility that some or all of the residual influxes via pathways a and c arc the results of exchange diffusion seems to be excluded by the fact that preincubation of the tissue in C1-free or Na-free solutions does not significantly affect the magnitude of these influxes (Schultz et al., 1967b; Nellans et al., 1973). Further, treatment of the tissue with ouabain or metabolic inhibitors, which markedly increase the intracellular Na concentration, minimally increase Na influx (Chez et al., 1967; Nellans et al., 1973). Thus neither Na nor C1 influxes appear to be significantly influenced by transconcentration effects. The possibility that the large residual influxes are consequences of the influx technique that employs inulin as a measure of the extracellular, adherent radioactive mucosal solution must be considered. If there are extracellular spaces that are not equilibrated with inulin during the brief (45- to 60-second) exposure of the mucosal surface of the tissue to the radioactive medium, or if there are large extracellular spaces that are

SODIUM AND CHLORIDE TRANSPORT

255

accessible to small ions but not to inulin, the calculated influxes of Na and CI would be artifactually high. This possibility has been raised by Sallee et al. (1972), but the likelihood that it seriously affects our influx measurements is minimized by the following observations: (1) the “inulin space” does not increase with time betwecn 0.2 and 2 minutes (Frizzell and Schultz, 1970b) ; ( 2 ) autoradiographic studies have indicated that inulin completely pendrates the intervillous spaces within 1 minute (Kinter and Wilson, 1965) ; and (3) 24 m M SCN inhibits approximately 90% of C1 influx in the presencc of 20 m M Cl (Frizzell et al., 1973a) (undoubtedly, a higher SCN/Cl ratio would result in an even greater inhibition). These observations, particularly the last, are difficult to reconcile with the notion that the calculated influxes are increased by inadequate estimation of the extracellular spaces. At least 90%, of the calculated CI influx must have traversed a barrier by means of a mechanism that is subject to competitive inhibition; a n unequal tlistribiitioii of c‘l and inulzn i n extracellular spaces cannot account for thi.5 .finding. A similar argumcnt can be extended to the case of Na, since at lead 50(% and perhaps as much as 85y0 of the N a influx across the rnucosal membranes is subject to inhibition by Li (Frizzell and Schultz, 1972). These Observations also make it highly unlikely that edge daniagr is responsible for the large residual fluxes of Na and CI. Having excluded exchange diffusion and experimental errors as reasonable explanations for the large residual C1 and Na influxes, it seems likely that most of these fluxes enter cells or compartments that are not involved in transepithelial transport, and that the predoniinant pathways involved in active transepithelial transport of Na and C1 are pathways b and c. This notion is supported by observations discussed in Section V, E. E. Relations Among Na and Ci influxes and Transepitheliai Transport

The effects of a Na-free medium and a C1-free medium on transepithelial fluxes of Na and C1 across short-circuitcd, stripped preparations of rabbit ilcum arc given in Table V. In the prcwnce of Ringer’s solution containing 10 m M HCO, (pH 7.2), both Na and C1 arc actively transported from mucosa to serosa, and the ratc of transport of N a significantly exceeds that of C1. There is a small rnsidual current of approximately 1 pmolccm-2. hr-l, which approximatcs the rate of HCO, production by this preparation. In light of the findings of Dietz and Field (1973), as well as the numerous observations in znuo cited above, it seems reasonable to attribute this residual current to HCOBsecretion. Replacement of Na with cholinr abolishes active C1 transport as a result of a decrease in JgL alone. At the same time, the transepithelial PD is

256

STANLEY G. SCHULTZ AND PETER F. CURRAN

TABLE V

TRANSEPITHELIAL FLUXES OF SODIUM AND CHLORIDE ACROSS S I I O R T - C I R C U I T E D RABBIT ILEUMa

Ringer's solution C1-free Na-free

12 10

-

8

_

8

-

4

10

8

2

2

_

_

_

8

8

0

3

2

1-2 0

1 0

All fluxes and the I,, are expressed in pmoles-cm-2.hr-1; qmS is in millivolts. All numbers have been rounded off, but the differences reported are statistically significant. (Data from Nellans et al., 1974.)

abolished. In contrast, replacement of C1 with SO, reduces, but does not eliminate, active Na transport; once more, the reduction is a result of a decline in JZ: with no significant change in J,",". The transepithelial P D also declines, but i s not abolished, and parallels the rate of active Na transport. Finally, it should be noted that the decreases in Jg: and J::t in a Na-free medium are almost exactly equal to the decreases in J,": and JZEt observed in a C1-free medium. These findings strongly suggest (1) that active C1 transport is entirely a consequence of the coupled NaCl influx process (pathway b, Fig. 2 ) , and that the residual C1 influx (pathway a, Fig. 2) is not involved in transepithelial C1 transport; and (2) active Na transport is in part a consequence of the coupled entry of NaCl and in part the result of a C1-independent entry process (pathway c, Fig. 2 ) . Further, in the absence of Na, the I,, does not differ significantly from zero, so that either HCOI secretion is also abolished or is accompanied by a cation (in all likelihood, H ) ; this remains to be clarified. Finally, it should be stressed that the rate of C1-independent transepithelial N a transport is only a small fraction of the C1-independent Na influx, suggesting that the bulk of the latter represents influx into cells or compartments that are not involved in transepit helial movements. Recent findings by Nellans et al. (1974) also strongly suggest that the coupled NaCl influx process is at least in part responsible for the secretory state elicited by theophylline or cholera toxin. I n the absence of Na, the transepithelial potential difference (close to zero) is unaffected by the presence of theophylline or cholera toxin, and active C1 secretion is not observed. These findings support the notion proposed by Taylor et al. (1968), Norris et al. (1969), and Powell et al. (1972, 1973) that anion

257

SODIUM AND CHLORIDE TRANSPORT

secretion by mammalian ileum is the result of a Na anion (C1 or HC08) neutral secretory process which does not contribute directly to the transepithelial PD. Further, in the absence of C1, active Na absorption is observed in theophylline-treated tissues. Similar findings have been reported by Powell et al. (1973) for short-circuited, stripped rabbit ileum treated with cholera toxin. Studies on Na and C1 transport across short-circuited stripped rabbit ileum by Binder et al. (1973) and Powell et al. (1973) have yielded results that differ from those of Nellans etal. (1974) (Table V) and are summarized in Table VI. These investigators found that in the presence of Ringer’s solution containing 25 mM HCOa (pH 7.4), active Na transport from mucosa to serosa was quite small, and they did not detect any net transport of C1. However, in the absence of C1, or HCOI and C1, activeNaabsorption was enhanced. The discrepancies between the observations reported in Tables V and VI may be resolved by the models illustrated in Fig. 5. The central features of these models are the presence of a C1-independent, CAMP-insensitive Na entry mechanism plus a coupled NaCl transport process that mediates NaCl influx and NaCl (or NaHC03) eMux across the brush border. As discussed above, the neutral NaCl influx process is inhibited by CAMP. Assuming that the effect of CAMP is restricted to this influx process, the data of Nellans et al. (1974) given in Table V are consistent with model a. In the presence of theophylline (model b), the neutral NaCl influx process is abolished, active Na absorption is abolished, and active C1 secretion is observed, mediated by the neutral brush border efflux mechanism. In essence, the C1-independent Na entry across the brush border is “recycled” by the neutral NaCl efflux process, and transepithelial Na movement is abolished. However, removal of C1 clearly would prevent the recycling of Na across the brush border and restore active Na absorption, as has been observed by Nellans et al. (1974) in theophylline-treated preparations, and Powell et al. ( 1973) in choleragenTABLE VI TRANSEPITHELIAL FLUXESOF SODIUM AND CHLORIDE ACROSSRABBITILEUMO

Ringer’s solution HC03, C1-Free Na-free

10 14

9

1

12

-

.-

2 -

7 7

7 7

0 0

A11 fluxes are expressed in fimoles.cm-2-hr-’ and have been rounded off, but t,he differences reported are statistically significant. (Data from Binder el al., 1973.)

258

STANLEY G. SCHULTZ AND PETER F. CURRAN

HIGH CAMP

LOW CAMP N

;

v

V

T

4

N

;

T

r

J

O

No CI

2

No

(bl

(0)

INTERMEDIATE

MUCOSAL SOLUTION

CI

CI

NO

CAMP

SEROSAL SOLUTION

No 05

No (C)

FIG.5 . Models illustrating the possible effects of CAMPon net movements of Na and C1 across the brush border and the epithelium via the transcellular route. Bold arrows designate net fluxes.

treated preparations. Model c represents an intermediate instance in which the neutral NaCl influx process is only partially inhibited, leading to a reduced rate of active Na absorption and a very low rate of net transepithelial C1 movement; removal of C1 from the bathing media would clearly enhance Na absorption. The purpose of this exercise is to illustrate the wide range of transport activities that can be accommodated by an epithelial tissue that features (not necessarily within a single cell type) opposing mechanisms for transepithelial Na transport, a rheogenic mechanism that extrudes Na from the cell across the basolateral membranes and a neutral Na anion mcchanism that extrudes Na from the cell across the mucosal membrane. The observed behavior of Na and C1 under control conditions, and the response to ion replacement, can clearly vary over a wide spectrum, depending on the balance between these opposing absorptive and secretory processes. Although these models can, in principle, explain many of the apparent discrepancies that have emerged from studies on stripped rabbit ileum, they raisc many questions that remain to be resolved. These involve the nature of the mechanism responsible for C1 movement across the basolateral membranes, the factors that influence the selectivity and provide the driving force for the neutral Na anion secretory process, and the effect of theophylline and/or cholera toxin on the transport properties of the basolateral membranes. Finally, it is well established that cholera

259

SODIUM AND CHLORIDE TRANSPORT

toxin stimulates HCO, secrction by in vivo mammalian ileum but apparently does not affect HCO, transport by in vitro rabbit ileum (Dietz and Field, 1973). The reason why in vivo and in vitro preparations differ with respect to the predominant anion secreted in response to cholera toxin is unknown. F. Effects of Acetazolamide on N a and CI Influxes and Transepithelial Fluxes

In 1956 Parsons reported that acetazolamide markedly inhibited Na, C1, and water absorption from in vivo loops of rat ileum perfused with HCO, (25 m M ) Ringer's solution and reversed the direction of HCO, movement from secretion to absorption. Subsrquently, Kinney and Code (1964) reported that acetazolamidc abolished active C1 absorption by in vivo canine ileal loops; analyses of bidirectional fluxes indicated that this effect was dur entirrly to a reduction in J:: (lumen to plasma) with no significant change in JFA (plaarna to lumen). The decrease in active C1 absorption was linearly related to a dccrease in water absorption and, in the absence of C1 absorption, water absorpt,ion did not differ significantly from zero ; these findings strongly suggest that acetazolamide inhibited Na absorption, as ~vellas that of C'1, and are in accord with the findings of Parsons ( 1956). More recently, Turnbrrg et (11. (1970) demonstrated that acetazolamide inhibits both Na and CI absorption by normal human ileum, and Phillips and Schnialz ( 1970) demonstrated that this agent inhibits Na, CI, and water absorption by zn vivo loops of rat colon. I n each instance the movement of HCO, was such as to maintain electroneutrality in the luminal solution so that a reduction in CI absorption in excess of the redurtion in N a absorption was associated with a reduction in HC03 secret ion, Studirs on the effect of acetazolamidc on Na and CI movcments in rabbit ileum in vitro have indicated that t,his agent ( I ) inhibits and J:, (2) reduces the intracellular concentration of C1, and (3) has no effect on the electrical potential diffrrencc across the mucosal membrane (Rose and Scliultz, 1971; Nellans et al., in prrparation; Frizzell et al., 1973a). Further, this agent only minimally inhibits J:\ in the presence of theophyllinc (7040% of its inhibitory cffrct on CI influx is exerted on the theophylline-sensitive pathu-ay). Thus the effects of acetazolamide on Na and CI absorption, like that of theophylline, appear to be attrihutablr to inhibition of the coupled NaCl influx process (pathway b, Fig. 2). Finally, acetazolamide abolishes active C1 absorption by short-circuited rabbit ileum and reduces, but does not complately abolish, active Na absorption (H. N. Nellans, R. A. Frizzell, and S. G. Schultz, unpublished observations). It is

Jzi

260

STANLEY G. SCHULTZ AND PETER F. CURRAN

of interest in this respect that acetazolamide decreases water, Na, C1, and HC03 secretion elicited by cholera toxin (Norris et al., 1969; Leitch et al., 1966), suggesting that these ion secretory processes may be mediated by the neutral mechanism illustrated in Fig. 4; this notion is consistent with the models for the spontaneous Na anion secretion proposed by Powell et al. (1972) for guinea pig ileum, and models for cholera-induced secretion in in vivo (Norris et al., 1969) and in vitro (Powell et al., 1973) rabbit ileum. The mechanism underlying the action (s) of acetazolamide are unclear, but at least three possibilities must be considered. First, acetazolamide inhibits Cl-HC03 exchange processes in various tissues that contain relatively high activities of carbonic anhydrase, so that its effect has customarily been attributed to the inhibition of intrai cellular formation of HC03 (cf Maren, 1967). Although there is good evidence for a link between C1 absorption and HCO3 secretion (Hubel, 1967, 1969) in mammalian ileum, available data indicate that the specific activity of carbonic anhydrase in small intestine is low compared with erythrocytes, gastric mucosa, or colonic mucosa, but is not negligible (Carter and Parsons, 1971, 1972). Thus the possibility that the action of acetazolamide on C1 absorption may be, a t least in part, a consequence of the inhibition of HC03 formation cannot be dismissed at present. Second, recently evidenced has been presented that acetazolamide inhibits 3’, 5’-cAMP-phosphodiesterase isolated from beef heart (Schultz and Senft, 1967). The inhibitor constant is approximately 6 mM, a value that is orders of magnitude greater than that needed to produce complete inhibition of carbonic anhydrase (Maren, 1967), but one that is well within the range of concentration employed to inhibit intestinal transport. Thus the effect of acetazolamide could be, at least in part, analogous to the effect of theophylline. However, recent studies by H. N. Nellans, R. A. Friezell, and S. G. Schultz (unpublished observations) indicate that acetazolamide does not increase CAMP levels in rabbit ileum. Further, as discussed below, the kinetics of the acetazolamide effect suggest that its action cannot be attributed to the inhibition of phosphodiesterase. Third, studies on the kinetics of the inhibitory effect of acetazolamide indicate that this agent behaves as a competitive inhibitor (Fig. 6 ) , on and that its effect is observed within 45 seconds of exposure to the mucosal surface of the epithelium (i.e., when it is present in the radioactive mucosal “test” solution alone) ; preincubation of the tissue with acetazolamide for 20-30 minutes prior to the determination of Cl influx increases the inhibitory effect on JEt by only l0-20%. Thus this agent behaves much like other competitive inhibitors of C1 influx such as SCN, N63, Br, and I. In this respect it is of interest that studies employing a catalytically active Co (11)-carbonic anhydrase have shown that this enzyme analog is in-

e\

26 1

SODIUM AND CHLORIDE TRANSPORT

281

0 O 24

0.01

0.02

003

I/[Cl](m

0.04

0.05

MI-'

FIG.6. Effect of acetazolamide on J::, plotted according to the method of Lineweaver and Burk. (From Frizzell el al., 1973a.)

hibited by monovalent anions, the sequence of inhibitory activity being NCO > NO, > I > Br > C1 > F (Lindskog, 1966). Spectral analysis suggested that these anions exert their inhibitory action by binding to the metal ion or to a nearby site. Acetazolamide also appears to exert its inhibitory action on this enzyme by binding to or near the same site as the inhibitory monovalent anions. The finding that the sequence of competitive inhibition of \:J by monovalent anions corresponds to that observed for Co (11)-carbonic anhydrase, and that acetazolamide conforms to the same kinetic inhibitory pattern, suggests that the effect of this agent may simply reflect direct interaction with the carrier mechanism at a weak cationic site which resembles the anion-acetazolamide binding site possessed by carbonic anhydrase ; carbonic anhydrase itself need not be implicated. However, Carter and Parsons (1971) demonstrated that 45% of the total carbonic anhydrase activity recovered from guinea pig colonic mucosa is particle-bound, and that 18% is localized to a particulate fraction consisting of nuclei and microvillous membranes; if the same obtains for rabbit ileum, the possibility that brush border carbonic anhydrase activity is somehow involved in the operation of thc neutral NaCl influx mechanism cannot be dismissed. In any event, it seems very likely that the effect of acetazolamide is the result of interaction with a brush border component

262

STANLEY G. SCHULTZ AND PETER F. CURRAN

directly involved in the influx process. The rapidity of its action, together with the fact that it is a competitive inhibitor of JE:, whereas theophylline is a noncompetitive inhibitor of this process, strongly mitigate against the possibility that the effect of acetazolamide is attributable entirely to inhibition of phosphodiesterase. In summary, current evidence suggests that acetazolamide inhibits the mechanism responsible for neutral NaCl influx but does not affect the C1-independent Na entry process. The relation between these brush border mechanisms and carbonic anhydrase activity remains to be determined. VI. SOLUTE-COUPLED TRANSPORT

There is abundant evidence that actively transported sugars and amino acids stimulate transepithelial Na transport across in vitro and in vivo preparations of small intestine from a wide variety of animal species (Schultz and Curran, 1970). This phenomenon cannot be attributed to an increase in the energy supply to the absorptive cells, since there is no reason to believe that in vivo preparations are energy-limited, and the enhancement of Na transport by in vitro preparations can be elicited by nonmetabolized sugars and/or amino acids. Further, theophylline does not affect sugar or amino acid-enhanced Na absorption (Field, 1971a,b), nor does replacement of C1 with a variety of other anions, so that these pathways must parallel the coupled NaCl influx pathway illustrated in Fig. 2. Thus, in addition to the pathways illustrated in Fig. 2, there are other pathways for Na influx coupled to the influxes of sugars and amino acids; these, in all likelihood, reside in the brush border of the villous absorptive cells that have been directly implicated in sugar and amino acid absorption (Kinter and Wilson, 1965). Na-coupled solute transport has been reviewed in detail (Schultz and Curran, 1970), so that the ensuing discussion focuses largely on observations and issues that have been disclosed, raised, or clarified recently. A. The Mechanism of Enhanced Na Transport

Three models have been proposed to account for the underlying mechanism by which actively transported sugars and amino acids enhance transepithelial Na transport by small intestine. Kimmich (1970) has suggested that the transport processes for sugars, amino acids, Na, and K all compete for a common energy source a t the brush border, namely, an ouabain-sensitive, Na ,K-activated ATPase. This ATPase would presumably supply energy directly, or indirectly through

SODIUM AND CHLORIDE TRANSPORT

263

another high-energy intermediate (E-P2), for the uphill movement of sugars and amino acids from the mucosal solution into the cell, active N a extrusion from the cell into the mucosal solution, and active K accumulation by the cell across the brush border. According to this model, sugar and amino acid transport would be inhibited by the depletion of intracellular Na and the inhibitory effect of ouabain on sugar and amino acid transport takes place a t the brush border. In addition, one would predict that the absence of extracellular K (mucosal solution) would also inhibit sugar and amino acid transport. Further, the active transport of sugars or amino acids would deplete some of the energy for active Na extrusion (J,","), which in turn would increase net movement of Na across the brush border and, under steady-state conditions, net transepithelial Na tranaport. Finally, this model accounts for the apparent mutual inhibitory effect between the active transport of sugars and amino acids observed under some conditions in in vilro preparations of small intestine (Schultz and Curran, 1970). In our view, Kimmich's model is unacceptable for the following reasons : 1. Depletion of cell Na does not affect sugar or amino acid influx when Na is present in the mucosal solution, but results in a marked inhibition of these influxes when the mucosal solution is Na-free. Thus these processes appear to depend on extracellular Na rather than intracellular Na (Schultz et al., 1967b; Goldner et al., 1969). 2. The presence of sugars and/or amino acids in the mucosal solution enhances JfrIE and JE,S (Schulta and Curran, 1970). Thus Na inJluz across the brush border is increased; there is no evidence that eflux of Na across . I : is; decreased. ) On the contrary, Curran et al. ( 1970) the brush border ( demonstrated that alanine accumulation by the cells increases Na e f h x across the brush border. 3. The coupled interaction between Na influx and the influxes of sugars and amino acids across the brush border is unaffected by ouabain or metabolic inhibitors (Rose and Schultz, 1971; Chez et al., 1967; Hajjar et al., 1970; Curran et al., 1970) ; according to the Kimmich model, the presence or absence of Na should be irrelevant if the cell is energy-depleted or the ATPase activity is inhibited by ouabain. 4. K influx (J&) is unaffected by sugars or amino acids under conditions where JzE is enhanced (Frizzell et al., 1973c) ; according to the Kimmich model, K influx should decrease. 5 . Finally, as discussed above, there is no compelling evidence for the presence of a ouabain-sensitive ATPase in the brush border. Studies on a purified brush border fraction from rabbit ileum (Frizzell and Schultz, unpublished observations) indicate that, whereas the brush border contains

264

STANLEY G. SCHULTZ AND PETER F. CURRAN

ATPase activity, there is no detectable stimulation of this activity by Na and K or inhibition by ouabain. Thus, on the basis of available evidence obtained on small intestine (as well as abundant evidence dealing with the interaction between Na and amino acid uptake by other cells), we consider the Kimmich model an intriguing hypothesis but one that is contradicted by a large body of data. In this respect it should be noted that Kimmich’s model is based on data obtained using a suspension of cells isolated from chicken small intestine. Not only are these cells unusually “leaky” (compared to other in vitro preparations of small intestine including that of the chicken), but the use of a cell suspension does not permit a distinction between the properties of the brush border and those of the basolateral membranes. The extent to which these two factors may influence Kimmich’s data is unclear. Finally, two points should be stressed. First, this critique has been concerned solely with the manner in which sugars and amino acids enhance transepithelial Na transport; the question of the adequacy of the Na gradient (or ion gradient) hypothesis with respect to active sugar or amino acid transport is incompletely resolved, but is not directly relevant to the topic of this article. Second, although in our opinion Kimmich’s model cannot be readily reconciled with a large body of experimental evidence, a definitive alternative explanation for his very interesting findings has not been proposed. The second model emerged from the studies of Fordtran et aZ. (1968) on human jejunum in vivo. These investigators suggested that enhanced Na absorption in the presence of glucose or galactose is a consequence of solvent drag in response to sugar-stimulated water absorption. The principle support for this hypothesis are the findings that human jejenum is highly permeable to Na, that net transepithelial Na movements are strongly influenced by solvent drag or the direction of net volume flow, and that glucose and galactose also stimulate what appears to be active urea transport from lumen to plasma. However, Turnberg (1971) has reported that K transport across normal human jejunum in vivo is also markedly influenced by solvent drag or the direction of water flow and, indeed, the apparent reflection coefficient for K calculated by Turnberg is smaller than that calculated by Fordtran et aZ. for Na ( U N , , / U K = 1.18). Frizzell and Schultz (1972) have reported that the permeability of the shunt pathway across rabbit ileum to K is greater than that to Na by a factor of 1.14. Nevertheless, whereas glucose, galactose, and alanine significantly stimulate Na influx across the luminal surface, simultaneously determined K influxes are unaffected (Frizzell et al., 1973~).Since in epithelial tissues characterized by low-resistance shunt pathways and high hydraulic con-

265

SODIUM AND CHLORIDE TRANSPORT

-60

I

p"

>

-

-50-

E

-40-

A

U v)

8

2

z U v)

-30-20-

K

-10-

->

..

E

0

n A

U

K

s

::! I 0-

t3

YU K I-

FIG.7 . Effect of alsnine ( A ) on the transmiicosal (\Erma) and transmural (\Erkms) PDs in rabbit ileum. (From Rose and Schultz, 1971, with the permission of the Rockefeller Ilniversity Press.)

ductivities, the extracellular route is likely to provide the principle pathway for solvent drag effects on electrolytes, we conclude that at least part of the enhanced Na transport is a consequence of the coupled movement of Na and sugars or amino acids across the mucosal membrane itself, as originally envisaged by Bihler et al. (1962) and modified by Schultz and Zalusky (Schultz and Zalusky, 1964b). This model is strongly supported by electrophysiological studies on rabbit ileum (Rose and Schultz, 1971), bullfrog small intestine (White and Armstrong, 1971), and the proximal tubule of the newt (Maruyama and Hoshi, 1972) which demonstrated that the addition of sugars and/or amino acid to the luminal solution results in a prompt depolarization of the PD across the luniinal membrane (Fig. 7). As discussed by Rose and Schultz (1971)) the observations on rabbit ileum cannot be attributed solely to the movement of Na through the shunt pathway, but are best accommodated by the notion of a rheogenic influx of Na across the mucosal membrane coupled to the influxes of sugars or amino acids. Finally, although the pathways for sugar- and amino acid-stimulated influx of Na across the brush border parallel pathways c and b, there is )

266

STANLEY G. SCHULTZ AND PETER F. CURRAN

currently no experimental evidence suggesting the presence of a pathway for Na efflux across the basolateral membrane in addition to pathway d which functions in the absence of sugars or amino acids. Thus, although several parallel processes appear to be capable of mediating Na influx into the cell (or cells) involved in transepithelial Na transport, a common mechanism mediated by ouabain-sensitive ATPase appears to be responsible for active Na extrusion out of the cell(s) across the basolateral membranes. B. Na-Dependent versus Na-Coupled Transport Processes

In our 1970 review of this subject (Schultz and Curran, 1970), we tabulated various solutes whose transport across the small intestinc is enhanced by the presence of Na and could therefore be considered Nadependent; in the past 3 years this list has grown significantly. At the same time, we attempted to distinguish between transepithelial transport processes that are enhanced by the presence of Na and processes that arc coupled to Na and result in a mutual or reciprocal stimulation of transport. This distinction has become increasingly important in light of recent evidence that the magnitude and direction of water movement across lowresistance epithelial tissues affects the width of the lateral intercellular spaces, and thereby the permeability of this extracellular pathway to small water-soluble organic solutes and ions (Loeschke et a!., 1970, 1971; Smulders et al., 1972). There is compelling evidence that water movement from mucosa to serosa, either spontaneous or induced bji an osmotic pressure difference, widens the interspaces and in some instances increases the tissue permeability to ions and other small solutes. In contrast, water movement from serosa to mucosa resulting from an osmotic pressure difference (Loeschke et al., 1970, 1971; Smulders et al., 1972) has the reverse effect on the dimensions of the spaces and tissue permeability. Thus the presence of Na in the mucosal solution may increase the permeability of the small intestine to small water-soluble solutes such as urea, thiourea, and acetamide (Esposito et al., 1969, 1970, 1972), as well as small ions, simply by virtue of the fact that water transport is for the most part contingent upon NaCl transport. A particularly striking illustration of this notion has recently been reported by Munck (1972) with respect to the effect of proline on the bidirectional fluxes of thiourea across in vitro rat jejunum. With 1 m M thiourea in the mucosal and serosal solutions, net transport of thiourea does not differ significantly from zero (Table VII). The addition of 20 mM proline to the mucosal solution results in an increase in the short-circuit current attributable to an in-

267

SODIUM AND CHLORIDE TRANSPORT

TABLE VII EFFECT OF PROLINE O N T ~ ~ U RTRANSPORT E : . ~ ACROSSRAT JEJUNUM^

Control Proline, 20 rnM

J,,

J,,

J,,

0.05 O.Ogb

0.05 0 .O G b

0 0.02b

Data from Murick (1972). All values are expressed in pmoles * cni-2 * hr-1, and have been rounded off to one signific:int figure. b Statistically significant difference from control values.

creased JE,”; a significant net transport of thiourea from mucosa to serosa in the absence of a concentration difference, attributable to solvent drag; significant increases in the bidirectional fluxes of thiourea, suggesting an increase in the permeability of the tissue to this solute; and a decrease in transepithelial resistance. Whilc the net transport of thiourea and the permeability of the tissue to thiourea was injuenced by proline, the notion that thiourea transport is “proline-dependent” is ludicrous; all these observations can be readily explained by the effects of Na-coupled proline transport on water t,ransport, the width of the intercellular spaces, tissue resistance, and solvent drag. One can state with a high degree of certainty that the effects of Na-coupled glucose, galactose, or proline transport on the transport of urea or thiourea have the same mechanistic basis as the nonspecific effect of Na on the transport of acetaniide, and so on. Mayersohn et al. (1971; Mayerson and Gibaldi, 1970) dcmonstratcd that replacement of Na with other cations inhibited the passive transfer of a variety of solutes across clvertecl rat intestine; of the cations examined K was most effective with respect to this inhibitory effect. These investigators also noted that the degree of inhibition of passive solute transport was paralleled by a gain in tissue water; they concluded that extracellular channels provide the major route for the transfer of these solutes, and that cell swelling with resulting narrowing of these lateral intercellular spaces js responsible for the observed inhibitions. Nwdless to say, this conclusion is entirely consistent with thc hypothcsis that the enhancement of solute transport by Na or, conversely, the inhibition of solute transport by the replacement of Na with other cations, may in many (if not most) instances reflect changes in the dimensions of the lateral intercellular spaces and tissue permeability and does not constitute evidence for Na-coupled transport.

268

STANLEY G. SCHULTZ AND PETER F. CURRAN

In addition to enhancing the movement of some solutes by virtue of widening intercellular spaces, increasing solute permeability and possible solvent drag effects, water flow from mucosa to serosa could also influence solute absorption because of unstirred and poorly stirred regions that surround the epithelial cell layer. Thus water flow from mucosa to serosa tends to concentrate solutes in the unstirred layers adjacent to the mucosal membranes and dilute (or “wash out”) solutes in the lateral spaces, subepithelial tissues, the capillary network in the villous core, and possibly within the absorptive cells themselves. Preexisting concentration gradients may be increased, and presumably absent concentration gradients may be established between the solution adjacent to the mucosal membrane and either the serosal tissues and solution in vitro, or the capillary plasma in vivo. This concentrating and washing-out effect is observed in wellstirred artificial membrane systems, is inevitable in less well-stirred in vitro preparations of small intestine, and is likely to play a maximal role in in vivo studies employing techniques that provide minimal stirring. In light of these considerations, the simple observation of an increase in solute absorption in response to an increase in water absorption should not be indiscriminantly attributed to classic solvent drag. To date, whereas Na has been shown to enhance the transepithelial transport of a wide variety of organic solutes, only actively transported sugars and amino acids have been shown to enhance the unidirectional in$ux of N u into small intestinal epithelium and N u absorption; the term Nadependent (or preferably, Na-coupled) should be reserved for these agents if this term is to have any significant meaning with respect to underlying mechanism. In those instances in which Na appears to enhance the absorption of other solutes, it is essential to determine whether this is a direct effect of Na or an effect secondary to increased water absorption. Returning to the question of the mechanism of sugar- and amino acidenhanced Na transport across small intestine, the current picture may be summarized as follows. There is abundant evidence that these organic solutes specifically stimulate Na movement across the mucosal membranes and qmo are virtually into the cell; the effects of these agents on J:, 9,,, instantaneous, and it is unlikely that significant volume flow is established rapidly enough to complicate these findings by any of the mechanisms described above. However, in view of the evidence that the tight junctions are permeable to Na, K, and C1, it is quite possible that, once a fluid stream from mucosa to serosa is established, the transepithelial movement of Na m a y be further enhanced by solvent drag, wash-out effects, and so on. The extent to which Na absorption is secondarily enhanced by the stimulation of water absorption depends upon a variety of factors, such as the rate of water absorption, the permeability of the junctional complexes to

SODIUM AND CHLORIDE TRANSPORT

269

Na, the effect of enhanced water flow on tissue permeability, and the role of unstirred regions, and is therefore likely to vary with different regions of the small intestine, in vivo or in vitro preparations, and so on. In summary, the model suggested by Schultz and Zalusky (1964b) and confirmed by direct measurements of the effects of sugars and amino acids on Na influx (Schultz and Curran, 1970; Frizzell et al., 1973c), and that proposed by Fordtran et al. (1968) to explain sugar and amino acid stimulation of Na and water absorption, are not mutually exclusive; whereas a direct coupling betwem Na influx and the influxes of sugars and amino acids across the brush border seems certain, an additional contribution secondary to enhanced water absorption is not only feasible but quite likely.

VII. ION TRANSPORT AND THE ELECTROPHYSIOLOGY OF RABBIT ILEUM

Figure 1 depicts our working model for the analysis of ion fluxes across the two limiting membranes and the permselective extracellular shunt pathway that characterize the absorptive epithelium of small intestine. The equivalent electrical circuit that corresponds to this working model is illustrated in Fig. 8. Emis the elrct,romotive force (emf) across the mucosal membrane, R1 is the internal resistance of this battery, and R2 represents a shunt resistance across the mucosal membrane; E,, Ra, and R4 are the

FIG.8. Equivalent electrical circuit iiiodel of sriiall intestinal epithelium. (From Rose and Schultz, 1971, with the permission of the Rockefeller University Press.)

270

STANLEY G. SCHULTZ AND PETER F. CURRAN

corresponding parameters for the serosal membrane, and EL, RK,and R0 are those for the permselective shunt pathway; m, c, and s designate the mucosal solution, intracellular compartment, and serosal solution, respectively; and, all potential differences are with reference to that of the mucosal solution. The solution of this equivalent circuit for *ma and *mc is: *ma

=

[R&L(E,R~- EmRm)

*mc

=

-[EmRm(RsRs

and

+ ELRL(R1Rm + R,Rs)I/Rt

(10)

+ RKRL)+ (EaRa - E L R L ) R I R ~ ] / R(11) ~

where

and

Rt

=

R1Rm

+ R3Ra + R ~ R L

The orientations of the electromotive forces shown in Fig. 8 are included in this solution, so that the absolute values of Em,E., and EL are employed in Eqs. (10) and (11) ; the orientation of any electromotive force can be reversed by simply changing the sign preceding that emf in these equations. Finally, as indicated previously, all resistances are apparent resistances inasmuch as they are related to the serosal area as opposed to the area of the mucosal epithelial cell layer which may be 30 times greater than the former. Nevertheless, these considerations are irrelevant with respect to the electrophysiological behavior of the tissue, since it is clear from Eqs. (10) and (11) (where Rt appears in the denominator) that the characteristic that distinguishes a low-conductance tissue from a high-conductance tissue is the relative resistance of the shunt pathway compared to the transcellular pathway. Thus correction of apparent resistances for the true mucosal area through the use of a factor relating serosal area to mucosal area would in no way affect our argument. A. The Spontaneous Transepithelial Electrical Potential Difference

The spontaneous electrical potential difference across in vitro rabbit ileum in the absence of sugars or amino acids is small, ranging between 2 and 5 mV serosa positive, and for this reason has often been overlooked in the analysis of transmural ion movements. However, as pointed out in Section 111, the presence of high-conductance paracellular shunt pathways permits significant net transepithelial ionic diffusion in the presence of small driving forces, so that even small electrical potential differences cannot be ignored.

271

SODIUM AND CHLORIDE TRANSPORT

According to the rquivalent electrical circuit illustrated in Fig. 8 and Eq. ( l o ) , it is clcar that the magnitude of *,,,,is markedly affected by thc conductance of the shunt pathway anti in itself tells one little regarding thr electroinotive forccs a t thc two limiting membranes. Thus for rabbit ileum (unstripped) R j R ~ O.l&, SO that Eq. (10) rrduces to 9,,G 0.1 (E,R, - Elr,Rll,)

+ 0.9E~h’i.

(See Frizzcll and Schultz, 1972.) Thus : 1. Under thc conditions employcd in these studies, when both solutions ! ,I is only onc-tenth of have identical ionic compositions and ELRL = 0, P the diffcrcnce betwren E,R, and B,,R,,,, so that this difference must range between 20 and 50 mV.Thesc valucs arr i n reasonable agrecment t\ith thc transrpithrlisl PDs observed across cpit hdia that are not characterized by rrlatively high-conductance extracellular shunts such as frog skin, frog stomach, and toad urinary bladdcr (Fromter and Diamond, 1972). 2. 9,,, is strongly dcpendcnt upon RSRI,,‘R1.Thus procedures that alter even if both the shunt conductance may have profound cffects on E,R,,, and E,R, are unaffectrd. Such procedures include changes in the ionic composition of both bathing solutions, changes in the shunt dimensions which may follow alterations in tlie ratc and direction of water flow (either spontancous or drivcri by osmotic pressure differences) , and the presence of agents that affect the prrineability and,’or permselective properties of the shunt. For example, the addition of 2.5 m M EDTA to which is slowly, and the mucosal solution results in a rapid decline in only partially reversed by Ca (hut not by Mg) (Schultz and Zalusky, unpublished observations). Although thr possibility that EDTA affects transcellular ion transport cannot br ruled out, there is evidence that EDTA loosens intercellular junctions, rendering the tissue permeable to rathrr large molecules (Cassidy and Tidball, 1967). Thus at least part of the effect of EDTA on qmS can be safely ascribed to a reduction in R ~ R L I R ~ . 3. Finally, it is clear from Eq. (10) that ionic asymmetries due either to differences in the compositions of the mucosal and serosal bathing solutions or to asyininrtries hrtween the mucosal solution and the lateral interspaces may profoundly aff ect *,,, through the dominating influcnce of ELRL.This point is illustrated in Fig. 9 nliich shows the cffrct of graded replacement of NaCI in the niucosal solution with isoosmotic mannitol and graded replaccixcnt of Na in thc iriucosnl solution with Ti. The curves n-crc calculated using thc absolutc perineabilitirs given in Table I and the Goldman-Hodgkin-Katz constant-field equation. The good agreement between the predicted and observed data indicates that, in the presence of

*,,,

272

STANLEY G. SCHULTZ AND PETER

-14 -1 3 ’-

-10

> E

F.

CURRAN

r

1 I

’

-81

ul

*E - 6 -4

t +4

I

r 5

10 MUCOSAL

50

20

“a].

100

mM

FIG.9. Effect of replacement of Na with K and NaCl with mannitol in the mucosal solution alone on diffusion potentials across rabbit ileum. The effects of replacement with mannitol are given on the larger ordinate; the effects of replacement with K are given on the smaller ordinate. The solid curves were obtained wing the constant-field equation and the shunt permeabilities given in Table I. (From Frizzell and Schultz, 1972, with the permission of the Rockefeller University Press.)

ionic asymmetries, f , . largely reflects the permselective properties of the shunt pathway and provides little information regarding the properties of the limiting cell membranes. From the above considerations it is clear that the interpretation of , , is frought with uncertainties. Nevertheless, in spite of the changes in f fact that under control conditions the short-circuit current may be the composite result of active Na and C1 absorption plus a residual current is closely linked to the that is consistent with active HCOI secretion, fms ,, is rate of active NP extrusion across the basolateral membranes ( J F ).P rapidly diminisl ed by the presence of ouabain in the serosal solution or the replacement of Na in both bathing solutions with choline; in both instances the final qms, which is reached within 20 minutes, is less than 0.2 mV and more often does not exceed the small electrical asymmetries

SODIUM AND CHLORIDE TRANSPORT

273

between the calomel electrodes (ca. 0.1 mV) . As mentioned previously, the inhibition of net Na transport by ouabain is attributable entirely to a decline in J:. Further, the addition of Na to both solutions bathing a tissue initially bathed by Na-free, choline Ringer’s solution results in an immediate increase in Qme from a value close to zero to a value that is essentially linearly related to the final Na concentration (Schultz and Zalusky, 1964a). In contrast, although the removal of C1 from the bathing solutions results in a decline in q,,, the final value reached differs significantly from zero (serosa positive) and parallels the rate of C1-independent active transepithelial Na transport (Table V) . In addition, the stimulation of active Na absorption by the addition of actively transported sugars or amino acids to the mucosal solution results in an immediate increase in Q m S ; this response is dependent only on the presence of Na in the mucosal solution, is unrelated to the presence or transepithelial movements of other ions, and is paralleled by an increase in J:. I n summary, excluding conditions that result in significant changes in appear to parallel the magnitransepithelial resistance, q,, (and the tude of as reflected by J2,4 [see Eq. (2)], regardless of the direction or magnitude of net Na transport. Thus it appears that the direction and magnitude of net Na transport are determined by the interaction between (1) an electrically neutral coupled Na anion transport process a t the mucosal membranes which can result, in NaCl absorption and/or NaCl or NaHC03 secretion, and (2) a Na absorptive transport process a t the basolateral membranes which id rheogenic. The rate of Na extrusion across the basolateral membranes (J?) appears to determine the magnitude of Q, and I,,, even though the neutral secretory process may be greater than the rheogenic absorptive process and thereby result in net Na secretion (Norris et al., 1969; Taylor et al., 1968; Munck, 1972; Powell et al., 1972, 1973; Binder et al., 1973).

JF

6. The Electrical Potential Proflle across Rabbit Ileum

The intracellular dectrical potential of rabbit ileal cells averages - 36 mV with respect to the mucosal solution (Rose and Schultz, 1971). A histogram of values obtained following “blind” microelectrode impalements indicates a normal distribution about the mcan, and there is no conipelling evidence for the presence of two or more populations of cells with markedly different intracellular PDs. The electrical profile across the tissuc appears to comprise only two distinct steps as the microelectrode is advanced from the mucosal solution through the entire thickness of the tissue, an abrupt negative deflection as the microelectrode enters the cell

2 74

STANLEY G. SCHULTZ AND PETER

F.

CURRAN

followed by an abrupt positive deflection when i t passes through the basolateral membrane. As indicated by Eq. (11), the analysis and interpretation of qmo is seriously complicated by the low-resistance shunt pathway which permits electrical coupling among all the emfs operating in the system. Thus, as discussed by Rose and Schultz (1971), a change in qmc resulting from a change in the ionic composition of mucosal solution alone, thereby creating a transepithelial asymmetry, can reflect changes in E , R , as well as changes in response to identical changes in EaRaand ELRL. Even a change in qmc in the ionic compositions of both bathing solutions, so that ELRL= 0, can in general reflect changes in EmR, as well as E,R,. Thus the traditional technique for elucidating the permselective properties of membranes surrounding symmetrical cells such as nerve axon and muscle, as well as the limiting membranes of epithelia that are not complicated by high-conductance shunt pathways, such as frog skin, are not readily applicable in low-resistance epithelia. For this reason the permselective properties of the mucosal membrane, as well as other factors that could contribute to E,R,, remain unclear. C. The Effects of Actively Transported Sugars and Amino Acids on the Electrical Potential Profile and Implications with Respect to the Mechanism of Active Na Extrusion

As illustrated in Fig. 7, the addition of actively transported sugars or amino acids to the mucosal solution results in a prompt depolarization of Qmo (less negative) and an equally prompt increase in qm8; similar findings have been reported for bullfrog small intestine (White and Armstrong, 1971) and the proximal renal tubule of the newt (Maruyama and Hoshi, 1972). Although these findings might suggest that the increase in *'ma is a result of the depolarization of q,, as a result of the rheogenic entry of Na coupled to the influxes of sugars or amino acids, the results of studies on tissues poisoned with ouabain and/or metabolic inhibitors indicate otherwise. As shown in Fig. 10, the addition of actively transported sugars or amino acids to poisoned tissues still results in prompt depolarization of 9,,,, but the increase in qmc is minimal. The depolarization of *ma is not surprising, since the coupled influxes of Na and sugars or amino acids across the brush border are not affected by ouabain or metabolic inhibitors (Chez et al., 1967; Hajjar et al., 1970; Curran et al., 1970). The failure to observe a marked increase in qma may be attributed to two properties of the epithelium: 1. Because of the low-resistance shunt pathway, the effect of a change

275

SODIUM AND CHLORIDE TRANSPORT

PDs in Fro. 10. Effect of alanine (A) 011 the trarisniucosal (qmo) and transmural tissrie treated with ouabain and/or met;tbolic irihibitors. (From Rose arid Schultx, 1971, with the permission of the Rockefeller University Press.)

in qmc on qms is markedly attonuatcd. Thus it can bc readily shown that for a change in E,R, alone

-A q -m , Aqmc

1

1

+ (R.&/R&L)

so that if, as in the case of rabbit, ileum, R3Rs>> RLRL,*ma will be minimally The current ( d i n a t e is that R3R. is six to nine affected by a change in Pmc. times greater than RaRL, so that Aq,,,/A\k,, should range between 0.1 and 0.17 in responsc to a change in E,R,, alone. 2. The principal process rcsponsiblc for the increase in P,, in response to the addition of actively transported sugars or amino acids is a rhcogenic, ouabain-sensitive, energy-dependent process which brings about the active extrusion of Na across thc bnsolateral membranes and results in an increase in E,R,. The notion that the active Na extrusion mechanism is rheogenic and is responsible for the t ransepitholial PD was suggested by numerous earlier For example, observations on the effects of metabolic inhibitors on qmB. the addition of 2 ,4-dinitrophenol or other metabolic inhibitors to the

276

STANLEY G. SCHULTZ AND PETER F. CURRAN

bathing solutions results in an immediate decline in Qms, which reaches zero (or very close to zero) long before ionic asymmetries between the intracellular compartment and the bathing solutions are abolished (Schultz and Zalusky, unpublished observations). Similarly, rapid declines in qrn3 can be produced by sudden anaerobiosis or cooling of the bathing solutions; in each instance the onset of the decline in is immediate. The finding that the virtually instantaneous increase in in response to the addition of actively transported sugars or amino acids is not associated with a change in intracellular ionic concentrations (Schultz et al., 1966; Koopman and Schultz, 1969) and is dependent upon a ouabain-sensitive and energydependent increase in E,R,, together with the above observations, seems to establish the rheogenicity of the active Na extrusion mechanism beyond reasonable doubt. VIII. CONCLUDING REMARKS

As implied in the introduction, our understanding of the behavior of rabbit ileum with respect to transepithelial Na and C1 transport is in a state of rapid evolution. Consequently, any attempt to reconcile the existing data with a “single picture” is a t best highly speculative and a t worst hazardous. Thus the model illustrated in Fig. 11 should be viewed as a hypothetical working model which is consistent with present data but is equally requiring of considerable further exploration; its purpose is to raise questions and suggest avenues for future investigation, rather than to imply that the mechanisms involved in Na and C1 transport across the epithelium are resolved. E P I T H E L I A L C E L L (S)

MUCOSAL SOL UTlON Na

--.

N a = 4 0 mM K = 1 3 o m ~ CI = 5 8 m M

.

CI

SEROSAL SOLUTION

..

.-m

Na CI

omv

-36mV

FIQ.11. Model for transcellular Na and C1 transport across rabbit ileum.

SODIUM AND CHLORIDE TRANSPORT

277

According to this model, in the absence of sugars or amino acids, one process responsible for Na and C1 entry across the mucosal membrane that is ultimately involved in the transepithelial transport of these ions is the neutral, coupled NaCl influx process that is inhibited by theophylline, intracellular CAMP, and acetazolamidc: process A. There is also reason to believe that this neutral transport mechanism is reversible, and under some conditions may bring about the secretion of NaCl and/or NaHCO,. Indeed, in view of the evidence that HCO, is secreted by rabbit ileum in vitro, and by rabbit, rat, dog, and human ileum in vivo, it seems likely that process A may normally function as a CI-HCO, exchange mechanism, as well as a pathway responsible for net entry of Na and C1 into the cell from the mucosal solution. This mechanism may also be responsible for the Na-H and Cl-HCO, exchange processes postulated by Turnberg et al. (24) for normal human ileum in vivo. Finally, the finding that acetazolamide markedly inhibits Na and C1 absorption and HCOs secretion by zn vivo preparations of mammalian ileum may be attributed to the inhibitory action of this agent on process A. Clearly, the reversibility (i.e., secretory potential) and specificity of process A warrant further investigation. Active C1 absorption, as wl-ell as CAMP-induced secretion by in aztro rabbit ileum, appear to be entirely dependent upon proccss A, inasmuch as both processes are abolished in the absencr of Na. However, there is a significant rate of active Na transport across short-circuited rabbit ileum in tjhe absence of C1, suggesting the presence of process B, which may either represent Na diffusion acl'oss the mucosal membrane down an electrochemical potential difference or a carrier-mediated entry ; current evidence does not permit a definitive distinction between thcsc two possibilities. All the available evidence to date suggests that active Na extrusion from the cell across the basolateral membranes is mediated exclusively by a ouabain-sensitive ATPase (process D) which, by analogy with other cell systems, is probably coupled to the active uptake of I nd

FIG.1. (A) The equivalent circuit of the squid axon niembrane proposed by Hodgkin and Huxley. (€3) Cation-exchanger membrane separating mixtures of Na and K salts. Fluxes of two cations are indicated by the arrows.

286

ICHlJl TASAKI AND EMlLlO CARBONE

can be formally described by using time-dependent g N a and gK. However, as has been emphasized by Schlogl (1956a), g N a defined in this fashion depends not only on Na ion mobility, but also on K ionmobility, EK, and so on (see also Finkelstein and Mauro, 1963; Tasaki, 1968, p. 41). In this case a formal description by the use of these equations tells practically nothing about the physicochemical properties of the membrane. Although it is quite probable that some part of the axon membrane behaves like a cation exchanger (see Section 11, C) ,the excitable membrane of the nerve fiber is undoubtedly more complex than an ordinary cationexchanger membrane, It, is expected that an attempt to treat the process of nerve excitation on LL physicochemical basis is hampered by the difficulties arising from the lack of precise knowledge of the structure of the nerve membrane. In spite of these difficulties, biochemically or physicochemically oriented biologists are not satisfied with a mere formal description without knowing the physical nature of interaction between ions and membrane macromolecules. In this article we attempt to describe the process of nerve excitation from a physicochemical point of view. There is no doubt that our attempt will be unsatisfactory to many present-day axonologists who are accustomed to accurate numerical analyses of highly time-dependent electrochemical phenomena, because we are not advocating the use of simple equations. However, we hope that molecular-biologically oriented investigators will find the present approach more satisfying than the orthodox description of electrical events in terms of the two equations cited above. In the following section, we describe the phenomenon of abrupt depolarization. Next, we discuss the relationship between this phenomenon and hyperpolarizing responses. Then we consider the lyotropic series formed by anions and cations affecting the nerve membrane. In the last two sections, we examine what can be learned about the nerve membrane by the use of various optical methods. Throughout this article, the lability of the conformational state of the membrane macromolecules is emphasized.

11. ABRUPT DEPOLARIZATION

A. Discovery of Abrupt Depolarization by Osterhout

Since the time of Matteucci (1841) and Du Bois-Reymond (1843), it has been known that a difference in electric potential exists on the surface of a muscle or a nerve when it is examined with a pair of electrodes, one placed on an intact surface and the other a t an injured point. Hermann

A MACROMOLECULAR APPROACH T O NERVE EXCITATION

2 87

(1877) and others believed that the source of the potential difference between these electrodes is created by the injury and docs not preexist. Bernstein (1912), however, postulatrd that the potcntial difference preexists across the surface membrane of the nerve or the muscle, and that it is produced by the unequal distribution of K ions across the membrane. MacDonald (1900) was the first to examine the effect of varying the salt concentrat on on the observed potential diff rrence. I n 1938, when Osterhout and Hill examined the K concentration effect on the excitable plant cell Nitella, they encountered an unexpected phenomenon. The relationship between the K salt concentration and the potential difference did not follow the Henderson equation but showed a clear discontinuity a t one particular concentration. Figure 2 is reproduced from the original article published by Osterhout and Hill (1938). The concentration a t which this discontinuity occurs, namely, the critical concentration, was about 0.3 mM in this case. A continuous recording of the potential difference a t the critical concentration indicated that there is an abrupt potential jump which bears a close resemblance to the depolarizing phase of an action potential. In Fig. 2 (top) an example of the records obtained by these investigators

-

J IOSI

KCI (mM)

FIG.2. (Top) Effect of stepwise increase in the external KCI concentration on the membrane potential of Nilella. (Bottom) The relationship between the external KCl concentration and the membrane potential in Nilella. The broken line represents the Nernst slope for the external K ion. Note that between 0.316 and 1 mM the change in the membrane potential exceeds the theoretical slope. (Osterhout and Hill, 1938.)

288

ICHlJl TASAKI A N D E M l l l O CARBONE

is shown. There is, however, one fundamental difference between this phenomenon and the action potential. In an action potential the observed potential returns to the initial level spontaneously. The potential jump produced by application of K salt is not followed by a return of the potential to the initial level. It is important to note that, in the entire range of the K salt concentrations examined, an abrupt depolarization is encountered only at one particular KC1 concentration. As can be seen in the figure, the slope of the potential K concentration curve is different in the range below and above the critical concentration. Only in the range of concentrations above the critical value did the observed potential difference (PD) follow the Nernst slope:

PD (millivolts)

=

58 log[K],

+ constant

where [Klo is the external K salt concentration. The phenomenon of abrupt depolarization created a serious difficulty for investigators who tried to explain the potential difference in terms of Nernst or Henderson equation. (Note that the curve shown in Fig. 2 represents a steady-state relationship between the K concentration and the potential difference.) There was even a rumor that some of Osterhout’s friends questioned the reliability of his recording instrument. When a reliable experimental finding does not agree with the theory, it must of course be the theory and not the experimental finding that has to be either modified or abandoned. If we assume that the resting potential is not exclusively determined by K ions, and that the physicochemical properties of the membrane (i.e., ion selectivities and mobilities) can change abruptly in response to a continuous change in its environment, the phenomenon of abrupt depolarization ceases to appear to be a baffling mystery. B. Demonstration of Abrupt Depolarization in Squid Giant Axons

A long time after Osterhout’s discovery, abrupt depolarization was found to occur in the node of Ranvier when the action potential was prolonged by application of Ni ions (Tasaki, 1959). Later, this phenomenon was observed in squid giant axons under various experimental conditions (Tasaki et al., 1965; Inoue et al., 1973). An analysis of an electrochemical phenomenon is easier when the experimental conditions under which the phenomenon is examined are simpler. The method of intracellular perfusion considerably simplifies electrochemical studies of a squid axon membrane. Under continuous internal perfusion,

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289

the chemical composition in the axon interior is well defined and timeindependent. When such an axon is immersed in a continuously running fluid medium, there is no ambiguity in the ionic compositions of the solution to which the axon membrane is exposed. Furthermore, this method enables us to alter the chemical species and the concentrations of the ions on either side of the membrane a t will. Natural sea water contains a large number of salts, the predominant component being NaCl. The normal protoplasm in the axon interior contains a high concentration of I< ions and a low concentration of Na ions. It is now known that the squid axon membrane is capable of developing action potentials in a highly simplified ionic environment. One of the simplest ionic environments in which the membrane excitability can be maintained is : Electrode I Ca salt solution I axon membrane I Na salt solution I electrode This is very similar to the normal ionic environment of the axon membrane from which NaCl in the external medium and the K salt in the axon interior have been removed and replaced with nonelectrolytes (sucrose or glycerol). In the axon interior a dilute solution of Cs salt can be used in place of Na salt. It is important to note in this connection that the existence of a divalent cation salt in the external medium and its absence in the axon interior is an indispensable condition for the maintenance of excitability. It is easy to demonstrate abrupt depolarization under these highly simplified experimental conditions. Figure 3B illustrates an example of such demonstration. It is seen in the figure that addition of KCI to the external CaClz solution has brought about a sudden rise in the potential of the internal electrode relative to the potential of the external electrode. The critical concentration of KCI needed for production of abrupt depolarization was about 10 mM when the CaClz concentration was 100 mM. When the external KC1 concentration decreased following production of abrupt depolarization, a reverse process, abrupt repolarization, was observed. The critical concentration of liCl needed for the production of abrupt repolarization was much higher than that for abrupt depolarization; in other words, a distinct hysteresis loop was formed when the external KCl concentration was varied in a cyclic manner. I t has been shown by inserting a long wire electrode into the axon interior that the abrupt depolarization is associated with a simultaneous fall in the membrane resistance. Axonologists who are familiar with the equivalent circuit of the membrane (Fig. 1A) may find immediately that, as far as the experiment shown in Fig. 3 is concerned, a sudden increase in the membrane permeability,

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ICHlJl TASAKI AND EMlLlO CARBONE

A

B

inside: outside:

I5mMNaF 100mMCaCI2 + YCI

-

inside outside:

\

'

--f

'Fo::

15nMNiF

IOOnMWI~ + KCI

FIG.3. The effect of gradual changes in the external univalent/divalent cation concentration ratio on the potential difference across the squid axon membrane. ( A ) Oscillograph trace indicating the membrane potential. (B) Oscillograph trace showing the univalent cation concentration in the external medium. Note that the membrane potential changes abruptly a t critical univalent cation concentrations. (Taken from Inoue el al., 1973.)

specifically to K ions, can account for the abrupt potential rise. However, this explanation is not satisfactory, because other univalent cations such as R b or Cs can be used in place of K ions in this experiment. Addition of NaCl to the external medium can also produce abrupt depolarization, although the critical concentration for NaCl is much higher than for KCl (Fig. 3A). The effectiveness of alkali metal cations in producing this phenomenon decreases in the following order. [It should be noted that this series does not represent an ion selectivity series for these univalent cations. It represents the sequence of the critical points (expressed in equivalent fractions of these univalent cations in the medium) a t which the selectivity coefficients of these univalent cations over Ca ion change abruptly (see Section 11, C) . These coefficients are determined primarily by entropy change associated with ion-exchange processes (Flett and Meares, 1966) .] K

> Rb > Cs > Na

When the univalent cation concentration is slightly below the critical level, lowering of the CaClz concentration was found to produce abrupt depolarization. When the CaClz concentration is raised, there is a rise in the critical concentration for the univalent cation salt to produce abrupt depolarization. In squid giant axons internally perfused with dilute CsF

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solution, i t was shown that a 10-fold increase in the CaClz is associated with an approximately 3-fold increase in the critical KC1 concentration. The fact just mentioned can easily be understood if we assume that both univalent and divalent cations are competing for the negatively charged binding sites on the axon membrane. Most biological macromolecules have acidic side groups (--COO, -PO,, and so on), which are partially or totally ionized at neutral or alkaline pH. Polyvalent cations are preferred by such groups to univalent cations because of a stronger electrostatic interaction between the negative binding sites and cations with a higher valency (Helfferich, 1962, p. 156). Therefore thr divalent/ univalent cation concentration ratio within a macromolecule is in general higher than the corresponding ratio in the surrounding solution. There is strong experimental evidence indicating that such negatively charged binding sites exist at the external surface of the squid axon membrane (see Tasaki, 1968, p. 59). Rased on these considerations, it sec’ms reasonable to explain the phenomenon of abrupt depolarization in terms of ion-exchange processes occurring within the membrane. Initially, a continuous increase in the univalent,’divalent cation concentration ratio in the surrounding medium brings about a continuous increase in the corresponding ratio in the membrane. The fact that both the membrane potential and conductance change abruptly can be explained by assuming a drastic structural change in the membrane macromolecules at a critical concentration. The critical concentration of KCI in the mildiuni must then correspond to the concentration within the membrane at which an abrupt structural change occurs. C. Phase Transition in Membrane Macromolecules

In various inanimate cation exchangers, it is known that exchange of divalent cations for univalent cations often produces a drastic change in the structure of the exchanger. Clowes (1916) seems to be one of the first to suggest, the importance of such a structural change in biological phenomena. He showed that a mixture of crude olive oil and 0.1 N NaOH exhibits a dramatic change in its colloidal properties when a certain amount of CaClz is added to the mixture. Watrrman (1928) demonstrated a huge increase in the electric resistance in this mixture when the critical concentration of CaC12 was added. In synthetic crystalline cation exchangers, zeolites, the x-ray pattern is known to change rather suddenly at a certain ratio of two different cation species in the exchanger (Barrer and Falconer, 1956). I n ion-exchange

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ICHlJl TASAKI AND EMlLlO CARBONE

processes in zeolites involving cations of different valencies, a phase transition can take place at a particular degree of exchange (Olson and Sherry, 1968). In many kinds of cation exchangers including zeolites, replacement of Na ions a t the binding sites with Ca ions is associated with an enthalpy change of 2-3 kcal per equivalent of exchangers (Sherry and Walton, 1967; Cruickshank and Meares, 1957; Flett and Meares, 1966). Now structural changes in membrane macromolecules may be linked to the process of nerve excitation under simplified experimental conditions. We have already shown that a squid giant axon internally perfused with a dilute Na salt solution is electrically excitable in an external medium containing only a Ca salt and a trace of pH buffer. (Note that in these observations the tonicity of both the internal and external fluid media have to be adjusted with glycerol or sucrose.) The amplitude of the action potential observed under these conditions is usually 50-70 mV. If one assumes for the moment that a stimulating current pulse increases the permeability of the membrane to Na ions, then the sign of the action potential is expected to be opposite that of an ordinary action potential. However, the observed action potentials are of the ordinary (i-e., depolarizing) sign. Faced with difficulty, some of our colleagues have suggested that the external Ca ions pass through the channels that are normally traversed by Na ions, and that the internal Na ions travel through the channels normally used by K ions. We find that this ad hoc assumption cannot explain the following fact. Under these conditions, addition of Na ions to the external medium brings about a definite increase in the action potential amplitude (Watanabe et al., 1967; Tasaki et al., 1969d) and in the membrane conductance (I. Inoue, unpublished data). We then would have to introduce the additional (and quite meaningless) assumption that the external Na ions pass through the Na channels and the internal Na ions go through the K channels. Thus we arrive a t the conclusion that it is practically futile to try to explain these action potentials in terms of the equivalent circuit shown in Fig. 1A. It is not difficult to explain the genesis of these action potentials on the basis of structural changes in membrane macromolecules. In the resting state the axon membrane is assumed to be in a compact, Ca-rich state. An outwardly directed current drives intracellular Na ions into the membrane and decreases the fraction of the binding sites occupied by Ca ions. At a critical Na/Ca concentration ratio, a sudden structural change occurs, bringing about a large increase in the mobility of the Ca ions in the membrane. This structural change is considered to result from a cooperative ion-exchange process (see Tasaki, 1968, p. 120, and Carnay and Tasaki, 1971).

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D. Thermally Induced Phase Transitions in Axon Membrane

The following observations show that the phenomenon of abrupt depolarization can be initiated simply by lowering the ambient temperature (Inoue et al., 1973). A squid giant axon was internally perfused with a dilute solution (about 15 mM) of NaF or CsF. The external fluid medium contained both CaClz and NaCI. The temperature of the external medium was altered slowly and smoothly (see Fig. 4 ) . Initially, the temperature of the rapidly circulating fluid medium was about 20". As the temperature of the medium was lowered gradually, a small change in the membrane potential was observed. When the temperature reached about 5" under these conditions, a sudden, large depolarization was observed. Following the occurence of an abrupt depolarization, a further fall in temperature brought about only a smooth, continuous change in the membrane potential. When the temperature of this depolarized axon was raised gradually, an abrupt repolarization was seen a t about 15". The existence of this phenomenon is anticipated from studies of inanimate cation exchangers. We expect that replacement of Ca ions by Na ions a t the binding sites on the axon membrane is exothermic. The results of measurements of small temperature changes associated with production of an action potential (Abbott et al., 1958) also lead to the same conclusion. Then, lowering of the ambient temperature must favor replacement of Ca ions a t the sites by Na ions, leading to a transition from the resting (Ca-rich) state to the depolarized (Na-rich) state.

g-501

, 20

,

,

10 TEMPEA A T U R E I"CI

,

, 0

FIG.4. The effect of gradual changes in temperature on the membrane potential of a squid giant axoii. The axon under study was internally perfused with a dilute NaF solution. The external medium contained 100 mM CaCL and 400 mM NaC1. Note that a distinct hysteresis loop is formed tiy a cyclic change in the temperature. (Taken from Inoue el al., 1973.)

2 94

ICHlJl TASAKI AND EMlLlO CARBONE

At a transition point the heat capacity of such a system becomes very large. Reflecting this situation (see, e.g., Glansdorff and Prigogine, 1971, p. 104), the system becomes unstable a t a transition point, and the amplitude of fluctuation of the system increases enormously. In fact, a large fluctuation in the membrane potential has been observed immediately before a thermally or chemically induced phase transition is produced (Inoue et al., 1973).

111. INSTABILITY AND EXCITABILITY OF THE AXON MEMBRANE

A. Two Stable States of Membrane Macromolecules

The term stability is used to describe a particular behavior of a thermodynamic system in relation to perturbations. When a change in one of the state variables of the system (produced by a small perturbation) vanishes with time after removal of the perturbation, the system is called stable against the perturbation (see, e.g., Glansdorff and Prigogine, 1971). The squid axon membrane in the resting state is stable against small changes in the ionic composition, in the membrane potential (produced by an electric current), and so on. However, when one of these perturbations is intensified step by step, the membrane reaches an unstable state. A membrane in an unstable state tends to deviate from this state with time. When the perturbation is intensified further, the membrane approaches a new stable state. An example of records showing the effects of electric perturbation of the axon membrane is illustrated in Fig. 5 . In this example the internal

0.5 $11

FIQ.5. Oscillograph records showing stability of the squid axon membrane in the resting state (A) and in the excited state (C). The upper oscillograph trace showed the membrane potential and the lower trace the current through the membrane. The axon under study was internally perfused with a dilute NaF solution and was immersed in a 100 mM CaCla solution. (I. Inoue, Y. Kobatake, and I. Tasaki, unpublished.)

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perfusion solution contained NaF, and the external medium was a solution of CaC12. However, very similar records have been obtained in axons internally perfused with a solution of the salt of Cs, tetramethylammonium, choline, and so on. The concept of stability is very general and is applicable to thermodynamic systems in a nonequilibrium state. It seems reasonable to assume that the stability and instability of the axon membrane derive from the physicochemical properties of the macromolecular complex of which the axon membrane is composed. During the plateau of an action potential, it is possible to apply a weak current pulse and to demonstrate that the membrane is stable except near the end of the plateau. The axon membrane is regarded as being in one stable state during the plateau of the action potential, although there is a gradual change in the potential level and in the membrane resistance. (This gradual change may be attributed to a relaxation process associated primarily with an enhanced interdiffusion of cations across the membrane.) Toward the end of the plateau, the membrane becomes unstable. Large fluctuations which appear toward the end of an action potential (Inoue et al., 1973) are a definite sign of this instability. From one of the peaks of “giant” fluctuations at the end of the action potential, a spontaneous evolution toward the resting state of the membrane is initiated. In this connection it may be pointed out that the threshold membrane potential needed to bring the axon membrane to the unstable state is of the order of 25 mV. This value is close to k T / e , where k is the Boltzmann constant, e the electronic charge, and T the absolute temperature. This potential difference is strong enough to produce significant changes in the concentration profiles for ions in the membrane (see, e.g., Schlogl, 1956b). However, it is insufficient to alter the thermal motions of ionized side groups of membrane macromolecules significantly. (N.ote that the electric field E produced by 25 mV applied across a 100-A-thick membrane layer can not give rise to a significant reorientation of a polar molecule whose dipole moment p is of the order of 10 D, because p E is only one-tenth of kT.) This is the reason why we have emphasized the importance of cation distribution within the membrane macromolecules as the agent that brings about instability. B. Models of Excitable Membranes

Most inanimate systems that produce potential variations resembling action potentials of axons are endowed with properties that enable the system to undergo transitions between two stable states. A formal descrip-

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tion of excitation phenomena in various model systems from this point of view was presented by Franck (1972) a t a recent International Congress of Biophysics. The oldest and most elegant model of a nerve is the one suggested originally by W. Ostwald (see Heathcote, 1907) and popularized by Lillie (1924). The action potential in this model is produced by a transition, from an oxidized state to a reduced state, of the surface of an iron wire immersed in concentrated nitric acid. Teorell’s (1959) model of nerve membrane is remarkable because it involves transitions between two stable “dissipative structures.” In this model there are two stable concentration profiles separated by an unstable profile. Both Monnier (1968) and Yoshida et al. (1972) were successful in demonstrating the existence of two stable conformational states in a membrane composed of pure lipids. Mueller and Rudin (1968) used protein material called “excitability inducing material” (EIM) to demonstrate electrical excitability. Recently, Bean ( 1972) discovered an extremely interesting physicochemical property of EIM molecules. He found that, when a very small amount of E I M is added to a lipid bilayer membrane, the membrane conductance rises and falls spontaneously in a discrete (i.e., all-or-none) manner. The records shown in Fig. 6 are an example of this discontinuous character of the membrane conductance under these conditions. This can be regarded as the most direct demonstration of the fact that a small assembly of protein molecules can possess two distinct conformational states. Extending these observations, Ehrenstein ( 1971) and co-workers examined the effect of potential differences applied across the EIM-containing I 2.17fl

picoamp

J

2.25

7 2 0 seconds

7 )ypL2.00 --.~

FIQ.6. Conductance jumps during an early reaction of EIM with a lipid bilayer membrane. The electrolyte was 1 M KCl, and the temperature was maintained at 36”. The membrane was polarized a t 20 mV. Dashed line indicates zero current level. (Taken from Bean, 1972.)

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bilayer membrane. In accordance with Ohm's law, the conductance of a unitary assembly was not affected by the magnitude of the potential difference. However, the percentage of the time during which the assembly is in the high-conductance state was found to vary continuously with the voltage applied. This experimental finding provided rather strong support for the explanation of the current-voltage relationship of the axon membrane based on the two-stable state concept (see Tasaki, 1968, p. 135). It should be pointed out in this connection that the similarity between the electrical events in the axon membrane and those in models derives solely from the existence of two stable states in both systems. The physicochemical process contributing to the stability and instability of a model system can be quite different from that in axons. I n spite of this limitation, various models of the nerve membrane have made significant contributions to a better understanding of the process of nerve excitation. C. Hyperpolarizing Responses

The demonstration of a hyperpolarizing response was an example of the cases in which predictions based on the t\vo-stable state hypothesis were verified. According to this hypothesis, it should be possible to demonstrate an electrophysiological manifefitation of a transition from a depolarized state of the membrane to the repolarized (i.e., resting) state. An extensive search for transitions in the reverse direction resulted in discoveries of phenomena which can be interpreted as signs of such transitions. In the original demonstration of a hyperpolarizing response (Segal, 1958; Tasaki, 1959), K-rich saline solutions were used. When the axon in normal sea water was electrically stimulated with a pulse of outwardly directed current through the membrane, a large action potential was produced, accompanied by a simultaneous reduction in membrane resistance. Then, small quantities of isotonic KC1 solution was added to the external medium step by step, until the ability of the axon to produce action potentials was completely suppressed. When this apparently inexcitable axon was subjected to a long pulse of inwardly directed current, a definite sign of excitability was observed. Figure 7 shows an example of the original records. A large potential variation (roughly 150 mV in amplitude) is seen to appear a t and above threshold intensity of the applied current. It is also seen that a large change in the membrane impedance is associated with this all-or-none response of the reverse (i.e., hyperpolarizing) sign. Similar observations were made on myelinated nerve fibers of the frog. Later, when the technique of intracellular perfusion became available, it was found that an increase in the external K ion concentration is not

298

ICHlJl TASAKI AND EMlLlO CAREONE

FIG.7. Changes in the meinlrane potential and impedance produced hy an inwardly directed current applied to the membrane of a squid giant axon immersed in a K-rich medium. (From Tasaki, 1959.)

required to demonstrate a hyperpolarizing response. Responses of this type (associated with large changes in membrane conductance) were found to appear in axons internally perfused with a dilute Na salt solution and depolarized by addition of NaCl to the external CaClz solution. [For this reason, interpretation of the hyperpolarizing response in terms of potassium inactivation (Grundfest, 1971) is not adopted here.] As we have stated previously, the existence of a hyperpolarizing response is anticipated on the basis of the two-stable state hypothesis. When the membrane is in the depolarized (Le., univalent cation-rich) state, an inwardly directed electric current is expected to raise the Ca ion conccntration in the outer layer of the membrane and t o bring about a transition of the membrane t o the hyperpolarized (Le., Ca-rich) state. This Ca enrichment is considered to arise from the low transference number of Ca ions in the membrane, and from the appearance of negative charges on the surfaces of the membrane induced by the applied field. All the observations of hyperpolarizing responses made under internal perfusion are consistent with this interpretation. From these considerations it is expected that the axon membrane near or a t a critical point may undergo transitions between the two states repetitively. In fact, under internal perfusion with a dilute Cs or Na salt solution, spontaneous transitions between the two states have been observed very frequently.

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IV. INTERACTIONS BETWEEN MEMBRANE MACROMOLECULES AND SMALL IONS

A. Proteins and Phospholipids in Axon Membrane

There seems little doubt that the axon membrane is, like other cell membranes, composed of proteins and lipids. However, it is very difficult to carry out direct chemical analysis of the squid axon membrane because of its extreme thinness. The net weight of a 10-mm-long squid giant axon (approximately 0.5 mm in diameter) is roughly 2 mg. In such an axon the fraction of the material occupying a membrane about 100 A thick is expected to be less than 0 1% of the total weight When an attempt is made to analyze the squid axon membrane chemically, a large amount of Schwann cell membrane which surrounds the axon membrane may create a further complication. Recently, the molecular weights of various proteins in squid axons were determined by the method of enzymic radioiodination (Gainer el al., 1972). By internally perfusing an axon with a proper mixture of reagents after extensive mechanical removal of the axoplasm, it was possible to label tyrosine residues of the protcins near the membrane under conditions in which the excitability is maintained. It was found by this technique combined with gel electrophorwis that the major portion of the protein a t or near the membrane has a molccular weight of 12,000. In addition, there was a small amount of protein with a molecular weight of about 70,000. When the proteins in the axoplasm were analyzed by the same technique, a large variety of proteins with molecular weights between 10,000 and 100,000 were encountered. These observations seem to indicate that the protein molecules in the axon niembrane are quite distinct from those in the axoplasm. I t is quit SO4 > C1> Br > SCN > I This order is practically identical with those determined by other investigators using various kinds of macromolecules (Von Hippel and Wong, 1964; Hamaguchi and Geiduschek, 1962). The dramatic effect of the anions in the axon interior may be illustrated by the following experimental finding. Under internal perfusion with a

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30 1

400 meq per liter KHr solution, the excitability of the axon was suppressed in about 5 minutes; with a KC1 solution of the same concentration, the axon survived for about 30 minutes; with ICF, the axon was found to maintain its excitability for more than 6 hours. Furthermore, a t the moment when axon excitability was suppressed by internal perfusion with KBr or KCl, perfusion with KF solution was found to restore the excitability immediately. The significance of these findings is very clear. These anions are interfering with the water structure and/or the salt linkages in the membrane macromolecules, affecting the normal sequence of phase transition. As mentioned before, the maintenance of axon excitability depends on the existence of a labile (i.e., unstable) state between two stable conformational states. When the water structure and the salt, linkages are seriously modified the macromolecules no longer possess two distinct stable conformational states and the axon loses its excitability. An alternative interpretation of these effects is to ascribe the sites of action of anions to the macromolecular structures that are not directly involved in the production of action potentials. However, since not only anions but also cations in axons can be arranged to form a lyotropic series, it is extremely difficult to distinguish two classes of membrane macromolecules simply on the basis of electrophysiological measurements, i.c., those direct1y involved in the production of action potentials from those that influence the excitability indirectly. The lyotropic series for internally applied cations determined by taking the survival time of axons as an index is: Cs

> Rb > I< > Na

It is important to note that the lyotropic series for cations is strongly influenced by the index taken to determine the series. (This is in marked contrast to the series for anions, which is very insensitive to the nature of the index or the experimental conditions.) It is evident that this series is not related to the mobilitias of these cations. However, it is possible that this srrics is directly related to the selectivities of the cations for a particular class of negatively charged sites. The external surface of the squid axon membrane is very insensitive to the chemical species of the anions in the external fluid medium, while it is extremely sensitive to the difference in cation species. This fact can easily be interpreted on the basis of experimental findings indicating the existencr of a negative fixed charge of high density on the external surface. I n a cation-exchanger membrane with a high density of fixed charges, co-ions are excluded from the tnembrane phase by virtue of electrostatic forces. The fixed charge density on the inner surface of the axon membrane is very low (Tasaki, 1968, p. 61).

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ICHlJl TASAKI AND EMlllO CARBONE

C. Fluxes of Radioisotopes across Axon Membrane

It is relatively easy to measure fluxes of radioactive elements across the squid axon membrane. If we inject a small amount of the salt of ssRb into an axon immersed in normal sea water, we find that about 0.5% of the total radioactive material in the axon is transferred from the axon interior to the sea water during a period of 5 minutes. It is also very easy to demonstrate that this flux of radioactive Rb ion increases enormously during nerve excitation. The relative magnitude of the radioisotope flux determined under these conditions can be adopted as a measure of the “permeability” of various ions across the axon membrane. Radioisotopes of other alkali metal ions have been used in a similar manner. The effluxes of these radioactive elements are all of comparable magnitude. (To be more precise, there is a systematic decline in the magnitude of fluxes with increasing atomic weight, the flux of 134Csbeing the smallest.) There are, however, several problems associated with the results of flux measurements of this type. The main problems ore: ( 1 ) the time resolution of radioisotope measurements are quite limited as compared with the duration of an action potential; and (2) when this method is used to measure fluxes of normally existing ion species (e.g., Na ion), there is more than one way of interpreting the results obtained. It is obvious that the poor time resolution of the radioisotope technique imposed a serious limitation on the value of the information obtained by this technique. To illustrate the second problem, namely, the problem associated with radioisotope labeling of a normally existing ionic species, let us consider the case in which a dilute solution of NaCl is separated from a solution of KCl by an ideally permselective cation-exchanger membrane. Under these conditions, the flux of Na ions J N must ~ be equal in magnitude and opposite in sign to that of K ions JK. This relationship J K J N =~ 0 is valid as long as there is no (net) current across the membrane, regardless of the difference in mobility between Na and K ions in the membrane or of the concentrations of the salts in the solutions. ~ easily be determined by labeling with radioisotopes The value of J N can of Na ion. However, this value does not reflect the mobility of Na ions alone; it may also be affected by the rate-limiting mobility of K ions in the membrane. This problem arises from the fact that individual ion species are not an independent component in Gibbs’ thermodynamics (see, e.g., Robinson and Stokes, 1959, p. 26). The resting state of an intact squid giant axon is far more complex than an ideally permselective cation-exchanger membrane. There are continuous fluxes of metabolites across the membrane, and some chemical reactions occurring within the membrane. Furthermore, there are poly-

+

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303

electrolytes in the axon interior, which make the results of a dc potential measurement with an internal electrode somewhat unreliable (see Tasaki, 1968, p. 38). Measurements of ion permeability using radioisotopes may yield measures of permeability consistent only with a particular definition of permeability. The measure of permeability obtained by this method does not, in general, agree with the permeability determined by measuring the membrane potential or conductance combined with ion substitution. Conductance measurements yield information on the mobilities of the ions in the membrane. Potential measurements give us information about the selectivities and mobilities of the ions in the membrane. Since the external fluid medium always contains divalent cations, substitution of one univalent cation species for another in the external medium leads to a frightful complication arising from competition of these univalent cations with the divalent cations in the membrane. Hence most of the results obtained by this method (see, e.g., Hagiwara, 1972) have only limited physicochemical significance. D. Ca Ion Influxes

We have repeatedly emphasized that Ca ions play a crucial role in the process of nerve excitation. We have presented experimental evidence indicating that mobilization of Ca ions fixed to negatively charged binding sites triggers a transition of membrane macromolecules from their lowconductance conformational state to a high-conductance state. The following observations show that the mobility of Ca ions in the membrane is enormously enhanced during nerve excitation. Following the successful use of 45Cain intact squid giant axons by Hodgkin and Keynes (1957), influxes of radioactive Ca ions were determined under conditions of intracellular perfusion (Tasaki et al., 1967). In the experiment illustrated in Fig. 8, a small amount of 45Cawas added to the external sea water, and the influx of the isotope was determined by collecting the internal perfusion fluid through the outlet pipet used for perfusion. The radioactivity observed under these conditions is a measure of Ca influx across the membrane. It is seen in the figure that, upon repetitive stimulation of the axon a t a frequency of 50 times per second, the average influx of Ca ions was increased by a factor of about 6. If we make a reasonable assumption that this increased influx occurs only during the period of increased membrane conductance, the flux a t the peak of membrane conductance is estimated to be roughly 300 times as high as that in the resting state of the membrane. This enormous increase in Ca influx during

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ICHlJl TASAKI AND EMlLlO CARBONE

CPM,

FIG.5. The effect of repetitive stimulation of a squid giant axon on the Ca influx across the membrane. (Taken from Tasaki el al., 1967.)

nerve excitation may be regarded as indicating that Ca ions are almost fixed and immobile (see Oosawa, 1971) in the resting state of the axon, and that their mobility rises (and selectivity falls) to a great extcnt during nerve excitation. Similar measurement with radioisotopes of Na and K ions indicated that the fluxes of these ions increase during excitation by a factor of about 100. Baker et al. (1971) in England, and Hallett and Carbone (1972) in the United States, used a Ca-sensitive luminescent protein, aequorin, to trace the movement of Ca ions in the squid axon (see Section V, B). This method yielded many interesting results, but suffers from severe limitations due to its poor time resolution. Furthermore, there is uncertainty as to the relationship between Ca ion concentration and the intensity of light emitted by this protein. E. Electrochemical Analysis of Normal Action Potentials

From an electrochemical point of view, an intact axon immersed in natural sea water is quite complex. The action potentials observed under these conditions are very short and large. Although such action potentials can be analyzed by the use of the voltage-clamp technique without difficulty, electrochemical analyses of such an intact axon is extremely difficult even in the resting state of the axon membrane.

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Short and large action potmtials, which resemble those of an intact axon in natural sea water, can be observed under much simplified experimental conditions. An axon internally perfused with a K F solution develops short and large action potentials in an external medium containing only CaC12 and NaC1. Under these conditions the fluxes of individual cations can be traced directly by use of the radioisotope technique. Because a small calomel electrode can be inserted directly into the protoplasm-free axon interior, there is little ambiguity in the results of potential measurement. Membrane conductance can he measured either by the inipedance technique (Cole and Curtis, 1939) or thc voltage-clamp method (Cole and Moore, 1960). Even under these simplified conditions, only approximate determinations of several electrochemical quantities are possible. We may simplify our analysis by treating the internally perfused axon membrane as a cation exchanger and approximating the fluxes of anions to be equal to zero. Then the ion fluxes across the axon membrane a t rest (in the absence of net current) satisfy the following relation.

+ + 2Jc8

J N ~

JK

=

0

We note further that the flux of Ca ions is far smaller than the fluxes of Na ions. (This is true even a t the peaks of action potentials.) This leads to a very simple relationship: JN,,

JK

=

0

In the case of cation-exchanger membranes, the magnitude of interdiffusion flux I J N s I = I JKI is directly related to the membrane resistance r by virtue of the Nernst-Einstein relation. The following relationship holds good irrespective of the difference in mobility between the two interdiffusing cations.

R T / F is close to 25 mV a t room tempcrature. When the membrane rcsistance (expressed in ohm.cm2) is known, the fluxes (in eq.cm-2.sec-1) can be calculated by using this relationship. ( F is the Faraday constant, 96,500 coulomb/eq.) The average value of the membrane resistance r a t rest is approximately 2000 ohm.cni2. Then the magnitude of the interdiffusion flux is expected to be of the order of 10-l. e q - ~ n - ~ . s e c - lThe . observed value was found to be 150-200 x 10-12 eq-cm-2.scc-1, i.c.., approximately of the order of the expected value (Tasaki, 1968). The membrane resistance falls to about 1 '100 a t the peak of an action potential. Therefore we expect that the interdiffusion flux increases by

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the same factor. Although the poor time resolution of the radioisotope technique does not permit very accurate determination of the fluxes during nerve excitation, it was clear that the observed value agrees, within the uncertainty of the measurements, with the expected value (see Tasaki, 1968, p. 83). When there is an electric current through the axon membrane, the absolute value of J K is no longer equal to that of JN&.When the I R drop produced by the applied current is about 50 mV (twice RTIF) or more, either one of these fluxes is expected to become exceedingly small. I n this case an inward current is carried mainly by Na ions and a n outward current mainly by K ions. In the range of an I R drop between 0 and 50 mV, a gradual transition from one limiting case to the other is expected to occur. The results obtained by Atwater et al. (1969) do not appear to be in conflict with this expectation. In voltage-clamped axons the inward current is carried by Na ions well above 25 mV away from the reversal potential. The amplitude of the action potential is known to vary with the external Na ion concentration (Hodgkin and Katz, 1949). Under simplified experimental conditions the amplitude increases by about 58 mV for a 10-fold increase in Na ion concentration. From an electrochemical point of view, this finding may be interpreted as indicating either (1) the axon behaves like an ideal Na-sensitive membrane electrode a t the peak of an action potential, or (2) the above relationship is the reflection of a high selectivity for univalent cations (or a low selectivity for divalent cations). The former of these two alternative interpretations was adopted by Hodgkin and Katz. Which one of the two alternative interpretations is valid can be determined by examining the action potential amplitude as a function of internal Na ion concentration. Various measurements carried out under the conditions of intracellular perfusion gave strong support to the second interpretation. I n an experiment in which the [Na]/[K] ratio was altered and the sum maintained a t a constant level, the dependence of the peak of the action potential on the internal Na ion concentration was approximately one-third that in the external medium. This experimental result invalidates the assumption that the axon behaves like a Na-sensitive membrane electrode. If we accept the second interpretation, the peak of the action potential (AP) should vary in accordance with the following equation : AP

=

58 log [NaI0

+ constant

because Na is the only univalent cation abundant in the external medium. This is nothing else but what Hodgkin and Katz proved experimentally.

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(Note that they did not examine the effect of variation of internal Na ion concentration. ) Many electrophysiologists chcrish a special interest in conducting detailed numerical analyses of rapidly changing quantities as a function of time. Such analyses are possible if one accepts a t the outset the equivalent circuit model of the axon membrane. However, most electrochemists are very reluctant to analyze time-dependent phenomena because of various difficulties involved. (As an example illustrating the difficulties involved in analysis of time-dependent nlectrochemical phenomena, we may consider the transient variation in the membrane potential produced by the addition of CaClz to one of the two coinpartments shown in Fig. 1B.) Qualitatively speaking, the major factor that causes rapid variation in the membrane potential folloicing transition to the excited (i.e., depolarized) statc is the enormous enhancement of the interdiffusion flux. Because the influxes of Ca and Na ions and the efflux of K ions increase by a factor of 100 or more a t the peak of an action potential, the distribution of these cations in and near the membrane changes as a function of time. This change in ion distribution invariably alters the potential difference between electrodes placed a long distance away from the membrane. Under the conditions of the experiment shown in Fig. 5 , it is relatively easy to analyze the time dependence of both the membrane conductance and potential during the falling phase of the action potential.

V. OPTICAL STUDIES OF EXCITABLE MEMBRANES A. Advent of Optical Techniques

In the preceding sections we presented a historical background of the problems directly and indirectly connected with the phenomenon of nerve excitation. We briefly surveyed the various approaches and tools that scientists used to investigate the process of membrane excitation. Attention was primarily concentrated on techniques of recording electric events such as steady potential differences, transient potential changes, and ion movements. Researchers have only recently turned their attention to different techniques fast enough to follow events on the time scale of milliseconds and thus able to give new information about structural changes in the nerve membrane (see, e.g., Hubbell and McConnell, 1969; Calvin et al., 1969). The optical studies of excitable membranes, independently initiated by Cohen el al. (1968) in England, Tasaki et al. (1968) in the United States,

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and Berestovsky et al. (1969) in Russia, are the most relevant examples of these new methods. Since then rapid progress has been made in this field. These methods were later applied and extended to study fast phenomena in different biological and artificial preparations (Carnay and Barry, 1969; Dos Remedios et al., 1972; Conti and Malerba, 1972; Sherebrin, 1972). At this point it can be asked: Why did axonologists switch their attention to these new techniques? Furthermore, what kind of information can an optical measurement give about the structure of a membrane, or about the kinetics of the excitation process? The answer to these questions is straightforward. Light-scattering, fluorescence, and birefringence techniques have been applied for many years by biophysicists and biologists to studies of the secondary and tertiary structure of proteins and other macromolecules (Dickerson, 1963; Weber and Teale, 1963). Moreovkr, conformational changes in the structure of macromolecules and fast enzymic reactions can easily be detected and analyzed by such methods (Gibson, 1966; Schechter, 1970). Therefore it should not be surprising that these techniques can also be applied to investigate the process of nerve excitation which clearly involves a reversible physicochemical change in the conformational structure of membrane macromolecules. In the following paragraphs we present and discuss each technique, its results, and its major shortcomings. More emphasis is given to the most recent work done in the field. 6. Aequorin Bioluminescence

Aequorin is a protein extracted from the jellyfish Aeguorea forskulea following a rather complex series of purifications (Shimomura et al., 1962; Shimomura and Johnson, 1969). When it is bound to Ca or Sr ions, this protein becomes strongly luminescent (light emission occurring in the blue visible range). The origin and kinetics of the light-emitting reactions are not yet well understood at present (Shimomura and Johnson, 1970; Hastings et al., 1969). However, in recent years aequorin has been successfully used as a Ca-sensitive probe in a large variety of excitable tissues. This protein was used by Ashley and Ridgway (1970) to monitor intracellular Ca transients associated with the contraction phenomena in single muscle fibers of the barnacle (Balanus nubilis). Later, Kaminer and Kimura (1972) used a similar preparation to investigate the effects of D,O on calcium release during excitation-contraction coupling. Aequorin was also used to study the entry of Ca into presynaptic terminals following a long train of repetitive stimulation (Llinas et al., 1972).

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The first report of successful application in a nerve preparation came from Baker et al. (1971). Using short, trains of repetitive voltage-clamp pulses of different duration, they analyzed the time course of Ca influx during rxcitation in a squid axon preparation. I n their experiments the signal-to-noise ratio was too small to follow directly the time course of aequorin luminescence during a single action potential. Detection of changes in aequorin luminescence following a single action potential in squid axons was accomplished in 1971 (Hallett and Carbone, 1972). I n that experiment approximately 1 n1m3 of 0.5 m M aequorin was injected into a 3- to 5-mm-long zone of the axon. The luminescent light was collected with a small fiber-optics probe located on top of the injected area and detected with a photomultiplier tube. The output of the photomultiplier was amplified and led to the input of a CAT averaging computer. Figure 9 shows the type of record obtained after a long averaging time. The time course of the luminesoent response produced by an action potential could be calculated from this record, assuming that an identical luminescence change occurs after each stimulus. After correction for the time constant of the recording system was made, the exact time course of the luminescent response following a single action potential was found to be very similar to that of the aequorin-Ca reaction in riztro (Hastings et al., 1969). From these data we concluded that the concentration of Ca a t the site on the axon where the luminescence originates is a t least lop5M . The

A

0

FIG.9. (A) Computer record showing e n h a ~ ~ c e n ~ of e n tluminescence following individual action potential in squid giant axon injected with aequorin. The arrow indicates the time of delivery of a single electric stimulus. The photograph was taken after the optical responses to 1650 individual action potentials had been averaged. The magnitude of the response is expressed as the ratio of the change to the background light intensity; one vertical division represents a change of 0.025 times the background. One horizontal division is 250 msec. The axon (430 fi in diameter) was immersed in Ca-rich artificial sea water (20"). (B) Luminescence curve for a single action potential. The two thin lines represent the actual rerord from (A), enclosing approximately 80% of the computer points. The dashed line shows the level of the base line calculated from the magnitude of the tails of the preceding light responses. The heavy line represents a fitting of the luminescence curve by a double exponential similar to that which describes the behavior of aequorin in the stopped-flow apparatus. Calibration of the curve is idential to that for ( A ) . (From Hallett and Carbone, 1972.)

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time course of Ca influx could not be determined by this technique because of the slow rate of the aequorin reaction itself. C. light-Scattering Studies

Light-scattering studies on nerve membranes are carried out by illuminating a 3- to 6-mm-long zone of the nerve with monochromatic or white light and collecting the scattered light a t different angles with a photomultiplier. The output resistance of the phototube is capacity-coupled to a differential amplifier, the output of which is led to a n averaging computer. Under these conditions light-scattering signals of the order of lop6 times the background light intensity can be detected after about 10,000 repetitions. Changes in light scattering from crab nerve during propagated action potentials were first reported by Cohen and Keynes (1968). Their results were immediately confirmed and extended to other biological preparations by other investigators (Cohen el al., 1968; Tasaki el al., 1968). Lightscattering signals were found to be dependent on the angle of detection. Transient decreases in the forward scattering at 10" were found to reverse their sign when the angle of detection was changed to 90" (Cohen and Landowne, 1971). Theoretical considerations suggest that light-scattering signals may be produced by either a change in the thickness or in the refractive index of the cylindrical structure of the nerve membrane during excitation (Tasaki, 1970). I n both cases, however, a drastic change in the macromolecular structure in or near the nerve membrane is thought to be the cause of the signals. I n order to investigate the dimensions and the possible geometry of such a structure, analyses of the dependence of the optical signal on the wavelength of incident light, and measurements of the degree of polarization of the resting scattered light, were carried out (Tasaki et al., 1968; Tasaki, 1970; Cohen and Keynes, 1971). The results suggested that cylindrically arranged components of the nerve with dimensions larger than the wavelength of the light in the visible range appear to be responsible for the change in light scattering during excitation. A new approach to the study of light scattering from nerve membranes has been made only recently by Fritz (1973). Using an optical homodyning technique (Forrester, 1961) with a He-Ne laser as a light source, Fritz examined the spectrum of the scattered light from a lobster nerve in the resting and in the excited state (KC1-depolarized). The shape of the spectrum of the Rayleigh line was found to be Lorentzian for both states of the membrane. Only the width was found to be different: 36 Khz (half-

A MACROMOLECULAR APPROACH TO NERVE EXCITATION

31 1

width) in the resting state, and 51 Khz in the excited state. This implies the existence of exponential decaying processes in the axonal structure with a characteristic relaxation time on the microsecond time scale. Further investigations are needed in order to clarify the nature and kinetics of such processes. So far no attempts have been made to record changes in the lightscattering spectrum during nerve excitation. Improvement of the time resolution of these techniques is expected to bring new information about the structure and hydrodynamic properties of the excitable membrane. D. Birefringence Studies

For an object with a single optical axis (uniaxial), the refractive indices measured with light polarized in the parallel and in the perpendicular direction relative to that axk, differ from each other (Born and Wolf, 1965, p. 680). The difference betwcrn these two quantities is taken as a measure of birefringence of the specimen examined. Birefringence techniques mere readily adopted by biologists as a means of measuring the thickness of living cells, to study the conformational structure of macromolecules, and even to follow biological movement inside ceIls (Weber and Teak, 1963; Inoue and Sato, 1967). Only recently they have been applied to the study of the process of nerve excitation. Transient decreases in birefringence during nervous activity was detected from different nerve preparations (Cohen el al., 1968; Tasaki et al., 1968; Berestovsky el al., 1969). T h r causes of such changes are still a matter of discussion, but it, is generally agreed that a macromolecular structure in and/or near the nerve membrane is directly or indirectly involved in such changes. I n their work on squid axon.;, Cohen et al. (1970) gave detailed proof of the authenticity of the birefringence signals. They could also measure the time course of the birefringence signals during action potential. A good correlation between the optical change and the time course of the action potential was found. They analyzed birefringence changes from different zones of the axonal membrane by using a longitudinal slit placed in the image plane of the optical system, and thereby dctcrminc.d the distribution of the change in birefringence as a function of the distance from the center of the axon. The change in birefringence expressed as a rat,io A I / I ( I being the maximum light intensity observed under cross-polarization conditions at rest) could be detected only from the edges, not from the middle, of an intact axon. They concluded that a Kerr-effect type of action on a radially

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oriented macromolecular structure in or near the axon membrane was the main cause of such changes. A critical analysis of the method used by these workers suggests that their measurements suffered serious technical limitations. In order to overcome such limitations, experiments in our laboratory were carried out using a Kohler type of illumination and flattened axons (Sato et al., 1974). The Kohler type of illumination was used to obviate imaging problems (Born and Wolf, 1965, p. 166). Removal of the axoplasm by either enzymic or mechanical treatment and subsequent flattening of the axon were performed to reduce the amount of axoplasm in the middle of the axon, Results obtained from different zones of a mechanically treated axon are shown in Fig. 10. These findings indicate that a transient decrease in the refractive index measured with light with its electric vector oriented in the longitudinal direction of the axon can be the cause of changes in birefringence during nerve activity. This implies that under these conditions a longitudinal and not a radial macromolecular structure in and/or near the membrane is directly involved in the process of nerve excitation. Further investigations of the effect of enzymic digestion of the axoplasm on the peak of inward current during voltage-clamp also suggested that birefringence changes are likely related to a mechanism controlling the membrane conductance rather than to a potential difference. Finally, we would like to present the results obtained by a third group of investigators, which we think are worth consideration. Following a

FIG. 10. (Top) Schematic diagram showing the cross section of a squid giant axon flattened after mechanical removal of a large portion of the axoplasm. Approximately 80% of the axon membrane was parallel t o the surfaces of the glass plates. (Bottom) Computer records made from three different zones of an axon, showing changes in birefringence associated with action potentials. The measuring light was restricted to different zones by the use of Kohler's illumination. The vertical bar represents the ratio A I / I , I being the maximum light intensity ut rest observed under cross-polarization conditions. The percentage of the area illuminated is indicated above each record. Temperature was 11".(From Sato et al., 1974.)

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313

different approach, Berestovsky et al. ( 1970) investigated the birefringent propertics of lipid membranrs undcr the action of varying electric fields. Using a phospholipid preparation cxtractcd from thv brain of a bull, they incasured and compared tlic changcs i n birefringrnce of lipid bilayers undcr different conditions. They concluded that changes in birefringence from unmodified lipid bilayers under the action of an external electric field are too small to account for thc large changes occurring in excitable mcmbranes. Their conclusion was that, thc observed birefringence signals in excitablc membranes might, be c a u d hy a nioleculnr mechanism controlling the electrical conductivitv of the inernbranc itself.

VI. FLUORESCENCE STUDIES

A. Introductory Remarks

During the last 29 years fluoresccncc spectroscopy techniques have been applied by protein chemists to studies of thc relation h e t w e n the structure and function of proteins (Weber and Laurence, 1954; McClurc. and Edelman, 1966). These methods could in fact give valid information about proccsscs of protein denaturation, distances bctween side groups, and the extent of flexibility of macromolecular complexes (Strycr and Haughland, 1967; Yguerabide et al., 1970). Fluorescence techniques ar(1 now very popular also among biologists and biophysicists interested in the structural and functional properties of biological membranes (Gomperts and Stock, 1971 ; Gitler and Rubalcava, 1971; Tasaki et al., 1968). The reason for such popularity derives mainly from the fact that a fluorescence measurement givcs information at the molecular level concerning the structure of the membrane or macromolecule under examination. I n an extrinsic fluorescence npproach, the membrane is labeled at one or more specific sites with a dyc moleculc whose behavior in zlitro is generally well known. Measurements of the changes in one or more of the fluorescence dye properties, following external perturbations, then gives information about a structural change a t that particular site. An intrinsic fluorescence approach differs from an extrinsic one, because it rnakcs use of fluorescent amino acid residues already present in the protein part of the membrane. In the following sections we show how these methods were applied to the study of nerve membranes, and what new information they gave about the process of nervc excitation. Before starting a detailed prescntation of the major findings in the field, it is valuable to discuss briefly the theoretical

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basis of the process of fluorescence, emphasizing the physicochemical properties of a class of dye molecules known as aminonaphthalene derivatives. B. Theoretical Background

It is well known that a change in solvent polarity affects both the wavelength of maximum emission and the half bandwidth of 2-p-toluidinylnaphthalene-6-sulfonate (2,6-TNS) and other aminonaphthalene derivatives in a characteristic manner (McClure and Edelman, 1966; Brand and Gohlke, 1972). An empirical scale called the 2 value (Kosower, 1968) was used by Turner and Brand (1968) to express solvent polarity quantitatively. A red shift and an increased broadening of the emission spectra is observed when the solvent polarity (or 2 value) is gradually increased. The reason for this has been attributed to an increase in the dipole moment of the probe molecules in the excited singlet state (Seliskar and Brand, 1971). A stronger interaction of the probe molecule with the surrounding polar solvent molecules in the lowest excited singlet state is expected to lower the energy difference between this singlet state and the ground state. Lippert (1957) and Mataga et al. (1956) independently related the effects of solvent polarity on the wave number of emission and absorption by means of the following equation :

where h is Planck’s constant, c is light velocity (in vacuo), v a and v f are the wave numbers (in cm-’) of the peaks of the absorption and emission bands, respectively, A p is the difference between the dipole moment in the excited state and that in the ground state, a is Onsager’s cavity radius, ) a function of the dielectric constant E and the refractive and f ( ~ , n is index n of the solvent defined by: E - 1 nz-1 f ( e , n ) = -2~ 1 2n2 1

+

~

+

A plot of the Stokes’ shift (v, - vf) as a function of f(c,n) for solutions of 2,6-TNS, anilinonaphthalene sulfonate (1 ,&ANS), 2,6 and 1,8-aminonaphthalene sulfonate and N-methyl-2’6-ANS in different solvents reveals the existence of a linear dependence having different slopes in different regions of f ( ~ , n (Tasaki ) et al., 1973b). Kosower and Tanizawa (1972) have also shown the existence of a sharp break in the linear relationship between of and the solvent polarity expressed in the scale known as Et(30)

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315

(see Reichardt and Dimroth, 1968) by using solutions of 2,6-TNS dissolved in a series of dioxan-water mixtures. These results suggest that there are two different excited states of the probe molecules having different solvent polarity sensitivity. For instance, the second state of the excited 2,6-TNS molecule [f (c,n) varying between 0.3 and 0.321 appears to be more sensitive to the polarity of the solvent than the first state [f(e,n) varying between 0 and 0.31. Solvent polarity not only affects the wavelength of the peak of maximum emission but also reduces the quantum yield of fluorescence and the lifetime of the excited molecules by facilitating transition from the excited singlet state to the triplet state. It is well known that addition of molecules with large dipole moments (H20, formamide, acetsmide, etc.) to a n ethanol, solution of 2,6-TNS produces a red shift in the emission spectra, a decrease in quantum yield, and a reduction in the nanosecond lifetime of the excited singlet state. Other factors besides solvent polarity can also influence the quantum yield of fluorescence. A group of compounds, such as pyrmidine, pyridine, dipropylamine, and I-, for instance, are known to quench the fluorescence of 2,6-TNS without altering the wavelength of maximum emission. These compounds, called dynamic quenchers (Radda and Vanderkooi, 1972), are believed to inactivate fluorescent molecules by collision during their excited state. When the ratio of the fluorescence intensities in the absence and presence of the quencher Fo/F and the corresponding ratio of the lifetimes 70/7are plotted against the quencher concentration, a single linear relationship can be obtained for low concentrations of the quencher. The kinetics of this reaction is described by a linear equation known as the Stern-Volmer equation (Stern and Volmer, 1919). In a solution of macromolecules and fluorescence dye, a factor influencing the degree of binding of the dye to the macromolecules is also expected to influence fluorescence intensity. This type of proceis can affect fluorescence intensity without changing the lifetime of the excited molecule or the wavelength of maximum emission. An example of this phenomenon is the effect of CaC12 on the fluorescence of 2,B-TNS or 1,8-ANS in an aqueous solution of lysolecithin (Vanderkooi and Martonosi, 1969), or bound to erythrocyte ghost membranes (Gitler and Rubalcava, 1971). The fluorescence of some aminonaphthalene derivatives is also influenced by other factors such as temperature and solvent viscosity. Lowering of the temperature of 2,6-TNS glycerol solution from 20" to 8" was found to increase the fluorescence intensity by a factor of about 3, and to cause a 4-nm blue shift of the wavelength of maximum emission. Studies of nanosecond time-resolved emission spectra (Brand and Gohlke, 1972) indicate that this blue shift is related to the time-dependent aspect of the

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dielectric constant E in f (t , n ) . For this reason, the influence of viscosity is not treated here as an independent factor. C. Fluorescence Changes during Nerve Excitation

Extrinsic fluorescence studies during propagated action potentials in the crab nerve and the squid axon were initiated a t the same time as the studies of light scattering and birefringence (Tasaki et aZ., 1968). Nerves from lobster, crab, and squid were found to emit visible light when they were stained with 1,g-ANS and illuminated with ultraviolet light. Following stimulation the fluorescent light intensity was found to increase to its maximum and then gradually decrease to its original resting level. These results were obtained by means of a specially constructed spectrofluorometer able to follow events on a time scale of milliseconds. The top of Fig, 11 illustrates the optical arrangement of the machine used.

S

LI

AN ALY 2 E R PA R A L LE L

PERPENDICULAR

FIG.11. (Top) Diagram showing the experimental setup used to study fluorescence properties of squid giant axons stained internally with TNS. S, Xenon-mercury lamp; L1, spherical quartz lens; Lf and L8, cylindrical quartz lenses; F1, primary filter for 365 nm; P, polarizer; R, quartz cover slip; E and E', stimulating and recording electrodes; M I and Mz, multiplier phototubes for the main and reference light beams, respectively; A, analyzer; Fz,secondary filter. (Bottom) Averaged records showing the effect of electric stimulation on the fluorescence of a TNS-stained axon. The record on the left shows a transient reduction in fluoresrenre in response to electric stimulation; the analyzer was placed in such a manner that the electric vector of the transmitted light was parallel to the long axis of the axon. The record on the right was obtained after rotating the analyzer by 90". Brief stimulatiiig current pulses were delivered at the second vertical line. One vertical division represents a change in fluorescence of 4.3 X 10-4; one horizontal division corresponds to 7.8 msec. These rerords were obtained at 7'. (From Tasaki el al., 1971.)

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The incident light from a 200-W xenon-mercury lamp was focused onto a 12-mm-long portion of the nerve using quartz lrnses and optical filters, as shown in Fig. 11. The light arising from the nerve was passed through a secondary filter F, and then collected a t a 90" angle with a photomultiplier tube. When required, Polaroid sheets (P) and (A) were introduced in the optical pathway (see Fig. 11). The output signal from the photomultiplier was amplified and then fed into a CAT averaging computer. The early results obtained from 1 ,&ANS were soon extended to other dyrs and investigated undrr different elcctrophysiological conditions. Changes in extrinsic fluorescence from axons internally labeled with 1,8ANS tverr found to be opposite in sign from those obtained under external staining. Changes in fluorcsccnce intcnsity were also recorded from crab nerves and squid axons using Pyronine B, rhodaminr B and G, fluorescein isothiocyanate (Tasaki et al., 1969b), and acridine orange (Tasaki et al., 1969b). In order to investigate these phenomena in more detail, fluorescence changes in squid giant axons intern:tlly labeled with I ,8-ANS were studied under voltage-clamp. Fluorescence signals could be rrcorded during hyperpolarizing and depolarixing clamping pulses; the amplitude of the optical changes was found to follow almost linearly the amplitude of the applied voltage pulse (Conti and Tasaki, 1970). Later the fluorescent properties of axons internally labeled with 2,6-TNS (by injrction of the probe) were also studied rather extensively. 2,6-TNS immrdiately appeared to be a very interesting and promising dyr in studying the structure of the axonal membrane. Following stimulation a transient decrease in background fluorescencr intensity was detected from axons internally labeled with this dye (Watanahe et al., 1970; Tasaki et al., 1971). The results obtained with 2,6-TNS are discussrd in more detail in Section VI, D. Following these early results, several conclusions can be drawn about the meaning and cause of such phenomena. First, it is quite clear that during rxcitation the dye molecules are monitoring a change in the conformational state of the membrane macromolecules. Srcond, the transient decrease in fluoresccnce intensity of axons internally labelrd with 1 ,8-ANS and 2,6-TNS suggests, according to the arguments discussed in Section VI,B, a t least four possible mechanisms: (1) a suddcn incrrase in solvent polarity surrounding the dye molecule, ( 2 ) a transient increase in the effect of some unknown qurncher present in the menibranr, (3) a reduction in the number of binding sites, and (4) a sudden fall in local viscosity. Third, therc is no direct relationship lietween the sign of thr optical signal and the electric charge of the dye molecules. I n fact, a molecule like 1,8-ANS (ncgatively charged) gives similar results when it is externally applied as acridine orange which is positively charged.

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D. Fluorescence Polarization Studies

The degree of polarization of the fluorescent (i.e., emitted) light arising from a stained nerve is defined by P = (I’- I f f ) / ( I f I”),I’ and I” being the fluorescence intensities measured with the analyzer parallel and perpendicular, respectively, to the nerve. The electric vector of the incident (i.e., exciting) light can be oriented either longitudinally or perpendicularly to the nerve. I n a system like the squid giant axon, where only less than 0.1% of the total volume is involved in the process of nerve excitation, measurements of the degree of polarization a t rest tell very little about the extent of orientation of the dye molecules incorporated into the membrane macromolecules. Much more information can instead be gained from measurements of changes in fluorescent light intensity made with the analyzer in parallel and perpendicular directions relative to the axon. At first, studies of the degree of polarization of the light arising from internally and externally stained nerve were undertaken by using a large variety of dyes. The results and their interpretation, however, were neither easily obtained nor encouraging. A better understanding of the polarization properties of the stained axonal membrane was possible only after 2,6-TNS was internally injected into the squid axon. The changes in fluorescent light arising from these stained axons were in fact found to be nearly totally polarized (Watanabe et al., 1970; Tasaki et al., 1971). The records of these measurements are shown a t the bottom of Fig. 11. These findings indicate that the degree of polarization of the dye molecules responsible for the transient decrease in fluorescence intensity is close to unity. Studies of the in vitro polarization properties of 2,6-TNS by the method of Nishijima et al. (1966) revealed that such a high degree of polarization can be obtained easily when the dye molecules are incorporated in wellstretched sheets of polyvinyl alcohol (Tasaki et al., 1971). In these cases the degree of polarization is found to be very close to unity, indicating that the majority of the incorporated probe molecules have a definite orientation, with their absorption and emission oscillators parallel to the direction of stretching. The degree of polarization in stained unstretched sheets instead was found to be never higher than 0.5. These values are expected if the dye molecules are randomly oriented in the sheet. The early resuIts obtained with 2,6-TNS were later confirmed and extended to other 2,6-aminonaphthalene derivatives (Tasaki et al., 1973a). In these investigations the existence of different polarization properties in 1,8- and 2 ,6-aminonaphthalene derivatives were also observed. The fluorescent light from nerves internally and externally labeled with 1,&ANS, 1,8-TNS, and 1,&aminonaphthalene sulfonate appeared to be nearly

+

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319

totally unpolarized. An insight into the origin of this difference can be gained by comparing the absorption spectra of these molecules. On the basis of semiempirical molecular orbital theory, including configurational interactions, BabR and Suzuki (1961) showed that the lowest energy absorption band of 1-aminonaphthalene (320-330 nm in wavelength) corresponds to two well-separated absorption bands (280 and 348 nm) of 2-aminonaphthalene. The addition of phenyl and sulfonate groups to these molecules was found to cause a red shift in their absorption spectra without altering the relative positions of their lowest energy absorption bands. For these reasons excitation of 1,&ANS with light near the lowest energy absorption band can produce either one of the two lowest energyexcited singlet states. Excitation of 2,6-ANS (or 2,6-TNS) with light near the lowest energy absorption band instead produces only the excited singlet state with its transition moment oriented in the direction parallcl to the long axis of the naphthalene ring. From these considerations it becomes apparent that 2,6-derivatives are the more appropriate dyes among the aminonaphthalene derivatives for studies of fluorescence polarization. Their application to studies of the process of nerve excitation reveals that a highly oriented longitudinal macromolecular structure at or near the membrane is directly involved in the process of action potential production. These results are in good agreement with the birefringence studies discussed in Section 5,D. Examination of a live squid axon under a Normarski interfcrence-contrast microscope also reveals the existence of a fine rodlike structure near the membrane, oriented in an approximately longitudinal direction (Metuzals and Izzard, 1969). E. Spectral Analyses

As mentioned earlier, a complete knowledge of the microenvironment surrounding the dye molecules is possible only after all the parameters characterizing the fluorescence emission have been determined. I n our case, for instance, measurements of the change in fluorescence intensity alone do not tell us much about the macromolecular processes occurring in the membrane during excitation, or even if they do there is alwayF more than one process that accounts for the obscrved result. For this reason spectral analyses of the fluorescent light deriving from the stained nerve a t rest and a t the peak of nerve excitation were initiated. Analyses of the spectrum of the light contributing to the transient decrease in fluorescence intensity from squid axon internally labeled with 2,6-TNS and illuminated with light having its electric vector in a direction

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parallel to the longitudinal axis of the axon were first reported in 1970 by Watanabe et al. The axons were internally injected with a solution of 2,6-TNS in a K phosphate-glycerol mixture and illuminatcd with ultraviolet light (365-nm wavelength). The apparatus used was practically the same as the one described in Section VI, C. The cutoff filter F2was in the present case replaced with an interference filter of 10-nm bandwidth (the center band wavelength varying between 420 and 580 nm). Under these conditions it was found that the spectrum of the light contributing to the changes in fluorescence intensity was much narrower and sharper than the spectrum of the fluorescent light arising from the axon a t rest (Tasaki el al., 197313). These early findings with 2 , 8 T N S were later confirmed and extended to other dyes such as 2,6- and 1,8-ANS (Tasaki et al., 1973b). Figure 12 shows the results of the spectral analyses conducted by means of an improved spectrofluorometer on squid giant axons internally labeled with 2,6-ANS and 2,6-TNS. At the top of each diagram is shown the averaged optical signals obtained with different interference filters. To gain an insight into the causes of these phenomena, an attempt was made to reconstruct the spectra of these probe molecules in squid giant axons by using the spectra of the same dyes dissolved in a mixture of ethanol and water. This could be done because, in a wide range of solvent

FIG.12. (Left) Emission spectrum of 2,6-TNS in squid giant axons at rest I , indicated by open circles), and that of the portion of the fluorescent light that changes during nerve excitation ( A I , indicated by the squares). Computer records (taken from one axon) showing fluorescence changes associated with action potentials are presented at the top of the figure. The center band wavelength (for normal incidence) of the secondary filter used is indicated above each record. The number of trials averaged was the same for the three records. The vertical lines indicate 4-msec intervals. The broken and continuous lines are constructed by the procedure described in the text. (Right) Similar spectra for 2,6-ANS in squid giant axons.

A MACROMOLECULAR APPROACH T O NERVE EXCITATION

32 1

polarity, both the wavelength of maximum emission and the bandwidth of the spectrum are determined almost uniquely by solvent polarity (Turner and Brand, 1968). I t was found by this procedure that the emission spectrum of the light arising from axons a t rest labeled with 2,6-TNS could be reproduced by taking a linear combination of the spectra of the probe molecules dissolved in 100 and 40y0 ethanol. The sharp and narrow spectrum of the light contributing to the transient decrease in fluorescence intensity instead could not be fitted with any spectrum of the in uitro system. A reconstruction of such a spectrum was possiblc only by taking the difference between the spectra of the probe molecules dissolved in 100 and SOCY, ethanol, or in other solutions having the same 2 value as these two solutions. These results strongly suggested that the transient decrease in fluorescence intcnsity during nerve cxcitatiori is most, likely caused by a solvent polarity increase in the niicroenvironrrlcnt surrounding the probe molecules. I n terms of Kozower’s 2 value this change corresponds to an increase from 80 to 85 a t the peak of nerve cxcitation. Thc possible alternative that the change is produced by a transient increase in the effect of some unknown quencher in the membrane during nerve excitation was excluded. In fact, the spectrum of the fluorcwence intensity changes caused by a quencher should have, according to the arguments given in Section V, c‘, the same spectrum as that in the resting state. Another possible cause such as a reduction in the number of binding sites was also excluded, because such mechanisms would have yielded a broadcr spectrum than the one observed. Finally, a possible decrease in viscosity was ruled out for the same reason. We can conclude therefore that the transient decrease in fluorescence intensity from squid giant axons internally labeled with 2 ,&TNS and illuminated with light having its electric vector in a direction parallel to the longitudinal axis of the axon is most likely due to an abrupt increasc in the solvent polarity of the environment surrounding the dye molecules during excitation. We cannot, however, distinguish a t this stage between a possible transient increase in water molecules in the vicinity of the binding sites, or a sudden movement of some polar side groups of macromolecules toward the binding sites.

VII. SUMMARY 1. The significance of Osterhout’s phenomenon of abrupt depolarization in the process of nerve excitation was described. This phenomenon shows that a continuous change in the univalentidivalent cation concentration

322

ICHlJl TASAKI AND EMlLlO CARBONE

ratio in the medium produces an abrupt change in the macromolecular structure of the axon membrane. 2. Membrane macromolecules involved in the process of nerve excitation are considered to be capable of undergoing transitions between two stable conformational states in a manner similar to the behavior of the EIM molecules studied by R. C. Bean. 3. It was possible to induce abrupt transitions in electrochemical properties of the axon membrane by lowering or raising the temperature of the medium. 4. An attempt was made to explain the process of nerve excitation on a physicochemical basis. 5 . The results of recent experiments employing optical techniques were described, including the detection of changes in (1) light scattering, (2) birefringence, and (3) extrinsic fluorescence. 6. The effects of various physicochemical agents on the fluorescence properties of aminonaphthalene derivatives were described. A possible physicochemical basis for the production of fluorescence changes in nerves labeled with these derivatives was discussed.

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SUBJECT INDEX A Acidification, renal mechanism of, 20.5222 Acetazolamide, effects on Na and C1 influxes, 259-262 Aequorin, bioluminescence of, 308-310 Aerobacter, anion transport in, 43 Alanine, effect on ileal ion transport, 274276 Alkali metals, as metabolic cofactors, 4142 Amino acid transport ATP hydrolysis and glycolysis in, 156157 coupling and energy transfer in, 137-159 coupled-flow equations, 140-142 to ion flows, 144-158 sodium-linked, 143-144, 147-156 Amino acids, effects on ileal ion transport, 274-276 1-Anilino-%naphthalene sulfonate (ANS), in fluorescent study of cation tmnsport, 20-21 Anion transport, in bacteria, 42-43 Anions, lyotropic series of, 300-301 Arabinose transport system description of, 91-93 ATP, hydrolysis of, in amino acid transport, 156-157 ATPase mutants for, in E . colt, 77-78 in proton transport, 21-24 Axons, giant, of squid abrupt depolarization in, 288-291 instability and excitability, 294-298 phase transitions in, 293-294 proteins in membrane of, 299-300 radioisotope flux across, 302-303

B Bacillus megalerzum, cation transport in, 22

Bacteria cation transport in, 1-50 chemiosmotic hypothesis, 15-17 in cytoplasm, 5-7 ionophores and, 9-14 energy coupling of active transport in, 7 1-83 ion balance in, 2-8 ion gradients in, 43-44 membrane potential and proton transport in, 14-26 measurement, 18-21 membranes and binding sites in, 7-8 osmotic adaptation of, 43-44 proton transport in, 21-26 Bicarbonate, reabsorption of, hydrogen secretion and, 161-224 acidification coupling in, 176-183 C o n gradient method in, 167-172 pH disequilibrium method in, 165-167 in turtle bladder, 193-205 Bioluminescence, in studies of excitable membranes, 308-310 Birefringence, use t o study nerve excitation, 310-311 Bladder, of turtle, see Turtle bladder Brush border, sodium and chloride influxes across, 244-262

C Calcium ion, in nerve excitation, 303-304 Carbon dioxide, balance and permeability of, 190-193 Carbon dioxide diffusion method, in studies of renal acidification, 207-212 Carbon dioxide gradient method, in bicarbonate reabsorption studies, 167-172 CO, profiles from, 172-176 327

328

SUBJECT INDEX

Carbonic anhydrase, inhibition of, in studies of renal acidification, 213-215, 218-219 Carotenoids, in spectroscopic studies of membrane potentials, 21 Carrier proteins, 51-136 Cation transport in bacteria, 1-50 cell functions and, 40-44 Cations, lyotropic series of, 300-301 Cell, functions of, cation transport and, 4044 Chemiosmotic hypothesis of bacterial cation transport, 15-17 of energy coupling to active transport, 71-74 Chemotaxis, galactose-binding protein in, 122-123 Chloride ion influx of, across brush border, 249-250 residual, 254-255 Chlorophyll in spectroscopic studies of membrane potentials, 21 Cytoplasm, of bacteria, cation transport in, 5-7

D Distal tubule, acidification in, 215-221

E Electrophysiology, of rabbit ileum, 269276 Energy coupling of MeGal transport system, 78-83 to respiration, 74-77 Epithelium, of intestine Na and C1 influx across, 239-244, 255259 working model of, 227-230 Escherichia coli ATPase mutants in, 77-78 cation transport and balance in, 3, 50 potassium and sodium, 34-40 galactose-binding protein in, 94-97 MeGal transport system in, 57-60 Excitable membranes, models of, 295-297

F Fluorescence, use to study nerve excitation, 313-321

0 Galactose transport system for, see MeGal transport system kinetics, 60-71 Galactose-binding protein activity measurement of, 99-106 amino acid composition and stability of, 96-97 binding assay of, 100-102 in chemotaxis, 122-123 conformational changes in, 105-1 13 in E. coli cell envelope, 94-96 immunological assay of, 105 in MeGal transport, 116-122 coregulation of, 120 genetic aspects, 121-122 molecular weight of, 97197 properties of, 94-116 structural features of, 98-99 sugar transport via, 51-136 sugar effects on, 58 synthesis of, regulation, 123-125 working model for, 113-116 Glycolysis, in amino acid transport, 156157 Gramicidins, as ionophores, 13-14

H Halobacterium cation transport and balance in, 3-50 potassium and sodium, 40 Hydrogen ion, bacterial transport of, 1-50 Hydrogen secretion, bicarbonate reabsorption and, 161-224 leakiness t o H and HCOa in, 183-185 steady states of minimal luminal pH in, 186-190

1 Ileum of rabbit electrophysiology of, 269-276 sodium transport across, 225-281 shunt pathway in, 231-239

SUBJECT INDEX

329

Intestine, epithelium of. working model of,

N

227-230

Ion balance, of bacterial cells, 2-8 Ion transport, electrophysiology of rat )bit ileum and, 269-276 Ionophores, in cation transport studies, 9-14

Karlmark method for COs in lilniinal fluid, 209-212 Kidney, acidification mechanism of, 205222

1 Lactose transport system, 53 Light scattering, use t o study nerve excitation, 310-311 Lipids, unsaturated, in MeGal transport system, 93-!94 Lurninal fluid, acidification of, by turtle bladder, 193-205 Lyotropic series, of anions and cations,

Nernst equation, 18 Nerve excitation, 283-325 iibrnpt depolarixation in, 286294 discovery, 286-288 in squid axons, 288-291 haluminescence studies of, 30&310 birefringence studies of, 310-31 1 c2alciumion influx role in, 303-304 electrochemical analysis of normal action potentials in, 304-307 fluorescence studies o f , 313-32 I polarization studies, 318-319 theory, 314-316 hyperpolarizing responses in, 297-298 light-scattering studies of, 310-311 macromolecular approach to, 283-325 iiiemhrane inst,ability and excitability of, 294-298 optical studies of excitable membranes in, 307-313 phase transition in membrane macromolecules nf. 291-292 in axon membrane, 293-294 Nigericin, as ionophore, 12-13

0

300-301

Optical studies, of exritable membranes,

M M protein, in lactose transport system, 53 MeGal, galactose-binding protein in, 116-

307-313

Oxidat,ive phosphorylation, transport activity in mutants defective in, 81-83

P

122

MeGal transport system assay for, 60-G3 energy coupling in, 68-71, 78-83 in galactose entry, 66-71 genetics of, 8 6 9 4 partial transport by, 91 properties of, 57-94 temperature effects on, 63 unsaturated lipids in, R3-94 Membrane, of bacteria, in cation transport, 9-10 Membrane vesicles, isolated, active transport in, 83-86 Monactin, as potassium ionophore, 10-1 1 Monensin, as ionophore, 12-13

Periplasmic binding proteins carrier function of, 126-127 transport system mediated by, 55-57 pH, of cells, regulation of, 42 Phospholipids, in axon membrane, 299-300 Phosphotransferase system (PTS), 53 description of, 54-55 Potassium ion harterial transport of, lL.50 ionophores for, 10-11 Proteins, in axon membrane, 299-300 Proton, conductors for, 11-12 Proton transport in bacteria, ATPase in, 21-24

330

SUBJECT INDEX

R Radioisotopes, fluxes across axon membrane of, 302-303 Respiration, energy coupling to, 74-77 Respiratory acidosis, renal acidification and, 216-218 Respiratory chain, membrane potential and, 24-25 Rhodospirillum, cation transport in, 23 Rubidium ion, bacterial transport of, 2833

S Sodium ion bacterial transport of, 1-50 linkage to amino acid transport, 143144, 147-156 transport of across brush border, 244-262 across rabbit ileum, 225-281 solute-coupled, 262-269 transepithelial, 239-244, 255-259

Shunt pathway ionic permeability of, 234-237 in isolated rabbit ileum, 231-239 properties of, 237-239 Solute-coupled transport, of sodium, 262269 Streptococcus faecalis cation transport and balance in, 3-50 potassium and sodium, 27-33 Sugars, effects on ileal ion transport, 274276

T Thermodynamics, irreversible, quasichemical notation of, 140-144 Turtle bladder, acidification of luminal fluid by, 193-205

V Valinomycin, as potassium ionophore, 1011, 19

A 4 B 5 C 6

D 7 € 8

F 9 G H 1 J

O 1 2 3