Current Topics in Membranes and Transport, Volume 06

Current Topics in Membranes and Transport

Advisory Board

Robert W . Berliner Peter F. Curran (Deceased) I . S. Edelman I . M . Glynn Franpis Morel Aser Rothstein Philip Siekewitz Torsten Teorell Daniel C . Tosteson Hans H. Ussing

Contributors

W . McD. Armstrong Halvor N . Christensen Torben Clausen Mahendra Kurnar J a i n A . A. Lev H . J . Schatzmann

Current Topics in Membranes and Transport

VOLUME 6

Edited by Felix Bronner

Department of Oral Biology University of Connecticut Health Center Farmington, Connecticut and Arnort Kleinzeller

Graduate Division of Medicine University of Pennsylvania Philadelphia, Pennsylvania

1975

Academic Press

New York

San Francisco

London

A subsidiary of Harcourt Brace Jouanovich, Publishers

COPYRIGHT 0 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO 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, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWl

LIBRARY OF CONGRESS CATALOG CARDNUMBER:70-1 17091 ISBN 0-12-153306-9 PRINTED IN THE UNITED STATES OF AMERICA

List of Contributors, vii Contents of Previous Volumes, ix Peter Ferguson Curran, 1931-1974, xiii Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMAR JAIN

I. Introduction and Scope, 1 11. Distribution of Cholesterol in Biological Systems, 5 111. Solubilisation and Dispersion of Cholesterol, 13 IV. Correlative Relationships of Cholesterol Content, 16 V. State of Cholesterol in Organized Lipid Aggregates, 23 VI. Molecular Aspects of Organization of Cholesterol in Bilayers, 39 References, 41 Ionic Activities in Cells A. A. LEV AND W. McD. ARMSTRONG I. Introduction, 59 11. Definition of “Single Ion Activities,” 63 111. Experiments with Model Polyelectrolyte Systems as Supporting Evidence for the Physical Validity of Single Ion Activity Parameters, 66 IV. Microelectrodes for Measuring Intracellular Ionic Activities, 72 V. Techniques for Measuring Intracellular Ionic Activities, 84 VI. Intracellular Ionic Activities, 90 VII. Conclusion, 113 References, 113

Active Calcium Transport and Ca2+-ActivatedATPase in Human Red Cells H. J. SCHATZMANN

I. Calcium Transport, 126 11. Membrane ATPases Activated by Calcium in Human Red Cells, 142 111. Relationship between Calcium Transport and Calcium Magnesium-Activated ATPase, 149 IV. Relation between Calcium-Transport-ATPase and SodiumPotassium Transport-ATPase, 154 V. Comparison with Other Systems Transporting Calcium, 155

+

V

vi

CONTENTS

VI. Physiological Significance of Calcium Pumps, 157 References] 161

The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN

I. Introduction, 169 11. Preparations Used for the Study of Insulin Action on Sugar Transport, 170 111. Cellular Structures Involved in Sugar Transport, 172 IV. The Function of the Glucose Transport System, 178 V. Cellular Signals Controlling Glucose Transport, 196 VI. Mechanisms for the Mode of Act.ion of Insulin, 209 References] 211 Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN

I. Introduction. Summary List of Principal Transport Systems for the Amino Acids, 227 11. Description of the Neutral Systems, 229 111. The Cationic Amino Acid Systems, 232 IV. Extension to System ASC of Approaches to Site Description Taught by the Basic Amino Acids, 235 V. Efforts to Develop System-Specific, Nonmetabolizable Substrates for the Neutral Systems, 237 VI. System-Specific Substrates for the Transport System for the Cationic Amino Acids, 241 VII. Stimulation of Release of Pancreatic Hormones by Amino Ac?ds, 243 VIII. Concluding Discussion, 250 References, 255 Subject Index, 259

List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin. Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana (59)

W. McD. Armrfrong,

Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan (227)

Halvor N. Christensen,

Torbon Clauren,

Fysiologisk Institut, Aarhus Universitet, Aarhus, Denmark (169)

Department of Chemistry and Health Sciences, University of Delaware, Newark, Delaware (1)

Mahendra Kumar Join,

Institute of Cytology of the Academy of Sciences of the USSR, Leningrad, USSR (59)

A. A. Lov,

Institute of Veterinary Pharmacology, University of Bern, Bern, Switzerland (125)

H. J. Schalrrnann,

vii

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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 across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia coli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASERROTHSTEIN Molecular Architecture of the Mitochondrion DAVIDH. MACLENNAN Author Index-Subject Index Volume 2

The Molecular Basis of Simple Diffusion within Biological Membranes W. R. LIEB AND W. D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems B. CHANCEAND M. MONTAL Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDER TZAGOLOFF Mitochondria1 Compartments: A Comparison of Two Models HENRY TEDESCHI Author Index-Subject Index

X

CONTENTS

OF PREVIOUS VOLUMES

Volume 3

The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWARTZ, GEORGE E. 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 HODR~GUEZ DE LORESARNAIZAND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells : I n 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 Genet>icControl of Membrane Transport CAROLYN W. SLAYMAN Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN 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 Volume 5

Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF Pro and Contra Carrier Proteins; Sugar Transport via the Periplasmic Galactose-Binding Protein WINFRIED Boos

CONTENTS OF PREVIOUS VOLUMES

Xi

Coupling and Energy Transfer in Active Amino Acid Transport ERICH HEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption : Theory and Applications to the Reptilian Bladder and Mammalian Kidney AND THEODORE P. SCHILB WILLIAM A. BRODSKY Sodium and Chloride Transport across Isolated Rabbit Ileum STANLEY G. SCHULTZ AND PETER F. CURRAN A Macromolecular Approach to Nerve Excitation ICHIJI TASAKI AND EMILIO CARBONE Subject Index

Peter Ferguson Curran

Peter Ferguson Curran, 1931-1974 Historiographers may argue that a scientist’s contributions should be subjected to the tests of time and appraised by a detached reviewer-not by a still stunned and saddened collaborator barely two weeks after the death of his friend. Yet, it is a testimony to the high caliber of Peter F. Curran’s accomplishments that many of his contributions have already admirably weathered these tests and have been so widely acclaimed by objective critics that unnecessary, but inevitable, praise from friends and admirers may be forgiven. Curran’s first major publication (with A. K. Solomon), “Ion and water fluxes in the ileum of rats” (J. Gen. Physzol. 41, 143, 1957) heralded the caliber and set the theme of his lifelong scientific endeavors. The principal conclusion, that water absorption is a passive process coupled to solute transport, challenged the then-viable notion of “active” water absorption and, within a very few years, became a truism. However, the notion of “active” water transport could not be dismissed until a formal mechanism was developed which could account for both the stoichiometric coupling between water and solute flows and the fact that water can be absorbed against adverse hydrostatic and/or osmotic pressure differences. In 1961, Curran, drawing upon the work of Staverman, laid the foundation of the widely acclaimed “double (series)-membrane model” which was experimentally verified a year later in the now classic paper (with R. MacIntosh), “A model system for biological water transport” [Nature (London) 193, 347, 19621. The almost immediate recognition and acclamation of this model stemmed from its power, elegance, and simplicity-hallmarks of creative genius, and recurring themes of many of Curran’s subsequent contributions. Active transport of solute into a constrained intraepithelial compartment could bring about passive water absorption in the absence of, and against, transepithelial gradients of water activity, providing the barriers bounding this compartment have different passive permeation properties; such asymmetries are sufficient to effect net water transport without having to postulate a direct link to metabolic energy. In short, the major characteristics of transepithelial water transport can be mimicked by a simple system that consists of two different artificial membranes arranged in series. The need to invoke “active” water absorption could be dismissed and the conceptual foundation for the ultrastructural analysis of transepithelial water transport was laid. A milestone in the application of theory to the elucidation of biological processes had been achieved! The “double (series)-membrane model” was born only one year after Ora Kedem and Aharon Katchalsky brilliantly thrust the formalisms of irreversible thermodynamics onto the biological stage. Curran was captivated by the beauty and power of this relatively new discipline and by the sparkle and genius of Katchalsky; Katchalsky, in turn, was immediately attracted to this talented young investigator who had already employed the concepts of irreversible thermodynamics to “tame” a major biological problem. Katchalsky and Curran became lifelong friends and co-authored the book, “Nonequilibrium Thermodynamics in Biophysics” (Harvard University Press, 1965)-a treatise that glows with lucidity, insight, and logic and which is destined to become a classic. [It is sad and ironic that one piece of unfinished business a t the time of Pete’s death xiii

xiv

PETER FUROUSON CURRAN, 1931-1974

was a dedication to Katchalsky (assassinated in 1972) which was to be published in the Biophysical Journal.] Curran was a gifted theoretician and experimentalist. Between 1957 and 1974 he authored or co-authored more than eighty papers dealing with transport across artificial membranes, frog skin, and intestine; the interaction between sodium transport and the transport of sugars and amino acids by small intestine; and, most recently, the molecular characteristics of the amino acid transport mechanisms in small intestine. The common thread running through all of his contributions is the attempt to marry theory with observation successfully-to construct the simplest model that could explain and unify a set of apparently unrelated observations and thereby lay a logical foundation for subsequent studies. This credo is clearly set forth in the paper entitled “Coupling between transport processes in intestine’’ (Physiologist 11, 3, 1968) delivered in 1967, when he was honored by being chosen the American Physiological Society’s Twelfth Bowditch Lecturer. The past quarter of a century has witnessed exciting and significant advances in our understanding of solute and water transport across epithelial tissues, advances to which the name of Peter F. Curran will always be closely linked. Peter F. Curran was born on November 5, 1931 in Waukesha, Wisconsin. He received his B.A. from Harvard College in 1953 and his Ph.D. from Harvard University in 1958. During his professional life he was involved in a host of activities reflecting the high esteem in which he was held by his colleagues. He was Section Editor for Gastrointestinal Physiology of the American Journal of Physiology (1968-1971), Chairman of the Publications Committee of the American Physiological Society (1971-1974), served on the Editorial Boards of the Biophysical Journal, Biochimica et Biophysica Acta, and the Journal of General Physiology, and was on the Advisory Board of This Publication. He was President of the Society of General Physiologists (1972-1973) and a Council Member of the American Physiological Society and the Biophysical Society. He waa a member of the Molecular Biology Advisory Panel of the National Science Foundation and Chairman of Physiology Study Section of the National Institutes of Health. At the time of his death, he was Professor of Physiology and Director of the Division of Biological Sciences at Yale University. Peter F. Curran died a t the age of forty-two. His professional career was rich with achievement and recognition. We will never know the full potential of his talents nor the further heights to which this extremely gifted and energetic scientist might have risen. His untimely death is not only a tragic loss to those who knew him but t o the entire scientific community. STANLEY G. SCHULTZ

Current Topics in Membranes and Transport Volume 6

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Related Systems* MAHENDRA K U M A R J A I N Department of Chemistry and Health Sciences, University of Delaware, Newark, Delaware

1. Introduction and Scope

. . . . . . . . . . . . . . .

. . . . . . . . Solubilization and Dispersion of Cholesterol. . . . . . . . . . Correlative Relationships of Cholesterol Content . . . . . . . .

11. Distribution of Cholesterol in Biological Systems 111.

IV.

A. With Barrier Properties of Biomembranes . . . . . B. With Permeability of Model Systems . . . . . . C. With Electrical Properties of Bilayer . . . . . . V. State of Cholesterol in Organized Lipid Aggregates . . . A. In Monolayers. . . . . . . . . . . . . B. In Bilayers. . . . . . . . . . . . . . C. I n Biomembranes . . . . . . . . . . . . VI. Molecular Aspects of Organization of Cholesterol in Bilayers . References . . . . . . . . . . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . .

. . . . . . . .

1 5 13 16 16 18 22 23 23 27 38 39 41

We must be grateful to Lord Russell for the unequaled skill with which he has left the vast darkness of the subject unobscured. Alfred North Whitehead

1. INTRODUCTION AND SCOPE

For quite some time now, considerable attention has been focused on the role of cholesterol in arterial degeneration and on its role as an essential

* Abbreviations used : CTAB, cetyl trimethylammonium bromide; BLM, black lipid membrane; DSC, differential scanning calorimetry; ESR, electron spin resonance; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PE, phosphatidylethanolamine; RBC, red blood cell; SM, sphingomyelin. 1

2

MAHENDRA KUMAR JAlN

component of many cell organelles and body fluids. The turnover and the organizational and physical state of cholesterol and its esters in tissues, cells, and body fluids have been the subject of intensive study. I n the solid state, cholesterol displays considerable pleomorphism and is not readily dispersed in water or electrolyte solutions. Cholesterol has a maximum solubility in aqueous solutions of 4.7 pM and c.m.c. of 25-40 nM a t 25’ C (Haberland and Reynolds, 1973). Cholesterol can, however, be “solubilized” and carried in ternary systems with lecithin, or in quaternary systems containing bile salts and lecithin (Small et aE., 1966), or in certain proteins and phosophlipids (see below). I n these dispersed systems cholesterol enters the mesophase only in the unsubstituted forms. Plasma lipoproteins, however, can also carry some cholesterol esters (Table I). The fatty acid chain length of the amphipath does influence the order and stability of incorporation of unsubstituted cholesterol in the mesophase. TABLE I

PERCENTAGE COMPORITION OF SOME HUMAN BLOOD SERUMLIPOPROTEINS

Lipoprotein characteristic Average hydrated densit.y (gm/ml) Molecular weight Particle size (diameter, A) Shape Percentage composition (w/w) Protein Phospholipid Cholest,erol Free Ester Trigly ceride Free fatty acid Fraction of plasma lipids Cholesterol Phospholipid Trigly ceride Presence in blood

Very low density Low density High density ‘(VLDL) (LDL) (HDL) pre-6p-lipoa-lipoChylomicron lipoprotein proteins proteins <0.95

0.98-1.03

109 to 10’0 750-6000 Spherical

5-10 X 106 2 x 106 300-700 150-300 Spherical Spherical

4 7.5

8 19

5 5.5 78 4-0

6 11 55

85 After fat ingestion

Trace 10 After fat ingest ion

1.03-1.09

1.1-1.2 1 4 X lo6 90-350 Elongated

21 28

58 25

8

3 8 6 Trace

33 10 Trace 70 50 2 Usual

30 50

a Usual

3

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

TABLE I1 CHANGES IN CHOLESTEROL LEVELSASSOCIATED WITH VARIOUSSYNDROMEW Disorder

Changes

1. Obstructive jaundice

Increased level of LDLb-like proteins, decreased levels of HDL. Higher levels of free cholesterol and lower levels of esterified cholesterol in HDL and “LDL” from patients.

2. Nephrotic syndrome (several

Patients with hyperlipemia show increased levels of cholesterol, triglycerides, and phospholipids. Generally, the cholesterol/ PL ratio increases in VLDL, decreases in LDL and remains unchanged or slightly increased in HDL fractions

kidney diseases)

3. Aging

Increased LDL level with age in both males and females; however, the peak reached much earlier in males

4. Abetalipoproteinemia

Decreased LDL levels, presumably due to lack of specific protein component

5. Tangier disease

Complete lack of HDL and marked storage of cholesterol, especially in the retieuloendothelial tissues

6. Cancer

Significant decrease in HDL and increase in LDL has been noted in patients with various forms of cancer

7. Cerebrosidosis (Gaucher’s disease)

Patients have subnormal levels of HDL and LDL cholesterol and HDL phospholipid-presumably reflecting lower levels of HDL and LDL

8. Sphingornyelin lipidosis

Serum cholesterol and PL are elevated as a reflection of the substantial increase in LDL and probably VLDL and HDL

(Niernann-Pick disease) 9. Glycolipid lipidosis (Fabry’s disease)

Elevated LDL

10. Hyperlipoproteinemias

Greater than usual concentrations of lipoproteins in blood. Several kinds are known

11. Psychological straw

Increased cholesterol levels in serum. LDL increased and HDL decreased (Continued)

4

MAHENDRA KUMAR JAlN

TABLE I1 (Continued) Disorder

Changes

12. Atherosclerosis and its

Higher serum cholesterol. Substantial decrease in HDL cholesterol Lower HDL and increased LDL in hepat,it,is.High cholesterol and increased LDL in biliary cirrhosis. Elevation of VLDL in acute or chronic forms of fatty liver and from excessive intake of fats, or carbon tetrachloride, ethanol, etc.

complications 13. Other liver diseases

From Barclay (1972). HDL, High density lipoproteins; LDL, low density lipoproteins; VLDL, very low density lipoproteins. a

b

Various pathological conditions can affect the levels and composition of plasma lipids (Nestel, 1970; Carnie, 1973). I n most cases these alterations result from changes in some classes of lipoproteins and thus disturb the levels of “soluble” cholesterol. For example, an increase in the level of plasma low-density lipoprotein (LDL) brings about increases not only in the total amount of lipids, but also in absolute and relative amounts of cholesterol, since the LDL fraction is the major carrier of cholesterol. I n many diseases where the plasma lipid composition is altered, lipoproteins remain unchanged chemically. In others, however, such changes have been detected (Table 11). In some pathological states the concentration of cholesterol increased beyond the solubilization point. This leads to the deposition of cholesterol and the formation of gallstone (Admirand and Small, 1968). Other situations of phase equilibria of cholesterol that are disturbed are aging and induction of atherosclerosis (Stewart, 1969; Hamilton et al., 1971). In all these cases there is indication that cholesterol and phospholipid molecules are weakly associated with each other to form a complex. Stability of such complexes appears to differ in various systems and to depend upon the presence of other components. It is particularly interesting that the lipid molecules in plasma lipoproteins and plasma membranes are exchangeable (see below). Similar aspects of intermolecular association are also reflected in the composition-function correlation of biomembranes. In this review we shall examine the molecular aspects of interaction between cholesterol and phospholipids. Aspects relating to the surface chemistry of lipids have been extensively reviewed elsewhere (Shah, 1970b; Phillips, 1972). This review mainly relates to how these subtle interlipid interactions modify the gross functions of membranes.

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

5

11. DISTRIBUTION OF CHOLESTEROL IN BIOLOGICAL SYSTEMS

A wide variety of sterol molecules occur in nature. These molecules have the same general perhydrocyclopentenophenanthrene ring structure, but they differ in degree of unsaturation and branching. Unlike other lipids, sterols are not ubiquitous. Only fungi, plants, protozoa, invertebrates, and vertebrates contain sterols. Sterols are neither synthesized nor required by prokaryotic cells. Certain animals, plants, and fungi can synthesize sterols, whereas most insects have a general and specific requirement for sterols (Clayton, 1970). The general requirement seems to be for a membrane structural component, which can be supplied by a number of different sterols. A very small amount of cholesterol is specifically required, presumably for the biosynthesis of hormones, such as ecdysone. I n the house fly, cholesterol stimulates ovarian development and is essential for maintaining the production of viable eggs (Monroe, 1959, 1960; Robbins and Shortino, 1962). Sterols stored in the larval tissues are carried over to the adult stage and are deposited in the developing oocytes (Monroe et al., 1967). The hatchability of eggs declines significantly when their sterol concentration is reduced below about two-thirds of the normal level. Occurrence of a given sterol in an organism is no assurance that it is an essential membrane component or that it is even located in a membrane. Artifacts of membrane isolation arise from incomplete separation, differential washing of various components, and loosening of intermolecular association due to removal of divalent ions and proteins from the membrane interface. It is now fairly well established that membrane lipids are in a state of Aux and are constantly exchainging with other lipids present in the surrounding medium (Silber et al., 1969; also see below). Nevertheless, membrane lipid composition appears to be fairly constant. This constancy varies with diet, temperature, pH, ionic concentration, metabolic state, and other parameters related to growth (Ostwald and Shannon, 1964; Bruckdorfer et al., 1968a, b, 1969; Rothblat et al., 1966; Back et al., 1969; Ginter, 1973; Meca and Conner, 1971; Spohn and Davison, 1972; Spritz and Mishkel, 1969). Membrane lipid composition differs also in various physiological and pathological states (Roberts and Straus, 1965; Gresham et al., 1965; Hestorff et al., 1965; Jaffe and Gottfried, 1968; Green and Green, 1973; Dam, 1971; Csogor, 1972) including drug-induced lipidosis of the liver (Yamamoto et al., 1971) and heat exposure of erythrocytes (Kuiper et al., 1971). Cholesterol (but this is not true for other lipids) is lost from erythrocytes stored under conditions that are homeostatic for glucose; however, in the absence of glucose both cholesterol and phospholipids are lost (Cooper and Jandl, 1968). It has been suggested that the initial step in the loss of cholesterol during storage is the lecithin: cholesterol

6

MAHENDRA KUMAR JAlN

acyltransferase reaction. This reaction operates somewhat abnormally in vitro because of the altered ratio of free cholesterol to esterified cholesterol in plasma lipoproteins, which disturbs cholesterol exchange between the cell and plasma. The exchange of cholesterol and other membrane sterols with the plasma sterols is dependent upon pH and catalyzed by organic solvents, but temperature has little effect (Hagerman and Could, 1951; Dobiasora and Linhart, 1970; Graham and Green, 1970; Banik and Davison, 1971; Edwards and Green, 1972; Bruckdorfer and Green, 1967; Green and Green, 1973; d’Hollander and Chevallier, 1972a, b). This indicates that the exchange phenomenon is not simply a collision displacement process but may be mediated by exchange protein (Christian and Zilversmit, 1973) or lecithin : cholesterol acyltransferase (Glomset, 1968) or some other cholesterol-binding species (Torsvik et al., 1972). Bruckdorfer et al. (1968a) also noted that if the plasma or the source of cholesterol to be exchanged with the cell is deficient in cholesterol, the cells lose cholesterol to the medium. Some species difference has been observed in the exchange of cholesterol in erythrocytes. I n both liposomes and erythrocytes the order of ease of incorporation is cholesta-4,6-dien-3-one > cholesterol > campesterol > sitosterol (Bruckdorfer et al., 1868b, 1969). It may be pertinent to note that compared with animal sterols some plant and fungal sterols are poorly absorbed in the intestine. This specificity of absorption has been attributed to the ability of sterols to enter membranes and soluble lipoproteins of the intestinal cell (Glover and Green, 1957; Desai and Glover, 1963; Edwards and Green, 1972). Interestingly, cholesterol-deficient cells remove cholesterol from P-lipoproteins only when dimethylsulfoxide (DMSO) is added to the medium (Bruckdorfer et al., 1969). Exchange of cholesterol from a-lipoproteins occurs without DMSO. This suggests different boundary arrangements for cholesterol in a- and P-lipoproteins. With the cautionary note just described, one may examine the proportion of cholesterol in total lipids from a variety of proteolipids (Table I) and membranes (Table 111). The mole fraction of cholesterol seems to vary from 0 to only 0.5. Therefore, in contrast to phospholipids, cholesterol is not a necessary constituent of all membranes and thus must perform a particular, rather than a general, function. Cholesterol is well recognized as a prominent lipid constituent of many biological interfaces. Besides cholesterol, only sterols such as 7-dehydrocholesterol, lathosterol, and cholestanol have been reported in animal cells (Lesser and Clayton, 1966; Werbin et al., 1962; Glover and Green, 1957; Subbiah et al., 1971). Cholestanol (dihydrocholesterol) has been identified in fractions of brain from patients with cerebrotendinous xanthomatosis

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

7

TABLE I11 COMPOSITION OF CERTAINMEMBRANE TYPEW

Source

Cholesterol (mole % of total Total lipid (% dry weight) phospholipid)

References

Myelin-human central nervous system Myelin-beef M y e l i n s p i n a l root (beef) Platelet-human

78.7

40.1

O’Brien (1967)

75.9 76 56

39 37 25

Cow RBC

38

43

Rat RBC

40

42

Sheep RBC

30

40

Dog RBC Rabbit RBC

43 39

48 47

Chicken RBC

55

38

Guinea pig RBC

47

47

Goat RBC Horse RBC Pig RBC Cat RBC Elephant seal RBC Harp seal Human RBC Rat liver plasma membrane

48 39 38 50 36 36 46 40

40 50 47 46 47 44 42 26

Rat myometrium

59

41

Rat liver microsomes

32

8

Guinea pig brain microsomes

30-35

8

Retinal rod outersegment

41

4

Squid retinal axon

45

33

O’Brien (1967) O’Brien (1967) Barber and Jamieson (1970) Cornwell et al. (1968);Nelson (1967a) Cornwell et a2. (1968); Nelson (1967a) Cornwell et al. (1968); Nelson (1967a) Nelson (1967a) Cornwell et al. (1968); Nelson (1967a) Kates and James (1961) Reed and Roberts (1968) Nelson (196713) Nelson (1967b) Nelson (1967b) NeIson (1967b) Nelson (1970) Nelson (1970) O’Brien (1967) Benedetti and Emmelot (1968) Kidwai et al. (1971) Glaumann and Dallner (1968) Fleischer and Rouser (1965) Eichberg and Hess (1967) Fischer et a1 (1970)

(Continued)

8

MAHENDRA KUMAR JAlN

TABLE I11 (Continued)

Source

Cholesterol (mole % of total Total lipid (% dry weight) phospholipid)

Synaptic vesicles (guinea pig brain) Ehrlich ascites carcinoma cells Bovine heart mitochondria

34

Rat liver mitochondria

21

. .5 i

Guinea pig kidney mitochondria Guinea pig brain mitochondria

15

0.5

27-30

1

Pig lymphocyte

42

50

Rat intest,inal microvillus

38

63

HeLa cell

40

52

Bovine olfactory epithelium

30

13

Mycoplasma laidlawii B Azobacter azilis

3.5-37 10

0

Escherichia coli

10

0

Agrobacteriicm tumefaciens

10

0

33 24

5.6 12 1

0

References Eichberg et al. (1964) Wood et al. (1970) Fleischer et al. (1967) Fleischer and Rouser (1965) Rouser et al. (1968) Fleischer and Rouser (1965) Allan and Crumpton (1970) Forstner et al. (1968) Bosman et al. (1968) Koyoma et al. (1971) Razin (1963) Kaneshiro and Marr (1962) Kaneshiro and Marr (1962) Kaneshiro and Marr (1962)

a The calculations used in the preparation of this table have often required making the assumption that total lipid has molecular weight 750. Because of this and because of the variations in experimental results, the reader is advised that the tabulated values should be considered &s estimates only, presented to emphasize the mole proportions and their variation. Neutral lipids of erythrocytes (RBC) contain almost exclusively cholesterol.

(Stahl et al., 1971). The role played by these sterols is unknown. The cholesterol content of biological membranes is highly variable. I n a given cell, the cholesterol content (both relative and absolute) of various organelle membranes seems to vary by almost an order of magnitude. For example, in rat liver the molar ratio of phospholipid to cholesterol is 0.76 for plasma membrane, 0.24 for smooth endoplasmic reticulum, 0.12 for microsomes and outer mitochondria1 membranes, 0.06 for rough endo-

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

9

plasmic reticulum (Colbeau et al., 1971). The highest levels of cholesterol so far reported have been for myelin, followed (in that order) b y plasma membranes from a variety of sources, microsomes, outer and inner mitochondrial and nuclear membranes (Lesser and Clayton, 1966). This is also the order in which the proportion of lipids to total membrane dry weight decreases and the proportion of unsaturated acyI chains in membrane lipids increases. The highest mole proportion of cholesterol to phospholipids is always one or below. This suggests some correlation between lipid-cholesterol interaction and its dependence upon degree of unsaturation of fatty acid chains. A rationale for this correlation may be found in the nature of intermolecular association between cholesterol and phospholipids as described later on. However, this poses serious questions regarding the considerable difference in cholesterol content of plasma membranes as compared to membranes of subcellular organelles. This may be partially accounted for in terms of degree of unsaturation. As the membranes of subcellular organelles contain higher proportions of u6,u6 double bonds, their propensity for interaction with cholesterol would be considerably reduced (see below). In contrast, u6 bonds in plasma phospholipids would not interfere with lipid-cholesterol association. Membrane phospholipids with a higher proportion of unsaturated acyl chains or 1-unsaturated-2-saturated phospholipids (see below) would not only be a less effective barrier, but would also be incapable of incorporating cholesterol. Thus the ability of a phospholipid to interact with cholesterol in a biomembrane would parallel the occurrence of these phospholipids in nature. I n many animal tissues there appears to be a positive correlation between levels of cholesterol, sphingomyelin, and total lipids. For example, these three components tend to be high in myelin and erythrocytes (O’Brien, 1967; Coleman, 1968), in aged human arteries (Buck and Rossiter, 1952), and in plasma membrane (Ray et al., 1969). On the other hand, all three of these are relatively low in mitochondria (Fleischer et al., 1967; Parsons and Yano, 1967). Plant tissues contain neither cholesterol nor sphingomyelin. Patton (1970) has found a positive correlstion between the cholesterol/sphingomyelin content of mitochondria, nucleus, endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat hepatocytes. It has been suggested that membrane lipid class composition is determined a t least in part by type and number of protein binding sites (e.g., see Kramer et al., 1972). However, cholesterol and polar lipids do not substitute for each other. Thus the presence of cholesterol in membranes must be explained by binding to polar lipids rather than directly to protein. Since the mole fraction of cholesterol in membrane lipids is always less than 0.5, it appears that cholesterol does not bind to all polar lipids. e9,

w699J2

10

MAHENDRA KUMAR JAlN

Based on lipid composition data of erythrocytes from various mammalian species (Nelson, 1967a, b), it has been suggested that two molecules of cholesterol bind to one molecule of either PE, sulfatide, or acidic phospholipids. These authors also observed that the molar sum of PC, SN, cerebrosides, and acidic phospholipids (i.e., all polar phospholipids except P E and sulfatides) of human brain was almost exactly equal to the number of moles of cholesterol. This correlation is not applicable to subcellular organelles which do not have the same high level of cholesterol as would be expected from their polar lipid composition. It may, however, be noted that the nature and degree of unsaturation are an important factor in determining sterol-phospholipid interaction. A higher degree of unsaturation as observed in acyl chains from phospholipids of subcellular organelles would retard interaction of cholesterol with these polar lipids. Bacteria do not require sterols as membrane constituents. Some strains of Mycoplasma laidlawii such as T strain (Rottem et al., 1971) and strain 07 (Smith, 196413) require sterols. M . laidlawii strain B is not dependent on sterols but can incorporate sterols from the growth medium (Razin et al., 1966; Deliruyff et al., 1972). Thus the strains of Mycoplasma may have between 10 and 30% sterols in their membranes (Smith, 1967, 1968, 1969). Although sterol esters are also incorporated by these strains (23%), there is no evidence that the free sterol molecule is chemically altered by these organisms. The ratio of membrane lipid classes in Myccplasma laidlawii is not significantly affected by variations (induced by adding lipids to growth medium) in fatty acid composition or sterol content (RlcElhaney et al., 1970). However, when a sterol-requiring strain is made to grow in the absence of cholesterol, its polar lipids become more saturated (Rottem et al., 1973a). Also the total lipid from the adapted strain shows phase-transition (Rottem et al., 1973b). Other organisms such as Saccharomyces cerevisiae (Starr and Parks, 1962) and Pythium spp. (Haskins, 1965; Schlosser and Gottlieb, 1966; Sietsma and Haskins, 1967) can maintain viability a t high temperatures (45-50°C) if sterols are added to the growth medium. It thus appears that sterol incorporation into the membrane may protect the membranes against high temperature damage. Similar protection may be provided by certain pigments such as neurosporene and carotenoids, along with sterols. A detailed description of the nature, physiological properties, and requirements for sterols and pigments may be found elsewhere (Smith, 1969). Sterol esters cannot replace free sterols in growth media. I n fact, to support Mycoplasma growth (a) the sterol must be planar, i.e., the A and B rings must be trans fused; (b) it must have a n equatorial 3-p-hydroxyl group; and (c) there must be a hydrocarbon chain. Thus cholesterol, cholestanol, lanosterol, p-sitosterol, stigmasterol, and ergosterol can

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

11

support growth, whereas coprostanol, 7-dehydrocholesterol, epicholesterol, and cholestan-3-one cannot (Smith, 1969). However, there appears to be considerable difference in various strains of mycoplasma. T strain can incorporate cholesterol and p-sitosterol and, to a lesser extent 7-dehydrocholesterol, stigmasterol, ergosterol, and cholestanol (Rottem et al., 1971). For strain 07 it is observed that only cholesterol and cholestanol support growth (Smith, 1964a). Strain B cell can incorporate epicholesterol besides cholesterol (DeKruyff et al., 1972). Proof for membrane localization of these sterols in these organisms is obtained from the ability of digitonin to lyse sterol containing Mycoplasma spp. only (Smith and Rothblatt, 1960), whereas other nonspecific surfactants, such as soaps, alcohols, cationic and anionic detergents, are able to disrupt several Mycoplasma strains (Smith, 1964a). Tetrahymanol, a sterollike triterpenoid, has been identified as a constituent of various membranes of Tetrahymena pyriformis (Thompson et al., 1972). The tetrahymano1:phospholipid ratio is constant in growing and early stationary phase, but increases in various membranes from starved or senescent cells. Tetrahymanol is resistant to catalytic attack. Surviving cells of senescent cultures ingest fragments of dead cells, degrading the phospholipid, but accumulating the triterpenoid. This is consistent with the observation that ergosterol (Conner et al., 1971) or cholesterol (Conner et al., 1968), added to the cultures of Tetrahymena, blocks tetrahymenol synthesis and eventually replaces it in the growing cells. Interestingly, the addition of tetrahymanol itself does not inhibit tetrahymanol biosynthesis (Landrey et al., 1971). Variation in cholesterol content has also been observed in plasma membranes from cells of higher animals. Cholesterol content and lipid composition of red blood cells can be changed by altering growth conditions or diet or by incubation in plasma from cholesterol-fed guinea pigs. Earlier studies had demonstrated that aqueous dispersions of egg lecithin can remove cholesterol from erythrocyte membranes and that cholesterol can exchange between lecithin-cholesterol dispersions and erythrocyte ghosts (Bruckdorfer et al., 1968a). Also large proportions of membrane sterol can be replaced by other lecithin-solubilized sterols (Eruckdorfer et al., 196813). Thus a choIestero1 increase in the diet of hamsters is associated with the appearance of prominent, abnormal spicules on the surface of the red blood cell. Alterations in erythrocyte cholesterol and lecithin levels result in increased osmotic fragility and premature destruction, even when the cells are essentially normal in other respects (e.g., in ATPase level and glucose metabolism). This results in eventual decrease in the viability of the erythrocytes, presumably due to an increase in the interal viscosity of the bilayer. Similarly, a greater osmotic fragility of hamster erythrocytes has

12

MAHENDRA KUMAR JAlN

been observed following prolonged exposure of the animals to a n elevated ambient temperature. Among other effects, a decrease in cholesterol: phospholipid ratio from 1.07 to 0.92 has been observed (Kuiper et al., 1971). This lowering of cholesterol content may be responsible for a variety of effects besides the mechanical stability of the cells. For example, penetration of cells by psychomimetic drugs (Demel and Van Deenen, 1966), plant growth substances (Kennedy and Harvcy, 1972), carcinogens (Belmonte and Swarbrick, 1973), catecholamines (Salt and Iversen, 1972), vitamin A (Bangham et al., 1964), an azo-dye-orangc IV (Horton et ul., 1973), and polyene antibiotics (Norman 6t al., 1972a,b; Hsuchen and Feingold, 1973; Drabikowski et al., 1973; Kinsky et aE., 1968) may all be influenced by the presence of cholesterol in membranes. A decrease in “fluidity” and increased cholesterol content of unilamellar vesicles has been shown to decrease the fusion of mammalian cells by these vesicles (Papahadjopoulos et ul., 1973a). Similar processes may regulate the influence of cholesterol on blood coagulation (Sterzing and Barton, 1973). Also autoxidation of cholesterol has been shown to be influenced by the presencr of lecithin (Weiner et al., 1973). Saponins and polyene antibiotics sekctively lyse membranes containing cholesterol, and their lytic activity is antagonized by free cholesterol in the medium. The polyene antibiotics behave very much like saponins, although there are significant differences in the electron micrographs of negatively strained liposonies containing lecithin cholesterol (7 :1) treated with polyenes and saponins (Rinsky et al., 1966, 1967). The pits and rings observed in these two sets of electron micrographs are similar in form, but not in dimension. It is particularly important to note that erythrocyte membranes treated with saponin do not show the same substructuring as the liposomes containing equimolar lecithin and cholesterol. This may indicate that the mode of packing of cholesterol in these two membranes is different. Furthermore, the interaction of saponins and polyene antibotics with membrane-bound cholesterol is consistent with the fact that cholesterol molecules have a considerable degree of freedom of rotation and reorientation, a condition that would be necessary if these lytic agents were to form channels by aggregation. Similar forces may be involved in the interaction of tyrocidine B with BLM; the presence of cholesterol (1 :1 molar ratio) makes the kinetics bimolecular (Goodall, 1970, 1973). Although these effects may be somewhat specific, a n explanation requires a n understanding of membrane packing, permeability, and partition behavior on a molecular basis (see below). An increase in the levels of cholesterol has been shown to render membranes more lipophilic, less permeable, and more susceptible to mechanical abuse. The modifications of

+

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

13

macroscopic phase behavior of bilayers are reflected in such diverse, membrane-related phenomena as functioning of secretory vesicles (Keenan and Moore, 1970), myelin formation (Vandenheuvel, 1963; O’Brien, 1967), morphology and viability of the red cells (Ostwald and Shannon, 1964; Murphy, 1965; Sardet et al., 1972), photohemolysis (Lamola et al., 1973), ouabain-sensitive sodium transport (Kroes and Ostwald, 1971; Kroes et al., 1972), milk-fat globule secretion (Bargmann and Knoop, 1959; Patton and Fowkers, 1967) accumulation of lipids into atheromatous plaques (Eisenberg et al., 1969; Portman and Alexander, 1970; Forte et al., 1971; Hamilton et al., 1971), and membrane-bound enzymic activity (Cobon and Haslam, 1973). Among rather specific effects of cholesterol may be mentioned activation of Na K - ATPase (Noguchi and Freed, 1971; Jarnefelt, 1972), inhibition of catecholamine uptake by rat heart (Salt and Iversen, 1972), inhibition of 6-glucuronidase and glutamate dehydrogenase (Tappel and Dillard, 1967), and possibly tetrodotoxin binding in nerves (Villegas et al., 1970). This last possibility appears unlikely since squid axon membranes generally have about 108 cholesterol molecules/pm2, whereas only < 100 T T X binding sites/pm2 are present on various nerve membranes.

+

111. SOLUBlLlZATlON AND DISPERSION OF CHOLESTEROL

I n the solid state, cholesterol is not readily dispersed in water or electrolyte solutions which could occur naturally. Yet, since cholesterol is integrated into all cells and most body fluids in nonparticulate form, some very efficient methods of microdispersion and phase change must operate biologically (e.g., see Rohmer et al., 1972; Rampone, 1973). Solubilization of cholesterol can be achieved in ternary systems with lecithin as the amphiphile, in which case a complex, stable mesophase develops at a certain critical concentration. Also complete solution has been achieved in quaternary aqueous systems containing bile salt and lecithin (Small et al., 1966). In these amphiphilic systems, cholesterol enters the mesophase only in the unsubstituted state; cholesterol esters, irrespective of the chain length of the fatty acid tail, do not form ordered phases. On the other hand, the fatty acid chain length of the ester used as amphiphile does influence the order and stability of cholesterol in mesophase. This suggests that the packing of cholesterol molecules may induce a steric pattern entirely different from that of phospholipids in bilayers. Cholesterol exhibits polymorphism (Spier and Van Senden, 1965; Bernal and Crowfoot, 1933; Bulkin and Krishnan, 1971; Scanu and Tardieu, 1971), and

14

MAHENDRA KUMAR JAlN

forms liquid crystals when mixed with a variety of simple compounds (Zull and Sciotto, 1969; Zull et al., 1968; Rowel1 et al., 1965; Gibson and Pochan, 1973; Snart, 1967a, b). The principal structural requirement of the second component is that it be a n amphipath with a chain-length greater than twelve methylene residues. There is an upper limit to the proportion of sterol that can be introduced into liposomes (Table IV, see also Horwitz et al., 1971). A closer examination of data presented in this table reveals that the orientation of hydroxyl group a t position 3, ring junction A/B, the position of double bonds, unsaturation and length of side chain, all seem to determine the extent of incorporation of these sterols into bilayers. The pattern of incorporation TABLE IV

THESOLUBILIZATION OF STEROLS BY SONICATION WITH EGGLECITHIN" Sterol

Cholesterolb Cholestanolb Lat hosterolb 7-Dehydroch~lesterol~ Ergosterol Stigmasterolb Androstan-3p-olb p-Norcholesterolb Caprostanol* Epicholesterolb Androstan-3-cr-01~ Cholestan-3-one Cholest-4-en-3-oneb Cholest-5-en-3-oneb Choleste-4, 6-dien-3-oneb Testosterone crotonoatec Testosterone benzoatec Testosterone 2-octenoatec Testosterone 3-octenoatec Testosterone undecylenatec Testosterone 2-methylpropionate" Testosterone 4-methyl pentanoateC

Sterol :phospholipid (molar ratio) 1.04:l 1.11:l 1.07:l 0.55:l 0.3931 0.57:l 0.72:l 1.16:l 1.04:l 0.30:l 0.58:l 0.43:l 0.59:l 0.57:l 0.55:1 0.14:l 0.04:l 0.56:l 0.55:l 0.56:l 1.11:l

0.2.5 :1

* See also Bourges et al. (1967a, b). Demel et al. (1972a). Stevens and Green (1972) ;see also Kellaway and Sanders (1967).

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

15

does not seem to correlate with the lipophilic character of the sterols (Stevens and Green, 1972). These observations are best rationalized in terms of conformational adaptation of sterols into polymethylene chains. Within this limitation, optimum incorporation (maximum solubilization) would be obtained with the molecule giving van der Waals interactions, and so side chain structural requirements may be somewhat specific. Solubilization of sterols by lysolecithin (Gale and Saunders, 1971) and incorporation of sterols into phospholipid monolayers (see below) may not necessarily depend upon these considerations since the geometry of the aggregates would introduce serious restrictions for intermolecular association and organization. Bile salts also have the solubilizing capacity for cholesterol (Admirand and Small, 1968). However, the addition of lecithin decreases the dissolution rate even though lecithin increases the equilibrium solubility of cholesterol in these solutions. The reduction in rates caused by lecithin has been attributed to a large crystal (cholesterol)solution interfacial barrier (Higuchi et al., 1972); cholesterol is also “solubilized” by lysolecithin (Neiderhiser and Roth, 1972). Similarly a change in the type of bile salts does not markedly affect the quantity of cholesterol solubilized by added lecithin (Neiderhiser and Roth, 1968). Admirand and Small (1968, 1972; see also Mufson et al., 1972a) have shown that the solubility of cholesterol in human bile is determined by the relative concentration of bile salts and lecithin in the quaternary aqueous system. I n some pathological states they found that the concentration of cholesterol was increased beyond the point of solubilization. This led to the deposition of cholesterol and the formation of gallstones. Admirand and Smith’s findings amount to an attractively simple biophysical explanation of the occurrence of some forms of gallbladder disease especially in conditions of cholestasis, or when the excess of cholesterol is attributable to metabolic cholesteroisis. Of particular interest in relation to the aging process in humans is the partitioning of cholesterol in plasma lipoproteins (Stewart, 1959, 1961a). The total concentration tends to increase in middle age, but it is the distribution rather than the total amount that shows the most striking change. I n youth, cholesterol and its esters are carried in the high-density lipoprotein (cf. Table I) fraction. I n men from the mid-twenties onward and in women after menopause there is sharp increase in low-density lipoproteins which carry a relatively higher concentration of all forms of lipid. Normally, excess lipid is split hydrolytically, but this process is less effective against low-density lipoproteins. Thus the lipolysis fails when it is most needed and cholesterol remains in the blood stream or deposits in various tissues (cf. atherosclerosis). The relative immunity of women to atherosclerosis and its consequences during their reproductive years

16

MAHENDRA KUMAR JAlN

may find a qualitative biophysical explanation in the fact that the excess cholesterol is directed to or held in ovarian cells (corpora lutea), where it is a precursor in hormonal synthesis (Stewart, 1961b). Consistent with this hypothesis is the finding that, in diseases associated with signs of premature aging, like untreated diabetes, hypothyrodism, and chronic nephritis, the low--density lipoprotein is markedly increased and deposition of cholesterol in arteries and elsewhere is very conspicuous.

IV. CORRELATIVE RELATIONSHIPS OF CHOLESTEROL CONTENT A. With Barrier Properties of Biomembranes

Permeability of biomembrane bilayer for small solutes, particularly water, has been measured in a variety of systems (Table V). I n most cases permeability has been measured by osmotic swelling on the underlying assumption that the osmotic response (fragility and permeability) of whole cells, vesicles, and liposomes may reflect the lipid composition and organization. Replacement of cholesterol in erythrocyte membranes by lanosterol, 7-dehydrocholesterol, 0-norcholesterol, or by exchange with lecithinsterol dispersions shows that these sterols have a n effect on membrane permeability comparable to that of cholesterol (Bruckdorfer et al., 1969). Replacement of cholesterol by cholestan-3-one1 cholestene-4 ,6-dien-3-one in erythrocyte membranes results in an increased permeability. Permeability of lecithin liposomes is not affected, however, when a variety of other ketosteroids is incorporated. Similarly, cholesterol-enriched mitochondria are more resistant to swelling than control mitochondria (Graham and Green, 1970). Pythium mycelium gown in the presence of cholesterol has a decreased rate of release of metabolites (Child et al., 1969). I n comparison to other membrane systems, however, no change in nonelectrolyte permeability has been observed in pig erythrocyte depleted of 35% of cholesterol (Deuticke and Zollner, 1972). The permeability of the cholesterol-loaded erythrocytes for nonelectrolytes has the same activation energy as for the untreated cells (Kroes and Ostwald, 1971). A similar observation has been made for Mycoplasma (DeGier et al., 1969). This suggests that the added choIesterol lowered the permeability without affecting the threshold energy for the process. Since cholesterol in the bilayer is unlikely to influence the rate of interfacial solute transfer, it may affect the rate of solute permeation through the hydrocarbon region of the bilayer.

17

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

TABLE V P.4SSIVE

PERME.1BILITY OF BIOMEMBRANES AS A FUNCTION OF CHOLESTEROL CONTENT Source

Permean t

*

Effect

Mycoplaarna laidlawii B

Glycerol, erythritol

Cholesterol-containing membranes have lower permeability; epicholesterol does not affect permeability (DeKruyff et al., 1972, 1973a; McElhaney et al., 1973). No change in activation energy for transport

Pig and rabbit RBC

Glycerol, erythritol

Cholesterol-depleted cells show higher permeability (Bruckdorfer et al., 1969)

Cholesterol-fed guinea pig ItBC

Erythritol, thiourea, monoacetin, Na

Permeability decreases following incorporation of cholesterol (Kroes and Ostwald, 1971). Act.ivation energy for hemolysis remain unchanged. Ouabain-sensitive and unsensitive permeability of Na is also lowered

Heart froin cholesterol-fed rats

Catecholamine

Permeability is inhibited (Salt and Iversen, 1972)

Both the active (ouabain-sensitive) and passive (ouabain-insensitive) components of sodium efflux are decreased in cholesterol-loaded erythrocytes (Kroes and Ostwald, 1971). However, the binding of ouabain to normal and loaded cells was approximately the same. This implies that the decrease in sodium permeability of the cholesterol-loaded cells was not due to a decrease in the number of sodium pump sites. These results could arise from a decreased leakage of cholesterol-loaded cells as active efflux will also decrease in such a situation. It may be pertinent to note that in delipidated Naf I<+- ATPase from beef brain (Noguchi and Freed, 1971) and in electroplax of Electrophorus electricus (Jarnefelt, 1872) cholesterol can reactivate ATPase activity much more than phospholipids. I n contrast, a substantial proportion of neutral lipids including cholesterol can be extracted from sarcoplasmic reticulum without changing the ability

+

18

MAHENDRA KUMAR JAlN

of the reticulum to accumulate CaZc (Drabikowski et al., 1972). This may imply either that Ca-activated ATPase does not require cholesterol for its functioning or that loosely bound cholesterol does not play a direct role in Ca transport. B. With Permeability of Model Systems

The complexity of the composition and organization of the components of biomembranes limits the interpretation of permeability data presented in Section IV, A. Furthermore, the cholesterol content in biomembranes can be changed only to a limited extent. These limitations can be overcome by studies done on liposomes and BLII (Jain, 1972; Sessa and Weissman, 1968). Liposomes may be prepared from phospholipids that contain varying proportions of various sterols. The captured volume of vesicles depends greatly on the excess fixed charge of the phospholipids. This could be due to a flattened or perhaps a biconcave shape of uncharged liposomes. Larger captured volumes in liposomes are critical for permeaability studies as conducted by following the swelling or shrinking rate or the isotope leakage rates (cf. DeGier et al., 1968). From studies on model systems composed of phospholipids passive diffusion through lipid barrier appears to be highly dependent on the degree of packing and of thermal and chemical randomization of the apolar chains. Similar conclusions have been arrived at from the study of biomembranes as discussed earlier. The Permeability of liposomes is reduced more or less in proportion to the amount of cholesterol above 15 mole percent of cholesterol. In liposomes containing 2-10 mole percent cholesterol the permeability for water has been found to increase with increasing cholesterol content (Jain et al., 1973). This behavior is consistent with other observations suggesting that a t lower proportions (up to -10 mole percent) of cholesterol the bilayer thickness decreases (Ladbrooke et al., 196813; Johnson, 1973). Generally speaking, the decrease in the permeability of liposomes containing more than 15 mole percent cholesterol is larger in the case of the smaller permeants. The effect of low proportions of cholesterol is most pronounced in the liposomes of mixed lecithins and in those of egg yolk lecithin; in the dioleoyllecithin liposomes the effect is smaller, but still significant. A higher concentration of cholesterol in dioleoyllecithin liposomes causes their permeability to decrease appreciably (DeGier et al., 1968). The sterol has the greatest effect when liposomes are prepared from lecithins whose molecular area, as determined by monolayer studies (see below), is significantly reduced by cholesterol. Thus 18: 1-18: 1-,

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

19

18:1-18:0-, 18:0-18: 1-, 16:O-18:2-, 18:2-16:O-, 16:O-18:3-, and 16:O18:4-lecithins show a much larger decrease in water and nonelectrolyte permeability than 18:3-18: 3-lecithin liposomes in the presence of cholesterol (Demel et al., 1968, 1972a,b). Diether and diester phosphate lecithins have the same rate of water permeability and are affected by cholesterol to the same extent (Bittman and Blau, 1972). Furthermore, propylphosphonylcholine and butylphosphinylcholine analogs do interact with cholesterol. This interaction is reflected in reduced water permeability. The data collected in Table VI indicate that 3-p-sterols like cholesterol, cholestanol, lathosterol, etc., reduce the permeability of egg-lecithin liposomes for a variety of solutes by a factor of about 3-5. The plant sterols (ergosterol and stigmasterol) also reduce the permeability, but to a somewhat lesser extent than animal sterols (Demel et al., 1972a, b). Thus the presence of the double bond at C-22 and/or the ethyl or methyl groups, respectively, a t C-24 restricts the interaction with the lecithin molecule (see below). The length of the sterol side chain appears to be of critical importance for sterol-lecithin interaction and therefore for the reduction of the permeability properties of liposomes. Similarly, a 3-p-OH and trans A/B ring fusion is necessary not only to reduce permeability, but, as noted earlier, for the incorporation of sterols into liposomes. Thus seemingly subtle modifications a t the 3 position of cholesterol (e.g., 3a-cholesterol and 3p-thiocholesterol) produce marked changes in the initial rates of water permeability. This suggests that strict demands a t the lecithin polar head group must be met in order for the lecithin-cholesterol interaction to occur. Cholesterol decreases permeability independently of the lipid’s surface charge and particuIar head groups. This is true for negatively charged (phosphatidic acid/phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) as well as positively charged (stearylamine/phosphatidylcholine) membranes. The permeability of various liposomes and biomembranes differs significantly, depending on the cholesterol content and the degree of unsaturation of hydrocarbon chains. However, Arrhenius plots show that the activation energy for permeation of chloride* and nonelectrolytes increases only slightly with the degree of unsaturation or the presence of cholesterol in the bilayer (Papahadjopoulos and Watkins, 1967; DeGier et al., 1970; Papahadjopoulos et al., 1971). In contrast, the Arrhenius activation energies for cation diffusion decrease from 27-30 kcal/mole to 13-14 kcal/mole after incorporation of equimolar amounts of cholesterol. * It may be noted t,hat the 36C1 flux through BLM is electrically silent (Pagan0 and Thompson, 1968). This indicates that C1 is diffusing through the membrane in an uncharged state or as an ion-pair (see also Bangham, 1972).

20

MAHENDRA KUMAR JAlN

TABLE VI

PERMEABILITY OF MODELMEMBRANES AS

Membrane composition

A

FUNCTION OF CHOLESTEROL CONTENT

Permeant (maximal permeability change)

Remarks

Egg lecithin (BLILI)

Water (X4)

Permeability decreased monotonically with increasing cholesterol content (Finkelstein and Cass, 1967; Cass and Finkelstein, 1967, 1968)

Egg lecithin (liposomes)

Water (X3.5)

Same as above (Jain et al., 1973)

Brain lipids (BLM)

Nonelectrolytes (X2-4)

Same as above (Bean et al., 1968)

Egg lecithin (liposomes)

Glycerol, erythritol, glucose, inorganic phosphate

Decrease in permeability by cholesterol (Kinsky et al., 1967; DeGier et al., 1968)

Egg lecithin (liposomes)

Glycerol (X6), Rb

3-fi-Sterols reduce membrane permeability. Ergosterol and stigmasterol are less effective. Sterols lacking side chain, or with nonplanar nucleus, or with 3-a-hydroxyl group, or ketosteroids have no significant effect on permeability (Demel et al., 1972b)

Lipids from Achoplasma Eaidlawii grown on sterol-enriched medium (liposomes)

Erythritol ( X 5 ) and glycerol (X.5)

3-a-Cholesterol has little or no effect on permeability (DeKruyff et al., 1972)

Dioleoyl-, l-stearoyl-2oleoyl-, dilinoleoyl-, and egg lecithins

Glucose ( x 2)

Cholesterol reduces permeability except in dilinoleoyl-lecithin liposomes. Cholesterol esters have no effect (Demel et al., 1968)

Brain lipids (BLM)

Indole derivatives

Reduced permeability (Bean et aE., 1968)

ROLE OF CHOLESTEROL IN BIOMEMBRANESAND RELATED SYSTEMS

21

TABLE VI (Continued)

Membrane composition

Permanent (maximal permeability change)

Remarks

Phosphatidylcholine, Na and, K ( X4-18) phosphatidylserine, C1 (X2), glucose (X2) phosphatidylglycerol, and other lipids (BLM and sonicated single-layered vesicles)

Free energy for permeation is about 20-23.7 kcal for all solutes and is little affected by cholesterol (Papahadjopoulos and Watkins, 1967;Papahadjopoulos et al., 1971). For cations Arrhenius E, is considerably reduced by incorporation of cholesterol. E, for C1- or glucose is not affected significantly

Phosphatidylcholine (liposomes)

Cholesterol reduces the rate (Vanderkooi and Martonosi, 1971)

Ca2+

This could be due to temperature-dependent structural changes in the phospholipid bilayer and is probably reflected in a nonlinear graph of permeability vs 1/T. Cholesterol would tend to inhibit such temperaturedependent structural changes. Consequently the activation energy obtained from the same plot in the presence of cholesterol is substantially lower, and perhaps much closer to its true value. This would imply that these structural changes do not have an appreciable effect on chloride or nonelectrolyte diffusion. This difference in permeability following incorporation of cholesterol is presumably due to the fact that these mixed membranes do not show phase-transition characteristics. The evidence presented thus far suggests that the sterol promotes a tighter packing of the phospholipid in the bilayer and thereby increases the effective viscosity or partition characteristics of the apolar core of the bilayer. These conclusions are compatible with a cholesterol-produced decrease in molecular motion and an increase in packing of chains (see below). However, permeability decreases by a factor of 4-18 for cations and other small solutes like water, glycerol, etc. (cf. Table VI), but b y a factor of only 2 in the case of glucose and chloride in membranes containing cholesterol. This may imply that cholesterol decreases the “dissolution” of the solute into the membrane more than the diffusion of the solute through the membrane matrix. If diffusion through the membrane matrix

22

MAHENDRA KUMAR JAlN

were to be critically affected by the introduction of Cholesterol, one would expect a larger decrease in the permeability coefficient of the larger molecules, e.g. glucose, than in that of the cations (Papahadjopoulos et al., 1971; Papahadjopoulos and Watkins, 1967). In terms of the molecular theory for diffusion of solutes across bilayer (Trauble, 1971b), the cholesterol-induced reduction in permeability would imply a reduction in the number of “kinks” availabile in the bilayer. A reduction in the number of kinks or “the mobile structural defects” in the polymethylene chains is consistent with the hypothesis that the polymethylene chains become more rigid in the presence of cholesterol. C. With Electrical Properties of Bilayer

Anisotropic orientation of molecules that form a lamellar bilayer, such as BLM, and the consequent requirement for an overall cohesion pressure resulting from dispersion forces may imply that the physical state of the lipid molecules ranges between fairly narrow limits. As the hydrocarbon core has a relatively coherent structure, it will define the basic level of dielectric and associated properties such as electrical capacity and conductivity, and refractive index. These characteristics are likely to be affected both by the composition of the bilayer by the aqueous environment. I n BLM prepared from phosphatidylserine (with EDTA in the medium) and phosphatidylcholine (Hanai et al., 1965; Ohki, 1969: Papahadjopoulos et al., 1971) the membrane capacitance varies significantly as the cholesterol content of the BLM-forming solution is increased. Thus capacitance increases monotonically from 0.38 to 0.6 pF as cholesterol concentration increases from 0 to 65%. I n the absence of EDTA, phosphatidylserine membranes respond quite differently to cholesterol; there is very little increase in capacitance up to 50 mole percent cholesterol concentration, with a rapid increase a: higher concentrations. Temperature has no measurable effect on capacity of BLM prepared from lecithin in decane (Redwood and Haydon, 1969). When cholesterol was added to the system, capacity decreased by 13% for every 10°C increase in temperature. This difference has been interpreted as resulting from the steric interactions between lecithin and cholesterol which are temperature sensitive; however, the interchain interactions between lecithin molecules are not temperature sensitive (White, 1970). These observations imply that either the dielectric constant of the hydrocarbon core in the membrane is different from that of the BLM without cholesterol, or that the effective dielectric thickness of the hydrocarbon region is less than its physical dimension. From X-ray diffraction data it

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

23

appears that phospholipid bilayer membranes that contain about 10 mole percent cholesterol are thicker a t room temperature than pure phospholipid membranes (Rand and Luzzati, 1968; Lecuyer and Dervichian, 1969; see also below). A possible explanation of increased capacitance with increasing cholesterol content would be that there is a limited penetration of water into the hydrocarbon region. This is aided by the presence of cholesterol and fluctuations of the free volume in their vicinity which may extend perhaps as far as the first 1-2 methylene residues. The penetration of water might go as far as 2-3 8 beyond the plane of the glycerols. This would reduce the dielectric thickness by 4-6 8, to a value sufficient to account for the observed capacities. The maximal dc resistance of phosphatidylcholine BLM containing 60% cholesterol is approximately 10 times higher than that for pure phosphatidylcholine BLM. The resistance decreases monotonically as the cholesterol content increases from 0 to 70 mole percent. Stable BLM cannot be prepared from a solution containing more than 80% cholesterol. BLhl from phosphatidylserine cholesterol solution behave similarly to Iecithin-BLM only in the presence of 10 pM EDTA. However, in the absence of EDTA, addition of cholesterol to PS containing BLM has a small effect, usually decreasing the resistance (Papahadjopoulos et al., 1971).

+

V. STATE OF CHOLESTEROL IN ORGANIZED LIPID AGGREGATES A. In Monolayers

The observations summarized in the preceding sections suggest that the addition of cholesterol to phospholipids modifies their organizational characteristics in aqueous suspensions. The membranes formed from mixed lipids appear t o be more condensed than those made from pure phospholipids which form bilayers. Studies with mixed monolayers have demonstrated that the presence of cholesterol reduces the area occupied by phospholipid molecules. However, the degree of condensation varies with the liquidity of the phospholipid films. It is observable only with films that are neither fully condensed nor fully expanded (see Shah, 1970b, for a review of the technique). Measurements made on maximally condensed monolayers 2f cholesterol have shown that each molecule occupies an area of about 36 A2. Similarly, the area of egg phosphatidylcholine molecule appears to be about 63 bz. However, monolayers prepared from a mixture (equimolar) of cholesterol and phosphatidylcholine can be compressed until the sum of areas oc-

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cupied by one molecule of lecithin and one molecule of cholesterol is 8 2 i 2 , that is 25% less than the sum of individual molecular areas (Rand and Luzzati, 1968; Lecuyer and Dervichian, 1969). Ever since Leathes (1925a, b) first observed the condensing effect of cholesterol on lipid monolayers, several other authors have observed similar phenomena in mixed monolayers made up of cholesterol and fatty acids (Dervichian, 1964; Adam and Jessop, 1928), in saturated lecithins a t surface pressure below 30 dynes. cm-l (Shah and Schulman, 1967; Demel et al., 1967), in 1 ,2-dielaidoyl-(rac)-phosphatidylethanolaminebelow 15 dynes cm-' (Chapman et al., 1966), in egg lecithin (DeBernard, 1958; Shah and Schulman, 1965) and dimyristin (Cadenhead and Phillips, 1968). A necessary but not sufficient condition for this condensation effect is that the pure lecithin must exhibit an expanded pressure-area curve a t the air-water interface. In all these cases cholesterol decreases the surface area of phospholipid to a value below the area of the phospholipid molecule a t the collapse pressure. However, there appears to be considerable specificity with regard to the nature of lipid chains and sterol (see below). The surface potential of mixed cholesterol plus dicetylphosphate monolayer also deviates from the sum of area of individual molecules. The division has been found to be smaller in the presence of calcium chloride in the aqueous medium. Such observations indicate the existence of an ion-dipole interaction between a hydroxyl group of cholesterol and phosphate group. In turn, this changes the average dipole per molecule (see also Shah and Schulman, 1967). Surface potential of lecithin-cholesterol monolayers does not deviate from additivity. The reduction in average area per molecule (condensation) is attributed to an interaction between the components in the mixed monolayer, or to steric factors which may govern organization and packing of molecules in a two-dimensional matrix. Thus the stoichiometry for observed maximal effect does not necessarily imply complex formation between Iipid and cholesterol, but may represent an optimum geometrical packing arrangement. The condensing effect may be explained by a consideration of molecular cavities or vacancies which are caused by the thermal motion of polymethylene chains. The size of these cavities is influenced by the length and degree of saturation of the fatty acyl chains and by the extent of compression of the monolayer. Various types of interactions that occur in mixed monolayers are reflected in surface pressure, surface potential, surface fluidity, or surface dipole moment (cf. Shah, 1970b). Interestingly, all these parameters do not necessarily change simultaneously. I n fact, several examples are known in which only surface potential or surface area changes. The maximum deviations from the expected additive values of surface area in mixed films is usually found a t 0.4 to 0.66 mole fraction of lecithin.

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25

Such a reduction of area in mixed films could be ascribed to purely geometrical packing factors (Shah, 1971; however, see Weiner and Felmeister, 1970). Cholesterol does not always produce a significant condensation. I n fact, the extent of area reduction is markedly influenced by the type of fatty acids present in the phospholipid (Demel et al., 1967; Ghosh and Tinoco, 1972). It is also a function of the nature of the polar groups and of other variables such as pH, temperature, and the kind of divalent ions present (Chapman et al., 1966; Shah and Schulman, 1967; Rand and Luzzati, 1968; Lecuyer and Dervichian, 1969; Demel, 1968; Demel and Joos, 1968; Joos and Demel, 1969; Standish and Pethica, 1967). Systematic studies have been carried out to work out the features of the molecular structure of the phospholipid molecule that may be responsible for interaction with cholesterol a t the air-water interface. Studies on surface properties of individual molecular species of lecithin indicate that cholesterol would condense in monolayer with lecithins containing one double bond peracyl chain and that those lecithins with more than one double bond per chain would not condense (Van Dernen, 1966; 1972; Demel et al., 1972~).So far, the only long-chain fatty acid lecithins that have shown condensation with cholesterol in monolayers have been the naturally occurring structures, namely, 18:O-18: 1-, 16:O-16:O-, 16:O18:2-, 16:0-18:3-, 16:0-20:4-, 18:0-18 :2-, 18:2-16:0-, 16 :0-22:6lecithins (Demel et al., 1967, 1972c; Chapman et al., 196913). Lecithins with two or more double bonds per chain would condense with cholesterol provided the unsaturated chain was in the 2-position. This occurs in nature (Tinoco and XlcIntosh, 1970; Ghosh et al., 1971, 1973; Demel et al., 1972~) .The unnatural or extremely rare 18 :0-18 :0-, 18:2-18 :2-, 18:3-18: 3-, and 18:2-18 :0-lecithins show little or no interaction with cholesterol. However, Demel et al. (1972~)have reported that both structural isomers, 16:0-18:2- and 18:2-16:0-lecithins, show striking reductions in mean molecular area after mixing with cholesterol. Also for 16:O-20:6lecithin, a reduction of the mean molecular area with cholesterol is found only a t room temperature. At physiological temperature no deviation from additivity is observed; 18 :2-18 :2- and 18:3-18: 3-lecithins do not show any interaction even at room temperature. Interestingly enough, mixed monolayers of 18:0-20: 3-phosphatidylethanolamine and cholesterol follow the additivity rule a t 20°C but not a t 34°C (Chapman et al., 1966). These data suggest that the 1-saturated-2-unsaturated lecithins, the most abundant species in nature, interact with cholesterol. This interaction might be important for their function in vivo. Furthermore, the temperature dependence of interaction of highly unsaturated lecithins with cholesterol may reflect the half-life of these complexes. The results relating lipid specificity in lecithin-cholesterol interaction

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in monolayers can be interpreted in purely steric and geometrical arrangement of these molecules for packing in mixed monolayer. I n a very expanded fiIm such as that produced by 16:&18:3-lecithin, there must be much unoccupied space since the fatty acid chains themselves occupy no more volume than those of 18: 18:O-lecithin which does not condense with cholesterol. The shape of the unoccupied space is the determining factor. Cholesterol can fit into part of this space if the lecithin has the l-saturated%unsaturated structure, but apparently it cannot fit as well if the 1position is occupied by an unsaturated fatty acid such as 18:2. It is also possible that various lecithins that differ in substituents a t the 1- and 2positions do not form a 2-dimensional matrix in which all the molecules are oriented identically. Thus the position of an unsaturated chain on the glycerol moiety as well as the position of the double bond in a given chain is of critical importance in determining the interaction of lecithin with cholesterol. Indeed, studies on correlation of condensing effect of chollesterol on lecithin also show that the effect is strongest with those molecules that have a segment of saturated hydrocarbon chain extending nine or more carbons from the carboxyl end (Ghosh et al., 1973). (For effect of polar groups on lipid cholesterol interaction, see DeKruyff et al., 1973b.) When the fatty acyl chains of lecithin are shorter (e.g., 10: 10-lecithin), the intermolecular cavity is smaller than that of the cholesterol molecule. Hence this type of mixed monolayer follows the additivity rule. The various geometrical constraints that apply to the organization of lecithin molecules in monolayers are also not reflected in a possible asymmetry inherent in D- and L-lecithins, both of which behave in a very similar fashion (Ghosh et al., 1971). Sterols interact not only with lecithins in monolayers, but also with a variety of other amphipaths (Abramson and Katzman, 1968; Colaccico and Rapport, 1968; Novak and Swift, 1972; Tinsley et al., 1971). One of the theories of action of steroid hormones postulates that after interaction with the hormone the structure of the cell membrane is altered so as to affect the specific permeability of the metabolites (Willmer, 1961). On the basis of monolayer studies it appears that steroids do not strongly associate with lipid films (Gershfeld and Heftman, 1963) even though steroids affect the hydration layer (Pak and Gershfeld, 1967), or markedly alter the mechanical properties of lipid films (Gershfeld and Pak, 1968) of the monolayers. Interaction of progesterone, testosterone, androsterone, and etiocholanone with insoluble lipid films of cholesterol and long-chain alcohols, amines, esters, etc., has been studied by surface pressure, surface potential, and radiotracer measurements. I n general steroids form mixed films but compression of the insoluble films to their most condensed state leads to complete ejection of the absorbed steroid in all cases except mixed

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

27

amine films where a small amount of steroid is still retained (Gershfeld and JIuramatsu, 1971). All these studies were conducted on monolayers of the amphipaths, which have little or no structural resemblance to lipids found in biomembranes. Nonetheless, one may conclude that the steroid-lipid interaction is nonspecific, even though structural changes depend on the chemical nature of the lipid film. These conclusions are in general accord with results obtained from steroid hormone-phospholipid interactions in liposomes (Bangham et al., 1965). The interest in this system is enhanced by recent experiments which indicate that the activity of steroid hormones is connected with the interaction of these molecules with cell membranes. For example, i t has been shown that steroid hormones play a role in the interaction of ribosomes with endoplasmic membranes (Sunshine et al., 1971; Blyth et al., 1971). Mixed monolayers of lecithin with several steroids exhibit deviations from additivity rule for the molecular area (Snart, 1967b). Mixed monolayers of cholesterol and lecithin interact with aromatic hydrocarbons and contribute to the area of the monolayer at mole ratios of lecithin to hydrocarbon below 3:l and a t cholesterol to hydrocarbon mole ratios of 1:l. This may be explained in terms of hydrocarbon solubility in a two-dimensional solution. Similarly, when OsOl interacts with 1:1 mixed monolayers of lecithin and either cholesterol, retinal, or tocopherol, egg lecithin is stabilized by the presence of these additives in the monolayers (Dreher et al., 1967; see also Shah, 1970a). Demel et al. (1972~)and Gnosh and Tinoco (1972) have studied the effect of variation in sterol structure on interaction with various lecithins. I n general, cholesterol and 0-sitosterol give greater condensation than do ergosterol and dihydrocholesterol. The planar nucleus and a 3-P-OH group are important factors in the interaction between lecithins and sterols in monolayers. Side chains have only a small, but noticeable, effect under similar conditions. Thus 5-a-androstan-3-0-01 produces smaller condensation than that produced by cholesterol and 0-sitosterol and resembles dihydrocholesterol or ergosterol more closely. Several autoxidation products of cholesterol also show marked condensation effects, even though these derivatives yield films that are more expanded than the cholesterol film (Kame1 et al., 1971a,b). However, if cholesterol in a biomembrane is subjected to air oxidation, the permeability characteristics and other properties of the membrane may not be altered significantly. B. In Bilayers

Evidence for the interaction of cholesterol with phospholipids to modify a bilayer has come from a variety of sources. Studies on sonicated disper-

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sions of phospholipid in water indicate that the dispersions containing cholesterol are heavier, more asymmetrical, less hydrated, and more rigid (Horwita et al., 1972; Johnson and Buttress, 1973). These changes are reflected in various properties of the liposomes as shown in Fig. 1 (also see Cherry et al., 1971; Green et al., 1971; Parsons and Akers, 1969; Trauble and Haynes, 1971). The effect of cholesterol on phospholipid dispersions in benzene is not so significant. However, available evidence does suggest that cholesterol is incorporated into the phospholipid aggregates (Janson et al., 1973). The evidence concerning possible interaction of cholesterol with phospholipids in the aqueous phase has come, however, from the use of physical techniques as described below. The membranes of many organisms show a thermotropic order-disorder transition in which the acyl chains of their membrane lipids are transformed from an ordered gel-like state to an ordered liquidlike state. This transition, which has been detected by calorimetry, NMR, ESR, and X-ray diffraction, has nearly the same enthalpy and occurs at approximately the same temperature in both intact membranes and protein-free membrane lipid dispersions (liposomes). Studies with pure phospholipids using a variety of physical techniques have shown that each phospholipid, on heating, exhibits a transition from a crystalline to a liquid crystalline phase. The transition temperature depends upon the type of fatty-acid chains in the lipid. The addition of water to a given phospholipid lowers this transition temperature until it reaches a characteristic limiting value at a concentration that corresponds to the maximum uptake of bound water by the phospholipid dispersion (Chapman et al., 1968). The ability to form smectic mesophases and to disperse lipids occurs above this characteristic temperature (cf. Fig. 1). The temperature at which the phase transitions occur are a function of the head group, the hydrocarbon chain length, the degree and type of unsaturation, and the nature of various additives (cf. Oldfield and Chapman, 1972b). Direct evidence for cholesterol-induced conformational change in phospholipid head group has been derived from infrared studies (Verma and Wallach, 1973). Cholesterol in dehydrated form shows a small endotherm at 35-40°C, which is not seen in hydrated samples (Mufson et al., 1972b). The biological significance of this endotherm is, however, not clear. I n contrast, DSC measurements show that cholesterol has dramatic effects on the phase transition characteristic of lecithins (Ladbrooke et al., 1968a, b; Ladbrooke and Chapman, 1969). The DSC curve for 1,2-dipalrnitoyl-~lecithin shows a sharp endothermic transition between 39.5 and 40.5% As the concentration of cholesterol is increased in the lecithin-water phase, the transition becomes broad and the transition temperature decreases and reaches a value of about 30" at a cholesteterol concentration of about 30 mole percent. When the concentration of cholesterol reaches 50 mole

I

0

1

a2

I

1

0.4

I

1

0.6

I

I

I

0.8

1.0

Cholesterol (mole fraction)

FIG.1. Various membrane-associated phenomena as a function of mole fraction of cholesterol in bilayers of different forms. Curve a: Transition temperature for hydrated dipalmitoyllecithin (DPL); 1 division (div.) = 3°C (range 40-30°C). Curve b: Heat absorbed during above transition; 1 div. = 3 cal (range 3-12 cal/g lipid). Curve c: Heat absorbed in the ice transition a t 0°C for the above transition; 1div. = 3 cal (range 20-40 cal per gram of total mixture). Curve d: X-ray long spacing a t 25°C for hydrated DPL; 1 div. = 30 A (range 60-83 A). Curve e: Permeability of egg lecithin liposomes for water; 1 div. = 10-3 (range 0.8-2.9 x 10-3 cm sec-1). Curve f : Permeability of egg (range 0 . 9 4 . 2 X lecithin black lipid membrane (BLM) for water; 1 div. = 3 X cm sec-I). Curve g: Maximum glucose released from egg lecithin liposomes after 1 hour a t 41OC; 1 div. = 1.5% (range 15-36%). Curve h: Molecular areas in mixed film! of cholesterol and 18:&18:4 lecithin at 5 dynes em-'; 1 div. = 30 (range 1 2 0 4 0 A*). Curve i : Hyperfine splitting in multibilayers of phosphatidylethanolamine (PE) ; 1 div. = 1.5 gauss (range 6.75-8.4 gauss). Curve j : Ratio of the low field peak-height to center peak height in multibilayers of P E ; 1 div. = 0.3 (range 0.79-0.98). Curve k: Electrical resistance of egg lecithin BLM, 1 div = x1.5 (range 7 to 17 X lo6Qcm2). Curve 1: Electrical capacitance of egg lecithin BLM; 1 div. = 0.15 pF ern+ (range 0.39-0.60 PF cm+). Curve m: Michaelis-Menten rate constant Vmsxfor the action of phospholipase A (bee venom) on egg lecithin; 1div. = X 2 (range 800-2100 &f/mg/min).

Az

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percent, no endothermic peak is observed (cf. Fig. 1). Upon addition of androstanol the transition temperature decreases only a little. However, at a lower temperature there occurs an additional pretransition (Ladbrooke and Chapman, 1969). These results can be best rationalized if it is assumed that after cholesterol becomes incorporated there ensues a condition of “intermediate disorganization.” Below the phase transition temperature, T,, the chains are less organized in the presence than in the absence of cholesterol. The opposite is true above T , when the steroid nucleus effectively prevents intermolecular interaction of the lipid hydrocarbon chains. Below T,, the presence of the sterol prevents the chains from crystallizing into the gel state. Although the transition from a gel to a liquid crystalline state is not observed with the aid of the DSC in the presence of 50 mole percent of cholesterol, laser Raman spectroscopic evidence indicates that a transition still takes place over a wide temperature range (Lippert and Peticolas, 1971). The disappearance of phase transition in hydrocarbon chains following incorporation of cholesterol into the lecithin bilayer also is accompanied by a change in the polar group region of the bilayer. The heat absorbed in the ice transition a t O’C, for example, is at a minimum at 50 mole percent cholesterol. This would indicate that the bound water is a t a maximum a t this composition (Ladbrooke et al., 1968b). Similarly, changes in the dissociation of polar groups of phospholipids has been noted following incorporation of cholesterol. Titration of sphingomyelin or lecithin alone shows very little reaction with protons in the acid range down the p H 3.5. However, mixtures of sphingomyelin and sulfatide or lecithin and sulfatide show the presence of a weak acid group not found in any of these lipids alone. The titration curves of these mixed lipids closely resemble that of a phospholipid containing a monoprotic phosphate group such as phosphatidylinositol. This presumably is due to the fact that the strong acid group of the sulfatide is able to form an ionic bond with the positively charged trimethylaniino group of the neighboring molecule of sphingomyelin or lecithin. As a result, the phosphate of PC is released and reacts as a weaker acid, uniting with protons t o a greater extent than when in the zwitterionic structure. Incorporation of cholesterol in 1 :1 sulfatide sphingomyelin suppressed some of the phosphate groups in titration curves (Abramson and Katzman, 1968). On increasing the mole percent of cholesterol in the mixture, the fraction of the total titrable phosphate decreases until a maximum of 50 mole percent cholesterol is incorporated. It may be reasoned that the cholesterol occupies spaces between the other two lipids, and with increasing content of cholesterol the average distance between the other two lipids increases. The ionic interaction of the sulfatide with the quarternary amine is decreased and the availability of the phosphate groups for titration is lessened.

+

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31

The disappearance of a sharp, gel-liquid crystalline transition in the presence of cholesterol has also been observed for sphingomyelin, cerebrosides (Clowes e2 al., 1971; Oldfield and Chapman, 1972a; see also Mickel and Hill, 1972), and Achoplasma laidlawii membrane lipids (DeKruyff et al., 1972). With A . laidlawii membrane lipids epicholesterol, 5a-androstan-3/3-01, and choleste-4,6-dien-3-one did not reduce the height or width of the endothermic peak to the same extent as cholesterol did. These results recall the effect of these steroids in reducing permeability of liposomes. However, the solubilization of sterols in the lecithin bilayer is dissimilar (cf. Table 111, see also below). It is particularly interesting to note that cholesterol enhances the rate of water permeability of liposomes derived from saturated lecithins below their transition temperatures (Jain, e2 al., 1973; Bittman and Blau, 1972). The rate of phase change in dipalmitoyllecithin has been investigated by a temperature-jump method (Trauble, 1971a). The relaxation time is markedly reduced by the presence of cholesterol in the lipid phase and further reduced when Caz+ is present in the aqueous solution. This may represent a new role for cholesterol, namely, one of enabling the membrane lipids to undergo a rapid phase change. Thus, below the phase transition temperature of the phospholipid, cholesterol would appear to decrease viscosity or the order of the hydrocarbon chains in the bilayer. Such a disordering effect is probably best observed when liposomes are subjected to freeze-fracturing, as cholesterol affects the fracture pattern of specimens (Verkleij et al., 1972). Above the phase transition temperature the fracture faces consistently appear to be smooth, but below it the bands become less clear with increasing cholesterol concentration and disappear when more than 20 mole percent of the cholesterol has bccome incorporated into lipid. These results indicate that cholesterol preferentially interacts with lipids in liquid crystalline state. Consistent with this hypothesis is the effect of cholesterol upon the crystalline 4 liquid crystalline phase transitions in a codispersion of 18: 1, 18: l-lecithin and 18:0, 18:O-lecithin (DeKruyff et al., 197313). At low concentrations (less than 25 mole percent) cholesterol preferentially associates with 18:1-18 :l-lecithin. At higher concentrations cholesterol interacts with 18 :0-18 :O-lecithin as well. Above the phase transition temperature of phospholipid, the incorporation of cholesterol has a ‘Lcondensing’’effect, i.e., the core of mixed bilayers seems to have a higher viscosity (Darke et al., 1971). Interestingly, in the mixed micelles of detergents with cetyl alcohol or cholesterol the microscopic viscosity of the interior is strikingly increased as measured by fluoresence depolarization (Shinitzky et at., 1971). The interior of a CTAB micelle has viscosity and fusion activation energy of 0.19 poise and 9.6 kcal/mole, respectively. Addition of 5 mM cholesterol changes the re-

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spective values to 3.57 poises and 5.1 kcal/niole. The results suggest that the micelle interior is quite liquidlike and that in the absence of any additive the fluorescence label in a CTAB micelle is partially exposed to the effect of the charge as judged by the high fusion activation energy. Addition of the apolar alcohols, like cholesterol, exposes the label to a more hydrocarbon-like environment of much higher viscosity. The methylene chains in bilayer are highly mobile above the phase transition temperature (Van Putte et al., 1968; Darke et al., 1971; Oldfield and Chapman, 1972a), as determined by the sharpness of the corresponding NMR signals. Following incorporation of cholesterol, there is a decrease in the intensity and a broadening of the chain proton signals in lecithin sphingomyelin and cerebrosides. Below the phase transition temperature, however, the spectrum of mixed liposomes is rather similar to that obtained above the phase transition temperature. Under these conditions the membrane seems to be fluidized by the incorporation of cholesterol. Interaction of cholesterol with sphingomyelin and cerebroside, the major components of myelin which have phase transition temperatures of 32-48' and 60-67"C, respectively, leads t o a "fluidized" membrane. It is this fluidization that provides the basis for an important biological role of cholesterol. The results also suggest that in the physiological tempcrature range the hydrocarbon chains in the presence of cholesterol are in a state of conformational restriction that is intermediate to that prevailing in the gel and liquid crystal phases. Nuclear magnetic resonance spectra of various biological as well as model bilayer systems are diffuse and their lines are broad (Chapman and Penkett, 1966; Chapman et al., 1968; Lee et al., 1972). However proton(Finer et aE., 1971; Finer, 1972) and 13C-NMR(Sears, 1971; lVIetcalfe et aE., 1972) spectra of sonicated lecithin are sharp and well resolved. However, in all these cases above the phase transition temperature one observes significant line broadening for the nuclei located between the polar interface and first few (8-10) methylene residues. Furthermore, cooling the lecithin-cholesterol system below the T o for the lipid alone results in a slight decrease in mobility as detected by quadrupole splitting of deuterium nuclei in perdeuterated lecithin (Oldfield et al., 1971). There is in fact a dependence of width of the hydrocarbon proton resonances on frequency, which, it is conjectured, arises from anisotropic modes of motion rather than adventitious effects like field inhomogeneity. The narrow lines are consistent with the relatively small size of the sonicated vesicles, which tumble sufficiently rapidly t o give rise to averaged dipolar interactions. I n these sonicated particles, a sufficient concentration of the steroid completely represses the transition to the liquidlike state that normally occurs on raising the temperature. From the relative effects on the various lecithin

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

33

proton resonances it is inferred that only part of the lecithin molecule makes contact with cholesterol (Darke et al., 1972). Whereas the hydroxylic region of the lecithin displays sharpening and thus increased mobility, the region giving rise to the NMe3signals is broadened. This is consistent with the view that cholesterol and lecithin are hydrogen bonded. Furthermore, when the mole proportion of cholesterol is appreciably less than one, the NMR spectra give evidence that part of the phospholipid exists in complexed, and the rest in uncomplexed, form. Such clusters could also exist in biomembranes. That cholesterol brings about ordering, condensing, as well as fluidizing of membranes has been deduced from results obtained also with other techniques, especially fluorescence and electron spin labels. Fluorescence of various probes [like retinol, 1-anilinonapththalene-8-sulfonate(ANS)] is enhanced in the presence of sonicated lipid dispersions. A decrease in rotational mobilities of these probes is also noted, as evidenced by the increase in the apparent period of rotation obtained from fluorescence polarization and lifetime measurements. I n the presence of cholesterol the fluorescence intensity of retinol (Radda, 1971; Sackman and Trauble, 1972a, b ; Papahadjopoulos et al., 1973b; Cogan et al., 1973; Badley et al., 1971; Trauble, 1971a) in the dispersion is further enhanced. This enhancement must be attributed to increased environmental constraints around the probe molecule. I n contrast, ANS shows a decrease in fluorescence intensity in cholesterol-containing liposomes. The increased fluorescence of the uncharged probe is attributed to an increase in the “rigidity” of the hydrocarbon interior of the bilayer, while the decrease in ANS fluorescence probably means that the ‘(interface” region of the dispersion has been opened up, allowing an effectively better penetration of water molecules to the probe binding site. A nitroxide label attached to a variety of molecules that are incorporated into a bilayer has been used to explore membrane organization to a depth of approximately 15 A (see Smith, 1971, for a review; Lapper et al., 1972; Libertini et al., 1969). ESR spectra are a function of the orientation of the nitroxide label with respect to the film and magnetic field and depend upon the tumbling rate. A cautionary note may be in order, however. Lack of knowledge of precise localization of the probe in the membrane plane, preferential localization of the probe into a more fluid domain, and perturbation of the local environment by the probe are some of the weaknesses of this technique. It may also be noted that 3-nitroxide of cholestane, a spin label used as probe for bilayers, does not show any condensing effect with lecithin monolapers. Nevertheless, control experiments and results obtained with other techniques have qualitatively confirmed the ESR results.

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The influence of cholesterol on the organization of egg lecithin liposomes has been studied by Hsia et al. (1970a, 1971; Hsia and Boggs, 1972). They used a stearamide label in which the nitroxide group is located a t the polar end of the long chain. This provides the possibility of independent motion relative to the chain. They also used a cholestane nitroxide in which the nitroxide group is locked onto a steroid backbone. Dramatically different effects of cholesterol were observed for the two labels. With the cholestane spin label the presence of cholesterol led to reduced mobility of the label. With the stearamide spin label, not only were the ESR lines broader in the presence of cholesterol, but the relative widths of the low field and center lines were reversed. This latter effect could have resulted from the spin label changing its location to a region of asymmetric polarity. An explanation for the absence of this ESR spectral effect with the cholestane spin label probably lies in the inability of its nitroxide moiety to move independently of the lipid backbone. Thus the entire steroid must be moved if the nitroxide is to sample a region of different polarity. A variety of agents, including cholesterol, has been found to have a significant effect on the “solubility” of small labels in liposome. Thus, butanethiol, octylamine, and tetracaine increase the solubility of TEMPO and other labels, whereas cholesterol in liposomes or Ca in the aqueous medium reduces the solubility. Presumably the latter effect is due to the ordering of the lecithin molecules (Hubbell and McConnell, 1968; Rutler et al., 1970a). Addition of cholesterol to dioleoyllecithin containing dinitrophenylnitroxide also causes a reduction in spin label mobility (Rarratt et al., 1969). It is particularly interesting that the rigid spin labels (such as the oxazolidone derivative of 5a-androstan-3-one), when incorporated into oriented lipid membranes, exhibit rotational motion preferentially about their long axis, whereas the same label without the OH group shows little or no anisotropy. Since the five-membered nitroxide label ring of the label is rigidly linked to the steroid nucleus, a rotational motion of the label indicates that the entire molecule undergoes rotational motion. This motion may be correlated directly with the environmental viscosity (to a depth of 12 A from the interface). Indeed it has been demonstrated that the correlation time T , characteristic of the tumbling motion of the steroid label in dipalmitoyllecithin bilayers, is 10-8 second a t 20°C. When the sample is heated above the phase transition temperature, the value decreases by a factor of 3-4 (Sackman and Trauble, 1972a, b ; Trauble and Sackman, 1972). The ESR spectra suggest that the organization of the androstane-lecithin membranes depends critically upon the ratio of label to lecithin. When the molar ratio exceeds 0.03, the steroid molecules below the phase transition temperature start to aggregate into closely packed submicroscopic clusters. The size of clusters increases with increasing label

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

35

concentration, whereas the density of the clusters is independent of the molar proportion of sterols. Above the phase transition temperature, the solubility of steroid molecules is considerably larger. Up to fairly high concentrations (0.1 mole fraction), the system consists of a homogeneous mixture of steroid, label, and lecithin molecules (Devaux and McConnell, 1972; Shimshick and McConnell, 1973). The hopping frequency for the steroid molecules within the lipid membrane is calculated to be about 3X106 sec-'. A useful model membrane system consists of stacks of phospholipid bilayers deposited on a flat plate. This system is similar to liposomes in many ways but has the advantage of lamellar symmetry. I n dry egg lecithin film the cholestane label undergoes isotropic rotational diffusion, characterized by a correlation time of 10-8 second. This indicates that the immediate environment of the label is liquidlike and that the label has no preferred orientation (Hsia et al., 1970b; Boggs and Hsia, 1972; Long et al., 1970). Exposure of the film to an aqueous salt solution resulted in orientation of the spin label and led to angular-dependent spectra. The maximum and minimum hyperfine splitting observed were 16.5 Gauss (G) and 9.5 G, respectively. These deviate from the values of 32 and 6 G expected for complete immobilization and orientation of the label. They also deviate for 19 and 6 G expected for complete orientation of the long axis of the cholestane label, with rapid rotation about the axis to average A,, and A,, [1/2 (Axx Azz) = 19 GI. When the lecithin films contained cholesterol, however, the behavior was entirely different. For a dry film containing cholesterol a t lecithin: cholesterol ratios of 1 :2, maximum and minimum splittings of 32 and 6 G were observed. This indicated perfect orientation of the spin label and suggested a rigid and highly organized structure for the film. After hydration the mixed lipid film gave splittings of 19 and 6.5 G, respectively. This demonstrated that in the presence of an aqueous phase the cholesterol-containing films are still highly organized. As a result the long axis of the cholestane spin label is oriented perpendicularly to the plane of the film, but remains sufflciently fluid so that orientation about the long steroid axis of the label can take place a t a rate greater than the frequency difference between A,, and A,,, i.e. > 73 MHz. The same effect occurs a t much lower cholesterol: lecithin ratios. The ordering effect has been analyzed in terms of the spin labels being oriented about a cone, the axis of which is perpendicular to the plane of the film. Increasing the cholesterol content decreases the solid angle contained by the cone. The highest degree of orientation was observed a t 25 mole percent cholesterol. This corresponds to a deviation of the long axis of the spin label from the perpendicular by 10 =t3". Between 25 and 50 mole percent the degree of order is constant (Lapper et al., 1972; Schreier-Muccillo et al., 1973;

+

36

MAHENDRA KUMAR JAlN

Long et al., 1971). The structural features of the steroid that can effect order in spin-labeled multibilayers are: a planar nucleus, a 3p-OH group, and a tail a t C-17 (Butler et al., 1970b). Sterols in the bilayer also affect the mobility gradient along the hydrocarbon chain. When spin-labeled fatty acids or lecithins are incorporated into egg lecithin (EL) bilayers, the decrease in order parameters S ( = 0 for an isotropic liquid and =1 for a perfect crystalline solid) is greater than logarithmic as the number of methylene groups separating the label from the polar head group increases. The data can be reconciled by assuming that a net tilt of 30" is present in the head group region. In the presence of cholesterol (EL:cholesterol, 2:1), it has been found that the first eight carbon atoms from the bilayer surface can be thought of as a rigid rod, with the remaining carbons greatly increasing their motion toward the center of the bilayer (Hubbell and XcConnell, 1971; Oldfield and Chapman, 1971; Seeling, 1971). I n dry egg lecithin film the cholesterol label undergoes isotropic rotational diffusion characterized by a correlation time of 10-8 second. Thus the immediate environment of the label appears to be liquidlike. I n hydrated lecithin film, the steroid label is oriented with its long axis approximately perpendicular (24" tilt) to the plane of the film, with the label undergoing rapid rotational reorientation about the long steroid axis at a rate of about lo8 sec-' (Hsia et al., 1970a). I n dry lecithin cholesterol films there is a considerable degree of spectral anisotropy. The long axis of the steroid label is perpendicular to the plane of the dried film, with no motion about the long axis of the label. In hydrated lecithin cholesterol films the steroid label has rotational diffusion about the long axis a t a rate of about lo8 sec-'. There appears to be almost perfect orientation of the label: the long axis is perpendicular to the plane of the multilayer film (Lapper et al., 1972). Thus cholesterol has a significant effect on the orientation of chains in the bilayer. Large amounts of cholesterol cause a stiffening of the hydrocarbon chains. Hydration of mixed lipid film induces a high degree of order, with a spacing between the hydrocarbon chains sufficiently large to allow rotation of the steroid label about its long axis. Similar conclusions have also been arrived a t from the study of brain and erythrocyte phospholipids (Butler et al., 1970b). Using fatty acid spin labels, Hsia et al. (1970a,b, 1971) found that cholesterol not only restricts probe motion, but moves the probe to a new and more symmetrical polar environment, with the tilt angle of acyl-chains decreased from 28' to 10'. Also, addition of cholesterol reduces the rotational freedom for the probes (Barrett et al., 1969; Hubbell and McConnell, 1968). These studies also indicate that the polarity of the hydrocarbon

+

+

ROLE OF CHOLESTEROL IN BIOMEMBRANESAND RELATED SYSTEMS

37

region dccrcascs as onc moves the label toward trrminal methyl group (Hubbell and McConnell, 1971). I n addition, as one goes from the outside to the ccntrr of the bilaycr, freedom of motion increases markedly. Low-angle X-ray diffraction data suggest that for a given hydration level the domains in the egg lecithin-cholesterol hydrocarbon region are better oriented than those in the hydrocarbon region (Levine and Wilkins, 1971). At 21% water content, the terminal methyl groups in egg lecithin lipsomes are distributed over a wide region. I n the presence of cholesterol, however, the terminal methyl groups appear to be well localized. It is particularly significant that the peak-to-peak distance across the hydrocarbon region for egg lecithin bilayers decreases from 39.6 A (at 14% water content) to 3 6 . 8 i a t 21% water content. I n contrast, for egg lecithin cholesterol bilayers, the peak-to-peak distance remains almost constant a t 42 A. These observations indicate that addition of cholesterol reduces the molecular motion in the bilayer and extends the chains so that the membrane thickness increases. This conclusion is consistent with the studies on thermal coefficient of expansion (Rand and Pangborn, 1973) and sedimentation coefficient (Johnson, 1973) of liposomes measured as a function of cholesterol mole fraction in lecithin. The 1,2-dipalmitoyllecithin-cholesterol-water systems show integral orders of a principal long spacing in the low-angle region (Ladbrooke et al., 196813). With increasing cholesterol content, the long spacing increases initially and reaches a maximum a t 7.5 mole percent cholesterol (Fig. 1). A parallel increase has been observed in the water permeability of liposomes that contain low (-10 mole percent) concentrations of cholesterol. On addition of further cholesterol the long spacing gradually doecreases to 64 a t mole percent. The sharp high-angle spacing of 4.2 A increases to 4.45 A and become diffuse. These results are consistent with the view that cholesterol causes a reduction in the cohesive forces between the adjacent hydrocarbon chains of lecithin. Nevertheless the passage of various permeants is reduced through cholesterol-containing liposomes. The results presented in this section show that the chains in bilayers exhibit considerable orientation and that their free ends are near the center. With increasing hydration the chains become disoriented and their ends localized or less mobile. Addition of cholesterol reduces the molecular motion in the bilayer and extends the chains so that they are well oriented with their terminal methyl groups well localized. From the analysis of ESR data, it has been suggested that below the phase transition temperature the spin-labeled steroid molecules are present as clusters in the bilayer (Trauble and Sackman, 1972). The density of clusters appears to be independent of the molar ratio of steroid :lipid, but the size of clusters increases with increasing steroid concentration. Above the phase

+

dO

38

MAHENDRA KUMAR JAlN

transition temperature the clusters dissolve and a homogeneous mixture is formed. C. In Biomembranes

Although cholesterol is a major component of plasma membranes of eukaryotic cells, very Iittle is known about the state of cholesterol in these membranes. It is certain that cholesterol in biomembranes is localized anisotropically with its long axis perpendicular to the plane of the membrane. The conclusions derived from studies on model bilayers may be applicable to biomembranes. The natural abundance 13C-NP\IR spectra of various biomembranes show very broad signals. This suggests that the correlation times for molecular motion are a t least an order of magnitude slower than those observed by the use of ESR and fluorescent probes in biomembranes and model membranes. Rothman and Engelman (1972) have shown that in liposomal membranes that contain cholesterol lipids, the lower region of the phospholipid chains will be in a “liquidlike” state and the upper region will be more ordered. This is because the cross-sectional area of the cholesterol side chain is only about half that of the ring system. X-ray diffraction studies suggest that biomembranes from a variety of sources contain extended regions of lipid bilayer (Wilkins et al., 1971). Membranes that contain high concentrations of cholesterol, such as in erythrocyte and myelin, have their chains extended and fairly perpendicular to the membrane plane. These membranes show little disorder (Chapman et al., 1968, 1969a; Williams et al., 1973). However, the broad band a t 4.6 seen in the X-ray diffraction pattern suggests that no long range order is present in the plane of the bilayer in these membranes. I n membranes containing little or no cholesterol, such as in mycoplasma and retinal rod mcmbranes, disorder of the chains is relatively significant. The electron density curves obtained from rabbit optic and sciatic membrane profiles are similar, but the profile from frog sciatic nerve membrane has a narrower central minimum and thus presumably shorter hydrocarbon chains (Casper and Kirschner, 1971). Furthermore there appears to be a slight asymmetry in the bilayer, which these authors interpret as being caused by an uneven distribution of cholesterol. The data indicate a n approximate equimolar ratio of cholesterol and polar lipid on the outer side of the hydrocarbon layer, and a ratio of about 3 :7 on the inner side. This interpretation accounts for the measured cholesterol content (40% mole percent, cf. Table 111). These X-ray measurements indicate that the hydrocarbon chains are predominantly close packed in the “steroid step regions” (it is up to ninth carbon atom, which is the most frequent position

+

ROLE OF CHOLESTEROL IN BIOMEMBRANES AND RELATED SYSTEMS

39

for the double bond). They also indicate that the ends of the chains near the bilayer center are pliant and disordered. The asymmetric cholesterol distribution presumably results from (or in) specific interactions with protein. However, the protein does not seem to modify significantly the local lipid packing arrangement. These and other (Oldfield and Chapman, 1972b; Marsh and Smith, 1973) observations suggest that cholesterol may have a dual role of preventing formation of crystalline gel areas in some membranes, while also inhibiting the motion of hydrocarbon chains in more fluid, liquid crystalline, regions.

VI. MOLECULAR ASPECTS

OF ORGANIZATION OF CHOLESTEROL IN BILAYERS

The biomembranes are probably as variable in composition as the number of different cells and organelles which they delimit; perhaps even more so. Nevertheless, the close packing of polymethylene chains (C16-C22)in two parallel arrays form the essential structural feature of biomembranes (Jain, 1972). Since cholesterol is an integral component of plasma membranes and is present in the same concentrations as phospholipids, the mode of packing of the polymethylene chains in a bilayer is definitely affected by the penetration of cholesterol. This is shown by monolayer studies, passive permeability measurements, phase transition characteristics, and various physicochemical studies. The experimental data suggest that, in general, the presence of cholesterol tends to condense a liquidcrystalline film in the sense that the combined molecules take up less space than that predicted for them from simple additivity of molecular cross sectional areas. The observed departure from additivity could be due to either formation of some stoichiometric phospholipid/cholesterol complex or may arise from the incorporation of cholesterol in the cavities (or void volume) between phospholipid molecules. The experimental data available so far does not eliminate either of these possibilities. Since most plasma membranes have both phospholipids and cholesterol as their main constituents, a study of the nature of intermolecular interactions in the mixed lipid bilayers is of interest. The effect of cholesterol on orientation, clustering, rotation, and lateral mobility of lipid molecules may be biologically significant. Incorporation and localization of cholesterol into phospholipid bilayer appear to be a consequence of the extended hydrophobic overlap between hydrocarbon chains of phospholipid and the planar ring system of cholesterol. If it is assumed that the cholesterol packs closely alongside or even forms a complex with the fatty acid chains or

40

MAHENDRA KUMAR JAIN

polar groups of phospholipids, such an interaction would be expected to hinder any rotational movements which the pure lecithin molecules may have. It would also allow a closer packing of the molecules. Various characteristics of bilayer show changes as a function of mole proportion of cholesterol present in the bilayer. Most of these changes are observed until the cholesterol mole proportion reaches a maximum of 0.5. At higher mole proportions, cholesterol crystallizes in the bilayer. However, not all bilayer properties show a maximum or minimum a t 50 mole percent cholesterol. I n fact, studies on phase transition characteristics of dipalmitolylecithin with varying mole proportions of cholesterol have yielded conflicting results. Ladbrooke et al. (1968b) reported that the enthalpy of transition vanished at 50 mole percent cholesterol. I n contrast Hinz and Sturtevant (1972) have reported that the transition enthalpy vanished a t 33 mole percent cholesterol. A model consisting of cholesterol surrounded by seven lecithin molecules in the membrane plane has been suggested (Engelman and Rothman, 1972) to account for the loss of transition enthalpy a t 33 mole percent cholesterol. This model is based on the assumptions that the two hydrocarbon chains of lecithin are freely mobile and that the phospholipid/cholesterol pair does not show any specificity of interaction or orientation. These assumptions are contrary to studies discussed earlier in this chapter. There is ample evidence for the involvement or modification of surface group region following incorporation of cholesterol in a bilayer (Abramson and Katzman, 1968). Other models consistent with a maximum incorporation of 50 mole percent cholesterol also suffer from several limitations. It appears that successively higher concentrations of cholesterol bring about changes in the packing and/or orientation of phospholipid chains in at least two (maybe three) different regions. These different orientation and packing characteristics of hydrocarbon chains or polar groups may be present a t about 10, 35, and 50 mole percent (for a hypothetical model see Jain and Cordes, 1974). All these changes, of course, may not be reflected by the use of a single technique. Thc molecular states of lipids corresponding to these three cholesterol concentrations may be distinct. As a result different regions of the bilayer may be affected as thc cholesterol molr proportion in the bilayer increases. Also a t certain mole proportion (lower range) cholesterol may be present as a distinct stoichiomrtric aggregate with lipids or floating as a n island in the bilayer of excess lipid. At higher cholesterol concentrations (30 mole percent or more), however, it is almost certain that cholesterol is randomly distributed among lipid molecules in the plane of the bilayer and thus has a disrupting effect on the “cooperative islands” of lipids. Also in biomembranes containing various phospholipid species one would expect that certain lipids (saturated species for example) would tend to accumulate

ROLE OF CHOLESTEROL IN BIOMEMBRANESAND RELATED SYSTEMS

41

in the vicinity of cholesterol. Highly unsaturated phospholipids would, however, tend to segregate cholesterol. Thus the effect of cholesterol on molecular orientation and organization of phospholipid would be sensitive to structural and compositional variation in hydrocarbon chains. A physiologically important question regarding the biological role of cholesterol pertains to a possible relationship between membrane-bound and plasma solubilized cholesterol. Questions relating to the mode of exchange of cholesterol between membranes and surrounding plasma, its physiological and biochemical regulation, possible presence of specific cholestero1 binding and/or exchange proteins, and the role and significance of various lipoproteins are yet to be answered. The relationship of this exchange process on the structural and organizational states of lipid molecules (fluidity, rotational, translational, and lateral mobility) may yield relevant information. Finally, it may be pertinent to note that the cholesterol-induced variation in membrane fluidity on the function of membrane-bound proteins (Fourcans and Jain, 1974) may have a farreaching regulatory role. ACKNOWLEDGMENT

I wish to thank Mmes Davis and Bergner for help in preparing this manuscript. REFERENCES Abramson, M., and Katzman, R. (1965). Ionic interactions of sulfatide with choline lipids. Science 161, 576-577. Adam, N., and Jessop, W. G. (1925). Structure of thin films. Part X I I : Cholesterol and its effect in admixture with other substances. Proc. Roy. Soc., Ser. A 120, 473-482. Admirand, W., and Small, D. M. (1968). The physicochemical basis of cholesterol gallstone formation in man. J . Clin. Invest. 47, 1043-1052. Admirand, W., and Small, D. M. (1972). The effect of modifications of lecithin and cholesterol on the micellar solubilization of cholesterol. Biochim. Biophys. Acta 270, 407413. Allan, D., and Crumpton, M. J. (1970). Preparation and characterization of the plasma membrane of pig lymphocyte. Biochem. J. 120, 133-143. Back, P., Hamprecht, B., and Lynen, F. (1969). Regulation of cholesterol biosynthesis in rat liver: Diurnal changes of activity and influence of bile acids. Arch. Biochem. Biophys. 133, 11-21. Badley, R. A., Schneider, H., and Martin, W. G. (1971). Organization and motion of a fluorescent probe in model membranes. Bzochem. Biophys, Res. Commun. 45, 174-183. Bangham, A. D. (1972). Lipid bilayers and biomembranes. Annu. Rev. Biochem. 41, 753-776. Bangham, A. C., Dingle, J. T., and Lucy, J. A. (1964). Studies on the mode of action of excess of vitamin A. Penetration of lipid monolayers by compounds in the vitamin A series. Biochem. J . 90, 133-140. Bangham, A. D., Standish, M. M., and Weissman, G. (1965). The action of steroids and

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osmotic fragility and glycerol permeability of erythrocytes. Biochim. Biophys. Acta 183, 334-343. Buck, R. C., and Itossiter, R. J. (1952). Lipids of normal and atherosclerotic aorta. Arch. Pathol. 51, 224-237. Rulkin, B. J., and Krishnan, K. (1971). Vibrational spectra of liquid crystals. 111. Raman spectra of crystals, cholesterir, and isotropic cholesterol esters, 28003100-cm-l region. J . Amer. Chem. SOC.93, 5998-6004. Burke, J. A., and Schubert, W. K. (1972). Deficient activity of hepatic arid lipase in cholesterol ester storage disease. Science 176, 309-310. Butler, K. W., Dugas, M., Schneider, M., and Smith, I. C. P. (1970a). Cation-induced organization changes in a lipid bilayer model membrane. Biochem. Biophys. Res. Commun. 40, 770-776. Butler, K.W., Smith, I. C. P., and Schneider, M. (1970b). Sterol structure and ordering effects in spin-labelled phospholipid multibilayer structures. Biochim. Biophys. Acta 219, 514-517. Cadenhead, D. A., and Phillips, M. C. (1968). Molecular interactions in mixed monolayers. Advan. Chem. Ser. 84, 131-148. Carnie, J. A. (1973). Diet and heart disease. Sci. Progr. (London) 60, 231-230. Casper, D. L. D., and Kirschner, D. A. (1971). Myelin membrane structure a t 10 b resolution. Nature (London),New Baol. 231, 46-52. Cass, A., and Finkelstein, A. (1967). Water permeability of thin lipid membranes. J . Gen. Physiol. 50, 176.3-1784. Cass, A., and Finkelstein, A. (1968). Permeability and electrical properties of thin lipid membranes. J. Gen. Phgsiol. 52, 145s-173s. Chapman, D., and Penkett, S. A. (1966). Nuclear magnetic resonance spectroscopic studies of the interaction of phospholipids with cholesterol. Nature (Londun) 211, 1304-1305. Chapman, D., Owens, N. F., and Walker, D. A. (1966). Physical studies of phospholipids. 11. Monolayer studies of some synthetic 2,3-diacyl-o~-phosphatidylethanolamines and phosphatidylcholines containing trans double bonds. Biochim. Biophys. Acta 120, 148-155. Chapman, D., Kamat, V. B., DeGier, J., and Penkett, S. A. (1968). Nuclear magnetic resonance studies of erythrocyte membranes. J . Mol. Biol. 31, 101-114. Chapman, D., Barratt, M. D., and Kamat, V. B. (1969a). A spin-label study of erythrocyte membranes. Biochim. Biophys. Acta 173, 154-157. Chapman, D., Owens, N. F., Phillips, M. C., and Walker, D. A. (196913). Mixed monolayers of phospholipids and cholesterol. Biochim. Biophys. Acta 183, 4.58465. Cherry, R. J., Graham, D. E., and Chapman, D. (1971). The effect of cholesterol on the optical properties of lecithin bilayers. Chem. Phys. Lipids 5, 125-130. Child, J. J., Defago, G., and Haskins, R. H. (1969). The effect of cholesterol and polyene antibiotics on the permeability of the protoplasmic membrane of Pythium PRL 2142. Can. J. Microbiol. 15, 599-603. Christian, E., and Zilversmit, D. B. (1973). Exchange of various phospholipids and of cholesterol between liposomes in the presence of highly purified phospholipid exchange protein. J . Biol. Chem. 248, 1719-1724. Clayton, R. B. (1970). The chemistry of non-hormonal interactions: Terpenoid compounds in ecology. I n “Chemical Ecology” (E. Sondheimer and J. B. Sirnone, eds.), pp. 235-280. Academic Press, New York.

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Clowes, A. W., Cherry, R. J., and Chapman, D. (1971). Physical properties of lecithincerebroside bilayers. Biochim. Biophys. Acla 249, 301-317. Cobon, G. S., and Haslam, J. M. (1973). The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondria1 ATPase. Biochem. Biophys. Res. Commun. 52,320-326. Cogan, U., Shinitzky, M., Weber, G., and Nishida, T. (1973). Microviscosity and order in the hydrocarbon region of phospholipid and phospholipid-cholesterol dispersions determined with fluorescent probes. Biochemistry 12, 521-528. Colaccico, G., and Rapport, M. M. (1968). Lipid monolayers: Effect of phosphatidylcholine and cholesterol on the interaction of dihydroceramide lactoside with rabbit 7-globulin. Aduan. Chem. Ser. 84, 157-168. Colbeau, A., Nachbaur, J., and Vignais, P. M. (1971). Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 249,462-492. Coleman, R. (1968). Some features of the lipid composition of rat liver surface and cytoplasmic membranes. Chem. Phys. Lipids 2, 144-146. Conner, R. L., Landrey, J. R., Burns, C. H., and Mallory, F. B. (1968). Cholesterol inhibition of pentacyclic triterpenoid biosynthesis in Tetrahymena pyriformis. J. Protozool. 15, 600-605. Conner, R. L., Mallory, F. B., Landrey, J. R., Ferguson, K. A., Kaneshiro, E. S.,and Ray, E. (1971). Ergosterol replacement of tetrahymanol in Tetrahymena membranes. Biochem. Biophys. Res. Commun. 44, 995-1000. Cooper, R. A., and Jandl, J. H. (1968). Physiologic and pathologic alterations of red cell lipids, membrane area, and shape. I n “Metabolism and Membrane Permeability of Erythrocytes and Thrombocytes” (E. Deutsch, E. Gerlach, and K. Moser, eds.), pp. 376-384. Thieme, Stuttgart. Cornwell, D. G., Meikkila, R. E., Bar, R. S.,and Biagi, G. L. (1968). Red blood cell lipids and the plasma membrane. J . Amer. Oil Chem. SOC.45, 297-304. Csogor, S. I. (1972). Diffusible fraction of serum cholesterol and atherogenesis. Nature (London), New Biol. 238, 287-288. Dam, H. (1971). Determinants of cholesterol cholelithiasis in man and animals. Amer J. Med. 51, 596-613. Darke, A., Finer, E. G., Flook, A. G., and Phillips, M. C. (1971). Complex and cluster formation in mixed lecithin-cholesterol bilayers. Cooperativity of motion in lipid systems. FEBS (Fed. Eur. Biochem. SOC.), Lett. 18, 326-330. Darke, A., Finer, E. G., Flook, A. G., and Phillips, M. C . (1972). Nuclear magnetic resonance study of lecithin-cholesterol interactions. J . Mol. Biol. 63, 265-279. DeBernard, L. (1958). Molecular associations between lipids 111. Lecithin and cholesterol. Bull. SOC.Chim. Biol. 40, 161-170. DeGier, J., Mandersloot, J. G., and Van Deenen, L. L. M. (1968). Lipid composition and permeability of liposomes. Biochim. Biophys. Acta 150, 666-675. DeGier, J., Mandersloot, J. G., and Van Deenen, L. L. M. (1969). The role of cholesterol in lipid membranes. Biochim. Biophys. Acta 173, 143-145. DeGier, J., Haest, C. W. M., Mandersloot, J. G., and Van Deenen, L. L. M. (1970). Valinomycin-induced permeation of **Rb*of liposomes with varying composition through the bilayers. Biochim. Biophys. Acta 211, 373-375. DeKruyff, B., Demel, R. A., and Van Deenen, L. L. M. (1972). The effect of cholesterol and epicholesterol incorporation on the permeability and on the phase transition of intact Acholeplasma laidlawii cell membranes and derived liposomes. Biochim. Biophys. Acta 255, 331-347.

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Ohki, E. (1969). The electrical capacitance of phospholipid membranes. Bwphys. J. 9, 1195-1208. Oldfield, E., and Chapman, D. (1971). Effects of cholesterol and cholesterol derivatives on hydrocarbon chain mobility in lipids. Biochem. Biophys. Res. Commun. 43, 610-616. Oldfield, E., and Chapman, D. (1972a). Molecular dynamics of cerbroside-cholesterol and sphingomyelin-cholesterol interactions. Implications for myelin membrane structure. FEBS (Fed. Eur. Biochem. SOC.),Lett. 21, 303-306. Oldfield, E., and Chapman, D. (197213). Dynamics of lipids in membranes. Heterogeneity and the role of cholesterol. FEBS (Fed. EUT.Biochem. SOC.),Lett. 23, 285-297. Oldfield, E., Chapman, D., and Derbyshire, W. (1971). Deuteron resonance: A novel approach to the study of hydrocarbon chain mobility in membrane systems. FEBS (Fed. Eur. Biochem. SOC.),Lett. 16, 102-104. Oldfield, E., Keough, K. M., and Chapman, D. (1972). The study of hydrocarbon chain mobility in membrane systems using spin label probes. FEBS (Fed. Eur. Biochem. SOC.),Lett. 20, 344-346. Ostwald, R., and Shannon, A. (1964). Composition of tissue lipids and anaemia of guinea pigs in response to dietary cholesterol. Biochem. J. 91, 146-154. Pagano, R., and Thompson, T. E. (1968). Spherical lipid bilayer membranes: electrical and isotopic studies of ion permeability. J. Mol. Biol. 38, 41-57. Pak, C. Y. C., and Gershfeld, N. L. (1967). Steroid hormones and monolayers. Nature (London) 214,888-889. Papahadjopoulos, D., and Watkins, J. C. (1967). Phospholipid model membranes. 11. Permeability properties of hydrated liquid crystals. Biochim. Biophys. Actu 135, 639-652. Paphadjopoulos, D., Nir, S., and Ohki, S. (1971). Permeability properties of phospholipid membranes : effect of cholesterol and temperature. Biochim. Biophys. Actu 266, 561-583. Papahadjopoulos, D., Poste, G., and Schaeffer, B. E. (1973a). Fusion of mammalian cells by unilamellar lipid vesicles: influence of lipid surface charge, fluidity and cholesterol. Biochim. Biophys. Acta 323, 2342. Paphadjopoulos, D., Jacobson, D., Nir, S., and Isac, T. (1973b). Phase transitions in phospholipid vesicle. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol. Biochim. Bwphys. Actu 311,330-348. Parsons, D. F., and Akers, C. K. (1969). Neutron diffraction of cell membranes (myelin). Science 165,1016-1018. Parsons, D. F., and Yano, Y. (1967). The cholesterol content of the outer and inner membranes of guinea pig liver mitochondria. Biochim. Biophys. Actu 135,362-364. Patton, S. (1970). Correlative relationship of cholesterol and sphingomyelin in cell membranes. J . Theor. Biol. 29,489491. Patton, S., and Fowkes, F. M. (1967). The role of the plasma membrane in the secretion of milk fat. J. Theor. Biol. 15, 274-281. Peters, J. H., and Hausen, P. (1971). Effect of phytohemagglutinin on lymphocyte membrane transport. I. Stimulation of uridine uptake. EUT. J. Bwchem. 19, 502-508. Phillips, M. C. (1972). The physical state of phospholipids and cholesterol in monolayers, bilayers, and membranes. Progr. Surfuce Membrane Sci. 5 , 139-221. Portman, 0. W., and Alexander, M. (1970). Metabolism of sphingolipids by normal and atherosclerotic aorta of squirrel monkeys. J. Lipid Res. 11, 23-30.

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A . A . LEV and W. McD . ARMSTRONG Institute of Cytology of the Academy of Sciences of the USSR. Leningrad. USSR. and Department of Physiology. Indiana Unicersity School of Medicine. Indianapolis. Indiana

I . Introduction . . . . . . . . . . . . . . . . . 11. Definitjion of “Single Ion Activities” . . . . . . . . . . . 111. Experiments with Model Polyelectrolyte Syst.ems as Supporting Evidence for the Physical Validity of Single Ion Activity Parameters . . . . IV . Microelect.rodes for Measuring Intracellular Ionic Activities . . . A . Glass Membrane Microelectrodes . . . . . . . . . . B. Liquid Ion Exchanger Microelectrodes . . . . . . . . . C . Metal Microelectrodes . . . . . . . . . . . . . D . Other Microelectrodes . . . . . . . . . . . . . V . Techniques for Measuring Intracellular Ionic Activities . . . . . A . Calibrat~ionof Ion Selective Microelectrodes . . . . . . . B. Measurement of the Intracellular Activity of a Single Ion . . . C . Simultaneous Measurement of t.he Intracellular Activities of Two Ions, e.g., Kf and Na+ . . . . . . . . . . . . . VI . Intracellular Ionic Activities . . . . . . . . . . . . . A. Intracellular H+ Activity-Intracellular pH . . . . . . . B. Intracellular Na+, K+, and C1- Activities and Their Relationship to Cellular Function . . . . . . . . . . . . . . VII . Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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66 72 72 79 81 83

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84

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85 87

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88 90 90

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1 INTRODUCTION

The role of inorganic ions in a variety of cellular functions. including such bioelectric phenomena as membrane. resting. and action potentials. has usually been interpreted in terms of conceptual models of the cell 59

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that are based on certain assumptions concerning intracellular ionic concentrations. These assumptions are that all the apparent intracellular water* acts as a solvent for intracellular solutes and that small intracellular ions are uniformly distributed throughout this water volume. Such models require the further assumption that the intracellular activity coefficients of small ions are essentially the same as their activity coefficients in the bathing medium. I n recent years the theoretical and experimental limitations of these models have become increasingly clear. The cell interior is a highly organized system that is extremely heterogeneous, both structurally and chemically. Biopolymers (e.g., proteins, polysaccharides, nucleic acids), mainly of a polyelectrolyte nature, are of prime importance in determining the structure and function of cytoplasm and cytoplasmic organelles. Lipids and phospholipids are not polyelectrolytes in the strict sense, but, when they form lamellar and/or micellar aggregates, they may in many respects be regarded as polyelectrolyte structures (Luzzati et al., 1969). As discussed below, the theoretical description and experimental determination of the thermodynamic state of small ions, even in homogeneous polyelectrolyte solutions, is not a simple problem. In heterogeneous multicompartment polyelectrolyte systems the problem becomes much more complex. Despite these complications, it has long been apparent that the influence of the polyelectrolyte components of cytoplasm on the physical state of intracellular ions may be highly significant and should be taken into consideration. On the basis of electrostatic interactions alone one would expect the thermodynamic state of mobile inorganic ions to be altered in the presence of polyelectrolyte molecules or phospholipid aggregates with a high surface or volume charge density. The extent of such interactions may be expected to depend on such factors as the nature and spacing of the fixed charges on the polyelectrolyte molecules and the distance of closest approach to them of the mobile ions. Variations in degree of electrostatic interaction which arise from such causes may be interpreted as differences in the “binding” of inorganic ions by polyelectrolyte systems. The above concept has been extensively developed in a number of hypotheses concerning solute distribution between the cell interior and the external medium. Notable among these are the “phase” or “sorption” hypothesis (Nasonov and Alexandrov, 1940, 1943; Troschin, 1956, 1961) and the association-induction hypothesis (Ling, 1952, 1960, 1962). I n these hypotheses specific association between inorganic ions and fixed

* This is normally computed as total tissue water minus that fraction (the “extracellular volume”) which equilibrates with an external nonpenetrating solute such as inulin, mannitol, polyethylene glycol (Conway, 1957).

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charge groups in cytoplasmic macromolecules is considered to be the sole reason for the marked discrepancies in ionic distribution between extracellular and intracellular fluid. This now appears unlikely in view of the vast and growing body of evidence for the existence in living cells of transmembrane active transport processes, in particular the ubiquitous “sodium pump.” Nevertheless the problem of inorganic ion interaction with cytoplasmic macromolecules or specific ion binding in cytoplasm continues to be an important one in relation to ion transport and electroselectivity in cells. For example, the difference in cytoplasmic binding between a divalent cation such as calcium and a monovalent ion such as potassium seems beyond reasonable doubt. Also the Donnan effect in the distribution of inorganic ions between the cell interior and the external medium may be regarded in a general sense as a “phase” property of the cytoplasm. Thus, a knowledge of the physical state of intracellular cations, particularly in the vicinity of the plasma membrane, is of obvious importance in relation t o bioelectric phenomena. The existence of discrete intracellular organelles creates further possibilities for inhomogeneity in the distribution of intracellular ions. Kleinzeller et al. (1969) introduced the terms structural and chemical compartmentation to describe different types of intracellular organelles which may differ in their ionic content. Organelles surrounded by membranelike diffusion barriers (e.g., endoplasmic reticulum and its derivatives, mitochondria, chloroplasts, vacuoles, lysosomes) and in which the internal ionic content is largely determined by active ion transport processes were designated structural compartments. Subcellular organelles not separated from the main intracellular space (hyaloplasm or ground cytoplasm) * by membrane structures were defined as chemical compartments. Such compartments include myofibrils, neurofibrils, tonofibrils, fibrils of flagella and cilia, ribosomes, lipid granules, and perhaps membrane structures themselves. In contrast to structural compartments, the ionic composition of chemical compartments should be a function of the ionic composition of the ground cytoplasm, a t least within the limits of the constancy of the polyelectrolyte content of the corresponding subcellular organelles, and the distribution of ions between ground cytoplasm and a given chemical compartment should depend primarily on the relative charge density of their polyelectrolyte components. If the ability to discriminate between different ions with the same sign of charge is not significant in either compartment, the distribution of mobile ions between them should obey the Donnan principle. *The existence within the cell of such a phase as “ground cytoplasm” is open to argument., but there is no doubt that, as an assumption, i t is extremely useful at present in constructing electrochemical models to explain bioelectric phenomena.

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If the mobile ions have significantly different affinities for the fixed charge groups in different compartments, their equilibrium distribution between these compartments is still subject to the restriction that their electrochemical potentials must be the same in all chemical compartments and in the ground cytoplasm, i.e., Pi,g.o.

=

Pi.1

= pio = pio

=

pi.2

=

+ RT In

* . a

-

Pi,n

+ RT In + z,Fpl + RT In a i , 2+ ziFpz = ... = pio + RT In a i , , + ziFpn ai,g.c.

= pio

where p i , g . c . , P ~ J p, i . 2 - . * p i , n are the electrochemical potentials of the i t h ion in the ground cytoplasm and chemical compartments, a i , g . c . , ai,2 a i , na r e its corresponding activities, pio its chemical potential in the standard state, z i its valence, and p1p2 * pn are the phase boundary potentials between the ground cytoplasm and chemical compartments 1,2. It follows from Eq. (1) that, a t equilibrium, the concentrations, and indeed the activities, of an ion may be different in different chemical compartments. Thus it is clear that intracellular ionic concentrations calculated from chemical or isotopic determinations of tissue water and ion contents are only a first approximation to real intracellular ionic concentrations. Methods in which ground cytoplasm is separated from whole cells and analyzed for its ionic content (see, e.g., Bruce and Marshall, 1965) inevit,ably involve tissue or cell homogenization, during which profound changes may occur in the ionic composition of many if not all cytoplasmic fractions. At present the only direct method available for determining the ionic composition of the ground cytoplasm of cells is the measurement of intracellular ionic activities with ion-sensitive microelectrodes. The position, with respect to cellular ultrastructures, of the tip of the ion selective electrode following impalement of a cell is usually not exactly known, and one cannot prove beyond doubt that the medium surrounding the electrode tip is in fact ground cytoplasm. However, it is clear that the position of these microelectrodes is exactly the same as that of ordinary capillary microelectrodes used for transmembrane potential difference measurements. Therefore ion activities found by the method of ion-sensitive microelectrodes are plausible for the description of electrochemical plasma membrane properties and transmembrane ionic fluxes.

... a n .

-

IONIC ACTIVITIES IN CELLS

63

II. DEFINITION OF "SINGLE ION ACTIVITIES"

The concept of "single ion activity" is somewhat uncertain from a strict thermodynamic viewpoint. This uncertainty arises from the negation in classical thermodynamics of the physical reality of ions of one sign of charge as independent components of macroscopic systems. Actually, ions of one sign of charge may not be added in macroscopic quantities to a system without simultaneous addition of an equivalent amount of oppositely charged ions or removal of an equivalent amount of another ion of the same charge. Thus the principle of macroscopic electroneutrality forces one to use such parameters as single ion activities and single ion activity coefficients only in combinations capable of being transformed into other parameters which are characteristic of electrolytes as a whole (Guggenheim, 1929, 1933). This situation is paradoxical in that real physical components of electrolyte systems (ions with one sign of charge) are under a forma.1restriction in the rigorous thermodynamic approach to the electrochemistry of solutions. In fact some investigators consider determinations of single ion parameters (including single ion activities and activity coefficients) to be in certain respects invalid. Hence it is not surprising to find attempts aimed at finding new theoretical and experimental approaches to the definition of single ion activities and activity coefficients and the provision of a physical meaning for these parameters (Rabinovich, 1964; Rabinovich et al., 1960, 1967; Manning, 1969; Manning and Zimm, 1966; Frank, 1967; Nikerov and Rabinovich, 1968; Lev et al., 1971). If the physical validity of single ion activities as parameters of thermodynamic systcms may be assumed, then there are two main experimental restrictions to their exact determination. The first concerns the question of standardization. This problem is the same as that which arises in pH measurements (Bates, 1973), since a pH measurement is a particular case of a single ionic activity determination. The second restriction originates from the difficulty of estimating precisely the liquid junction potential which necessarily arises when galvanic cells with transference are used in electrochemical measurements. We shall discuss briefly the importance of these two restrictions in the determination of single ionic activities. The mean activity of a salt in solution is a thermodynamically rigorous parameter which may be defined by several independent methods, including potentiometric measurements with the aid of galvanic cells without transference (Robinson and Stokes, 1965). For such measurements it is sufficient to have a pair of electrodes, one of which is reversible for the cation and the other for the anion of the salt under investigation. A commonly used method for standardizing the mean activity of the salt is based on the

64

A. A. LEV AND W. McD. ARMSTRONG

assumption of equality between salt activity and salt concentration in very M ) solutions. dilute (in practice, say less than To determine the activity of a single ion, one requires a galvanic cell consisting of an electrode which is reversible for the cation or anion in question and a reference electrode connected to the solution by a salt bridge. For the moment we shall postpone discussion of the liquid junction potential between the solution in the salt bridge and the solution under investigation and regard this potential as known, or a t least as constant. I n this case the problem of single ion activity standardization becomes simply a question of common agreement on a primary standard. This may be a solution of any salt or acid provided reversible electrodes are available for both its cations and anions. For example, one may choose a KC1 solution since highly selective electrodes reversible to K+ ions are now available (e.g., Orion model 92-19 liquid ion exchange potassium electrodes). An Ag/AgCl or calomel electrode may be used as the C1- reversible electrode. On the basis of the Debye-Huckel limiting law, one may assume that in a dilute KC1 solution the K+ ion activity ( U K ) is equal to the C1activity ( u c ~ and ) that both are equal to the mean activity ( u K c ~ ) of KC1 in the reference solution. If it is assumed that the liquid junction potential remains constant for all solutions used, one may then extend this method to provide secondary standards for other ions; e.g., a galvanic cell with transference which includes a -C1- reversible electrode can be used with a dilute NaCl solution to obtain a reference standard for Naf activity. If the cell is first calibrated for C1- activity in the primary KCl standard solution it can be used to determine the C1- activity in a dilute NaCl solution. If the mean NaCl activity of this solution is known, its Na+ activity is readily calculated from the relationship 2

UNs = uNaCl/uCl

(2)

Data thus obtained from a series of NaCl solutions can then be used to calibrate a reversible Na+ electrode. In like manner, secondary standards can be obtained for other cations and anions.* As already mentioned, the above standardization procedure is correct under the assumption of constant liquid junction potential for all solutions used (the actual magnitude of this potential is relatively unimportant). Unfortunately this assumption is usually not justified. Further, estimation of the probable size of the liquid junction potential and of the amount by

* pH standards conforming to an accepted convention (Bates and Alfenaar, 1969) map be used as primary standards from which secondary standards for the activities of other ions may be derived.

65

IONIC ACTIVITIES IN CELLS

which it may change under the different experimental conditions employed is not easy. Liquid junction potentials may be regarded as a particular case of phase boundary potentials, and it is well known that the potential difference between two phases is not theoretically determinable. Our inability in principle to determine the liquid junction potential for a given set of conditions implies a similar inability to determine its constancy or lack of constancy under varying conditions. I n the most general approach, the liquid junction of diffusion potential between two solutions can be represented by the following equation :

where t i and t, are the transference numbers of the ith ionic species and the solvent respectively, ai‘ and a:’ are the ith ion activities in the two solutions in contact with each other, and a,’ and a:’ are the corresponding solvent activities. Equation (3) can be integrated only under certain assumptions. The problem has been considered by several authors (Planck, 1890a,b; Behn, 1894; Henderson, 1907, 1908) who obtained satisfactory results subject to certain specific limitations. These limiting conditions depend on the model chosen for the boundary between the mixing solutions. The most popular integrated form of Eq. (3) is the diffusion equation of Henderson (1907, 1908).

RT

c ui(c:’-

Ci’)

c

UiZiCi’

n

(4) n

n

This equation was obtained quasithermodynamically with the diffusion zone regarded as a continuous series of mixtures of solutions (’) and (”). Ionic mobilities were supposed to be independent of concentration, and single ion activity coefficients of unity were assumed. A more rigorous determination of the diffusion potential difference requires a knowledge of the actual profile of single ion activities in the zone of solution mixtures. However, as already pointed out, determination of these parameters depends on the possibility of determining the liquid junction potential. Thus, the range of unsolved questions is closed. Fortunately, this state of affairs does not mean that approximate estimations of diffusion potentials, from Eq. (4) for example, are useless, especially in the case of contacts between simple salt solutions containing cations and anions of nearly equal mobilities (e.g., such pairs as K+ and C1- or NH4+and NOa-) a t low concentrations. Under these conditions, diffusion potentials estimated from the

66

A. A. LEV A N D W. McD. ARMSTRONG

Henderson Eq. (4) range from a few tenths of a millivolt to 2-3 mV. These limits of the estimated diffusion potentials introducc an uncertainty of approximately 5-150/, in the determination of single ion activities in M to lo-' ill salt solutions when the method of standardizing described above is used. Because of the unccrtainties in the assumptions underlying Eq. (4), this estimate of the error inherent in single ion activity determinations is not quite certain, even for simple salt solutions. The situation with respect to liquid junction potentials in polyelectrolyte solutions is much less sure. Hence the reliability of estimates of single ion activities in such solutions is considerably reduced, and a similar situation holds for corresponding estimates in cytoplasm. Obviously, additional criteria are required to establish the validity of ionic activity measurements in polyelectrolyte solutions and, by inference, in cytoplasm. Such criteria can be found in the following ways: (a) comparison of experimental data on single ion activities in polyelectrolyte solutions with theoretically predicted values : (b) comparison of single ionic activities found in polyelectrolyte solutions with other physical parameters (found by independent methods) in these solutions. Some results of such comparisons are given in the next section.

111. EXPERIMENTS WITH MODEL POLYELECTROLYTE SYSTEMS AS SUPPORTING EVIDENCE FOR THE PHYSICAL VALIDITY OF SINGLE ION ACTIVITY PARAMETERS

Some experimental evidence that the physical state of inorganic ions in solutions containing biological polyelectrolytes differs from their state in aqueous solutions of simple salts was found many years ago (Hammersten, 1924; Teunissen van Zijp and Bungenberg de Jong, 1938; Creeth and Jordan, 1949; Shack et al., 1952). Convincing quantitative data bearing on this problem, however, were obtained only after the development of ion-sensitive electrodes (Scatchard et al., 1957). In a series of studies on the state of Na+ ions in DNA solutions (Botre et al., 1958; Ascoli et al., 1959, 1961; Vorobjev et al., 1971), a marked decrease in the activity coefficient of Na+ was noted in solutions containing native DNA. The converse of this, an increase in Na+ activity (i.e., a decrease in counterion binding) was observed during thermal or dilution denaturation of DNA. The state of small inorganic ions (counter- and co-ions) in solutions containing polyelectrolytes of known structure was considered theoretically by Manning and Zimm (1966) and by Manning (1969). The equations obtained by these authors can be applied to biological systems because they

67

IONIC ACTIVITIES IN CELLS

were derived for solutions containing concentrations of polyelectrolyte and simple salt which are comparable to those found in cytoplasm. Those obtained earlier by Katchalsky et al. (1966) are less applicable since they are based on a consideration of salt-free polyelectrolyte solutions or polyelectrolyte solutions containing a large excess of salt. I n deriving cquations for counter- and co-ion activity in polyelectrolyte solutions, Manning (1969) made certain assumptions. Among these is a model of polyelectrolyte molecules as linear chains with uniformly distributed surfacc charges. Interactions between charged groups on the polyelectrolyte molecule were ignored, and the dielectric constant of the polyelectrolyte solution was considered to be the same as that for a simple salt solution. Also, the Debye-Hiickel approximation was applied to all mobile ions except those in the region of ion condensation. Manning's (1969) equations for singly charged counter- and co-ions (in the case of DNA and polyacrylate solutions these will be cations and anions, respectively) are yi = y(f-lx

+ 1)(x + l)-l exp[YA

Y ~ A=

y2(E-'X

= y exp[- &'x/(E-'

+ 1) ( X + l)-'

&lx/(t-l

+ 2)l

+ 2)1

exp[ - €-'X/(t-'

(5) (6)

+ 211

(7)

In these equations y i is the activity coefficient of the counterion, Y A that of the co-ion and Y ~ Ais the mean activity coefficient of the (univalent) salt; X = n,/n, where n, is an equivalent concentration of the charged group of the polyelectrolyte (e.g., the concentration of phosphate groups in the case of DNA) ; n, is the concentration of uni/univalent salt added to the polyelectrolyte solution and E = e2DkTb where e is the unit charge, D is the dielectric constant (80 for water), k is Boltzmann's constant, T is the absolute temperature, and b is the distance between charges on the polyelectrolyte molecule (projected onto its axis). The factor y (Manning and Zimm, 1966) was introduced to take account of interactions between small inorganic ions in the solution and is given by the equation

where X = 4re2/DkT = (8.76 x lo-' cm for aqueous solutions) and C, is the total concentration of inorganic ions in the solution. The above equations permit one t o calculate activity coefficients for polyelectrolyte solutions containing a known concentration of polyelectrolyte and different concentrations of NaC1. These can then be compared with the experimentally observed values of these parameters under the same conditions. We have made such a comparison for DNA and poly-

68

A. A. LEV AND W. McD. ARMSTRONG

acrylate solutions containing NaC1. DNA and polyacrylate were chosen for this study because their charge distribution is well known. Galvanic cells with transference were used to measure Na+ and C1activities. These cells consisted of a sodium-sensitive glass electrode and an Ag/AgCl electrode. The mean NaCl activity was either determined directly with a galvanic cell without transference or taken as the geometric mean of u N a and ucl. In both cases the results should be the same. Since the concentration of NaCl added to the polyelectrolyte solution was known, experimental values for the activity coefficients of Naf (yNa), C1- (ycl), and the mean activity coefficient of NaCl (yaScL)were readily obtained from the activity data. These were then compared with the theoretical values for these parameters calculated from Eqs. ( 5 ) , (6), and (7). The results obtained for native sodium-DNA and sodium-polyacrylate solutions are shown in Figs. 1 and 2. The quantitative agreement between calculated and observed activity coefficients for DNA (Fig. 1) is not very good although the mode of change of the experimental values and their general position on the graph are in accordance with theoretical predictions. Agreement between theoretical and observed values is better for the polyacrylate/NaCl solutions (Fig. 2) although there is some scatter in the experimental points. The deviation from theory of the y N a points for DNA

0.6

*

0.4

.

0.2

.

0

Y

{O-’

40-3

(0-2

AO-4

CNaCL

FIG.1. Dependence of YNa, yc1, and Y N ~ C Ion NaCl concentration in solutions of native DNA. The curves are theoretical values calculatedofrom Eqs. ( 5 ) , (6), and (7), the parameter b in these equations being taken as 7 A. The points are experimentally determined activity coefficients. The DNA concentration ranged from 0.159 to 0.171 gm/100 ml.

69

IONIC ACTIVITIES IN CELLS

0.8 Y

0.6 0.4 0.2

t

i

!O-'

10-2

10-4 CNoCL

FIG.2. Data as in Fig. 1, but for sodium polyacrylate solutions (0.05 g m / l O O ml). The degTee of neutralization of the polyacrylic acid was 95%. Parameter b was taken as 2.55 A.

solutions (Fig. 1) may be due, in part a t least, to some denaturation with time of the polymer a t room temperature resulting in changes in the mean intercharge spacing, 6. An important feature of the data shown in Figs. 1and 2 is that the differences between theoretical and experimental values for mean activity coefficients appear to be very similar to the differences between predicted and observed values for single ion activity coefficients. This similarity is particularly apparent in Fig. 1. The mean activity coefficient of a salt (yfNaa in Figs. 1 and 2) is a rigorously defined thermodynamic parameter. As such it is free from the theoretical objections which are sometimes raised against the concept of single ion activities (page 63). Furthermore, mean activity coefficients can be determined without the complications connected with liquid junction potentials. The fact that mean activity coefficients measured in this way proved to be in no better agreement with theory than experimentally determined single ion activity coefficients can be interpreted as an indication that the liquid junction potential did not vary significantly over the whole range of salt concentrations employed in these experiments (in the experiments with DNA this was to lo-' M ) . This conclusion is important supporting evidence for the validity of single ion activities measurements and of standardization procedures (page 64). Further support for the validity of single ion activity determinations was obtained during an investigation of the conditions necessary for the so-called isoionic dilution of polyelectrolytes. It is known that in the

70

A. A. LEV AND W. McD. ARMSTRONG

measurement of some hydrodynamic and optical parameters of polymer solutions (e.g., characteristic viscosity determination and measurement of rotatory diffusion by flow birefringence) extrapolation to zero polymer concentration must be used. This extrapolation can be done most precisely under conditions where the measured parameter (reduced viscosity when characteristic viscosity is determined, or the x angle when rotatory diffusion is studied) shows a linear dependence on polymer concentration. To obtain such linear dependence in polyelectrolyte solutions it is necessary to dilute the initial solution with the simple salt solution at a Concentration which does not alter the screening of polyelectrolyte charged groups by counterions. This is the condition of isoionic dilution. Its importance is that the dimensions (and hence the intercharge spacing) of the polyelectrolyte molecules are held constant during dilution. Where the thermodynamic state of the counterions is not influenced by the polymer (i.e., in solutions of uncharged polymers), isoionic dilution is quite simply obtained by adding simple salt a t the same concentration as that in the initial sample of polymer solution. If, as in the case of polyelectrolyte solutions, the activity of counterions may be significantly changed by “ion-binding,” then, to ensure that dilution is isoionic, the activities rather than the concentrations of counterions in the polyelectrolyte solution must be known. Dranitskaya et al. (1967, 1974) and Lev et al. (1971) approached thc problem of isoionic dilution of a number of biopolymers and synthetic polyelectrolytes in solutions containing NaCl as follows: Reduced viscosity and counterion activity were measured in the initial sample of polymer solution. As expected, counterion activities were frequently much smaller than their corresponding concentration. For example, in solutions of native DNA, Y N was ~ found to be as low as 0.25, and in sodium polyacrylate solutions Y N appeared ~ to be between 0.3 and 0.4 when a minute amount or no NaCl was added. Polymer solutions with measured counterion activities were diluted successively with increasing amounts of salt solutions having the same counterion activity, this activity being measured independently in the salt solution. Reduced viscosity was determined a t each dilution state and plotted as a function of polymer concentration. A linear relationship between reduced viscosity and polymer concentration was taken as an indication that dilution was in fact isoionic. In the majority of cases studied, it was found that when the measured counterion activity of the diluent solution was adjusted to equal its measured activity in the initial polymer sample, the reduced viscosity was in fact a linear function of polymer concentration. This is illustrated in Fig. 3 which shows the results of an experiment with solutions of sodium polyacrylate containing NaCl a t initial mean activities ranging from 3.4 t o 7.2 X M. I n all

71

IONIC ACTIVITIES IN CELLS

30

10

I

1

0.01

0.02

0.03 CPA

0.04

0.05

(Oh)

FIG.3. Dependence of reduced viscosity, (7 - l ) / C N a p A on the concentration of sodium polyacrylate ( C ~ A(MW ) 5.1 x 106) in samples with different initial concentrations of NaCl. Isoionic dilutions (judged by a linear dependence of reduced viscosity on polymer concentration) were obtained when the NaCl solutions used for dilution (the “solvent”) had the same UN* as that in the initial samples of polyeleetrolyte. The broken line shows the dependence when a N s in the “solvent” was the same as a N s c l in the I for the initial polyelectrolyte solution. The numbers near the lines are the U N ~ C values initial polyelectrolyte solutions.

cases where the diluting solution (“solvent”) had the same aNaas the initial polymer sample a linear dependence of reduced viscosity on polymer concentration was obtained. It is highly significant that in the one instance where dilution was performed by adding NaCl at the same mean activity (aNaci) as that of the initial sample (dashed line in Fig. 3) the plot of reduced viscosity versus polymer concentration was not a straight line. Also, a differenceof 10-15% between aNa in the “solvent” and the initial sample =as sufficient to cause a significant deviation from linearity. Essentially similar results have been obtained for natural and reconstituted nucleohistone complexes (Frisman et al., 1970a,b), ribosomal RNA solutions (Schagina et al., 1969), and solutions of polymethacrylic acid (Dranitskaya et al., 1974). The significance of these results in the present context may be summa.rized as follows: Single ion activity measurements performed with the aid of galvanic cells with transference permit one to define the conditions

72

A. A. LEV A N D W. McD. ARMSTRONG

for constant screening of polyelectrolyte charged groups in solution. Constancy of screening under these conditions can be checked by an absolutely independent hydrodynamic method. If the screening of polyelectrolyte charged groups in solution is a physically real phenomenon, then single ionic activities in solution have the same physical reality. Furthermore, these experiments strongly support the concept that ionic activity determinations in solutions containing polymers have the same validity as corresponding measurements in simple salt solutions.

IV. MICROELECTRODES FOR

MEASURING ACTIVITIES

INTRACELLULAR IONIC

The use of ion selective electrodes for the determination of ionic activities on a macroscale is well established as a routine analytical method. A large and rapidly growing variety of these electrodes is now commercially available and the list of their application to biological and nonbiological systems is almost bewildering (see, for instance, Beljustin and Lev, 1965; Eisenman, 1967a; Feder, 1968; Durst, 1969). To date, the number of types of electrodes used and the extent of their use in intracellular studies, although rapidly increasing, is much more modest, so that it is still possible to review this subject within a reasonable space. For convenience, we have classified the different kinds of microelectrodes so far used for measuring intracellular ionic activities under four general headings. These are, glass membrane electrodes, liquid ion exchanger microelectrodes, metallic microelectrodes, and other microelectrodes. A. Glass Membrane Microelectrodes

1. SIMPLIFIED THEORY OF THE GLASSELECTRODE

A rigorous discussion of the theory of glass membrane electrodes (commonly called glass electrodes) is outside the scope of this review. An excellent account of their properties is that of Eisenman (1965), on which much of the brief outline presented below is based (see also Nicolsky et al., 1967). A glass electrode consists of a thin glass membrane enclosing a solution of known ionic composition. When it is inserted in an external solution of different composition a potential difference develops across the membrane. This potential difference can be measured by immersing reversible reference electrodes (e.g., Ag/AgCl or calomel half-cells) in the inner and outer solutions, respectively. Depending on the composition of the glass mem-

73

IONIC ACTIVITIES IN CELLS

brane, glass electrodes show various selectivities; that is, they respond preferentially to a specific ion. This is invariably a cation because glass is a fused mixture of the oxides of elements with a valence of 1+ or 2+ and oxides of elements with a valence of 3f or more and univalent cations are the only readily mobile ions in the fused mixture. Bivalent cations have much smaller mobilities and the mobility of anions is essentially zero (Anderson and Stuart, 1954). Also, because of the high concentration of fixed negative charges in the glass network, its concentration of small free anions is very low. Thus, glass membranes function essentially as univalent cation exchangers (Eisenman, 1965). The detailed relationship between the composition of glasses and their ionic selectivity is discussed by Eisenman (1965, 196713, 1969), HBbert (1969), and Bates (1973). Depending on its composition, the selectivity of a glass membrane may be so great as to be virtually absolute in practice. In other words, cations other than those to which the glass is primarily responsive will, under normal conditions of use, have only negligible effects on the total electrode potential. Perhaps the best-known example of this behavior is the H+ glass electrode routinely used in pH measurements. Alternatively, one or more ions, in addition to the preferred ion, may contribute significantly to the potential recorded by the electrode in solutions containing mixtures of cations. Examples are electrodes made from K+ or Na+ selective glasses (e.g., NAS 25-4 K+ selective glass or NAS 11-18 Na+ selective glass*). Both of these are sensitive to H+ ions and, in addition, +(I selective glasses show significant sensitivity to Na+. The latter fact, as discussed below, has some interesting consequences in the determination of intracellular Na+ and K+ activities. If a glass membrane separates two solutions containing an ion i to which it is sensitive, the potential across it is given by the Nernst equation

E

=

(RT/F) ln(ail/a:’)

(9)

where air and a:’ are the activities of ion i on the two sides of the membrane. If one of these activities, e.g., a:’ is held constant, then

E

=

const.

+ (RTIF) lna,’

(10)

and, following appropriate calibration, this equation may be used to determine the activity of i in solutions in which it is the only cation. Further,

* These and similar codes for the composition of glasses are interpreted as follows (Eisenman et al., 1957) : The letters NAS indicate that the glass is made from a mixture of NazO, AlzO,, and SiOz. The numbers following the letters are the moles percent of NazO and Alzo, respectively; e.g., NAS 27-4 contains 27 moles percent of Na202, 4 moles percent of AlzOaand, hence, 69 moles percent of SiOz.

74

A. A. LEV A N D W. McD. ARMSTRONG

if the electrode type and the conditions are such that i is the only ion which contributes significantly to the overall potential (e.g., the pH glass electrode under ordinary conditions), or, if interfering cations are present in minute amounts compared to i, Eq. (10) can also be applied. Nicolsky (1937) has shown theoretically that if a glass electrode is immersed in a solution containing two univalent cations to which it is sensitive in different degrees, the total electrode potential is given by the equation E = E, (RT/F) U a , k,,aJ (11)

+

+

where i is the preferred cation.* E, corresponds to the constant term in Eq. (10);and i t is the potential registered by the electrode in a mixture of i and j, for which the term in parentheses is unity. k , , is a selectivity coefficient which expresses the selectivity of the electrode for i compared to j. It is readily determined from the potentials recorded by the electrode in solutions of i a n d j , respectively, when a, = a,. Under these conditions Ink,, = F(E,

- E,)/RT

(12)

where Ej and E , are the potentials observed in the solution containing j only and in that containing i only. Note that the value of k , , is inversely proportional to the selectivity of the glass for i relative to j. A k , , of 0.1 means that the glass is 10 times more sensitive to i than to j.

When an electrode is sensitive, in varying degrees, t o a number of ions, Eq. (11) takes the general form

E

=

E,

+ (RT/F)ln(a, + kt,a, +

k,,a,)

(13)

if the electrode is immersed in a solution containing all these ions and on the assumption that the solution approximates ideal behavior. The physical meaning of the selectivity coefficient k , , in Eq. (11) deserves comment. Nicolsky’s (1937) original derivation of this equation was for the response of glass electrodes to Na+ and H+ and had its origin

* The experimental validity of

Nicolsky’s equation has been confirmed by Eisenman al. (1957), who also found that an even wider range of phenomena is adequately described by the empirical equation et

E

=

Ea

+ ( n R T / F )In

+ (kij

~ j ) ” ~ ]

(114

where n is a constant for a specific glass and a given pair of cations. Obviously, when n = 1, Eq. ( l l a ) reduces to Eq. (11). This usually turns out to be the case for Na+ or K + selective glasses when the conditions are such that Naf and K + are the only ions contributing significantly to the overall electrode potential. Hence, Eq. (11) has proved adequate for the determination of intracellular Na+ and K+ activities with K + selective glass electrodes (see, e.g., Lev, 1964; Armstrong and Lee, 1971).

75

IONIC ACTIVITIES IN CELLS

in earlier studies that sought to explain the well known “alkaline error” of pH glass electrodes (Hughes, 1922).* Nicolsky assumed a reversible exchange of Na+ and H+ between glass and solution with the same population of sites available to both ions. It was further assumed that the activities of Na+ and H+ in the glass phase m r e proportional to their mole fractions therein and that the total electrode potential was the sum of the phase boundary potentials a t the two glass/solution interfaces. No account was taken of possible contributions from diffusion potentials within the glass membrane. More recently it has been shown (Karreman and Eisenman, 1962; Conti and Eisenman, 1965a,b, 1966) that the glass electiode potential, like the potentials generated across other fixed site ion-exchange membranes (Helfferich, 1956, 1959, 1962; Mackay and Meares, 1960), is a composite of diffusion potentials within the glass membrane together with equilibrium phase boundary potentials a t the glass/solution interfaces so that one may write kt,

=

kt(u,/u,) = P,/Pa

(14)

where ual u,,P a , P , are the mobilities and permeability coefficients of i and j in the glass membrane and k,“,is the ion exchange equilibrium constant. The fact that k,,, as determined, for example, from electrode potential measurements using Eq. (12), is a composite of exchange equilibrium and nonequilibrium kinetic components can impose important practical restrictions on the selectivity of glass membrane electrodes. For instance, the K+/Na+ selectivity of NAS 27-4 glasses as determined by electrode potential measurements is about 8-10-fold. Eisenman (1965) has shown that the equilibrium selectivity of such glasses is in the general range of 50-80. The much smaller overall selectivities are due to the fact that U N * in these glasses is 5-10 times greater than U K . When an electrode is responding, under conditions of zero current flow, in ideal fashion to a given ion, i.e., in a pure solution of that ion or where the effects of other ions on the electrode potential are negligible (cf. the linear portions of the curves in Fig. 5), changes in total electrode potential are determined by changes in the phase boundary potential alone. Under these conditions the diffusion potential is constant, maximal, and is given by RT log uK/uNain the case under discussion (Eisenman, 1965). This explains why Eq. ( l l ) , which is based on simple ion-exchange theory, can successfully predict the behavior of K+ selective glass electrodes in many situations. The condition of zero current flow is effectively that under which intracellular ionic activities are normally measured. * For a review of early work on the reiationship between the composition of glass and its relative sensitivity to H + and Na+ ions, see Eisenman (1965).

76

A. A. LEV AND W. McD. ARMSTRONG

2. DESIGNOF GLASSMEMBRANE MICROELECTRODES FOR MINATION OF IONIC ACTIVITIES

THE

DETER-

Glass membrane microelectrodes have been used successfully for the measurement of intracellular H+, Na+, and I<+activities. It is not our intention here to describe in detail the fabrication of individual types of electrodes,* but rather to discuss the general features of the major varieties and to evaluate their relative advantages and disadvantages. Basically, glass membrane microelectrodes consist of a capillary tube, drawn out to a fine taper a t one end which is then sealed to form a thin membrane. The capillary is filled with an appropriate electrolyte solution which is connected to a reversible half-cell (Ag/AgCl or calomel), the circuit being completed by a second (usually identical) half-cell which serves as a reference electrode. There is one major difference between electrodes designed for intracellular work and those intended for use in simple solutions. With intracellular microelectrodes, while the sensitive glass element should obviously make contact with the cytoplasm, it should most emphatically not be in contact with the medium outside the cell membrane. Thus, apart from the immediate region of the tip, the sensitive glass capillary (most of which of necessity remains outside the cell) must be isolated electrically from its surroundings. In addition, with small cells it is usually necessary to impale the cell under investigation directly through its membrane. This means that the junction between the sensitive glass and the material used to insulate the body of the electrode must also pass through the cell membrane without damaging it unduly. Therefore the diameter of the electrode a t the junction should be small enough to permit this. In other words, the electrode should be insulated almost to the tip and the region of transition from sensitive glass to outer insulating material should be smooth and not involve an abrupt increase in diameter. In general, two methods have been employed t o achieve these ends. One is to coat the ion selective glass capillary to within a short distance of the tip with an insulating material such as shellac or polystyrene which adheres well t o glass. The other is t o encase the ion-selective capillary in a slightly larger capillary tube made from nonsensitive glass. This capillary is drawn to a taper with an opening somewhat larger than the tip of the ion-selective capillary. The junction between the two is effected either by a direct glass-to-glass seal or by a plug of nonconducting material such as

* Detailed accounts of the methods of construction of various kinds of glass microelectrodes are given by Hinke (19.59, 1969), Lev and Buzhinsky (1961), Khuri (1969), Kleinzeller et al. (1969), and Lee and Armstrong (1974).

77

IONIC ACTIVITIES IN CELLS

beeswax or paraffin. Both types of electrode are shown schematically in Fig. 4. The earliest glass microelectrodes to be used successfully, the p H microelectrodes of Caldwell (1954, 1958), were of the shellac-coated type. They were designed for use with cells having a largediameter, such as fibers from lobster or crab leg muscles or giant squid axons. Furthermore, in axons they were inserted parallel to the major axis of the cell. Thus their dimensions (tip diameter 50-80 pm; exposedtip length about 0.5 mm) barely qualify for the prefix “micro-” in contemporary parlance. Much smaller shellac microelectrodes (tip diameter 1-5 pm; exposed tip length about 10 pm) were devised by Kostyuk and his co-workers (Kostyuk and Sorokina, 1961; Kostyuk et al., 1969) for intracellular p H measurements in frog sartorius fibers (impalement being made perpendicular to the long axis of the fibers). These workers, and Hinke (1969), have emphasized that coated electrodes, although easy to make, are somewhat unsatisfactory in that they frequently deteriorate within a few days of their construction. This is because the insulating material tends to peel away from the junction where it meets the exposed glass tip. As a result the area of the exposed tip can vary unpredictably with the age of the electrode. Moreover, fluid may be trapped between the sensitive glass and the insulator. Either case may lead to serious perturbations in the electrical response of the electrode. The design of Carter et al. (1967a; Rector et al., 1965), in which a ceramic

b

-e

c

D

E

FIG.4. Schematic representation of five basic designs for glass microelectrodes; a, cation-sensitive glass; b, shellac or polystyrene insulation; c, nonsensitive insulating glass; d, glass-to-glass seal; e, wax or paraffin seals (see text for further details).

78

A. A. LEV AND W. McD. ARMSTRONG

glaze is baked onto the outside of pH glass capillaries before pulling, would seem to be less prone to deterioration. Hinke (1959) was the first to describe glass-shielded microelectrodes for the measurement of intracellular Na+ and I<+activities. Like Caldwell’s (1954), Hinke’s electrodes were intended for use with the large cells of crab, lobster, or barnacle muscle fibers into which the electrode could be inserted coaxially and tied in place like a microcannula (McLaughlin and Hinke, 1966). These electrodes had tip diameters in the range of 10-30 pm. * Hinke’s designs (shown schematically in Fig. 4B and C) were of crucial importance to subsequent technical developments. He used the Na selective NAS 11-18 and the K selective KAS 27-5 glasses of Eisenman et al. (1957), encased in EP-1 lead glass insulating tubes. With NAS 11-18 electrodes the tip of the insulating pipette was fused directly to the sensitive glass (Fig. 4B), a technique subsequently employed (Hinke, 1969) for p H glass microelectrodes. Because of its unusual thermal properties NAS 27-5 could not be successfully fused to EP-1 lead glass. Instead, dental cement was used to form a water-tight junction (Fig. 4C).1. Hinke’s design for a NAS 27-5 microelectrode formed the basis of a number of other successful electrodes (Lev and Buzhinsky, 1961; Kostyuk et al., 1969; Lee and Armstrong, 1974) used to measure K+ and Na+ activity in a variety of cells. These include frog skeletal muscle fibers (Lev, 1964; IZostyuk et at., 1969; Armstrong and Lee, 1971), giant neurons of the molluscs Hetis and Planorbis (Kostyuk et al., 1969), and epithelial cells of frog small intestine (Lee and Armstrong, 197213). It has been possible to fabricate successful electrodes of this type with tip diameters of the order of 0.5 pm and exposed tip lengths of less than 2 pm (Fig. 4D). Methods for fabricating ultrafine electrodes of this kind are described in detail by Kleinzeller et al. (1969) and by Lee and Armstrong (1974). At present it would seem that the lower size limit of these electrodes (and thus, effectively, of the cells in which they can be used) will be set, not primarily by the tip diameter of the selective glass membrane, but rather by the minimum diameter of the insulating junction and the minimum length of exposed tip that can be reproduced with consistency. Further, although glass-to-glass seals are undoubtedly superior to others in terms of longevity, and electrodes in which the insulating junction is formed as in Fig. 4C and D may with time be subject to junction failure (as already discussed for

* Hinke’s (1961) Na+ and K+ electrodes for longitudinal impalement of Loligo axons were even larger, having tip diameters of 70-90 pm and exposed tip lengths of 2-4 mm. t Later, Hinke (1969) described a process in which K + selective, lead glass, and Pyrex capillaries were fused together to form a glass junction. The process is, on Hinke’s own admission, complex and difficult for his relatively large electrodes and would, in our opinion, prove excessively so in ultrafine work.

IONIC ACTIVITIES IN CELLS

79

shellac-coated electrodes), it appears that carefully constructed electrodes of the type illustrated in Fig. 4D can be stored for periods of a t least two months without significant deterioration (personal communication from Dr. C. 0. Lee). TWOfurther variants of Hinke’s basic design merit comment. Kostyuk and Sorokina (1961) and, later Hinke (1969), showed that glass-insulated and glass-to-glass sealed microelectrodes could be made by fixing a pH glass capillary inside a nonsensitive glass capillary (by cementing or fusing their ends together) and drawing both together in an electrode puller. At first glance this would appear to be the ideal method of fabrication. Unfortunately, because of the rather complex combination of factors required for success, the method so far has been confined to pH glass. Further, the percentage of successful pulls is low (Hinke, 1969), and an exposed tip length of 10-15 pm seems to be the lower limit presently obtainable. An ingenious modification of Hinke’s design (Fig. 4B) for an NAS 11-18 microelectrode is due to Thomas (1970, 1972) and is shown in Fig. 4E. It should be pointed out that ultrafine NAS 27-5 microelectrodes frequently have anomalously low resistances (about 0.5 to 1 X lo9 ohm, about two orders of magnitude less than one would predict from the bulk resistivity of the glass and the tip dimensions). Lev (1969) has suggested that this is a consequence of “incomplete sealing,” i.e., the existence of small aqueous pores in the glass tip. An alternative explanation, based on the idea that in electrodes with fine tips the wall thickness is such that the glass is fully hydrated throughout the wall and the tip conductance is dominated by the cation conductance of the hydrated glass membrane (Eisenman, 1965), has been proposed by Lee and Armstrong (1972a, 1973, 1974). In either event, this phenomenon permits one to use these electrodes in fairly conventional electrophysiological circuits, provided the input impedance of the voltage follower connected to the electrode is sufficiently high (about 1013 to 10’4 ohm). With other glasses, e.g., NAS 11-18, the resistance of microelectrodes with exposed tip lengths of the order of 2 pm may be inconveniently high (1012 to 1013 ohm). Thomas’ (1970) design, in which the tip of an NAS 11-18 microelectrode is fixed inside a nonsensitive open-tip electrode by a fused glass seal, combines the advantages of a relatively large tip areawith those of a very small tip diameter, highly effective shielding, and the ability to impale small cells successfully. Thomas (1972) has used these electrodes to determine Na+ activities in snail neurons. B. liquid ion Exchanger Microelectrodes

Ross (1967) described a liquid ion exchanger for the determination of Ca2+ion activity on a macro scale. Since then development of such electrodes has been quite intensive, and a number of them are now available

80

A. A. LEV AND W. McD. ARMSfRONG

commercially. A good summary of their composition, properties, and mode of operation is that of Ross (1969). At the same time the theory of liquid ion exchangers has also received considerable attention (Conti and Eisenman, 1966; Sandblom et al., 1967a,b; Eisenman, 1969) and has been extended t o include the transport of ions across lipid layers by neutral ionophores such as macrolide antibiotics and cyclic polyethers (Eisenman et al., 1973; Andreoli et aZ., 1967; Mueller and Rudin, 1967; Lev and Buzhinsky, 1967). Liquid ion exchangers are solutions of organic electrolytes in waterimmiscible solvents with low dielectric constants. The organic ion itself should be relatively insoluble in water. Depending on the composition and sign of charge of the organic ion (Ross, 1969), liquid ion exchangers can be made selective toward cations or anions, respectively, and can exhibit varying degrees of selectivity toward individual ions. I n essence, a liquid ion exchanger electrode consists of a layer of exchanger interposed between two aqueous solutions, one of which is a reference solution of defined, constant composition. In these circumstances, the electrode potential is described by an equation analogous to Eq. (11). The physical meaning of the measured selectivity coefficient, however, is somewhat different from its meaning in fixed-site exchange systems. An inorganic ion in the aqueous phase will enter the nonaqueous layer only after by an organic ion of opposite charge which, because of its solubility, is virtually restricted to this phase. Both ions diffuse across the nonaqueous layer as an uncharged ion pair. Thus, in the expression for the coefficient k , j (Eq. 14) it is the mobilities, within the lipid phase, of the ion pair complexes rather than those of the free ions that must be considered (cf. Eisenman et al., 1973). Walker (1971) has successfully adapted electrodes whose sensitive elements are composed of K+ and C1- selective liquid exchangers to intracellular activity measurements. These electrodes are made from conventional open tip micropipettes (tip diameter about 0.5 pm) by the following procedure. The pipette is drawn, and the tip is made hydrophobic to a length of about 200 pm by applying an organic silicone compound to it. This is done by briefly immersing the electrode tip in a solution of the silicone in a suitable nonaqueous solvent and then drying it in an oven. The tip is then immersed in the appropriate exchanger (Walker uses commercial Corning code 477317 K+ exchanger or code 477315 chloride exchanger) for sufficient time to fill the tip to a length of 100-200 pm. A fine syringe is used to fill the micropipette with the reference solution (e.g., 0.5 M KCl) through the open end. Filling is accomplished under 100-fold magnification, and air bubbles are removed with a fine glass needle. Finally, a drop of mineral oil is introduced into the open end of the micropipette

IONIC ACTIVITIES IN CELLS

81

to prevent evaporation of the filling solution during storage. During use, a fine chloridized silver wire makes contact with the filling solution. Walker’s electrodes have many advantages for measuring steady state intracellular activities. They are comparatively simple to make and have excellent selectivities. For example, in solutions of 0.1 M ionic strength, his K+ electrode has a selectivity ratio of 50:l for K+ with respect to H+ and Na+, and his C1- electrode has a selectivity of 20:l with respect to HCO3- (Walker, 1971). In addition, the tiresome problem of tip insulation is completely eliminated. Some drawbacks are the comparatively high resistances of these electrodes (109 to 1O1O ohm) and the fact (Walker, 1971) that the plug of liquid ion exchanger is sometimes lost within 2-3 hours of filling. Liquid ion exchanger electrodes for C1-, Ca2+, and Mg2+have also been discussed by Orme (1969). Orme’s electrodes are not dissimilar in general construction from those described by Walker (1971), but the process of fabrication appears to be more cumbersome. The Ca2+microelectrode is of obvious interest from the point of view of intracellular investigations. The liquid ion exchanger employed is the commercial Orion code 92-20-02 Ca2+ exchanger in which the organic ion is a high molecular weight organophosphoric acid. Although Orme’s electrodes have rather high resistances (-loll ohm) and have relatively slow response times (20-30 minutes), they yield satisfactory voltage/activity curves in CaClz solutions. However, intracellular use of these electrodes is severely restricted by the fact th a t K+ at the activity level normally encountered in cytoplasm, and also Mgz+, markedly affect the overall electrode potential (see also Walker, 1971). If present limitations can be overcome, the Ca2+ microelectrode could prove t o be a tool of major importance in the investigation of cellular function. C. Metal Microelectrodes

Various forms of metallic microelectrodes have been used for the determination of intracellular pH. Thus micro-antimony (Buytendyk and Woerdeman, 1927) and micro-platinum/hydrogen (Dorfman, 1936) electrodes have been used to record p H changes during the development of amphibian eggs. Caldwell(l954) described a micro-tungsten electrode made from a 25 pm tungsten wire embedded in shellac or paraffin inside a glass capillary (exposed tungsten tip length about 200 pm) which he used to determine intracellular p H in crab muscle fibers. There is a serious question concerning the H+ specificity of these electrodes (they may, for example, respond t o intracellular pOz as well as intracellular pH) and Caldwell (1954), noting that his tungsten electrodes frequently gave anomalously

82

A. A. LEV AND W. McD. ARMSTRONG

high intracellular p H values, concluded that they were not satisfactory for this purpose. Kurella and his co-workers (Kurella, 1969) have made extensive use of glass microcapillaries filled with antimony in investigations of the intracellular p H of Nitella and state that if the current density through the electrode is kept sufficiently low (e.g., by the use of ultrahigh resistance amplifiers), reliable readings can be obtained in the p H range of 3.5 t o 8. The practical lower limit for the tip diameter of these electrodes appears to be about 3 pm (Kurella, 1969). Below this size, electrode resistance becomes a formidable problem. At the present time, metal microelectrodes seem to offer few advantages over well constructed glass microelectrodes for intracellular p H measurement. However, further studies of their properties appear to be necessary before their status can be fully evaluated. Several metal electrodes for the determination of intracellular C1- activities have been described. With very large cells such as squid giant axons or barnacle muscle fibers satisfactory C1- electrodes can be made in which the working tip is a chloridized silver wire inserted directly into the cytoplasm (Mauro, 1954; Strickholm and Wallin, 1965; Hinke, 1969). As with microelectrodes made from cation-sensitive glasses, the problem of isolating the sensitive element from the extracellular medium also arises with Ag/AgCl microelectrodes. Hinke (1969) has devised a n interesting solution to this problem. In his intracellular C1- electrode a fine Pt wire (diameter 20 pm) is sealed inside a tapered glass capillary, leaving a short length of the wire protruding from the tapered end. The Pt tip is then trimmed t o a length of a few microns and coated with moist AgZO, which is then reduced to metallic Ag in a microforge. Finally AgCl is fused onto the Ag tip by immersing it in a bead of molten AgCl. An ingenious method of “sealing” the tip of an Ag/AgCl microelectrode was devised by L. N. Vorobiev for studies on Nitella cell sap (Vorobiev and Kurella, 1966; Kurella, 1969). In this method a n empty glass capillary is first inserted into the cell. Because of capillary action and the turgor pressure of the cell contents, a small amount of cell sap enters the capillary after a few minutes. To measure C1- activity, a fine Ag wire, electrolytically tapered and chloridized, is inserted into the open end of the glass capillary until it makes contact with the cell sap in the LLmicrofistula.”If a convential open-tip electrode is inserted into the sap in the “microfistula,” the membrane potential can also be measured. Kurella and his co-workers (Kurella, 1969) have also used this method for recording intracellular K+ activities in Nitella with K+ glass microelectrodes. The method is of interest for investigation of large cells under conditions where intracellular activities are constant. It has obvious limitations with smaller cells. It is also of little use under conditions where intracellular activities may be

IONIC ACTIVITIES IN CELLS

83

changing since, according to Kurella (1969), once the L‘microfistula’’is formed there is little if any further exchange between it and the bulk of the cell sap. Kerkut and Meech (1966a,b) have described an electrode with a metal tip suitable for measuring C1- activities in small cells (e.g., brain cells of the snail Helix aspera with diameters about 60 pm). An open-tip Pyrex microcapillary is filled with a solution of ammoniacal silver nitrate, and its tip is then immersed in a 20% formaldehyde solution for about 10 hours. When successful, this results in the tip of the microelectrode being blocked by a small plug of metallic Ag. Several electrodes of this type have been fabricated in the laboratory of one of us (W. l\/lcD. A.). The successful ones have shown a good response to the C1- activity of KCI solutions, but we have not used them to impale cells. D. Other Microelectrodes

Two kinds of microelectrode may be considered under this heading. Vorobiev (1968) and Vorobiev and Khitrov (1971) have described K+sensitive microelectrodes prepared by blocking the tip of an open-tip micropipette with a precipitate of K cobaltinitrite or K dipicrylamine. This can be accomplished either by first filling the microelectrode with a IlCl solution and immersing its tip in the reagent solution or vice versa. In either case, the formation of precipitate is observed under the microscope, care being taken not to allow excess precipitate to form on the outside of the tip. When properly prepared, these electrodes have resistances in the range 107-109 ohm and response times of 10-30 sec. Vorobiev and K h i t r w (1971) believe that K+ precipitate microelectrodes function by virtue of the ion exchange properties of the precipitate. The possibility exists, however, that their K+ selectivity arises from virtual but incomplete blocking of the orifice of the micropipette. This would be analogous to the phenomenon of “incomplete sealing” in glass membrane microelectrodes (Lev, 1969) and to the cation selectivity of open tip microelectrodes with ultrasmall diameters (see below). This possibility is suggested by the fact that the k K N s values obtained by Vorobiev and Khitrov (1971) for I(+precipitate microelectrodes (generally about 0.2) are close to those found with incompletely sealed electrodes (Lev, 1969). Theoretical considerations (Lev, 1969) lead one to predict that, in electrodes in which the sensitive element is equivalent to a very narrow pore or pores, electrode conductance will depend more on the concentration and mobility of cations in the double layer adjacent to the charged glass surface than on the bulk selectivity of the glass (see also Agin, 1969; Lavalke and Szabo, 1969). This seems to be the reason why microelectrodes made from Na+-selective

84

A. A. LEV AND W. McD. ARMSTRONG

NAS 20-10 glass and which display anomalously low resistances (i.e., are probably “incompletely sealed”) exhibit K+ selectivity rather than the normal Na+ selectivity observed in high resistance electrodes made from the same glass (Lev, 1969). This question could probably be settled by constructing Na+ precipitate electrodes similar to the K+ precipitate electrodes of TJorobiev and Khitrov and observing their K-Na selectivities. An “anomalous” Kf selectivity would be strong evidence in favor of the pore theory. * Studies of the tip potential of open-tip microelectrodes (Adrian, 1956; Agin and Holtzman, 1966; Agin, 1969; Lavallke and Szabo, 1969) showed that when the tip diameter is reduced to extremely small dimensions (as judged by electrode resistance) these electrodes acquire cation selectivity. For example, Agin (1969) reported that a very high resistance (-lo9 ohm) Pyrex glass microelectrode filled with 1 M KCl exhibited almost perfect K+ exchange properties when immersed in external KC1 solutions of different concentrations. Lavallke (1964) and Lavallke and Szabo (1969) observed a dependence on external K+ and H+ of the electrode potential of open-tip microelectrodes drawn from pH sensitive and Pyrex (Corning code 7740) glasses. These findings raise the question whether, by appropriate manipulation of the tip diameter and other factors, it is possible to make open-tip microelectrodes capable of measuring intracellular activities. Lavallke (1964) has in fact utilized the H+ sensitivity of the tip potential in microelectrodes drawn from a H+ selective glass (Corning code 0150C) to measure intracellular pH in fibers of isolated rat atria. His results are in reasonably good agreement with those obtained with the microelectrode technique by other workers in muscle fibers from other animal species (see Section VI,A), although his electrodes showed highly variable and disappointingly low values of the slope dV/dpH (31.2 mV f 36.5 SD per pH unit in 102 measurements with 17 electrodes). It is possible, however, that more extensive studies of open-tip microelectrodes with very small effective tip diameters may lead to the development of highly effective electrodes for measuring the activities of intracellular cations. Such microelectrodes would have the advantage of relative ease of construction and would not require special techniques for isolating the working tip. V. TECHNIQUES FOR MEASURING INTRACELLULAR IONIC ACTIVITIES

In this section some general principles underlying present day techniques for measuring intracellular ionic activities will be briefly reviewed. No at* Vorobiev (1968) commented briefly on precipitate electrodes which showed Na or Ca2+selectivity, but did not give quantitative data.

85

IONIC ACTIVITIES IN CELLS

tempt will be made to discuss experimental details. These can be found in individual papers concerned with these measurements. As a general rule it may be said that intracellular activity measurements do not require any drastic changes in the electrical circuitry routinely used for other intracellular recording techniques (Bures et al., 1967). A voltage follower with sufficiently high impedance to give reliable recordings from microelectrodes with resistances in the range 108-1010ohms should be employed. A number of these are commercially available at present. A. Calibration of Ion Selective Microelectrodes

The electrical potential (E,) registered by an electrode designed to measure the activity of an ion in solution should (when the electrode is filled with a solution containing i a t a constant activity) be given by an equation of the form

Et

=

E,

+ S log (a;),

(15)

where (a,), is the activity of i in the outer (test) solution and E , is the standard potential of the electrode, i.e., its potential in a solution for which (aJo= 1. Equation (15) is identical with Eq. (lo), except that the empirical value S of the slope dEt/d log (a,), is substituted for its theoretical value (2.303 R T / F ) a t the same temperature. The essential point is that E t should be a linear function of log (a,),. Because of variations in the fine structure of individual microelectrodes, S may differ from its theoretical value (this is particularly true of glass membrane microelectrodes where small changes in the structure of the glass tip can occur during the process of drawing out and sealing the glass). However, as long as the linear relationship between Et and log (a;), predicted by Eq. (15) is obtained, the electrode is usable, although values of S that are markedly less than its theoretical value result in lowered electrode sensitivity. The first step in calibrating an electrode sensitive to an ion i is therefore to establish the values of E , and S by recording the potentials observed a t different values of (a%),,.If i is univalent, this is conveniently done by using a set of solutions containing a uni/univalent salt of i for which ai can be obtained from the mean activity of the salt according to Eq. (2). A practical example of this procedure is illustrated in Fig. 5, which shows calibration data in KCl solutions for a K+ selective glass microelectrode made from NAS 27-5 glass (Lee and Armstrong, 1974). In the example shown in Fig. 5, S was very close to its theoretical value (59.2 mV) a t 25%. Figure 5 also shows the procedure for determining k,, ( k K N a in the example shown) for an ion j which is likely to interfere with the electrode

86

A. A. LEV AND W. McD. ARMSTRONG

-210

-180

/

-150

KC1

- 120 P.D. (mv) -90

-60

-30

0

1

0

2 - L O G

3

a

FIG.5 . Calibration data for Ktselective glass (NAS 27-5) microelectrodes a t 25°C. The observed responses ( 0 )of the electrode potential to pure KCl solutions, pure NaCl solutions, and mixtures of these are shown. These are compared to the calculated of the electrode under the same conditions, obtained as follows: Data for responses (0) pure KC1 were obtained by inserting the experimentally observed values of S and Eo (intercept of the KC1 curve with t h e y axis) into Eq. (15). Those for NaCl were calculated S log k K N a c ~ N * ,using experimentally determined values from the equation El = EL for S , ~ K N * ,and Ei (intercept of the line for pure NaCl with the y axis). Data for mixtures were calculated from the equation Et = E, S log (UK ~ K N ,U N ~ ) .

+

+

+

response to i under the proposed conditions of use. As noted above for S, k i j for individual microelectrodes may differ significantly from the value predicted from the known properties of the sensitive element. Again, this is particularly true for ion selective glass microelectrodes, where the selectivity coefficients for individual electrodes can (and usually do) differ

IONIC ACTIVITIES IN CELLS

87

from each other and from the “bulk” selectivity coefficient for the glass. As shown in Fig. 5, kij is determined by recording the response of the electrode potential to log (aj),. If this response meets the necessary criteria, k i j is then calculated from Eq. (12). An obvious requirement is that E , be a linear function of ( ~ j as) well ~ as of (a,),. In addition, the condition that a single value of kij be applicable over the range of activities to be studied requires that the slope dE,/d log a be the same for solutions of i and j over this range. In practice, some difference in these slopes is frequently observed. The question then is to decide what degree of difference can be tolerated without introducing excessive error into the final results. This will obviously be inversely related to the magnitude of k i j . 8. Measurement of the lntracellular Activity of a Single Ion

When a microelectrode sensitive t o an ion i is inserted into a cell, the total electrode potential is given by

or where Emis the membrane potential of the cell and (ai), is the intracellular activity of the ion i. It is a t once apparent that, to solve this equation, Em must be known as well as E , and S. With large cells (Caldwell, 1954; Hinke, 1961, 1969) it is a simple matter to insert a reference microelectrode into the cell containing the ion-specific electrode and record the difference between the potentials registered by the two electrodes. With smaller cells, less direct methods may be necessary. In this case a representative sample of membrane potentials is recorded with the reference microelectrode. After this a number of penetrations are made with the ion-selective electrode. The difference between the average value of the potentials recorded with the ion-specific microelectrode and the average value obtained for the membrane potential can then be inserted in Eq. (16a). Because of the design limitations imposed by the necessity for providing some form of electrical shielding for the sensitive tip of the ion-selective electrode, the above procedure is often the only feasible one when cationselective glass microelectrodes are used t o measure cation activities in small cells. With liquid ion-exchanger microelectrodes other approaches are possible. For example, Khuri et at. (1972) have succeeded in making a double-barreled microelectrode in which one barrel is a Walker (1971) type liquid exchanger K+ electrode, the other being an open-tip reference

88

A. A. LEV AND W. McD. ARMSTRONG

microelectrode. With this electrode they have obtained measurements of intrafiber I<+ activity in frog sartorius muscle which are in good agreement with earlier data obtained with I<+-selective glass microelectrodes (Lev, 1964; Armstrong and Lee, 1971). Whichever method is used to record electrode potentials, the measurements should conform to certain criteria. The tip potential of the reference electrode should not change significantly during impalement. For this reason reference electrodes with low tip potentials should be selected. Also the characteristics of the cation selective electrode should not change during the experiment. This can be checked by recalibrating the electrode a t the end of the intracellular measurements. * Furthermore, it is obvious that an electrode designed to measure the intracellular activity of a single ion should have a sufficiently high selectivity for that ion so that the results obtained are not biased by effects due to interfering ions. Thus, for example, the K+ and Na+ selectivities of an intracellular H+ microelectrode and H+ selectivity of K+ and Na+ microelectrodes should be such that, under the experimental conditions employed, undesired interference is negligible. C. Simultaneous Measurement of the lntracellular Activities of Two Ions, e.g., K+ and Na+

Many cellular processes invoke interrelated changes in the intracellular content or coupled transmembrane transport of pairs of ions. Na+ and K+ are noteworthy in this respect. Increases or decreases in cell K+ are usually accompanied by converse changes in cell Na+ and vice versa. A well known example of a membrane transport mechanism that depends on the concentration of both these ions is the Na+ pump linked to the activity of membrane (Na+ and K+) activated ATPase (Skou, 1965). Similarly, in terms of the constant field equation (Goldman, 1943; Hodgkin and Katz, 1949), one can anticipate that variations in the resting potential will sometimes reflect changes in both the intracellular Na+ and K+ activities as well as in the membrane permeabilities to both these ions. For these reasons, simultaneous measurements of intracellular Na+ and I<+activities under control and experimental conditions will often yield much more informa-

* A useful rule of thumb emerged from Lee and Armstrong’s (1974) studies of Na+ and K + activities in frog skeletal muscle. These authors found that when the potential registered by a K+ selective glass microelectrode in the bathing medium did not change by more than f 1mV during an experiment, constancy (within the limits of experimental error) of the electrode characteristics could be assumed.

89

IONIC ACTIVITIES IN CELLS

tion about the cellular mechanisms involved than can be obtained from determinations of the intracellular activities of one of these species. The current range of K+ and Na+ selective microelectrodes permits several approaches to this problem. One could, for example, combine a Na+ glass microelectrode, such as that of Thomas (1970, 1972), having a relatively high Na+/K+ selectivity with a liquid ion exchanger K+-selective electrode such as that of Walker (1971) and solve the appropriate forms of Eq. (16a) for Na+ and K+ activities. Alternatively, one could use a highly selective microelectrode in combination with a less selective one, e.g., a liquid exchanger K+ selective microelectrode and a relatively poorly K+ selective glass (NAS 27-5) microelectode and derive K+ and Na+ activities from Eqs. (17) and (18).

EI/S = log Eg/S

=

1%

[(aK)c

(UK)~

f

k K N a (aNa)cl

(17) (18)

where El and E , represent the response as defined by Eq. (16a) of the liquid ion exchanger and glass microelectrodes to intracellular K+ and Na+. A third approach, used successfully in a number of investigations (e.g., Lev, 1964; Armstrong and Lee, 1971), takes advantage of the fact that NAS 27-5 glass microelectrodes not only are rather poorly selective for K+ over Na+, but also exhibit quite variable individual selectivities. * Within a batch of electrodes prepared from the same sample of capillary glass, individual k K N a values may vary over as wide a range as 0.1-0.4. If one chooses two microelectrodes with widely different selectivities one obtains a system that can be described by Eqs. (19) and (20).

E'/S'

=

log

(aK

+

kkNsaNn)

(19)

and

E"/S"

=

log

(UK

+

kffNnuNa)

(20)

from which, after experimental determination of the parameters E', El', s',s",k k N a , and k&,, U K and U N are ~ readily obtained. It is apparent that, in principle, all the above methods for the simultaneous determination of K+ and Na+ activities could be extended to cover three or more ions (e.g., Hf, K+, and Na+). In practice, however, the number of measurements involved would rapidly become prohibitive. * It is important to note that the H + selectivity of these electrodes (Kleinzeller et al., 1969: Lee and Armstrong, 1974) are such that there is no appreciable effect of this ion on the electrode potential at p H values in the range t o be expected in the cell interior (Waddell and Bates, 1969).

90

A. A. LEV A N D W. McD. ARMSTRONG

VI. INTRACELLULAR IONIC ACTIVITIES A. lntracellular H+ Activity-lntracellular pH

I n accordance with established convention, data obtained from intracellular measurements with H+ selective microelectrodes are almost invariably reported in terms of intracellular pH. This parameter is operationally defined as pH(,) = pHo)

+ (E, - E,)F/RT In 10

(2 1)

where E, and E, are the potentials registered by a p H cell when its electrodes are immersed in a standard solution S whose pH is defined and in a solution X whose p H one wishes to determine. However, the exact relationship between pH as defined by Eq. (21), and the hydrogen ion activity of the unknown solution has been the subject of considerable discussion (Waddell and Bates, 1969; Bates, 1973). This is a special case of the general problem already discussed in Section I1 of relating potentials recorded by ion specific electrodes to single ion activities. Hence, subject to the restrictions noted in that section, the potentials recorded by intracellular H+ selective microelectrodes (and intracellular p H values derived therefrom) can be considered to reflect the H+ activity of the cell to the same degree as that in which analogous measurements with K+-, Na+-, or C1-sensitive microelectrodes give estimates of the intracellular activities of these ions. Among the questions on which accurate information concerning intracellular pH could be expected to shed light is the problem of the mechanism of H+ transport across the cell membrane. If the distribution of H+ between the cytoplasm and the extracellular fluid is entirely “passive,” then, according to the Donnan relationship, one would expect to find the following relationship between intracellular and extracellular p H (pHo - pH,)

=

log

(aHz)/(aHo)

= EmF/RT

(22)

where a H z and a H o are the intra- and extracellular H+ activities and Em is the resting membrane potential. On the other hand, if the maintenance of the intracellular/extracellular H+ activity ratio involves active pumping of H+ ions across the membrane the relationship between intracellular and extracellular pH could be quite different. Caldwell’s (1954) initial glass microelectrode measurements of intrafiber pH in isolated muscles of the crab Carcinus maenas were addressed in part to this problem. With muscles immersed in a saline medium (Fatt and Katz, 1953), approximating the composition of crab blood and having a

IONIC ACTIVITIES IN CELLS

91

bulk pH of 7.53 (not very different from his recorded value of 7.48 for crab hemolymph), Caldwell found a mean intrafiber p H of 6.9. No significant effect of membrane potential (over the range 0-55 mV) on intrafiber p H was noted. It iyas observed, however, that, in the absence of perfusion the p H of the saline medium in the immediate neighborhood of the fibers dropped to 7.06. From these latter two findings, Caldwell (1954) concluded that the intrafiber p H was not consistent with a Donnan distribution of Hf ions across the fiber membrane. Although his estimate of intrafiber p H in crab muscle was in reasonably good agreement with earlier values obtained by entirely different methods for crab muscle (Cowan, 1933) and frog skeletal muscle (Fenn, 1928; Stella, 1929). Caldwell’s results were criticized by Conway (1957) on the grounds that insertion of an electrode with a tip diameter in excess of 100 pm into a fiber with a diameter of 600 pm probably resulted in significant cellular injury. Caldwell himself (1954) was evidently aware of this possibility, since he noted that the membrane potentials he recorded (in no case exceeding 55 mV) were significantly less than the average membrane potential (70 mV) reported for these fibers by Fatt and Kate (1953). I n a much more extended study, Caldwell (1958) confirmed his earlier value of about 7 for the intrafiber p H of Carcinus muscle. He also showed that the pH measured in intact fibers u7as close to that observed in preparations of minced muscle. Under normal conditions the intrafiber p H in much larger muscle fibers (about 2 mm diameter) from the spider crab M a i a squinado and giant nerve axons from the squid Loligo forbesi was also shown t o be close to 7 (in the case of squid axons the p H obtained from intracellular recordings was in good agreement with that measured in extruded axoplasm). This is about 0.5 p H units higher than would bc predicted from Eq. (22) on the assumption that H+ is distributed across the fiber membrane according to the Donnan relationship. I n addition, Caldwell (1958) showed that, in all three preparations, the response of intrafiber pH t o changes in the p H of the bathing medium was never close (except when pH was changed by saturating the medium with COz) to that predicted by Donnan theory and that complete depolarization of Maia muscles and Loligo axons with KC1 induced only slight changes (about 0.1 pH unit) in intrafiber pH. Caldwell concluded that the internal p H of muscle and nerve is regulated primarily by the buffering capacity of the cytoplasm and possibly by active extrusion of H+ ions across the fiber membrane, rather than by Donnan forces. Caldwell’s (1954, 1958) estimate of about 7 for the normal value of cytoplasmic pH has been well supported by subsequent microelectrode measurements as well as by data obtained by other methods (Caldwell, 1956; Waddell and Bates, 1969). Thus, Spryopoulos (1960) using glass

92

A. A. LEV AND W. McD. ARMSTRONG

microelectrodes similar to those of Caldwell found an average in situ of 7.35 for the axoplasmic pH in axons from the squid Loligo pealii. The pH of extruded cytoplasm was 6.9-7.1. Iiostyuk and Sorokina (1961) and Kostyuk et al. (1969)’using much smaller glass microelectrodes, examined the intrafiber pH in frog skeletal muscle. Their average value for muscles immersed in a Ringer solution of pH 7.36 (pH of frog blood = 7.42) was 7.12. An identical value was obtained for the intrafiber pH of frog muscles in situ. Intrafiber pH was remarkably stable and unaffected by changes in membrane potential, changes in extracellular pH within the range 5-10, or metabolic inhibitors. Summarizing later worli from their laboratory (see, e.g., Sorokina, 1965), Kostyuk et al. (1969) reported average values of 7.34 and 7.26 for the intracellular pH of rat skeletal muscle and giant neurons of mollusca (Helix and Planorbis). Again, these values were relatively independent of such factors as the membrane potential and extracellular pH, and Kostyuk et al. concluded that in their studies intracellular pH was not consistent with an equilibrium distribution, in accordance with Eq. ( 2 2 ) , of H+ ions across the plasma membrane. Lavallbe (1964) found an average value of 6.9 (which fell to 6.6 under acute anoxia) for the intrafiber pH of isolated rat atria beating a t a rate of 200 per minute a t 30°C in a Krebs-Ringer solution of pH 7.4, and Hinke and McLaughlin (1967) reported that the intrafiber pH of isolated barnacle muscle between 7 and 30°C lies within the approximate range 7.2-7.6. Thus the majority of glass microelectrode measurements of intracellular pH in muscle and nerve tend to confirm Caldwell’s (1954) conclusion that intracellular pH is usually close to 7 and is not the result of a simple Donnan distribution of H+ ions across the fiber membranc. Intracellular pH measurements by other techniques provide considerable support for this point of view (Waddell and Bates, 1969). In sharp contrast to these results, however, are those reported by Carter et al. (1967b) for the intrafiber pH of rat skeletal muscle. Although this group had earlier obtained a value for this parameter (about 6.8, see Carter, 1961) which was in reasonably good agreement with those summarized in the preceding paragraph they subsequently reported that the average pH of fibers for which Em was between 85 and 93 mV was 5.99. Also intrafiber pH was found to vary with Em (under both hyperpolarized and depolarized conditions) in accordance with the predictions of Eq. (22). Carter et at. (1967b) concluded that, in rat skeletal muscle, H+ ions are in a state of electrochemical equilibrium across the fiber membrane as suggested by Conway (1957; see also Conway and Fearon, 1944). The discrepancy between the results of Carter et al. (1967b) and those of other investigators cannot be unequivocally explained a t present. It is true that in many of Caldwell’s (1954, 1958) measurements Emwas anom-

IONIC ACTIVITIES IN CELLS

93

alously low. If there is in fact a dependence of pH, on Em of the kind predicted by Eq. (22) this would result in values for pH, above the normal value. However, the results of Kostyuk et al. (1969) with frog skeletal muscle and the work of LaVallee (1964) with rat atria are not subject to this criticism, and the fact that the former workers obtained identical pHC values for isolated fibers and fibers in situ suggests that the discrepancy is not due to any intrinsic difference between in vivo and in vitro pHi values. More recently, Carter (1972) compared the values obtained for the intracellular p H of giant muscle fibers from the barnacle Balunus nubitus with double-barreled (i.e., combined micro p H and open tip, tip diameter <1 km) microelectrodes (Carter et al., 1967a), and a modified Caldwell (1954, 1958) microelectrode. The fibers were maintained in barnacle Ringer solution (McLaughlin and Hinke, 1966) a t p H 7.4. With the doublebarreled microelectrode, the fluid at the surface of the fiber was found to have a pH (7.05) significantly below that of the bath as a whole (cf. Caldwell, 1958). The mean measured p H of 253 fibers was 6.12 f 0.03 (SE). The calculated intrafiber pH, from Eq. (22), was 6.09 f 0.03. This suggests that H+ is in electrochemical equilibrium across the fiber membrane. On the other hand, longitudinal impalement of four fibers with a modified Caldwell microelectrode gave a mean pH of 7.0. When a double-barreled microelectrode was placed in the hole made by prcvious impalement with the Caldwell microelectrode, the same average p H was recorded. The average internal p H of these fibers, calculated from Eq. (22), was 6.2. By contrast, when three of these fibers were impaled radially (below the zone of previous electrode injury), the average valuc found was 6.2, which was identical with the corresponding value calculated from Eq. (22). On the basis of these results and of further experiments in which intrafiber p H was calculated from the equilibrium distribution of the weak acid 5,5-dimethyl-2 ,4-oxazolidinedione and the weak base nicotine, Carter (1972) concluded that the muscle fiber probably contains two or more internal compartments with different p H values. One of these, the cell cytoplasm, normally has a p H of about 6 and is the region in which p H is normally sensed by very small p H microelectrodes. In Carter’s view, impalement of the cell by a relatively large microelectrode may result in mechanical disruption of more alkaline cellular compartments, with the result that the fluid surrounding the tip of the microelectrode has a p H higher than the true cytoplasmic pH. Although the idea that the internal p H is not homogeneous throughout the cell seems intuitively reasonable, two criticisms of Carter’s (1972) conclusions may be advanced. First, it is puzzling that no evidence for an acidic cytoplasmic compartment was reported by Kostyuk et al. (1969) or by Lavallbe (1964). Second, the quantitative models developed by Carter

94

A. A. LEV AND

W. McD. ARMSTRONG

(1972) on the basis of the above hypothesis lead to the conclusion that the cytoplasmic compartment accounts for only 2-3% of the total cell volume. Clearly, the discrepancy between the results of Carter et al. (1967a,b) and those of other workers require further investigation. pH glass microelectrodes have also been used fairly extensively to measure the p H of cell sap in higher plant cells. Hirakawa and Yoshimura (1964) used a glass-to-glass sealed microelectrode, 60-80 pm in diameter, to measure the p H in the sap of internodal cells of Nitella JEexilis (400-600 pm in diameter) and found an averagc value of 5.63. Kurella and his associates (Kurella, 1969) have used both pH glass microelectrodes and antimony-filled microelcctrodes in their investigations. They reported p H values for the cell sap of Nitella ranging from 4.2 t o 5.3 depending on the age of the cells, the season, and the growing conditions. The p H of the cell sap appeared t o be consistently lower than that of the culture medium, but did not appear to be particularly sensitive to transient changes in extracellular pH or Em. B. lntracellular Na+, K+, and CI- Activities and Their Relationship to Cellular Function

By the 1950’s radiotracer studies of the kinetics of Na+ and I<+exchange in skeletal muscIe (Harris and Burn, 1949; Harris, 1950; Carey and Conway, 1954; Simon et al., 1957) had provided evidence that fiber Na+ and I<+in this tissue are not uniformly distributed throughout the apparent fiber water. For example, on the basis of such evidence Conway (1957) considered that, of the 10.4 meq IKa+/kg wet weight found by him and his associates in sartorius muscles of the frog Rana temporaria (after having made allowance for extracellular Na+) only 2.6 meq could be designated “internal fiber Na.” Because of its more rapid rate of exchange with external solutions containing K+ ions, Conway (1957) assigned the remaining 7.8 meq fiber Na+/kg muscle to a special external region of the fibers (tentatively identified as the sarcolemma) in which Na+ ions were supposed to be “immobilized” by fixed anionic charges. A similar conclusion concerning the low intrafiber Na+ content of frog sartorius muscle emerged from the kinetic measurements of Keynes and Swan (1959). More recent kinetic studies have provided strong supporting evidence for the inhomogeneity of fiber Na+ in frog sartorius (Beaug6 and Sjodin, 1968; Keynes and Steinhardt, 1968) and other muscles (Allen and Hinke, 1970, 1971). At the same time a relatively small but highly articulate group of workers had espoused the viewpoint that the well-known ability of muscle fibers to discriminate between I<+ and Na+ is due to the ion exchange properties

IONIC ACTIVITIES IN CELLS

95

of the myoplasm as a whole rather than the operation of special membrane transport processes (Nasonov and Alexandrov, 1940; Simon, 1961; Troschin, 1961; Ungar, 1961; Ling, 1962; Ernst, 1963; Ling and Cope, 1969; Ling et al., 1973). Explicit quantitative hypotheses were developed to account for the steady-state levels of intrafiber K+ in these terms. The “sorption theory” of Troschin (1961) and Ling’s “association-induction hypothesis” (1962) are perhaps the most widely known of these. Additional evidence suggestive of intracellular Na+ compartmentation was found in chemical and autoradiographic studies of Na+ distribution in whole cells and subcellular organelles (Naora et al., 1962; Dick e2 al., 1970; Sorokina and Kholodova, 1970). Nevertheless, the exact role of intracellular Na+ compartmentation in relation to such electrophysiological parameters as the Na+ equilibrium potential and the reversible work required to transport Na+ across the fiber membrane remained conjectural. Therefore total fiber Na+, determined by conventional techniques, continued in general use in the calculation of such parameters (e.g., the socalled “critical energy barrier” of Conway et ul., 1961). In this situation, the importance of direct methods for the determination of internal Na+ and K+ activities in muscle fibers and other cells is self-evident. Hinke (1959) successfully adapted glass microelectrodes [fabricated from the Na+ and K+ selective NAS 11-18 and NAS 27-8 glasses of Eisenman et al. (1957)l to the measurement of Na+ and E(+ activities in large (200-500 pm in diameter) isolated muscle fibers from the crab Carcinus maenas and the lobster Homarus vulgaris maintained in a suitable Ringer solution (Fatt and Kata, 1953). In these experiments the average molar Na+ activity (aNa)of Carcinus fibers was found to be 0.0135. a N a in HOmarus fibers was about 0.015. The E(+ activity of Homarus fibers was 0.084. Assuming that all the fiber water acts as “normal” solvent water for Na+ and K+ (see page 100 for further discussion of this point), flame photometric analysis gave a mean apparent molar Na+ concentration (C,,) of 0.0516 for Carcinus and 0.055 for Homarus. Thee orresponding K+ concentration (C,) for Homarus was 0.153. When these are compared with the activity data quoted above it is evident that both the intrafiber Na+ and K+ activities are lower than would be predicted on the assumption that all the apparent intrafiber Ka+ and I(+can be assigned an activity coefficient equal to the mean ionic activity coefficient of the myoplasm, and that the latter parameter can be taken as approximating the mean ionic activity coefficient of the bathing medium (about 0.7). This discrepancy is particularly marked with Na+. Later studies with several species from various animals have amply confirmed Hinke’s (1959) observation concerning the relationship between a N , and C N ~Table . I summarizes a number of such observations. Com-

96

A. A. LEV AND W. McD. ARMSTRONG

TABLE I AVERAGEMEASURED MOLARNA+ ACTIVITIES(aa.), CONCENTRATIONS ( C N ~ )AND APPARENT ACTIVITY COEFFICIENTS ( Y N a = aNa/CNa) IN VARIOUS CELL SPECIESCOMPARED WITH MEANCYTOPLASMIC IONIC ACTIVITY COEFFICIENTS y,* (ASSUMED EQUALTO THE MEANIONIC ACTIVITY COEFFICIENT OF THE MEDIUM) Cell species Nerve axon

Animal Loligo jorbesi

aNa

C N ~

YN*

Reference

0 . 0370a 0.147" 0 .0275b 0.0418 0.0915 0.014 0.081

0.40

0.67 Hinke (1961)

0.46 0.17

0.0065 0.029"

0.19

0.67 Hinke (1961) 0.65 McLaughlin and Hinke (1966) 0 . 77h Lev (1964)

Rana pipiens

0.0091 0.0238 0.0073p 0.0188 0.0065 0.020'

0.38 0.39 0.33

R a m esculenta

0.0216

Nerve axond Loligo Depressor muscle Balanus fibers nubilus Sartorius muscle fiber Sartorius muscle fiber Sartorius muscle fiber Epithelial cells of urinary bladder Oocyte

Rana temporaria Frog'

Bujo bujo

0.0093 0.0258

0.36

Giant neuron

Helix pomatin

0.013

0.031

0.42

Giant neuron

Planorbis corneus Helix aspera Rana culesbeiana

0.008

0.138

0.46

0.0036i 0.014 0.029

0.48

Large neuron Epithelial cells of small intestine

yc*

-

-

0.77h Kostyuk et al. (1969) 0 . 77h Armstrong and Lee (1971) 0.76 JanAEek et al. (1968) 0.75 Dick and McLaughlin (1969) - Kostyuk et al. (1969) - Kostyuk et al. (1969) - Thomas (1972) 0 . 8 3 Lee and Armstrong (1972b)

In sea water. In artificial sea water. CN*determined in cytoplasm extruded from axons after measuring activities. Extruded axoplasm from freshly isolated axons. Extracellular volume taken as 10% of muscle wet weight. f Species unspecified. Nonisolated muscles. Calculated from published data on ionic composition of frog sartorius myoplasm using an extended form of the Debye-Huckel equation (Lee and Armstrong, 1974). Extracellular volumes determined for individual muscles studied. i This is an intracellular "free" Na concentration, assuming the cytoplasmic activity coefficient equal to that of an equivalent KCl solution. 0

97

IONIC ACTIVITIES IN CELLS

parable observations on the relationship between U K and CK are listed in Table 11. It is clear from Table I that, in every example listed, U N ~ meas, ured with Na+ or K+ selective gIass electrodes (see Section IV,B), is substantially lower than C N a determined by conventional analytical techniques. The observed differences between U K and CK (Table 11) are consistently much smaller. It is generally assumed that U N and ~ U K reflect the intracellular concentrations of “free” Na+ and K+, herein designated Ch,, and CK’, and that these latter parameters can be obtained from the relationships U N ~ / Y $ and U K / Y ~ * , where (the mean ionic activity coefficient of the cytoplasm) is the same or virtually the same as the mean activity coefficient of the bathing medium. The converse of this argument is that if all the chemicaIly determined cell Na+ or K+ is “free” or osmotically active, the and Y K (calapparent intracellular Na+ and K+ activity coefficients, N ~U K / C Krespectively), , should within experimental culated as U N ~ / Cand limits be identical to yc*. The wide discrepancy which, in the case of Na+, is consistently observed between these two parameters (Table I) is often interpreted as indicating that a large fraction of the apparent intracellular Na+ is “bound” or sequestered in one or more cellular regions that are inaccessible to a microelectrode. They are therefore considered excluded from the extracellular fraction of tissue Na+ estimated by conventional methods (e.g., the volume of distribution of chemical or isotopic markers assumed to equilibrate with all of the extracellular water, but not to penetrate the cell membrane). Conversely, the fact that the observed value of Y K is frequently close to the assumed or calculated value for yc* and sometimes exceeds it (Table 11) is frequently taken as evidence against significant “binding” or sequestering of intracellular K+ (Caldwell, 1968). On this basis the fraction of “bound” or sequestered intracellular Na+ can be calculated by means of equations such as the following (McLaughlin and Hinke, 1966; Lee and Armstrong, 1972b; see also Dick and McLaughlin, 1969). a = (~,*-CK)/UK (23) ~~f

PNa

= 1-

(a’aNa)/Ye*CNa

(24)

where a: is the fraction of cell water that is lLfree” or “solvent” water (McLaughlin and Hinke, 1966). Such calculations have been made by a number of authors. McLaughlin and Hinke (1966) concluded that some 43y0 of intracellular water and 8570 of intracellular Na+ are bound or compartmentalized in Balanus muscle fibers. Dick and McLaughlin (1969) calculated that about 50% of the cell Na+ (and about 3% of the K+) in toad oocytes is sequestered. Lee and Armstrong (1972b) estimated the percentages of “bound” water and

98

A. A. LEV A N D W. McD. ARMSTRONG

TABLE I1 MOLARKf (A) AND Cl- (B) ACTIVITIES,CONCENTRATIONS, AND APPARENT ACTIVITYCOEFFICIENTS IN VARIOUSCELLSCOMPARED WITH ESTIMATED MEANCYTOPLASMIC ACTIVITYCOEFFICIENTS AS IN TABLE I) (SYMBOLS

A. Cell species Nerve axon Nerve axond

Animal

Loligo forbesi

Loligo forbesi

arc

CK

Y I<

yo*

0.20W 0.369" 0.170* -

0.56 -

0.67

Reference Hinke (1961)

-

0.223

0.370

0.60

-

Hinke (1961)

Depressor muscle Balanus fibers nubilus

0.193

0.168

1.15

0.65

McLaughlin and Hinke (1966)

Sartorius muscle Rana fiber temporaria

0.094i 0.099i 0.097

- 0.751 0.1256 0.796 0.127 0.76

0.770 -

Lev (1964)

Sartorius muscle Frogs fiber

0.094 0.096

0.1328 0.71 0.1355 0.71

0.779

Kostyuk et al. (1969)

Sartorius muscle Rana pipiens fiber

0.090

0.123h 0.73

0.77g

Armstrong and Lee (1971)

Sartorius muscle Rana fiber ridibunda

0.105

0.142

0.74

0.770

Khuri et al. (1972)

Bladder Rana esculenta epithelial cells

0.067

-

-

0.76

Jan4Eek et al. (1968)

Oocyte

Bufo bufo

0.082

0.113

0.73

0.75

Dick and McLaughlin (1969)

Giant neurons

Helix pomatia Planorbis corneus

0.073 0.039

0.093 0.053

0.78 0.73

-

Kostyuk et al. (1969)

Giant axon

Procambarus clarkii

0.210

0.265

0.79

-

Cornwall et al. (1970)

Epithelial cells of small intestine

Rana catesbeiana

0.085

0.086

1.0

Runa pipiens Sinus venosus Atrial fibers Ventricular fibers

0.064 0.080 0.086 ~ 0 . 0 8 -1.0 1~

Amoeba proteus

0.0183- 0.019 0.019

0.961.o

-

0.83

0.92

Lee and Armstrong (1972b) Walker and Ladle (1973) A. S. Zubov"

99

IONIC ACTIVITIES IN CELLS

TABLE I1 (Continued)

B. Cell species

Animal

Squid giant axon Loligo forbesi Depressor muscle fiber

Balanus nubilus

1-I and D cells of Helix aspera subesophageal ganglia

acl

CCI

YCI

y ~ f

-

-

0.108

0.7

0.023

0.035

0.66

0.65

0.01 1

-

-

-

Giant axon

Procambarus clarkii

(H)i 0.025 (D)’ 0.025 0.013’ -2.0 0.014 - -1.0

Giant cells (abdominal ganglion)

Aplysia californica

0.028”’ 0.041”

-

-

0.72

-

Reference Keynes (1963) Hinke and Gayton (1971) Kerkut and Meech (1966b) Strickholm and Wallin (1965) Cornwall et aZ. (1970) Brown et al. (1970)

In sea water. In artificial sea water. CNAdetermined in cytoplasm extruded from axons after measuring activities. Extruded axoplasm from freshly isolated axons. Species unspecified. f Nonisolated muscles. Calculated from published data on ionic composition of frog sartorius myoplasm using an extended form of the Debye-Huckel equation (Lee and Armstrong, 1974). Extracellular volumes determined for individual muscles studied. This is an intracellular “free” Na concentration, assuming the cytoplasmic activit,y coefficient equal to that of an equivalent KC1 solution. i Minimal and maximal estimates from the same data. Computed from the data of Armstrong et al. (1969) for isolated ventricle. From Wallin (1967). Data for two sets of cells differing in their electrophysiological responses to [Sol (see text for details). * Personal communication to A. A. Lev. Activities measured for several minutes in 5 mM K.2304 solution. Q

b

Na+ in epithelial cells of bullfrog small intestine t o about 16 and 50%, respectively. Even if the “excess” cell Na+ which frequently appears in chemical determinations and which cannot readily be accounted for as “free” intracellular Na+ is truly intracellular, quantitative estimates of “bound” Na+ are open to question. C N depends ~ on the estimated extracellular space, a

100

A. A. LEV AND W. McD. ARMSTRONG

value that varies widely even in the same tissue under similar conditions. For example, the extracellular space in frog skeletal muscle has been reported t o vary from 8 to 40oJ,, depending on the method of measurement (Tasker et al., 1959; Kernan, 1972). Armstrong et al. (1969) studying isolated frog ventricle, found that apparent extracellular volumes ranged from 12 t o 42 m1/100 gm wet weight, depending on the extracellular marker used. I n principle, estimated values of CN,,, CK,and C C Iwill all be affected by inaccuracies in the determination of extracellular water and consequent errors in the amounts of Na+, K+ and C1- considered as extracellular when C N ~CK, , and C C Lare computed from measurements of the total tissue content of these ions. The error is particularly serious for ions like Na+ and C1-, which are mainly extracellular in virtually all animal tissues. Two additional uncertainties beset estimates of “bound” intracellular Na+ from equations such as (23) and (24) above. These are the extent to which intracellular water can be regarded as normal solvent water, and the degree, if any, of intracellular K+ binding or sequestration. It is generally agreed that, in the light of what is known of complex polyelectrolyte solutions, all the intracellular water is not free to act as a solvent for ions and small molecules. In addition, evidence for “bound” intracellular water has been known for many years (e.g., Overton, 1902; Rubner, 1922; Hill, 1930), and its existence has been reconfirmed by a variety of techniques. These include osmotic (Dydynska and Wilkie, 1963; Reuben et al., 1963; Bozler, 1965; Kleinzeller et al., 1967; Hinke, 1970), microelectrode (Hinke, 1970), and nuclear magnetic resonance measurements (Cope, 1967, 1972; Hazelwood et al., 1969; Cooke and Wien, 1971; Civan and Shporer, 1972; Chang et al., 1972; Outhred and George, 1973) in addition to studies on the equilibrium distribution of solutes like urea (Kotyk and Kleinzeller, 1963; De Rruijne and van Steveninck, 1972) and propylene glycol (Rybovh, 1965). There seems to be universal agreement that intracellular water is heterogeneous, but estimates of how much of it is highly ordered vary from a few percent (Hazelwood et al., 1969) to virtually all (Cope, 1967). The exact relationship between “structured,” “osmotically inactive,” and “nonsolvent” cell water also remains to be clarified, although Hinke (1970) has presented evidence that, in Balanus muscle fibers a t least, the latter two terms may be synonymous. A useful summary of current findings and views on water structure in biological systems can be found in a recent symposium edited by Hazelwood (1973). I n the light of these considerations it is evident that estimates on “nonsolvent” cell water based on relationships such as those expressed by Eqs. (23) and (24) should be regarded as highly tentative. Their principal justification a t present is that they fall within the modal range of values obtained by a variety of techniques (Hinke, 1970; Kleinzeller, 1972). The

IONIC ACTIVITIES

IN

CELLS

101

problem is further complicated by the fact that Eqs. (23) and (24), and similar equations (McLaughlin and Hinke, 1966) assume that the fraction of “bound” K+ in cytoplasm is essentially zero. I n view of the numerical relationship between Y K and ye* found in many investigations (Table 11), this assumption (which maximizes the value of a obtained from Eq. 23) is, a t first glance, plausible. However, it is evident from the above discussion that it, in turn, depends on the amount of nonsolvent water in the cell. Thus the frequently observed agreement between Y K and T ~ could * be fortuitous. This again points to the fundamental importance of the physical state of cell water for any interpretation of the physical states of intracellular ions that is based on activity measurements. Dick and McLaughlin (1969) and Kostyuk et al. (1969) have argued against too ready acceptance of the general agreement between Y K and the mean activity coefficient of the bathing medium as evidence that little or no cytoplasmic K+ is sequestered. They claim that in polyelectrolyte solutions the activity coefficients of small ions may be altered because of electrostatic interactions. In support of this Kostyuk et al. (1969) reported two series of experiments. In one of these, the apparent activity coefficients of Na+ in polymer solutions were found to be significantly lower following polymerization than in the corresponding monomer solutions. In the second Y N * was followed as a function of degree of polymer hydrolysis and was shown to increase as hydrolysis progressed. However, as pointed out above (page SO), such variations in the activity coefficients arising from variations in the density of the electrostatic field surrounding charged polymer groups can in fact be interpreted as differences in the degree of “binding” of the counterion. The converse observation, a lack of change in the activity coefficient of a small inorganic ion in the presence of a polyelectrolyte can, with equal justice, be interpreted as indicating the absence of “binding.” Despite the quantitative uncertainties involved, the measurements of intracellular K+ activity reported so far (Table 11) strongly suggest that the greater part of cytoplasmic K+ behaves as if it were in free solution.* A similar conclusion emerges from studies of the diffusion coefficient and

* As pointed out by Spanswick (1968) and by Dick and McLaughlin (1969), the results obtained from microelectrode measurements of intracellular K+ and Na+ activities (Tables I and 11) are difficult to reconcile with mechanisms for ionic accumulation in cells such as those proposed in Troschin’s (1961) “sorption hypothesis” or Ling’s (1962) “association-induction hypothesis.” These hypotheses would seem to require a preferential “binding” of K + rather than Na+ by cytoplasm. Similarly, Ling’s (1969) criticism that the measurement of intracellular ionic activities by cation-selective microelectrodes may be invalid because the electrode could cause release of ions (specifically K+) bound to cytoplasmic proteins in the immediate region of the electrode tip seems sufficiently answered by experiments with polyelectrolyte solutions such as those discussed by Kostyuk et al. (1969) and in Section I11 of this review.

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A. A. LEV AND W. McD. ARMSTRONG

electrophoretic mobility of cytoplasmic d2K (Hodgkin and Keynes, 1953; Harris, 1957). However, recent evidence (Armstrong and Lee, 1971) points t o the existence of a significant fraction of bound or sequestered K+ in a t least one cell species, frog sartorius muscle, and indicates that this sequestered I<+plays an important role in net Na+-I<+ exchange. The evidence in support of this conclusion may be summarized as follows: Sartorius muscles which had been stored for 24 hours a t 5°C in a Kf-free medium containing 120 m M Na+ were compared with companion muscles that had been allowed t o equilibrate for 2 hours a t 25°C in a Ringer solution containing 105 m M Na+ and 2.5 mM K+. As expected (Desmedt, 1953; Carey et al., 1959), the muscle fibers gained a considerable amount of Na (27 f 2 mM; mean f SE: n = 8) and lost an equivalent amount of K+ during storage a t 5°C. However the increase in fiber Na+ activity (4.7 f 0.4 mM) and the decrease in fiber I<+activity (8.4 f 1.3 mM) were much smaller than would be predicted as a result of replacing 27 mM osmotically active I<+by an equivalent amount of osmotically active Na+. Furthermore, during storage at 5"C, Y K increased from 0.73 t o 0.84 despite the loss to the medium of some 21% of the total fiber K+. Since there is no evidence for any external region in sartorius fibers which might contain this relatively large amount of I<+,it was concluded that most of the fiber I<+lost under these conditions was originally complexed or sequestered within the fibers and that the Na+ which replaced it was for the most part similarly complexed or sequestered. The existence in frog sartorius fibers of a significant fraction (about 20% of the whole) of complexed or sequestered I<+was later confirmed in further studies (Lee and Armstrong, 1972a, 1974). Two explanations have been offered for the relatively large discrepancies usually found between observed values of U N * and C N (Table ~ I). One assigns an intracellular location to the cytoplasmic Na+ which is not detected by intracellular cation-sensitive electrodes. The other (Caldwell, 1968) relates rather specifically to muscle and suggests that the low U N values found in this tissue (Table I) are due to localization of much of the apparent fiber Na+ in extrasarcoplasmic structures, specifically in the central elements of the triad system which are in direct communication with the exterior of the muscle fiber (Huxley, 1964). Supporting evidence for binding of Na+ to specific sites on cytoplasmic macromolecules or cytoplasmic organelles (e.g., the sarcoplasmic reticulum in muscle) or sequestration of Na+ within intracellular organelles, such as the nucleus or mitochondria (Kostyuk et al., 1969), has been obtained in a number of studies. Lewis and Saroff (1957) demonstrated preferential binding of Na+ over K+ by myosin. Hinke and McLaughlin (1967) showed that when Balanus fibers shortened irreversibly between 37°C and 40°C,

~

IONIC ACTIVITIES IN CELLS

103

fiber Naf activity increased rapidly. Fiber K+ activity was relatively unaffected, but fiber H+ activity increased. Hinke and McLaughlin interpreted the increase in Na+ activity that occurred under these conditions as a release of Na+ ions from myosin due to disruption of the myofilaments. The same authors (McLaughlin and Hinke, 1968) reported large and reversible decreases in optical density when Balanus fibers were transferred from normal Ringer solution t o a solution in which NaCl was isosmotically replaced by sucrose. Similar reversible decreases in optical density were observed when I<+ or Tris were used as substitutes for Na+. These changes in optical density were interpreted as being due to a reversible release and binding Na+ by negatively charged groups on cytoplasmic macromolecules. Isolated cytoplasmic structures, such as the lipoprotein membranes of sarcoplasmic reticulum in muscle (Carvalho and Leo, 1967) and nuclei and mitochondria (Naora et al., 1962; Siebert et al., 1965; Siebert and Langendorf, 1970; Sorokina and Kholodova, 1970), have been shown to bind small inorganic ions. Furthermore, nuclear magnetic resonance studies of Na+ in muscle and other cells have been widely interpreted as providing evidence for Na+ binding (Cope, 1967, 1970; Martinez et al., 1969; Ceeisler et al., 1970), although an alternative interpretation, involving a nuclear quadrupolar interaction rather than the division of cellular Na+ ions into two discrete populations, has been proposed for the 23Naspectrum of biological tissues (Shporer and Civan, 1972). Several other lines of investigation provide supporting evidence for heterogeneous distribution of fiber Na+ in muscle, although the exact interpretation of these results in terms of intra- and extracellular fractions is not clear. Thus, Troschin (1961) and Sorokina (1964) found that a substantial fraction (about 30%) of the fiber Naf in frog sartorius muscle exchanged very slowly with external 2*Na, and Beaug6 and Sjodin (1968) and Keynes and Steinhardt (1968) obtained kinetic evidence for a t least two Na+-Li+ exchange pathways in this tissue. Allen and Hinke (1970) found that 22Nainflux and efflux in Balanus fibers both showed a rapid and a slow component. Since its size (58% of the fiber Na+) agreed fairly well with the fraction of fiber Na+ detectable by Naf selective microelectrodes (51.6%), these authors assumed that the rapidly exchanging fraction represented ‘lfree” myoplasmic Na+. In later experiments (1971) the same authors studied Na+-Li+ exchange in these fibers and reported two kinetic components for this process. A comparison of a N a and C N a in the fibers during immersion in Lit Ringer solutions showed that G‘N~ decreased more rapidly than a N s l indicating that, under these conditions, the slow moving fraction of the fiber Na+ was associated with “free” myoplasmic Naf identified by activity measurements. Allen and Hinke (1971) suggest that the slow phase of Naf-Li+ exchange and the fast rate of Na+-Naf ex-

104

A. A. LEV AND W. McD. ARMSTRONG

change previously observed (Allen and Hinke, 1970) are due to identical intrafiber Na+ fractions. However, the mechanism of their assumed “inversion” of Na+ exchange rates, depending on whether the exchanging ion in the external medium is Na+ or Li+, is difficult to visualize. It seems equally plausible to propose that, in both cases, the rapid phase of Na+ exchange observed by Allen and Hinke (1971) is associated with (lbound” or complexed fiber Na+, part a t least of which could be located extracellularly. The results of Sjodin and Beaug6 (1973) also deserve mention in the present context. In the course of an extensive study of Na+ and K+ fluxes in frog sartorius muscle these authors carefully considered the estimation of intrafiber Na+ from kinetic data. According to them, their most reliable estimates of this parameter were obtained by extrapolation to zero time of the slowest exponential component of Na+ efflux into a K+ and Na+ free medium containing 10-4 M ouabain. Thirty-one experiments of this kind gave a mean intrafiber Na+ concentration of 10.6 f 2.9 (SD) mmoles/ kg fiber water. Assuming a mean myoplasmic activity coefficient of 0.77 (Table II), measurements of U N in ~ twenty “normal” muscles (Lee and Armstrong, 1974) yielded an average value of 9.4 f- 0.7 meq/kg fiber water for Cg,, the apparent “free” myoplasmic Na+ concentration in this tissue. These two estimates do not differ significantly a t the 0.05 confidence level. Thus it is evident that while measurements of intracellular Na+ activity have contributed to a clearer understanding of the amount of cell Nai- that may be considered as “free” or “osmotically active” Na+, many questions remain concerning the amount and location of apparent cell Na+ not detected by cation selective intracellular electrodes. Recent studies (Lee and Armstrong, 1974) have revealed that Ca2+ions play an important regulatory role in the exchange of “bound” and “free” K+ respectively with external Na+ (Table 111). The results obtained with muscles immersed in media containing 1.8 mM can be summarized as follows: A C N and ~ - ACK are significantly greater than AC&, and - ACK‘, respectively. This indicates that loss of K+ and uptake of Na+ during immersion involves a t least two cellular compartments only one of which is detected by activity measurements with cation selective microelectrodes. The relative proportions of Kf lost and of Na+ taken up by these two compartments can be calculated from the data given in Table I11 (Lee and Armstrong, 1974). The results indicate that the major fraction of K+-Na+ exchange during 24 hours of immersion involves loss of K+ from and uptake of Na+ by a cellular pool or pools which are inaccessible to a K+ selective microelectrode. During 48 hours of immersion, there appears t o be an approximately equal involvement of “active” and “inactive” cellular fractions in K+-Na+ exchange.

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IONIC ACTIVITIES IN CELLS

TABLE I11 OBSERVED ( A c ) AND CALCULATED (A(?') CHANGES I N INTR.4FIBER SODIUM AND CONCENTRATION, TOGETHER WITH OBSERVED INTRAFIBER POTASSIUM ACTIVITYCHANGES ( A a ) AND MEMBRANE POTENTIALS (Em), FOLLOWING IMMERSION OF FROG SARTORIUS MUSCLESAT 5°C IN K+-FREE RINGERSOLUTIONS CONTAINING 120 m M N A + ~ . ~

A B C D

8 2 7 f 2 4.7*0.4 6 3 0 f l 13.lZkl.O 6 4 2 f 3 30.8Zk2.0 7 7 6 f 2 47.8f1.6

6fl 17fl 40f3 63f2

26f2 30f3 51f3 85f2

8.4f1.3 10.5f1.6 29.lf2.9 49.5f1.3

l l f 2 100.3f2.2 1 4 f 2 100.2f0.7 3 8 f 4 42.9f0.6 6 4 f 2 42.0f1.4

Data from Lee and Armstrong (1974). Rows A and B are for muscles immersed for 24 and 48 hours, respectively, in media containing 1.8 m M Ca2+. Rows C and D are for muscles similarly immersed in media containing 0.09 mM Ca2+.Mean value (meq/kg fiber water except for Em)f standard error is shown for each parameter listed.

Quite different results emerge from the data given in Table I11 for muscles immersed a t 5°C in K+-free media containing 0.09 mM Ca2+. Following 24 hours immersion ACN,and ACh, were statistically identical. This indicates that all or virtually all the Na+ taken up under these conditions entered the fibers as osmotically active Na+. On the other hand, - ACK under these conditions was significantly greater than - ACK' ( P < 0.05). This suggests a loss of osmotically inactive K+ which was not replaced by external Na+. After 48 hours of immersion when the net loss of K+ and net gain of Naf were more extensive, both ACNa and -ACK were significantly greater than ACha and - ACK', respectively. However, the difference in magnitude between - ACK and - ACK' was significantly greatcr than that between ACN, and ACh, indicating that while some of the osmotically inactive K+ lost under these conditions was replaced by external Na+, this replacement was not complete. These results suggest two effects of Ca2+ on Na+-K+ exchange. First, the virtual restriction of Na+-K+ exchange to the osmotically active fiber compartment during 24 hours of immersion a t 5°C in a K+-free medium containing 0.09 mM Ca2+ (in contrast to the apparently predominant exchange of bound or sequestered fiber K+ for external Na+ under similar conditions but with 1.8 mM Ca2+ in the medium) indicates that Ca2+ plays an important regulatory role in controlling the permeability barrier between this compartment and the external medium. In media containing normal concentrations of Ca2+a relatively low permeability of this barrier

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A. A. LEV AND W. McD. ARMSTRONG

to Naf could be the rate-limiting factor in Na+-K+ exchange across it. An increased Na+ permeability in media low in Ca2+ could increase the rate of such exchange. If the properties of this barrier are the principal determinants of the membrane potential, the marked depolarization in fibers exposed to low Ca2+media (Table 111) is also readily understood in terms of an increase in Na+ permeability. A second effect of prolonged immersion at low temperature in low Ca2+ media is suggested by the data of Table 111. After 24 hours of immersion under these conditions, there appeared to be a net loss of bound or scquestered I<+ which was not balanced by an equivalent gain of Na+. After 48 hours of immersion there was a significant increase in the “bound” Na+ content of the fibers, which was, however, significantly .less than the total amount of I<+ lost from this compartment. This could be interpreted as arising from a change in the cation-binding affinities of a population of intracellular binding sites. In turn this could reflect conformational changes in intracellular macromolecules induced by depletion of myoplasmic Ca2+ under these conditions. As already noted for intracellular I<+, direct measurements of acl yield values for the apparent intracellular C1- activity coefficient (ycl) in a number of cells which are consistent with the idea that intracellular C1is mainly in a “free” or osmotically active state (Table 11). Hinke (1970) has utilized the relationship between the water content of the fiber and the free myoplasmic C1- concentration (derived from direct microelectrodc measurements of acl) as one method for determining the amount of solvent water in Balanus muscle fibers. In his method, water content (V) and free myoplasmic C1- concentration (CL-) are followed as a function of time following immersion of the fiber in hypotonic barnacle Ringer. On the assumption that, during the first few minutes of osmotic stress, the total free C1- in the myoplasm remains constant and the change in cell volume d V is equal to aV, (the increase in osmotically active water), the fraction of solvent water (a,) in the myoplasm can be calculated from the relationship dV/d(Cl,-)

=

-

(a,.

V/(Cl-),

(25)

The value 0.64 for a,,,thus obtained was in excellent agreement with the overall average for this parameter (0.65) derived from this and six other methods employed (Hinke, 1970). Hence a, = 0.65 can be assumed in computing the fraction of bound C1- in Balanus muscles. On this basis Hinke et al. (1973) estimated bound C1- in this tissue to be 33 mmoles/kg dry weight, or about 50% of the total fiber C1-. The exact location of this fraction of fiber C1- cannot be specified with certainty a t present. Gayton and Hinke (1971) found approximately equal fast and slow compartments

107

IONIC ACTIVITIES IN CELLS

for W I - cxchange and net C1- efflux into a low C1- medium and concluded that the fast component corresponded to “free” myoplasmic C1-. In another series of experiments Hinke and Gayton (1971) examined the relationship between a K , uc1, and Emin Balanus muscle fibers under three sets of conditions. I n the first of these, [KO]and [CI,] were varied but [KO]X [Cl,] and [KO] “a,] were maintained constant; in the second, [KO]was varied but [Cl,] and [KO] “a,] were held constant; and, in the third [KO],[Cl,], and total osmolality were varied. The results showed that under all three conditions the steady-state relationship

+

+

aK,,/aK

= acl/uc1, = exp (Em F I R T )

(26)

was obeyed.* On the other hand, no such simple relationship was found to hold between Em and the corresponding ionic concentration ratios. These results are consistent with inhomogeneous distribution of K+ and C1within the fibers and indicate the importance of transmembrane actix-ity gradients rather than concentration gradients in determining such electrophysiological parameters as Em. Walker and Brown (1970) examined the relationship between acl and electrical responses to increased CO2 in giant neuronal cells of the abdominal ganglion in Aplysia california. The effect of C02 was considered t o arise from a concomitant decrease in extracellular pH. Some cells were hyperpolarized and others were depolarized under these conditions. I n both cases, membrane resistance decreased by 40-50%. This was due to a marked increasc in C1- conductance (K+ conductance was reduced by about 25y0).As indicated in Table IT, these cells fell into two groups with respect t o U C I . For those with the lower value of acl (Table II), the calculated E C Iranged from -57 to -70 mV. The average resting potential was -56.4 mV. In two of these cells, decreased external p H produced a hyperpolarization as would be predicted from an increased Cl- conductance. In the group of cells with high acl (Table II), E C Iranged from -49 to -57 mV, and the mean Emwas -58 mV. Two such cells were depolarized (as one would predict from Em, E C I ,and increased C1- conductance) by decreased external pH. In further studies Brown et at. (1970) showed that when [Cl0-] was decreased by one half the hyperpolarizing response became a depolarization and that a similar effect was observed when [Cli-] was increased iontophoretically. Earlier, Kerkut and Meech (1966a) found a somewhat similar result. Cells in the subesophageal ganglion of Helix asperu which are hyperpolarized by acetylcholine (H cells) have lower ucl values than cells which are * I n this discussion activity symbols with the subscript “0” refer to ionic activities in the extracellular medium. Those without subscripts represent intracellular activities.

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depolarized by this agent (D cells). As in the studies of Walker and Brown (1970) and Brown et al. (1970), Kerkut and Meech concluded that, during inhibitory postsynaptic potentials, enhanced C1- conductance across the membrane drives Em in the direction of E C I(hyperpolarization). On the other hand, experiments with D cells (Table 11) which involved removal of C1- and of Na+ from the bathing medium indicated that during excitatory postsynaptic potentials (depolarization), Na+ conductance as well as C1conductance is increased. An earlier demonstration of the utility of intracellular Na+ and K+ activity measurements in the interpretation of electrophysiological phenomena was given by Hinke (1961). The theory of the nerve action potential developed by Hodgkin and Huxley (1952a,b) predicts that the limiting amplitude of the action potential is the sum of the resting potential and the Na+ electrochemical potential gradient across the fiber membrane. Working with Loligo axons immersed in artificial sea water, Hinke showed that within the range of experimental error, this limit was in no instance exceeded. Further, after repetitive stimulation (30 per second for 30 minutes) a N a was increased and a K decreased in accordance with the predictions of Hodgkin-Huxley theory. Following a subsequent period of quiescence a N a and a K tended to return toward normal values. Hinke’s data permitted calculation of the net Na+ influx and K* efflux during a single impulse. The averagc values obtained (3.9 pmole/cm2 for Na+ and 5.8 pmole/cm2 for K+) agreed with similar estimates derivcd from isotope studies (Grundfest and Nachmansohn, 1950; Rothenberg, 1950; Keynes and Lewis, 1951). It is of interest that, in these experiments, the average resting potential across the axonal membrane was found to be about 20 mV less negative than the value of E K computed from the measured ratio aK,/aK.

Walker and Ladle (1973) have explored in some detail the relationship between E K and Em in frog heart muscle. In sinus venosus, atrium, and ventricle maintained in Ringer solution the observed Em was found to be significantly less than E K (calculated from the measured a K , / a K ratio), indicating the need for active K+ uptake in these tissues. The relationship between Em and loglo a K and that between exp (- E m F / R T ) and a K was also studied for all three preparations. * In ventricle, the observed slope of Emvs loglo a K a t 20-22°C (56.6 mV) did not deviate significantly from the value of 58 mV predicted from the Nernst equation. In sinus venoms and atrium, the observed slopes (29.4 and 32.3 mV) were significantly lower

* Because the cells were too small to permit simultaneous impalement with open-tip microelectrodes and liquid ion exchanger K+ selective microelectrodes, average values of Em and U K were taken for each preparation used.

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IONIC ACTIVITIES IN CELLS

than the theoretical value. Although the range of U K values was too small to permit a rigorous analysis, Walker and Ladle concluded that the low slopes observed with sinus venosus and atrium did not in fact represent anomalously low linear slopes but were, rather, consistent with the predictions of the constant field equation (Goldman, 1943; Hodgkin and Katz, 1949) in the form

-Em = RT/F log, ( A U K+ B )

+

+

+

(27)

+

ffaNao @CI) and B = (aQNa @cI,)/(QK, where A = l / ( a K , aaNa, @‘%I), a = P N a / P K , @ = PCI/PK. With these assumptions, approximate estimates of (Y and p were made. These estimates suggest the following conclusions. a is small and of the same order of magnitude (about 10-3) in atrium and ventricle so that Na+ does not contribute materially to Em in either tissue. On the other hand, P(w7 X lop2in ventricle and 7 x lo-’ in atrium) is large enough in both to contribute significantly to Em,but since it is an order of magnitude larger in atrium than in ventricle, it can account for the much larger deviation from ideal I(+electrode behavior observed in the former tissue. In sinus venosus a (about 10-3 to 10-2) and p(-7 x 10-l) are both relatively large, so that Na+ as well as C1- contributes significantly to the overall value of Em. Thomas (1972) utilized microelectrode measurements of u N s * in neuronal cells of Helix asperu t o monitor continuously the effect of a number of experimental manipulations on the activity of the Na+ pump in these cells. His results may be summarized as follows: inhibition of the Na+ pump by ouabain increased “a,] a t a rate of 0.54 mdd per minute. This corresponds to a passive influx of Naf similar to that observed by Hodgkin and Keynes (1955) for squid axons. Changes in [KO]in the range 1-8 mM had little effect on “a,] and hence presumably on pump rate. With [KO] a t zero or 0.25 mM, “a,] increased indicating a decreased rate of Na+ pumping. Hyperpolarization of the membrane by 90 mV increased ”a,], but this was attributed to an increased Na influx, not to pump inhibition. Reducing “a,] appeared not to affect the rate of Na+ pumping. From these and additional results, Thomas (1972) concluded that the rate of Na+ pumping in snail neurons is mainly controlled by “a,] providing [KO]is held a t an adequate level. A membrane depolarization of about 3 mV in the presence of ouabain and a hyperpolarization of about 10 mV observed when [KO]was restored to normal after exposure of the cells t o a

+

* Since Thomas calibrated his electrodes in terms of [Na,], the parameter he measured corresponds more closely to C ‘ N than ~ to U N in ~ our terminology (see also Kerkut and Meech, 1966a).

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A. A. LEV AND W. McD. ARMSTRONG

K+ free medium was taken as evidence that the Na+ pump is electrogenic at normal and subnormal “a,] levels. Since the increase in “a,] induced by ouabain wps linear with time, Thomas concluded that, if there were any binding of Na+ in the cells, it must be proportional to “a,]. “Ion gradient” hypotheses, i.e. the concept that the downhill transmembrane electrochemical gradient for an ion can be utilized as the sole energy source for uphill transmembrane movement of another ion or an uncharged molecular species have been much invoked in recent discussions of coupled transport in a variety of cells (Schultz and Curran, 1970; Heinz, 1972). An essential (though not sufficient) condition for the validity of such hypotheses is that the energy available from the downhill movement of the driving ion be greater than or equal* to the work required to achieve the observed steady-state transmembrane accumulation ratio of the driven species. A specific ion gradient hypothesis which has been widely investigated is the Na+ gradient hypothesis for sugar accumulation in epithelial cells of small intestine (Crane, 1962). According to this, the chemical or electrochemical Naf gradient across thc mucosal membrane of the cells provides all the energy required for accumulation of sugar in the cell interior. There has been considerable disagreement (Schultz and Curran, 1970; Kimmich, 1973) concerning the relationship between the transmucosal Na+ gradient expressed in terms of the ratio [Na,]:[Na,] and thc extent of sugar accumulation. In view of the uncertainties inherent in measuring “a,] and the fact that a considerable fraction of the internal Na+ in these cells is probably bound or sequestered (Lee and Armstrong, 1972b; see also Table 11) and therefore not contributing to the thermodynamic Naf gradient across the membrane, this is not too surprising. Direct measurements of U N in ~ epithelial cells of small intestine (Lee and Armstrong, 1972b) can contribute to a better definition of the electrochemical Na+ gradient and its role in intestinal sugar transport. I n preliminary studies (Armstrong et al., 1973a,b) in which the reversible work (Apgal) required for steady-state accumulation of D-galactose by isolated strips of small intestine immersed a t 25°C in an oxygenated sodium sulfate Ringer solution was compared to the chemical ( A ~ N and ~ ) electrochemical ( A F N ~ transmucosal ) Na+ gradient determined from the measured a N ao/aN a ratio and the resting potential (Em)of the cells under the same conditions, the results were as follows. Apgal (in joules mole-’) was 3400, A ~ was N ~ 2700, and A j i was ~ ~ 5800. If one assumes a 1:l coupling ratio for Na+dependent sugar transport (Goldner et al., 1969)) it is evident that A p N a alone is not sufficient to account for the steady-state cel1:lumen galactose * The latter condition assumes the rather unlikely condition of 100% efficiency for the coupling process.

IONIC ACTIVITIES IN CELLS

111

concentration ratio achieved. This finding is accentuated when one considers that the intracellular galactose concentration used in the above estimate of Apgal was calculated on the assumption that all the apparent cell water was available as solvent water for the sugar. This is unlikely (Lee and Armstrong, 1972b). A , i i ~is~ theoretically sufficient to provide enough energy for sugar accumulation under these conditions but, for a 1:1 coupling ratio, would necessitate an efficiency (>50%) which may be highly unlikely (Geck et al., 1972). It must again be emphasized that these results are preliminary. In particular the Na :galactose coupling ratio in bullfrog intesting under these conditions may not be the same as the Na+: sugar coupling ratio found by Goldner et at. (1969) for isolated rabbit ileum. However, they illustrate the potential utility of intracellular activity measurements in evaluating the energetic adequacy of ion gradient hypotheses for coupled transport. I n summary, direct measurement of intracellular ionic activities seems in many instances to be the best method currently available for determining transmembrane electrochemical potential gradients. This in turn permits more rigorous evaluation of such parameters as the equilibrium potentials of intracellular ions and the energetic requirements for ion transport across cell membranes than can be obtained from conventional estimates of intracellular ionic concentrations. I n combination with a variety of other techniques, such as nuclear magnetic resonance, radiotracer kinetic studies, and cell fractionation procedures, intracellular activity measurements are helping to provide further insights into the complexities of intracellular ionic distribution and are contributing to the development of more realistic electrophysiological models of cell function. In the latter connection, however, it must be admitted that certain fundamental questions remain to be resolved. I n particular, as Kleinzeller (1972) has pointed out, a more sophisticated approach to the interpretation of ionic events a t the subcellular level requires a much fuller understanding of the physical state and organization of cell water than is presently available.

NA+,K+, AND CL- ACTIVITIES IN PLANT CELLS Vorobiev (1967) reported a series of intracellular K+ activity measurements in two species of alga, the green fresh water species Chara australis and the red marine alga Grifithsia. In these studies Vorobiev employed his “precipitate” K+ selective microelectrode described above (page 83). aK was measured both in the cytoplasm and in the vacuolar sap. I n Chara cells, the average cytoplasmic U K was 115 mM; vacuolar aK was 48 mM. Y K in the vacuole of Chara was estimated by measuring vacuolar acl

112

A. A. LEV A N D W. McD. ARMSTRONG

(using the “microfistula” method discussed on page 82), which was found to be 84 mM, and assuming that the mean ionic activity coefficient in the vacuole was 0.75, equal to that of a KC1 solution corresponding in C1concentration t o vacuolar acl. When this value of Y K was applied to the analytically determined value of C K in the vacuole (60 mM) a calculated U K of 45 mM was obtained, in excellent agreement with the observed value. This strongly suggests (Spanswick, 1968) that vacuolar K+ in Cham is essentially in “free” solution. It is of interest that U K , in Vorobiev’s experiments was only 9.6 X M. Except that the relationship between cytoplasmic (153 mM) and vacuolar U K (343 mM) was reversed, essentially similar results were obtained with G‘rifithsia ( U K , was 6-8 mM in the artificial sea water bathing these cells). Vacuolar C K was 546 mM in this species, whence, if one assumes Tc* equal to T* for the medium (0.65), a calculated value of 370 mM is obtained for vacuolar U K . Vorobiev (1967) also examined the relationship between E K (calculated from the I<+activity ratio) between the medium and either the cytoplasm or the vacuole for Chara and Grifithsia, respectively, and the observed potential, Eel between these phases. For Cham both cytoplasmic (173 mV) and vacuolar (155 mV) Eel values were virtually identical with E K (178 and 156 mV, respectively) suggesting that K+ is in electrochemical equilibrium between the three phases. In Grifithsia cytoplasmic E K and Eel (78 and 80 mV) also agreed but vacuolar Eel (50 mV) was significantly less than the corresponding EK (99 mV). This difference corresponds to a APK of 4600 joule mole-’ from vacuole to cytoplasm and indicates that there must be an active transport of K+ in the opposite direction. A number of workers have reported on U K and ac 1 in the vacuolar sap of Nitella and in fact, this system seems to enjoy a certain popularity as a “calibrating solution” for new types of ion-selective microelectrodes destined for use in animal cells. Thus, Kerkut and Meech (1966a) reported an average U K (or CK’) of 180 mM in the vacuolar sap of internodal cells of Nitella opaca. This compares with 170 m M found by chemical analysis in NitelEa translucens (MacRobbie, 1964). Cornwall et al. (1970) and Brown et al. (1970) give the following values for the vauolar sap of internodal cells of Nitella (the exact species is not identified and the data given in both these papers are apparently for the same set of experiments), UK 60.6 mM, CK68.5 mM, acl96.9 mM, Ccll30.8 mill. Kurella (1969) quotes a value of 125.6 mM for acl in the vacuolar sap of Nitella (JEexilis and/or mucronata). As pointed out by Kurella (1969), vacuolar ionic activities in giant plant cells are very dependent on the composition of the culture medium. Thus direct comparison between values cited by different groups of workers is difficult. Nevertheless, the results so far obtained demon-

113

IONIC ACTIVITIES IN CELLS

strate the utility of intracellular ionic activity measurements in the study of ion transport and bielectric phenomena in plant, as well as in animal cells. VII. CONCLUSION

This review has attempted to outline the rationale, methodology, and current applications of the measurement of intracellular ionic activities with ion-selective microelectrodes. We hope that it will serve to emphasize the potential importance of this technique in general cellular and electrophysiology. Since the pioneer experiments of Caldwell (1954, 1958) and Hinke (1959, 1961) notable advances have been made. I n particular, the development of ultrafine microelectrodes has extended the useful range of the technique to include small cells, such as cardiac muscle fibers and epithelial cells. One feels confident that the intensive work currently in progress in the field of ion-selective macroelectrodes will lead to the introduction of novel kinds of intracellular electrodes in the near future and to a rapid expansion of their use in the investigation of such problems as the physical state of intracellular ions, the ionic basis of electrophysiological phenomena, and the mechanisms of ion transport across cell membranes. ACKNOWLEDGMENT One of us (W. McD. A.) wishes to acknowledge the support received from the U.S. Public Health Service (grants AM 12715 and HL 06308) in studies described herein and in the preparation of this review. REFERENCES Adrian, R. H. (1956). The effect of internal and external potassium concentration on the membrane potential of frog muscle. J . Physiol. (London) 133, 631-6.58. Agin, D. P. (1969). Electrochemical properties of glass microelectrodes. I n “Glass Microelectrodes” (M. LavallBe, 0. F. Schanne, and N. C. Hbbert, eds.), pp. 62-75. Wiley, New York. Agin, D. P., and Holtzman, D. (1966). Glass microelectrodes: The origin and elimination of tip potentials. Nulure (London) 211, 1194-1195. Allen, R. D., and Hinke, J. A. M. (1970). Sodium compartmentalization in single muscle fibers of the giant barnacle. Can. J . Physiol. Phurmacol. 48, 139-146. Allen, R. D., and Hinke, J. A. M. (1971). Na+-Li+ exchange in single muscle fibers of the giant barnacle. Cun. J . Physiol. Pharmucol. 49, 862-866. Anderson, 0. L., and Stuart, D. A. (1954). Calculation of activation energy of ionic conductivity in silica glasses by classical methods. J . Amer. Cerum. SOC.37,573. Andreoli, T. E., Tieffenberg, M., and Tosteson, D. C. (1967). The effect of valinomycin on the ionic permeability of thin lipid membranes. J . Gen. Physiol. 50,. 2527-2545.

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Naora, H., Naora, H., Izawa, M., Allfrey, V. G., and Mirsky, A. E. (1962). Some observations on differences in composition between the nucleus and cytoplasm of the frog oocyte. Proc. Nat. Acad. Sci. U.S. 48, 853-859. Nasonov, D. N., and Alexandrov, V. Ja. (1940). “Living Substance Reaction on External Influence.” Moscow (in Russian). Nasonov, D. N., and Alexandrov, V. Ja. (1943). Principles of diffusion and distribution in the problem of cell permeability. Usp. Sovrem. Biol. 16,577-598. Nicolsky, B. P. (1937). Theory of the glass electrode. I. Actu Physicochim. URSS 7,597. Nicolsky, B. P., Schultz, M. M., and Lev, A. A. (1967). Recent developments in the ion-exchange theory of the glass electrode and its application in the chemistry of glass. In “Glass Electrodes for Hydrogen and Other Cations” (G. Eisenman, ed.), pp. 174-222. Dekker, New York. Nikerov, A. E., and Rabinovich, V. A. (1968). On thermodynamical characteristics of single ion species in electrolyte solutions. Zzv. Leningrad. Elektrotekh. Znst. 64, 183-188. Orme, F. W. (1969). Liquid ion-exchanger microelectrodes. I n ‘‘Glass Microelectrodes” (M. LavallBe, 0.F. Schanne, and N. C. HBbert, eds.), pp. 376-395. Wiley, New York. Outhred, R. K., and George, E. P. (1973). Water and ions in muscles and model systems. Biophys. J . 13, 97-103. Overton, E. (1902). Beitrage zur allgemeinen Muskel-und Nervenphysiologie. Arch. Gesamte Physiol. Menschen Tiere 92, 115-280. Planck, M. (1890a). Ueber die Erregung von Electricitat und Warme in Electrolyten. Ann. Phys. Chem. 131 39, 161-186. Planck, M. (1890b). Ueber die Potentialdifferenz zwischen zwei verdunnten Losungen binarer Electrolyte. Ann. Phys. Chem. [3] 40, 561-576. Rabinovich, V. A. (1964). Electromotive force of galvanic cell and thermodynamical activity of single ions in connection with compensatory effect conception. Zh. Fiz. Khim. 38, 1331-1334. Rabinovich, V. A., Nikerov, A. E., Rothshtein, V. P., and Sokolov, P. N. (1960). On the question of possibilities in determination of thermodynamical activities of single ions. Vestn. Leningrad. Univ., Fiz., Khim. 4, 101-105. Rabinovich, V. A., Nikerov, A. E., and Rothshtein, V. P. (1967). On real thermodynamic single-ion activity in electrolyte solutions. Electrochim. Acta 12, 155-172. Rector, F. C., Jr., Carter, N. W., and Seldin, D. W. (1965). The mechanism of bicarbonate reabsorption in the proximal and distal tubules of the kidney. J . Clin. Invest. 44, 278-290. Reuben, J., Lopez, E., Brandt, P. W., and Grundfest, H. (1963). Muscle volume changes in isolated fibers. Science 142, 246-248. Robinson, R. A., and Stokes, R. H. (1965). “Electrolyte Solutions.” Butterworth, London. Ross, J. W. (1967). Calcium-selective electrode with liquid ion exchanger. Science 156, 1378-1379. Ross, J. W., Jr. (1969). Solid-state and liquid membrane ion-selective electrodes. In “Ion-Selective Electrodes” (R. A. Durst, ed.), pp. 57-88, Nat. Bur. Stand., Washington, D.C. Rothenberg, M. A. (1950). Studies on permeability in relation to nerve function. 11. Ionic movements across axonal membranes. Biochim. Biophys. Actu 4, 96-114. Rubner, M. (1922). Uber die wasserbindung in Kolloiden mit besonderer Berucksichtigung des quergestreiften Muskels. Abh. Preuss. Akad. Wiss., Phys.-Math. K l . 1, 1-70.

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Rybovb, R. (1965). The free and xylose space in the rat diaphragm and the effect of potassium ions on it. Physiol. Bohemoslov. 14,412-416. Sandblom, J. P., Eisenman, G., and Walker, J. L., Jr. (1967a). Electrical phenomena associated with the transport of ions and ion pairs in liquid ion-exchange membranes. I. Zero current properties. J . Phys. Chem. 71,3862-3870. Sandblom, J. P., Eisenman, G., and Walker, J. L., Jr. (196713). Electrical phenomena associated with the transport of ions and ion pairs in liquid ion-exchange membranes. 11. Nonzero current properties. J . Phys. Chem. 71, 3871-3878. Scatchard, G., Coleman, J. S., and Shen, A. L. (1957). Physical chemistry of protein solutions. VII. The binding of some small anions to serum albumin. J . Amer. Chem. SOC. 79, 12-20. Schagina, L. V., Richter, D., Frisman, E. V., Vorobjev, V. I., and Lev, A. A. (1969). Studies of structure of ribosomal RNA in solutions of different ionic strength. Abstr. All-Union Conj. Neurodyn. Gen. Biophys. p. 24 (in Russian). Schultz, S. G., and Curran, P. F. (1970). Coupled transport of sodium and organic solutes. Physiol. Rev. 50, 637-718. Shack, J., Jenkin, R. J., and Thompset, J. M. (1952). The binding of sodium chloride by calf thymus deoxypentose nucleic acid. J . Biol. Chem. 198, 85-92. Shporer, M., and Civan, M. M. (1972). Nuclear magnetic resonance of sodium 23 linoleate water. Basis for an alternative interpretation of sodium 23 spectra within cells. Biophys. J . 12, 114. Siebert, G., and Langendorf, H. (1970). Ionenhaushalt im Zellkern. N ~ ~ u r ~ i ~ s e ~ c h u ~ ~ e n 57, 119-124. Siebert, G., Langendorf, H., Hannover, R., Nitz-Litzow, D., Pressman, B. C., and Moore, C. (1965). Hoppe-Seyler’s 2. Physiol. Chem. 343, 101. Simon, S. E. (1961). Is the concept of active transport significant in the maintenance of the ionic pattern of the resting cell? I n “Membrane Transport and Metabolism’’ (A. Kleinzeller and A. Kotyk, eds.), pp. 148-154. Academic Press, New York. Simon, S. E., Shaw, F. H., Bennett, S., and Muller, M. (1957). The relationship between sodium, potassium, and chloride in amphibian muscle. J . Gen. Physiol. 40,753-777. Sjodin, R. A., and Beaug6, L. A. (1973). An analysis of the leakage of sodium ions into and potassium ions out of striated muscle cells. J. Gen. Physiol. 61, 222-250. Skou, J. C. (1965). Enzymatic basis for active transport of Na+ and K + across cell membrane. Physiol. Rev. 45, 596-617. Sorokina, Z. A. (1964). On the state of the main inorganic ions inside the muscle fibres. Tsitologiya 6, 152-161. Sorokina, Z. A. (1965). Measurement of the hydrogen ion activity inside and outside of nerve cells in mollusc ganglia. Zh. Evol. Biokhim. Fiziol. 1, 343-350. Sorokina, Z. A,, and Kholodova, Y. D. (1970). The content of inorganic ions in subcellular fractions of skeletal muscles. Biofisika 15, 844. Spanswick, R. M. (1968). Measurements of potassium ion activity in the cytoplasm of the Churaceae as a test of the sorption theory. Nature (London) 218, 357. Spyropoulos, C. S. (1960). Cytoplasmic p H of nerve fibers. J . Neurochem. 5, 185-194. Stella, G. (1929). The combination of carbon dioxide with muscle; its heat of neutralization and its dissociation curve. J . Physiol. (London) 68, 49-66. Strickholm, A., and Wallin, B. G. (1965). Intracellular chloride activity of crayfish giant axons. Nature (London) 208,790-791. Tasker, P., Simon, S. E., Johnstone, R. M., Shankly, K. H., and Shaw, F. H. (1959). The dimensions of extracellular space in sartorius muscle. J . Gen. Physiol. 43,39-53. Teunissen van Zijp, P. H., and Bungenberg de Jong, H. G. (1938). Negativ, nicht

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amphotere Biokolloide als hochmolejulare Elektrolyte. 11. Reihenfolgen der Kationen bei der Umladung mit Nuetralsalzen. Analogien mit den Reihenfolgen der Loslichkeit von entsprechenden kleinmolekularen Elektrolyten. Kolloid-Beih. 48, 33-92. Thomas, R. C. (1970). New design for sodium-sensitive glass electrode. J . Physiol. ( L o d o n ) 210, 82P-83P. Thomas, R. C . (1972). Intracellular sodium activity and the sodium pump in snail neurones. J . Physiol. (London) 220, 55-71. Troschin, A. S. (1956). “Problems of Cell Permeability.” Moscow, USSR (Oxford Univ. Press, London and New York, 1968). Troschin, A. S. (1961). Sorption properties of protoplasm and their role in cell permeability. I n “Membrane Transport and Metabolism” (A. Kleinzeller and A. Kotyk, eds.), pp. 45-53. Academic Press, New York. Ungar, G. (1961). Transport of Na+ and K + across resting, stimulated, and insulintreated rat diaphragm. I n “Membrane Transport and Metabolism” (A. Kleinzeller and A. Kotyk, eds.), pp. 160-162. Academic Press, New York. Vorobiev, L. N. (1967). Potassium ion activity in the cytoplasm and the vacuole of cells of Chara and Grijtthsia. Nature (London) 216, 1325-1327. Vorobiev, L. N. (1968). Potassium selective microelectrodes with precipitate in the tip. Nature (London) 217,4.50-451. Vorobiev, L. N., and Khitrov, Yu. A. (1971). A new type of K+-sensitive microelectrode. Stud. Biophys. 2 6 , 4 9 4 6 . Vorobiev, L. N., and Kurella, G. A. (1966). Role of polyelectrolyte structures of plant cells in selective ion accumulation. Abh. Deut. Akad. Wiss. Berlin, KZ. Med. 4, 309-3 13. Vorobjev, V. I., Dranitskaya, M. I., Lev, A. A., Matveeva, A. I., and Schagina, L. V. (1971). Investigations on the state of low molecular salt components in biopolymer solutions. I. Activity coefficients of sodium and chloride ions and mean activity coefficients of sodium chloride in solutions of native and denatured DNA. MoZ. Biol. 5, 171-186. Waddell, W. J., and Bates, R. G. (1969). Intracellular pH. Physiol. Rev. 49, 285-329. Walker, J. L., Jr. (1971). Ion specific liquid ion exchanger microelectrodes. Anal. Chem. 43989A-93A. Walker, J. L., Jr., and Brown, A. M. (1970). Unified account of the variable effects of carbon dioxide on nerve cells. Science 167, 1502-1504. Walker, J. L., Jr., and Ladle, R. 0. (1973). Frog heart intracellular potassium activities measured with potassium microelectrodes. Amer. J . Physiol. 225, 263-267. Wallin, B. G. (1967). Intracellular ion concentrations in single crayfish axons. Acta Physiol. Sand. 70, 419430.

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Active Calcium Transport and Ca2+-Activated ATPase in Human Red Cells H . J . SCHATZMANN Institute of Veterinary Pharmacology. University of Bern. Bern. Switzerland

I . Calcium Transport . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . B. Calcium Concentration in Human Red Cells . . . . . . . C . Calcium Binding to the Membrane . . . . . . . . . . D. Passive Permeability of the Membrane for Calcium . . . . . E. Active Calcium Transport . . . . . . . . . . . . I1. Membrane ATPases Activated by Calcium in Human Red Cells . . . A . Introduction . . . . . . . . . . . . . . . . B . Different Calcium-ATPases . . . . . . . . . . . . C . Characteristics of the Calcium Magnesium-Stimulated Membrane ATPase . . . . . . . . . . . . . . . . . . 111. Relationship between Calcium Transport and Calcium MagnesiumActivated ATPase . . . . . . . . . . . . . . . . A . Common Features . . . . . . . . . . . . . . . B . Stoichiometry . . . . . . . . . . . . . . . . C. Thermodynamic Considerations . . . . . . . . . . . D . A Possible Model . . . . . . . . . . . . . . . IV. Relation between Calcium Transport-ATPase and Sodium-PotassiumTransport-ATPase . . . . . . . . . . . . . . . . V . Comparison with Other Systems Transporting Calcium . . . . . A. ATP-Dependent Systems . . . . . . . . . . . . . B. Sodium-Calcium Heteroexchange . . . . . . . . . . VI . Physiological Significance of Calcium Pumps . . . . . . . . . A . Muscle . . . . . . . . . . . . . . . . . . B. Red Cells . . . . . . . . . . . . . . . . . C. Epithelia . . . . . . . . . . . . . . . . . D . Secretory Cells . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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1. CALCIUM TRANSPORT

A. Introduction

It has long been known that human red cells contain little calcium (Kramer and Tisdall, 1922). Amazingly the obvious fact that the calcium concentration per milliliter of cells lies far below the concentrations of total and ionized Ca in blood plasma did not arouse much interest until recently. Those workers who studied the Ca content of red cells mainly stressed the point that the cell membrane exhibits low permeability for Ca, which admittedly explains why a Ca gradient across the membrane is not readily dissipated, but they left unresolved the mystery of how the gradient is created. B. Calcium Concentration in Human Red Cells

As analytical procedurcs successively improved with the advent of emission flame photometry, complexometric titrations, and atomic absorption flame photometry, reported values for total Ca concentration in red cells kept decreasing. In a careful study Harrison and Long (1968), using the techniques of dry ashing and absorption flame photometry, found 0.0158 pmole/ml cells and were able to demonstrate that Ca associated with the cell membrane fully accounts for this figure so that virtually no Ca is left which might be ascribed to the intracellular space. If one assumes that the authors could have detected a difference of two standard errors between the result obtained with whole cells and membranes, their finding means that 0.003 pmole/ml cells a t best is present in the intracellular space. Concentration of Ca2+ will certainly be less than total Ca concentration since soluble proteins and small molecules like ATP, diphosphoglycerate, triose phosphates, etc., will bind a considerable fraction of the total Ca content (Schatzmann, 1973). The finding that all the Ca that can be detected by chemical analysis is present in the membrane fraction was recently confirmed by Lichtman and Weed (1973). There are limitations to the detection of Ca in biological material because Ca is a ubiquitous element and contamination becomes a problem below 5 X l O P M in the solutions to be analyzed. We shall see later that indirect evidence suggests that intracellular Ca2+ concentration is less than M . Thus, as the plasma Ca2f concentration is l.X10-3 to 1.5X10-3 M , a gradient of lo3 to lo4 exists across the cell membrane. It is out of question that this behavior can be explained by any passive mechanism: binding inside the cell cannot account for it simply because there is

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a considerable gradient for total Ca and the membrane potential of 9 mV (inside negative) can cause only a slight gradient in the opposite direction. C. Calcium Binding to the Membrane

A number of interesting studies on isolated and disrupted membranes concerning this problem is available. Gent et al. (1964) gave a very comprehensive account, which can be summarized by saying that under physiological conditions of ionic strength and pH the number of binding M . Long and sites is 5.9 pmoles/gm dry weight and the KdisB is 2.8 X Mouat (1971) showed that in sucrose solution three sites of different affinity can be distinguished and that ghosts bind 2.5 times as much Ca as whole cells; this indicates that a large fraction of these sites is accessible for Ca only from the inside. LaCelle et aE. (1972), have found several classes of Ca binding sites of different affinities in a spectrinlike preparation. The lowest which they identified was 0.33.10-6 M . It seems that the external sites are mostly accounted for by the sialic acid residues. The internal sites are in equilibrium with the low free intracellular Ca concentration, and occupancy of at least some of them has profound functional effects (see later). Forstner and Manery (1971b) showed that ATP reduces Ca binding to the membrane, probably by complexing Ca in the solution. Conversely, Duffy and Schwarz (1973), working with membranes prepared in a way not likely to yield inside-out vesicles, found that 1 mM ATP plus [Mg2+]>2mM stimulated the 45Cauptake into the protein fraction of the membranes by about 200%. Calcium (0.2 nmole per milligram of protein) was taken up from a 10 pM CaClz medium in a rather slow process (requiring 20 minutes a t 37°C). Trypsin treatment liberated 45% of the bound Ca, whereas neuraminidase was without effect. Ruthenium red and M completely abolished the Ca binding. It is well ethacrynic acid possible that this type of binding has some connection with the Ca-transport system (see below). Forstner and Manery (1971a) demonstrated that blocking -COOH groups abolishes binding to proteins but does not affect binding to membrane lipids. D. Passive Permeability of the Membrane for Calcium

Diffusion of Ca across the intact membrane is extremely slow. Rummel et al. (1962) incubated red cells in isotonic Ca solutions and were able to demonstrate a moderate increase of the Ca content of the cells to some millimoles per liter of cells. However, it is questionable whether this treatment leaves the membrane structure intact.

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Schatzmann and Vincenzi (1969) exposed red cells to labeled Ca ("Ca) in physiological concentrations for 1 week a t 2°C and found that the 45Caconcentration per milliliter of cells reaches only a few percent of that of the medium after this time. This must be compared with the behavior of Na and K, which nearly equilibrate during this time a t 2°C. In energydepleted cells stored a t 37"C, the picture is different. A net Ca uptake can be observed (Weed, 1968). Schatzmann (1969) found that during incubation of whole blood a t 37°C under sterile conditions about 0.06 mmole of Ca was taken up by 1 liter of cells per hour. This rapid phase, however, began only about 10-12 hours after the start of the incubation (Fig. 1). The uptake of 60 pmoles/liter per hour in the experiment of Fig. 1 is larger than the values found by Lew (1971) and Jenkins and Lew (1973). I n fresh, washed cells in a low K medium, the uptake due to depletion with iodoacetamide (5 mM) and inosine (5 mM) was about 12 pmoles/liter per hour (Lew, 1971). It may be that cells in serum behave differently. How-

Time (hours)

FIG.1. Single experiment. Whole defibrinated human blood incubated in sterile conditions a t 37°C. Disappearance of Ca from serum was followed by measuring total Ca in trichloroacetic acid filtrates of serum. Ca having entered the cells (ordinate) was calculated from original hematocrit, hematocrit a t experimental time, and total Ca concentration. Hemolysis after 25 hours was 2.3%. Notice the rapid entry after 12 hours, when ATP content of cells had reached a low level. (From Schatzmann, 1969, reproduced by courtesy of Maemillan Publishers.)

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ever, the possibility that Ca phosphates were precipitated in the experiment of Fig. 1 (Passow, personal communication) cannot be ruled out with certainty. It is of considerable interest that the entry of Ca into depleted cells is inhibited by high external K concentration (Lew, 1971). It is not known whether this Ca uptake is mediated by preformed channels or whether it is initiated because deprivation of metabolic energy leads to structural alterations in the membrane. In experiments with depleted and resealed cells, Porzig (1970) came to the conclusion that Ca which enters the cells causes the membrane to become impermeable to Ca itself so that equilibrium between inside and outside is seemingly not reached. An alternative explanation for his observations is that the depletion of ATP is not carried to the point where the pump activity ceases completely. Porzig (1973) further found a Ca-Ca exchange diffusion, exhibiting saturation kinetics, and possibly countertransport in starved cells which were resealed after osmotic hemolysis in the cold. The rate of these Ca movements was rapid compared to the uptake of 45Cain intact cells so that the question arises whether reversal of hemolysis or energy depletion caused the phenomenon. This would be rather interesting because the movements observed were obviously not diffusion through a leak channel but clearly carrier-mediated transport. From the work of Lee and Shin (1969), who also used resealed cells, no such Ca-Ca exchange can be inferred. Romero (1972) has presented evidence for a possible exchange between Ca2+ and Hf ions across the membrane. E. Active Calcium Transport

1. METHODS FOR DEMONSTRATING ACTIVECALCIUM TRANSPORT The unequal distribution of Ca on the two sides of the red cell membrane strongly suggests the existence of an active extrusion mechanism for Ca. This transport system surely would have to satisfy the requirement that it moves Ca2+uphill against the chemical and electrical potential gradient. However, it might be either directly coupled to the energy-supplying metabolism of the cell or dependent on the movement of another actively transported species, for instance Na, in analogy to secondary transports like those of glucose, amino acids, or iodide (in the thyroid). In order to demonstrate either uphill net Ca movement out of the cell or unequal unidirectional fluxes in the absence of a gradient as proof for active transport, one must be able to introduce Ca into the cells. To this end several methods are available; all of them, however, may be accused of not leaving the membrane in its original state. a. Reversal of Hemolysis. This technique, first introduced by Straub

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(1954), was found to be of great help in the study of Na-K transport of red cells (Whittam, 1962). It was used in the first demonstration of active Ca extrusion from red cells (Schatzmann, 1966) and is still the most reliable and versatile method for work with Ca, if properly modified from the original version. Washed cells are lysed in hypotonic solution containing Ca2+ as chloride or, if control of intracellular Ca2+ concentration is desirable, a Ca-EGTA buffer of known Ca2+concentration. In addition, the lysing solution contains ATP as the immediate energy source, Mg in a concentration equal to or exceeding the ATP concentration and a 5-10 mM pH buffer. After a short time ( 2 minutes) isotonicity is restored by the addition of an appropriate amount of concentrated solution of KC1 (or any other salt). At this point the majority of cells are still leaky to K, Na, etc. (see Bodemann and Passow, 1972) and the intracellular salt concentration achieved is similar to that in the external fluid used to wash the cells. Sealing to K and Na occurs only after prolonged incubation at 37°C whereas sealing to Ca occurs immediately. It was found that about 80% of cells are sealed to Ca (and ATP and EGTA) if Ca concentrations in the range of 10+ to M buffered with EGTA are used (Schatzmann, 1973). We carry out the hemolysis at room temperature. After addition of KC1, 3-5 minutes are allowed, then the sample is cooled to O"C, the desired external Ca concentration is established by adding CaC12, and the cells are washed in the incubation medium in the cold. In our hands, the Ca concentration per milliliter of cells achieved is usually considerably above that of the lysing medium (by a factor 1.5) if loading is performed with CaCL M, and equal or below the expected concentration if Ca-EGTA buffers 5 mM with [Ca2+]in the range of 10-6 to 10-5 M are employed. At M) revery low Ca2+ concentrations in the lysing medium (below sealing to Ca and ATP is poor. The experiments are started by heating the cell suspension to the desired temperature; 28°C is a convenient temperature because Ca transport is too rapid a t 37°C to be followed by separation of cells and medium by conventional centrifugation. For analysis the cells are packed without washing or washed in Ca-free cold solutions. b. Treatment with p-Ch2ormercuribenzenesulfonic Acid (PCMBS). Garrahan and Rega (1967) showed that exposure of red cells to this mercurial brings about a reversible leakiness for Na and K such that intracellular K and Na concentration can be adjusted to a desired level within hours in the cold. The mercurial is removed by thiol compounds, such as cysteine. This technique is also applicable to Ca loading. Starved cells are exposed to 5 mM CaClz and 0.05 mM PCMBS in an isotonic medium for 15 hours a t 2°C. Sizable amounts of Ca enter during this time, and upon removal of the mercurial Ca transport activity is at least partially recovered (Schatzmann, 1973).

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c. Isotonic C a Solution. As mentioned before, Rummel et al. (1962) showed that Ca is taken up from isotonic CaClz solution. To the best of my knowledge no use has been made of this method in transport studies so far. d. Depletion of Ener.gy Stores. Thoroughly starved cells fairly rapidly take up Ca from the surrounding medium. Depletion is achieved by incubation of washed cells in glucose-free media a t 37°C for 15 hours or longer (Weed, 1968; Schatzmann, 1969). It is not clear whether this phenomenon is explained simply by the arrest of the Ca pump or whether energy depletion deprives the cell of the possibility to maintain the integrity of the membrane structure. Be that as it may, the Ca extrusion is recovered when energy-yielding substrate is offered either as glucose or as inosine plus adenine, the latter combination being particularly effective (Romero and Whittam, 1971). e. Increase of p H . Romero and Whittam (1971) have shown that in old cells (stored for one month or longer a t 2OC) a very slight upward shift of the pH of the medium is sufficient to make the membrane leaky for Ca. A pH of 7.6 or 7.8 caused the cells to take up 1 mmole/l of Ca in a few hours from a solution containing 10 mM CaCl2. We were able to confirm this observation but, contrary to the authors, failed to revive Ca transport after this treatment. In the majority of studies on Ca transport that are available, method (a) was applied. Measurements of net Ca movements have prevailed so far. Experiments with labeled Ca vere reported (Lee and Shin, 1969; Porzig, 1970; Schatzmann, 1969), but simultaneous measurements of unidirectional fluxes in both directions under transport conditions are still lacking. Cha et al. (1971b) and Weiner and Lee (1972) have used the technique of Steck et al. (1970) to produce inside-out vesicles from human red cells, which seem to transport Ca from the medium to the intravesicular space, as expected. The difficulty is that one obtains a mixture of inside-out and right-side-out vesicles, which must be separated. 2. EVIDENCE FOR REDCELLS

THE

EXISTENCE OF ACTIVECA EXTRUSION FROM HUMAN

In 1966 some indication for uphill, ATP-dependent Ca extrusion from red cells appeared (Schatzmann, 1966). Figures 2 and 3 show the results of experiments in which net Ca movements in cells resealed with CaClz or Ca-EGTA were followed. It is obvious that, in the presence of ATP (and Mg) inside the cells, a rapid movement of Ca from the cells to the medium takes place, whereas in the absence of ATP the gradient established by the loading procedure is maintained. As the net Ca concentration in the medium

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FIG.2. Changes of Ca concentration in cells and medium and Pi release from ATP in resealed cells. Hemolysis in water containing 2 mM Tris-ATP, 5 mM Tris-C1, 4 mM MgC12, and 1 or 2 mM (in three experiments in the ATP-free sample) CaC12. Reversal of hemolysis in presence of KCI. Previous to hemolysis starvation for 17 hours a t 37°C in glucose-free solution (130 mM Na, 5 mM K, 20 mM Tris, 155 mM Cl). Medium: 130 mM Na, 5 mM K, 20 m M Tris, 1 mM Ca, 157 mM C1, 10-4 g/ml ouabain. Temperature 37°C. Hematocrit: ATP sample, 0.249; ATP-free sample, 0.255. 0 , With ATP in the cells; 0 without ATP. Arrow in panel A: mean concentration measured in whole suspension. Four experiments, vertical bars 2 X SE of mean. (From Schatamann and Vincenai, 1969, reproduced by courtesy of Physiological Society. Cambridge University Press.)

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FIG.3. Extrusion of Ca from starved resealed human red cells at 28°C. The cells were loaded with Ca-EGTA buffer of [Ca2+]= loTGM ; NazATP, 2 mM; MgC12,4 mM; imidazole-C1, 5 mM. Resealing was in 100 mM KCl 50 mM imidazole-Cl, p H 6.8. Notice that ATP-free cells retained less Ca. Medium: NaC1, 100; KCl, 5 ; MgCl,, 4 ; CaC12, 0.1; imidazole-Cl, 50 mM; p H 6.8. Ordinate: total cellular [Ca] measured by atomic absorption flame photometry. Ca2+movement is uphill in ATP-containing cells; no movement in ATP-free cells. (0)2 mM ATP; ouabain ( A ) 10-4 g/ml, has no effect on Ca transport (Schatzmann, unpublished). It was shown that what appears in the M; medium is C$+ rather than Ca-EGTA (Schatzmann, 1973). [CaZ+]td,i= [ C a Z + I t ~= , . 10-4 M .

+

rises above that in the cells, and even that in the cell water, there can be no doubt that the movement is uphill (Schatzmann and Vincenzi, 1969). Figure 4 shows the dependence of outward Ca movement on ATP, but does not exclude the possibility of ATP increasing the passive permeability or inducing a Ca-Ca exchange across the membrane. The possibility that even in spite of the demonstrated uphill character the movement was not due to ATP supplying chemical energy for the transport, but that ATP or any of its hydrolysis products carried Ca in complexed form across the membrane and that Ca uphill movement was thus due to a downhill movement of these compounds could be ruled out by

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T I M E IN MINUTES

FIG.4. Movement of "Ca during incubation of resealed red cell ghosts. With ATP: lysing solution contained 2 mM ATP; Without ATP: lysing solution did not contain ATP. With ATP: cell, X-X ; medium, 0-0.Without ATP: cells, 0-0; medium, A-A. (Redrawn from Lee and Shin, 1969, reproduced by courtesy of Rockefeller University Press.)

adding to the external medium concentrations of these compounds (Pi, ATP, ADP, AMP) exceeding those that could possibly arise inside the cells. The external Ca2+ concentration was equal to or higher than the internal Ca2+ concentration (Schatzmann and Vincenzi, 1969). This did not abolish the Ca movement. Olson and Cazort (1974) have recently confirmed this finding by direct measurement of these compounds during Ca transport. Their measurements clearly show that the magnitude of Pi or nucleoside phosphate shifts does not account for the Ca movements. Since energy-depleted cells were used in the experiments of Figs. 2 and 3, the only energy source for Ca uphill movement is the added ATP. It is easily shown that in these cells ATP is hydrolyzed (Fig. 2). An indirect way

ACTIVE CALCIUM TRANSPORT AND CaZf-ACTIVATED ATPase IN HUMAN RED CELLS

135

of putticg the chemical energy of ATP hydrolysis to use in the Ca transport might be by way of Na-K-transport, if a Na-Ca exchange existed and if the Na pump maintained a Na (and K) gradient across the membrane. This possibility can be ruled out on the following grounds : as stated above, the cells do not reseal to Na and K without prolonged incubation at 37°C. In fact in experiments of the type of Fig. 2 no alkali cation gradient was detected. Furthermore, the experiments were carried out in the presence of high concentrations of ouabain gm/ml), sufficient to block the Na-K pump completely. Finally, Ca is invariably transported regardless of whether the system is nominally made Na and K free or contains Na and K (Fig. 5 ; Schatzmann, unpublished), Thus the Ca transport seems to depend directly on the energy liberated in the hydrolysis of ATP. OF ACTIVECA TRANSPORT IN HUMAN RED CELLS 3. CHARACTERISTICS

a. Dependence on Intrucellular ATP. This dependence seems to be established (see above). Palek et al. (1971a) have shown that extracellular ATP is not hydrolyzed by red cells. b. Dependence on Intracellular Mg Concentration. In Fig. 6 (Schatzmann, 1969) two experiments are shown which indicate that the Ca transport requires Mg inside the cell, as was shown by Lee and Shin (1969). In order to interpret experiments with varying Mg concentration correctly, one must realize that manipulation of the Mg concentration in the presence 0

-

E

K 6.5rnM No: 135rnM

0.03rnM

38.5rnM

0.09mM

1.5. Medlum

Medlym

<

O

r

--. 0

C

V 0

U

0

15

30

3 15 30 TIME (minutes )

FIG.5. Ca extrusion from human red cells loaded by lysis in 20-fold volume of a solution with CaClz 0.75, Mg-ATP 4, MgClz 4, Tris-C1 5 mM. Medium: NaCl 130, KCI 5, MgCL 2, CaCL 1, Tris-C1 20, or: choline-Cl 85, KCI 50, MgClz 2, CaClz 1, Tris-C1 20, or: choline-Cl 135, MgClz 2, CaClZ 1, Tris-C1 20 mM; p H 7.4; temperature B"C, hematocrit approximately 0.25. Figures above the curves are concentrations measured in the total suspension. Notice that removal of Na or Na K, or absence of Na and increase of K concentration do not abolish Ca transport. As initial rate was not measured, this experiment is not suitable to detect small changes in rate (Schatsmann, unpublished).

+

H. J. SCHATZMANN

136

[c$+]

= 1.1 pnoIe/mL cells [ASP,],

= 216

[Mg2'],

= 0.005

v)

u 2E

:-\ Ex

'I,,

= 1.3

[ATP,],

[ C O * + ] ~ = 1.02 [ATP,],

= 2.07

I 0

I

10 Time (min)

I 20

= 2.24

[Mg2+], = 0.97

[Mg2+], = 1.60

0.

= 0.04

[Mg2+],

\

i

[Ca2

2-

= 1.50

E

\

0 -

pu

= 1.5 [ATP,],

"-\

-

r u -7

[Ca2+],

0

I 0

1

10

I

20

Time (rnin)

FIG.6. Two single experiments with two different blood samples showing Mg requirement for Ca transport. Citrated blood, cells washed four times and starved for 15 hr in the absence of glucose. Hemolysis in 10-fold volume of water in the presence of Tris-ATP (pH 7.2) and Ca with or without added Mg. Total [Ca] (ordinate) was measured by flame photometry in the cells after deproteinization with an equal volume of 10% trichloroacetic acid. Incubation a t 28°C in approximately %fold volume of medium containing (mM) 130 Na, 5 K, 1 Ca, 2 Mg, 20 Tris, 161 CI; p H 7.3. The medium for the low Mg cells contained no Mg. Figures near the curves give values a t zero time for total cellular [ATP], free [Ca*+]and free [Mg2+]calculated from measured total cellular concentration and stability constant of the ATP-metal complexes a t p H 7.3. Notice marked decrease of Ca-extrusion rate in low-Mg cells a t similar or higher cellular [Caz+]. (From Schatzmann, 1969, reproduced by courtesy of Macmillan Publishers.)

of ATP (which is the most important complexing agent for divalent cations in the system) affects the Cazf concentration, because the formation constants of Mg-ATP and Ca-ATP are of the same order. In the experiments of Fig. 6, therefore, all three components were varied in the hemolyzing fluid, and measured in the resealed cells until, by trial and error, concentrations were found that gave either very low or high [Mg] in the presence of about equal free [Caz+],which was calculated in the customary way from the total concentrations (see Wolf, 1973b). It may be seen that Ca movement was nearly completely abolished if the Mg concentration

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPase IN HUMAN RED CELLS

137

was drastically reduced and that this was certainly not due to a lack of Ca2+ inside the cells. It is quite probable, although not proven, that Mg-ATP rather than free ATP is the substrate for the transport system (see Section 11) much as in the case of the Na-K pump (Epstein and Whittam, 1966). c. Specificity with Respect to Metal Ions. Figure 7 shows that if both Mg and Ca are present inside the cell, Mg is not transported, but in fact is maintained a t the level introduced during resealing. This shows that if Mg has an affinity for the transport site it must be very much lower than that for Ca. However, Sr can take the place of Ca (Schatzmann and Vin-

Mg cells

7

0 )

I.5

6

sE

I

.-

-& E

5 -

c

c

0

0 .c

0

0 c L

c L

c

4:

0)

0

0

c

c 0

0

1

0

0

3;

u ca

cells

2

0.5 1

10

20

30

TIME ( m i n )

FIG.7. Absence of Mg movement in resealed human red cells which transport Ca. Mean of 7 experiments of the type in Fig. 2. Ca and Mg were measured in the same samples. ATP concentration in lysing solution of 2 mM. Notice that the considerable Mg- gradient from cells to medium is maintained, whereas that. for Ca is.reversed. (Unpublished experiments carried out during ICRO-EMBO Training Course on Membrane Biophysics, Bern, 1972; see Schatzmann and Vincenzi, 1969.)

138

H. J. SCHATZMANN

cenzi, 1969; Olson and Cazort, 1969), and it was shown that intracellular Ca and Sr compete for the transport system. The relative affinities have not been determined, but qualitative experiments show that they might be of the same order of magnitude. Mn2+, which partially activates ATPase in the presence of Mg2+ (see below), is not transported from resealed cells, and does not inhibit Ca transport when both metal ions are present inside the cells a t a concentration of 1 mmole per liter of cells (Schatzmann, unpublished). Cu2+a t a concentration of M did not inhibit Ca transport and therefore is probably not transported itself. Ho3+ and Pr3+ (trivalent cations of the lanthanide group) not only inhibit Ca transport but also Ca-ATPase, and for the latter reason are probably not transported. For their inhibitory action see below. d. Specificity with Respect to Nucleoside Triphosphates and Other Phosphutes. Olson and Cazort (1969) and Lee and Shin (1969) tested different nucleoside triphosphates and agree that GTP and I T P can replace ATP, although they are less effective. Lee and Shin (1969) came to the conclusion that CTP and UTP are quite as effective as ATP in maintaining Ca transport. Olson and Cazort (1969) demonstrated that pyrophosphate and acetyl phosphate are unable to support any Ca movement. In their work with inside-out vesicles from red cells, Cha et al. (1971b) were able to show that among ATP, GTP, ITP, and CTP present in the medium bathing washed vesicles, only ATP sustained 45Ca uptake and was hydrolyzed rapidly. However, when cell contents (membrane-free hemolysate) were added to the medium all four nucleoside triphosphates were active. It seems therefore, that there is high specificity for ATP as the ultimate energy source for Ca transport. e. Activation Energy. Lee and Shin (1969) and Schatzmann and Vincenzi (1969) studied the temperature dependence of the Ca transport and presented values for the activation energy which, however, differ considerably. The former authors found 13.6 kcal/mole, the latter 25.03 kcal/mol. The second figure would correspond to a Qlo of about 3.5. Notwithstanding the discrepancy by a factor of nearly two and the theoretical difficulty in interpreting activation energy in a transport system of high complexity, the strong dependence on temperature is in accord with an active transport involving the making and breaking of chemical bonds. f. p H Dependence. Lee and Shin (1969) in experiments on undirectional Ca movements showed that what possibly was active transport took place at pH values as extreme as 5 and 9.5, but was somewhat larger between 7.5 and 8.5 than a t the extremes. As these experiments do not necessarily demonstrate uphill movement of Ca, they must be interpreted judiciously. 9. Kinetics. The original experiments (Fig. 2) were carried out with total intracellular Ca concentrations in the 1 mM range. Lee and Shin’s

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPasc IN HUMAN RED CELLS

139

work (1969) suggested that lowering the intracellular Ca concentration to 0.1 mM did not affect the rate of transport. A serious inherent difficulty in the study of dependence of transport rate on cellular Ca2+ concentration is that the cellular Ca2+ concentration is unknown after resealing, on account of binding to ATP and other cell constituents, and that the uncertainty about [Ca2+]ibecomes more formidable at low total Ca concentrations. Possibilities for predicting this binding are very limited because data on binding to stroma proteins (Gent et al., 1964; Long and Mouat, 1971; Forstner and Manery, 1971a, b) do not allow one to distinguish quantitatively between inside and outside facing sites; pH and ionic strength are not equal in binding studies and work with resealed cells; and soluble cell constituents are diluted in an unpredictable manner in the hemolysisresealing process (hemoglobin, for instance, does not equilibrate while the cells are open). Another obstacle is that the dissociation constant of the transport system for Ca is rather low at the internal surface, as will be shown presently. This makes it all the more hopeless to attempt to measure total Ca in the cells as a rough approximation to [Ca'+]. In the interesting range of Ca concentrations, this is certainly no feasible approach to the determination of the dependence of transport rate on internal Ca concentration. It was found that Ca2+concentration in the red cell can be controlled with Ca-EGTA buffer solutions because EGTA enters during hemolysis and is retained after resealing (Schatzmann, 1973). At pH values near 7 the apparent dissociation constant of EGTA for Ca lies in the neighborhood of M . Hence the effects of other Ca complexing agents in the cells are overruled, provided that a sufficient concentration of the buffer is incorporated (say 5 mM). The affinity of EGTA for Mg is approximately l o 5 times less than that for Ca. Thus, high Mg2+ concentrations in the presence of Ca-EGTA are feasible and do not markedly disturb the Ca2+ concentration. Experiments with Ca-EGTA-loaded cells (Figs. 8 and 3) showed that the Ca transport system has a Ko.5 (concentration for halfsaturation) of 4 ~ 1 0 M - ~ or less on the internal surface of the membrane (Schatzmann, 1973). As the resealed cells become leaky for ATP a t very low Ca2+concentrations inside, only an upper limit for K0.6 can be given. Cells containing Ca buffers of 0.5X10-5 to 1x10-5 M Ca2+ pumped Ca into solutions of 3x10-7 to 5 ~ l O -M~ Ca'f concentration at about the same rate. The rate at saturation with internal Ca was about 0.15 pmole per milliliter of cells per minute at 28°C. The highest gradient which was tested and against which the cells transported Ca at the same rate as they did in the downhill direction was 700-fold. It is obvious that the transport system must change its affinity for Ca on the outward journey at least by a factor corresponding to this large gradient. These experiments teach

H. J. SCHATZMANN

140

I

0

5 10x10-6 Mean intracellular [Ca2+] (mole/ liter cells)

FIG.8. Seven experiments (different symbols) of type of Fig. 3. Different cellular [Cazf] set by different Ca:EGTA ratios in lysing solution. Initial rate was measured during 5 minutes at 28°C; 100% on ordinate corresponds to 0.148 rmole per milliliter of cells per minute. Mean cellular [Ca2+]calculated from initial [Caz+]and Ca transported in 5 minutes. [Caz+]for half-saturation approximately 4 x 10-6 M . (From Schatzmann, 1973, reproduced by courtesy of Physiological Societ,y, Cambridge University Press.)

that the directional selectivity of the system is excellent and that, even in resealed cells, the back leakage through diffusional pathways in parallel to the pump channel must be negligible; this is in accord with the observed low Ca permeability in intact cells. These experiments show that the standard experiment of Fig. 2 is misleading with respect to the efficiency of the system. The very negligible gradient of 3 achieved in these experiments is explained by the fact that the final Ca concentration in resealed cells was overshadowed by Ca accommodated within leaky cells since cells were not washed before analysis. So far, no indications have been forthcoming that Ca transport might be coupled to the movement of other cations in the opposite direction. Mg, which might be a candidate can be ruled out (Schatzmann, 1967) (see Section 11, C, 1) (Lee and Shin, 1969). The same is true for Na and K since their omission does not affect the transport of Ca (Fig. 5). This

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPase IN HUMAN RED CELLS

14 1

argument may not seem stringent because rigorously Na- and K-free media were not achieved (Fig. 5) and affinities for alkali cations might be high. However, the possibility of affinities exceeding those of the Na-K pump by orders of magnitude seems rather remote. h. Inhibitors. A number of inhibitors acting on other Ca-transporting systems (sarcoplasmic reticulum, mitochondria) or interfering with other active transport mechanisms have been tested. An organic molecule with multiple bonding requirements and/or a peculiar steric conformation showing high specificity comparable to that of ouabain for the Na-K pump has not yet been found. Sulfhydryl reagents : Mersalyl (salyrgan), well known from work on muscle, is strongly inhibitory at 5X1Ow4M (Schatzmann and Vincenzi, 1969), but its action is complicated because it also increases the passive permeability for Ca, which is revealed when the experiments are prolonged; Ca may leak in along the gradient after long exposure. As mentioned earlier, PCMBS possesses this property to a very pronounced degree. The question whether this leak is through the Ca pump channel or in parallel to it, is unanswered. Vincenzi (1968) showed that ethacrynic acid was inM , when long-standing exposure of starved cells preceded hibitory at the experiment. Chlorpromaxine, which was fairly active in sarcoplasmic reticulum (Balzer et al., 1968), gave only partial inhibition at a concentration of M even when present on both sides of the membrane (Schatzmann, 1969). Tetracaine has an inhibitory effect at elevated pH, and propranolol seems to be an effective inhibitor if millimolar concentrations are used (Porzig, personal communication). Oligomycin up to 5x10-5 M proved ineffective (Schatzmann and Vincenzi, 1969; Lee and Shin, 1969). All authors agree that ouabain is completely ineffective (Fig. 3). Cu2+,caffeine, imidazole, have no effect (Schatzmann, unpublished). D 600, methoxyisoptin (obtained by courtesy of Knoll AG, Ludwigshafen), which interferes with the Ca current in the action potential of cardiac muscle fibers (Kohlhardt et al., 1972), was ineffective at lo-* M (Schatzmann, unpublished). Sodium azide, fluoride (Lee and Shin, 1969), and dinitrophenol (Schatzmann, 1969) have no effect. Ruthenium red, a large 6-valent cation, which was effectively used to block Ca transport in mitochondria, gave very marginal if any effectswhen present on both sides (Schatzmann, unpublished). As it inhibits the Ca+ Mg stimulated ATPase (see below), this point needs further elucidation. Commercial ruthenium red may contain several other Ru compounds in large amounts, and any of these might be inhibitory and it appears that the onset of action is slow. The two lanthanides Ho3+ (holmium) and P?+ (praseodymium) were tested because they are effective in mitochondria

142

H. J. SCHATZMANN

(Mela and Chance, 1969; Mela, 1968a, b). They gave full inhibition with a .KO.&of about 10-4 M without causing a leak for Ca (Schatzmann and Tschabold, 1971). They are particularly interesting because they seem to act from the outside in resealed cells (Schatzmann, unpublished). It is unlikely that they penetrate into Ca-tight cells, and the experiments with Ho3+were done on Ca-EGTA loaded cells so that any holmium entering the cell would have been captured by EGTA, which has a formation constant for the Ho-complex of loL5(Sillen and Martell, 1964). Ho3+therefore seems to attack the Ca site when it faces the external side, where it has low Ca affinity. Ho3+has some theoretical interest because its radius in the unhydrated state is similar to that of Ca, and the charge density is higher because it is trivalent.

II. MEMBRANE ATPares ACTIVATED BY CALCIUM IN HUMAN RED CELLS A. Introduction

The studies on Ca transport quite convincingly show that ATP hydrolysis is the immediate source of energy for the uphill movement of the cation. ‘It seems logical to assume that Ca, when transported, will activate the ATP splitting, in the same way as Na does, while crossing the membrane by way of the pump. Furthermore any ATPase activity related to a pump should be membrane bound. In fact, different ATPases which require Ca2+ can be found in isolated red cell membranes, provided that those are broken up so that ATP and activators have access to the interior surface. This can conveniently be done by freezing and thawing. They are characterized by requiring only Ca or Ca plus other cations and by different kinetic parameters. It cannot be decided whether the different activities reflect different proteins or different behavior of one protein. Wolf (1973~)seems to favor the second possibility. However, from a functional point of view the first alternative seems more plausible. The membranes should be “white,” that is free of hemoglobin when-as is currently done-activity is referred to protein rather than lipid content. B. Different Calcium-ATPaser

1. CA + MG-ACTIVATED ATPASE The largest fraction, which at full activation has several times the intensity of the Na K stimulated activity, is an entity characterized by

+

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPase IN HUMAN RED CELLS

143

5

L i

0.I

0.5

I

CO'+

2

5

r10-4

concentration ( M )

FIG.9. ATPase activity stimulated by Ca in red cell membranes prepared according to Garrahan et al. (1969). Medium: Tris-C1 60, choline-C1 40, ATP 2, MgC12 (when present) 4 mM; pH 7.0. Incubation 90 min, temperature 37°C. 0 , A: [Cae+]fixed with 0.5 mM Ca-EGTA buffers; 0, A:[Calf] calculated from added CaC12. Blank: 0.5 m M EGTA. Notice small activity in the absence of Mg. Two components with different Ca affinity in Ca Mg-ATPase probably due to preparation procedure (compare with Fig. 11) (Schatzmann, unpublished).

+

requiring Mg in addition to Ca (Fig. 9) (Dunham and Glynn, 1961; Nakao et al., 1963; Wins and Schoffeniels, 1966b; Wolf, 1970, 1972a, b; 1973~; Schatzmann and Rossi, 1971). Mg must be present at least in the same molar concentration as ATP to give full activity. 2. CA

+ MG + (NAOR K) ACTIVATEDATPASE

When an alkali cation is present together with Mg and Ca, an additional activity appears that amounts to about one-fourth of the main activity (Schatzmann and Rossi, 1971; Bond and Green, 1971). The Kdiss for Na M ; and for K , 5 X M . The Kdiss for Ca is approximately is 33 X 0.2 x M (Schatzmann and Rossi, 1971). Whatever the significance of this activity may be, it is unlikely that it plays a role in Ca transport, because we have seen that transport does not require Na or K, a t all events not in concentrations as high as these. 3. CA-ACTIVATED ATPASE There is a very small fraction of enzyme activity not requiring Mg, whose kinetics have not been studied in detail in whole membranes (Fig. 9). Rosenthal et al. (1970) have isolated a protein from human red cells show-

144

H. J. SCHATZMANN

ing ATPase activity stimulated by Ca alone whose K d i s s for Ca is about M and which is inhibited by Mg. I t is well possible that it is identical with the protein spectrin described by Marchesi and Steers (1968), which seems to make up the material seen in electron microscopic pictures as an irregular layer adhering to the internal surface of the membrane proper. It is a linear protein which is solubilized at low ionic strength and forms strands under the influence of Ca and ATP. In view of the low affinity of this ATPase for Ca in the isolated state, and, since Ca transport requires Mg, this ATPase seems not to be related to transport. It might be a contractile protein involved in the morphological behavior of red cells. For reasons mentioned above, it is most likely that the Ca+Mg-requiring activity is associated with Ca transport. C. Characteristics of the Calcium

+ Magnesium-Stimulated

Membrane

ATPase

1. KINETICS

In media containing excess Mg the enzyme activation depends on Ca2+ concentration, as is obvious from experiments with Ca-EGTA buffers. The M dissociation constant of the Ca enzyme complex is of the order of Ca2+ (Wolf, 1970, 1972a,b, 1973c; Schatzmann and Rossi, 1971; Schatzmann, 1973) (Fig. 10). For some time it was claimed that the enzyme had two receptors of different affinity for Ca or that the affinity for Ca changed with varying Ca concentration (Fig. 9) (Schatzmann and Rossi, 1971; Bader, 1971; Wolf, 1 9 7 3 ~ )the ; second, low affinity component, however, seems to be an artifact created by the preparation of the membranes (Fig. 11). Long exposure to solutions containing potent Ca chelators seems to be detrimental (Wolf, 1972a, 1973c; Scharff, 1972; Schatzmann, 1973; Bramley and Coleman, 1972). M (Wolf, The K , for ATP (viz. Mg-ATP) was found to be 4 to 5 X 1970, 1972b). The same author also demonstrated that Mg-ATP rather than free ATP is the substrate for the enzyme. If resealed cells are used in which the external and internal membrane surface can be exposed to different solutions, the enzyme shows a characteristic sidedness or asymmetry. ATP is not split if present on the external side (Palek et al., 1971b), Ca (Schatzmann and Vincenzi, 1969) and Mg (Schatzmann, 1967) must be present on the internal surface in order to activate (Fig. 12), and Pi is liberated on the internal side of the membrane (Schatzmann, unpublished; Olson and Cazort, 1974) (phosphate leaks across the membrane, but, a t full activation of the Ca+Mg, ATPase production of Pi is much faster than the loss from the cell).

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPare IN HUMAN RED CELLS

t ~ - ~[ / ~

*+a I

145

M-'

FIG.10. Dependence of rate (8)of ATP hydrolysis on [Calf]. l / v vs l/[Caz+]plot. Membranes prepared by hemolysis in 20 mM sucrose, glycine buffer pH 9.5. Washing in 2 mM sucrose, 50 mM NaCl, buffer pH 9.2. Medium: Na 100 mM, Mg2+ 2 mM, CaEGTA buffer 0.4 mM; pH 7.0. Mg-ATP: 0 1 mM, 0.4 mM, A 0.2 mM, A 0.1 mM, 7 0.04 mM, X 0.02 mM. (Redrawn from Wolf, 1972b, reproduced by courtesy of Biochimica Biophysica Acta, Elsevier Publishing Company.)

m

In disrupted membranes the interaction of Ca and Mg has been studied (Dunham and Glynn, 1961; Schatzmann and Vincenzi, 1969). Ca in excess of 10-4 M concentration is inhibitory, and this inhibition is counteracted by raising the Mg concentration. Since Mg is instrumental in binding ATP to the enzyme (Wolf, 197213, 1973c), it is possible that Ca competes with Mg for ATP and that Ca-ATP cannot occupy the site on the enzyme or that it can take the place of Mg-ATP but that this complex is inactive. Wolf (1972b) has shown that in the absence of Ca the enzyme forms an inactive Mg-ATP complex whereas Cha et al. (1971a) claimed that in the absence of Ca a phosphorylated intermediate is formed. 2. CHEMICAL CONSTITUTION OF

THE

ACTIVESITE

The system contains two ionizable groups with pK, values of 5.8, presumably imidazole nitrogens-one necessary for substrate binding, and one for substrate splitting-and a group with a pK, of 8.2 necessary for

146

H. J. SCHATZMANN

Co concentrotion ( M )

+

FIG.11. Membrane Ca Mg-ATPase. Dependence on [Caz+]. From the same cells membranes were prepared in two different ways: A, hemolysis in 30 mM Tris-C1 p H 7.1, 1 mM EDTA, washing in 15 mM tris; 0, hemolysis in 20 mM Tris-C1, 20 mM sucrose, 10 mM NaCl pH 7.45, washing in 2 mM sucrose, 1 mM NaCl, 1 mM KCl, 2 mM Tris-C1, p H 7.7. Both preparations frozen before use. Notice that the second method prevents appearance of a low affinity component and enhances the activity a t low Ca concentration. Medium: 30 mM imidazole-C1, 70 mM choline-Cl, 2 m M Mg-ATP, 4 mM MgCl,, 0.5 mM Ca-EGTA buffer (up to 10-5 M [Ca2+]). Blank 0.5 mM EGTA (compare with Figs. 9 and 10) (Schatzmann, unpublished).

substrate splitting, probably an a-amino group of an amino acid (Wolf, 197213). Wolf (1973a, c) applied a number of group-specific inhibitors and concluded from this study that the active center contains an -NHT, at least two S H , at least two histidine, and at least two carboxyl groups and one methionine group.

3. SPECIFICITY WITH RESPECT TO DIVALENT CATIONS

Sr2+is as effective as Ca2+in activating the enzyme when Mgz+is present (Schatzmann and Vincenzi, 1969; Pfleger, quoted in Wolf, 1973~).In experiments without metal buffers, we found that Mn2+, Co2+, 3n2+, and Ni2+ cannot fully replace Caz+ when equal concentrations (2X10-5 M and 2 X M ) are compared. When Ca and any of the heavy metals were present simultaneously in equal concentration (2 X lo-* M ) and Mg was in excess, the activity was less than with Ca+Mg alone. Mn2+ lo+ M , had no inhibitory effect on the activation by M Ca2+.Pfleger (quoted in Wolf, 1973c), using metal buffers came to the conclusion that the heavy metals mentioned activate the enzyme but that V,,, reached under their influence is only about 50% of what is achieved by Ca or Sr. Their affinity for the system exceeds that of Ca. In recent experiments, we found that V,,, obtained with Mn2+ is about one-third to one-fourth of that resulting with Ca2+and that half-saturation is achieved with M Mn2+.

147

ACTIVE CALCIUM TRANSPORT AND CAZf-ACTIVATED ATPase IN HUMAN RED CELLS

However, Mn2+does not inhibit the action of Ca2+ as one would expect from a partial agonist at one single enzymic site. Moreover, Mn2+is not transported and does not inhibit Ca transport (see above; Schatzmann, unpublished). There are two possibilities that might explain this observation: either activation by Mn2+ is altogether foreign to the Mg CaATPase, or the enzyme has two sites that accept metals, one being the “ATPase site” (a), and the other the transport site (b); Ca2+has access to both sites, and Mn2+only to (a). Full activation results when (a) and (b) are occupied by Ca2+or when (a) is occupied by Mn2+and (b) by Ca2+. Presence of Mn2+at (a) leads only to partial activation. Ba2+ and Pb2+were also less effective than Ca2+ and Sr2+in activating the enzyme (Pfleger, quoted in Wolf, 1973~).

+,

Ca inside+outside

1

r

No C a

d 0

15 Timr (min)

30

FIG.12. Asymmetry of membrane ATPase activation by Ca. Resealed cells; hemolysis in the presence of 2 mM Na2-ATP, 5 m M Tris-C1 (pH 7.4),1 mM CaCl2, 2 mM MgC12. Medium as in Fig. 4, with 10-4 g of ouabain per milliliter. Ordinate: Pi released per milliliter of cells. Ca was omitted from medium or hemolyzing fluid as indicated in the figure. Three experiments; vertical bars 2 X SE of mean. Notice that Ca activates ATPase only if present inside the cells. (From Schatzmann and Vincenzi, 1969, reproduced by courtesy of Physiological Society, Cambridge University Press.)

148

H. J. SCHATZMANN

4. SPECIFICITY WITH RESPECT TO NUCLEOSIDE TRIPHOSPHATES Watson et al. (1971a) have demonstrated beyond doubt that specificity for ATP is extremely high. Ca2+-activated hydrolysis of CTP, UTP, and GTP was below the level of detectability, whereas the observed activity in the presence of ITP was quite negligible. This is in accord with the high specificity for ATP of Ca transport mentioned before and makes it probable that Ca transport in resealed cells is sustained by nucleoside triphosphates other than ATP because this preparation is able to transfer a phosph&grouplenzymically from these to ADP (Mourad and Parks, 1966). 5 . INHIBITORS' Watson et al. (1971b) have demonstrated that ruthenium red inhibits the enzyme by 90% a t a concentration of 6X10-5 M and that there is a certain specificity insofar as Na-K-activated and Mg-activated ATPase are hardly affected by this concentration. The compound was successfully used to block Ca uptake in mitochondria (Moore, 1971). It is a bulky 6-valent cation of the conjectural structure (Cotton and Wilkinson, 1966) [(NH~)~Ru-O-RU(NH~)~-O-RU(NH~)~~~~ Its commercial forms are highly impure, containing large and variable amounts of several other Ru compounds (Luft, 1971). Weiner and Lee (1972) demonstrated in inside-out ghosts that to 4XlO+ M La3+ inhibits the Ca-ATPase by 50%. Holmium and praseodymium, which are active in mitochondria1 Ca transport (Mela and Chance, 1969), effectively block the enzyme. At 0.06 mM Ca2+ concentration 5Oy0 inhibition is achieved by 0.07 mM Ho3+ (Schatzmann and Tschabold, 1971), which is a higher concentration than that needed in mitochondria. The effect is not specific, in that Na+K-activated ATPase is also inhibited and shows the same affinity (Kdiss-10-5 M ) (Schatzmann, unpublished), whereas the Mg-activated ATPase seems not to be affected. In resealed cells, however, a difference between Na-K transport and Ca transport is apparent. Ho3+ blocks the Ca transport in resealed cells if applied outside, whereas Na-K transport is not affected in whole cells when Ho3+is present in the medium in a concentration of M (which gives about 7oY0 inhibition of Ca transport). Wins and Schoffeniels (1968) pointed out that inhibitors of flavoprotein enzymes involved in oxidoreductions, like 2,6-dichlorphenolindophenol or amytal, reduce the activity of the enzyme. Wolf (1973a, c) has tested a variety of group-specific inhibitors which cannot be enumerated here (see Section 11, C, 2).

ACTIVE CALCIUM TRANSPORT AND Ca*+-ACTIVATED ATPase I N HUMAN RED CELLS

149

Ouabain does not affect the enzyme in concentrations that block Na-Kactivated ATPase completely. 6. PHOSPHATASE ACTIVITY AS

AN

ASPECTOF

THE

C A + M GATPASE

Pouchan et al. (1969) and Garrahan et al. (1970) described a ouabaininsensitive p-nitrophenyl phosphatase in human red cell membranes which is characterized by its requirement for Mg Ca ATP and whose activity is strongly increased by K. In a recent study Rega et al. (1973) were able to show that (a) Na can replace K but has a lower affinity for the system; (b) Ca and ATP must be present on the internal membrane surface in order to activate p-nitrophenyl phosphatase; (c) the affinity for Ca2+ is high (Kdiss = 8X10-6 M ) ; and (d) ATP cannot be replaced by other nucleoside triphosphates. They further showed that 40mM p-nitrophenyl phosphate inhibits both Ca extrusion and Ca-dependent ATPase by 50% or more. The similarity between this phosphatase and the Ca-dependent ATPase is indeed striking, and the authors propose that both are properties of the same system. They suggest that p-nitrophenyl phosphate hydrolysis requires only part of the pathway necessary for ATP hydrolysis. This is consistent with the idea that ATP hydrolysis is a multistage process involving intermediate compounds, some of which may be replaced by p-nitrophenyl phosphate.

+ +

7. AN ACTIVATOR PROTEIN

Bond and Clough (1973) have recently presented evidence for the existence of a factor present in membrane-free hemolysates dialyzed against water, which considerably increases ,,T ,i of the enzyme in a concentrationdependent fashion. The factor is completely destroyed by trypsin treatment or by exposure to temperatures above 54°C for 10 minutes. It is specific in the sense that it enhances the activity of Ca+Mg-ATPase and C a + Mg K-ATPase, but not that of Mg-ATPase and Na K Mg-ATPase.

+ +

+

111. RELATIONSHIP BETWEEN CALCIUM TRANSPORT AND CALCIUM MAGNESIUM-ACTIVATED ATPase

+

A. Common Features

+

It may be noticed that the Ca Mg enzyme and active Ca transport share a number of characteristics: (1) the dissociation constant for Ca is very similar; (2) both require Mg; (3) both are insensitive to ouabain;

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(4)both accept strontium instead of Ca; ( 5 ) both are inhibited by the same concentration of holmium. B. Stoichiometry

It seems warranted therefore to conclude from this-admittedly circumstantial-evidence that Ca transport and the Ca Mg-stimulated ATPase activity are two aspects of the same system. This amounts to saying that the Ca transport system behaves as an ATPase in which the transported species is the activator for the energy-yielding hydrolysis of the terminal phosphate bond of ATP, whereby the arrangement is such that the energy liberated is used to do osmotic and electrical work on the transported Ca ion. Because these two phenomena appear linked one may expect a quantitative relation between the ATPase reaction and the Ca transport. The question can then be asked which is the coupling ratio or stoichiometric relation between Pi liberation and Ca transport. The problem is complicated by the existence of other ATPases in the membrane. Na-K-ATPase can be excluded by working in Na- and K-free Mg Na or K)solutions and/or in the presence of ouabain. (Ca ATPase can be minimized by choosing low Ca2+ concentrations and the Mg-ATPase (whose significance is somewhat enigmatic) can be measured and deducted. This was done by running a Ca-free sample or by using EGTA in the external medium which did or did not contain ATP; in this way Mg-ATPase was assessed in the fraction of leaky cells and the value obtained applied to the fraction of tight cells (Schatzmann, 1973). Both methods have disadvantages and were used in combination. The result was a Ca:ATP ratio of 0.86 in the first method and 1.39 and 1.27 in uphill or downhill transport experiments with the second method. These figures are statistically very significantly different from 2. It may tentatively be concluded, therefore, that the true relation is one, rather than any higher number of Ca ions transported per pyrophosphate bond of ATP split. This is different from the case of the sarcoplasmic reticulum, in which a Ca:ATP ratio of 2 has been found (Makinose and Hasselbach, 1971).

+

+

+

C. Thermodynamic Considerations

On the assumption of a Ca:ATP ratio of unity and by proposing a plausible value for the normal intracellular Ca2+ concentration the energy requirement for the Ca transport may be calculated. The total intracellular Ca concentration is below measurable values and therefore even with a better understanding of the binding properties of celluhr material than that available the [Caz+], would be unknown. However, a n estimate of the

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPare IN HUMAN RED CELLS

15 1

upper limit of this value may be obtained by the following reasoning: I n viable cells containing a saturating Ca2+ concentration (2 X M) extrusion of lmmole of Ca2+per liter of cells a t 37°C is a matter of minutes. In energy-depleted cells, penetration of 1 mmole Ca into 1 liter of cells takes many hours. Consequently pump and leak fluxes differ a t least by a factor of 100. Half-saturation of the pump sites on the internal surface of the membrane is achieved by approximately M Ca2+.From the leak and pump principle, steady-state concentration is attained when influx and eflux are equal. Thus, to reach the steady state the pump rate a t half saturation may be reduced at least 50-fold by about a 50-fold reduction of the intracellular [Caz+]. This will bring the steady-state Ca2+ concentration inside the cells into the vicinity of or even below lo-’ M . For the sake of argument, we choose 5XlO--? M which might be a conservative figure. With 1.5X1OP3M for the Ca2+concentration in blood plasma and 5 x lo-’ M inside the cells 5.35 kcal per mole of Ca2+ is obtained for the work done in the Ca transport. This must be compared with the energy liberated in the ATP hydrolysis reaction. Assuming a standard free energy change of 7.2 kcal/mole under physiological conditions of pH and ionic strength and taking the respective concentrations of ATP, ADP, and Pi in human red cells as 1.5 mM, 0.32 mM and 0.36 mM, the change of free energy of the reaction turns out to be 13.04 kcal/mole (Schatzmann, 1973). This figure is larger than the work done on 1 mole of Ca moving out, which means that the coupled reaction of ATP hydrolysis and Ca movement is thermodynamically feasible under the chosen conditions. The argument must not be reversed; thermodynamics shows that under the chosen conditions Ca moves through the pump channel, but does not by any means prove that the concentration of 5 x 10-7 M Ca2+can be maintained by the pump. For Na-K transport in red cells (Glynn and Lew, 1969, 1970; Lew et al., 1970; Ellory and Lew, 1970; Whittam et al., 1970) and for Ca transport into vesicles of the sarcoplasmic reticulum (Makinose and Hasselbach, 1971), it could be shown that the pump can be reversed by setting up a large cation gradient in opposition to the pump flux and by lowering the ratio [ATP]:[ADP] [Pi] with the result that the cation gradient is used to synthesize ATP. A similar experiment with Ca in red cells failed for technical reasons; it was not possible to maintain high phosphate concentrations inside the cells without high phosphate concentration in the medium which in turn prevented the use of large Ca2+ concentrations outside (Schatzmann, unpublished).

-

D. A Possible Model

It might be profitable to discuss any possible model in comparison with the germane systems for Na-K transport in red cells and Ca transport into

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the sarcoplasmic reticulum. Na-K transport has been extensively studied and agreement as to the main points of the mechanism involved has been reached. Between Ca transport in sarcoplasmic reticulum and red cells striking similarities are apparent and therefore the insight gained in muscle physiology might be applicable to red cells. Ca transport in red cells differs from alkali cation transport in that it implies no coupling between movements of two cations in opposite direction. Quite recently, however, Lew et al. (1973) have presented evidence to the effect that a fraction of the active Na transport in red cells is uncoupled from K movement, so that this difference is perhaps less fundamental than hitherto thought. I n the Na-K system the structural basis is a protein which apparently is in contact with the internal and external bulk fluid and which requires for its functioning the presence of phospholipids. Phosphatidylserine is particularly active in this respect (Wheeler and Whittam, 1970; Priestland and Whittam, 1972). However, the recent demonstration by De Pont et al. (1973) that membrane phosphatidylscrine can be transformed into phosphatidylethanolamine by enzymic decarboxylation without loss of ATPase activity cases somc doubt on the unique position claimed for phosphatidylscrine. On the internal surface the protein accepts one phosphate molecule from ATP in a group transfer reaction at an acyl group. This requires Mg and Na. In a second reaction this primary phosphorylated intermediate is transformed either by internal transphosphorylation or by a conformational change. This reaction is blocked by N-ethylmaleimide (NEM) or oligomycin. The second form of the phosphorylated intermediate is decomposed in the presence of K. This reaction is blocked by ouabain. The dephosphorylation restores the original situation and the cycle can start again. The conformational change implies a change in affinity for Na and K and a movement whereby the alkali cations are transferred across the boundary (for a review see Post et al., 1969; Glynn, 1968; Albers et al., 1968; Whittam and Wheeler, 1970). In the sarcoplasmic membrane a phosphorylated intermediate is formed in the presence of Ca (and Mg) on the external surface. It remains stable if the internal Ca concentration is high, and it decomposes if the internal Ca concentration is kept low with oxalate (Makinose, 1969). Formation of the phosphoprotein, ATP-splitting, and phosphate exchange between the phosphoprotein and ADP (the reversal of the protein phosphorylation) show strictly the same dependence on external Ca concentration (Makinose, 1969). Conformational change and Ca transfer seem to occur in analogy to the Na-K system. The only difference is that dephosphorylation is spontaneous, i.e., does not require a second cationic activator, which may not be a very decisive point (Lew et al., 1973). Such a reaction scheme would be compatible with the finding

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of Wolf (1972b, 1973c) in red cell membranes, that without Ca but in the presence of Mg the enzyme forms an inactive ATP-Mg complex. From this, one would guess that also in red cells (internal) Ca triggers the formation of a phosphoprotein. Recently Knauf et al., (1974; see also Katz and Blostein, 1973) were indeed able to show that Ca (0.5 mM) a t low ATP and Mg concentrations induces formation of a phosphoprotein in human red cells membranes. The denaturated membrane proteins were separated by electrophoresis, and it was demonstrated that, under the influence of Ca, phosphorylation takes place in a protein fraction which can be distinguished from that involved in Na-induced phosphorylation. The reaction scheme therefore is MgtCs

ATPfE

E-P+ADP

E-P--+E+Pi

where E stands for the transport protein and Pi for inorganic phosphate. A short report by Cha et al. (1971a) had claimed that Ca is not necessary for phosphoprotein formation but for dephosphorylation. This is conceivable, but seems improbable in the light of the reported findings. Figure 13 makes an attempt to summarize the possible sequence of events. Two things are clear: (1) The protein must have two conformational

inside

---

outside

FIG.13. A possible scheme for the cyclic changes in the transport protein. EI and EP symbolize two conformational states of the protein, in which the Ca binding site faces the internal and external bulk phase, respectively. The dashed line symbolizes the boundary between inside and outside with respect to Ca and Ca binding site, ATP, ADP, and inorganic phosphate (Pi). Ca is necessary for phosphorylation, and phosphorylation P have high affinity, E2 and EP P induces the conformational change. E1 and El have low affinity for Ca. EB -+ El is spontaneous. N

-

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H. J. SCHATZMANN

states, El and Ez, with the Ca site facing the cell interior and the external medium, respectively. ( 2 ) In the El conformation the Ca Site must have high affinity for Ca, in the Ez conformation low affinity for Ca. Some indications for conformational changes have been given by Bond (1972). If one assumes that the Ca ion triggers phosphorylation when bound to the transport site, the sequence of events might be as depicted in Fig. 13. In the El conformation, the affinity for Ca is high; and in the EP conformation, low. Phosphorylation brings about the conformational transition E1-Ez. Dephosphorylation is spontaneous once Ca has left its binding site. In the absence of ATP equilibrium (E1%E2) favors the El configuration. Since the position of equilibria in the cycle is immaterial for the argument, all reactions are symbolized by one arrow only. The dashed line represents the boundary between inside and outside of the membrane with respect to Ca and its binding site on the transport protein and indicates that ATP and ADP are located inside and that Pi is released into the intracellular space. IV. RELATION BETWEEN CALCIUM TRANSPORT-ATPase AND SODIUMPOTASSIUM TRANSPORT-ATPase

The question may be raised whether the two cation transport systems are separate entities or simply two different aspects of one protein molecule or even of one single transport site. The fact that maximal activation yields different activities in the two functions is no argument against a single protein for both. If a single system existed for both transports, Na K activation should disappear when activation by Ca takes place. It is true that Ca inhibits the Na K activated system (Dunham and Glynn, 1961; Davis and Vincenzi, 1971; Dunn, 1974), but Epstein and Whittam (1966) have presented good evidence for the claim that this is due to competition of Ca with Mg for ATP; formation of Ca-ATP is inhibitory since Mg-ATP is the substrate for the N a + K system. Furthermore, considerably higher Ca concentrations are required to inhibit Na KATPase than to activate Ca Mg-ATPase (Davis and Vincenzi, 1971). The insensitivity of the Ca Mg-ATPase toward ouabain is not what one would expect if the N a + K activated system were able to transport Ca, although one might argue that Ca destroys the conformation favorable for ouabain binding; but a finding by Schon et al. (1970) seems to indicate that precisely the contrary is true: much as Naf does, Ca2+ induces the form of the N a + K enzyme which accepts ouabain. The experiments pertaining to phosphoprotein formation described in Section 111, D, show that two different proteins are responsible for the two transport

+

+

+

+

+

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functions. The possibility remains, however, that they have certain aspects in common. Finally, if the two functions were exerted by the same protein, measurements of both in different individuals should correlate positively. This investigation was carried out in cattle cells. In bovines cellular K concentration, Na K-ATPase, and Na-K pump activity vary widely among different individuals (Ellory and Tucker, 1970; Christinaz and Schatzmann, 1972). No positive correlation was found between Ca-ATPase and Na K-ATPase (in fact a significant negative correlation was obtained) (Schatzmann, 1974). I n summary, several points speak against the possibility that the two cation pump activities are due to a single molecular species.

+

+

V. COMPARISON WITH OTHER SYSTEMS TRANSPORTING CALCIUM A. ATP-Dependent Systems

1. SARCOPLASVIC RETICULUM

The Ca transport in red cells has a great similarity to that in sarcoplasmic reticulum (SR) (for reviews see Ebashi and Endo, 1968; Martonosi, 1972) except for the direction which is into the corpuscle in SR and out of the corpuscle in red cells. Both systems utilize the energy of ATP hydrolysis directly, and both achieve very high gradients across the membrane; this means that transport is rapid compared with leak flux through the pump itself or through channels in parallel to the pump. The molecular machinery, however, seems not to be identical in the two systems. The stoichiometric relation between Ca and ATP is 2 in the SR and probably 1 in red cells. 2. LIVERCELLS,L CELLS

For liver cells van Rossum (1970) has presented evidence for a Caextruding mechanism that depends on energy expenditure of the cell and seems not to be related to the Na-K transport. Lamb and Lindsay (1971) have convincingly demonstrated that active Ca extrusion which depends directly on ATP exists in cultured L cells. They found a Ca:ATP ratio of 1.8. 3. SMOOTH MUSCLE Casteels et al. (1973) have presented evidence for a Ca extrusion from taenia coli smooth muscle which seems to depend directly on ATP.

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4. SYSTEMS WITH UNKNOWN MECHANISM Borle (1969) found that, in HeLa fibroblasts in culture, Ca extrusion depends on metabolism, but he did not establish whether the movement depended on concomitant Na movements or directly on ATP utilization. A variety of cells, among which many epithelia, are capable of transporting Ca from one cell surface to another cell surface. This transcellular Ca transport may reflect the combination of an area of the cell surface provided with Ca pump sites with areas of high Ca permeability in the opposite membrane. By analogy with red cells and liver cells, it would seem likely that the active transport in this case is from cell to medium. However, more complicated systems with two pumps at both cell surfaces or even active mechanisms within the cell body are conceivable. Transcellular Ca transport occurs in the intestinal mucosa (Melancon and DeLuca, 1970), in kidney tubules (Frick et al., 1965; Parkinson and Radde, 1971; Rorive and Kleinzeller, 1972), probably in the mammary gland and the shell gland of the oviduct of birds. Moore et al. (1974) have recently shown that in the kidney both the plasma membrane and microsomes exhibit ATP dependent Ca-pump activity. In the intestinal mucosa a calcium binding protein was found, whose abundance depends on the amount of vitamin D available to the animal (Wasserman et al., 1968, 1971) and therefore parallels the activity of the intestinal Ca pump. Its precise role in the transport process has not been assessed. The likelihood of its being part of the transport machinery in the membrane is reduced by the fact that it is a soluble protein. A particularly intriguing case is represented by bone cells in which active Ca transport may be involved in either bone Ca deposition or resorption (see volume edited by Comar and Bronner, 1969).

5 . MITOCHONDRIA

A special case is Ca accumulation into mitochondria. There is no doubt that it is energy linked and may be sustained both by ATP hydrolysis and the electron transport chain. This suggests that a phosphorylated or nonphosphorylated unknown high energy compound may serve as the immediate source of energy. The transport takes place a t the inner membrane, and considerable accumulation of Ca is achieved in the matrix (Chance, 1965; Carafoli, 1967; Mela and Chance, 1968; Cittadini et al., 1973). The free Ca2+ concentration in the matrix, however, is unknown; therefore the work done by this transport system remains to be determined.

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B. Sodium-Calcium Heteroexchange

In excitable cells Ca extrusion has been found which differs fundamentally from the Ca transport in SR or red cells. Baker et al. (1967, 1969) demonstrated that an exchange of Ca for Na take&place across the external membrane of the squid axon (for review see Baker, 1972). An analogous system was detected by Reuter and Seitz (1968) in guinea pig atrial cells and further described by Glitsch et al. (1970). The system is an equilibrating carrier transport mechanism and accepts Na and Ca apparently a t the same site. By countertransport, such a system is able to move one species uphill if the other species moves downhill in the opposite direction. Since the Na pump maintains an inwardly directed Na gradient this coupled system will-under physiological conditions-transport Ca out of the cell. This Ca movement depends via the Na pump on the energy supply of the cell. On account of its kinetic peculiarities this system cannot generate a Cao:Cai ratio which is larger than the Naz0:Na2iratio. Since in excitable cells the Ca ratio (> 10,000) across the membrane exceeds the Na2 ratio (10x10) (Portzehl et al., 1964; Baker, 1972), this heteroexchange will not account for the internal Ca2+ concentration a t physiological external Ca concentrations. The possibility exists that the mechanism operates on a cellular compartment underneath the plasma membrane in which the Ca2+concentration is raised by another type of Ca pump to a level above that in the bulk cytoplasm (Reuter, 1973; see below). For the giant axon of the squid, Baker and Glitsch (1973) leave the possibility open that in addition to the exchange mechanism an ATP-driven pump may exist.

VI. PHYSIOLOGICAL SIGNIFICANCE OF CALCIUM PUMPS A. Muscle

Calcium-sequestering systems within the cell (SR, mitochondria) have undoubtedly the task of rapidly restoring low intracellular Ca2+ concentration in the cytosol when it. has been raised by the events during excitation. In skeletal muscle the SR is the ‘Lrelaxingfactor” (see Ebashi and Endo, 1968) which in 20-100 msec removes Ca released by an action potential from the space where the actomyosin is located, regardless of the source from which Ca entered this space during the action potential. I n skeletal muscle this source may be the SR itself whereas in cardiac muscle the main source may be the external medium. At all events these seques-

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tering systems do not account for the steadily maintained high Ca gradient between external medium and cytosol unless they can discharge their content to the external medium. For mitochondria any such possibility seems remote and in SR it is a moot point whether a communication between the lumen of the SR and the transverse tubules exists. An alternative is an efficient Ca pump located in the plasma membrane. For quantitative reasons the Na-Ca heteroexchange cannot possibly take this role alone, but it might be one component in a two-step transport. This possibility seems not too far-fetched in cardiac muscle where an anatomical substrate seems to exist for a Ca-rich space underneath the membrane and for which electrophysiological data suggest the existencc of such a compartment (Reuter, 1973).

B.

Red Cells

1. INFLUENCE ON ALKALICATION PERUEABILITY Whereas in muscle relaxation affords a good reason for mechanisms keeping intracellular [Ca] low, these contrivances seem a mere luxury in noncontractile cells. But apart from the fact that contractile processes seem to play a role in many cells, maintenance of low Ca concentrations is necessary for the proper functioning of the cell membrane. I n this respect, red cells of certain species (including man) (Jenkins and Lew, 1973) display a behavior which might be present and important in more highly organized cells, too. Gardos (1958, 1959) demonstrated that metabolic depletion of human red cells increases the K permeability of the membrane provided that Ca is present in the medium. Since its discovery this effect has been confirmed by Hoffman (1962), Whittam (1968), Kregenow (1962), Kregenow and Hoffman (1972), Romero and Whittam (1971), Lew (1970, 1971), Blum and Hoffman (1971), and Dunn (1974), and there seems to be full agreement that the cause for the very dramatic rise in K permeability is a slight rise in intracellular Ca2+ concentration when ATP is missing. Lew (1970) has indicated that an increase in cellular [Caz+]by lop6 M is sufficient to raise the K permeability very markedly. Blum and Hoffman (1971) were able to show that the Ca-induced K outflow exhibits saturation kinetics, and they seem to be inclined to think that internal Ca modifies the Na-K pump into a shuttling carrier for K. In a recent report Jenkins and Lew (1973) showed that the K effect is present in human, rat, guinea pig, and the coypu red cells, but not in sheep, cattle, and goat red cells, although the ruminant red cells also take up Ca when starved. As mentioned before, it is not clear whether the Ca entry is due only to the arrest of the Ca pump or whether the energy-depleted

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state causes structural changes in the membrane that favor the inward leakage of Ca. 2. INFLUENCE ON MECHANICAL BEHAVIOR AND SHAPE

Entry of Ca into human red cells leads to conspicuous alterations in the mechanical properties and morphological behavior of these cells (Dunn, 1974). Weed (1968), Weed et al. (1969), and Weed and Chailley (1973) have shown that starved red cells with extremely low ATP content become more rigid and that this alteration is strictly paralleled by an increase in cellular Ca content (rigidity is quantitated by measuring filterability or viscosity of cell suspensions or by observing the deformability of a single cell sucked partially into a microcapillary). The effect is reversible if the cells are allowed to pump Ca out again and can be prevented by Ca chelators. I n the depleted state the cells undergo disk-sphere transformation by passing through a phase of crenated disks-crenated spheres (echinocytes). Nakao et al. (1960), Wins and Schoffeniels (1966a), and Palek et al. (1971a, b) have pointed out that Ca2+ can induce shrinking of red cell ghosts. This effect can be attributed only to a Ca action on the internal side of the membrane. There is, however, no agreement as to the role of ATP in this shrinking. Wins and Schoffeniels are inclined to ascribe the effect t o ATP-consuming contractile proteins of the actomyosin type. The existence of a contractile protein was claimed by Ohnishi (1962), and it might be identical or associated with the protein spectrin, which seems to be arranged on the internal membrane surface and was solubilized by Marchesi and Steers (1968). The latter authors showed that their protein forms filaments visible in the electron microscope if ATP and Ca or Mg are present in concentrations between 0.1 and 1 mM. This in turn might be the morphological substrate of Ca-stimulated ATPase activity isolated from ghosts by Rosenthal et al. (1970). Palek et al. (1971b) obtainedmarked shrinking of ghosts in the presence of Ca when isotonicity was restored in the absence of ATP. Weed and Chailley (1973) have considered the possibility that the crenation is due to local activation of contractile processes in membrane areas where Ca pump sites are scarce. The cells become spherical and turn rigid under conditions allowing entry of Ca. Both alterations are unfavorable from a functional point of view. It follows that Ca cntry must be prevented during the lifespan of a red cell, and the Ca pump seems to be an appropriatc mechanism to this end. On the other hand, it seems quite plausible that these processes are initiated when the energy-librrating processes decline in aged cells, thus precipitating their mechanical destruction in the body (LaCelle et al., 1973).

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It is quite possible that in certain hemolytic anemias a disturbance in the Ca transport is a t the basis of the increased vulnerability of cells. I n a recent report, Eaton et al. (1973) demonstrated quite convincingly that in sickle cell disease red cell Ca permeability is profoundly disturbed. The Ca content of red cells was 7.6 times that found in cells from healthy subjects, sickled individual cells had higher Ca content than the patient’s cells exhibiting normal morphology, and the 45Cainflux into cells of patients was 9.3 times that of cells of normal subjects in the oxygenated state and 48.5 times larger than in normal cells in the 02-deprived state. It seems that the defect consists mainly in an increased passive permeability for Ca. The function of the Ca transport system and of Ca-ATPase was not tested. As one would expect, the sickle cells also exhibit excessive permeability to alkali cations. Horton et al. (1970) found that in cystic fibrosis patients Ca-ATPase activity of red cells is reduced. However, no functional impairment seems to ensue in this case. It is tempting to speculate that the normal disk shape of red cells is somehow caused by the contractile system mentioned, and that intracellular Ca2+ concentration is kept low by the pump in order to ensure partial rather than maximal activation of the contractile system. However, it is extremely difficult even to come to a coherent view that might be put to a reasonable test. The first difficulty is that the ATPase in the isolated protein has a K d i s s for Ca of lov3M , which is orders of magnitude away from the intracellular Ca concentration. Further, there is no morphological evidence for protein strands running across the intracellular space. Finally, Bull (1973) has shown that a red cell firmly fixed to a glass surface can be rolled by hydraulic force like a caterpillar track so that the convexities and concavities change place on the cell surface, which seems incompatible with any structural elements running across the cell space. However, it is conceivable that activation of a very small fraction of the contractile protein is sufficient to maintain the biconcave shape, and that the width of the protein strands is below the resolution of the electron microscope. If the connection between loose ends of protein chains extending into the lumen of the cell from opposite parts were due to ATP-activated bonding, the mechanism could catch whenever two opposite cell surfaces are brought sufficiently near each other by external forces, such as in Bull’s experiment. A mechanism of this type is compatible with the experiment of Palek et al. (1971b) showing that transient osmotic shrinking with NaCl solutions of ghosts that were probably leaky for alkali cations led to a permanent reduction of volume. Here again the osmotic shrinking might have brought loose ends into contact. But the possible role of a contractile protein in the preservation of the bioconcave disk shape of red cells is highly debatable and requires further experimentation. Bull (1973) has proposed

ACTIVE CALCIUM TRANSPORT AND Ca2+-ACTIVATED ATPase IN HUMAN RED CELLS

16 1

an ingenious model of the membrane which predicts the disk shape without resorting to any contractile mechanism. C. Epithelia

From the work of Loewenstein (1967; Olivera-Castro and Loewenstein, 1971), it is known that high permeability for alkali cations, but also for organic molecules at junctional membranes of epithelia, is abolished if the Ca2+ concentration is allowed to rise inside the cells. It seems, therefore, that maintenance of low intracellular Ca2+ concentration is necessary for the cellular communication in epithelia, and perhaps also a t the nexus of smooth muscle and at intercalated disks in cardiac muscle. D. Secretory Cells

Discharge or exocytosis of storage vesicles in presynaptic nerve endings (Harvey and MacIntosh, 1940; Katz and Miledi, 1967, 1969; Blaustein, 1971), mast cells (Foreman et at., 1973), certain endocrine and possibly exocrine glands is a consequence of Ca entry into the cytosol, whence it follows that low intracellular Ca2+ concentration ensures the stability of these vesicles a t rest and is a prerequisite for a regulatory function of Ca in the release process (for review see Rubin, 1970). REFERENCES Albers, R. W., Koval, G. S., and Siege], G. J. (1968). Studies on the interaction of ouabain and other cardioactive steroids with sodium-potassium-activated adenosinetriphosphatase. Mot. Pharmacol. 4, 324-336. Bader, H. (1971). Two (Ca++)-activated ATPases in human erythrocyte ghosts. Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, 545. Baker, P. F. (1972). Transport and metabolism of calcium ions in nerve. Progr. Biophys. M o ~Biol. . 24, 177-223. Baker, P. F., and Glitsch, H. G. (1973). Does metabolic energy participate directly in the Natdependent extrusion of Ca2+ ions from squid giant axons? J . Physiol. (London) 233,44P. Baker, P. F., Blaustein, M. P., Hodgkin, A. L., and Steinhardt, R. A. (1967). The effect of sodium concentration on calcium movements in giant axons of Loligo forbesi. J . Physiol. (London) 192,43P. Baker, P. F., Blaustein, M. P., Hodgkin, A. L., and Steinhardt, R. A. (1969) The influence of calcium on sodium efflux in squid axon. J . Physiol. (London) 200, 431458. Balzer, H., Makinose, M., and Hasselbach, W. (1968). The inhibition of the sarcoplasmic calcium pump by prenylamine, reserpine, chlorpromazine and imipramine. N u u n y n Schmiedebergs Arch. Pharmakol. Exp. Pathol. 260, 444-455.

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The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN Fysiologisk Institut Aarhus Universitet. Aarhus. Denmark

I . Introduction . . . . . . . . . . . . . . . . . I1. Preparations Used for the Study of Insulin Action on Sugar Transport A Skeletal Muscle . . . . . . . . . . . . . . B . Heart . . . . . . . . . . . . . . . . . C. Smooth Muscle . . . . . . . . . . . . . . I11. Cellular Structures Involved in Sugar Transport . . . . . . A The Localization of the Glucose Transport System . . . . B. The Role of Membrane Lipids . . . . . . . . . . C. The Role of Membrane Proteins . . . . . . . . . IV. The Function of the Glucose Transport System . . . . . . A. The Basal State . . . . . . . . . . . . . . B . The Effect of Insulin . . . . . . . . . . . . . C . The Effects of Other Activators . . . . . . . . . . D The Effect of Inhibitors . . . . . . . . . . . . V Cellular Signals Controlling Glucose Transport . . . . . . . A Enzymic Processes and Metabolites . . . . . . . . . B The Binding, Transport, and Distribution of Electrolytes . . VI Mechanisms for the Mode of Action of Insulin . . . . . . References . . . . . . . . . . . . . . . . .

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The fact that the present review concerning the effect of one hormone on one parameter in one cell type comprises 310 references without being complete is paradigmatic for the degree of specialization reached in the biological sciences. The action of insulin on glucose transport is the classical example of metabolism being controlled by the permeability of membranes. and the many investigations have been rewarding for promoting understanding of basic patterns in structures. functions. and signals related to 169

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transport and hormone action. This may justify that the following text concentrates on such common denominators and leaves out a number of specific details. Several recent reviews may be helpful for those who want the complete picture of this corner of nature (Krahl, 1961; Park et al., 1968; Morgan and Neely, 1972; Morgan and Whitfield, 1974; Clausen, 1974). II. PREPARATIONS USED FOR THE STUDY OF INSULIN ACTION ON SUGAR TRANSPORT A. Skeletal Muscle

The mere size of this major target for the action of insulin would indicate that it plays an obvious role in blood glucose homeostasis, and numerous preparations have been proposed for the study of how insulin controls glucose transport in skeletal muscle cells. An early approach to the problem was made with the demonstration of insulin effects in eviscerated cats (Best et al., 1926) and followed up by several studies of the distribution of nonmetabolized sugars in eviscerated dogs (Levine et al., 1949), rabbits (Wick and Drury, 1953), and nephrectomized rats (Helmreich and Cori, 1957). More recently, the hindquarter (Mahler et al., 1968; Ruderman et al., 1971) and the hemicorpus of the rat (Jefferson et al., 1972) have been shown to provide sensitive tools for the evaluation of insulin effects in major intact and composite structures of skeletal muscle. The effect of insulin on glucose consumption in human peripheral skeletal muscle has also been assessed by measurements of the arteriovenous concentration difference in the forearm (Andres et al., 1962). This method has yielded clear-cut evidence that glucose uptake is a function of the insulin concentration in the physiological range (Christensen and grskov, 1968), but since it is complicated, it has not been used in many laboratories. Although perfused tissues have several advantages with respect to integrity and insulin sensitivity, they are often heterogeneous and technically difficult to work with. A series of isolated muscle preparations have been developed in order to obtain more versatile tools for the study of details in the mechanisms of glucose transport and insulin action. Since the work of Gemmill (1940), the isolated rat diaphragm muscle [which was originally proposed by Meyerhof and Himwich (1924)l has become the classical choice for such investigations, being easy to prepare and sufficiently thin to allow rapid equilibration of oxygen and substrates between the muscle cells and the incubation medium. With the purpose of

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preserving the integrity of the muscle fibers, various modifications have been devised in which the muscle is incubated with its attachments to the rib cage (Kipnis and Cori, 1957; Kono and Colowick, 1961; Creese and Northover, 1961 ; Creese, 1968). Whereas such “intact” diaphragm muscles have decisive advantages in studies of sugar accumulation, the adhering central tendon, cartilage, and cut intercostal fibers represent a major difficulty when such parameters as glucose uptake, the production of metabolites, or the efflux of various solutes are to be evaluated. Because of these limitations together with the fact that the muscle is rhythmically contracting up to the moment of its isolation, it is often desirable to consider alternative preparations, which are perhaps more representative 01 peripheral skeletal muscle. The levator ani muscle of small rats is easier to prepare and has been well characterized (Arvill, 1967), but, owing to the presence of adhering tissues, it suffers from part of the disadvantages of the intact diaphragm. The sartorius muscle of frogs has been shown to be suitable for sugar transport studies (Narahara et al., 1960), but seasonal variations and some discrepancies from mammalian muscle present yet another set of limitations to the conclusions that may be drawn from experiments with this preparation. When using baby rats, the extensor digitorum longus (Pain and ManChester, 1970; Rogus and Zierler, 1973) and the soleus muscle (Chaudry and Gould, 1969; Kohn and Clausen, 1971) can easily be prepared with intact fibers and are quite convenient for most in vitro studies of glucose transport and metabolism as well as the measurement of ionic fluxes (Clausen et al., 1973). It should be noted, however, that diffusion through the interstitial space represents a rate-limiting factor for the exchange of solutes in these preparations, and that spontaneous contractures may occur if the muscles are not thoroughly oxygenated. Attempts to prepare intact isolated muscle cells by mechanical separation of fibers from large mammalian muscles have to some extent been successful, but not without appreciable loss of insulin responsiveness (Beatty et al., 1960). Similar problems may explain why isolated human skeletal muscle fibers do not respond at all to insulin in witro (Holm and Schersten, 1972). Quite a long time ago, it was demonstrated that quarter diaphragms have a smaller response to insulin than do hemidiaphragms (Groen et al., 1952; Liebecq, 1956). B. Heart

Some of the earliest studies of insulin actions i n witro were done with the isolated perfused heart of rabbits (Hepburn and Latchford, 1922).

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Since the work of Bleehen and Fisher (1954) and Morgan et al. (1959), the isolated rat heart has become one of the standard preparations for the investigation of glucose transport and insulin action in muscle cells. It has the advantage of being able to maintain a considerable degree of functional integrity and insulin sensitivity, of being easy to prepare for larger series of experiments, and of allowing an evaluation of the role of contractile activity for basal and insulin-stimulated sugar transport. Information about glucose metabolism and insulin action in the human heart has been obtained in measurements of arteriovenous concentration differences (Rudolph et al., 1969). Whole nonperfused hearts of fetal rats (Clark, 1971) and chickens (Guidotti et al., 1961) have turned out to be convenient for the study of the appearance of insulin sensitivity during fetal life. Digestion of fetal hearts with trypsin and hyaluronidase may yield viable muscle cells, which can be cultured, but these cells show a variable and modest response to insulin (Clark, 1971; Dunand et al., 1972). C. Smooth Muscle

The small and variable effect of insulin on glucose uptake in smooth muscle cells may have been discouraging for the study of this otherwise important tissue element. In the isolated aorta of rabbits (Mulcahy and Winegrad, 1962) or rats (Wertheimer and Ben-Tor, 1962) insulin was found to produce no or only a very modest stimulation of glucose uptake. A somewhat better response was found in taenia coli, which contains a larger proportion of smooth muscle cells (Grossmann and Manchester, 1966); and also in bladder wall (Bower and Grodsky, 1963) and the detrusor muscle of rats (Bihler et al., 1971), insulin could be shown to stimulate glucose uptake and the transport of nonmetabolized sugars. However, only recently a more systematic study has revealed that in the aorta of rabbits and rats, in rabbit colon, and in bovine mesenteric arteries insulin has a clear-cut, although delayed, stimulating effect on the transport of both glucose and nonmetabolized sugars (Arnquist, 1973).

111. CELLULAR STRUCTURES INVOLVED IN SUGAR TRANSPORT A. The Localization of the Glucose Transport System

It is generally assumed that the plasma membrane constitutes the major and rate-limiting barrier for the access of glucose to the cytoplasm. Several

THE EFFECT O F INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

173

studies have demonstrated that the system of transverse tubules of the sarcoplasmic reticulum is an extension of the outer sarcolemma and thus adds a potential surface available for transport, which is 5-7 times larger than the sarcolemma (Peachey, 1965). Although this electron microscopic evidence has been available for more than a decade, almost no transport studies have taken into consideration the complexity and heterogeneity of the membranes which may be involved in the uptake of glucose. Electron microscopy of tissues which had been exposed to hyperosmolarity or hypoosmolarity has shown that the volume of the sarcoplasmic reticulum is increased or, respectively, decreased under these conditions (Huxley et al., 1963; Girardier et al., 1963; Freygang et al., 1964; Sperelakis and Schneider, 1968; Rapoport et al., 1969; Birks and Davey, 1969). This indicates that the membranes lining the sarcoplasmic reticulum are participating in the exchange of water, and that the lumen of both the transverse and the longitudinal elements is available to sucrose. This was already suggested by Harris (1963), and the observation that, in rat diaphragm muscle, inulin occupies a smaller space than sucrose or mannitol (Kipnis and Parrish, 1965; Hider et aE., 1971) is compatible with the existence of a compartment which is available to compounds of low molecular weight, but from which inulin and larger structures are excluded. This has been suggested to be identical with the lumina of the longitudinal sarcoplasmic tubules, but direct evidence is not available (Rogus and Zierler, 1973). In frog sartorius muscle, the difference between the spaces available to fructose and inulin was found to vary with osmolarity in accordance with the above mentioned changes seen in electron micrographs (Vinogradova, 1968). Several reports indicate that at least the wall of the transverse tubules is permeable to K+ ions (Adrian and Freygang, 1962; Harris, 1963; Hodgkin and Nakajima, 1972; Almers, 1972), and it was recently proposed that they are also of significance for the exchange of amino acids (Hider et al., 1971) and Na+ ions (Rogus and Zierler, 1973). Hyperosmolarity stimulates the transport of glucose and 3-O-methylglucose in skeletal muscle (Kuzuya et al., 1965; Clausen, 1968a; Clausen et al., 1970; Nikolsky et al., 1971), and hypotonicity suppresses or abolishes the stimulating effect of insulin, work, 2, 4-dinitrophenol, and trypsin on sugar transport (Kohn and Clausen, 1972). On this basis, it was suggested that the sarcoplasmic tubules participate in the exchange of sugars, and that the rate of transport is in part a function of the accessibility (diameter) of the transverse tubules (Clausen and Kohn, 1972). (For alternative interpretations, see Section IV, c, 5.) Too little is known about the relative participation of various membrane elements in the transport of glucose and insulin action, but the information

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available about structural heterogeneity suggests that kinetic analysis can be meaningful only if most of these processes take place a t the sarcolemma. Nothing is known about the effect of insulin on the configuration of the sarcoplasmic reticulum, but cells in which this system is poorly developed (fetal and smooth muscle cells) are relatively insensitive to insulin (Clark, 1971 ; Arnquist, 1973). The observations that significant proportions of the enzymes controlling glycolysis (Fahimi and Karnovsky, 1966; Karpatkin and Braun, 1971) and glycogen metabolism (Andersson-Cedergren and Muscatello, 1963; Meyer et al., 1970) are associated with the membranes of the sarcoplasmic reticulum [around which the density of glycogen granules is particularly high (Wanson and Drochmans, 1968)] indicate that glucose entering via the sarcoplasmic tubules a t least has ready access to appropriate metabolic machinery. Compartmentalization of glucose metabolism in muscle has been demonstrated several times and was recently reinvestigated and discussed by Kalant and Beitner (1971).

B. The Role of Membrane lipids Owing to the hydrophilic nature of the glucose molecule, the lipid matrix of the plasma membrane constitutes an important barrier, and in membranes prepared from extracts of the lipids in red cell ghosts, the permeability to glucose was found to be four orders of magnitude lower than in intact cells (Jung, 1971). The same study showed that the permeability of these artificial membranes to various sugars in no way paralleled that found in red cells. Thus, 3-O-methylglucose, which is more lipophilic than glucose, had a 13-fold higher permeability. LeFevre et al. (1968) suggested that membrane phospholipids are not directly involved in the binding and transport of glucose, but could be of importance for the mobility of the glucose carrier. With phospholipase C it is possible to split off phosphorylcholine, which is the hydrophilic component of the phospholipids available from the outside of the plasma membrane. Rodbell (1966) demonstrated that this enzyme stimulates glucose metabolism in fat cells, and in frog sartorius muscle it produced an increase in the Vmax of 3-O-methylglucose transport, thus mimicking the action of insulin, which was proposed to induce an increase in overall mobility of the glucose carrier (Weis and Narahara, 1969). The fluid mosaic model proposed by Singer (1971) would predict that the mobility of membrane proteins (and therefore presumably also the glucose carrier) is determined by the fluidity of the lipid matrix, which is again influenced by temperature, ionic milieu, chemical composition and a number of drugs. In cultured muscle fibers, membrane proteins labeled

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

175

with a fluorescent probe showed a temperature-dependent lateral movement in the plane of the plasma membrane (Edidin and Fambrough, 1973). Further, although indirect, evidence for the involvement of membrane lipids in the transport of glucose comes from studies with membrane stabilizrrs. This general term was suggested for a wide variety of compounds that reduce the excitability, cation permeability, and osmotic fragility of the plasma membrane (Shanes, 1958). Some of the most potent membrane stabilizers, the local anesthetics, were shown to increase the lateral pressure in monomolecular films of lipids (Skou, 1961). This effect was correlated to their anesthetic potency, and it was proposed that these compounds occupy the interspaces in the ordered array of lipid molecules leading to lateral expansion and increased overall rigidity of the membrane. Several different membrane stabilizers (local anesthetics, barbiturates, psychotropic and anticonvulsant drugs) have been shown to inhibit the transport of glucose, galactose, 3-O-methylglucose, and sorbose in rat diaphragm muscle (Rafaelsen, 1961; Bihler and Sawh, 1971c), soleus muscle, adipocytes, and erythrocytes of the rat (Clausen et al., 1973) as well as in human erythrocytes (Baker and Rogers, 1973). It should be noted that the inhibitory effects of these compounds are seen only within a certain concentration range, beyond which a nonspecific leakage of the plasma membrane is produced with ensuing loss of potassium and an apparent stimulation of sugar transport (Bihler and Sawh, 1971c; Clausen et al., 1973). Conversely, veratrine or exposure to a calcium-free environment, which has been shown to labilize the plasma membrane (Shanes, 1958; Sperelakis and Pappano, 1969), were found to accentuate the effect of insulin on the transport of 3-O-methylglucose in rat soleus muscle (Table I). The observation that ions with a high charge density (La3+,Ni2+,Zn2+, Mn2+, and Co2+) inhibit the insulin-induced rise in the accumulation of 3-O-methylglucose in rat diaphragm muscle suggests that electrostatic forces between the negative charges of the surface of the plasma membrane can be of significance for the activation and the mobility of the glucose transport system (Bihler, 1972). The same study showed that calcium could overcome this inhibitory effect and that exposure to a calcium-free environment led to a (progressive) decrease in insulin responsiveness. In rat soleus muscle, lanthanum was found to suppress the stimulating effect of insulin on the efflux of 3-O-methylglucose (Table I). Further studies of the time course of these phenomena are required before the data can be encompassed into a meaningful whole, and direct effects of these ions on membrane proteins will have to be identified. However, the collective evidence suggests that the configuration and composition of polar heads and hydrophobic elements of the plasma

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TABLE I

EFFECT OF TETRACAINE, THIOPENTAL, VERATRINE, INSULIN

cAzi,

AND LAS+ ON

RESPONSIVENESS OF RAT SOLEUS MUSCLE^

Additions

Percent of rnethylglu~ose-~~C released per minute

Pb

Control Tetracaine (2 mM) Insulin (1 mU/ml) tetracaine (0.5 mM) tetracaine (2.0 mM) thiopental (1.0 mM)

0.21 0.19 1.62 0.73 0.20 0.82

f 0.03 f 0.04 f 0.16 f 0.08 f 0.02 f 0.03

(4) (4) (4) (3) (3) (4)

< < <

0.01 0.001 0.005

Control Veratrine (10-5 M ) (10-4 M ) Insulin (1 mU/ml) veratrine (10-6 M ) veratrine (10-4 M )

0.38 0.43 0.41 1.14 1.81 2.41

f 0.04 f 0.07 f 0.04 f 0.06 f 0.23 f 0.24

(4) (4) (3) (11) (7) (7)

< <

0.005 0.001

Control EGTA (0.5 mM); Ca omitted Insulin (1 mU/ml) EGTA; Ca omitted

0.36 0.46 1.23 2.07

f 0.03 (4) f 0.09 (4)

<

0.001

Control LaC13 (0.5 mM) Insulin (2 mU/ml) LaC13 (0.5 mM) Insulin (100 mU/ml) LaCI3 (0.5 mM)

0.40 0.52 2.83 0.98 4.63 2.90

f 0.07 f 0.03 f 0.27 f 0.09 f 0.52 f 0.10

<

0.05

<

0.05

+ + + + +

+

+ +

f 0.11 (10) f 0.15 (10)

(3) (3) (3) (3) (3) (3)

The efflux of 3-0-methylglucose-14C from preloaded intact soleus muscles was determined in the absence of, or 20 minutes after, the addition of, the compounds listed. lost per minute f SEM The data are given as the fraction of 3-O-methylgl~cose-~~C with the number of observations in parentheses. For experimental details, see Kohn and Clawen (1971). The significance of the differences between the experimental groups and the groups given insulin alone.

membrane lipids are as decisive for glucose transport and insulin action as for excitatory events and ionic permeability in muscle cells. C. The Role of Membrane Proteins

Whereas the study of relationships between membrane lipids and glucose transport has been somewhat neglected, numerous experiments with

177

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

insulin-sensitive tissues have been performed on the basis of the genera1 assumption that the glucose transport system is of protein nature. This contention derives in part from the close similarity between the kinetics of enzymic processes and sugar transport (Wilbrandt and Rosenberg, 1961). Studies with D-glycosyl isothiocyanate and cytochalasin B have given some idea about the number of units involved in glucose transport in erythrocytes (Taverna and Langdon, 1973a,b). However, it has not yet been possible to isolate or determine the number of hypothetical glucose TABLE I1 EFFECTOF CYTOCHALASIN B AND INSULIN ON THE UPTAKE OF 3-0-METHYLGLUCOSE AND ~-AYINOISOBUTYRIC ACID I N RAT SOLEUSMUSCLE^

Nanomoles per gram wet weight/GO minutes

Additions 3-0-Methylglucose

Control Insulin (100 mU/ml) Cytochalasin B (5 d m l ) Insulin (100 mU/ml) cytochalasin B (5 d m l )

+

a-Aminoisobutyric acid

Control . Insulin (100 mU/ml Cytochalasin B (5 pg/ml) Insulin (100 mU/ml) cytochalasin B (5 pg/ml)

+

Significance of difference between control and experimental

111 f 27 (6) 323 i 15 ( 4 )

<

0.001

32 f 21 (7)

<

0.02

8 f 15 (4)

<

0.01

0.81 f 0.05 (3) 1.22 f 0.10 (3) 0.89 f 0.05 (3)

< >

0.025

1.59 f 0.17 (3)

<

0.020

0.10

Soleus muscles prepared from fed rats (GO-70 gm) were incubated for 60 minutes in Krebs-Ringer bicarbonate buffer containing either 1 mM 3-0-methylgl~cose-~~C (0.1 pCi/ml) or 10-6 M a-aminoisobutyric acid-lJ4C (0.1 pCi/ml) without or with the additions indicated. The amount of sugar or amino acid taken up in the space not available to sucrose is expressed as nanomoles per gram wet weight f SEM with the number of observations in parentheses. For experimental details, see Kohn and Clausen (1971).

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TORBEN CLAUSEN

transport systems in muscle cells. In muscle, phlorizin seems to have relatively low affinity for the glucose transport system and may not be bound directly to the carrier site, but cytochalasin B (Czech et al., 1973), which is active a t minute concentrations (Table 11) and is known to interact with the proteins of microtubules, may be a more specific marker. Many agents that modify the structure of proteins have been shown to affect glucose transport, but it is difficult to determine whether such actions are specifically exerted on the glucose transport system. The inhibitory effects of SH-reagents have repeatedly been demonstrated in muscle and are commented on in Section IV, D. Neuraminidase, trypsin, and chymotrypsin have been found to stimulate glucose transport in rat diaphragm (Rieser and Rieser, 1964) and both the influx and the efflux of 3-O-methylglucose in frog sartorius muscle (Weis and Narahara, 1969) and rat soleus muscle (Kohn and Clausen, 1971). It seems reasonable to assume that these ef'fects are the outcome of a looser structure of the membrane in general or an increased accessibility or flexibility of the glucose transport system. Kahlenberg et al. (1972) suggested that the stimulating effect of trypsin on glucose transport in red cell membranes could be the result of exposure of latent sites capable of binding and transporting glucose. On the basis of the detection of an extremely low proteolytic activity in insulin it was proposed that this could be of significance for its action on sugar transport (Rieser, 1967).

IV. THE FUNCTION OF THE GLUCOSE TRANSPORT SYSTEM

The processes by which glucose is metabolized are limited by the availability of certain key enzymes and are controlled by feedback via metabolites (Garfinkel and Hess, 1964). Under basal conditions, however, their overall capacity exceeds that of the processes by which glucose gains access to the cytoplasm, and both in skeletal muscle (Cori et al., 1933; Lundsgaard, 1939; Kipnis et al., 1959; Chaudry and Gould, 1969), heart (Morgan et al., 1961a, 1964), and smooth muscle (Arnquist, 1972) the concentration of free glucose in the cytoplasm is close to zero. This constitutes the basis for both endogenous and exogenous control of the rate of glucose consumption being exerted at the plasma membrane. Whereas endogenous control may be assumed to function in accordance with the demands of the cell per se, the exogenous control exerted by insulin and other factors is rather in accordance with the needs of the whole organism as well as with some more long-term requirements for the storage of readily available substrate in each individual cell.

THE EFFECT OF INSULIN O N GLUCOSE TRANSPORT IN MUSCLE CELLS

179

Most if not all of the situations where the endogenous demand for metabolizable substrate is increased would be expected to elicit a rise in glucose permeability. Therefore, conditions where the ATP-demand is augmented owing to contractile activity, active transport of ions, and processes of synthesis offer opportunities for identifying the signals by which endogenous control of glucose transport is exerted. Since these signals may in part be the same as those triggered by the exogenous factors known to control glucose transport, an attempt is made to analyze the action of insulin by comparing it with a variety of factors and conditions that mimic or interfere with the effect of the hormone on glucose transport. A. The Basal State

Owing t o variations in endogenous energy demands, the integrity of the plasma membrane, and the previous fate of the preparation used, it is difficult t o define a set of conditions under which the processes of glucose transport can be said to operate a t a basal rate. This may explain why inhibition or stimulation of glucose transport found in one system cannot be detected in another, but also makes it complicated to determine whether glucose transport may be mediated by more than one system (or various states of the same system). As in the following, the basal state can only be defined operationally as the condition where no inhibitory or stimulatory factors have been introduced. As early as in 1939, Lundsgaard showed that the rate of glucose consumption shows saturation a t high concentrations, and such phenomena have later been found in several preparations of skeletal muscle (Norman et al., 1959; Narahara et al., 1960; Chaudry and Gould, 1969) and heart (Post et al., 1961; Guidotti et al., 1966) and in smooth muscle (Arnquist, 1972). The demonstration of stereospecificity and countertraneport makes it reasonable to conclude that most of the glucose transport across the plasma membrane is mediated by a carrier structure (Morgan and Neely, 1972). I n the heart (Morgan et al., 1964) and in smooth muscle (Arnquist, 1972), the distribution of L-glucose does not exceed the space available to sorbitol, either in the absence or in the presence of insulin, indicating that only minute amounts of glucose penetrate the plasma membrane b y free diffusion and that the carrier is the basic structure in regulat,ion. Another analog, 3-O-methylglucose, however, shows a faster rate of diffusion, perhaps owing to its more lipophilic nature (Jung, 1971). Because of their low temperature coefficient, galactose, 2-deoxygalactose, and D-xylose have been claimed to penetrate the plasma membrane by diffusion (Kipnis and Cori, 1959). The lack of competition between these

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TORBEN CLAUSEN

sugars and 2-deoxyglucose and glucose led to the suggestion that the membrane may contain two systems for sugar transport. Others have also found that galactose transport is not interfered with by glucose (Battaglia and Randle, 1960), but] both D-xylose and L-arabinose may share the glucose transport system, according to Fisher and Lindsay (1956) and Battaglia and Randle (1960). Studies with erythrocytes have shown that a C-1 chair conformation in the sugar molecule is the preferred structure for carrier-mediated transport (LeFevre and Marshall, 1958), and this stereospecificity of the glucose transport system has led to the deliberate designing of synthetic glucose analogs (3-0-methylglucose and 2-deoxyglucose). These sugars have been found to have a high affinity for the glucose transport system. A t variance with 2-deoxyglucose, 3-O-methylglucose is neither phosphorylated nor metabolized, and it causes no interference with carbohydrate metabolism in muscle (Csaky and Wilson, 1956; Narahara and Ozand, 1963; Kohn and Clausen, 1971). Furthermore, its transport is not modified by labeling with 3H or I4C (Narahara and Ozand, 1963). One of the difficulties in evaluating results obtained with nonmetabolized sugars is related to the fact that these compounds are readily released from the cytoplasm, and usually only the net result of influx and efflux is measured. Under basal conditions, the return of already accumulated sugar becomes a significant error when the intracellular concentration exceeds 16% of the extracellular (Narahara and Ozand, 1963). Since an increase in influx is associated with a similar acceleration of efflux, the effect of stimuli to the sugar transport system may be considerably underestimated if only the net uptake is measured. Although these problems may partially be solved by reducing the incubation period, the time interval which ought to be used is often too short to allow an even distribution of the sugar in the extracellular space. In frog sartorius muscle and rat soleus muscle, the equilibration of mannitol and sucrose, respectively, was found to require 40-60 minutes (Narahara and Ozand, 1963; Law, 1967), and in the diaphragm and soleus muscles of the rat the concentration of glucose in the interstitial space was found to be lower than that of the incubation medium (Randle and Smith, 1958; Chaudry and Gould, 1969). Therefore, it is difficult to ascertain that all the cells in a muscle preparation are exposed simultaneously to the sugar, and that the concentration in the interstitial space is uniform. The use of double-labeling for the simultaneous determination of the space available t o a sugar and an extracellular marker of similar molecular weight has increased precision in the measurement of sugar uptake (Narahara and Ozand, 1963; Bihler, 1968). However, the above-mentioned

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

181

systematic sources of error have not been overcome, and the values obtained for kinetic constants (in particular in the presence of insulin) are as yet only of operational significance and are not a very reliable basis for models describing the effect of insulin on the properties of the glucose transport system . Measurements of efflux from tissues which have been preloaded with a labeled nonmetabolized sugar may yield values that over longer intervals of time are more representative of transport in mainly one direction (Morgan et al., 1961b; Narahara and Ozand, 1963; Young, 1965; Saha et al., 1968; Kohn and Clausen, 1971). Thus various stimuli for 3-0-methylglucose transport in soleus muscle of the rat were found to produce a considerably larger rise in the rate of efflux than in the rate of uptake. The same study provided some evidence that the individual cells in this muscle may differ considerably with respect to sugar permeability and insulin responsiveness-a conclusion also supported by studies of the incorporation of 14C-labeled glucose into glycogen in muscle cells of the tongue (Coimbra, 1968). The rate of glucose consumption depends on the relative content of white and red fibers (Bocek et al., 1966), and both in the diaphragm (Libbecq, 1954) and soleus muscle of the rat (Chaudry and Gould, 1969) it is inversely related to the weight. For the utilization and the penetration of glucose in frog sartorius muscle, Qlo values of 4.0 and 1.7, respectively, were found (Narahara et al., 1960). In rat gastrocnemius, the Qlo for penetration and phosphorylation were, respectively, 2 and 2.5 (Kipnis et al., 1959). At low temperatures, the penetration may exceed the rate of phosphorylation (Fuhrman and Fuhrman, 1964), and at 12°C free glucose was detected in the cytoplasm (Park et al., 1955). B. The Effect of Insulin

It is generally assumed that the first step in the action of insulin consists in a contact or binding between the hormone and some specific receptor. Various muscle preparations can bind appreciable amounts of 1311-labeled insulin (Stadie et al., 1949; Garratt et al., 1966; Wohltmann and Narahara, 1966), but, since it is rapidly degraded by insulinase (Piazza et al., 1959; Brush, 1971) upon contact with this tissue, such studies have not yielded precise information about the number of insulin receptors. In fact, it is still not ascertained that the specific (displaceable) binding is related to the action of insulin and not merely to the first step in the inactivation of the hormone (Wohltmann and Narahara, 1966). The recent observation that trypsin treatment of rat diaphragm muscle leads to a (reversible) upheaval

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TORBEN CLAUSEN

of insulin sensitivity indicates that, as in fat cells, an insulin receptor of protein nature is situated a t the surface of the muscle cell membrane (Renner, 1973). Studies with frog sartorius muscle illustrate the time lag between the binding of insulin (which could be detected within 15 minutes) and the onset of the rise in sugar permeability, which in this preparation is only apparent after 30 minutes of exposure to the hormone and becomes maximal 3 hours later (Narahara and Ozand, 1963). The same study showed that insulin-treated muscles maintain increased sugar permeability during several hours of washing in insulin-free buffer. A long time lag for the onset of the insulin effect on sugar transport was also found in smooth muscle (Arnquist, 1973), and these types of responses offer possibilities‘for studying the processes by which the glucose transport system is activated. In the perfused rat heart, the human forearm, and isolated rat soleus and diaphragm muscles, the stimulating effect of insulin on the transport of glucose and nonmetabolized sugar can be detected within the time resolution of the systems used, i.e., less than 5 minutes after the first exposure t o the hormone (Bleehen and Fisher, 1954; Williams, 1959; Morgan et al., 1961b; Andres et al., 1962; Mahler et al., 1968; Kohn and Clausen, 1971). Conversely, when a rat heart, after exposure to insulin, is perfused with insulin-free medium, the rate of glucose uptake returns to basal levels with the same speed as insulin is washed out (TI12 of 3-5 minutes) (Bleehen and Fisher, 1954), indicating that the “resetting” of the glucose transport system can take place a few minutes after the removal of the hormone. During fetal life, the basal rate of glucose uptake is gradually decreasing, and sensitivity towards insulin develops rather late, apparently coinciding with the appearance of insulin in the pancreas (Guidotti et al., 1961; Clark, 1971; Felix et aE., 1971). Unfortunately, no information is available about the possible role of insulin or other factors in inducing a receptor and the activation mechanism which solves the problems related to the gradual decrease in basal sugar permeability. Once developed, the capacity for responding to insulin reaches a level where an up t o 15-fold increase in the rate of glucose transport can be induced with maximal concentrations of the hormone (Ruderman et al., 1971). I n the isolated rat hemidiaphragm, a stimulating effect of insulin can be detected down to concentrations below 10 MU/ml (Groen et al., 1952), which is close to the level measured in human plasma with immunoassay. This preparation has been the most widely used for the bioassay of insulinlike activity in plasma (Vallance-Owen and Wright, 1960) and the study of insulin action. However, since the magnitude of the maximal response

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

183

depends on the integrity of the cells, several other preparations are to be preferred for the latter purpose. In the forearm of normal human subjects, the glucose consumption was significantly stimulated by infusion of insulin into the brachial artery to a final concentration of 40 pU/ml in the blood (Zierler and Rabinowitz, 1964), and a positive correlation was found between the immunoreactive insulin levels and glucose consumption in the fasting state (Christensen and Prskov, 196s). In guinea pig taenia coli (Grossman and Manchester, 1966), rabbit colon, and rat aorta (Arnquist, 1971), the effect of insulin on the uptake of glucose and D-xylose is modest and seen only with very high concentrations of the hormone. In bovine mesenteric arteries, a definite doseresponse relationship was found in a concentration range two orders of magnitude above that measured in parallel experiments with rat diaphragm (Arnquist, 1973). More pronounced effects may be obtained if the tissues are preincubated with insulin, and it seems likely that insulin plays a role in the more long-term regulation of glucose transport in smooth muscle cells. Quite a long time ago, the glucose consumption in rabbit aorta was found to be suppressed by alloxan diabetes (Yalcin and Winegrad, 1963), but this was found to be related to a decrease in hexokinase activity. In the rat detrusor muscle, the facilitated transport of 3-O-methylglucose was reported to be insulin sensitive (Bihler et al., 1971). A major argument for the idea that the action of insulin on glucose transport can be separated from the processes of phosphorylation was obtained by the demonstration that the hormone increases the space available to D-galactose, L-arabinose, and D-xylose in the nephrectomized dog (Levine et al., 1949). This concept has been corrobated by numerous experiments with eviscerated animals, preparations of isolated hearts, skeletal and smooth muscle cells, and nonmetabolized sugars have become important tools for the characterization of insulin action (for reviews, see Park et al., 1959; Levine, 1965; Park et al., 1968; Morgan and Whitfield, 1974; Arnquist, 1973). In muscle cells, insulin increases the transport of several naturally occurring sugars : D-galactose (Fisher and Lindsay, 1956; Resnick and Hechter, 1957; Young, 1965), D-arabinose (Carlin and Hechter, 1961), L-arabinose (Carlin and Hechter, 1961; Park et al., 1961; Fisher and Gilbert, 1970), L-xylose (Carlin and Hechter, 196l), D-xylose (Kipnis and Cori, 1957; Fisher and Gilbert, 1970; Arnquist, 1971), D-fructose (Nakada, 1956), and D-glucosamine (Nakada et al., 1955; Beloff-Chain et al., 1970; Chambaut et al., 1969). Insulin also accelerates the transport of the synthetic glucose analogs 3-O-methylglucose, 2-deoxyglucose, and 2-deoxygalactose in the heart (Morgan et al., 1964), skeletal muscle (Narahara and &and, 1963; Kipnis et al., 1959; Parrish and Kipnis, 1964;

184

TORBEN CLAUSEN

Kipnis and Parrish, 1965; Kohn and Clausen, 1971), and smooth muscle (Arnquist , 1973). These sugars differ considerably with respect to rate of transport and kinetic constants, but when used for the evaluation of the sensitivity to insulin and the time course of insulin action, the results are essentially the same as those obtained with glucose. The effect of insulin on galactose transport in the heart shows rather marked seasonal variation (Young, 1965), but it is not known whether this is related to changes in the intrinsic properties of the glucose transport system. The effect of insulin on glucose uptake is temperature dependent, and considerably suppressed a t 13"C, where free glucose accumulates in the cytoplasm (Park et al., 1955). In frog muscle, the stimulating effect of insulin (but not that of contractile activity) on 3-0-methylglucose transport is abolished at O"C, indicating that the insulin-induced signals for activation of the glucose transport system are suppressed (Holloszy and Narahara, 1965). In rat diaphragm muscle, the uptake of galactose, D-xylose, and 2-deoxygalactose was found to have a higher & l o in the presence of insulin than in its absence (Kipnis and Cori, 1959; Parrish and Kipnis, 1964). Theoretically, insulin may stimulate sugar transport either by modifying the properties of the system mediating the transfer of sugars under basal conditions, or by bringing new (and perhaps kinetically different) transport systems into function. The information available about kinetic constants does not allow any clear distinction between these possibilities. Insulin has been reported to produce an increase (Fisher and Zachariah, 1961; Fisher and Gilbert, 1970; Post et al., 1961; Morgan et al., 1961a), a decrease (Norman et al., 1959; Guidotti et al., 1966; Chaudry and Gould, 1969), or no change (Narahara and Ozand, 1963) in the apparent K , for sugar transport. The V,,, was either unaltered (Norman et al., 1959; Chaudry and Gould, 1969; Guidotti et al., 1966) or increased (Post et al., 1961; Fisher and Gilbert, 1970; Narahara and ozand, 1963). I n view of the structural complexity and heterogeneity discussed in Section IV, A. this is not so surprising, but the discrepancies may also be the result of differences in preparations and sugars used for the evaluation. On the basis of the carefully designed experiments with perfused hearts (Post et al., 1961; Fisher and Gilbert, 1970) and frog sartorius muscle (Narahara and ozand, 1963) it seems reasonable to conclude that the maximal capacity of the sugar transport system is increased by insulin. Narahara and Ozand (1963) suggested that this could reflect an increased mobility of the glucose carrier, and the inhibitory effects of membrane stabilizers and cooling seem to support this conclusion (see Section 111, B).

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

185

C. The Effects of Other Activators

1. CONTRACTILE ACTIVITY

Contractile activity is associated with a rise in the permeability to glucose and other sugars, which has been demonstrated both in Vivo and in vitro with a variety of skeletal muscle preparations and the heart. In the perfused hindquarter and the isolated soleus muscle of the rat, the stimulating effect of contractile activity is slightly smaller and somewhat later in onset than that of insulin (Ruderman et al., 1971; Kohn and Clausen, 1971). In the isolated frog sartorius muscle, the uptake of 3-O-methylglucose was correlated to the frequency of stimulation, not to the amount of work performed or the rate of lactate production. A t maximum levels of stimulation, the effects of insulin and electrical stimulation were neither significantly different nor additive, and the kinetic constants showed the same change (increased V,,, and unaltered apparent K,). This indicates that the two stimuli act on the same glucose transport system, but, at variance with insulin, electrical stimulation leads to a prompt rise in the uptake of 3-O-methylglucose which could be detected even at 0°C (Holloszy and Narahara, 1965). Muscular work may lead to localized relative hypoxia or substrate depletion, but this cannot account for the rise in sugar permeability in frog sartorius muscle (Holloszy and Narahara, 1965) or the heart (Neely et al., 1967). Among the processes eliciting contraction, the depolarization of the plasma membrane does not seem to play any essential role in the activation of the glucose transport system. Thus, caffeine, which may induce contractions without producing action potentials, accelerates the uptake of 3-O-methylglucose in frog sartorius muscle, an effect that has been related to an increase in the concentration of Ca2+in the myoplasm (Holloszy and Narahara, 1967; Skopicheva, 1972). In rat soleus muscle, caffeine (10 mM) was found to stimulate the efflux of the same sugar, but this was only seen 40 minutes after the rise in tension and 45Ca-release produced by the drug (Clausen et al., 1974b). This may be the result of the inhibitory effect of caffeine discussed in Section V, A, 3. Contractions elicited by K+-rich buffer lead to a stimulation of sugar transport (when measured after return to normal K+ concentration) which is dependent on the extracellular concentration of calcium (Holloszy and Narahara, 1967; Nikolsky et al., 1971). Since the glucose transport system seems to be activated for, up to several hours after the cessation of contractile activity (and the reestab-

186

TORBEN CLAUSEN

lishment of a low cytoplasmic level of free Ca2+),the suggested role of Ca2+ as a trigger for the activation of the glucose transport system may also be indirect or combined with other factors (Holloszy and Narahara, 1965; Gould and Rawlinson, 1966a; Arvill, 1967; Chapler and Stainsby, 1968). During contraction, a variety of muscular tissues (including uterus and intestine) have been shown to release a labile humoral factor that stimulates the transport of glucose and other sugars in diaphragm and adipose tissue (Goldstein et al., 1953; Levine and Goldstein, 1955; Goldstein, 1961; Havivi and Wertheimer, 1964; Bihler et al., 1970). This factor is nondialyzable and precipitable with ammonium sulfate (Havivi and Wertheimer, 1964). It also differs from insulin immunologically (Bihler et al., 1970). I n pancreatectomized, eviscerated dogs, muscle work still stimulated galactose transport (Goldstein et al., 1953); this indicates that insulin is not required for this effect. Training was found to augment the response of the glucose transport system to the stimulus of exercise (Gould and Rawlinson, 1966b) and that of insulin (Lipman et al., 1972). Therefore, the stimulating effect of contractile activity on sugar transport must be considered not only as a n acute phenomenon, but also as a sometimes more persistent modification of the properties of the glucose transport system that will enable the muscle cells to cover not only the immediate, but also subsequent, metabolic demands. 2. ANOXIAAND METABOLIC POISONS Randle and Smith (1958) demonstrated that in muscle cells inhibition of energy production by exposure to anoxia or metabolic poisons leads to stimulation of glucose uptake and the transport of nonmetabolized sugars. Several others have described similar effects in rat diaphragm muscle (Lotspeich and Wheeler, 1962; Kono and Colowick, 1961; Bihler, 1968), monkey sartorius muscle (Beatty et al., 1966), rat soleus muscle (Chaudry and Gould, 1969; Kohn and Clausen, 1971), frog sartorius muscle (ozand et al., 1962; Vinogradova et al., 1968; Kudryavtseva et al., 1972), and the isolated perfused rat heart (Morgan et al., 1959, 1961a). The stimulation was found t o be somewhat slower in onset than that induced by insulin, and owing to its progression with time (Kohn and Clausen, 1971) it is difficult t o compare the effects of the two stimuli with respect to absolute magnitude and to determine whether they are additive (Ozand et al., 1962; Kono and Colowick, 1961). During inhibition of energy production, glucose uptake shows saturation kinetics (Chaudry and Gould, 1969), and phlorizin suppresses the increase in permeability to nonmetabolized sugars (Lotspeich and Wheeler,

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

187

1962; Kohn and Clausen, 1971); this indicates that the effect is due to a n activation of the glucose transport system, not a nonspecific overall increase in the permeability of the plasma membrane. At variance with insulin, anoxia was found to increase the Vmaxfor glucose uptake in rat soleus muscle (Chaudry and Gould, 1969). Transport stereospecificity is the same as with insulin (Kono and Colowick, 1961). Lack of metabolizable substrate in the perfusate was found to increase the permeability to pentoses in the heart (Henderson et al., 1961; Bihler et al., 1965), I n rat soleus muscle the basal rate of 3-0-methylglucose transport was almost doubled by 18 hours of fasting (Kohn and Clausen, 1971). Muscles from winter frogs, which are more sparsely provided with endogenous energy sources, are more permeable to 3-0-methylglucose than muscles from summer frogs (Narahara and Ozand, 1963). Increased sugar permeability seems to be induced when the energy production can no longer meet the metabolic demands of the cell. Since this condition is associated with a change in the concentration of a considerable number of metabolites and electrolytes, it is difficult to identify those which might be of significance in eliciting the activation of the glucose transport system. Already very early, A T P was proposed as a regulatory substance that could limit the activity of the glucose transport system by maintaining a postulated protein in a phosphorylated state (Smith et al., 1961). However, under conditions where the A T P content was not detectably altered, anaerobiosis was found to produce a marked rise in glucose uptake (Ozand et al., 1962; Chaudry and Gould, 1970). In rat soleus muscle and frog muscle, the stimulating effect of anaerobiosis and metabolic inhibitors on the transport of n-xylose was not correlated with the change in ATP content (Chaudry and Gould, 1970; Kudryavtseva et al., 1972; Kono and Colowick, 1961). Metabolic inhibitors were reported to induce rigor in muscles (Randle and Smith, 1958), and there is evidence that inhibition of energy production leads t o a rise in the cytoplasmic concentration of Ca2+ions in a variety of tissues (Rojas and Hidalgo, 1968; Blaustein and Hodgkin, 1969; Clausen, 1970; Case and Clausen, 1973) including skeletal (Paul, 1961; Kohn and Clausen, 1971; Clausen et aE., 1974b) and smooth muscle (Van Breemen et al., 1973). As discussed in Section V, B, Ca2+ ions may be considered as an alternative to ATP for the control of glucose transport during energy depletion as well as under a wider variety of conditions.

3. COMPOUNDS STRUCTURALLY RELATED TO INSULIN Proinsulin is a single-chain polypeptide in which the middle portion, the C-peptide, links the A- and the B-chain of insulin (Steiner et al., 1972).

188

TORBEN CLAUSEN

It has been found to produce the same pattern of metabolic effects as insulin in a variety of preparations, but with a potency ranging from 2 to 25y0 of that of insulin (Rubinstein et al., 1972). It has been suggested that this effect is preceded by a proteolytic conversion into insulin (Shaw and Chance, 1968; Lazarus et al., 1970)) but other studies with muscle have not yielded any evidence in support of this mechanism, and it seems likely that proinsulin has definite intrinsic biological activity (Brush, 1971; Narahara, 1972). The data are compatible with the idea that proinsulin interacts with the same receptor as insulin, but with considerably lower affinity. Like proinsulin, the A-chain has been found to produce hypoglycemia (Fenichel et al., 1968) and to stimulate glucose uptake in the isolated rat diaphragm (Volfin et al., 1964; Surmaczynska and Metz, 1969). I n witro, the potency of the A-chain is about 1/1000 that of insulin, but since the synthetic A-chain has a similar activity, the stimulating effect on glucose uptake cannot be accounted for as due to contamination with insulin. Although the isolated B-chain does not stimulate glucose uptake in muscle (Mahler et al., 1968; Surmaczynska and Metz, 1969), several derivatives of arginine (Bzz) have been found to mimic the effect of insulin in augmenting glucose uptake and glycogen deposition in rat diaphragm, but only when present at very high concentrations (Weitzel et al., 1971). The presence of disulfide bridges in the insulin molecule has given rise to some speculation that these structures might be of importance for the binding or biological action of the hormone. Sulfhydryl compounds have been found t o stimulate glucose uptake in rat hemidiaphragm, but the late onset of the effect together with the simulta,neous acceleration of glycogenolysis suggest a mechanism of action similar to that of metabolic inhibitors (Haugaard et al.. 1972). The above-mentioned results emphasize the specificity of the insulin receptor. For a further analysis of the active components of the insulin molecule, comparisons between the biological activity and the affinity for binding to the insulin receptor seem to be required. 4. ELECTROLYTES Although it has not been possible to demonstrate an active accumulation of sugars coupled to ionic gradients in muscle, electrolytes, or the distribution of electrolytes across the plasma membrane may influence both basal and insulin-stimulated sugar transport quite markedly (Clausen, 1972). The iso-osmolar replacement of NaCl by sucrose or mannitol leads to an increase in glucose uptake or the influx and efflux of nonmetabolized

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

189

sugars in diaphragm (Bhattacharya, 1961; Ilse and Ong, 1970; Bihler, 1972) and soleus muscle of the rat (Kohn and Clausen, 1972). This was associated with an increase in the uptake of Ca (Ilse and Ong, 1970). Substitution of Na+ by Li+ stimulates the uptake of glucose and arabinose in rat diaphragm muscle (Bhattacharya, 1961; Clausen, 1968b) and 3-O-methylglucose transport in rat soleus muscle (Kohn and Clausen, 1972). These effects are seen down to Li+ concentrations of 5 mM (Clausen, 1968b), and their increase with the time of exposure to Li+ suggests that the intracellular concentration of the ion is of significance. Li+ inhibits the uptake of calcium in microsomes prepared from skeletal muscle (de Meis, 1971) and may thus produce a rise in the cytoplasmic Ca2+ level. In a buffer where Na+ had been replaced by Lif, the resting tension of the frog rectus abdominis muscle was increased (Irwin and Oliver, 1970), and in rat soleus muscle the influx of calcium was stimulated by 43% (T. Clausen, unpublished observations). The effects of K+ are described in Sections IV, C, 1 and D, 2. The role of calcium and membrane stability is discussed in Sections 111, B and V, B, 4. Inhibition of the active Na+-K+ transport with cardiac glycosides or by incubation in K+-free buffer was found to stimulate glucose uptake (Kypson et al., 1968) and 3-O-methylglucose transport in rat diaphragm muscle (Bihler, 1968). This effect was rather closely correlated to the intracellular Na+:K+ ratio; it was not the immediate consequence of inhibited Na+-K+ transport (Bihler and Sawh, 1971a, b). Others have not been able to detect any early effects of these compounds (or K+ omission) on the uptake of glucose, 2-deoxyglucose, or galactose in diaphragm muscle (Kipnis and Parrish, 1965; Clausen, 1965, 1966) or the transport of 3-O-methylglucose in soleus muscle (Kohn and Clausen, 1971, 1972). The variability and time lag of the insulinlike effect produced by reducing the Na+-K+ gradients across the plasma membrane would suggest that other factors are involved. Similar conditions have been found to induce contractures in the rectus abdominis muscle of the frog (Shigei et al., 1963; Irwin and Oliver, 1970), indicating a rise in the cytoplasmic level of Ca2+.In frog sartorius muscle, K+ omission or ouabain was found to increase the uptake of labeled calcium, and the calcium content was correlated with the Na+ content (Cosmos and Harris, 1961). In nerve axon (Baker, 1970), cardiac muscle (Reuter and Seitz, 1968), aortic wall (Reuter et al., 1973), and adipose tissue (Clausen, 1970), the transport of Ca2+ across the plasma membrane seems to be linked to Na+ transport. A rise in the intracellular calcium content might be induced by an increase in the Naf level, and this would again depend on the Na+ fluxes and the exchange-

190

TORBEN CLAUSEN

ability of the calcium accumulated in various cellular pools. Furthermore, Na+ (40-120 mM) has been found to inhibit the uptake of calcium in microsomes from skeletal muscle (de Meis, 1971). I n slices of heart muscle, cardiac glycosides were found to stimulate the uptake and metabolism of glucose (Wollenberger, 1947). Digoxin (lop7 mole/kg) was reported to stimulate glucose utilization (Kien and Sherrod, 1960) and galactose uptake in the heart of normal dogs (Kien et al., 1960). Studies with isolated perfused hearts from rats and guinea pigs have shown that ouabain increases glucose consumption and arabinose uptake (Kreisberg and Williamson, 1964; Hoeschen, 1971 ; Elbrink and Bihler, 1973). This may be secondary to the inotropic effects of these drugs and related to a change in intracellular Ca2+.The stimulating effect of ouabain on glucose metabolism was found to depend on the concentration of calcium in the perfusate (Kreisberg and Williamson, 1964). Toxic concentrations of ouabain, however, seemed to stimulate arabinose transport markedly, independent of contractile activity (Elbrink and Bihler, 1973). Ouabain has also been found to mimic the effect of insulin in reducing the blood sugar (Triner et al., 1968) and accelerating the disappearance of intravenously injected g ala~ to se-~(Kien ~ C et al., 1960). This may in part be the result of increased release of insulin (Milner and Hales, 1967), since it is not seen in pancreatectomized dogs. 5. HYPEROSMOLARITY Kuzuya et al. (1965) found that the addition of NaC1, sucrose, or mannitol in the concentration range 50-200 mOsm caused a considerable increase in the uptake of glucose in rat hemidiaphragm. The addition of a rapidly penetrating solute, urea, produced no change. Studies with the same preparation (Clausen, 1968a) and frog sartorius muscle (Nikolsky et al., 1971) confirmed these observations, and in rat soleus muscle, hyperosmolarity was found to stimulate both the influx and the efflux of 3-0methylglucose (Clausen et al., 1970; Kohn and Clausen, 1971, 1972). Hyperosmolarity may stimulate glucose uptake and 3-0-methylglucose transport to approximately the same level as a supramaximal concentration of insulin, but with a slightly slower rate of onset (Clausen et al., 1970). The effect of hyperosmolarity on sugar transport is not affected by insulin antibody (Kuzuya et al., 1965), but suppressed by factors which also interfere with the effect of insulin on sugar transport (phlorizin, high extracellular K+ and membrane stabilizers) (Kohn and Clausen, 1972; Clausen et al., 1973). The close similarity between the effects of hyperosmolarity and insulin on the time course and kinetic constants for glucose utilization in fat

191

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

cells (Clausen et al., 1970) prompted a more systematic comparison between these two factors. From Table III it can be seen that in rat soleus muscle hyperosmolarity mimics the effect of insulin on 3-0-methylglucose transport, the incorporation of glucose into glycogen, Na+ efflux, and the uptake of 32P.However, as earlier found by Isaacson (1969), hyperosmolarity stimulates the rate of 45Carelease, and as can be seen from Fig. 1, a contraction is induced. This is also seen in a calcium-free buffer (Clausen et al., 1974b) and probably reflects an increase in the cytoplasmic level of Caf2 following a release from cellular organelles. I n microsomes isolated from skeletal muscle (de Meis, 1971) or aortic wall (Baudouin-Legros and Meyer, 1973) the uptake of calcium was inhibited by Na+ and K+ in the concentration range 50-100 mM, and studies with isolated plasma membranes indicate that K+ ions can displace bound calcium. Hyperosmolarity TENSION

Igrn) MANNITOL

3'0

I

1200 rnM) (10 r n U i r n l I

INSULIN

I

,,.SULIN

I

/ f

In,. A I Jl"

/

0

,

/

P

-*,

co.

01

0

1.0

-

0.oL

CONTROL

I

!

0

20

40

60

MINUTES

FIG.1. Effect of hyperosmolarity and of insulin on tension in soleus muscle. Soleus muscles were prepared from 48-hour fasted rats (60-70 gm) and placed vertically in plastic holders with the proximal end fixed and the distal tendon attached to an isometric force transducer. Tension was recorded with a Servograph pen recorder (REA 310). The muscles were immersed a t 30°C in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 0.1 gm of albumin per 100 ml, 1.27 mM calcium, and no metabolizable substrates, and were continuously gassed with O2 : CO, (95% : 5%). Two groups of muscles were exposed to hyperosmolarity by the addition of mannitol (200 mM); one served as a control and the other WBS given insulin as indicated. The bars indicate 2 X SEM. Data are from Clausen et al. (197413).

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TORBEN CLAUSEN

TABLE I11 EFFECTS OF INSULIN AND HYPEROSMOLARITY~ ~

~

~~

Parameter

~~

Insulin (0.1 IU/ml)

Control

3-O-Methylgl~cose-~~C53 f 14 uptake* (nmoles/gm wet wt./60 min)

(5)

3-O-Methylgl~cose-~~C 0.21 f 0.01 (3) efflux* (% released per minute)

3.59 f 17

~

Mannitol (200 mM) (10)

160 f 16

(5)

4.80 f 0.10 (3)

1.88 f 0.22 (3)

8230 f 1020 (3)

7570 f 820

(3)

GluCose-"C incorporation into glycogenc (nmoles/gm wet wt./120 minutes)

790 f 50

(3)

BNa effluxd (% released per minute)

4.4 f 0 . 3

(3)

8 . 4 f 0.7

(3)

8.1 f 0.9

(4)

22Naeffluxd in the presence of ouabain (1 mM) (% released per minute)

1.7 f 0.1

(3)

2.5 f 0 . 1

(3)

4.2 f 0 . 2

(3)

=Na effluxdin the presence of phlorizin (5 mM) (% released per minute)

4.6

(2)

7.8

(2)

uptakee (nmoles/gm wet wt./60 minutes)

685 f 151 (6)

uptake" in the presence of 2,4dinitrophenol (0.lmM) (nmoles/gm wet wt./60 minutes)

119 f 27

3zPi

32Pi

'6Ca-effluxf (% released per minute)

(3)

0.50 f 0.05 (3)

1463 f 113 (6)

119 f 21

(3)

0.60 f 0.06 (3)

1444 f 31

(3)

163 f 19

(3)

0.79 f 0.06 (3)

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

193

induces a rise in the intracellular ionic strength, which may thus favor a redistribution of calcium with an ensuing rise in the cytoplasmic Ca2+ level. In adipocytes, hyperosmolarity was found to stimulate calcium influx (Clausen et al., 1974a) and the rate coefficient of 45Carelease (T. Clausen, unpublished observations). Similar findings in the isolated rat pancreas (Case and Clausen, 1973) indicate that hyperosmolarity may be used as a tool for the study of the role of cytoplasmic Ca2+in contraction, secretion, and other cellular processes.

D. The Effect of Inhibitors 1. THEDISSOCIATION OF VARIOUS INSULIN ACTIONS The major result of studies with factors inhibiting the action of insulin on different parameters is that it has been possible to dissociate these effects. Thus phlorizin and phloretin, which are relatively specific inhibitors of sugar transport in a variety of tissues (Crane, 1960), have been shown to suppress the effect of insulin on the transport of glucose (Battaglia et al., 1960) and other sugars (Battaglia and Randle, 1960; Bihler et al., 1965; Kohn and Clausen, 1971) in skeletal muscle and heart, without interfering with its effect on amino acid transport (Battaglia et al., 1960) or Naf efflux (Table 111). Cytochalasin B, which was found to inhibit sugar transport in several cell types (Czech et al., 1973), can suppress basal and insulin-stimulated 3-0-methylglucose transport in rat soleus muscle without preventing the effect of insulin on a-aminoisobutyric acid accumulation (Table 11). Conversely, puromycin and actinomycin D do not interfere with the effect of insulin on the uptake of glucose and D-xylose in rat diaphragm (Eboue-Bonis et al., 1963; Carlin and Hechter, 1963; SZvik, 1965), or in bovine mesenteric arteries and rabbit colon (Arnquist, 1973).

Isolated rat soleus muscles were prepared from fed Wistar rats weighing 60-70 gm and incubated in Krebs-Ringer bicarbonate buffer without or with insulin or mannitol as indicated. Glucose (10 mM) was present only in the experiment where glycogen deposition was measured. All results are given with SEM and the number of observations in parentheses. Experimental details are described in the references listed in the footnotes. Kohn and Clausen (1971). Clausen (1966). Dahl-Hansen and Clausen (1973). Measured as the total amount of 32Pi activity taken up in the space not available to sucroseJ4C. f Clausen et at. (l974b).

194

TORBEN CLAUSEN

This indicates that the synthesis of proteins or RNA is unlikely to be of significance for the immediate effect of insulin on the glucose transport system. An analogous example is the finding that total inhibition of active coupled Na+-K+ transport with cardiac glycosides does not prevent insulin from stimulating the transport of galactose, 2-deoxyglucose (Kipnis and Parrish, 1965), glucose (Clausen, 1966), or 3-O-methylglucose (Bihler, 1968; Kohn and Clausen, 1971). Many years ago, LeFevre (1947) showed that sulfhydryl inhibitors (p-chloromercuribenzoate, Hg and Cu ions) diminished the rate of efflux and influx of glycerol and glucose in human erythrocytes. These effects were reversed by gluthathione and cysteine. Later, p-chloromercuribenzoate and N-ethylmaleimide (NEM) were shown to inhibit insulin-stimulated D-XylOSe uptake in the intact rat diaphragm (Battaglia and Itandle, 1960), and for some time it was believed that the effect of NEM on insulinstimulated sugar transport was the outcome of interference with the binding of insulin (Cadenas et at., 1961). However, treatment with NEM does not prevent the stimulating effect of insulin on glycogen deposition or the incorporation of phosphate into nucleotides, even when the effect on sugar transport is abolished (EbouB-Bonis et at., 1967). NEM also suppressed the stimulating effect of ouabain and K+-free environment on 3-O-methylglucose uptake in the intact hemidiaphragm (Bihler, 1968). This indicates that this compound is not specifically interfering with insulin action, but rather has a very general inhibitory effect on sugar transport. This seems to be apparent only under conditions where the transport is stimulated, and in skeletal muscle and heart no inhibition is seen under basal conditions (Cadenas et al., 1961; Carlin and Hechter, 1962; Bihler, 1968). However, these findings are difficult to interpret, since longer exposure to NEM leads to a stimulation of glucose (Cadenas et aE., 1961) and Dxylose transport (Carlin and Hechter, 1962). NEM penetrates the plasma membrane and inhibits a number of energy-yielding enzymic processes (Webb, 1966). Since it may thus mimic the action of the metabolic inhibitors described above (Section IV, C, 2), it is not the most reliable tool for the characterization of sugar transport. The SH-blocking agent p-hydroxymercuribenzoate stimulates sugar transport (Kono and Colowick, 1961).

2. THEDIFFERENTIATION OF BASAL AND STIMULATED GLUCOSE TRANSPORT Apart from allowing the exclusion of certain relationships, the work done with inhibitors has led toward a more positive identification of processes involved in the stimulation of glucose permeation and brought out some differences between the function in the basal and the activated state.

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

195

In some instances, increased permeation of glucose or other sugars may be the outcome of nonspecific leakage of the plasma membrane (Clausen et al., 1973). This possibility may be excluded if an inhibitory effect of phloriein, phloretin, NEM, or cytochalasin B can be demonstrated. Early studies on the effect of phlorizin and phloretin would suggest that these compounds preferentially inhibit sugar transport in the presence of insulin, being ineffective under basal conditions (Keller and Lotspeich, 1959; Lotspeich and Wheeler, 1962; Bihler et al., 1965; Bihler, 1968). However! others have found an inhibitory effect on basal transport also (Park et al., 1959; Weis and Narahara, 1969; Kohn and Clausen, 1971), and it seems doubtful whether they can be used for distinguishing different properties of the glucose transport system in the absence and in the presence of insulin. K+-rich incubation medium was found to suppress or abolish the stimulating effect of insulin and various other factors on the uptake of 2deoxyglucose in rat diaphragm muscle (Kipnis and Parrish, 1965) and on 3-O-methylglucose transport in rat soleus muscle (Kohn and Clausen, 1972; Clausen and Kohn, 1972). This would suggest some qualitative differences between the basal and the activated state of the glucose transport system. However, inhibitory effects of K+-substitution on the basal uptake of glucose, xylose, and 3-O-methylglucose have been found in rat diaphragm (Bhattacharya, 1961; Menozzi and Polleri, 1961; Clausen, 1968a; Bihler and Sawh, 1971b) and soleus muscle (Could and Chaudry, 1970). This evidence is suggestive of some similarity with the process of sugar accumulation in intestine and kidney, where K+ seems to exert a rather specific inhibitory action. On the other hand, the addition of up to 100 mM or KC1 in excess of the other components of the incubation medium caused no change in the insulin-stimulated efflux of 3-O-methylglucose in rat soleus muscle (Clausen and Kohn, 1972). Iso-osmotic K+ substitution may cause considerable swelling of muscle cells (Kipnis and Parrish, 1965; Mobley and Page, 1971; Kohn and Clausen, 1972; Clausen and Kohn, 1972). When swelling is induced merely by reducing the tonicity of the incubation medium, the stimulating effect of insulin, trypsin, and 2,4dinitrophenol is inhibited to about the same extent as in Kf-substituted media, and the basal rate of transport remains unaffected (Clausen and Kohn, 1972). These findings argue against any direct or specific effect of K+ ions on the glucose transport system. An alternative interpretation is that the inhibition is related to a rise in the cellular content of cyclic adenosine monophosphate (CAMP) (see Section V, A, 3). Like Kf-rich medium and hypoosmolarity, membrane stabilizers were found to exert a rather selective inhibitory effect on the suprabasal com-

196

TORBEN CLAUSEN

ponent of glucose uptake and 3-O-methylglucose transport in rat soleus muscle (Clausen el al., 1973). However, chlorpromazine and diphenylhydantoin, which also have membrane-stabilizing properties, inhibited the basal uptake of glucose (Rafaelsen, 1961) and 3-O-methylglucose in rat diaphragm muscle (Bihler and Sawh, 1 9 7 1 ~ ).These discrepancies may be related to the above-mentioned difficulties in defining basal conditions for the function of the glucose transport system (see Section IV, A). It was pointed out that in the lieart fatty substrates have little effect on basal sugar transport, but suppress the stimulating effect of insulin and increased work load (Morgan and Whitfield, 1974). Several investigators have failed to find any inhibitory effect of such compounds on glucose uptake in skeletal muscle (Jervell, 1965; Schonfeld and Kipnis, 1968; Beatty and Bocek, 1971; Jefferson et al., 1972), but recently, it was reported that when present at a very high concentration (8 mM), palmitate suppresses the stimulating effect of insulin, contractile activity, and ouabain on 3-O-methylglucose uptake in rat diaphragm, without altering the basal rate of transport (Bihler, 1972). I n the heart (Neely et al., 1969) and in rat diaphragm muscle (Bihler, 1972), the inhibitory effect of fatty substrates was not seen under anaerobic conditions, and it was reduced slightly by 2-bromostearate (Bihler, 1972), an inhibitor of fatty acid oxidation (Randle, 1969). Thus, the phenomenon seems to depend on the metabolism of the fatty acids, a conclusion also supported by the inhibitory effect of intermediates in fatty acid metabolism (Neely et al., 1969; Morgan and Whitfield, 1974). An alternative interpretation is suggested by the finding that free fatty acids have membrane-stabilizing properties (Roth and Seeman, 1971). Like the other membrane stabilizers described in Section 111, B, they may contribute to the inhibition of suprabasal sugar transport by acting directly on the plasma membrane. I n vivo, insulin acts in concert with numerous other interfering hormones and factors, which have been ascribed various roles in the pathophysiology of diabetes mellitus. Recent reviews have described the effects of anti-insulin factors in man (Ensinck and Williams, 1972) and in isolated tissues (Morgan and Whitfield, 1974).

V. CELLULAR SIGNALS CONTROLLING GLUCOSE TRANSPORT

I n the following, a series of factors are described and analyzed with the purpose of determining their potential role as signals in the control of sugar transport exerted by insulin. It should be possible to define a set of basal requirements to such signals: (1) They should be rapid in onset and

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

197

readily turned off when insulin is removed. (2) They should be elicited from the outer surface of the cell membrane and have ready access to this structure. (3) They should be specific and produce a minimum of disturbance in other cellular functions. (4) On the other hand, since little is gained from an isolated increase in sugar permeability per se, they are likely to be associated with signals that can facilitate the further metabolism of glucose. A. Enzymatic Processes and Metabolites

1. HEXOKINASE AND

GLUCOSE

6-PHOSPHATE

In extracts of skeletal muscle at least three types of hexokinase can be isolated chromatographically, all with K , values considerably lower that the apparent K , of the glucose transport system (Katzen and Schimke, 1965). However, in the intact tissue, the apparent K , for glucose phosphorylation seems to be appreciably higher (azand et al., 1962; Morgan et al., 1961a), indicating that part of the cellular hexokinase is separated from its substrate. Ozand et al. (1962) proposed that this might be due to the existence of intracellular permeability barriers, which could maintain the glucose concentration in the milieu directly in contact with the hexokinase at a level lower than the average cytoplasmic level calculated on the basis of measurements of the total glucose content. The idea of compartmentalization is supported by the finding that up to 45% of the hexokinase activity in muscle was found to be associated with cell organelles (Katzen et al., 1970), and the reversibility of this binding (Karpatkin and Braun, 1971) suggests that changes in the overall phosphorylating capacity may be regulated by redistribution of the enzyme among various pools. In the normal rat heart, insulin was found to produce only a modest increase in the rate of phosphorylation (Morgan et al., 1961a). More recently, insulin was found to augment the fraction of hexokinase type I1 bound to mitochondria, and this has been suggested to allow for a more efficient energy generation (Bessman, 1972). Although this phenomenon does not appear to explain the action of insulin on glucose transport, it may represent a signal allowing the cells to cope with the increased availability of glucose in the cytoplasm. In skeletal muscle and the heart isolated from diabetic rats, the phosphorylating capacity was found to be decreased (Kipnis et al., 1959; Kipnis and Cori, 1960; Park et al., 1961; Morgan et al., 1961b). In the perfused diabetic rat heart, insulin had no immediate effect on phosphorylation, but anoxia plus insulin restored the rate of phosphorylation

198

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to around 80% of the level measured in insulin-treated aerobic normal hearts (Morgan et al., 1961a, and b). This was taken to indicate that, in the diabetic animal, the phosphorylating system was inhibited and that the enzyme content was not necessarily deficient. More recently, streptozotocin-induced diabetes was reported to be associated with a 30-40Oj, decrease in the total activity of hexokinase isolated from muscle. This was probably mostly accounted for by an apparently selective suppression of type 11. The administration of insulin led to restoration of activity within a few hours, and even supranormal levels could be achieved (Katzen et al., 1970). However, in view of the fact that the transport of glucose (which is inhibited in the diabetic animals) is rate-limiting for its utilization, the reported decrease in phosphorylating capacity may not be of major regulatory significance. In the aerobic diabetic rat heart, the intracellular concentration of free glucose was only slightly higher than in the normal control hearts (Morgan et al., 1961b). It has been suggested that the membrane-associated hexokinases may play a role in sugar transport, possibly as carrier structures (Katzen, 1969), but if this hypothesis should account for the transport of nonphosphorylated sugars also, more detailed information about the relative affinities of these compounds is required. The elevation of intracellular glucose 6-phosphate produced by epinephrine (Kipnis et al., 1959; Newsholme and Randle, 1961; &and et al., 1962) may account for the inhibitory effect of this hormone on glucose uptake. When tested with nonmetabolized sugars, the rate of transport rather seems to be augmented, indicating that glucose 6-phosphate is not inhibiting the glucose transport system per se (Newsholme and Randle, 1961; Saha et al., 1968). 2. ATP

It has not been possible to detect any significant effect of insulin on the total ATP content in rat diaphragm (Sacks, 1952; Clauser et al., 1962; Ebou6-Bonis et al., 1967; Walaas et al., 1969), heart (Williamson and Kreisberg, 1965), or soleus muscle (Chaudry and Gould, 1970). The turnover of 32Pinto ATP is increased, and it cannot be excluded that this is associated with a redistribution of ATP leading to localized changes in its concentration (Clauser et al., 1962; Walaas et al., 1969). However, externally added ATP (which was shown to penetrate into the cells) did not suppress the stimulating effect of insulin or metabolic inhibitors on glucose uptake in rat soleus muscle (Chaudry and Gould, 1970). Although the same study demonstrated a relatively specific inhibitory effect of ATP on the acceleration of glucose uptake in anaerobiosis, the major part of the evidence

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

199

argues against the proposed role of ATP as a common regulator of glucose transport (see Section IV, C, 2). 3.

CYCLI CYCLIC AMP

Insulin produces a decrease in the cAMP content of isolated fat cells (Kono and Barham, 1973), possibly because of a stimulation of the phosphodiesterase degrading the nucleotide (Loten and Sneyd, 1970). It has not been possible to detect any significant effect of insulin on the total content of cAMP in isolated rat diaphragm (Goldberg et al., 1967; Craig et al., 1969; Walaas et al., 1969), but the rise induced by epinephrine was diminished (Craig et al., 1969). The adenyl cyclase activity of sarcolemma isolated from skeletal muscle was not altered by insulin (Drummond et al., 1972), but the phosphodiesterase purified from beef heart was stimulated, and in diabetic rats the injection of insulin produced a rise in the activity of the diesterase in skeletal muscle (Senft et al., 1968). In frog muscle, insulin was reported to accelerate the degradation of labeled cAMP (Woo and Manery, 1973). Furthermore, Walaas et al. (1973) reported that, in rat diaphragm, insulin decreased the ratio between the CAMPindependent and the CAMP-dependent forms of a protein kinase, indicating that the hormone had either induced a localized decrease in the cAMP level or stimulated the production of a factor interfering with the action of CAMP. The recent discovery that the total cAMP level in the heart shows considerable fluctuations with the contractile cycle indicates that the state of the muscle may be decisive for the possibilities of detecting clearcut effects of various factors (Wollenberger et al., 1973). Since cAMP penetrates slowly into the cytoplasm and induces a marked modification of several metabolic processes, it is difficult to determine whether it has any direct effect on the glucose transport system. I n the rat diaphragm muscle, dibutyryl cAMP was found to inhibit glucose uptake both in the presence and in the absence of insulin. Although basal galactose uptake was not affected, the effect of insulin on this parameter was blocked by dibutyryl cAMP in combination with theophylline (Chambaut et al., 1969). In isolated fat cells, dibutyryl cAMP and caffeine were also found to inhibit glucose metabolism (Kitabchi et al., 1970). From Fig. 2 it can be seen that caffeine suppresses the stimulating effect of insulin on 3-0-methylglucose efflux from rat soleus muscle. Another condition known to produce a considerable rise in the cAMP content of skeletal muscle is exposure to a Kf-rich environment (Lundholm et al., 1967), and, as discussed in Section IV, D, 2, this is associated with amarked suppression of suprabasal sugar transport.

200

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(MIN)

-1 CAFFEINE

0.050 I

I N S U L I N (100 m U l m l )

0.040 + z 3 Y

-

I a

(L

W

+

VI

s

-

qu

0.030

VI Y

0 V

3 2

2 L

k

I

+

0.020

T Y

0

z

2

z

< ( Y L

0.010

0.000

J

I 80

1

I

1

100

120

140

(MINI

DURATION OF WASHOUT

FIG.2. The effect of insulin and caffeine on 3-0-methylglucose-*4C efflux from rat soleus. Soleus muscles from fed rats weighing 60-70 gm were preloaded by incubation for 60 minutes a t 30" in Krebs-Ringer bicarbonate buffer containing 1 m M 3-0-methylg l u ~ o s e - ~(2 ~ CpCi/ml). Each individual muscle was then attached to a polyethylene cannula (which served a t the same time as gas inlet) and transferred through a series of tubes containing 3 ml of buffer without 3-0-methylglucose. Insulin and caffeine was included in the washout medium from the time indicated by the arrows. The fraction of 3-0-methylglucose-1% released during each washout period was determined as described elsewhere (Kohn and Clausen, 1971). Each curve represents the mean of two observations. Data are from Clausen et al. (1974b).

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

20 1

CAMP was found to induce a marked release of Ca from isolated mitochondria (Borle, 1973b), and this may modify its effect on sugar transport. The collective evidence suggests that the rate of glucose transport can be determined by the relative levels of CAMP and Ca2+ ions, but further studies of the time course and the localization of changes in the concentration of these two compounds are required before their role in insulin action can be determined. B. The Binding, Transport, and Distribution of Electrolytes

None of the electrolytes normally available in the extracellular milieu seem to be essential for the stimulating effect of insulin on glucose transport (Chaudry and Could, 1969; Clausen, 1972). Therefore, it seems unlikely that insulin accelerates this process solely by inducing a change in the rate of influx of one of these ions. However, it cannot be excluded that changes in the binding, efflux or intracellular concentration of electrolytes may be of importance. 1. SODIUM Insulin stimulates the efflux of Na+ by an ouabain-resistant mechanism (Zierler, 1966; P.G. Kohn and T. Clausen, unpublished observations) (Table 111). Although the resulting decrease in the intracellular Na+ concentration (Creese and Northover, 1961) may in part account for the stimulation of the influx (Hider et al., 1971) and inhibition of the efflux of amino acids (Pozefsky et al., 1969) induced by insulin, it is not likely to account for the rise in sugar permeability. A decrease in intracellular Na+ if anything reduces the rate of sugar transport (Bihler and Sawh, 1971b), and even when the intracellular level is considerably elevated by exposure to ouabain, the stimulating effect of insulin is not diminished (Clausen, 1966; Bihler, 1968; Kohn and Clausen, 1971). The fact that metabolic inhibitors suppress Na+ efflux, together with the observation that insulin augments glucose transport under these conditions argues that the effect of the hormone on Naf efflux is not directly related to its stimulating effect on sugar transport (see also Section IV, D). 2. POTASSIUM

Even at minute concentrations, insulin favors the retention of K+ in the human forearm (Zierler and Rabinowitz, 1964), and experiments with isolated rat diaphragm muscle show that the intracellular concentration of K+ may be slightly increased (Creese and Northover, 1961). Again, the

202

TORBEN CLAUSEN

above-mentioned experiments with ouabain, as well as the observations that the effect on K+ retention is not correlated to that on glucose uptake and is seen in the absence of glucose (Zierler, 1972), argue that this signal cannot account for the action of insulin on glucose transport. 3. MEMBRANE POTENTIAL In skeletal muscle, insulin augments the resting membrane potential (Zierler, 1957; Bolte and Luderitz, 1968; Otsuka and Ohtsuki, 1970), perhaps due to a preferential favoring of Na+ efflux. The relatively slow onset of this effect (Zierler, 1959), together with the observation that even when acute elevation of the membrane potential is produced by the omission of Kf there is no immediate rise in sugar permeability (Kohn and Clausen, 1972), indicates that this change per se is not the cause of the prompt rise in sugar permeability induced by insulin. The removal of extracellular Ca2+ by the addition of EDTA leads to depolarization in muscle (Jenden and Reger, 1963), but, as can be seen from Table I, this does not prevent insulin from stimulating sugar transport. 4. CALCIUM

Like CAMP, Ca2+ has been shown to play a prominent role in the mechanism of action of a wide variety of hormones (Rasmussen, 1970), and its potentialities as a cellular signal are further illustrated by its pronounced effects on membrane permeability (Manery, 1966), metabolic processes (Bygrave, 1967), and contractile (Weber et al., 1964) or secretory activity (Douglas, 1968). Insulin has from time to time been reported to induce a small rise in the concentration of calcium in blood (Davies et al., 1926; Brougher, 1927; Valencia, 1954; Hammarsten and Smith, 1956), but this effect has been difficult to reproduce. It was suggested that it is secondary to the concomitant decrease in the concentration of inorganic phosphate in serum (Brougher, 1927; Mellerup, 1974). Insulin has been found to decrease the binding of calcium to artificial phospholipid membranes (Kafka and Pak, 1969) and plasma membranes isolated from the liver (Marinetti et al., 1972). In epididymal fat pads, insulin was found to accelerate the rate of 45Ca release (Clausen, 1969b); and in isolated fat cells, an even larger effect (up to 60% rise) could be demonstrated with concentrations down to 10 pU/ml (Martin et al., 1973; Clausen et al., 1974a). In isolated soleus muscles, it has not been possible to detect any significant effects of insulin on the influx or the efflux of * T a , but as can be seen from Fig. 1, in a hyperosmolar environment, insulin produces a rise in tension, indicating that the hormone may

Thf EFFECT OF INSULIN

ON GLUCOSE TRANSPORT IN MUSCLE CELLS

203

induce an increase in the cytoplasmic concentration of free Ca2+ions. This effect was seen in the absence of metabolizable substrate in the buffer and was completely blocked by insulin antibody (Clausen et al., 1974a). In the normotonic buffer, insulin did not produce any significant change in tension, and it seems unlikely that the hormone under basal conditions induces any major generalized increase in the cytoplasmic Ca2+ level. I n analogy with what has been suggested for other hormones (Borle, 1973a), insulin may rather be imagined to induce a shift of calcium from one cellular pool to another, and this may occur via a limited region of the cell interior. The major problem in detecting such changes is related to the fact that in muscle cells, the cytoplasmic concentration of free Ca2+ions is around lo-' M (Hellam and Podolsky, 1969; Winegrad, 1973) and a comparison with the total exchange of calcium between muscle cells and the extracellular phase indicates that this cytoplasmic pool has a theoretical turnover time of a few milliseconds. Apart from calcium in the form of various complexes in the cytoplasm, by far the greater part of it is retained in three pools: in the mitochondria, in the sarcoplasmic reticulum, and bound to the plasma membrane. As originally proposed by Holloszy and Narahara (1967), Ca2+ions may be of importance in the activation of the glucose transport system, and a logical test of this hypothesis would consist in considering all the factors that may induce a rise in the concentration of Ca2+a t the inner surface of the plasma membrane. As reflected in a rise in tension and the rate of 45Carelease from preloaded muscles, calcium is transferred into the cytoplasm from the pool residing in the sarcoplasmic reticulum either by electrical stimulation, K+ depolarization or caffeine (Bianchi, 1961; Clausen et al., 1974b). All these factors were reported to produce an immediate or delayed rise in the permeability to glucose and nonmetabolized sugars (see Section IV, C, 1). As discussed in Section IV, C, 5 , hyperosmolarity induces a rise in tension and the release of 45Ca, which is associated with a stimulation of sugar transport. Several reports indicate that in the heart (Fehmers, 1967; Patriarca and Carafoli, 1968; Horn et al., 1971) and skeletal muscle (Patriarca and and Carafoli, 1969), the mitochondria accumulate substantial amounts of calcium. Isolated mitochondria were found to accumulate calcium from an environment containing down to 10-7 M , and this organelle may therefore (in particular in red muscle fibers) play a significant role in the control of the cytoplasmic level of free Ca2+ions (for review, see Lehninger, 1970). The accumulation of Ca in mitochondria is energy-dependent, and in rat diaphragm muscle, several metabolic poisons were found to inhibit the

204

TORBEN CLAUSEN

uptake of 45Caand favor its release from the preloaded tissue (Carafoli et al., 1969). Randle and Smith (1958) noticed that those metabolic poisons that were shown to stimulate sugar transport also induced rigor in the diaphragm muscles, and similar observations have been made by others (Paul, 1961; Kohn and Clausen, 1971; Clausen et al., 1974b). Also, in epididymal fat pads, metabolic poisons were found to induce a rise in the rate coefficient of 45Ca release, which was associated with a stimulation of 3-0-methylglucose transport (Clausen, 1969a,b, 1970). The stimulation of sugar transport seen during curtailment of energy supply has been considered as a rather direct outcome of a decrease in the cellular level of ATP (see Section IV, C, 2 ) . However, the accumulation of calcium in the mitochondria depends on the energy supply, and ATP is required for the uptake of calcium into the sarcoplasmic reticulum (Hasselbach et al., 1970), the binding of calcium to the sarcolemma (Severson et al., 1972), and (directly or indirectly) for the extrusion of calcium into the extracellular phase (Baker, 1972; Reuter and Seitz, 1968). Therefore, it is difficult to exclude that various states of energy depletion lead to a rise in the cytoplasmic level of Ca2+ ions, and that this could exert a direct effect on the sugar transport system. Experiments with kidney cells indicate that cooling leads to an accumulation of calcium in the cytoplasm, probably due to the decrease in extrusion across the plasma membrane and the accumulation in cell organelles (Borle, 1972). Immediately upon return to 37"C, the rate coefficient for the release of 45Ca was found to attain levels appreciably above those recorded in the steady-state experiments performed at the same temperature. From Fig. 3A it can be seen that a similar peak in the rate coefficient for the release of 45Cafrom rat soleus muscles can be detected, and when the same experiment is performed with measurement of 3-0-methylglucose -14C efflux, a concomitant sharp rise in the rate coefficient for sugar release is found (Fig. 3B). The magnitude of both peaks depends on the duration of the cooling, indicating that the accumulation of labeled calcium in the cytoplasm is progressive. The rise in 3-0-methylglucose efflux was suppressed by phlorizin (5 mM), indicating that it is not the outcome of nonspecific impairment of plasma membrane integrity. With the recognition that the extrusion of calcium from the cytoplasm depends on the concentration gradient for Naf ions, further possibilities for influencing the cytoplasmic calcium level have become available. As discussed in Section IV, C, 4,the exposure of muscle cells to cardiac glycosides, Kf-free environment or Naf-depleted media may induce tension or increased uptake of calcium (Cosmos and Harris, 1961; Ilse and Ong, 1970). This is in several instances associated with an increase in the transport of glucose or other sugars.

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

(MINI

-1

0.01:

205

30'

r

1

E

z 3

z CL

Y LL

c

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v,

0.01c

L 1 V c

0; FROM dJO la', 0 FROM 60 TO 120

4: V

P

&

0.005

z

0

V I4

E

0.000

A 100

1lO

140

160

180

(MIN)

DURATION OF WASHOUT

FIG.3. The effect of cooling on the washout of '6Ca and 3-O-methylglucose-14Cfrom rat soleus. Loading and washout were performed essentially as described in the legend to Fig. 2. In order to obtain a uniform distribution of 46Cain the preparation, the tendons were carefully dissected without damaging the muscle fibers. All loading took place a t 30°C, but as indicated the washout of some groups of muscles was performed a t 0°C for 60, 120, or 180 minutes. From the time indicated by the arrow, the washout was in some cases allowed to continue a t 30°C. Each curve represents the mean of t,hree to four observations with bars indicating 2 x SEM, where this exceeds the size of the symbols. (A) The washout of 45Ca. Data from Clausen et al. (1974b).

The collective evidence is compatible with the idea that a rise in the concentration of Ca2+ions in a specific cellular compartment constitutes a common signal for the activation of the glucose transport system. The demonstration of a similar relationship in adipocytes (Clausen, 1970), together with the observations that in lymphocytes, phytohemagglutinin stimulates both calcium uptake (Whitney and Sutherland, 1973) and 3-0-methylglucose transport (Peters and Hausen, 1971) indicates that the phenomenon is not restricted to muscle cells. The observation that cytochalasin B inhibits sugar transport in adipocytes (Czech et al., 1973), smooth muscle cells (Gorski and Raker, 1973), and soleus muscle (Table 11) suggests that contractile filaments are involved in the function of the glucose transport system.

206

TORBEN CLAUSEN

(MIN)

0.03(

-1

30'

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+ 3 Y

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160

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FROM 0' TO 120'

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FROM 50' TO 120' 30' T H R O U G H O U T I

180

(MIN)

FIG.3(B) The washout of 3-0-methylglucose-1%. See Fig. 3(A) legend for details. Data from Clausen et al. (1974b).

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

207

Several membranes have been found to contain filaments which contract in the presence of calcium (Jahn and Bovee, 1969), and if such structures are associated with the glucose carrier, Ca2+ might convert the system into a conformation with a more efficient function. This may only be maintained as long as Ca2+is present and revert back to the basal state-either when Ca2+is removed or under the influence of CAMP. In this connection it is interesting that calcium inhibits adenylate cyclase (Birnbaumer et d., 1969; Severson et al., 1972) at concentrations down to M and stimulates phosphodiesterase at concentrations of around M (Kakiuchi and Yamazaki, 1970; Teo and Wang, 1973). 5. MAGNESIUM

Insulin was reported to produce a slight, but significant increase in the magnesium content of skeletal muscle and heart i n Vivo (Aikawa, 1960), as well as in isolated rat uterus (Lostroh and Krahl, 1973). Insulin was recently reported to stimulate the uptake of zsMg in isolated rat hemidiaphragm (Mellerup, 1974). In rat hemidiaphragm, magnesium was found to be required for the stimulating effect of insulin on glucose uptake (Bhattacharya, 1961), but this was not readily confirmed in experiments with rat soleus muscle (Chaudry and Gould, 1969). Later, the same group reported that in muscles that had been treated with EDTA in a calciummagnesium-free medium, the insulin response was lost and was restored by the addition of magnesium (Gould and Chaudry, 1970).

PHOSPHATE 6. INORGANIC The classical observation that insulin can induce a drop in the concentration of inorganic phosphate in blood (Harrop and Benedict, 1924; Kerr, 1928) and stimulate the uptake of phosphate in perfused hind limbs (Pollack et al., 1934) was for a long time taken to indicate some relationship between the transport of glucose and phosphate. However, experiments with the isolated intact diaphragm muscle (Walaas et al., 1969) and the perfused rat heart (Kaji and Park, 1961; Sarkar and Ottaway, 1962) have demonstrated that insulin stimulates the uptake of phosphate in the absence of glucose (see also Table 111),and that there is no stoichiometric relationship between the increases in the uptake of these two compounds (Sacks, 1952). In isolated tissues, the uptake of 32Pis determined by the relative rates of influx and efflux, and the observation that insulin decreases the rate of 32P release (Kaji and Park, 1961; Sarkar and Ottaway, 1962) indicates that its stimulating effect on the net accumulation is not the result of an

208

TORBEN CLAUSEN

increased permeability of the plasma membrane to inorganic phosphate. A decrease in the rate of 3zP release suggests that the cytoplasmic concentration of inorganic phosphate is lowered. This could result from a stimulation of the mitochondria1 accumulation of phosphate and would favor the net influx of phosphate from the extracellular phase. In the mitochondria, inorganic phosphate is taken up together with calcium by an energy-requiring process (Lehninger et al., 1967). I n rat soleus muscle, 2,4-dinitrophenol produces a marked decrease in the total laccumulation of "P (Table 111),and in isolated fat cells, 32Puptake was found to be energy dependent and stimulated by calcium (Martin et al., 1975). Hyperosmolarity, which induces a rise in the cytoplasmic concentration of Ca2+ (Isaacson, 1969; Clausen et al., 1974b), stimulates the accumulation of 32P (Table 111). These results are compatible with the idea that, in the intact cell, the net uptake of 32Pis to a large extent dependent on a Ca2+-stimulated energy-requiring accumulation in the mitochondria. In the heart, anoxia was reported to abolish the effect of insulin on phosphate uptake (Kaji and Park, 1961) and in rat soleus muscle, 2,4-dinitrophenol suppresses the stimulating effect of both insulin and hyperosmolarity on the total accumulation of 32P(Table 111). The inorganic phosphate content of muscle cells is considerably higher than that of the extracellular milieu, and during incubation in the presence of 32P,the labeling of cellular phosphate is a relatively slow process (Walaas et al., 1969). This may explain the observation that even though insulin doubled the rate of labeling of inorganic phosphate in intact diaphragms (Walaas et al., 1969), the hormone produced no detectable increase in the total content of inorganic phosphate in this muscle during incubation periods of 30-60 minutes (Sacks, 1952; Clauser et al., 1962; Ebou6-Bonk et al., 1967; Walaas et al., 1969). However, since part of the cellular inorganic phosphate is sequestered in the mitochondria, it is difficult to draw conclusions about the cytoplasmic concentration of inorganic phosphate on the basis of determinations of total cellular content. In the isolated rat hemidiaphragm, the consumption of glucose is stimulated by high levels of inorganic phosphate in the incubation medium (Villee et al., 1949; Randle and Smith, 1958), but little is known about its effect on the processes of glucose transport. The omission of phosphate from the incubation medium does not prevent the stimulating effect of insulin on glucose uptake (Chaudry and Gould, 1969) or on 3-O-methylglucose transport in rat soleus muscle (T. Clausen, unpublished observations). It cannot be excluded, however, that the cytoplasmic level of inorganic phosphate can directly or indirectly influence the transport of glucose or be of importance for its stimulation by insulin.

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

209

VI. MECHANISMS FOR THE MODE OF ACTION OF INSULIN

Although it is the duty of the reviewer to avoid biased evaluation of the data available, their interpretation and integration constitute an understandable motivation for his work of compilation. Consequently, this review is concluded and summarized in a hypothetical scheme for the mode of action of insulin; the scheme a t the moment appears to be compatible with much of the evidence described above (Fig. 4).Although more detailed than previous models, it leaves open certain possibilities that should be accessible experimental testing. 1. The binding of insulin to its receptor is supposed to induce a change in the quaternary structure, which is propagated in the plane of the plasma membrane as a "pressure wave" in the lipid matrix. As pointed out by Singer (1971), such a succession of compression and decompression I

MIT.

-/A Insulin Na* Ca"

I

Glucose

FIG.4. Hypothetical diagram of cellular elements in the basal state (above) and under the influence of insulin (below). Segments of the plasma membrane with insulin receptor, glucose transport system, and adenyl cyclase (Ad. Cyc.) are shown with sarcoplasmic (Sarc.) reticulum and mitochondria (MIT.) containing large deposits of calcium. Det,ails of the cellular events induced by insulin are described in the text.

210

TORBEN CLAUSEN

may be transmitted for an appreciable distance and induce transient changes in the quaternary structure of the proteins and in the distances between adjacent lipid molecules. 2. If this is associated with a decrease in the relative affinity for calcium and monovalent cations, Ca2+ions could be released from the inner surface of the plasma membrane into some compartments of the cytoplasm (see Section V, B, 4). In artificial phospholipid monolayers, calcium bound to the acidic polar heads can be displaced by Na+ and K+ in the concentration range found in cells (Rojas and Tobias, 1965), and in the calcium-associated state the membrane lipids are assumed to be condensed and less mobile (see Dawson and Hauser, 1970). 3. The ouabain-resistant rise in Na+ efflux induced by insulin may be another reflection of an increase in the relative affinity for Na+ and Ca2+ ions. An increase in ionic strength displaces Ca from membranes, and when such a change is induced in the cytoplasm by exposure to hyperosmolarity, Na+ efflux is stimulated and other insulinlike effects are produced (see Section IV, C, 5 ) . 4.A preferential stimulation of Na+ efflux may explain the decrease in intracellular Na+ concentration, the rise in membrane potential, and the favoring of K+ retention seen in the presence of insulin (see Section V, B). 5 . A decrease in the intracellular Na+ concentration favors the accumulation and retention of those amino acids which are transported by mechanisms directly or indirectly dependent on the transmembrane Na+ gradient. 6. Owing to the rapid accumulation in mitochondria and sarcoplasmic reticulum, the rise in the cytoplasmic level of free Ca2+ ions is probably limited. Since the energy-linked mitochondria1 accumulation of calcium is accompanied by an uptake of inorganic phosphate (Lehninger et al., 1967), it is conceivable that a localized lowering of the cytoplasmic concentration of this ion may occur, thus favoring influx and reducing efflux across the plasma membrane (see Section V, B, 6). 7. A further comequence of the suggested transfer of Ca from the plasma membrane into the mitochondria is activation of the Ca2+-sensitive phosphatase (Pettit et al., 1972), converting pyruvate dehydrogenase from a n inactive to an active form. Insulin has been found to induce such a conversion in normal adipose tissue (Denton et al., 1972) and in the heart of diabetic rats (Wieland et al., 1971). Conditions that induce a rise in the uptake of caIcium (hyperosmolarity, ouabain, or K-free medium) were found to augment the activity of pyruvate dehydrogenase in adipose tissue (Martin et al., 1973). 8. Phosphodiesterase isolated from the bovine heart was found to be calcium dependent (Teo and Wang, 1973), and a rise in the Ca2+ion level may therefore reduce the concentration of CAMP a t certain loci (see

THE EFFECT OF INSULIN ON GLUCOSE TRANSPORT IN MUSCLE CELLS

21 1

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of diabetes, fasting, and refeeding on pyruvate dehydrogenase interconversion. Arch. Biochem. Biophys. 143, 593-601. Wilbrandt, W., and Rosenberg, T. (1961). The concept of carrier transport and its corollaries in pharmacology. Pharmacol. Rev. 13, 109. Williams, G. R. (1959). A continuous flow method for the determination of glucose uptake by excised rat diaphragm. J. Gen. Physiol. 42, 1139. Williamson, J. R., and Kreisberg, R. A. (1965). Effect of insulin on the levels of oxidized and reduced nicotineamide-adenine dinucleotides in the perfused rat heart. Biochim. Biophys. Acta 97, 347-349. Winegrad, S. (1973). Intracellular calcium binding and release in frog heart. J. Gen. Physiol. 62, 693. Wohltmann, H. J., and Narahara, H. T. (1966). Binding of insulin-lSlI by isolated frog sartorius muscles. Relationship to changes in permeability to sugar caused by insulin. J. Biol. Chem. 241, 49314939. Wollenberger, A. (1947). Metabolic action of the cardiac glycosides. 111. Influence of ouabain on the utilization of C14-labeledglucose, lactate, and pyruvate by dog heart slices. Naunyn-SchmiedebergsArch. Exp. Pathol. Phrmakol. 219, 408-419. Wollenberger, A., Babskii, E. B., Krause, E.-G., Genz, S., Blohm, D., and Bogdanova, E. V. (1973). Cyclic changes in levels of cyclic AMP and cyclic G M P in frog myocardium during the cardiac cycle. Biochem. Biophys. Res. Commun. 55,446452. Woo, Y.-T., and Manery, J. F. (1973). Cyclic AMP phosphodiesterase activity a t the external surface of intact skeletal muscles and stimulation of the enzyme by insulin. Arch. Biochem. Biophys. 154, 510-519. Yalcin, S., and Winegrad, A. I. (1963). Defect in glucose metabolism in aortic tissue from alloxan diabetic rabbits. Amer. J. Physiol. 205, 1253-1259. Young, D. A. B. (1965). Hypothalamic (photoperiodic) control of a seasonal antagonism to insulin in the rat heart. J . Physiol. (London) 178, 530-543. Zierler, K. L. (1957). Increase in resting membrane potential of skeletal muscle produced by insulin. Science 126, 1067-1068. Zierler, K. L. (1959). Effect of insulin on membrane potential and potassium content of rat muscle. Amer. J . Physiol. 197,515. Zierler, K. L. (1966). Possible mechanisms of insulin action on membrane potential and ion fluxes. Amer. J . Med. 40,735-739. Zierler, K. L. (1972). Insulin, ions, and membrane potentials. I n “Handbook of Physiology” (Amer. Physiol. SOC.,J. Field, ed.), Sect. 7, Vol. I, p. 347. Williams & Wilkins, Baltimore, Maryland. Zierler, K. L., and Rabinowite, D. (1964). Effect of very small concentrations of insulin on forearm metabolism. Persistence of its action on potassium and free fatty acids without its effect on glucose. J . Clin. Invest. 43, 950-962.

Recognition Sites for Material Transport and Information Transfer* HALVOR N . CHRISTENSEN Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan

1. Introduction. Summary List of Principal Transport Systems for the Amino Acids . . . . . . . . . . . . . . . . . 11. Description of the Neutral Systems . . . . . . . . . . . . 111. The Cationic Amino Acid Systems . . . . . . . . . . . . IV. Extension to System ASC of Approaches to Site Description Taught by the Basic Amino Acids . . . . . . . . . . . . . . V. Efforts to Develop System-Specific, Nonmetaboliaable Substrates for the Neutral Systems . . . . . . . . . . . . . . . . VI. System-Specific Substrates for the Transport System for the Cationic Amino Acids . . . . . . . . . . . . . . . . . VII. Stimulation of Release of Pancreatic Hormones by Amino Acids . . . VIII. Concluding Discussion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

1. INTRODUCTION.

227 229 232 235 237 241 243 250

265

SUMMARY LIST OF PRINCIPAL TRANSPORT SYSTEMS FOR THE AMINO ACIDS

When researchers began t o examine the molecular basis of specific membrane transport, they anticipated quite naturally that a single agency would be found to produce the transport of any given metabolite. The

* The portions of the work discussed here that come from the author’s laboratory have received support from Grant C-2645, National Cancer Institute, and in recent years, Grant HD01233, Institute for Child Health and Human Development, National Institutes of Health, U. S. Public Health Service, Attention is called to the importance of contributions from my co-authors cited in the bibliography. 227

228

HALVOR N. CHRISTENSEN

cases where many organic solutes of a given class (e.g., monosaccharides, neutral amino acids) were found each to inhibit the transport of the others taught us that a given transport system can show a wide scope, i.e., that the structural requirements for acceptance of an analog may allow considerable latitude. The cases where only one member of the class showed any reactivity with the transport system showed that in contrast the structural requirements could be quite sophisticated, although usually it has proved possible to construct at least one analog that shares the transport with the natural substrate. Another lesson that has come harder to the student of transport, however, is to appreciate that, in a given context, a metabolite may simultaneously be transported by two, three, or more apparently independent agencies. To take an example, the amino acid, alanine, enters many animal cells by two different Naf-dependent systems and one Na+-independent system. In each of these systems the transport is shared with a different group of amino acids. For a given bacterial cell, a sugar or an amino acid may chance to be taken up by a system of broad reactivity, by another of intermediate specificity, and a third of almost complete specificity to that amino acid. The full extent of the molecular heterogeneity that characterizes membrane transport has probably by no means been discovered. When we appreciate not only that a metabolite of a given class may share a, transport route with several other metabolites, but that this sharing may concern two or more transport systems, the degree of sharing being different for each system and for each pair of metabolites, then we realize that in a given case the problem of codification of the several systems involved may be quite complicated. A complex, quantitative inhibition analysis, requiring experimental modification of the environmental conditions, such as I have described elsewhere (Christensen, 1966, 1969), becomes necessary. Support for the discriminations thus reached comes from differences in the developmental history of these systems, also from differences in their genetic determination, inducibility, repressability, and sensitivity to hormonal stimulation, as reviewed elsewhere (Christensen, 1973a). By the application of these discriminatory techniques to the transmembrane migration of the amino acids, a number of transport systems have now been discerned for various organisms. Such studies reveal a microcosm of transport systems, parallel to but different from the microcosm of enzymes. In contrast to the situation for enzymes, however, we are still largely unable to isolate each transport system chemically for study. Hence we are often forced to study the systems in admixture. Each of these systems can be characterized by its receptor site, i.e., by the detailed structure for which we obtain a complementary picture by the study of the

229

RECOGNITION SITES FOR MATERIAL TRANSPORT AND INFORMATION TRANSFER

TABLE I AN APPROXIMATE SURVEYOF THE REACTIVITIES OF a-AMINOACIDSWITH SEVERAL TRANSPORT SYSTEMS" System Amino acid GlY Pro Ala, Ser Thr, Homoser a-Aminobutyric GluNH2 Norval Norleu, Met His, Tyr, T r p Val Leu, Ile, Phe

Glu

A

ASC

L

+++

++ +++

f f

f

In some cases 0 0

0 0

0 0 0 0 0

+++ +++

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

+++

++++ +++ ++ +0 0

0 0

0

+

++ f + ++ +++ ++++ ++ ++ ++++

a Reactivities are roughly compared &s observed in the Ehrlich ascites tumor cell and in various red blood cells-see comment in text on differences to be expected in other tissues. A zero implies that little or no reactivity was observed under the ordinary conditions of comparison; under selected conditions significant reactivity may yet be observable. The four systems listed a t the top receive further definition in the text and in the review by Christensen (1969).

effects of every possible change in the structure of the substrate. The concept thus approached has been referred to as the concept of the receptor site (or the reactive site) for biological transport. To introduce the ideas implied by the title, I want to review a list of some of the most conspicuous transport systems for the amino acids observed in cells of the higher animal. Table I indicates the approximate range of the reactivity to transport of several of the well characterized systems, using mainly the results with the Ehrlich cell to compare in an approximate way how much each amino acid is transported by each of the several systems for neutral amino acids.

II. DESCRIPTION OF THE NEUTRAL SYSTEMS

A Naf-dependent system largely specific to glycine (Gly) is listed first in Table I. This system we may define from its description for the pigeon red blood cell by Vidaver (1964). It is strictly Na+-dependent. Sarcosine

230

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also serves as a substrate and N-ethylglycine reacts with it, but the ordinary amino acids other than glycine show only small inhibition of glycine uptake. To what degree these inhibitory effects apply instead to a slow uptake of glycine by another system (ASC; Eavenson and Christensen, 1967) has not been established. The results so far show that the glycinespecific system tolerates only the addition of a methyl or an ethyl group to the nitrogen atom of glycine. Two systems with broad reactivities known as System A and System L, participate together in the transport of the neutral amino acids. The broad scope of Systems A and L is apparent from Table I. Almost all ordinary neutral amino acids show appreciable transport by both systems, and often by still another system. Amino acids with short, polar, or linear side chains tend to be preferred by System A ; those with branches or rings on the side chain, by System L. It would be incorrect, however, to speak of any natural amino acid, say alanine, as a System A substrate, or of phenylalanine as a System L substrate (Oxender and Christensen, 1963). The toleration by System A of an N-methyl group (or, in analogy, of the imino acid structure of the prolines; see Fig. 1) is one feature differentiating it from System L (Christensen et al., 1965). The sharing of this feature with the glycine system is one of several circumstances suggesting that System A and the glycine system may be extreme variants of each other. For example, my colleagues and I have not yet encountered a cell in which the two exist together; in the erythrocyte (Eavenson and Christensen, 1967) and the rabbit reticulocyte (Winter and Christensen, 1965), the glycine system seems to take the place of System A as seen in the Ehrlich cell. Furthermore, the so-called iminoglycine system of the renal tubule, absent in the genetic disease, iminoglycinuria, lies almost midway in between the two in its scope, including as it does glycine, the imino acids, and the model substrate a-aminoisobutyric acid (AIB). For these reasons we find interesting the possibility that these systems may have arisen from a common precursor, perhaps by gene duplication, or, in the two occurrences illustrated at the left in Table I, through differentiation. The most striking feature differentiating System L from the other sysI

CH,- COOI

H,C -NH: Sarcosine

H,C-CH, I I H,C,+,C, N H COOH2 Proline

H,C-C-COOI

H,C- NH:

2- (Methylamino)isobutyric acid

FIG.1. Three imino acids. In these the N atom is secondary.

RECOGNITION SITES FOR MATERIAL TRANSPORT AND INFORMATION TRANSFER

NH: I H,C- CH - COO-

Alanine

NH:

HOC&-

I CH-COO-

Serine

231

NH: I

HSCHz- CH- COOCysteine

FIG.2. Three defining substrates of the ASC system. Another carbon atom in the linear structure enhances affinity, and two more carbon atoms are acceptable. As can be illustrated by comparison of threonine and homoserine, the fourth carbon atom need not be added a t the end of the chain.

tems listed is, of course, its Naf-independence. This feature, together with the intense exchange traffic that takes place by this system in most but not all cells, has led to a serious misinterpretation of it as purely an exchanging agency, presumably because it does not have access to the energy stored in alkali-metal gradients. This interpretation appears wrong on every count, as we have discussed elsewhere (Christensen et al., 1973): 1. Net uptake and exodus occur by this system, and uphill transport by it can under experimental conditions be intense and unequivocal. In the pigeon red blood cell, System L shows little or no exchange (Eavenson and Christensen, 1967). 2. Despite the dependence of System A on the presence of Na+, and despite the usual transfer of energy from Naf gradients to amino acid gradients by way of this system, it can also produce intense uphill transport with minimal associated Naf movement (Christensen et al., 1973).

The other Na+-dependent transport system for neutral a-amino acids, System ASC, is limited to a much narrower range of neutral amino acids (Table I ; see also Fig. 2). It can be differentiated from System A most readily by its not tolerating N-methylation of the amino acid substrate. From its apparently absolute specificity to Na+ as the cosubstrate for transport, and from its unusually high stereospecificity, we may judge that the basis of the recognition of the amino acid side chain differs, as shown below, from that of the other systems considered so far. Despite this circumstance, we have not yet identified or designed an amino acid whose transport is limited to this system. In the rabbit reticulocyte and the pigeon erythrocyte, System ASC appears largely limited to transport by exchange, although the stoichimetry of the exchange, i.e., as to number of sodium ions accompanying each amino acid molecule, depends on the structures of the exchanging amino acids (Wheeler and Christensen, 1967 ; Thomas and Christensen, 1971; Koser and Christensen, 1971).

232

HALVOR N. CHRISTENSEN

111. THE CATIONIC AMINO ACID SYSTEMS

The transport of cationic amino acids appears for the most part t o be satisfactorily accounted for by a single system (Lyf) in the animal cells studied, given that we recognize a variable ability of diamino acids to be transported by the neutral systems. This system requires a positively charged group on the side chain. It is not enough that the group be positively charged in neutral aqueous solution; the diamino acids so far studied have values for pKza less than 8.7 are poor substrates (Christensen and Liang, 1966; Christensen, 197313). On the other hand, the presence of a distinctly cationic structure on the side chain is not necessarily sufficient either; the quaternary base, 4-amino-1-dimethylpiperidine-4-carboxylic acid, shows only a very slow, apparently nonsaturable uptake by the Ehrlich cell, even though the transport site accepts 4-amino-1-guanylpiperidine-4-carboxylic acid (Fig. 3), which as may be noted, should require somewhat more room a t the site than the dimethyl analog. a-Trimethy1-Llysine (Fig. 4) inhibits only weakly the system for cationic amino acids. In the liver mitochondrion (Gamble and Lehninger, 1973) and in bacteria (Rosen, 1973; Quay and Christensen, 1974), transport systems are known that distinguish between arginine and ornithine or lysine. We believe we understand how discrimination can be made by such systems in favor of the guanidinium group over the protonated amino group, from the observation that certain 4-hydroxyamino acids (homoserine, trans-4coo-

coo-

MPA

coo-

MzPA

FIG.3. The piperidine analogs of the basic amino acids. The first structure, 4-amino1-methylpiperidine-4-carboxylicacid (MPA), unexpectedly is mainly transported by neutral System L. Adding a second methyl group to the piperidine nitrogen, to quaternize it, led to a cationic amino acid for which no mediated uptake could be detected. The guanyl group on the piperidine N leads in contrast to a model substrate, 4-amino-1guanylpiperidine-4-carboxylic acid (GPA), for System Ly +.

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233

NH: I

C-N H2N‘

- CH2CH2CHZCHZCH-COO-

E-N- Methylhomoorginine

NH: I

y 3

CH3-+N

- CH2 CH2 CHzCHzCH - COO-

I CH3 E-N -Trimethyllysine

FIG.4. An example of the specific recognition of an amino acid. Top: The arrows point to the features we believe to be recognized a t the receptor site of the arginine-

H specific system. Encircled is the essential -Ngroup, which is lacking in the other two structures, cN-methylhomoarginine and e-N-trimethyllysine. Quay and I propose that structure to be the cationic structure (arrow a t left) must be separate from the -NHrecognized at the arginine-specific site.

hydroxyproline but not its cis isomer) serve as inhibitors of arginine uptake but not as substrates (Quay and Christensen, 1974). We reason that an appropriately directed hydroxy group is recognized by a feature of the site that ordinarily recognizes the secondary nitrogen atom of the guanidine group (for arginine the N on the delta carbon), or perhaps the hydrogen atom on that nitrogen. To obtain transport, an adjoining feature of the site must presumably also be satisfied, namely by a cationic group lying on the side chain of the substrate. In analyzing the bimodal uptake of the basic amino acids by Salmonella typhimurium, homoserine served to eliminate selectively arginine uptake by the arginine-specific system, whereas histidine served to eliminate arginine uptake by the general cationic system. Kinetic analysis under such conditions can go much further in describing the dichotomy than a mere demonstration of a bent Lineweaver-Burk curve.

H Our interpretation that the -&-structure of the guanidine group needs to be recognized for transport by the arginine-specific system of Salmonella typhimum’um was confirmed by the synthesis of €4-methyl+

234

HALVOR N. CHRISTENSEN

homoarginine (Fig. 4).’This amino acid lacks the hydrogen atom of the H -fi- structure, and therefore fails to inhibit the arginine-specific system. Figure 5 illustrates a two-component recognition subsite for the guanidinium group, one not responsive to a single terminal amino group as in lysine. In the Ehrlich cell and the rabbit reticulocyte, however, the site recognizing the cationic group of the amino acid side chain is less sophisticated. Even though it refuses the dimethylpiperidine structure, e-N-methylhomoarginine is an effective substrate, and the system can be “fooled” by relatively high concentrations of Na+ or one other alkali metal ion (Li+ in the Ehrlich cell; Kf in the rabbit reticulocyte, Thomas et al., 1971), along with a selected neutral amino acid. The most effective neutral amino acids have a hydroxyl group on carbon 5 (the position optimal for the Ehrlich cell) or on carbon 4 (optimal for the rabbit reticulocyte). In combination with Na+ these amino acids are effective inhibitors of the uptake of homoarginine or arginine, and effective stimulators of the exodus of the previously accumulated basic amino acids (Christensen and Hand-

FIG.5 . A representation of a possible distribution of the subsites participating in the recognition of arginine analogs for transport. I n addition to subsites 1 and 2, recognizing the a-carboxyl and the a-amino groups, there is 3, a subsite recognizing the cationic structure on the side chain, and 5, a region responding to the apolar mass of the side chain. Besides these four, also shown in Fig. 6, Quay and Christensen (1974) propose for an arginine-specific system a subsite 4, lying in a trans relation to subsite f. Subsite 4 is held to recognize the H atom on the secondary N of the guanidinium group, perhaps by hydrogen bonding. Some of the steric details suggested here go beyond the available evidence, e.g., the precise positions of subsites 3 and 5.

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235

FIG.6. Scheme suggesting how a neutral amino acid plus Na+ reacts with the cationic amino acid transport site. Note contrast with Fig. 5 in that only one subsite is needed to recognize the cationic structure on the side chain. The neutral amino acid (lower diagram) shows maximum Natdependent reactivity, both for inhibition of cationic amino acid uptake and for stimulating the exodus of a previously accumulated basic amino acid, when the chain has five carbon atoms in a linear relation, and when an oxygen or sulfur atom is exposed on carbon 5 . In the present instance, only four carbon atoms are present, but homoserine is also an effective substrate. (From Christensen, 1970, reproduced with permission.)

logten, 1969; Christensen et al., 1969a; Thomas et al., 1971). The requirement for the alkali-metal ion differentiates this action of the hydroxy amino acid from that already described for S. typhimurium. Figure 6 shows how we suppose such combinations of a neutral amino acid plus an alkali metal ion fit at the site of System Ly+.That the electrons of the oxygen atom may actually enter the Na+ orbital we argue from an analogy with System ASC, to be described below. We may add that the selectivity among the alkali-metal ions is sharply accentuated when the hydroxyl group lies on the appropriate carbon atom. It is an interesting detail that the effect of chain length observed with a combination of a hydroxy amino acid and Na+ yields a much sharper indication of the space between the subsites pictured in Fig. 6 than does the chain length of the basic amino acids themselves. Thus, the position of the hydroxyl group is much more critical than that of the distal amino group. No doubt the ‘(two-piece substrate” holds together at the site only when the steric strain is minimal. IV. EXTENSION TO SYSTEM ASC OF APPROACHES TO SITE DESCRIPTION TAUGHT BY THE BASIC AMINO ACIDS

The observation that Na+ plus a neutral amino acid can mimic the Na+independent transport reactivity of a basic amino acid led us to wonder

236

H A L V O R N. CHRISTENSEN

whether conversely a cationic amino acid might mimic the transport reactivity of Na+ and a neutral amino acid in a Naf-dependent system. Arginine proved indeed to be a Na+-independent inhibitor, although not a substrate, for System ASC (Thomas and Christensen, 1971). This finding led to a study of the effects of amino acid structure on the transport cooperativity between the amino acid and Na+. The optimal position of the hydroxyl or sulfhydryl group for cooperativity lies on carbon 4 (carbon 3 is next best) in both the rabbit reticulocyte and the pigeon red blood cell. Furthermore, the direction of the bond to the oxygen atom can be decisive;

\

L

\

FIG.7. Diagrammatic representation to show both Na+ and hydroxyproline in the relative positions they are believed to take a t the site for System ASC. A part of the interaction between the two cosubstrates is mediated by the receptor structure, but when an appropriately oriented hydroxyl or sulfhydryl group is present on carbon 3 or 4 of the amino acid side chain, evidence is obtained for a further strong interaction between the two cosubstrates. If the hydroxyl group is instead in the cis orientation, no favorable effect occurs. (From Thomas and Christensen, 1970, reproduced with permission.)

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237

the trans hydroxyprolines are highly effective, but their cis isomers are less effective than proline itself (Thomas and Christensen, 1970, 1971). These results led us to the diagram of Fig. 7, which proposes geometric relations taken by the amino acids and the sodium ion in their normal binding at the ASC transport receptor site. Another signal for the ability of the amino acid to assume the most favorable relation to Na+ is seen in the change in the stoichiometric ratio between the fluxes of Wa+ and the amino acid (Koser and Christensen, 1971). By reference to Fig. 7, the reader may note that a cis hydroxyl group on the ring structure of proline would project upward, to engage an entirely different portion of the receptor site. From the unfavorable effect of the cis hydroxyl group, and the favorable effect of the methyl and ethyl side chains of alanine and 2-amino-n-butyric acid, we may reason that an apolar structure at this aspect enhances the transport cooperativity between the amino acid and Na+. In contrast, our results show that a trans hydroxyl group on carbon 4 (as illustrated) produces optimum cooperativity. The reciprocal relations between Systems Ly+ and ASC in these interactions between a basic amino acid on the one hand, and a neutral amino acid plus Waf, on the other, adds materially to the force of the arguments, as summarized previously (Thomas and Christensen, 1971; Thomas et al., 1971). The possibility that the two systems might instead be manifestations of a single transport acceptor site can, however, be ruled out. The K , and Ki values are grossly inconsistent with that interpretation, the “normal” substrates being almost fifty times as reactive as the surrogate substrates in both systems. The optimal distance between the amino and the hydroxyl group is different in the two systems. Furthermore, System Ly+ survives in the maturing rabbit reticulocyte after System ASC can no longer be detected.

V. EFFORTS TO DEVELOP SYSTEM-SPECIFIC, NONMETABOLIZABLE SUBSTRATES FOR THE NEUTRAL SYSTEMS

During these studies, the design of model substrates largely specific to a single transport system became both a means toward systems discrimination and a product of the research. Table I1 shows the model amino acids presently recommended as nearly system-specific. For System A , 2-(methylamino)-isobutyric has emerged as the most promising. Specificity for System L has called for bulkiness of the side chain of the substrate in both lateral dimensions, a property best obtained so far with 2-aminonorbornane2-carboxylic acid, one of the four isomers of this amino acid, as discussed

238

HALVOR N. CHRISTENSEN

TABLE I1 SOMEMODELSUBSTRATES RECOMMENDED FOR THEIR RESTRICTED TRANSPORT REACTIVITY" Transport systems Substrates

P

Glycine Taurine MeAIB (-)b-BCH MPA GPA DCG

+++

0

0 0 0 0 0

GlY

A

++++ +++ 0 0 +++++ 0 0 0 0 0

f f 0 0

ASC

L

Lg+

f

f 0 0

0 0 0 0

0 0 0 0 0 0

+++++ ++++ 0 0

+ ++ 0

Reactivities are roughly compared for the Ehrlich cell and various red blood cells. A zero means that little or no reactivity has so far been observed, whereas a f means that small but definite transport is believed to take place. Hazards in extending the use of these model substrates to new contexts may be illustrated by observations that DCG is accumulated by the liver in situ and by brain slices, in each case by an agency resembling System L.MeAIB = 2-(methy1amino)isobutyric acid; (-)b-BCH = the levorotatory isomer of 2-(endo)aminonorbornane2-carboxylicacid; MPA = 4-amino-l-methylpiperidine-4-carboxylic acid; GPA = 4-amino-l-guanylpiperidine-4-carboxylicacid; DCG = a,a-dicyclopropaneglycine. (1

below, being most generally transport reactive (Christensen et al., 1969b). In Escherichia coli K12, this isomer is the only one for which cellular uptake could be observed, and it is also by far the most strongly bound by the binding protein associated with the system ( L I V ) for the branched-chain amino acids in that organism. Kinetics identify the uptake with System LIV, which has, however, been found on further study to have broader reactivity, for example including alanine (Robbins and Oxender, 1973; cf. Tager and Christensen, 1971a). The same isomer of the norbornane amino acid also serves as a substrate for a somewhat similar system in yeast. Among the isomers it shows the strongest uptake by the Ehrlich cell, the human and pigeon red blood cells, and segments of the rat small intestine. It undergoes a Na+-independent uptake also by the isolated, intact rat diaphragm, an uptake which incidentally is stimulated neither by insulin nor glucagon (Harrison and Christensen, 1971). Uptake of the selected isomeric form of the norbornane amino acid shows in our hands very little slowing on replacement of the sodium ion of the medium with choline, indicating little uptake by Na+-dependent systems. With longer incubation and a t higher substrate levels, the effect of Na+

RECOGNITION SITES FOR MATERIAL TRANSPORT AND INFORMATION TRANSFER

239

replacement on net uptake grows larger, but mainly through loss into the choline medium of the amino acid already accumulated rather than from the slowing of its further uptake (Christensen el al., 1973). At high levels of the substrate one can exaggerate any uptake by a high K , system and minimize that by System L ; by allowing longer intervals, one can also reinforce that emphasis. We are, nevertheless, at a loss to understand the considerable fraction of Na+-dependent uptake recently reported in one study for this amino acid even at fairly low levels and during short intervals (McClellan and Schafer, 1973). Caution in interpreting results with unfamiliar strains of cells must be recommended for the present until this discrepancy is understood. Another principle may prove useful for intensifying specificity to System L. Azaleucine [P-(dimethylamino)-alanine] proves rather more specific than leucine to System L (Christensen et aE., 1973). This compound, along with other diamino acids, has a high rate of uptake by System L (Fig. 8) and a high asymmetry between influx and efflux-properties that we are seeking to explain (Christensen et al., 1973; Christensen, 1974). The higher specificity to System L may well be associated with these properties. Perhaps a model substrate combining the bulk of the norbornane ring and these effects of a second amino group would prove to be uniquely specific c 5._

-

[L-aza~eu], m M FIG.8. Apparent division of the uptake of ~-azaleucine-Me-'~Cby the Ehrlich cell among three transport systems. The major portion, from 96 to 70% in the concentration range 0.2-5 mM, is inhibitable by the norbornane amino acid ( A ) a t 20 mM, and is therefore attributed to System L. The maximal velocity of this component ranged from 4 to 6 mmoles per kilogram of cell water per minute, two or three times the usual values. Despite the low pK2 of 6.8, a substantial component is sensitive to inhibition by homoarginine (0)at 10 mM, and is attributed to the cationic amino acid system. A smaller component was inhibited by 20 m M MeAIB (0) or by omission of Na+ from the suspending medium. Total uptake ( 0 ) .

240

HALVOR

N. CHRISTENSEN

to System L. Application of the chloromethylketone analogs of the natural amino acids as transport inhibitors (Glover et al., 1972) is now suggesting a basis for recognizing the carboxyl group in the Na+-independent system differing from that for the Na+-dependent systems. So far we have not succeeded in codifying the transport system of the liver parenchyma by direct inhibition analysis. A study of amino acid uptake by the liver in situ shows, however, that, among model substrates for the neutral systems, uptake of the norbornane amino acids escapes stimulation by glucagon or theophylline, whereas uptake of AIB and MeAIB does not (Harrison and Christensen, 1971). In the diaphragm, a corresponding unresponsiveness to humoral effects is associated -with an essentially Na+-independent, mediated transport. Therefore, we conclude that a system corresponding to System L contributes to hepatic transport of amino acids. Quite regularly, the transport system subject to hormonal regulation, to repression and derepression (Franchi-Gazzola et al., 1973), to increase after fertilization of the egg (Epel, 1972) and during a stage of mitosis (Sander and Pardee, 1972) appears either to be System A , per se, or an unidentified Na+-dependent system closely resembling System A . System A may be a strategic one on which to apply regulation, because much of the energy for uphill amino acid transport appears primarily to be applied to this system and then distributed among other amino acids by exchange through other systems. Hence, the energy invested in amino acid transport may be controlled at this point. We have already indicated, however, that we should not suppose that energy is applied only via System A or only by Na+ comigration. Crowding at the side chain area of transport sites was intensified experimentally in an attempt to measure and exceed the spatial limits. The presence of two ethyl groups in a,a-diethylglycine succeeds in greatly slowing uptake by the Ehrlich cell, but exodus is slowed even more, with the unexpected result that this amino acid is gradually accumuIated to a high degree, still by the Na+-independent agency (Christensen and Liang, 1965; Christensen et al., 1973). Furthermore, a linked flux of H+ could be shown during uptake of this amino acid, an effect not obtained when further crowding was produced in the test of a,a-dicyclopropylglycine(Christensen et al., 1973), which shows very slow, probably unmediated uptake and exodus in the Ehrlich cell (Christensen and Handlogten, 1968). A similar but more weakly linked flux of H+ may occur during uptake of ordinary -substrates of System L. The unexpected properties of diethylglycine are still under study. In the tissues of the intact rat, diethylglycine appears largely to escape mediated transport, whereas oddly enough the much bulkier dicyclopropyl-

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24 1

FIG.9. Diagram of the molecular structure of a,a-dicyclopropylglycine. Although this amino acid is a structural isomer to the norbornane amino acids, it presents four rather than three carbon atoms a t the level of the @-carbonatom of an amino acid, and is excluded from System L in some of its occurrences.

glycine is accumulated by the liver by a methionine-inhibitable process, and gradually by bra'in slices in vitro, by a process inhibited by the norbornane amino acid, i.e., via System L. The two cyclopropyl groups yield the same mass as the norbornane ring system, but they present four, rather than three, gamma carbon atoms (Fig. 9), and hence require greater space in a slightly different geometric distribution. These results warn us to anticipate a degree of variability in the amount of space available a t the site in various occurrences of a transport system.

V1. SYSTEM-SPECIFIC SUBSTRATES FOR THE TRANSPORT SYSTEM FOR THE CATIONIC AMINO ACIDS

The directions taken in the work summarized above to design systemspecific substrates also provided amino acids that almost completely escape metabolic attack in several biological systems. This objective was pressed by avoiding the presence of a hydrogen atom on the a-carbon and by avoiding chain ends through the introduction of cyclic structures. Homoarginine, a model substrate that has served us well for System Ly+, is subject to slow breakdown by arginase, and hence by no means escapes all metabolic attack. Its affinity for transport in the Ehrlich cell is superior to that of arginine, a consequence, no doubt, of the greater apolar mass it presents (Christensen et al., 1969a). Even the small susceptibility to arginase attack could be eliminated by introducing an e-N-methyl group, in an analog already mentioned. The residual low metabolizability by other routes still to be expected from the presence of an a-hydrogen atom was avoided through the synthesis of 4-amino-1-guanylpiperidine-4-carboxylic acid (Christensen and Cullen, 1973). This amino acid we may think of as analogous to the lower homolog of arginine, a-amino, 7-guanidinobutyric acid, except that the two-carbon side chain is repeated to generate a

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piperidine ring. From other experience, we anticipated that some crowding of site Ly+ would arise from the presence of two beta carbon atoms in this amino acid. Its higher K,, relative to arginine (Table 111), shows the loss we accept in the apparent transport affinity for the sake of greater resistance to metabolism. Interestingly, the arginine analog built on the piperidine ring shows very little transport or inhibition of transport in Salmonella typhimurium, either by System LAO, not specific to any one of the basic amino acids, or by the arginine-specific system (Quay and Christensen, 1974). From comparison with e-N-methylhomoarginine, we already understand that the absence of a hydrogen atom on the proximal guanidine nitrogen atom here may be sufficient to prevent recognition. The inhibition of arginine transport by certain hydroxy amino acids that are not transported by the same system (Quay and Christensen, 1974; see also this chapter, p. 233) deserves special comment as a phenomenon that is occasionally overlooked. A number of precedents show the same phenomenon, i.e., that the receptor site can be occupied by an analog to the natural substrates, even though the analog lacks a certain feature required if transport is to follow. Earlier cases are, e.g., the action of phlorizin on the active sugar transport in the intestine [phlorizin competes for the sugar binding site (Alvarado, 1967) but does not enter the cell (Sterling, 1967)]; the Na+-independent inhibition of the ASC system by arginine, methionine, or norleucine (Christensen et al., 1969c) ; and the TABLE I11 COMPARISON OF ARGININE AND SOMEARGININE ANALOGS AS TO TRANSPORT RE.4CTIVITY IN THE EHRLICHCELL .4ND AS TO AUGMENTATION OF THE LEVELOF IMMUNOREACTIVE INSULIN AND GLUCAGON IN THE PLASMA OF THE RAT^

Compound Arginine Homoarginine 4-Amino-1-guanylpiperidine4-carboxylic acid

K , for uptake by the Ehrlich cell A Insulin A Glucagon (mM) (mU/liter plasma) (ng/ml plasma) 0.25 0.06 2.2

7.1 6.0 3.9

0.45 0.78 0.51

Uptake w&s measured at 37", pH 7.4 during 1 minute; serum hormone levels 27 minutes after administration of 8 mmole/kg doses to male rats of about 100 gm. The increases in the hormone levels over those produced by 0.15 N NaCl are in each case significant. Because the immunizing hormones were not homologous, these increases should he considered relative rather than ahsolute.

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Naf-dependent inhibition of the glucose transport system in the intestine by L-fucose (Caspary et at., 1969). Table I11 summarizes the progress so far made in discovering or designing model amino acid substrates for the transport systems of the several cells under study. We hope the amino acids on this list may prove applicable in circumstances not yet studied with them, and that the principles illustrated in their design will apply also to other situations.

VII. STIMULATION OF RELEASE OF PANCREATIC HORMONES BY AMINO ACIDS

This background of explanation of the progress in the discrimination of separate transport systems, especially in animal cells, and the incompletely successful effort to discover a model substrate specific to each system, probably has provided the reader with no stronger sense of expectation than we had that the same amino acids designed as specific transport models might prove to mimic their natural analogs in stimulating the release of the pancreatic hormones. This parallelism was entirely unexpected. Fajans and his associates have reviewed the history of the discovery that protein as well as carbohydrate feeding can stimulate the release of insulin from the pancreatic islet (Fajans et al., 1967), and that the various amino acids contribute to variable degrees to the total effect of protein feeding. At first this effect was supposed to apply only to patients with a n idiopathic hypoglycemia and to cases of tumor-associated hyperinsulinism. Faj ans and his associates (1960) showed, however, that the phenomenon has a more general physiological importance. They also showed that the stimulatory amino acids could be placed in two classes with different characteristics, the leucinelike group and the lysine-and-arginine-like group. The amino acids of the latter group also stimulate the release of glucagon from the alpha cells. We were engaged in attempting to codify the transport systems of various tissues in the intact rat including those of the renal tubule, by challenging them with large doses of various metabolism-resistant amino acids. In the course of these experiments, control tests with adrenalectomized animals became desirable. These animals, surprisingly, were prostrated by substantial doses of the norbornane amino acid (Christensen and Cullen, 1969). The preparation used had been synthesized via the corresponding spirohydantoin, which yields a product about 95% in the ( f)-amino-endo form, also called ( = t ) - b (Christensen et al., 1969b). Figure 10 illustrates the isomerism of this compound. By the Strecker synthesis, i.e., via the cor-

HALVOR N. CHRISTENSEN

244

I

Optical Isomers

I

FIG.10. The isomers of 2-aminonorbornane-2-earboxylic acid. Isomer (111) is the one showing transport into all organisms tested, including yeast and Escherichiu coli, and also the one stimulating the release of insulin from the pancreatic islet. (From Tager and Christensen, 1972, reproduced with permission.)

responding cyanohydrin, we could obtain mainly the (&)-a form, i.e., the other racemic mixture. As Fig. 11 shows, the latter shows no hypoglycemic action on the rat, whereas the ( & ) - b isomer had a larger and more prolonged action than that of leucine. These experiments were made in rats primed by five doses of tolbutamide, administered at 12-hour intervals. The hypoglycemic effect of amino acids acting in the mode of leucine is considerably magnified by priming with the sulfonylureas. The stimulation of insulin release from the surviving fetal pancreas by 2-aminoisobutyric acid was observed in pioneering experiments by Lambert et al. (1971), even before we had traced the hypoglycemic action of the norbornane amino acid to an insulin-releasing action. Such an effect has not, however, been observed for 2-aminoisobutyric acid in the intact animal. The four analogs whose structures are compared with those of leucine and the norbornane amino acid in Fig. 12 proved not to produce hypoglycemia in the rat under the same conditions. These amino acids lack a t least one bond and up to two of the carbon atoms present in the norbornane amino acid. The results suggest that the hypoglycemia action requires a detailed imitation of the positions taken by the side chain of the effective amino acids.

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I

I

I

I

245

I

2

3

4

Time, hr

FIG.11. Hypoglycemic action of the geometric isomers of the norbornane amino acid in the tolbutamide-primed rat. Dose, 8 mmoles of amino acid per kilogram of body weight. A similar comparison showed that the activity of the geometric isomer identified as b is entirely due to its levorotatory form. (From Tager and Christensen, 1971b, reproduced with permission.)

/’

L’,

b;;;

Cycloleucine

3-Methylcycloleucine

&:&;:: ;; ‘J

Cyclopentylglycine

Cyclopentyl-

alanine

FIG.12. Amino acids structurally related to the norbornane amino acid and leucine (top row), which fail to produce hypoglycemia in the tolbutamide-primed rat. BCH, 2-(endo)aminobornane-2-carboxylic acid, (Unpublished results of H. Tager and H. N. Christensen, 1969.)

246

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Tager subsequently resolved the two racemic preparations of the norbornane amino acid into their optical antipodes. Of these only the levorotatory b form proved hypoglycemic (Tager and Christensen, 1972). I n the first such test by Tager, I can well recall the dramatic contrast in the appearance of four rats, each of which had received a separate isomer. One was in a state of hypoglycemic shock, while the other three were normally active. Through the collaboration of Doctors Fajans and Pek we were able to show that the level of immunologically reactive insulin in the plasma of the rat was sharply increased during hypoglycemia (Christensen and Cullen, 1973). Subsequently, the norbornane amino acid was compared with leucine as to its insulin-releasing action in the dogs, in a continuing collaboration with Fajans and his associates (1971). In this species, the effect was somewhat less than that of an equimolar quantity of leucine (Fig. 11). Nevertheless, the action shared a series of characteristics with leucine : It was strongly enhanced by priming with sulfonyl urea; it was blocked by diazoxide. The effect was limited to insulin release and did not affect the circulating level of glucagon. Furthermore, as illustrated in Fig. 12, the prior and concurrent infusion of mannoheptulose does not block the action of either leucine or the norbornane amino acid; on the contrary, the action of the latter amino acid appears to be enhanced (Fajans et al., 1971). This result implies that the utilization of glucose is not necessary to the action of these amino acids, nor does glucose need to participate in the signal for insulin release. Comparison of Fig. 13 with Fig. 14 shows a striking contrast between the action of leucine or the norbornane amino acid, on one hand, and arginine or lysine, on the other. As shown in Fig. 15, the infusion

40

-

;

'1,

$=-A '\

duc 2 0 -

-

$

I )

, I

:o-

b

::

; 'L

-+.".d

I

'..

,~-. -m

-. \,----*

----+---

0-8

FIG.13. Effect on the plasma level of immunoreactive insulin of the intravenous infusion of leucine ( 0 - - - 0 ;30 mmoles) or of the b geometric isomer of 2-aminonorbornane-Bcaroxyloic acid;.--.I 60 mmoles = 28 mmoles (-) b-BCH] in a chlorpropamide-treated dog. (From Fajans et al., 1971, reproduced with permission.)

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60 Dog M. 10kg. F

1

OLL-'

-30

1

1

-15

0

30 40

15

50

60

75

90

Minutes

FIG.14. Effect on plasma levels of insulin of the intravenous infusion of the norbornane amino acid without ( 0- - - 0 , saline BCH) and with mannoheptulose (0-0; MH-MH.BCH; M H = 0.5 grn/kg) in a chlorpropamide-treated dog. (From Fajans et al., 1971, reproduced with permission.)

of mannoheptulose not only lowers sharply the background circulating insulin concentration, but also prevents the arginine-induced increase. These results show that the norbornane amino acid is a functional analog of leucine, not of arginine, in its action on the pancreatic islet. Table 111 shows the plasma insulin and glucagon levels observed 30 minutes after injection into the rat of each of the three cationic amino

80 -

I I S&

; ',

603

5

5

I

L

I

\

I I

5

40-

, I

\,

'

&-4,

FIG.15. Effect on the plasma levels of insulin of the intravenous infusion of arginine without ( 0 - - 0 ; saline-argine, 30 mmoles) and with mannoheptulose (0-0; MH-MH-arginine; M H = 0.5 gm/kg) in a dog. (From Fajans et al., 1971, reproduced with permission.)

-

HALVOR N. CHRISTENSEN

24 i x p i a s r n a glucagon I1 sharply elevated

20

I1

L

a

a

II i I

I

ii

I 1

I 1

I6 .-c m 13 C

- 8 0

E

h

4

0‘ -30

0

-15

30 Minutes

15

60

45

75

90

FIG.16. Effect of intravenous infusions (20 mmoles) of 4-amino-l-guanylpiperidine-4carboxylic acid on plasma insulin in a female, 17.2 kg, dog, in the absence and in the presence, (0.5g/kg as 10% solution) of mannoheptulose. The marks a t the top identify the intervals when the plasma glucagon showed peak values. (Experimenk of Fajans et al., 1974,reproduced with permission.)

Soline

Dog L, I76 Kg, F

I

-30

-15

GPA

MH.GPA

1 . 1

I

I

I

0 5 D 1 5 2 0 Minutes

1

I

30

40

I4c4c4WcI

50607590120

FIG.1-7. Effect of intravenous infusion of 4-amino-I-guanylpiperidine-4-carboxylic acid ( 0 - - - 0 ; saline-GPA, 20 mmoles) without and with mannoheptulose (0; MH = 0.5 g/kg) on the plasma glucagon in a dog. (From Fajans et al., 1974,reproduced with permission.)

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acids, arginine, homoarginine, and the arginine analog built on the piperidine ring, along with the K , values of these three amino acids for transport (Christensen and Cullen, 1973). Figure 16 shows the action of the latter analog on the circulating levels of immunoreactive insulin on infusion into the dog, a.lso the elimination of this response in the presence of mannoheptulose. The time a t which peaks occurred in the simultaneous plasma level of glucagon are also represented at the top of the figure (Fajans et al., 1974). The time intervals required for the effects on the two types of secretory cells correspond so closely as to greatly diminish the possibility that the release of one of these hormones mediates the release of the other. Figure 17 shows that the stimulation of glucagon by the arginine analog is not, in contrast, affected by the presence of mannoheptulose. These results show that the guanylpiperidine amino acid is a functional analog of arginine in its stimulation of the release of the pancreatic hormones. TABLE I V

EFFECT OF

THE LEUCINEANALOG BCH, AND TIIE ARGININE ANALOG GPA, ON INSULIN RELEASEQ*'

Insulin (ng) released per dry islet (pg) Glucose (mM) : First 5 min 0 10 Subsequent 60 min 0 10

Control

.5 mM b(+)BCHc

5 mM b ( -)BCH

0.12 f 0.03 0.82 f 0.11

0.19 f 0.06 0.63 f 0.13

0.37 f 0.08 0.80 f 0.17

0.33 f 0.06 6.05 f 0.83

0.36 f 0.08 5.94 f 0.52

1.00 f 0.34 8.24 f 0.87

5 mM GPA

10 mM GPA

0.15 f 0.05 0.88 f 0.24

0.21 f 0.05 1.07 f 0.47

0.18 f 0.08 0.91 f 0.17

0.34 f 0.08 5.60 f 1.68

0.50 rt 0.18 5.11 f 0.48

0.48 f 0.16 9.13 f 1.16

First 5 min 0 10 Subsequent 10 min 0 10

From Christensen, Hellman et al. (1971). The value after f is the standard error for 8-10 experiments. The effect of the levorotatory b isomer of BCH was significant in both the early and the late phase, and both in the presence and the absence of glucose, The effect of GPA was significant only in the late phase in the presence of glucose. c BCH, 2-(endo)aminonorbornane-2-carboxylic acid; GPA, 4-amino-1-guanylpiperidine-4-carboxylic acid.

250

HALVOR N. CHRISTENSEN

Subsequently to making the experiments just described in intact animals, we had the happy opportunity to see the effects on insulin release confirmed for isolated pancreatic islets from obese diabetics strain of mice in the Laboratory of Professor Bo Hellman a t the University of Umeh, Sweden (Christensen, Hellman et al., 1971). These microdissected islets are composed to the extent of at least 90% of beta cells; hence, their use further minimizes any possibility that the release of insulin could be a secondary consequence of a stimulation of glucagon release and shows that effects of the nonmetabolizable amino acids are direct effects on the beta cells. Table I V shows that the guanylpiperidine amino acid is stimulating to insulin release in vitro only during the so-called late phase and only if glucose is present in the suspending medium. In contrast, the norbornane amino acid was equally stimulating in the presence and in the absence of glucose. VIII. CONCLUDING DISCUSSION

It seemed to us a remarkable circumstance that the synthetic amino acids designed as model substrates for two transport systems should mimic so closely the action of some typical substrates of these systems in causing hormone release. First of all it seems to settle unequivocally the question whether amino acids need to be catabolized to have these effects. (Fajans et al. had already demonstrated that the action of leucine could not be accounted for by the direct action of any known leucine breakdown product.) Accordingly, we may say that this behavior corresponds well to the lock-and-key analogy of Emil Fischer-it corresponds better indeed than does ordinary enzyme action, in that the “key” is destroyed by the enzyme each time it is used, whereas the metabolite that signals for hormone release when certain levels are exceeded need not be modified. Both in transport and in information transfer the “key” can be used over and over again. During our study so far of the structural requirements for the stimulation of hormone release by amino acid analogs, we have thought of the following possible relations among the recognition sites concerned : 1. That the filling of the transport receptor site per se ca,uses a message to be transmitted to the cell interior to produce hormone release. The idea that the transported amino acid itself serves as the message seems unattractive, because then a second, sequential recognition site would be needed. 2. That the accelerated operation of the transport system yields the signal. Under this heading we thought particularly of a well known asym-

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metry in the orientation of the recognition site or carrier that can be produced when a substantial load of substrate is placed a t the external surface of the cell, the asymmetry believed to produce trans stimulation. This mode of signal production would require that fairly rapid transport be produced for the substrate; otherwise such an asymmetry in orientation would be small or absent. 3. That a sufficient intensity of the transport event produces a characteristic change in cellular metabolism, which leads to hormone release. 4. That the same recognition structure serves for both transport and signaling for hormone release, but that a different effector apparatus completes the process in the two cases. Indications of such a relation between transport and chemotaxis will be considered below. 5. That the two recognition sites are similar, but not identical, and have a common differentiative or evolutionary origin.

A final choice among these alternatives cannot yet be made. The results of Fig. 18 are unfavorable to the first hypothesis. This figure shows that the two optical isomers of the amino-endo norbornane amino acid are transported into the isolated rat islets a t almost the same rate and to similar steady-state levels. Hence both isomers ought to give the signal if it is based either on the filling of the site or the operation of the transport system ; yet only the levorotatory isomer causes insulin release. In the absence of a specific codification of the transport systems of the

E

INCUBATION TIME (MIN )

FIG.18. Uptake of the levorotatory and the dextrorotatory isomer of the norbornane amino acid in 14C-carboxyl-labeledform by pancreatic islets from an obese, diabetic strain of mice. The medium was 1 mM in the amino acid. Mannitol served for the measure of and correction for extracellular space in the tissue. The vertical lines show the standard errors for 4-13 observations. (From Christensen, Hellman et al., 1971, reproduced with permission.)

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HALVOR N. CHRISTENSEN

TABLE V EFFECTS

(ME-HOMOARG) ON PANCREATIC ISLETW~

OF E-~-METHYL-L-HOMOARGININE

FROM

Test substance None (control) Me-Homoarg None (control) Me-Homoarg Homoarg Arg MeHomoarg Arg Homoarg MeHomoarg Homoarg Arg

+

+ +

Glucose concentration (mM) 3 3 10 10 10 10 10 10 10

INSULIN

RELEASE

Insulin release (ng/pg dry islet) p Value for effect 0.41 0.71 2.72 5.92 7.38 9.84 8.99 9.61 8.98

f 0.15 f 0.31 f 0.41 f 0.53 f 1.06 f 0.73 f. 1.37 f 1.36 f 1.47

>0.03

<0.OOd <0.001 <0.001

a After a first 40 minutes of incubation in Krebs-Ringer bicarbonate medium containing 3 mM glucose and 1 mg/ml albumin, the islets (from fed ob/ob mice) were incubated a further 60 minutes in the same medium containing 3 or 10 mM glucose and 10 mM of the amino acid as shown in the table. Eig$t experiments in each case. Unpublished experiments by Bo Hellman and Ake Lernmark, The University of Ume%,Sweden.

beta cell, we might argue, not very plausibly, that the dextrorotatory isomer is transported by a system different than that serving for the levorotatory isomer. A finding of Hellman et al. (1973) is antagonistic to that explanation. These investigators noted that D- and L-leucine compete for transport, even though only the L isomer stimulates insulin release. We might nevertheless propose that only the L isomer is transported rapidly enough to cause a sufficient asymmetry in the orientation of the carrier site. But if that were true, D-leucine at elevated concentrations should prevent the insulin-releasing action of the L isomer, an action it does not show (Hellman et al., 1973). Another case is illustrated by Table V, which shows that E-N-methylL-homoarginine is somewhat less effective than homoarginine itself in stimulating insulin release in the presence of 10 mM glucose. Nevertheless, the presence of the N-methyl derivative showed no tendency to decrease the effectiveness of either arginine or homoarginine. We are still looking for amino acid analogs which bind to the receptor sites for hormone release to block the stimulation of release by ordinary amino acids, an effect that should be observable if some secondary event, not mere occupation of a recognition site, is required for the hormone-releasing signal.

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One metabolic consequence of the presence of the norbornane amino acid has been noted by Matschinsky et al. (1974)-namely, a stimulation of lactate production. This stimulation is produced by a number of amino acids including both the levo- and the dextrorotatory isomers of the reactive form of the norbornane amino acid. Hence this metabolic effect does not parallel precisely the insulin-releasing potency of these amino acids. Another metabolic effect of the amino acids can, however, be correlated with the insulin-releasing activity. Panten and Christians observed that a prompt change in the fluorescence attributable to NADH and NADPH occurs on adding the levorotatory but not the dextrorotatory isomer of the norbornane amino acid to perifused islets. Glucose addition also produces a change in fluorescence, but arginine addition does not. These authors speculated that the prompt change in metabolism indicated by this characteristic change in fluorescence might arise from an ion redistribution (Panten and Christians, 1973). Freinkel et al. (1974) have in the meantime observed a stimulation of the pulselike release of inorganic phosphate (“phosphate-flush”) only by the levorotatory, amino-endo form of the norbornane amino acid, the one that causes insulin release. Before closing the question of the relation between the two types of recognition sites, let me briefly bring into the discussion another type of information transfer from the cellular environment, namely chemotaxis. The studies of Hazelbauer and Adler (1971; Adler, 1969) showed that the ability of motile, ciliated E. coli cells to respond to gradients of a metabolite in their environment can be associated with the presence of the same binding protein already implicated in the transport of the metabolite. The loss of this binding protein can be associated with the loss of the chemotactic response. Furthermore, the addition of purified binding protein to the depleted cells has been reported to restore the chemotactic response, even if the specific transport function is not restored by the same procedure (Hazelbauer and Adler, 1971). Hence occupation of the binding protein seems able somehow to signal increased or decreased concentrations of the nutrient that it binds. A physical position permitting the binding protein t o function in such information transfer may not, however, be suitable to permit it to serve in material transport. The role of the binding proteins in chemotaxis is receiving intense study in several laboratories with results of remarkable interest. Perhaps we should be satisfied for the present with this contrast between the service of binding proteins in transport and in chemotaxis : The binding protein may lie in a position permitting chemotaxis without necessarily being able to serve in transport. The receptor sites for metabolite sensing and metabolite transfer may have a common evolutionary or diff erentiative

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HALVOR N. CHRISTENSEN

origin even though their specificities or functional positions may not be identical. It is important to note that none of the numerous binding proteins so far implicated in transport or in chemotaxis has been found to destabilize the specifically bound solute. Such destabilization is, in contrast, a defining characteristic of enzymes. A binding protein for tasted sugars has also been found in association with functional taste buds, without indications of enzymic action on the substrate bound (Hiji and Sato, 1973). We have noted correlations between the substrate preference of the recognition sites for transport and the recognition sites for information transfer. This common ground deserves special emphasis to help us to refute a tendency of some enzymologists to feel that the sites for transport must necessarily be the reactive sites of enymes. Under this tendency, the whole field of transport tends occasionally to be preempted for a process of perhaps parallel biological significance, namely group translocation. The bias is generated that models to account for uphill transport have really failed to grapple properly with the problem until they have proposed the generation of small-moleculed intermediates from the transported solute, e.g., phosphosugars for sugar transport, or peptides for amino acid transport. Wherever such substances are indeed obligatory intermediates, they must be discovered; but it seems absurd to feel that progress is made only by proposing them, whether or not they actually serve. In the meantime, the alternative proposal must be considered that the forms of the transport substrate bound to the mediating structure of the membrane may prove to be the sole intermediates in the transport process, a situation that appears to hold for Na+ and I(+ transport. The bias for the enzymic site as the recognition site for transport is encouraged by the circumstance that many nonenzymologists have not had to think hard about the difference between the reactive sites of enzymes and the other categories of binding sites on protein molecules. Therefore, when they hear it said that the substrate for a specific transport must of course be recognized by the reactive site of an enzyme, they may acquiesce without realizing all that is implied. They overlook the dubious implication that the substrate must be destabilized so that it will react with another structure in the environment to form an intermediate. The biological phenomenon of transport seems usually not to include this defining characteristic of the reactive site of an enzyme, that is, that the transport system must destabilize the molecule it binds. Support for the questionable view that enzymic sites serve for the recognition of the transported solute comes also from more sophisticated considerations. When an oligomeric complex situated in a biological membrane is able to produce uphill transport, it is likely to act as an enzyme

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with regard to an apparently untransported substrate, for example ATP. Hence, the whole complex can appropriately be called an enzyme. Accordingly, to insist that energy-transducing protein oligomers are not enzymes may aIso be absurd. We would be mislead, however, if we gathered from this circumstance that the substrate is grasped by the reactive site of an enzyme, for example, that Na+ is grasped by a site on the ATPase able to destabilize the alkali metal ion. We should recall that enzymes often have a second class of recognition sites, besides their reactive sites-namely, the modifier sites. When these are occupied, the behavior of the reactive site undergoes quantitative modification. We may regard it as certain that the influence between the reactive site and the modifier site goes in both directions, i.e., that the catalytic action also has an effect on the modifier site. Because conformational changes appear t o be inherent in the action of the modifier site, it j s not unlikely that the modifier site regularly is caused to move several Angstrom units by the events occurring at the reactive site of the enzyme. Such effects could in the final analysis be basic to transport. Perhaps this relation may permit us to deal successfully with the confusion about what we ought to call the recognition sites for ordinarly uphill transport, by suggesting that they may sometimes be modifier sites to enzymes. Even if the future may prove that we are not uniformly correct t o call them modifier sites to enzymes, that conceptualization may nevertheless break the dubious habit of looking on these recognition sites as the reactive sites of enzymes. REFERENCES Adler, J. (1969). Chemoreceptors in bacteria. Science 166, 1588-1597. Alvarado, F. (1967). Hypothesis for the interaction of phlorizin and phloretin with membrane carriers for sugars. Biochim. Biophys. Acta 135, 483-495. Caspary, W. F., Stevenson, N. R., and Crane, R. K. (1969). Evidence for an intermediate step in carrier-mediated sugar translocation across the brush-border membrane of hamster intestine. Biochim. Biophys. Acta 193, 168-178. Christensen, H. N. (1966). Methods for distinguishing amino acid transport systems of a given cell or tissue. Fed. Proc., Fed. Amer. Soc. Exp. Biol. 25, 8.50-853. Christensen, H. N. (1969). Some special kinetics problem of transport. Aduan. Enzymol. Relat. Areas Mol. Biol. 32, 1-20. Christensen, H. N. (1970). Introduction to symposium on transport,. (Fed.Eur. Biochem. Sac.), Symp. 20, 81-85. Christensen, H. N. (1973a). On the development of amino acid transport systems. Fed. Proc., Fed. Amer. SOC.Exp. Biol. 32, 19-28. Christensen, H. N. (1973b). On themeaning of effect.sof subst,ratestructure on biological transport. J. Bioenerg. 4, 31-61. Christensen, H. N., and Cullen, A. M. (1969). Behavior in the rat of EL transport-specific bicyclic amino acid. J . Biol. Chem. 244, 1521-1526.

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Christensen, H. N., and Cullen, A. M. (1973). Synthesis of metabolism-resistant substrates for the transport system for cationic amino acids; their stimulation of the release of insulin and glucagon, and of the urinary loss of amino acids related to cystinuria. Biochim. Biophys. Acta 298, 932-950. Christensen, H. N., and Handlogten, M. E. (1968). Modes of mediated exodus of amino acids from the Ehrlich ascites tumor cell. J . Biol. Chem. 243, 5428-5438. Christensen, H. N., and Handlogten, M. E. (1969). Reactions of neutral amino acids plus Na+ with a cationic amino acid transport system. FEBS, Fed. Eur. Biochem. SOC.),Lett. 3, 14-17. Christensen, H. N., and Handlogten, M. E. (1974). A deprotonation-reprotonation cycle energizing transport? Proc. Nut. Acad. Sci. U.S., in press. Christensen, H. N., and Liang, M. (1965). An amino acid transport system of unassigned function in the Ehrlich ascites tumor cell. J. Biol. Chem. 240, 3601-3608. Christensen, H. N., and Liang, M. (1966). Transport of diamino acids into the Ehrlich cell. J. Biol. Chem. 241, 5542-5551. Christensen, H. N., Oxender, D. L., Liang, M., and Vatz, K. A. (1965). The use of N-methylation to direct the route of mediated transport of amino acids. J . Biol. Chem. 240,3609-3616. Christensen, H. N., Handlogten, M. E., and Thomas, E. L. (1969a). Nai-facilitated reactions of neutral amino acids with a cationic amino acid transport system. Proc. Nut. Acad. Sci. U.S. 63, 948-953. Christensen, H. N., Handlogten, M. E., Lam, I., Tager, H. S., and Zand, R. (1969b). A bicyclic amino acid to improve discriminations among transport systems. J. Biol. Chem. 244, 1510-1520. Christensen, H. N., Thomas, E. L., and Handlogten, M. E. (1969~).Features of amino structure enhancing or obstructing cosubstrate reactivity of Na+ in transport. Biochim. Biophys. Acta 193, 228-230. Christensen, H. N., Hellman, B., Lernmark, Sehlin, J., Tager, H. S., and Taljedahl, I. B. (1971). I n vitro stimulation of insulin release by non-metabolizable, transportspecific amino acids. Biochim. Biophys. Acta 241, 341-348. Christensen, H. N., decespedes, C., Handlogten, M. E., and Ronquist, G. (1973). Energization of amino acid transport studied for the Ehrlich ascites t,umor cell. Biochim. Biophys. Acta, Rev. Biomembranes 300, 487-522. Eavenson, E., and Christensen, H. N. (1967). Transport systems for neutral amino acids in the pigeon erythrocyte. J. Biol. Chem. 242, 5386-5396. Epel, D. (1972). Activation of an Na+-dependent amino acid transport system upon fertilization of sea urchin eggs. Exp. Cell Res. 72, 74-89. Fajans, S. S., Power, L., Gwinup, G. W., Knopf, R. F., and Conn, J. W. (1960). Studies on the mechanism of leucine hypoglycemia. J . Lab. CZin. Med. 56, 810-811. Fajans, S. S., Floyd, J. C., Jr., Knopf, R. F., and Conn, J. W. (1967). Effects of amino acids and proteins on insulin secretion in man. Recent Progr. Horm. Res. 23,617-662. Fajans, S . S., Quibrera, R., Pek, S., Floyd, J. C., Jr., Christensen, H. N., and Conn, J. W. (1971). Stimulation of insulin release in the dog by a non-metabolizable amino acid. Comparison with leucine and arginine. J . Clin. Endocrinol. Metab. 33, 3 5 4 1 . Fajans, S. S., Christensen, H. N., Floyd, J. C., Jr., and Pek, S. (1974). Stimulation of insulin and glucagon release in the dog by non-metabolizable arginine analogs. Endocrinology 94, 230-233. Franchi-Gazzola, R., Gazzola, G. C., Ronchi, P., Saibeni, V., and Guidotti, G. C. (1973). Regulation of amino acid transport in chick embryo heart cells. Biochim. Biophys. Acla 291, 545-556.

w.,

RECOGNITION SITES FOR MATERIAL TRANSPORT AND INFORMATION TRANSFER

257

Freinkel, N., El Younsi, C., Christensen, H. N., and Dawson, R. M. C. (1974). Effects of non-metabolizable amino acids on “phosphate flush” from isolated pancreatic islets. Program of the 56th Annual Meeting of the Endocrine Society, Atlanta, Ga., p. a119. Gamble, J. G., and Lehninger, A. L. (1973). Transport of ornithine and citrulline across the mitochondria1 membrane. J . Biol. Chem. 248, 610-618. Glover, G., Stenmark, S., and Jensen, R. (1972). Characterization of tyrosine transport in Bacillus subtilis. Fed. Proc., Fed. Amer. SOC.Exp. Biol. 31, 860 (abstr). Harrison, L. I., and Christensen, H. N. (1971). Evidence for a hepatic transport system not responsive to glucagon or theophylline. Biochem. Biophys. Res. Commun. 43, 119-125. Hazelbauer, G. L., Adler, J. (1971). Role of the galactose binding protein in chemotaxis of Eschericia coli toward galactose. Nature (London), New Biol. 230, 101-104. Hellman, B., Sehlin, J., and Tiiljedal, I. B. (1973). Transport of tleucine and D-leucine into pancreatic 8-cells with reference to the mechanisms of amino acid-induced insulin release. Bwchim. Bwphys. Acta 266, 436443. Hiji, Y.,and Sato, M. (1973). Isolation of the sugar-binding protein from r a t taste buds. Nature (London),New Biol. 244, 91-93. Koser, B., and Christensen, H. N. (1971). Effect of substrate structure on coupling ratio for Na+-dependent transport of amino acids. Biochim. Biophys. Acta 241, 9-19. Lambert, A. E., Kanazawa, Y., Orci, L., Burr, I. M., Christensen, H. N., and Renold, A. E. (1971). Stimulation of insulin release in oitro by non-metabolized amino acid analogues. PTOC. SOC.Exp. Biol. Med. 137, 377-381. McClellan, W. M., and Schafer, J. A. (1973). Transport of the amino acid analog, 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid, by Ehrlich ascites tumor cells. Biochim. Biophys. Acta 311, 462-475. Matschinsky, F. M., Ellerman, J., Pace, C. S., and Stillings, S. (1974). Interactions between glucose and amino acids in rat pancreatic islets. Personal communication. Oxender, D. L., and Christensen, H. N. (1963). Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem. 238, 3686-3699. Panten, U., and Christians, J. (1973). Effect of 2-endo-aminonorbornane-2-carboxylic acid upon insulin secretion and fluorescence of reduced pyridine nucleotides of isolated perifused pancreatic islets. Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol. 276, 55-62. Quay, S. C., and Christensen, H. N. (1974). Molecular basis of transport discrimination between arginine and lysine or ornithine in Salmonella typhimurium. J. Bwl. Chem., in press. Robbins, J. C., and Oxender, D. L. (1973). Transport systems for alanine, serine and glycine in Escherichia coli K-12. J . Bacteriol. 116, 12-18. Rosen, B. P. (1973). Basic amino acid transport in Escherichia coli. 11. Purification and properties of an arginine-specific binding protein. J . Biol. Chem. 248, 1211-1218. Sander, G.,and Pardee, A. B. (1972). Transport changes in synchronously growing CHO and L cells. J. Cell. Physiol. 80, 267-271. Sterling, C. E. (1967). High-resolution radioautography of phloriah3H in rings of hamster intestine. J. Cell Biol. 35, 605418. Tager, H. S., and Christensen, H. N. (1971a). Transport of the four isomers of %aminonorbornane-2-carboxylic acid in selected mammalian systems and in Escherichia coli. J. Biol. Chem. %6, 7572-7580. Tager, H. S., and Christensen, H. N. (1971b). Hypoglycemic action of 2-aminonorbornane-2-carboxylic acid in the rat. Bwchem. Biophys. Res. Commun. 44, 185-191.

258

HALVOR N. CHRISTENSEN

Tager, H. S., and Christensen, H. N. (1972). 2-Aminonorbornane-2-carboxylic acid. Preparation, properties, and identification of the form isomers. J. Amer. Chem. Soc. 94, 968-972. Thomas, E. L., and Christensen, H. N. (1970). Indications of spatial relations among structures recognizing amino acids and Na+ a t a transport receptor site. Biochem. Biophys. Res. Commun. 40,277-283. Thomas, E. L., and Christensen, H. N. (1971). Nature of the cosubstrate action of Na+ and neutral amino acid in a transport system. J . Biol. Chem. 246, 1682-1688. Thomas, E. L., Shao, T.-C., and Christensen, H. N. (1971). Structural selectivity in interaction of neutral amino acids and alkali metal ions with a cationic amino acid transport system. J. Biol. Chem. 246, 1677-1681. Vidaver, G. A. (1964). Transport of glycine by pigeon red blood cells. Biochemistry 3,662-667. Wheeler, K. P., and Christensen, H. N. (1967). Interdependent fluxes of amino acids and sodium ion in the pigeon red blood cell. J. Biol. Chem. 242,3782-3788. Winter, C. G., and Christ,ensen, H. N. (1965). Contrasts in neutral amino acid transport by rabbit erythocytes and reticulocytes. J. Biol. Chem. 240, 3594-3600.

A Abetalipoproteinemia, cholesterol in, 3 Aging, cholesterol in, 3 Amino acids pancreatic hormone release by, 243-250 transport systems for, 227-229 Anoxia, muscular glucose transport and, 186-187 ASC system, in amino acid transport, 235-237 Atherosclerosis, cholesterol in, 3 ATP in calcium transport systems, 155-157 in muscular glucose transport), 198-199 in red cell Ca transport, 135-137 ATPase, calcium-activated in red blood cells, 126-168 comparison to other types, 154-155 magnesium in, 144-157 stoichiometry, 150 systems compared to, 155-157 thermodynamic aspects, 150-151 types, 142-149

B Bilayer, electrical properties related to cholesterol content, 22-23, 3 9 4 1

C Calcium in muscular glucose transport,, 202-207 in red blood cells, 126-127 Calcium pumps, 157-161 in epithelia, 161 in muscle, 157-158 in red cells, 158-161 in secretory cells, 161

Calcium transport, in red blood cells, 126-168 CAMP, in muscular glucose transport, 199-201 Cancer, cholesterol in, 3 Cationic amino acids transport systems for, 232-235 system-specific substrates, 241-243 Cellular ionic activity, 59-123 Cerebrosidosis, cholesterol in, 3 Chlorpromazine, a9. calcium t.ransport inhibitor, 141 Cholesterol in bilayers, 39-41 in biological systems, 5-13 in biomembranes, 1-57 barrier-properties, 16-18 composition, 7-8 electrical properties, 22-23 permeability, 17-22 role, 1-57 in disease syndromes, 3-4 in lipid aggregates, 23-39 in bilayers, 27-38 in biomembranes, 38-39 in monolayers, 23-27 solubilieation and dispersal of, 13-16 Contractile act.ivity of muscle, glucose transport and, 185-186 Cytochalasin B, effect on glucose transport, 177

E Electrolytes, effect on muscular glucose transport, 188-190 Enzymes, in muscular glucose transport, 197-201 259

2 60

SUBJECT INDEX

Epithelia, calcium pumps in, 161 Erythrocytes, see Red blood cells Ethacrynic acid, as calcium transport, inhibitor, 141

F Fabry’s disease, cholesterol in, 3

G Gaucher’s disease, cholesterol in, 3 Glass membrane microelectrodes, for cellular ionic studies, 72-79 Glucose-6-phosphate, in muscular glucose transport, 197-198 Glucose transport system in muscle cellular signals controlling, 196-208 electrolyte effects, 201-208 function, 178-196 inhibitors of, 193-196 insulin effects on, 169-226 mechanisms, 209-211 Glycolipid lipidosis, cholesterol in, 3

H Heart, glucose transport in, insulin effects on, 171-172 Hexokinase, in muscular glucose transport, 197-198 Ilyperlipoproteinemias, cholesterol in, 3 Hyperosmolality, effect on muscular glucose transport, 190-193

liquid ion exchanger microelectrodes for studies of, 79-81 measurement of, 84-89 of single ion, 87-88 of two ions, 88-89 metal microelectrodes for study of, 8183 microelectrodes for measurements of, 72-84 calibration, 85-87 design, 76-84 in plant cells, 111-113 polyelectrolyte model systems for, 66-72 single ion activities, 63-66

J Jaundice, cholesterol in, 3

1 Lanthanides, as calcium transport inhibitor, 141 Lanthanum, effect on glucose transport, 176 Lecithin, cholesterol solubilixation by, 14 Lipid aggregates, cholesterol in, 23-39 Lipids, in membranes, role in glucose transport, 174-176 Lipoproteins, composition of, 2 Liver, diseases of, cholesterol in, 4 Liver cells, calcium transport in, 155

M

I Information transfer, recognition sites for, 227-258 Insulin compounds related to, effects on glucose transport, 187-188 effect on muscular glucose transport, 169-226 Ion exchangers (liquid) microelectrodes, for cellular ionic activity studies, 79-81 Ionic cellular activity, 59-123 cellular function and, 94-111 intracellular, 90-113 pH activity, 90-94

Magnesium in membrane ATPase, 144 in muscular glucose transport, 207 Material transfer, recognition sites for, 227-258 Membrane potential, in muscular glucose transport,, 202 Membranes cholesterol in, 1-57 lipids in, role in glucose transport, 174176 proteins in, role in glucose transport, 176-1 78 Mersalyl, as calcium transport inhibitor, 141

26 1

SUBJECT INDEX

Metabolic poisons, effect on muscular glucose transport, 187-188 Metal ions, in red cell Ca transport, 137138 Methyl-L-homoarginine, effects on pancreatic insulin release, 252 Microelectrodes, for cellular ionic activity studies, 72-84 Mitochondria, calcium transport in, 156 Muscle calcium pumps in, 157-158 calcium transport in, 155 glucose transport in, insulin effects on, 169-226

N Nephrotic syndrome, cholesterol in, 3 Neutral systems, in amino acid transport, 229-231 system-specific, nonmetabolizable substrates, 237-241 Niemann-Pick disease, cholesterol in, 3 Nucleoside triphosphates, in red cell Ca transport, 138

0 Open-tip microelectrodes, for ionic cellular activity studies, 84

P

A

c

6 D 7 G H 1 1

O 1 2 3

Pancreatic hormones, insulin release by, 243-250 Phosphatase, activity of, in calciummagnesium ATPase, 149 Phosphate, in muscular glucose transport, 207-208 Plant cells, ionic activities in, 111-113 Potassium, in muscular glucose transport, 201-202 Potassium precipitate microelectrodes for ionic cellular activity studies, 83-84 Proinsulin, effect on glucose transport, 187-1 88 Psychological stress, cholesterol in, 3

R Recognition sites, for material transport and information transfer, 227-258 Red blood cells calcium-binding to membrane in, 127 magnesium role, 144-157 calcium concentration in, 126-127 calcium pumps in, 158-161 alkali cation permeability, 158-159 mechanical behavior, 159-161 calcium trsnsport in, 126-168 active, 129-142 inhibition, 141-142 passive permeability of membrane for calcium, 127-129 Rutrhenium red, as calcium transport inhibitor, 141-142

5 Sarcoplasmic reticulum, calcium transport in, 153 Secretory cells, calcium pumps in, 161 Skeletal muscle, glucose transport in, insulin effects on, 170-171 Smooth muscle, glucose transport in, insulin effects on, 172 Sodium, in muscular glucose transport, 201 Sodium-calcium heteroexchange, in cells, 157 Sphingomyelin lipidosis, cholesterol in, 3 Sugar transport, insulin effects on, in muscle, 169-226

T Tangier disease, cholesterol in, 3 Tetracaine as calcium transport inhibitor, 141 effect on glucose transport, 176 Thiopental, effect on glucose transport, 176

V Veratrine, effect on glucose transport, 176

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