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 (Ry