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Distribution and fluxes of sodium and hydrogen in crustacean muscle cells 1980

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DISTRIBUTION AND FLUXES OF SODIUM AND HYDROGEN IN CRUSTACEAN MUSCLE' CELLS by MICHAEL REALD MENARD B.Sc, University of B r i t i s h Columbia, 1971 M.Sc, U n i v e r s i t y of Toronto, 1972 M.D., Un i v e r s i t y of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Anatomy We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1980 ©Michael Reald Menard, 1980 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of Anatomy The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date A p r i l 22, 1980 ABSTRACT A new technique was devised for measurement of the u n i d i r e c t i o n a l sodium e f f l u x from sin g l e s t r i a t e d muscle c e l l s of the giant barnacle, Balanus nubilus. It involves the continuous measurement of the a c t i v i t y of sodium i n the myoplasm with an i n t r a c e l l u l a r sodium-specific microelectrode, during the c o l l e c t i o n of radiosodium from the same c e l l by a standard method. Changes i n the s p e c i f i c a c t i v i t y inside the c e l l , which are larger than had been .. thought previously, can be calculated d i r e c t l y . Thus the sodium e f f l u x can be calculated accurately. It i s assumed i n these c a l c u l a t i o n s that the only pool of i n t r a c e l l u l a r sodium of appreciable s i z e which exchanges r a p i d l y with the e x t r a c e l l u l a r s o l u t i o n i s the sodium i n s o l u t i o n i n the myoplasm. Several experiments which test this assumption, together with r e s u l t s from the l i t e r a t u r e , are consistent with the hypothesis that most of the sodium associated with the c e l l yet not detected by the i n t r a c e l l u l a r sodium-specific microelectrode resides i n the e x t r a c e l l u l a r space i n a s s o c i a t i o n with the glycocalyx. I n t r a c e l l u l a r microinjection was used to load the myoplasm of s i n g l e c e l l s with radiosodium. I t was necessary to take into account the longitud- i n a l d i f f u s i o n of tracer inside the c e l l from i n j e c t e d to noninjected regions. Use of the new technique to measure the sodium e f f l u x from i n t a c t s i n g l e muscle c e l l s revealed several new r e s u l t s . Saturation of the e f f l u x into normal Ringer's s o l u t i o n was not apparent even i n c e l l s with very high sodium content. However, saturation of the e f f l u x into potassium-free s o l u t i o n and into ouabain-containing s o l u t i o n occurred at r e l a t i v e l y low l e v e l s of i n t r a - c e l l u l a r sodium. The e f f l u x into sodium-free s o l u t i o n was s i m i l a r to that into normal Ringer's s o l u t i o n . The decline i n the sodium e f f l u x reported by other workers to occur i n this s i t u a t i o n was found to be due to the rapid decline of the sodium content of the myoplasm which occurs. No 'sodium-sddium i i i exchange' was found. Most of the sodium e f f l u x under normal conditions appears to be due to a mechanism which i s not s e n s i t i v e to external ouabain or potassium. The sodium e f f l u x i n barnacle muscle was shown to be electrogenic. A c o r r e l a t i o n between the measured values of the active sodium e f f l u x and the electrogenic portion of the membrane p o t e n t i a l was found. The c o r r e l a t i o n was consistent with the predictions of a phenomenological extension of the leading model for the membrane p o t e n t i a l , the Goldman-Hodgkin-Katz equation. The e f f l u x of hydrogen ions from the c e l l can only be measured i n d i r e c t l y , from changes i n the i n t r a c e l l u l a r pH. Measurements of the i n t r a c e l l u l a r pH with an i n t r a c e l l u l a r pH-specific glass microelectrode revealed no 'pH transients' of the type reported by other workers i n d i f f e r e n t preparations of barnacle muscle. Measurements of the i n t r a c e l l u l a r pH made with the micro- electrode and with an i n d i c a t o r method were i n close agreement. However, the d i s t r i b u t i o n of the i n d i c a t o r DMO (5,5-dimethyl-2,4-oxazolidinedione) exhibited unusual behavior not previously reported. A refinement of the DMO method which takes this behavior into account i s described. i v TABLE OF CONTENTS Abstract L i s t of Tables L i s t of Figures Acknowledgements Section 1. General Introduction: A. Scope of the thesis B. H i s t o r i c a l notes , 11 . . v i . v n ..x ...4 Section 2. Transmembrane Fluxes of Sodium and Hydrogen Ions: A. General considerations B. States of water and ions i n c e l l s C. The sodium e f f l u x ( i ) normal Ringer's s o l u t i o n ( i i ) potassium-free s o l u t i o n ( i i i ) sodium-free s o l u t i o n (iv) ouabain-containing s o l u t i o n D. Membrane p o t e n t i a l E. Summary of problems to be addressed F. Summary of models (i ) sodium e f f l u x from a whole c e l l ( i i ) steady state d i s t r i b u t i o n of cations ( i i i ) electrogenic membrane p o t e n t i a l The States of Sodium i n C e l l s ; Introduction General methods A. Increase of c e l l sodium B. Decrease of c e l l sodium Section 4. M i c r o i n j e c t i o n of Radiosodium into Single Muscle Cell s Methods Results Discussion Section 3. Section 5. Section 6. Section 7, Survey of the Sodium E f f l u x from Single Muscle C e l l s Methods Results: ( i ) normal Ringer's s o l u t i o n ( i i ) potassium-free s o l u t i o n ( i i i ) sodium-free s o l u t i o n (iv) ouabain-containing s o l u t i o n Discussion Comparison of Sodium Electrode and Radiosodium Measurements Methods Results Discussion Electrogenic Sodium Transport Methods Results Discussion ,10 .19 .31 .33 .34 .36 .44 .48 .56 .59 .69 .71 ...75 ...77 .. .85 ...95 ..100 ..107 ..117 ..133 . .137 . .138 ..141 ..145 ..148 ..153 ..157 ..172 ..173 ..174 ..184 ..187 ..189 ..189 ..196 V Section 8. D i s t r i b u t i o n of Hydrogen Ions During Steady Conditions ...201 Methods ...2 02 Results ...204 Discussion ...212 Section 9. Comparison of the I n t r a c e l l u l a r pH Measured by DMO and by microelectrodes ...217 Methods ...218 Results ...223 Discussion ...230 Section 10. Significance of the Results and Suggestions for Further Work ...237 Bibliography ...242 LIST OF TABLES Table I Composition of solutions. Table II a. Summary of measurements on passively- loaded c e l l s b. Ion content of the myoplasmic and nonmyoplasmic compartments. Table I I I I n t r a c e l l u l a r pH and membrane p o t e n t i a l i n crustacean muscle. Table IV Mean water and e l e c t r o l y t e content of test c e l l s , Table V Cal c u l a t i o n of flux j m from the data of F i g . 29. Table VI S e n s i t i v i t y of pH(DMO) to errors i n measurement. LIST OF FIGURES Figure 1. Models of the c e l l used i n c a l c u l a t i o n of ion fluxes. Figure 2. Insertion of microelectrodes into c e l l . Figure 3. Changes i n the sodium content of c e l l s during immersion i n sodium-free lithium-substituted Ringer's s o l u t i o n . Figure 4. M i c r o i n j e c t i o n apparatus. Figure 5. Perfusion apparatus. Figure 6. Vacuum system. Figure 7. E f f l u x of microinjected radiosodium from a c e l l into normal Ringer's s o l u t i o n . Figure 8. 'Slope Ratio' for a c e l l versus myoplasmic sodium a c t i v i t y . Figure 9. E f f l u x of passively-loaded radiosodium from a c e l l into normal Ringer's s o l u t i o n . Figure 10. Summary of the raw data and reduced r e s u l t s for a t y p i c a l experiment. Figure 11. E f f l u x of sodium from a c e l l into normal Ringer's solut i o n , uncorr sodium a c t i v i t y . ected for Na£^.^, versus myoplasmic Figure 12. E f f l u x of sodium from a c e l l into normal Ringer's solut i o n , corrected for Na* versus myoplasmic \- cell J r sodium a c t i v i t y . Figure 13. E f f l u x of sodium from a c e l l into potassium-free solut i o n , uncorrected for ®acen> versus myoplasmic sodium a c t i v i t y . Figure 14. E f f l u x of sodium from a c e l l into potassium-free solut i o n , correc sodium a c t i v i t y . ted for Na" 1 l 5 versus myoplasmic c e l l J Figure 15. The e f f e c t of sodium-free solutions on M^ a < Figure 16. E f f l u x of sodium from a c e l l into sodium-free solut i o n , uncorr sodium a c t i v i t y . ected for ̂ a' c e^> versus myoplasmic Figure 17. E f f l u x of sodium from a c e l l into sodium-free so l u t i o n , corrected for Na* ,., versus myoplasmic j . • • c e l l sodium a c t i v i t y . V l l l LIST OF FIGURES (cont'd) Figure 18. E f f l u x of sodium from a c e l l into normal Ringer's s o l u t i o n which contains ouabain, versus myoplasmic sodium a c t i v i t y . ...155 Figure 19. Summary of results for e f f l u x experiments. ...162 Figure 20. F a l l of the myoplasmic sodium a c t i v i t y upon exposure of the c e l l to sodium-free l i t h i u m - substituted s o l u t i o n . ...175 Figure 21. Size of the rapid f a l l i n the myoplasmic sodium a c t i v i t y upon exposure of the c e l l to sodium- free lithium-substituted s o l u t i o n . ...177 Figure 22. Rate of f a l l of the myoplasmic sodium a c t i v i t y immediately a f t e r exposure of the c e l l to sodium- free s o l u t i o n , versus the myoplasmic sodium a c t i v i t y . ...178 Figure 23. Ratio of the sodium e f f l u x to the rate of loss of sodium from the myoplasm, for e f f l u x into sodium- free s o l u t i o n . ...182 Figure 24. Change i n membrane po t e n t i a l on exposure of the c e l l to potassium-free or ouabain-containing s o l u t i o n . ...190 Figure 25. Relationship between the change i n membrane po t e n t i a l and the change i n sodium e f f l u x which occur i n response to ouabain. ...193 Figure 26. Resting membrane p o t e n t i a l of c e l l s loaded with sodium by microinjection, versus myoplasmic sodium a c t i v i t y . ...195 Figure 27. Response of membrane p o t e n t i a l and pH^ to C02 _ Ringer's s o l u t i o n . ...207 Figure 28. Response of membrane p o t e n t i a l and pH. to NH^- Ringer's s o l u t i o n . ...208 Figure 29. Relationship between transmembrane gradient of pH and the r e s t i n g membrane p o t e n t i a l . ...211 Figure 30. Model of the transmembrane d i s t r i b u t i o n of DMO. ...220 Figure 31. Uptake of indi c a t o r compounds i n normal Ringer's s o l u t i o n . ...224 Figure 32. Uptake of i n d i c a t o r compounds i n CO^ Ringer's s o l u t i o n . .. .226 Figure 33. Uptake of indi c a t o r compounds i n NH^ Ringer's s o l u t i o n . . . .227 Figure 34. C o r r e l a t i o n between pH(DMO) and pH(electrode). ...229 LIST OF SYMBOLS 2 A area of membrane (cm ) Page f i r s t us ( a ^ a ) m myoplasmic sodium a c t i v i t y (mM) c(x) concentration of cation at distance x from o r i g i n cpm counts per minute of r a d i o a c t i v i t y dpm disinte g r a t i o n s per minute of r a d i o a c t i v i t y E membrane p o t e n t i a l ( m i l l i v o l t s ) m F charge of a mole of electrons (96,520 coulomb/mole) conductivity to potassium ions (coulomb2/joule-cm^-sec) I current of potassium ions (coulomb/cm^-sec) K. j( x ) ion flux at distance x from o r i g i n ; j^=passive, j m = a c t i v e k rate constant (units depend on context, usually min"-'-) M„ u n i d i r e c t i o n a l sodium e f f l u x (mole/cm2-sec) Na 2 M net cation e f f l u x i n electrogenic transport (mole/cm -sec] m„ ,m„,m • net passive flux across membrane (mole/cm^-sec) Na K Cl mc m i l l i c u r i e of r a d i o a c t i v i t y mV m i l l i v o l t "k 22 Na moles Na c o l l e c t e d i n 5 min i n an e f f l u x experiment * 22 Na „ moles Na inside the c e l l c e l l * 22 Na .moles Na i n s o l u t i o n i n the myoplasm m 2 3 Na ,, moles Na inside the c e l l c e l l (Na) , = Ba , ., /V : apparent concentration of Na i n c e l l ' c e l l c e l l r o.d. outside diameter (mm or |j) pes picomoles/cm^-sec P^ permeability of membrane to species x (cm/sec) (R. coupling r a t i o of ouabain-sensitive Na-K exchange R gas constant per mole (8.3 x lO'' erg/mole-°K) SA s p e c i f i c a c t i v i t y of sodium Slope Ratio T absolute temperature (°K) t time u mobility (erg-cm/mole-sec) U numerator of l n term of Goldman-Hodgkin-Katz equation V volume of region indicated by subscript W denominator of l n term of Goldman-Hodgkin-Katz equation z valence of ion 6 p a r t i t i o n c o e f f i c i e n t between s o l u t i o n and membrane y + mean i o n i c a c t i v i t y c o e f f i c i e n t X m i c r o l i t r e "j electrochemical p o t e n t i a l (erg/mole) u micrometre p i m i c r o l i t r e $ e l e c t r i c a l p o t e n t i a l (joule/coulomb) ( ) concentration (mole/litre) subscripts: i = i n t r a c e l l u l a r o,e = e x t r a c e l l u l a r m = myoplasmic X ACKNOWLEDGEMENTS The work described i n this thesis was c a r r i e d out i n 1973-1976 as part of a combined M.D.-Ph.D. program. I wish to thank my thesis supervisor Dr. J.A.M. Hinke for the advice and encouragement he offered throughout the course of this program. I also wish to thank Dr. S.M. Friedman and Dr. V. Palaty for t h e i r assistance, and for the support they provided a f t e r Dr. Hinke moved to the U n i v e r s i t y of Ottawa,, S k i l l e d technical assistance with the DMO experiments and some of the flame photometry was provided by Ms. Edwina Nee Wong and Mr. Laurie N i c o l . 1 SECTION 1. GENERAL INTRODUCTION A. SCOPE OF THE THESIS A p r i n c i p a l function of the c e l l membrane i s the tra n s l o c a t i o n of ions and molecules. I t has been estimated that between o n e - f i f t h and one-third of the r e s t i n g energy production of the c e l l is devoted to the mechanism which transports sodium and potassium ions alone (Brinley & Mullins 1968; Whittam 1975; but see Chinet, Clausen, & G i r a r d i e r 1977). The transmembrane d i s t r i b u t i o n of ions and molecules is far d i f f e r e n t from that which would occur i f they a l l were in equilibrium. In p a r t i c u l a r , the e x t r a c e l l u l a r medium i s poor i n soluble organic molecules r e l a t i v e to the i n t r a c e l l u l a r medium. Altogether, there i s always present a force which tends to move water and e l e c t r o l y t e s into the c e l l . For c e l l s which lack r i g i d walls, the amount of osmotically a c t i v e i n t r a c e l l u l a r solute must be regulated i f osmotic l y s i s is to be prevented and the c e l l is to be enabled to ex i s t . The a c t i v e extrusion of sodium i s f e l t to be the major control of the water content of the c e l l (eg. Tosteson 1964; MacKnight & Leaf 1977). The transmembrane d i s t r i b u t i o n of sodium i s also a store of energy, sui t a b l e for u t i l i z a t i o n by energy-requiring reactions and processes at the c e l l membrane. Important examples are those processes which e f f e c t trans- port of substances across the c e l l membrane, and those which r a p i d l y transmit signals to another part of the c e l l or to a d i f f e r e n t c e l l i n order to t r i g g e r chemical reactions. Of course, whatever the other functions i t serves, the regulated i o n i c composition of the i n t e r i o r of the c e l l appears to be required for the e f f e c t i v e functioning of the metabolic machinery of the c e l l . This thesis i s concerned with the experimental measurement of ion 2 transport, and with c e r t a i n aspects of the transport of sodium and hydrogen ions i n whole c e l l s of s t r i a t e d muscle. Interpretation of transport data is much more straightforward with i s o l a t e d membrane preparations than with whole c e l l s . However, with current techniques, i t is only from a few c e l l s that i n t a c t membranes can be i s o l a t e d for study: red blood c e l l s , and giant axons such as that of the squid. There are many s i m i l a r i t i e s between the transport properties of these two c e l l types, but there are also many differences. Of necessity, then, whole c e l l s must be examined so the nature and importance of the various transport processes can be discovered. In addition, there are p o s i t i v e reasons for examining membrane trans- port properties i n whole c e l l s . The c e l l s of a s p e c i a l i z e d t i s s u e w i l l possess a r e p e r t o i r e of transport mechanisms s u i t a b l e for the tissue's function. A p a r t i c u l a r transport mechanism, present i n most c e l l types, might be prominent i n a p a r t i c u l a r c e l l type and so be more e a s i l y studied there. The chemical species which mediates a given aspect of ion transport can only be i d e n t i f i e d i n i s o l a t i o n i f i t s behavior is known. This behavior must be deduced from study of the behavior of the whole c e l l . Once the d e t a i l e d properties of an i d e n t i f i e d species are known, the part that species plays i n the complete transport system of the c e l l can be deduced. Then the behavior due to other transport species can be deduced and an attempt made to i s o l a t e them. Eventually i t is hoped that a l l species which contribute to ion transport w i l l be characterized, and t h e i r behavior when acting i n concert understood. F i n a l l y , abnormalities of the transport systems can be a cause of or a feature of pathology of the t i s s u e ( B o l i s , Hoffman, &•Leaf 1976). As with other areas of physiology, a d e t a i l e d knowledge of c e l l u l a r transport mechanisms can lead to the formulation of a r a t i o n a l treatment plan. An 3 example i s the use in the treatment of cholera of one transport mechanism to bypass another which is deranged ( F i e l d 1977). There are many reasons, then, why the c a p a b i l i t y to study transport i n whole c e l l s and m u l t i c e l l u l a r preparations should be developed. Several problems are addressed i n th i s thesis. The f i r s t i s the tech- n i c a l problem of measuring the trans-membrane f l u x i n whole c e l l s . The main concern i s the measurement of sodium fluxes. The measurement of hydrogen ion fluxes presents d i f f e r e n t problems, of both a conceptual and a p r a c t i c a l nature. I t is of secondary concern here. In order to resolve the technical problem of measuring the flux, an in v e s t i g a t i o n of the states of water and ions inside the c e l l had to be ca r r i e d out. This is an act i v e area of research i n i t s own r i g h t . The technique developed for fl u x measurement, which involves simulta- neous use of an i o n - s p e c i f i c i n t r a c e l l u l a r microelectrode and radioisotopes, was applied to a b r i e f overview of the k i n e t i c s of sodium transport i n a whole c e l l . Then, two s p e c i f i c problems were investigated: the sodium e f f l u x into sodium-free solutions, of the type previously seen i n frog s k e l e t a l muscle; and the electrogenic properties of the sodium transport. F i n a l l y , i n d i r e c t measurements of the extrusion of acid by the c e l l and of the heterogeneity of the i n t r a c e l l u l a r pH were made. In this context, the use of an indicato r for the measurement of pH was evaluated. The l a t t e r is a question of great p r a c t i c a l i n t e r e s t . The c e l l chosen for th i s work i s the very large s t r i a t e d muscle c e l l of the giant barnacle Balanus nubilus. This crustacean was described by Darwin i n 1854, but i t was only i n 1963 that Hoyle and Smyth described i t s neuromuscular physiology and suggested that i t would be a valuable prepara- t i o n for further such study. Since then,, work has been published on i t s 4 u l t r a s t r u c t u r e , on the states of i t s water and ions, on the permeability and e l e c t r i c a l properties of i t s membranes, and on i t s ion transport mechanisms. Its large s i z e makes i t e s p e c i a l l y suitable for impalement by micro- electrodes (which i t tolerates for long periods) and for microinjection. It was desired to use these techniques to sample the c e l l i n t e r i o r s elec- t i v e l y and to load the c e l l i n t e r i o r with radioisotope s e l e c t i v e l y , as w i l l be explained more f u l l y l a t e r . B. HISTORICAL NOTES The study of the movement of substances into and out of c e l l s i s almost as old as the c e l l theory i t s e l f . The c e l l membrane cannot be seen with the l i g h t microscope, but permeation of solutes and osmotic e f f e c t s are r e a d i l y demonstrated. Nageli, a student of Schleiden's, inferred the presence of a c e l l membrane from his studies of plant c e l l permeability (Nageli & Cramer 1855). P f e f f e r (1877) proposed from h i s work with a r t i f i c i a l semipermeable films that a f i l m with s i m i l a r properties surrounded the c e l l . Overton (1899) measured the permeability of c e l l s to many substances, and proposed that a layer of l i p i d on the surface of the c e l l was the p r i n c i p a l b a r r i e r to penetration. It was the plant physiologists who led the way i n these studies on s i n g l e c e l l s . They found that plant c e l l s a c t u a l l y accumulated c e r t a i n substances, and seemed to e x i s t in a 'non-equilibrium condition' i n this respect (Hoagland & Davis 1929; Brooks 1929). Osterhout (1931) rejected the Donnan e f f e c t as the cause of the accumulation of ions, and proposed 5 that the continuous production of a c i d by the c e l l led to the passive inflow of potassium and chloride. Brooks (1938) appears to have been the f i r s t to employ radioisotopes i n 1 the study of ion accumulation by i n d i v i d u a l c e l l s . He employed a r a d i o i s o - tope of potassium to quantitate the accumulation of potassium by a c e l l , and expressed the i n t r a c e l l u l a r concentration i n terms of the t o t a l c e l l water (the d i f f e r e n c e between the wet and dry weights of the 'protoplasm'). He observed a rapid penetration of potassium against the gradient of potassium concentration. He a t t r i b u t e d this to ion exchange (Brooks 1940). Steinbach (1940) noted that the potassium accumulation theories then current required low permeability to sodium, while experiments had shown that sodium pene- trates the c e l l quite r e a d i l y . -He remarked that "there must be some mechanism present for pumping out the sodium which wanders into the proto- plasm." He f e l t that the ion d i s t r i b u t i o n as a whole was due to a "physico-chemical balance between the protoplasm and the medium, with the permeability c h a r a c t e r i s t i c s of the membrane playing only a subordinate s t r u c t u r a l r o l e . " The e f f l u x from c e l l s of a radioisotope of sodium was measured by L e v i and Ussing (1949) and l a t e r by others. Ussing (1949) derived a r e l a t i o n which should be obeyed by passive fluxes. Hodgkin and Keynes (1954) found that according to Ussing's r e l a t i o n , sodium was a c t i v e l y expelled from nerve c e l l s . """The f i r s t use of radioisotope i n uptake studies was much e a r l i e r . Hevesy (1923) measured the uptake by plants of an isotope of lead obtained as a natural breakdown product,of thorium. The use of radioisotopes became more common a f t e r the development of the cyclotron and rad i o a c t i v a - t i o n i n about 1936. It was E.O. Lawrence of the Univ e r s i t y of C a l i f o r n i a at Berkeley and Niels Bohr of the I n s t i t u t e of Theoretical Physics i n Copenhagen who supplied l o c a l physiologists with radioisotopes of phosphorous and potassium. 6 Keynes and Lewis (1951) e x p l i c i t l y formulated the 'bag model' of the animal c e l l for flux studies, wherein the i n t r a c e l l u l a r region was assumed to comprise a sin g l e homogeneous compartment within a closed s e l e c t i v e l y - permeable membrane. The re s u l t s of th e i r experiments on squid axon seemed to be consistent with t h i s formulation, but the re s u l t s for muscle c e l l s were more d i f f i c u l t to interpret. The trend has been to employ more compli- cated models having several c e l l u l a r compartments among which ions can move (for example, Keynes & Steinhardt 1968; Rogus & Z i e r l e r 1973). An enzymatic basis for the a c t i v e transport of sodium and potassium across the c e l l membrane was discovered by Skou (1957) i n the form of a sodium- and potassium-activated, magnesium-dependent adenosine triphosphate phosphohydrolase (the (Na + K)ATPase). This enzyme has come to be c a l l e d "the sodium pump" (Glynn & K a r l i s h 1975). Other mechanisms for the trans- port of sodium have been postulated, as w i l l be discussed l a t e r , and many mechanisms for the transport of other species have been postulated. The a b i l i t y of tissues to generate an e l e c t r i c p o t e n t i a l d i f f e r e n c e has been recognized for well over a hundred years (Matteucci 1840; Du Bois- Reymond 1843). The early work involved rather gross i n j u r y to the tissues, and the p o t e n t i a l differences were c a l l e d 'injury p o t e n t i a l s ' . They were thought to be due to the freeing of inorganic ions through chemical reactions i n the injured tissue. The equilibrium theory of Donnan (1910) provided one model for the o r i g i n of the po t e n t i a l difference across the c e l l membrane, while the t h e o r e t i c a l d e s c r i p t i o n by T e o r e l l (1935) and Meyer and Sievers (1936) of the p o t e n t i a l difference at the boundary between two solutions of d i f f e r e n t composition or concentration provided another. Osterhout (1931) measured e l e c t r i c a l p o t e n t i a l differences across the 'protoplasm' of s i n g l e plant c e l l s , and formulated a model for t h e i r o r i g i n as d i f f u s i o n potentials c h i e f l y due to potassium. He f e l t that phase 7 boundary potentials and the Donnan po t e n t i a l would be too small to account for his measured values, and that oxidation-reduction potentials would not be measured with his apparatus. He stated that the equations of Nernst and Henderson describing d i f f u s i o n potentials "enable us to predict poten- t i a l differences with s u f f i c i e n t accuracy to j u s t i f y t h e i r use quite aside from a l l other considerations." He i d e n t i f i e d differences i n i o n i c mobil- i t i e s as the key feature of d i f f u s i o n potentials, and set.out to test the model by measuring the m o b i l i t i e s of the ions i n the "nonaqueous laye r s " (membranes) of c e l l s . In the t h e o r e t i c a l d e s c r i p t i o n formulated by T e o r e l l (1935) and Meyer and Sievers (1936) for the concentration p o t e n t i a l developed across membranes separating two e l e c t r o l y t e solutions, therewas a Donnan po t e n t i a l at each interface and a d i f f u s i o n p o t e n t i a l i n the membrane. Steinbach (1940) noted that the maintenance of a d i f f u s i o n p o t e n t i a l requires "continued production of e l e c t r o l y t e s , and as such i s linked to the metabolism of the c e l l . " Boyle and Conway (1941) analyzed the accumulation of potassium by muscle and concluded that i t must be due to a Donnan equilibrium, while the sodium permeability of the muscle c e l l membrane must be extremely low. Goldman (1943) applied the theory of d i f f u s i o n potentials to s i m p l i f i e d models of the c e l l membrane, and obtained a good q u a l i t a t i v e d e s c r i p t i o n of the r e c t i f i c a t i o n and membrane po t e n t i a l i n squid axon. Hodgkin and Katz (1949) used Goldman's equations under the assumption that the r e s t i n g squid axon membrane was more permeable to potassium than to sodium, while the sodium permeability could increase greatly to bring about the reversal of p o l a r i z a t i o n of the membrane which occurs during an action p o t e n t i a l . The passage of sodium across the membrane was proposed to occur " i n combination with a l i p o i d - s o l u b l e c a r r i e r i n the membrane 8 which is only free to move when the membrane is depolarized." They regarded t h e i r expression for the value of the membrane po t e n t i a l as "no more than a rough approximation." However, i t was s u f f i c i e n t l y simple and f l e x i b l e that i t could be applied to almost any r e s u l t under quite reasonable assumptions. Goldman's equation and va r i a t i o n s of i t continue to be used to describe the membrane pot e n t i a l (eg. Schwartz 1971). C e l l water came under scrutiny very early on, because of the f a i l u r e of c e l l s to act as perfect osmometers (for example, Overton 1902). I t was f e l t that "a considerable portion of the water i n the c e l l or body is ph y s i c a l l y 'bound' i n the c o l l o i d a l structure of the protoplasm and must be considered an i n t e g r a l part of the l i v i n g system" (Sharp 1934). The presence of 'bound ions' was indicated by the presence of slowly-exchanging fractions i n ion uptake and depletion studies. With the construction of i o n - s p e c i f i c electrodes small enough to be placed into s i n g l e c e l l s (Taylor & Whitaker 1927; Caldwell 1954; Hinke 1959; Walker 1971) i t became possible to study the i n t e r i o r of the c e l l d i r e c t l y , and i t was apparent that not a l l of the ions measured by chemical analysis of the c e l l were present i n free s o l u t i o n inside the c e l l . Some workers have concluded that the asymmetrical ion d i s t r i b u t i o n s and osmotic behavior of the c e l l are due to the a s s o c i a t i o n of the ions with fixed charge groups i n cytoplasmic macromolecules and to organization of the c e l l water, with the membrane playing only a passive r o l e (Troschin 1961; Ling 1962; Ling, M i l l e r & Ochsenfeld 1973). A problem addressed by many present-day investigators is the elucida- t i o n of the de t a i l e d mechanism of ion transport i n c e l l s . Most attention is addressed to the c e l l membrane, but for the in t e r p r e t a t i o n of studies of the transport properties of the membrane i n whole c e l l s , i t is necessary to 9 characterize the states of the i n t r a c e l l u l a r water and ions. Why t h i s is so i s described i n the next section. 10 SECTION 2. TRANS-MEMBRANE FLUXES OF SODIUM AND HYDROGEN IONS A. GENERAL CONSIDERATIONS Almost a l l of the observed passage of sodium ions and indeed of a l l l i p i d - i n s o l u b l e inorganic ions across the c e l l membrane i s associated with membrane proteins. The permeability of a pure phospholipid b i l a y e r to sodium is several orders of magnitude lower than that of a c e l l membrane (Jain 1972; Lauger & Neumcke 1973). The tra n s l o c a t i o n of the ions can thus be regarded as an enzyme-mediated reaction i n which one product i s the translocated ion. The e f f l u x of sodium from c e l l s normally involves a considerable increase i n the electrochemical p o t e n t i a l of the translocated ions, and so requires energy. This energy must come ul t i m a t e l y from metabolism. (Exchange and the passive u n i d i r e c t i o n a l f l u x are considered below.) In theory, the d i r e c t source of energy for sodium e f f l u x could be other ions (or sodium) which pass spontaneously to a region of lower electrochemical p o t e n t i a l , or i t could be the hydrolysis of 'high-energy' phosphate compounds or other products of metabolism. A mechanism involving the cytochromes of the electron transport system d i r e c t l y has also been proposed (M i t c h e l l 1969). Experiments have indicated that most and perhaps a l l of the metabolism- dependent sodium e f f l u x depends d i r e c t l y on adenosine triphosphate (ATP) for energy (Dunham 1957; Whittam 1958; Caldwell 1960; Hoffman 1960). Inosine triphosphate (ITP), guanosine triphosphate (GTP), u r i d i n e triphos- phate (UTP), phosphoarginine (PA), c y t i d i n e triphosphate (CTP), a c e t y l phosphate (AcP), phospho(enol) pyruvate (PEP), D-glyceraldehyde-3-phosphate (G-3-P), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) do 11 not support sodium e f f l u x , while deoxyadenosine triphosphate (d-ATP) supports about h a l f of the normal sodium e f f l u x (Hoffman 1960; Bri n l e y & Mull ins 1968). When the ATP concentration i n squid axon i s reduced to very low le v e l s by i n t e r n a l d i a l y s i s , the sodium e f f l u x approaches the rate expected from passive mechanisms (Brinley & Mullins 1968). I f the axon is treated with cyanide (2 mM. CN for 1 - 3 hours) and then dialyzed, the ATP concentration is reduced to about 2 JAM. and the sodium e f f l u x is reduced to the value estimated for passive movement (Brinley & Mullins 1967). CN alone has l i t t l e immediate e f f e c t on the sodium e f f l u x (Keynes & Maisal 1954; Hodgkin & Keynes 1956; Carey, Conway, 6c Kernan 1959). An ATPase found i n the c e l l membrane and activated by sodium and potassium has come to be regarded as being l a r g e l y i f not e n t i r e l y respon- s i b l e for the metabolism-dependent sodium e f f l u x (Skou 1965), at least i n 1 red blood c e l l s . I t is curr e n t l y being r e f e r r e d to as "the" sodium pump (Glynn & K a r l i s h 1975). Five modes of behavior have been described for this (Na+K)ATPase: (i) exchange of in t e r n a l sodium for external potassium, r e q u i r i n g ATP and accompanied by a net hydrolysis of ATP; ( i i ) r eversal of ( i ) ; ( i i i ) exchange of i n t e r n a l sodium for external sodium, r e q u i r i n g ATP and ADP but accom- ^Sodium has also been alleged to be involved i n the e f f l u x of calcium from nerve and muscle, where i n t e r n a l calcium i s exchanged for external sodium with no net hydrolysis of ATP (Baker 1972; Requena, DiPolo, Brinley, & Mullins 1977), and i n the e f f l u x of magnesium, where once again sodium enters and there is no net hydrolysis of ATP (Baker 6e Crawford 1972; Ashley 6c E l l o r y 1972; Mullins, Brinley, Spangler, 6c Abercrombie 1977). Sodium e f f l u x apparently is associated with the transport of sugars (Schultz 6e Curran 1970; Kimmich 1972) and amino acids (Colombini 6c Johnstone 1974; Johnstone 1974). A sodium-hydrogen exchange has been suggested but is not c l e a r (Keynes " 1965; Biro 1965; B i t t a r e t a l . 1973, 1976). An a s s o c i a t i o n of sodium and iodide transport has also been suggested (Skou 1965). 12 panied by no net hydrolysis of ATP; (a separate sodium-sodium exchange found i n nerve and muscle i s discussed l a t e r ; i t i s thought to comprise as much as h a l f of the sodium e f f l u x in. muscle c e l l s and is i n s e n s i t i v e to i n h i b i t o r s ) ; (iv) exchange of in t e r n a l potassium for external potassium, re q u i r i n g inorganic phosphate and ATP but accompanied by no net hydrolysis of ATP; th i s apparently is associated with operation i n mode ( i ) ; and (v) e f f l u x of sodium without coupling to i n f l u x of another ion, re q u i r i n g ATP and probably accompanied by a net hydrolysis of ATP (or much less e f f e c - t i v e l y , CTP, ITP, or UTP: this mode is much less fastidious than the others) (Glynn & K a r l i s h 1974, 1975). The contribution of a given mode of the (Na+K)ATPase or of other trans- port mechanisms to the net fl u x might be d i f f i c u l t to deduce from simple experiments, since a l t e r a t i o n of the substrate concentrations (ions and ATP) should cause a r e d i s t r i b u t i o n among a l l of the modes, rather than simply an a l t e r a t i o n of the contribution of a si n g l e mode. An example from the l i t e r a t u r e is. that i n the absence but not i n the presence of external potassium, both the i n f l u x and e f f l u x of sodium i n frog s k e l e t a l muscle are reduced by ouabain (Keynes & Steinhardt 1968). That i s , removal of external potassium disables the sodium-potassium exchange but unmasks a ouabain- s e n s i t i v e sodium-sodium exchange not apparent i n solutions which contain potassium. This type of occurrence must be acknowledged i n the interpreta- t i o n of experiments. Nevertheless, experiments i n which the i n t r a - or e x t r a c e l l u l a r sodium or potassium ion concentration i s a l t e r e d and the e f f e c t on the sodium e f f l u x i s observed are of value i n the study of the sodium e f f l u x . Results from such experiments and va r i a t i o n s on them are known for many c e l l s , and by consideration of th i s abundance of data i t i s hoped that the general features of the d i f f e r e n t routes of sodium e f f l u x can be deduced. 13 L i k e the e f f l u x of sodium, the e f f l u x of hydrogen ions from c e l l s involves an increase i n the electrochemical p o t e n t i a l of the translocated ions, and so requires energy. I t is only r e l a t i v e l y recently that con- vincing evidence for s p e c i f i c transport mechanisms has been found. The study of the e f f l u x mechanism for hydrogen ions is more d i f f i c u l t than is the case for sodium because processes equivalent to the expulsion of protons appear to be operating, i n addi t i o n to the expulsion of protons themselves. Also, the c e l l i s not conservative with respect to hydrogen ions. (Further d i f f i c u l t i e s of a more technical nature w i l l be discussed l a t e r . ) M i t c h e l l (1969) proposed a model, o r i g i n a l l y for mitochondria, wherein the electron transport system of aerobic metabolism was embedded i n the membrane, and the energy derived from the flow of electrons through this system was used d i r e c t l y to expel protons to the outer side of the membrane. He assumed that there was a membrane-bound ATPase which had the c a p a b i l i t y of e x p e l l i n g protons as i t hydroly.zed ATP. I t was thought to 'run back- wards' as protons passed into the c e l l through i t , and ATP was thus formed from ADP and inorganic phosphate. This is c a l l e d the chemiosmotic hypothesis. Rehm (1972) proposed that the a c i d i f i c a t i o n of the lumen of the stomach was due to an electrogenic hydrogen ion pump directed toward the lumen. In addition, a chl o r i d e pump was thought to be directed toward the lumen and a sodium pump toward the blood. A c i d i f i c a t i o n would occur when the hydrogen ion pump became act i v e and the sodium pump became inactive. Coupling of the pumps was not r i g i d . The resp i r a t o r y chain was invoked d i r e c t l y to pump protons, as i n the M i t c h e l l model, and i t was thought that ATP was not d i r e c t l y involved. Stoeckenius and co-workers (Osterhelt & Stoeckenius 1973; Danon & Stoeckenius 1974; Stoeckenius 1976) found that the 'purple membrane' of the 14 bacterium Halobacterium halobium could expel protons from the bacterium when exposed to l i g h t , and that the action spectrum for ATP production i n response to l i g h t was s i m i l a r to the absorption spectrum of the purple membrane. The bacterium could also expel protons i n the dark under aerobic conditions. Stoeckenius proposed that the bacterium contained three membrane- bound systems capable of e x p e l l i n g protons; ( i ) a purple membrane pr o t e i n which could expel protons when exposed to l i g h t ; ( i i ) a re s p i r a t o r y chain which could expel protons using the energy from aerobic metabolism; and ( i i i ) an ATPase which could expel protons using the energy from the hydro- l y s i s of ATP, but which usually operated i n the opposite sense, synthesizing ATP from ADP and inorganic phosphate using the energy stored i n the 'proton gradient' created by the other two systems. This seems to be a unique case, but i t provided evidence that an ATPase capable of causing proton transport per se ex i s t s . Nature has tended to employ extended and improved versions of p r i m i t i v e c e l l u l a r mechanisms i n the more s o p h i s i t i c a t e d c e l l s which evolved l a t e r . I t would not be too s u r p r i s i n g i f the 'proton pump' of nucleated c e l l s is found to be b u i l t on these mechanisms. A sodium-hydrogen exchange was suggested by many workers (Keynes 1965; Biro 1965; B i t t a r et a l . 1973, 1976) but only recently has the dependence of the a l k a l i n i z a t i o n of the c e l l on e x t r a c e l l u l a r sodium been demonstrated, i n mouse s k e l e t a l muscle c e l l s (Aickin & Thomas 1977), s n a i l neurone (Thomas 1977), and barnacle muscle c e l l s (Boron & Ross 1978). F i n a l l y , a chloride-bicarbonate exchange has been proposed. The exchange of i n t e r n a l chloride for external bicarbonate would be equivalent to the extrusion of HC1, since at a given CC^ tension excess bicarbonate would quickly combine with a proton to y i e l d carbonic a c i d and then, under c a t a l y s i s by carbonic anhydrase, water and CC^- The CC^ would leave the c e l l passively. Similar systems had been proposed for the cerebrospinal 15 f l u i d (eg. review by Si e s j o 1972). Good evidence for chloride-bicarbonate exchange has been found in s n a i l neurone (Thomas 1976), squid axon (Boron & DeWeer 1976a), mouse s k e l e t a l muscle (Aickin & Thomas 1977), and barnacle muscle (Boron & Roos 1978). A p a r t i c u l a r mechanism for sodium or hydrogen transport i s characterized by i t s k i n e t i c behavior. Much more work has been done on the sodium e f f l u x k i n e t i c s than on the proton e f f l u x k i n e t i c s . The connection between the e f f l u x and the k i n e t i c s of each of the sodium transport systems w i l l be discussed i n part (C) of t h i s section. In the general case, the connection i s made v i a a chemical reaction model i n which the substrate S binds with an enzyme E to form a complex ES, which then dissociates into enzyme and products Pr, with no back reaction: k- k 1 2 E + S s. ES E + Pr V T -1 The o v e r a l l rate, and hence the e f f l u x rate i f t h i s models the dominant mechanism for sodium extrusion, i s proportional to the concentration of the complex, (ES). I t is assumed that the d i s s o c i a t i o n of this complex to y i e l d products i s so much slower than the-reactions which form i t that the reactions preceding t h i s d i s s o c i a t i o n are e s s e n t i a l l y at equilibrium (steady state assumption). The reaction mechanism for the (Na+K)ATPase has been characterized i n some d e t a i l through the use of ixi v i t r o preparations of the enzyme, but a s i m p l i f i e d model w i l l be adopted here. Only one parameter, (S) = (Na), is assumed to be var i a b l e , and the rest is concealed i n the rate constants. In t h i s , the elementary 'Michaelis-Menten' model of enzyme k i n e t i c s , (ES) can be expressed in terms of (S) and the equilibrium constant for the rea c t i o n which forms the complex, and the o v e r a l l rate, eg. of sodium e f f l u x M^a, depends on (Na) as 16 M = M / (1 + k ) 'Na max For three sodium ions binding successively to E at equivalent indepen- dent s i t e s , a r e a l i s t i c model of the (Na+K)ATPase (Glynne & K a r l i s h 1975), a s i m i l a r treatment y i e l d s (Na) (Na) (Na) where k of course has a meaning d i f f e r e n t from that i n the previous case. I f the three sodium ions bind simultaneously, the r e l a t i o n i s (Na) 3 (Mullins & Frumento 1963), where again k has a d i f f e r e n t s i g n i f i c a n c e . More complicated versions w i l l be described l a t e r . I t i s conceivable that the e f f e c t i v e number of binding s i t e s i s d i f f e r e n t at d i f f e r e n t sodium concentrations, for example. It should be noted at t h i s point that Baker, Blaustein, Keynes et al_. (1969) and Garay and Garrahan (1973) appeared to take a d i f f e r e n t approach, i n that they assumed instead that three sodium ions had to bind to equiva- lent independent s i t e s on the enzyme, and that the e f f l u x was proportional to the f r a c t i o n of the independent enzyme units which were f u l l y occupied by three sodium ions. This f r a c t i o n they took to be the cube of the p r o b a b i l i t y of having an enzyme un i t occupied with sodium at one s i t e . This p r o b a b i l i t y i s (E) + (ES) + (ES 2) + (ES 3) but they took i t to be I E S 1 (E) + (ES) 1 + k/(S) for k = (S)(E)/(ES). Since 17 (S) (E) (S) (ES) (S) (ES„) k = = (ES) (ES 2) (ES 3) then (E) + (ES) + (ES 2) + (ES 3) = (E) T l + x§I + ( X§1 ) 2 + ( i s i ) 3 L k k k J Writing (ES) = (S) (E) / k, one sees that the p r o b a b i l i t y i s a c t u a l l y 1 k + 1 + <£t + ( <£L ) 2 (S) k " k for having one s i t e occupied by sodium, while the p r o b a b i l i t y of having a l l three s i t e s occupied is (ES3) (E) + (ES) + (ES 2) + (ES 3) 1 1 + J L . + ( J L _ ) 2 + ( J i _ ) 3 (S) (S) (S) or just as for the Michael is-Menten case for three equivalent s i t e s . This had to be so, of course, since the assumptions were equivalent. The fact that a good f i t to the experimental data was obtained with the incorrect model i l l u s t r a t e s the ease with which a smooth curve can be approximated by a polynomial, and the l i m i t a t i o n s of t h i s sort of modelling. Continuing i n t h i s vein, the a p p l i c a b i l i t y of this type of model to the (Na+K)ATPase can be considered. In r e a l i t y , the steps followed by the enzyme to transport sodium out of the c e l l v i a the (Na+K)ATPase involve the binding of ATP, magnesium, and "n" sodium ions. The enzyme becomes phosphorylated and the conformational changes required to make the sodium a v a i l a b l e to the outside of the c e l l occur. I t has generally been assumed that these steps of conformational change w i l l be rate l i m i t i n g , so that the steady state approximation can be applied. Recently, Mardh and Post (1977) found evidence that with each binding of ligand to E, the conformation s h i f t e d s i g n i f i c a n t l y towards the "potent" 18 complex which can proceed to phosphorylation of E. That i s , the i n i t i a l rate of phosphorylation of the enzyme was increased over that when ATP, magnesium, and sodium were a l l made a v a i l a b l e at once, i f one or two of the ligands were added f i r s t , and then the missing ligands were added. This suggests that the steady state assumption cannot be applied with impunity i n the case of the sodium e f f l u x . Nevertheless, i t should be possible to obtain semi-quantitative agree- ment with the data i f the basic notion of a 'dominant mode' with n sodium ions binding is correct. Thus, the approach has been to compare the k i n e t i c curves of the sodium transport out of c e l l s with the curves produced by k i n e t i c models such as the above. Even with just two adjustable parameters, k and i t is u s u a l l y easy to get a reasonable f i t for data over most of the concentration range. I t i s at low substrate (sodium) concentration that differences between models are most apparent. I t i s important, then, to measure as accurately as possible the concentration of the reactant (eg. sodium) i n the transport reaction. (In fact, the quantity of i n t e r e s t is the chemical p o t e n t i a l , but discussion of such refinements w i l l not be presented here.) This is the concentration at the i n t e r n a l reaction s i t e s of the transport enzymes, and i t is on t h i s point that a complication a r i s e s . As noted above, the c e l l i s a heterogeneous structure, and i t has been found that the water and solutes inside the c e l l do not behave as i f they were in a simple aqueous s o l u t i o n bounded by a p r o t e i n - l i p i d membrane. Before the experimental work on fluxes could be done, i t was f e l t that the current notions about the states of water and sodium ions inside the barnacle muscle c e l l had to be c l a r i f i e d . In p a r t i c u l a r , the amount and d i s t r i b u t i o n of the c e l l u l a r sodium which w i l l p a r t i c i p a t e in flux studies, 19 and the concentration of sodium i n the s o l u t i o n which bathes the i n t e r n a l surface of the c e l l membrane must be known. This i n t e r e s t i n g general problem i s reviewed next. B. STATES OF WATER AND IONS IN CELLS The current wiew of the state of water and ions inside l i v i n g c e l l s can be summarized b r i e f l y as follows (Tait & Franks 1971; Hinke, C a i l l e , & Gayton 1973; Palaty & Friedman 1973; Cooke & Kunta 1974; Berendsen 1975; Lee & Armstrong 1974; Edzes & Berendsen 1975; Lev & Armstrong 1975). Water i n c e l l s behaves l a r g e l y as i t does i n bulk s o l u t i o n s . 75 to 90% of the water has normal l i q u i d properties as far as d i f f u s i o n of ions and molecules, osmotic e f f e c t s , and s o l v a t i o n are concerned, and responds l i k e bulk water i n NMR, i n f r a r e d spectroscopy, and x-ray and neutron d i f f r a c t i o n studies. About 1% i s t i g h t l y bound to macromolecules as ' s t r u c t u r a l water'. The remaining 8 - 24% i s influenced by the macromolecules and the s t r u c t u r a l water, apparently through the formation of s h o r t - l i v e d extended clusters of water molecules, by means of hydrogen bonding, i n the s o - c a l l e d 'hydrophobic i n t e r a c t i o n ' . This f r a c t i o n i s of s p e c i a l i n t e r e s t i n that i t i s conceivable that i t s behavior is d i f f e r e n t from that of bulk water. There i s disagreement about i t s exact s i z e . Techniqes which r e f l e c t the freedom of motion of i n d i v i d u a l water molecules, such as NMR y i e l d the lower estimates, while measurements of solvent properties y i e l d the higher estimates. Water passes the c e l l u l a r membranes very r e a d i l y , and quickly flows to or from any region of the c e l l where i t s chemical p o t e n t i a l deviates from that of the rest of the c e l l , or indeed of the v i c i n i t y of the c e l l . Because the c e l l membranes can transport substances and are selectively-permeable, however, this i s not the case for the major inorganic ions. They can be 20 confined within or barred from the c e l l or membrane delimited organelles inside the c e l l . Further, i n a given compartment inside the c e l l or i n the e x t r a c e l l u l a r space they can be i n free s o l u t i o n , or associated with large or small organicrjnolecules v i a s p e c i f i c or nonspecific binding. F i n a l l y , even though an ion can pass from a free state to one of the non-free states l i s t e d , i n response to a non-uniformity i n i t s chemical p o t e n t i a l , the c h a r a c t e r i s t i c time of the exchange might be very slow r e l a t i v e to that of other c e l l processes, notably d i f f u s i o n i n bulk s o l u t i o n and transmembrane transport. The a c t i v i t y of some ions inside the c e l l can be measured with ion- s p e c i f i c microelectrodes. These a c t u a l l y r e f l e c t the chemical p o t e n t i a l of the ion inside the c e l l , but the a c t i v i t y and concentration of the free ion can be estimated under reasonable assumptions about the solvent proper- t i e s of the water i n which the free ion i s thought to reside. I f the volume of d i s t r i b u t i o n of the free ion can be estimated, then the amount of free ion i n the c e l l can be calculated. This is the conceptual heart of the question. The ions i n s o l u t i o n are no more free than those p a r t i c i p a t i n g i n ion pairing, during steady conditions, i n the sense that the chemical p o t e n t i a l is the same for the two groups. The d i s t i n c t i o n i s made because both are measured by chemical analysis, while only the solvated ion i s assumed to be measured in microelec- trode studies. The assumption involved is that a l l of the free ion i s in a homogeneous compartment as far as concentration is concerned, although i t i s c l e a r that near charge inhomogeneities on membranes or macromolecules, considered as a group, the concentration w i l l be d i f f e r e n t from that i n the bulk even though the chemical p o t e n t i a l is the same. The t o t a l ion content of the c e l l , including that i n e x t r a c e l l u l a r locations, can be determined quite accurately from chemical analysis of the 21 ashed tissue. The ion content of the e x t r a c e l l u l a r space is often d i f f i c u l t to determine, because fixed negatively-charged s i t e s abound i n . the polysaccharide-rich glycocalyx. I f t h i s ion f r a c t i o n can be estimated, then by subtraction one can c a l c u l a t e the amount of ion which is t r u l y i n t r a c e l l u l a r but not i n free s o l u t i o n . This is e s p e c i a l l y d i f f i c u l t for sodium, which has a high e x t r a c e l l u l a r but low i n t r a c e l l u l a r concentration. The i n d i v i d u a l ions can be considered i n turn. They a l l can pass the r e s t i n g c e l l membrane, but at very d i f f e r e n t rates. Sodium i s a c t i v e l y expelled from the c e l l (that i s , during steady conditions the chemical p o t e n t i a l for sodium ions i s lower inside the c e l l than outside the c e l l ) . No l o c a l i n t r a c e l l u l a r accumulations of sodium have been found i n analyses of s u b c e l l u l a r f r a c t i o n s . (This w i l l be reviewed below.) However, a large amount is thought to be associated with fixed negative s i t e s on i n t r a c e l l u l a r macromolecules, i n competition with other cations. Hydrogen i s a c t i v e l y expelled from the c e l l . The i n t r a c e l l u l a r pH i s buffered by the i n t r a c e l l u l a r proteins, and by phosphate and bicarbonate. Hydrogen ions are also produced and consumed in many reactions in the c e l l . Potassium i s a c t i v e l y accumulated by the c e l l , but because the permea- b i l i t y of the c e l l membrane to potassium i s r e l a t i v e l y high the chemical p o t e n t i a l for potassium ions is about the same inside and outside many c e l l s . A quantity of potassium which is small r e l a t i v e to the amount of potassium in s o l u t i o n probably associates with fixed negative s i t e s inside the c e l l . Calcium i s a c t i v e l y expelled from the c e l l . It is also a c t i v e l y sequestered in mitochondria and in the sarcoplasmic reticulum of muscle. An a d d i t i o n a l b u f f e r i n g mechanism of very large capacity appears to e x i s t (Brinley, T i f f e r t , Scarpa, & Mull ins 1977). Magnesium is a c t i v e l y expelled from the c e l l . About, h a l f of the 22 i n t r a c e l l u l a r magnesium i s bound to ATP i n barnacle muscle (Brinley, Scarpa, & T i f f e r t 1977). The s i t u a t i o n with chloride is not c l e a r . Microelectrode measurements indicate that there i s a s l i g h t accumulation of chloride inside the c e l l (see also Bolton & Vaughan Jones 1977; Dulhunty 1978). D i f f u s i o n studies show l i t t l e c h loride associated with fixed i n t r a c e l l u l a r s i t e s , but large bound fractions have been reported. Chloride, l i k e sodium, is abundant in the e x t r a c e l l u l a r space, and t h i s makes accurate a l l o c a t i o n of chloride to compartments d i f f i c u l t . The s i t u a t i o n i n a given c e l l type often d i f f e r s i n d e t a i l from these generalizations. This thesis i s p r i m a r i l y concerned with sodium and hydrogen i n barnacle muscle c e l l s . The hydrogen ion exemplifies the problems of i n t e r p r e t a t i o n of measure- ments of the chemical p o t e n t i a l .discussed above. The hydrogen ion d i f f e r s i n several fundamental respects from the sodium ion i n l i v i n g systems. F i r s t , the c e l l is not conservative with respect to hydrogen: hydrogen ions p a r t i c i p a t e as reactant and product i n many chemical reactions i n the c e l l . Changes i n these reactions might occur with any manipulation of the c e l l or i t s environment. Second, hydrogen i s buffered by the bicarbonate-C02 system and, more importantly inside the c e l l , by the phosphate system and the protein system. Only 0.001% of the a v a i l a b l e hydrogen ion i s free i n s o l u t i o n at pH 7.0 (Waddell & Bates 1969). To c a l c u l a t e changes i n the amount of hydrogen ions with any manipulation, i t i s not s u f f i c i e n t to measure only pH changes. The. buffering capacity at each stage of the manipulation must be known. Third, hydrogen i s a l a b i l e part of the solvent (water) i n which the e n t i r e c e l l u l a r system is embedded. The e f f e c t i v e t r a n s l o c a t i o n of hydrogen ions can occur by the forming and breaking of hydrogen bonds and hydrogen-oxygen bonds in the water. This is 23 much more rapid than the s e l f - d i f f u s i o n of hydrogen, and means that no radioisotope can be used to measure fluxes. F i n a l l y , the concentration of hydrogen ions i n so l u t i o n i n the c e l l is usually about 10^ times smaller than that of sodium. The question of the r e l a t i o n s h i p between measurements of pH (which is defined i n terms of the po t e n t i a l d i f f e r e n c e developed i n a standard e l e c t r o - chemical c e l l ) and 'hydrogen ion concentration' troubles even the physical chemists, and they maintain that the quantity of p r a c t i c a l i n t e r e s t i n almost a l l contexts i s the.chemical p o t e n t i a l (Waddell & Bates 1969). Nevertheless, processes equivalent to the movement of hydrogen ions do occur across the c e l l membrane, and i t i s reasonable to c a l c u l a t e the e f f e c t i v e quantity of hydrogen ions involved. The reasonable approximation that pH is the negative of the logarithm of the hydrogen ion a c t i v i t y w i l l be adopted i n these q u a l i t a t i v e discussions. The question of the inhomogeneity of the pH inside the c e l l arose early because measurement of the d i s t r i b u t i o n of an indicator (weak acid or weak base) was the usual technique for measuring pH (Fenn & Maurer 1935). When these measurements indicated that the pH was too high inside c e l l s , the existence of a l k a l i n e organelles was suggested as the cause. Otherwise the a c t i v e extrusion of protons from the c e l l would have had to be postulated. Recently, Garthwaite (1977) examined the di f f e r e n c e i n the pH measured by a c i d i c (DM0 - see section 9) and basic (nicotine) indicators i n various tissues, with reference to the number of mitochondria present. A weak a c i d w i l l y i e l d a pH value closer to the higher pH i n the inhomogeneous tissue, and a weak base w i l l y i e l d a pH value closer to the lower pH (Waddell & Bates 1969). The difference ( p H a c i d - p H b a s e ) i n the pH res u l t s of the two indicators was 1.0 i n brown fat, which has many mitochondria, about 0.8 in 24 most c e l l s , and about 0.08 In mature red blood c e l l s , which have no organelles. Mitochondria are thought to have a high pH. Further, i n most c e l l s the diff e r e n c e i n the measured pH values was reduced by exposure to DNP (dinitrophenol). This would uncouple oxidative phosphorylation i n mitochondria and presumably prevent them from maintaining a high i n t e r n a l PH. For barnacle muscle c e l l s , the pH measured with the a c i d i c indicator DMO has been reported to be higher than that measured with the basic i n d i - cator methylamine by about 0.1 (Boron & Roos 1976). These values were lower than the value measured with an i n t r a c e l l u l a r electrode, which i s consistent with the existence of an a c i d i c i n t r a c e l l u l a r compartment. This phenomenon presents a p r a c t i c a l problem, i n that the i n t r a c e l l u l a r pH for most tissues can only be measured with indicators, and the meaning of what they measure is not c e r t a i n . For this reason, experiments were done, as part of the work presented' i n this thesis, i n which the pH was measured with DMO, as a measure of the pH of the whole c e l l i n the sense discussed above, and with a pH microelectrode, as a measure of the pH of the major aqueous i n t r a c e l l u l a r compartment, over a wide range of i n t r a - c e l l u l a r pH, in identically-prepared barnacle muscle c e l l s . The r e s u l t , discussed i n section 9, was that pH(DMO) was c o n s i s t e n t l y higher than pH(electrode). This in i t s e l f i s consistent with the existence of an a l k a l i n e i n t r a c e l l u l a r compartment, but the technical d i f f i c u l t i e s of the indicator method are such that t h i s cannot be stated with any degree of ce r t a i n t y . Attention w i l l now be turned to the states of water and sodium i n barnacle muscle. Hinke (1970) adopted a working hypothesis for s i n g l e barnacle muscle c e l l s wherein the i n t r a c e l l u l a r water was divided into two f r a c t i o n s : one 25 ("ideal water") was completely l i k e bulk water; the other was not behaving as bulk water i n that i t did not act as solvent for sodium, potassium or chloride, and was not osmotically a c t i v e . His experiments indicated that the bulk water comprised about 68% of the water i n a blotted c e l l , which is about 73%, of the i n t r a c e l l u l a r water since about TL of the c e l l water l i e s i n the e x t r a c e l l u l a r space. The measured s i z e of the e x t r a c e l l u l a r space depends on the technique of blotting.. In the same study, i t was found that the mean i o n i c a c t i v i t y c o e f f i c i e n t i n the myoplasm was 0.65, the value i n a bulk s o l u t i o n at the io n i c strength of normal barnacle Ringer's so l u t i o n . A s i m i l a r value can be deduced from the data of Hagiwara, Chichibu, and Naka (1964). The volume of d i s t r i b u t i o n of the free ion was assumed to be the volume of " i d e a l water", so free ion contents could be determined from microelec- trode measurements. I t was found that only a part of the i n t r a c e l l u l a r sodium, potassium, and chlo r i d e measured by chemical analysis of whole c e l l s i s i n free s o l u t i o n i n the " i d e a l water" (McLaughlin & Hinke 1966; Hinke, C a i l l e , & Gayton 1973). The "missing f r a c t i o n s " were t y p i c a l l y 13% of the potassium, 73% of the sodium, and 31% of the chloride (Hinke et al. 1973). One estimate was that f u l l y 83% of the i n t r a c e l l u l a r sodium could be inaccessible to the sodium microelectrode (Hinke 1969b). The r e s u l t s of microelectrode studies by other workers i n other c e l l s have been s i m i l a r (reviewed by Lev & Armstrong 1975). However, e x p l i c i t allowance for 'bound water' i s seldom made. Lee & Armstrong (1972;1974) made no allowance for 'bound water' i n t h e i r c a l c u l a t i o n s of free ion con- centrations i n frog s k e l e t a l muscle, although they acknowledge the concept and the physical meaning of the calculated concentrations. They based t h e i r conclusions about the existence of sequestered sodium and potassium on the observed changes i n the apparent a c t i v i t y c o e f f i c i e n t ( a N a ) / ( N a ) i when the 26 sodium content of the c e l l was altered, where (a^ a) 1 S the a c t i v i t y of sodium deduced from the microelectrode measurement and (Na)^ i s the quotient of the t o t a l analyzed c e l l u l a r sodium and the t o t a l water content of the c e l l , excluding the e x t r a c e l l u l a r space. The l o c a t i o n of the 'missing sodium' i n barnacle muscle c e l l s is not ce r t a i n , but some conclusions can be drawn from a c r i t i c a l review of morphological and p h y s i o l o g i c a l studies. The u l t r a s t r u c t u r e of the barnacle muscle c e l l was examined by Hoyle et ai.(/9?3).The structure is q u a l i t a t i v e l y s i m i l a r to that of vertebrate s t r i a t e d muscle, but there are several unusual features. The c e l l membrane i s deeply furrowed by an extensive, unordered system of c l e f t s . These were c l a s s i f i e d as "major c l e f t s " , deep furrows opening d i r e c t l y into the bathing s o l u t i o n a l l along t h e i r length, and "minor c l e f t s " , branches opening into the major c l e f t s or the bathing s o l u t i o n only at t h e i r ends. The c l e f t s contained "mucopolysaccharide-like" material, and comprised about 8% of the t o t a l c e l l volume as measured from micrographs. A system of flattened tubules oriented both l o n g i t u d i n a l l y and r a d i a l l y , and devoid of the 'mucopolysaccharide', comprised less than 17o of the t o t a l volume. The l a t t e r system was i d e n t i f i e d as the analogue of the transverse tubular system (TTS) of vertebrate s t r i a t e d muscle. The tubules open into c l e f t s or to the exposed surface of the c e l l . The sarcoplasmic reticulum i s small, comprising about 0.57o of the t o t a l c e l l volume. By comparison, i t is about 13% of the t o t a l c e l l volume for frog s a r t o r i u s muscle (Peachey 1965). There are lo n g i t u d i n a l and c i s t e r n a l elements, and d i a d i c (rarely: t r i a d i c ) contacts are made with TTS. Mitochondria and n u c l e i are located just under the exposed sarcolemma and the membrane of the major c l e f t s . Together the l a t t e r organelles probably comprise less than 1% of the c e l l volume. The remaining almost 90% of the c e l l volume is occupied by the 27 c o n t r a c t i l e proteins and the myoplasmic s o l u t i o n . I t is among a l l of these structures that the compartments of a f l u x model should f i n d counterparts. I f the sodium not detected i n the myoplasm by the microelectrode is i s o l a t e d i n the other i n t r a c e l l u l a r compartments, very high concentrations must be attained. Few studies on the i o n i c content of organelles are a v a i l a b l e . Size alone was considered to r u l e out the n u c l e i and mitochondria 2 of barnacle muscle c e l l s as s i g n i f i c a n t r e p o s i t o r i e s of sodium. The c e l l membrane i t s e l f i s probably s l i g h t l y more important, since sodium, potassium, magnesium, and calcium i n t e r a c t competitively with the membrane polar groups. The c l e f t system i s d i r e c t l y open to the bathing solution, and so the s o l u t i o n f i l l i n g i t w i l l have the sodium-rich composition of the bathing s o l u t i o n . The s o l i d material i n the c l e f t s i s the negatively-charged polysaccharide of the glycocalyx and w i l l have sodium associated with i t , perhaps in large quantities (Harris & Steinbach 1956; Brading & Widdicombe 1977). The amount of sodium associated or adsorbed w i l l depend on the concentration of sodium i n the bathing s o l u t i o n . The amount of glyco- protein i n barnacle c e l l s has not been determined. An i n d i c a t i o n of i t s binding capacity can be found i n experiments on smooth muscle, where at least h a l f of the e x t r a c e l l u l a r space cation content was found not to be in s o l u t i o n i n the sucrose space (Brading & Widdicombe 1977). Correction of the t o t a l analyzed c e l l sodium by assuming that a l l e x t r a c e l l u l a r sodium i s in a volume of f l u i d equal to the i n u l i n (or other o Some sodium is sequestered in the n u c l e i and mitochondria of frog s k e l e t a l muscle (Sorokina & Kholodova 1970) and i n the n u c l e i of rat hepatocytes (Hooper & Dick 1976). However, electron microprobe analysis has shown that nuclear and cytoplasmic sodium concentrations are the same i n toad oocytes (Dick 197 8), and some accumulation of sodium i n n u c l e i and mitochondria of thymus and l i v e r c e l l s has been reported i n d i f f e r e n t species (Itch & Schwarta 1957; Thiers, Reynolds, & Valee 1960). 28 marker) space, at the concentration of the bathing solution, thus probably is inadequate. The TTS of c r a y f i s h muscle has been shown to swell when chloride is caused to enter i t from the myoplasm (Girardier, Reuben, Brandt, & Grundfest 1963). This occurs when the e x t r a c e l l u l a r chloride or potassium concentra- t i o n is reduced so that potassium chloride is caused to leave the c e l l , but not during an osmotic stress when the product (K)bath x(^-'-)bath °^ concentra- tions i s kept constant so that potassium chloride does not leave the c e l l . The TTS therefore might comprise more than VL of the c e l l volume under some conditions. I t appears to open d i r e c t l y into the e x t r a c e l l u l a r solution, and indeed in frog s k e l e t a l muscle is accessible to e x t r a c e l l u l a r sucrose (Birks & Davey 1969). Flux studies by Harris (1963) had indicated that 15 - 307„ of the c e l l volume was a " s p e c i a l region" f r e e l y accessible to sodium, chloride, and sucrose. The work of Birks and Davey plus , that of many others (Vinogradova 1967, 1968; Vinogradova, Nikolsky, & Troshin 1967; Sperelakis, Shigenobu, & Rubio 1978; Rogus & Z i e r l e r 1973) indicated that this was the sarcoplasmic reticulum. However, N e v i l l e (1979) has shown from the k i n e t i c behavior of this " s p e c i a l region" that i t cannot be sarcoplasmic reticulum, and Somlyo, Shuman, & Somlyo (1977a) found no accumulation of sodium i n the sarcoplasmic reticulum on electron microprobe analysis of toadfish s t r i a t e d muscle. I t is possible that the s p e c i a l compartments of flux studies are a r t i f a c t s of analysis.. In any case, the TTS of the barnacle c e l l s used in the experiments described in t h i s thesis were subjected to treatment which would cause swelling i n only a few s p e c i a l cases. Altogether, i t is very u n l i k e l y that the TTS i n barnacle contains much of the 'missing sodium'. The c o n t r a c t i l e proteins form a large compartment, and have not yet been considered here. It might reasonably be expected that most of the 29 i n t r a c e l l u l a r sodium not detected by the electrode i s associated as counter- ion with the fixed anionic s i t e s on the proteins i n th i s compartment (Hinke e_t al_. 1973) . Myosin i s known to associate with cations (Szyent-Gyorgi 1947; Fenn 1957; Lewis & Saroff 1957), and is unique among the major proteins of the c e l l i n showing a modest preference for sodium over potassium. Studies of the d i f f u s i o n of ions and molecules inside barnacle muscle c e l l s have indicated that the muscle protein has s i t e s which can sequester cations but admit to very rapid exchange with the cations which are free i n so l u t i o n inside the c e l l ( C a i l l e & Hinke 1972, 1973, 1974). The t o t a l capacity of these s i t e s for sodium and potassium was estimated to be about 68 millimoles per kilogram of dry weight. Again, a simple model, with only very r a p i d l y exchanging s i t e s , was assumed, so the capacity might be larger i f some s i t e s have longer residence times. Experiments with a d i f f e r e n t time r e s o l u t i o n , i n which the c e l l membrane was destroyed and the protein allowed to e q u i l i b r a t e v i a a jacket of porous glass, with bathing solutions of d i f f e r e n t sodium and potassium content, yielded capacities about twice as large (Fenn 1957; McLaughlin 1968; Hinke e t al_. 1973). About h a l f of the dry weight of a barnacle muscle c e l l apparently is due to soluble organic molecules (M.E. Clark, personal communication), accounting for the larger apparent capacity, but the experiments with membrane-damaged c e l l s might r e f l e c t compartmentalization with l e s s - r a p i d exchange. From these morphological and ph y s i o l o g i c a l studies, then, i t seems reasonable to conclude that, i n barnacle muscle c e l l s , the c e l l membrane and membrane-delimited organelles'which sequester sodium and are not d i r e c t l y open to the e x t r a c e l l u l a r space contain only a small portion of the i n t r a c e l l u l a r sodium not detected by the microelectrode ( i n the model of the c e l l outlined above). Most of this small pool of sodium should.engage i n 30 rapid exchange with sodium i n free s o l u t i o n in the c e l l and so influence f l u x experiments, but i t appears that accurate measurement of the extra- c e l l u l a r sodium might be a more s i g n i f i c a n t problem. The plan of experimental i n v e s t i g a t i o n of this problem for this thesis was to follow changes in the sodium content of the compartment measured by the microelectrode, as the t o t a l sodium content of the c e l l was manipulated. It was found that indeed a great deal of the 'missing sodium' appears to reside i n the e x t r a c e l l u l a r space, but that there appears to be some sequestered inside the c e l l as w e l l . These experiments are described i n section 3. It was concluded from these experiments that the i n t r a c e l l u l a r sodium a c t i v i t y measured by a sodium-specific microelectrode was the most su i t a b l e parameter against which the sodium e f f l u x should be compared i n k i n e t i c studies. An a d d i t i o n a l advantage of the use of continuous measurements with the microelectrode was that rapid changes i n the i n t r a c e l l u l a r sodium a c t i v i t y could be followed. I t was a n t i c i p a t e d that these might occur i n sodium-free solutions under c e r t a i n conditions, as they had in frog s k e l e t a l muscle (White & Hinke 1976). It was also concluded that an attempt should be made to load the inside of the c e l l with radiosodium s e l e c t i v e l y , so that e f f l u x from the extra- c e l l u l a r space would not conceal any part of the transmembrane f l u x (White & Hinke 1976), although this is not the only way to accomplish t h i s end. This meant that a study of the e f f e c t s of microinjection had to be done, and this i s described i n section 4. M i c r o i n j e c t i o n studies can also y i e l d information about the states of sodium inside the c e l l , as w i l l be described l a t e r . 31 C. THE SODIUM EFFLUX As noted above, the general experimental approach to the sodium e f f l u x has been to compare the data with the predictions of k i n e t i c models i n hope of determining the general k i n e t i c properties of the sodium transport systems. The behavior of the transport systems when the c e l l s are in phy s i o l o g i c a l s a l i n e i n v i t r o should be close to that i n vivo. However, i t is l i k e l y that there i s more than one transport mode, so the r e s u l t s i n the ph y s i o l o g i c a l s a l i n e (normal Ringer's solution) probably r e f l e c t the contributions of several modes. It has been of int e r e s t to compare the k i n e t i c s i n normal Ringer's s o l u t i o n with the k i n e t i c s i n solutions where one possible mode has been al t e r e d . For example, a sodium-potassium exchange mode should be markedly reduced i f potassium i s omitted from the sa l i n e , and the contribution of other modes to the sodium e f f l u x seen i n this s i t u a t i o n w i l l be greater. It bears repeating that this maneuver possibly does not just reduce the sodium-potassium exchange mode, but rather causes i n addition a change i n the contribution of the other modes to the e f f l u x . Neither the s i z e of the sodium-potassium exchange mode nor the s i z e of the contribution of the other modes i n normal Ringer's s o l u t i o n can be measured. Nevertheless, i t i s possible to obtain information from experiments such as these. An example from the l i t e r a t u r e is the find i n g that i n invertebrate giant axons, the reduction i n the sodium e f f l u x which follows removal of external potas- sium can be appreciably greater than the s i z e of the potassium i n f l u x . This was strong support for the hypothesis that potassium ions act as ac t i v a t o r s of the sodium e f f l u x as well as engaging i n exchange for sodium (Hodgkin & Keynes 1955a; Sjodin & Beauge 1967; Mullins & Brinley 1967, 1969; Baker, Blaustein, Keynes, Manil, Shaw & Steinhardt 1969). 32 Examination of the k i n e t i c s of the t o t a l sodium e f f l u x t r a d i t i o n a l l y has been done for e f f l u x into sodium-free solutions, because some experi- ments indicated that a great deal of sodium-for-sodium exchange occurred across the c e l l membrane (Keynes & Swan 1959, but see Mullins & Frumento 1963). This was considered to be e n t i r e l y separate from the 'active e f f l u x ' , which was the e f f l u x of most int e r e s t (Keynes 1966, but see Keynes & Stein- hardt 1968). Such experiments defined the problem of the dependence of the e f f l u x on the i n t e r n a l sodium concentration, so they w i l l be reviewed b r i e f l y here with reference to th i s question. Keynes and Swan (1959) found that a plot, for selected experiments, of the e f f l u x of radiosodium from frog s k e l e t a l muscle versus the t o t a l amount 23 of radiosodium remaining i n the muscle, as radiosodium and Na were washed out into sodium-free lithium-substituted Ringer's solution, implied a power law r e l a t i o n s h i p between e f f l u x and sodium content. They suggested that the c a r r i e r i n the membrane can only operate when "n" sodium ions were bound to i t , where n appeared to be three. This was assumed to be indepen- dent of the sodium-sodium exchange seen i n normal Ringer's solution. This model was a t t r a c t i v e because i t required only one i n t r a c e l l u l a r compartment, hence no complicated exchanges of sodium between i n t r a c e l l u l a r compartments, and yet explained the data f a i r l y w e l l . Of course, a power law takes no account of saturation of the transport system, and should f i t best at very low values of the c e l l u l a r sodium content. Keynes and Swan found deviations at low rather than at high levels of sodium. Mullins and Frumento (1963) extended these experiments to higher values of sodium content. They found a "cube law" f i t best at low sodium concentration, but that the 'power1 decreased as the sodium content increased, and that saturation occurred. (At very high sodium content, a very rapid e f f l u x was seen. This w i l l be discussed with the sodium-free e f f e c t below.) They used a r e l a t i o n very 33 s i m i l a r to the Michaelis-Menten case of three sodium ions binding simul- taneously, discussed above, but did not use the Michael is-Menten model. Later, Keynes and Steinhardt (1968) recanted on the power law model because the large sarcoplasmic reticulum of the frog muscle c e l l had been described (Peachey 1965), providing a morphological basis for two-compart- ment models. In addition, the large f r a c t i o n of the i n t r a c e l l u l a r sodium not detected by microelectrodes had been described for frog muscle (Lev 1964). A " s e r i e s - p a r a l l e l " model with f i r s t - o r d e r k i n e t i c s was proposed, and explained some of the observations. However, the properties of the (Na+K)ATPase had been further elucidated i n the meantime, and i t appeared that about three sodium ions were trans- ported per ATP molecule hydrolyzed (Glynn 1962; Bonting & Caravaggio 1963; Sen & Post 1961, 1964). The mechanism suggested by Keynes and Swan thus could not be ignored. The k i n e t i c s of the various modes of the ATPase were v e r i f i e d (eg. Glynn & K a r l i s h 1975), but attempts to c l a r i f y the k i n e t i c s of the sodium e f f l u x from intact c e l l s other than erythrocytes tended instead to reveal added complications (eg. White & Hinke 1976). (i) Sodium e f f l u x into normal Ringer's so l u t i o n. The e f f l u x of sodium M^a into normal Ringer's solution, that i s , where the e x t r a c e l l u l a r sodium concentration has not been reduced, has been measured i n several c e l l types, and the question of how the e f f l u x varies with the i n t e r n a l sodium concentration (Na) has been considered. In the squid axon, M^a is a s t r i c t l i n e a r function of the i n t r a c e l l u l a r sodium concentration over the range of 1 to 220 mM (Hodgkin & Keynes 1956; Sjodin & Beauge 1967; Brinley & Mullins 1968). No saturation was seen. I t is known that nerve c e l l s have high concentrations of the (Na+K)ATPase (eg. Bonting, Simon, & Hawkins 1961), but i t i s not cl e a r why such a large 34 pumping capacity i s needed by squid axon. In s n a i l neurones, the rate of f a l l of the i n t r a c e l l u l a r sodium a c t i v i t y (ajj a) following iontophoretic i n j e c t i o n of sodium ions is an a f f i n e function of (a ), that i s , l i n e a r above a threshold value of (a,T ) (Thomas 1972b). v Na Na' v 7 In frog v e n t r i c u l a r muscle, no saturation was seen over the range of sodium content studied, but M^a rose as (Na)? for n between 1.0 and 1.6 in d i f f e r e n t experiments (Van der Kloot & Dane 1964). In red blood c e l l s (Garay & Garrahan 1973) and in frog s k e l e t a l muscle (Harris 1965) the r e l a t i o n s h i p between Mjj a and (Na) ̂  i s sigmoidal. In barnacle muscle c e l l s , B r i n l e y (1968) found saturation of M^a at high (Na)^, with the shoulder at ca. 20mM. The experiments to be described i n section 5 of. this thesis revealed that the true behavior of the sodium e f f l u x from barnacle muscle into normal Ringer's s o l u t i o n i s s i m i l a r to that from the squid axon and s n a i l neurone. Saturation does not occur at the low l e v e l found by Brinley (1968). ( i i ) Sodium e f f l u x into potassium-free sol u t i o n. Steinbach (1940).showed that when frog s k e l e t a l muscle is soaked i n potassium-free Ringer's solution, i t loses potassium and gains sodium. Return of potassium to the s o l u t i o n enables the c e l l s to extrude some of the accumulated sodium. The e f f e c t was ascribed to a reduction i n the sodium e f f l u x i n potassium-free media. A s i m i l a r e f f e c t was found i n red blood c e l l s by Harris and Maizels (1951), and in giant axons by Hodgkin and Keynes (1954). I t was not due to permeability changes or to changes i n the membrane po t e n t i a l . Harris and Maizels (1952)-proposed that the potassium i n f l u x and the sodium e f f l u x i n red c e l l s were linked. As described above, linked sodium and potassium transport was found to be the p r i n c i p a l mode of the 'sodium pump' under normal conditions. The stoichiometry of the coupling 35 appears to be fixed i n red c e l l s (Garrahan & Glynn 1967c) but to be v a r i a b l e in frog s k e l e t a l muscle (Cross, Keynes, Rybova 1965) and i n squid axon (Mullins & Brinley 1969). However, the decrease i n the sodium e f f l u x from squid axon upon removal of the external potassium can far exceed the magni- tude of the potassium i n f l u x , as discussed by Sjodin (1971). I t is implied that potassium ions act as a c t i v a t o r s for sodium transport. In barnacle muscles, too, removal of the e x t r a c e l l u l a r potassium r e s u l t s i n a decrease in the sodium e f f l u x (Brinley 1968; B i t t a r et,.al. 1972). Elevation of the potassium concentration of the bathing solution, to a value between 8 (the normal value) and 40 mM, had no e f f e c t on the sodium e f f l u x in barnacle, even though the c e l l s contract at concentrations greater than 20mM. ( B i t t a r et a l . 1972). However, further increases above 40 mM caused a marked stimulation of the sodium e f f l u x . A s i m i l a r e f f e c t was found i n frog s k e l e t a l muscle by Horowicz and Gerber (1965a,b), and they proposed that the increase i n sodium e f f l u x was mediated by the coincident a l t e r a t i o n s of the membrane po t e n t i a l due to the change i n the potassium concentration. Beauge and Sjodin (1976) have shown that for frog s k e l e t a l muscle c e l l s i n which E m i s made unresponsive to changes i n the external potassium concentration by prolonged incubation in potassium-rich solutions, changes i n the external potassium concentration between 0 and 10 mM a c t i v a t e the sodium e f f l u x along an a c t i v a t i o n curve almost i d e n t i c a l to that obtained for untreated control c e l l s . The potas- sium-activated sodium e f f l u x also was shown to d i f f e r from the azide- stimulated sodium e f f l u x , which also was independent of membrane pot e n t i a l changes. Beauge and Sjodin suggest that external potassium activates the sodium pump i n frog muscle by a l t e r i n g the transport enzyme d i r e c t l y , at l e a s t for external potassium concentrations between 0 and 10 mM. The s i t e at which a c t i v a t i o n by potassium occurs is d i s t i n c t from the c a t a l y t i c s i t e 36 for potassium transport. The a c t i v a t i o n at very high concentrations of external potassium remains to be explained. Only the e f f e c t of reduction of the external potassium concentration was examined experimentally i n the present work, as descirbed i n section 5. It was found that the e f f l u x into potassium-free solutions behaved much l i k e the e f f l u x into ouabain-containing Ringer's solu t i o n . ( i i i ) Sodium e f f l u x into sodium-free solu t i o n. The removal of sodium from the e x t r a c e l l u l a r medium reverses the gradient of electrochemical p o t e n t i a l which is the d r i v i n g force for 'passive' sodium ion movement across the c e l l membrane. This should make the e f f l u x of sodium from the c e l l less c o s t l y i n terms of energy, and so r e s u l t i n an increase both i n the passive and i n the a c t i v e e f f l u x . However, for enzyme- mediated transport of i n t r a c e l l u l a r sodium to occur, sodium might be re- quired at an external nontransport s i t e . I t has also been suggested that an exchange of i n t e r n a l for external sodium ions might occur, with no net fl u x of ions and no net consumption of energy. There are two classes of 'sodium-free e f f e c t s ' on the basis of the time course. The diffe r e n c e between the steady sodium e f f l u x into normal Ringer's s o l u t i o n and the steady sodium e f f l u x into sodium-free s o l u t i o n has been the one more studied. There i s also a transient rapid loss of sodium from the myoplasm of frog s k e l e t a l muscle and crab s t r i a t e d muscle and from the s n a i l neurone upon removal of sodium from the bathing so l u t i o n . The better-known e f f e c t w i l l be reviewed f i r s t . Ussing (1947, 1949) f i r s t suggested the p o s s i b i l i t y that a one-for-one exchange of i n t r a c e l l u l a r sodium for e x t r a c e l l u l a r sodium could occur, with no net expenditure of metabolic energy. This would complicate the interpre- t a t i o n of radiosodium fluxes. Subsequently i t was found (Keynes & Swan 37 1959) that the radiosodium e f f l u x from frog skeletal.muscle was r e v e r s i b l y reduced to about h a l f when the external sodium was replaced by l i t h i u m or choline, with external potassium unchanged. The f r a c t i o n a l reduction was less when the sodium content of the muscle was elevated by incubation i n an appropriate solution, but could be restored by lowering the sodium content again by a further incubation. External potassium did not a f f e c t the sodium-free response. In squid axon, however, choline- or lithium-substituted sodium-free solutions caused an increase i n the e f f l u x of radiosodium (Hodgkin & Keynes 1955; Mullins et a l . 1962). Mullins (appendix to Mullins & Frumento 1963) suggested that accumulation of incoming sodium near the in t e r n a l s i t e of an a c t i v e transport enzyme, due to r e s t r i c t e d d i f f u s i o n from t h i s l o c a t i o n to the bulk cytoplasm, could account for both of these observations, where the i n t r a c e l l u l a r sodium concentration (Na) was the determining factor. Subsequently i t was shown that removal of external sodium does indeed cause a decrease i n the radiosodium e f f l u x from squid axons of low sodium content (lowered by stimulation of the axon i n l i t h i u m solution) (Frumento & Mullins 1967; Mullins & Br i n l e y 1967; Sjodin & Beauge 1968a), and an increase i n the radiosodium e f f l u x from frog s k e l e t a l muscle of high sodium content (loaded by soaking for 20 hours at 2 deg.C i n potassium-free solution); (Keynes 1965; Beauge & Sjodin 1968). (The glycoside s e n s i t i v i t y of the e f f e c t d i f f e r s i n the two tissues (Sjodin & Beauge 1968b; Baker, Bl^ustein, Keynes, Manil, Shaw & Steinhardt 1969)). In toad oocyte, however, there was l i t t l e e f f e c t of removal of external sodium, whatever the sodium content (Dick & Lea 1964). A cardiac g l y c o s i d e - s e n s i t i v e sodium-sodium exchange of the type seen i n red blood c e l l s (Garrahan & Glynn 1967) (there i s no glycoside i n - s e n s i t i v e sodium-sodium exchange i n red blood c e l l s ) was thought to be 38 responsible for only part of the external sodium-dependent e f f l u x seen i n muscle since the e f f e c t s of glycosides and the removal of external sodium appeared to be independent and a d d i t i v e (Horowicz 1965; Keynes 1966; Sjodin & Beauge 1968a). Glycoside decreased both sodium e f f l u x and i n f l u x by about 207» i n frog muscle in potass ium-free so l u t i o n but the i n f l u x was unaffected by glycoside i n potassium-containing s o l u t i o n (Keynes & Stein- hardt 1968; Keynes 1966), as for red blood c e l l s (Garrahan & Glynn 1967). Lithium appeared to stimulate the a c t i v e sodium e f f l u x from frog s k e l e t a l muscle i n the same way that potassium, rubidium, and cesium do (Beauge & Sjodin 1968). The stimulation was abolished by cardiac glycosides. A "dual e f f e c t " of lithium-substituted sodium-free solutions on sodium e f f l u x was proposed: (a) removal of- e x t r a c e l l u l a r sodium prevents the sodium- dependent e f f l u x , and (b) l i t h i u m stimulates the sodium e f f l u x by acting l i k e potassium externally. Thus as the i n t e r n a l sodium content r i s e s , the pump rate w i l l r i s e , and so the stimulatory e f f e c t of l i t h i u m w i l l increase. Eventually this stimulated e f f l u x w i l l surpass the i n h i b i t o r y e f f e c t of the lack of external sodium. A complication of these studies i n whole muscle preparations i s that i n potassium-free solutions, potassium which leaves the c e l l s passively can accumulate i n the e x t r a c e l l u l a r space in s u f f i c i e n t amounts to stimulate sodium e f f l u x appreciably (Sjodin & Beauge 1973). Beauge (1975) estimated that almost h a l f of the-stimulation of the sodium e f f l u x seen i n sodium- free, potassium-free, lithium-substituted s o l u t i o n i n frog muscle was due to this reaccumulation of potassium. Sachs (1977) has shown that the e f f e c t s of external sodium on sodium extrusion by red blood c e l l s are consistent with' sodium acting as a "dead- end competitive i n h i b i t o r and as a heterotropic a l l o s t e r i c e f f e c t o r " on the (Na+K)ATPase. 39 It was found that a small glycoside-sensitive increase i n the sodium e f f l u x from frog s k e l e t a l muscle was obtained i f calcium or magnesium were used to replace external sodium, as.well as when l i t h i u m was so used (Horowicz, Taylor, & Waggoner 1970). I t was concluded that most of the g l y c o s i d e - i n s e n s i t i v e sodium e f f l u x required external sodium, but of the glyc o s i d e - s e n s i t i v e sodium e f f l u x , part can be i n h i b i t e d by external sodium (?sodium-sodium exchange). The former part of the gly c o s i d e - s e n s i t i v e e f f l u x appears when (Na)^ i s high, and the l a t t e r when (Na)^ i s low. Similar r e s u l t s were obtained by another worker (Sjodin 1971). That the glycoside- s e n s i t i v e e f f e c t s are due to the (Na+K)ATPase is supported by r e s u l t s on the i s o l a t e d enzyme (Robinson 1975). Further, Kennedy and DeWeer (1976) have demonstrated a strophanthidin-sensitive increase i n sodium e f f l u x r e q u i r i n g external sodium but not potassium, i n frog s k e l e t a l muscle i n which the ATP/ADP r a t i o had been lowered by poisoning. A strophanthidin-sensitive sodium i n f l u x of s i m i l a r s i z e also occurs under these conditions. In squid axons, Baker, Blaustein, Keynes et a l . (1969) saw sodium- sodium exchange under conditions of p a r t i a l poisoning, contrary to the r e s u l t s of Frumento and Mullins (1964). The exchange was glycoside-sensitive. Poisoning with CN or DNP, or i n j e c t i o n of apyrase (which hydrolyzes ATP) a l l g i v e r r i s e to an external-potassium-independent, external-sodium-dependent sodium e f f l u x (DeWeer 1970, 1974). Apparently the presence of ADP i n high concentrations i s the c r i t i c a l factor. This is s i m i l a r to the r e s u l t s i n red blood c e l l s (Garrahan & Glynn 1967b; Glynn & Hoffman 1971). Since the potassium-free and sodium-free e f f e c t s are highly correlated (Sjodin & Beauge 1969; DeWeer 1970), i t was concluded that the 98% of the sodium e f f l u x from squid axon which i s not passive is due to: (a) potassium- stimulated sodium e f f l u x , i n h i b i t e d by external sodium (ascribed to the sodium-potassium exchange mode of the pump), and (b) external-sodium- 40 stimulated sodium e f f l u x , i n h i b i t e d by external potassium (ascribed to the sodium-sodium exchange mode plus a d i f f e r e n t , g l y c o s i d e - i n s e n s i t i v e mechanism) . Sodium-sodium exchange conceivably could occur by a mechanism other than a c l o s e l y - l i n k e d one-for-one exchange. For example, i f from the measured sodium i n f l u x into frog muscle one subtracts the passive i n f l u x calculated i n the constant f i e l d approximation, there remains a component of the sodium i n f l u x which i s about 38% of the t o t a l ( i n normal Ringer's solution) (Venosa 1974). I f the c e l l is maintaining a constant sodium content, this must be balanced by sodium extrusion, (as i s the passive i n - f l u x ) . I f this i n f l u x is v i a an enzyme, the net r e s u l t is enzyme-mediated sodium-sodium exchange comprising 38% of the t o t a l sodium e f f l u x , but i t need not be one-for-one exchange by. one enzyme. The notion of one-for-one exchange of sodium ions eliminates the need to specify a counterion motion, but c l e a r l y creates other d i f f i c u l t i e s . I t might be that one enzyme mediates sodium i n f l u x and a nearby enzyme mediates sodium e f f l u x . The key question is how far apart can such a pair of d i f f e r e n t enzymes be yet operate in a 'thermodynamically permissible 1 manner. Perhaps another i n d i c a t i o n of the existence of a separate sodium transport enzyme is the e f f e c t of e t h a c r i n i c acid on muscle. E t h a c r i n i c a c i d appears to i n h i b i t the external-sodium-dependent sodium i n f l u x without reducing the glycoside-sensitive, external-potassium-sensitive sodium e f f l u x ( E r l i j & Leblanc 1971), but only i n glycoside-treated muscles: eth a c r i n i c a c i d alone stimulates the sodium e f f l u x . E t h a c r i n i c a c i d has no e f f e c t on the sodium e f f l u x from barnacle muscle normally, but prevents the increase i n sodium e f f l u x which usually follows exposure to (Danielson et^ a l . 1972). 41 In summary, i t appears that i n nerve (under some conditions) and i n muscle (normally), there probably i s a component of the sodium e f f l u x which requires external sodium. It i s usually regarded as s t r i c t one-for-one exchange of in t e r n a l for external sodium. On the basis of the e f f e c t of poisons, i t would appear that much of this exchange proceeds by a mechanism other than the (Na+K)ATPase, although sodium"sodium exchange of the (Na+K)ATP- ase type can be demonstrated under some conditions. Since removal of external sodium causes a decrease i n the sodium e f f l u x when (Na) i s low, but an increase when (Na)^ is high, there appears to be a mechanism by which external sodium can i n h i b i t the sodium e f f l u x . This could be the sodium- potassium mode o f the (Na+K)ATPase. The sodium-free e f f e c t i s the combined r e s u l t of (at least) these two ef f e c t s , and the net e f f e c t observed i n experiments i n v i t r o depends on the experimental conditions (Sjodin 1971). In frog s k e l e t a l muscle, 20 to 507=, of the sodium e f f l u x under normal conditions i s of the sodium-sodium exchange type. In squid axon, none of the sodium e f f l u x under normal conditions is of the sodium-sodium exchange type. The s i t u a t i o n i n barnacle muscle was not cl e a r . Brinley,(1968) found that replacement of external sodium by li t h i u m reduced the radiosodium e f f l u x by 677OJ while replacement by sucrose reduced i t by 477o. (Potassium- free solutions reduce the radiosodium e f f l u x by 517».) In c e l l s with higher sodium content, an increase i n e f f l u x was seen, but the absence of external sodium always caused a contracture, so the effects could not be measured. Experiments on this type of sodium-free e f f e c t are described i n section 5. Both an i n h i b i t o r y and a stimulatory e f f e c t of the removal of external sodium were seen i n barnacle muscle c e l l s , but on the whole the k i n e t i c c h a r a c t e r i s t i c s of the e f f l u x into sodium-free sol u t i o n were the same as those of the e f f l u x into normal Ringer's solution. That i s , much of the 'sodium-free e f f e c t s ' i n barnacle muscle is due to changes i n the sodium 42 content of the c e l l s which occur when they are placed in sodium-free solution. A second 'sodium-free e f f e c t - has been reported. Exposure of frog s k e l e t a l muscle to sodium-free solutions (substituted with l i t h i u m or t r i s ) causes a large rapid f a l l of the i n t r a c e l l u l a r sodium a c t i v i t y measured with an i n t r a c e l l u l a r microelectrode (White & Hinke 1976). A s i m i l a r rapid e f f e c t has. recently been reported i n crab muscle (Vaughan-Jones 1977), and a s i m i l a r but slower e f f e c t e n t i r e l y consistent with the continuation of the normal sodium e f f l u x i n the absence of i n f l u x , was found in s n a i l neurone (Thomas 1972b). For frog muscle (White & Hinke 1976), the time course of the f a l l could be f i t t e d by a sum of two exponential terms. The two rate constants were unchanged by ouabain treatment, but the 'capacity' of the k i n e t i c compartment defined by the more rapid rate was reduced. The slower rate was comparable to that for sodium i n f l u x , and was i d e n t i f i e d as "passive" leakage. The more rapid rate was i d e n t i f i e d as ac t i v e sodium extrusion, and the rate constant was the same as that found for the washout of l a b e l l e d sodium from the e x t r a c e l l u l a r space. With a muscle which had been loaded with radiosodium by passive uptake, the washout of radiosodium from the e x t r a c e l l u l a r space would mask such a rapid e f f l u x unless the e x t r a c e l l u l a r space was cleared of radiosodium before the muscle was exposed to sodium-free s o l u t i o n (White & Henke 1976). Chemical analysis of the muscles indicated that the f a l l i n myoplasmic sodium a c t i v i t y is due to movement of sodium ions out of the c e l l (White & Hinke 1976), but this has not been confirmed. Some e a r l i e r workers attached some s i g n i f i c a n c e to the i n i t i a l r a p id exchange of radiosodium i n whole frog muscle (Carey & Conway 1954). Others discarded the f i r s t twenty minutes of the isotope e f f l u x data from sin g l e muscle c e l l s , which they 43 quite reasonably assumed to represent washout of the e x t r a c e l l u l a r space almost e x c l u s i v e l y (Hodgkin & Horowicz 1959). The s i t u a t i o n i n crab muscle is quite d i f f e r e n t (Vaughan-Jones 1977). The rapid f a l l of the myoplasmic sodium a c t i v i t y on exposure of the c e l l to sodium-free lithium-substituted or t r i s - s u b s t i t u t e d s o l u t i o n was unaffected by ouabain, the removal of e x t r a c e l l u l a r potassium, calcium, or magnesium, or by changes i n the e x t r a c e l l u l a r pH. I t was blocked by manganese, cobalt, and lanthanum, which are known to block the movement of divalent cations across membranes, but not by D600 or Verapamil, which block calcium fluxes i n nerve. However, lanthanum and, to a le s s e r extent, manganese themselves often caused a rapid f a l l of the myoplasmic sodium a c t i v i t y i n the presence of external sodium. I f the rapid e f f l u x of sodium i s not accompanied by potassium i n f l u x , nor by ch l o r i d e e f f l u x (the myoplasmic chloride a c t i v i t y i s not changed i n low external sodium s o l u t i o n s ) , one would expect a considerable electrogenic c o n t r i b u t i o n to the membrane po t e n t i a l to occur. Only a s l i g h t depolariza- t i o n was found. Perhaps there is some counterion or co-ion transport, or the permeability to l i t h i u m might be greater than that to sodium and a concurrent depolarization thus r e s u l t i n the r e s t i n g membrane p o t e n t i a l , approximately countering the electrogenic e f f e c t . Calcium i n f l u x i s suggested by the r e s u l t s with manganese, cobalt, and lanthanum, and by the s i m i l a r time course of the r i s e of (Ca)^ in crab muscle and squid axon under s i m i l a r conditions. Removal of external calcium prevents the r i s e of the sodium e f f l u x normally seen i n squid axon when exposed to l i t h i u m solutions (Baker, Blaustein, Hodgkin et a l . 1969). However, with crab muscle removal of e x t r a c e l l u l a r calcium and/or magnesium had no e f f e c t . Further, a sudden rapid i n f l u x of calcium would t r i g g e r a contraction, and this did not occur. I t might be that the e x t r a c e l l u l a r 44 space of the whole-muscle preparation used was not completely cleared of calcium or potassium, but t h i s i s u n l i k e l y because long washout times were used. The s i m i l a r i t y of the response to lanthanum and removal of external sodium was noted above. Lanthanum can displace quite large quantities of membrane-bound calcium, and i n t r a c e l l u l a r calcium stimulates sodium e f f l u x from barnacle muscle ( B i t t a r et a l . 1972, 1973). Altogether, though, the processes which lead to a decline i n the myoplasmic sodium a c t i v i t y remain unknown. Measurements with the i n t r a c e l l u l a r microelectrode can only detect loss of sodium from the major i n t r a c e l l u l a r compartment. They cannot reveal the fate of the l o s t sodium. Therefore an experiment i n which the micro- electrode measurements were combined with radiosodium e f f l u x measurements was devised, as described i n se c t i o n 6. I t was found that a rapid f a l l i n the myoplasmic sodium a c t i v i t y s i m i l a r to that i n frog and crab muscle occurs i n barnacle muscle under c e r t a i n conditions, and that i t i s accom- panied by a rapid loss of sodium from the c e l l . As with s n a i l neurone, this was found to be due to the continuing normal operation of the sodium e f f l u x i n the absence of sodium i n f l u x . (iv) The e f f e c t of ouabain on the e f f l u x of sodium. Cardiac glycosides, p r i n c i p a l l y ouabain (g-strophanthin) and i t s aglycone strophanthidin, have long been known to i n h i b i t the transport of sodium and potassium i n red blood c e l l s and frog s k e l e t a l muscle (Schatzmann 1953; Matchett & Johnson 1954). Some such action had been suspected because toxic doses of the drugs were known to cause a loss of potassium from heart muscle (Schatzmann & Witt 1954). It was found l a t e r that cardiac glycosides a l s o i n h i b i t the a c t i v i t y of the (Na+K)ATPase (Skou 1965). The current theory of the act i o n of cardiac glycosides (Schwartz et a l . 45 1975; Glynn & K a r l i s h 1975) i s that they bind s p e c i f i c a l l y to a s i n g l e s i t e on the (Na+K)ATPase which is separate from the c a t a l y t i c s i t e s . The binding s i t e is exposed only at the external surface of the c e l l membrane, and there i s a p a r t i c u l a r conformation of the enzyme which favours binding of the glycoside. The binding i s very strong. The h a l f time for d i s s o c i a - t i o n of the i s o l a t e d enzyme-glycoside complex at 37°C is about 2.5 hours although the p h y s i o l o g i c a l e f f e c t of glycosides can be reversed much more ra p i d l y . The binding does not render the enzyme completely inactive. Although the sodium-potassium exchange appears to be prevented, some p a r t i a l or side reactions can s t i l l occur (Glynn et a l . 1974). I t is conceivable that the e f f e c t s of glycoside are d i f f e r e n t for the i s o l a t e d and the i n s i t u enzyme, however. In whole c e l l s , cardiac glycosides can promote some modes of ion flux. In squid.axons with low ATP content, strophanthidin increased the rate of sodium e f f l u x (Brinley & Mullins 1968). Strophanthidin also increases the potassium e f f l u x from frog s k e l e t a l muscle (Harris 1957; Sjodin & Beauge 1968a) and squid axons (Mullins & B r i n l e y 1969). The i n h i b i t i o n of the sodium e f f l u x by strophanthidin increases with increasing sodium content i n 'aged' frog sartorius muscle (Sjodin & Beauge 1968a) but decreases with increasing sodium content i n freshly-dissected frog sartorius muscle (Horowicz et al_. 1970) . Dependence of fluxes on ATP does not always c o r r e l a t e with the s e n s i - t i v i t y to glycosides (Mullins & B r i n l e y 1969). In ATP-depleted squid axon, strophanthidin causes a marked increase i n the sodium e f f l u x , against the gradient of electrochemical p o t e n t i a l , while leaving the sodium i n f l u x unchanged (Mullins 1972). Recent experiments on red blood c e l l ghosts (Bodeman & Hoffman 1976) revealed that i n the presence of external potassium, the rate at which 46 ouabain bound decreased when either the in t e r n a l sodium or the in t e r n a l potassium was raised. When external potassium was not present, such v a r i a t i o n s i n ion content had no e f f e c t on ouabain binding. In other experiments, the f i n a l amount of ouabain bound was not affe c t e d by such manipulations (Schwartz eit a l . 197 5), and i t i s not known i f an increase i n the i n t e r n a l ion concentrations can promote the d i s a s s o c i a t i o n from the enzyme of ouabain which is already bound to the enzyme. The effects of strophanthidin on the sodium e f f l u x from barnacle muscle c e l l s were studied by Brin l e y (1968). He found that there was l i t t l e or no -8 i n h i b i t i o n at 10 M. strophanthidin and that as the concentration of stroph- anthidin was increased to 10 ^M, the percent i n h i b i t i o n increased. The maximum i n h i b i t i o n was about 907o and occurred for concentrations of stroph- anthidin greater than or equal to 10 "'M. In one c e l l of very high sodium content, there was a delayed increase i n the sodium e f f l u x a f t e r an i n i t i a l -4 f a l l at 10 M strophanthidin. Ouabain appeared to be s l i g h t l y less e f f e c t i v e . The i n h i b i t i o n was less i n c e l l s which had a larger sodium content. Strophanthidin produced a greater reduction of sodium e f f l u x than did removal of external sodium and/or potassium, and removal of external sodium and potassium did not increase the i n h i b i t i o n i n strophanthidin- treated c e l l s . The e f f e c t s of ouabain on the sodium e f f l u x from barnacle muscle c e l l s were studied by B i t t a r et a l . (1973). They obtained dose-response curves by exposing i s o l a t e d c e l l s to increasing concentrations of ouabain, as Bri n l e y had done, and obtained a s i m i l a r curve but with a maximum i n h i b i t i o n of about 707o. They used the reduction i n the f r a c t i o n of radiosodium l o s t per unit time as t h e i r measure of i n h i b i t i o n , while Brinley had calculated _3 the s i z e of the sodium e f f l u x using an estimated value for (Na)^. At 10 M, a f t e r about twenty minutes of exposure to ouabain, the e f f l u x of radiosodium 47 began to r i s e again. The i n h i b i t i o n was greater i n c e l l s which had "slope r a t i o s " close to 1, that i s , i n c e l l s having a lower sodium content (see sec- t i o n 4). Inj e c t i o n of ouabain into the c e l l s caused no change i n the sodium e f f l u x . The increase i n the sodium e f f l u x caused by depolarization of the c e l l s , by r a i s i n g the external potassium concentration or by i n j e c t i n g CaC^, was not in h i b i t e d by ouabain. Nor was the increase i n the sodium e f f l u x caused by CO2 treatment. B i t t a r et a l . concluded that there are at least two separate sodium extrusion systems, located i n d i f f e r e n t parts of the membrane. The e f f e c t s of several cardiac aglycones on the sodium e f f l u x i n barnacle muscle c e l l s have also been studied ( B i t t a r & Brown 1977). They a l l appear to bind to the cardiac glycoside s i t e and to have the same ef f e c t s as cardiac glycosides. Only the potency d i f f e r s . Three aspects of the e f f e c t of ouabain on the sodium e f f l u x i n barnacle muscle were examined experimentally as part of the work reported i n th i s thesis. F i r s t , the e f f e c t on the dose-response curve of the use of the sodium electrode to measure the r i s i n g i n t r a c e l l u l a r sodium a c t i v i t y a f t e r ouabain begins to act was investigated. Impariment of the extrusion mechanism by ouabain should re- s u l t i n an immediate increase i n the sodium content of the c e l l as the passive i n f l u x continues unchanged, and the increased sodium content should be r e f l e c t e d i n the measured sodium e f f l u x . Second, the k i n e t i c c h a r a c t e r i s t i c s of the sodium e f f l u x i n c e l l s which are maximally i n h i b i t e d by ouabain were to be measured. I t turned out that this f l u x is an increasing function of the i n t r a - c e l l u l a r sodium a c t i v i t y at low levels of in t e r n a l sodium, but reaches a plateau at higher l e v e l s . These two aspects are described i n section 5. Third, an electrogenic c o n t r i b u t i o n of the sodium flux to the membrane po t e n t i a l was sought. Experiments which demonstrate the existence of an electrogenic poten- t i a l i n barnacle muscle w i l l be described i n section 7. Although the membrane po t e n t i a l has only now been mentioned i n connection 48 with the ion fluxes, i t is of course intimately involved with them. In much of the early work on ionic currents, the models of e l e c t r i c a l c i r c u i t s were adopted operationally. The r e s u l t has been a mixture of molecular physics and c i r c u i t theory, with emphasis on the l a t t e r . I t was f e l t to be worthwhile to review the orig i n s of the most commonly used models of the membrane poten- t i a l and ionic currents, and to indicate how they could be improved. This review is presented i n the next part of th i s section, and completes the in t r o - duction. I t i s followed by a summary of the problems to be addressed i n this thesis, and a de s c r i p t i o n of three models used l a t e r i n the in t e r p r e t a t i o n of experiments but gathered together for the convenience of the reader. D. THE TRANSMEMBRANE DIFFERENCE IN ELECTRICAL POTENTIAL In a l l c e l l s there is an e l e c t r i c a l p o t e n t i a l difference measurable across the c e l l membrane. Almost always, the e l e c t r i c a l p o t e n t i a l measured in the i n t e r i o r of the c e l l is negative with respect to that measured i n the bulk s o l u t i o n bathing the c e l l . In a number of c e l l s i t has been found that i f the rate of the sodium pump i s suddenly altered, there is an immediate change i n the re s t i n g mem- brane p o t e n t i a l . A decrease i n the pump rate, caused for example by applica- t i o n of ouabain, i s accompanied by a depolarization of the c e l l . That i s , the inside of the c e l l becomes less negative with respect to the outside. An increase i n the pump rate, caused for example by i n j e c t i o n of sodium into the c e l l i n t e r i o r , is accompanied by a hyperpolarization (see reviews by Kernan 1970; Thomas 1972; also DeWeer & Geduldig 1973; DeWeer 1974). Dif f e r e n t modes of the pump appear to have d i f f e r e n t degrees of 49 e l e c t r o g e n i c i t y (DeWeer 1974). The stoichiometry of the net sodium- potassium exchange by the transport enzymes appears to be fixed for red blood c e l l s at 3Na : 2K, but to vary from 1:1 to 3:1 in squid axon (Glynn & K a r l i s h 1975). A separation of charge such as i s implied by this could only be effected with a very large investment of energy. The question of how the charge separation necessary to produce the observed change i n membrane pot e n t i a l comes about is e s s e n t i a l l y the same as the question of how active sodium extrusion comes about. The development of ideas on the e l e c t r o g e n i c i t y of the sodium pump has been reviewed by Thomas (1972). Theories of the o r i g i n of the contribution of the electrogenic sodium pump to the r e s t i n g membrane po t e n t i a l are phenomenological extensions of the approach of Goldman (1943) and Hodgkin and Katz (1949). The notions of ionic permeability and conductance, and the ioni c mechanisms involved i n t h i s approach, often are employed i n other contexts. It is generally f e l t that the membrane pot e n t i a l E m can be regarded as b a s i c a l l y a d i f f u s i o n p o t e n t i a l , which arises i n nerve and s t r i a t e d muscle because of the s e l e c t i v e permeability of the c e l l membrane to potassium and the elevated i n t r a c e l l u l a r potassium concentration created by a c t i v e transport of ions. (Other l i k e l y contributions to the measured value are considered below.) KC1 tends to leak out of the c e l l across the membrane, but since the mobility i n the membrane of the potassium ions i s greater than that of the counterion c h l o r i d e (the m o b i l i t i e s are almost equal i n bulk s o l u t i o n ) , a small l o c a l charge separation occurs. This charge separation r e s u l t s i n an e l e c t r i c a l force which retards the potassium ion movement and promotes the chloride ion movement. A steady state is attained where the u n i d i r e c t i o n a l e f f l u x of K and Cl are equal. This i s ju s t d i k e _the f a m i l i a r l i q u i d junction-or d i f f u s i o n p o t e n t i a l (eg. • „ Lakshminarayanaiah 1969), but there i s no net loss of K because of the 50 sodium-potassium exchange pump, there, i s no net loss-of Cl at'the steady value of the r e s t i n g . p o t e n t i a l , and the d i f f u s i o n front i s fixed i n space by the membrane, l i k e the "constrained l i q u i d junction" of Planck (Lakshminarayanaiah 1969). From a model of the c e l l membrane as a homogeneous lamella, charac- t e r i z e d by m o b i l i t i e s (empirical r a t i o s of average d i f f u s i o n v e l o c i t y to d r i v i n g force) for each major ion which are much lower than the m o b i l i t i e s in bulk s o l u t i o n and in which the e l e c t r i c f i e l d is constant, Goldman (1943) derived an expression for the transmembrane f l u x of an ion by integrating, over the thickness of the membrane, a r e l a t i o n between the flux and the d r i v i n g force at each point i n the membrane (the Nernst-Planck equation). The i n t e g r a t i o n can be c a r r i e d out for sodium, potassium, and chloride ions, and when the t o t a l current i s zero, E _ RT l n J PK • ( K ) o f ?Na ' < N a )o + PC1 " ( C 1 ) i 1 m F (_P K . ( K ) i + P N a . (Na)i + P C 1 . ( C l ) 0 J (Hodgkin & Katz 1949), where subscript i refers to i n t r a c e l l u l a r and o to e x t r a c e l l u l a r , and P i s the permeability of the membrane to the ion (essen- t i a l l y the m o b i l i t y ) . (This w i l l be referred to as the 'GHK equation 1.) C l e a r l y t h i s model of d i f f u s i o n through a homogeneous slab does not coincide with the s i t u a t i o n i n a r e a l membrane. In r e a l i t y , the ions pass the membrane only at c e r t a i n locations, i e . in a s s o c i a t i o n with proteins. At the molecular l e v e l the movement of the ions bears no simple r e l a t i o n to that seen i n d i f f u s i o n in a bulk sol u t i o n . Yet the equation describes -the membrane p o t e n t i a l very well. Therefore the e s s e n t i a l physical basis of the equation must describe what i s occurring at the molecular l e v e l . The basis of the equation i s the Nernst-Planck equation, which simply states that the flux of an ion is proportional to the d r i v i n g force. The 51 d r i v i n g force for a system at constant and uniform pressure and temperature can be deduced from an expression for the energy change accomplished by the r e s u l t i n g flow, the flow being easy to define. The energy change is -dG (where G is the Gibbs free energy) and the force conjugate to a flow of ions is -^/u, wherey^= + RT"ln(a) + zF<f> is the electrochemical p o t e n t i a l for the ion. (a) = a c t i v i t y of the ion, <f> = e l e c t r i c a l p o t e n t i a l . (The d r i v i n g force can also be deduced i n the framework of i r r e v e r s i b l e thermo- dynamics, from the entropy production - eg. see Katchalsky & Curran 1967.) This "phenomenological force" i s an approximation, useful i n a macroscopic representation of a system i n which ' a l l gradients are s u f f i c i e n t l y gradual.' The thermodynamic functions cannot a c t u a l l y be defined at each point i n space, since they represent the interactions of a large number of p a r t i c l e s , and unless the system can be regarded as an aggregate of macroscopically- small volume elements, each containing a large number of p a r t i c l e s , the representation stated above cannot be applied with any expectation of success. Goldman (1943) stated that "the current c a r r i e r s pass through more or less randomly d i s t r i b u t e d i n t e r s t i c e s i n the structure (membrane), which is assumed uniform normal to the d i r e c t i o n of flow." The integration was along a d i r e c t i o n p a r a l l e l to the d i r e c t i o n of flow, passing through a pore. In the pore the ions were regarded as d i f f u s i n g as they do in bulk solut i o n , but i n one dimension and with much lower mobility. The r e s u l t , not s u r p r i s i n g l y , was s i m i l a r to that of Planck for a. 'constrained l i q u i d j u n c t i o n 1 . Hodgkin and Katz wrote the s o l u t i o n for the major ions and solved for the transmembrane p o t e n t i a l difference at zero net current, as stated above. A mechanistic model at the molecular l e v e l for the o r i g i n of the membrane pote n t i a l can be envisaged. The physical o r i g i n of the po t e n t i a l difference is indeed s i m i l a r to that in the case of a l i q u i d junction. In 52 the c e l l membrane, the protein channels through which cations pass are thought to be l i n e d with electronegative moieties, such as carbonyl or carboxyl groups, so that the c a t i o n i c charge can be p a r t i a l l y or completely balanced when i t i s in the channel. (These models are c a l l e d the "neutral polar pore" (Eisenman 1968; Mueller & Rudin 1967) and the " f i x e d charge pore" (Eisenman 1968)). There i s a f i n i t e concentration of cation i n the channel. When a cation enters the channel, i t leaves i t s counterion behind. This can occur only occasionally on the molecular scale of space and time, since a l o c a l concentration of negative charge would retard further egress of cation (via the channel or otherwise). Such an inhomogeneity of charge could be balanced by a movement of cations from the region to which the cations i n the channel are heading, i n a one-for-one exchange on average, but i t i s more l i k e l y that the anions w i l l be drawn a f t e r the cations i f a path is a v a i l a b l e . The anion c l e a r l y cannot r e a d i l y follow through the cation channel, so there i s assumed to be an anion channel nearby. The anions normally would pass through the channel i n a manner s i m i l a r to that of the cations, but the cations can do so more r e a d i l y ("higher mobility i n the membrane"). A separation of charge occurs, and an e l e c t r i c a l p o t e n t i a l difference r e s u l t s . The GHK equation has proved to be a good q u a l i t a t i v e and quantitative or semiquantitative d e s c r i p t i o n of the membrane p o t e n t i a l i n many s i t u a t i o n s . However, there are several ways i n which a quantitative r e l a t i o n between the p o t e n t i a l difference across the membrane and the d r i v i n g force for the flow of ions could be formulated. The net e f f e c t alone can be considered, so that the membrane channels are regarded as 'black boxes' characterized by a resistance or mobility. Then the current equals the quotient of the voltage and a resistance, or the fl u x v e l o c i t y equals the product of the net difference i n chemical p o t e n t i a l and a permeability (or mobility, 53 denoted u). Such a r e l a t i o n underlies the usual d e f i n i t i o n of membrane conductance (Hodgkin & Horowicz 1959b) and the usual conception of the 'electrogenic component' of the r e s t i n g membrane p o t e n t i a l (Hodgkin & Keynes 1955a). I f one regards the potassium flux i n th i s manner, for example, then 2 Mj, (moles/cm sec) = - u R . (K) ̂  . &jui = - u K . ( K ) ± . ( R-T-In ( R ) i + F E m ) 09 o 2 while Ij, (coulomb/cm sec) = g£ • ( Em ~ EK ) = S K • ( Em " ̂  * l n > F ( K ) i 2 and since Ij^ = Mjr . F, the conductance g^ would be F Ujr (K)^. In fact, Hodgkin and Horowicz (1959b) r e l a t e d g£ to Pj^ by s u b s t i t u t i n g from the constant f i e l d s o l u t i o n for Ijr i n Ijr = gjr ( E m - E K ) . They mixed t h e i r models, i n a sense, and the r e s u l t i n g r e l a t i o n s h i p between g^ and P^ i s a complicated function of E m and the concentrations. Thus the mobility i n the pure "net e f f e c t " model i s also a complicated function of E m and the concentrations, as might have been expected when the complications of the process are forced into the mobility as a p r o p o r t i o n a l i t y factor. The Goldman-Hodgkin-Katz (GHK) treatment, as already stated, regards the ions as d i f f u s i n g through a regime of reduced but constant mobility, a f t e r entering the channel by an unspecified process. The entry into the channel i s included i n the permeability P as a p a r t i t i o n c o e f f i c i e n t (^ ): the concentration of ion i n the membrane i s (3 times the concentration i n the bulk s o l u t i o n . A more d e t a i l e d model would treat entry into and e x i t from the channel as a mass-action s i t u a t i o n , again with d i f f u s i o n assumed to occur i n the channel. For example, T e o r e l l (1935) regarded t h i s as a Donnan equilibrium. 54 This sort of treatment was used by Eisenman et a l . (1968) to describe ion transport v i a neutral mobile c a r r i e r s i n the membrane, but the r e s u l t can be taken over to the channel model almost i n t a c t . The r e s u l t is the GHK equation plus an extra term, where the p a r t i t i o n c o e f f i c i e n t has been characterized e x p l i c i t l y . Perhaps a better example is the model proposed for the p o t e n t i a l developed i n a glass microelectrode ( a r t i c l e s by Doremus, by Eisenman, and by Nicolsky i n Eisenman 1967). The interactions at the surfaces, which provide the s e l e c t i v i t y , and the d i f f u s i o n through the matrix of the glass (described by a constant mobility) are treated separately, to y i e l d a r e l a t i o n l i k e the GHK equation,' ;although the counter- ion transport occurs v i a an external c i r c u i t . A s t i l l better representation would take into account the " s i n g l e - f i l e " e f f e c t s which must occur i n a pore (Hodgkin & Keynes 1955; Lakshminarayanaiah 1969). The mobility would be further characterized, but a l i q u i d - j u n c t i o n - type equation would s t i l l be obtained. The e s s e n t i a l feature of a l l of the models is the d i f f e r e n t mobility of the cation and anion. The usual approach taken to include electrogenic pumping i n the expression for E m has been to include the f l u x of ions which occurs through the sodium pump as a phenomenological term in a flux balance wherein the passive fluxes are described by the Nernst-Planck equation (eg. Mullins & Noda 1963; Moreton 1969; Schwartz 1971). The electrogenic p o t e n t i a l then arises i n the same way that the rest of the membrane pot e n t i a l a r i s e s . In a sense, the 'mobility' of sodium ions i n the membrane (for outward movement) is enhanced by the pump, so that when there i s an apparent net expulsion of cations, a p o t e n t i a l develops i n the manner described above as counterion movement occurs. I f t h is hypothesis for the o r i g i n of the electrogenic contribution to the r e s t i n g membrane po t e n t i a l i s e s s e n t i a l l y correct, a measurement of the 55 electrogenic p o t e n t i a l is a measurement of the net ion current through the sodium pump. S i m i l a r i l y , i n a voltage-clamped c e l l , the current required to keep E m steady i s a measurement of the net ion current through the sodium pump. With the simultaneous use of radioisotopes and i n t r a c e l l u l a r micro- electrodes, i t is possible to measure the sodium flux and the membrane po t e n t i a l simultaneously, and to detect the simultaneous changes i n the two when the sodium pump i s s e l e c t i v e l y impaired by exposure of the c e l l to ouabain. Such experiments are described i n section 7, and i t is shown that the two measurements can .be r e l a t e d i n a t h e o r e t i c a l model l i k e those described above, to y i e l d measurements of permeabilities or of the coupling r a t i o of the sodium pump. Voltage .clamp studies by other workers are also described i n section 7. Before concluding t h i s discussion of the membrane p o t e n t i a l , further mention must be made of the contribution of other potentials to the measured value of the membrane p o t e n t i a l . The l i q u i d junction and t i p potentials of micropipette electrodes are well known, and are technical problems. Several workers have found that an e l e c t r i c a l p o t e n t i a l d i f f e r e n c e q u a l i t a t i v e l y and q u a n t i t a t i v e l y l i k e a Donnan p o t e n t i a l can be measured i n muscle c e l l s which have been 'chemically skinned' by extraction with g l y c e r o l or with detergent ( C o l l i n s & Edwards 1971; Pemrick & Edwards 1974; J.A. Hinke - personal communication). The Donnan p o t e n t i a l due to fixed charges on the l a t t i c e of c o n t r a c t i l e proteins must be confined to a region of at most a few hundred Angstroms diameter around the charges, yet i t appears to influence the i n t r a c e l l u l a r microelectrode. The membrane po t e n t i a l measured with i n t r a c e l l u l a r tnicroelectrodes thus might be d i f f e r e n t from the e l e c t r i c a l p o t e n t i a l difference between the bulk i n t r a c e l l u l a r s o l u t i o n and the external s o l u t i o n . Tasake and Singer (1968) review several problems involved i n e l e c t r i c a l 56 measurements of b i o l o g i c a l systems. They remark that "no right-minded electrochemist would even attempt to perform meaningful measurements under the complex conditions which are required to maintain l i v i n g b i o l o g i c a l systems,!' but conclude that meaningful measurements can be made i f the "proper precautions are observed." E. SUMMARY OF THE PROBLEMS TO BE ADDRESSED The p r i n c i p a l object of t h i s thesis i s the measurement and interpreta- t i o n of the sodium and hydrogen ion e f f l u x from whole c e l l s . Hydrogen ion e f f l u x can only be measured i n d i r e c t l y ; most of the work to be described concerns the sodium e f f l u x . It is cu r r e n t l y believed that i t i s mainly the c e l l membrane which controls the ion content of the c e l l , by i t s passive permeability properties and i t s a b i l i t y to translocate ions against the forces which e f f e c t passive flow. The membrane transport reactions can be viewed as enzyme reactions and ch a r a c t e r i z a t i o n of the transport can be c a r r i e d out i n the context of enzyme k i n e t i c s . One enzyme system, the (Na+K)ATPase, has been i s o l a t e d i n an ac t i v e form. Discussion of sodium transport tends to be dominated by discussion of the (Na+K)ATPase, but there appear to be other membrane mechanisms by which sodium can be transported i n whole c e l l s . The char a c t e r i z a t i o n of the a c t i v i t y of the enzyme in the whole c e l l is required before a component of the measured flux can be ascribed to a p a r t i c u l a r enzyme which has been extracted and studied i n i s o l a t i o n . 57 The actual measurement of the sodium e f f l u x from whole c e l l s involves several problems. The state of^sodium inside the c e l l has not been well characterized, a prominent problem being the estimation of the amount of sodium which resides i n e x t r a c e l l u l a r regions, that i s , i n regions outside the c e l l membrane per se. The sodium concentration i n the s o l u t i o n which bathes .the i n t e r n a l surface of the c e l l membrane should be measured, because i t is the correct parameter to use for the i n t e r p r e t a t i o n of experiments i n the context of the enzyme k i n e t i c s model. An i n t r a c e l l u l a r sodium-specific microelectrode w i l l measure this parameter, even when i t changes r a p i d l y . In the l a s t part of t h i s section i s described a revised equation by which the sodium e f f l u x from whole barnacle muscle c e l l s can be calculated from simultaneous microelectrode and radioisotope measurements. Two other models, r e l a t e d to the steady state cation d i s t r i b u t i o n and to the r e l a t i o n between the sodium e f f l u x and the membrane p o t e n t i a l , are also described there. In section 3 are described measurements made with an i n t r a c e l l u l a r sodium-specific microelectrode on c e l l s whose sodium content had been al t e r e d . The r e s u l t s and those of other workers are consistent with a simple model of the states of sodium i n the c e l l which included both i n t r a c e l l u l a r and e x t r a c e l l u l a r pools of sequestered sodium. In section 4 i s described an i n v e s t i g a t i o n of the use of i n t r a c e l l u l a r microinjection. This was c a r r i e d out because other workers had interpreted t h e i r r e s u l t s on microinjected barnacle muscle c e l l s i n terms of a model for the states of sodium i n the c e l l which contradicts the model described i n section 3. An alternate i n t e r p r e t a t i o n of t h e i r r e s u l t s i s presented. M i c r o i n j e c t i o n was also studied because i t was desired to use i t to load the i n t e r i o r of c e l l s with radioisotope r a p i d l y and s e l e c t i v e l y , as a convenience in carrying out e f f l u x experiments. M i c r o i n j e c t i o n i s shown to 58 be r e l a t i v e l y , although not e n t i r e l y benign as far as the barnacle muscle c e l l is concerned. I t i s shown to be equivalent to a passive method for loading the c e l l with radioisotope, aside from i t s f a i l u r e to load the e x t r a c e l l u l a r space, once c e r t a i n corrections are applied. In section 5 a survey of the sodium e f f l u x from barnacle muscle c e l l s i s presented. It i s shown that the dependence of the sodium e f f l u x on the i n t r a c e l l u l a r sodium concentration is s i m i l a r to that i n squid axon and s n a i l neurone. Saturation of the e f f l u x into normal Ringer's s o l u t i o n does not occur over the wide range of sodium content studied. The e f f l u x into sodium-free s o l u t i o n is shown to be very s i m i l a r to that into normal Ringer's s o l u t i o n . The behavior of the sodium e f f l u x into potassium-free and into ouabain-containing solutions i s shown to be almost i d e n t i c a l . The re s u l t s for barnacle d i f f e r i n several respects from those of previous workers because of d i f f i c u l t i e s with the microinjection and radioisotope techniques which have not been recognized before. In section 6 the measurements with the sodium-specific microelectrode and with radiosodium are compared for e f f l u x into sodium-free solutions. Consistency of the model and techniques developed i n the preceding sections is demonstrated, and the nature of the sodium-free e f f e c t is considerably c l a r i f i e d . In section 7 an electrogenic contribution to the membrane p o t e n t i a l of barnacle muscle is demonstrated. The c o r r e l a t i o n between the electrogenic membrane po t e n t i a l and the a c t i v e sodium e f f l u x is measured, and is i n t e r - preted i n terms of an extension of the GHK model for the membrane pot e n t i a l . In section 8 the r e s u l t s of measurement of the i n t r a c e l l u l a r pH using pH-specific glass microelectrodes are presented. I t is shown that i n the barnacle muscle c e l l preparation used i n the present work, there are no "pH t r a n s i e n t s " of the type reported by other workers. Also, a r e l a t i o n s h i p 59 between the steady d i s t r i b u t i o n of hydrogen ions and the r e s t i n g membrane po t e n t i a l is described, and an estimate of the s i z e of the act i v e hydrogen ion e f f l u x i s made. In section 9 is presented a d i r e c t comparison of measurements of the i n t r a c e l l u l a r pH made with the microelectrode and with an indicator method Such a comparison can reveal the existence of su b c e l l u l a r compartments having a r e l a t i v e l y low or high pH, but the most important r e s u l t s of this study turned out to be those concerning the a p p l i c a b i l i t y of the indicator method. In section 10 i s presented a b r i e f discussion of the s i g n i f i c a n c e of the r e s u l t s and some suggestions for further work. F. SUMMARY OF MODELS Part of the development of the models presented here r e l i e s on res u l t s which have not yet been described. The reader might wish to proceed d i r e c t l y to section 3 and to re f e r back to this section when references to these models are encountered. (i) E f f l u x of sodium from a whole c e l l . It was desired to formulate an expression by which the sodium e f f l u x from a whole c e l l could be calculated from experimental measurements. In the model of the c e l l used by early workers (Keynes & Lewis 1951; Keynes 1951), i t was assumed that the i n t r a c e l l u l a r medium is a simple s o l u t i o n of s a l t s and organic molecules bathing the in t e r n a l surface of the c e l l membrane, and that only a n e g l i g i b l y small f r a c t i o n of the transmembrane 60 passage of ions occurs by simple d i f f u s i o n . In such a s i t u a t i o n , i f one could replace some of the i n t r a c e l l u l a r sodium with l a b e l l e d sodium ions, the u n i d i r e c t i o n a l e f f l u x of sodium ions from the c e l l (which i s d i f f e r e n t from the net efflux) could be deduced from the e f f l u x of the l a b e l l e d ions into a large bathing s o l u t i o n containing no l a b e l l e d sodium ions. I t is assumed that the l a b e l l e d ion behaves j u s t as the abundant ion does as far 22 23 as ion movements are concerned, an assumption s a t i s f i e d by Na and Na. Thus: the s p e c i f i c a c t i v i t y of sodium i n the c e l l can be approximated 22 _ moles Na i n the c e l l ^ A c e l l - 23 22 moles Na i n the c e l l + moles Na in the c e l l 22 A moles Na in c e l l . „ moles 2 3Na i n c e l l =  N a c e l l N a c e l l * 22 where N a c e l (moles of Na in the c e l l ) i s much smaller than Na c e-Q (moles of 2 3Na i n the c e l l ) i n prac t i c e . A short i n t e r v a l of time " t " i s considered. (The extension from the dis c r e t e c o l l e c t i o n i n t e r v a l s used i n pract i c e to a function applicable at any instant i s assumed i m p l i c i t l y i n this development.) I f , i n time t, Na moles of radiosodium leave the c e l l with no backflux of radiosodium, then the t o t a l number of moles of sodium leaving the c e l l is Na / SA c e-Q and the e f f l u x density is Mjj a (moles/cm -sec) = Na . 1 . 1 S A c e l l A t ...(1) where A is the area of the membrane across which the sodium ions pass. I f V i s the volume of the i n t r a c e l l u l a r f l u i d , then Y_ • Na* . (Na) ...(2) M = _JL_ • _£S_ • v»«v Cell Na A-t N * n i  w a c e l l where ( N a ) c e l l = 'Na c e l l/V. 61 In an actual experiment, Mjj a as a function of time i s determined by c o l l e c t i o n of Na for each of a succession of time periods. The value of • ^ a c e l l a t t* i e beginning of each c o l l e c t i o n period is found by "back- * a d d i t i o n " of the Na values to the r a d i o a c t i v i t y l e f t i n the c e l l at the end of the experiment. I t is required that (Na) C £-Q be known, and that i t not change much during a c o l l e c t i o n period. The r e s u l t s of experiments on squid axons seemed to be consistent with t h i s formulation. The rate at which radiosodium came out of the c e l l f e l l o f f as a simple exponential with time, and was approximately equal to the i n f l u x rate, which can be measured d i r e c t l y from separate isotope uptake experiments (Keynes & Lewis 1951; Keynes 1951). The r e s u l t s for muscle c e l l s are more d i f f i c u l t to interpret. The trend has been to employ more complicated models having several c e l l u l a r compartments which can exchange sodium ions, such as that i l l u s t r a t e d i n F i g . 1(b) (eg. Keynes & Steinhardt 1968). These interpretations have not been completely successful, but recognize in part the current view of the heterogeneous structure of the c e l l and the state of ions and water inside the c e l l which would seem to make the above model untenable for most c e l l s . The features which probably give r i s e to error are as follows. F i r s t , the relevant concentrations are those of the 'myoplasm', the large aqueous compartment inside the c e l l which is much l i k e a bulk aqueous s o l u t i o n and which is assumed to bathe the i n t e r n a l surface of the c e l l membrane. (Any s t r u c t u r a l layer deep to the 'bimolecular l e a f l e t ' w i l l be assumed to be part of the 'functional membrane'.) ( N a ) c e | ^ must be replaced by the concentration of sodium i n free s o l u t i o n i n the myoplasm (Na) . The l a t t e r m can be measured r e l i a b l y as (Na) m = (ajja)m / tf+ • The value for y is taken to be 0.65. This is j u s t i f i e d by the concept of the myoplasmic compartment as a s o l u t i o n l i k e a bulk solution, and by an experimental determination 62 Figure 1, Models of the c e l l used i n c a l c u l a t i o n of ion fluxes. (A) Model on which equation (2) is based. A l l of the i n t r a c e l l u l a r sodium is i n s o l u t i o n i n a l l of the i n t r a c e l l u l a r water, and can only exchange with the e x t r a c e l l u l a r compartment (arrows). (B) Current model. I n t r a c e l l u l a r sodium can reside i n a nonmyoplasmic compartment exchanging only with the myoplasmic compartment, or i n a nonmyoplasmic compartment which also exchanges with the e x t r a c e l l u l a r compartment. 'Nonparticipatory' sodium and water are not shown, nor are a l l possible exchanges for a multicompartment system. 63 (Hinke 1970). It i s suggested by previous work (and confirmed by the present experi- ments) that Mj^a i s a nondecreasing function of ( a j j a ) m . Continuous monitor- ing of ( a j j a ) m during e f f l u x experiments, e s p e c i a l l y those in which (aj\j a) m changes, should y i e l d a more r e l i a b l e measurement of M^a than i s possible from chemical analysis of t o t a l c e l l u l a r sodium and back-addition of changes due to transmembrane transport. Na* i s r e a d i l y and unequivocally measured as the amount of radioisotope c o l l e c t e d i n the s o l u t i o n ..bathing the c e l l during a given time i n t e r v a l . However, N a ^ n i s a problem. As far as can be measured with the present techniques, injected radiosodium i s deposited only i n the myoplasm and i n a small compartment which exchanges sodium with the myoplasm very quickly. This was concluded from the close s i m i l a r i t y of the sodium e f f l u x from c e l l s loaded with radiosodium by microi n j e c t i o n and from c e l l s loaded by immersion i n normal Ringer's s o l u t i o n which contained radiosodium, as described i n section 4. Morphological and p h y s i o l o g i c a l studies r u l e out the membrane-delimited organelles as important s i t e s of sequestration of sodium. They do not have a large enough capacity, nor. rapid enough exchange with the myoplasm i n barnacle muscle c e l l s . The c o n t r a c t i l e proteins are known to bind sodium and to exchange r a p i d l y with the myoplasm. Their t o t a l capacity, ca. 68 millimoles per kg 3 dry weight for r a p i d l y exchanging s i t e s , would l a r g e l y be taken up by potassium and hydrogen since they show only a modest preference for sodium over potassium and the i n t r a c e l l u l a r concentration of potassium i s much moles Na , 70water moles Na kg dry weight kg c e l l water 1 - 70water 64 greater than that of sodium. They c e r t a i n l y should bind some sodium, though. From experiments in which the t o t a l sodium content of barnacle muscle c e l l s was a l t e r e d (section 3 ) , i t was concluded that of the t o t a l amount of sodium associated with the c e l l a f t e r account had been taken of e x t r a c e l l u l a r sodium by the usual techniques, a large amount of e x t r a c e l l u l a r sodium was s t i l l included. I f this i s taken into account, then about 3 0 7 o of the i n t r a - c e l l u l a r sodium i s not accessible to the microelectrode. About 2 0 7 o exchanges so slowly as to be nonparticipatory i n the flux experiments discussed here. The most generous estimate was that less than 1 5 7 o (and probably less than 1 0 7 o ) of the i n t r a c e l l u l a r sodium is in rapid exchange with the free sodium, where the free sodium represents about 7 0 7 ° of the sodium which i s t r u l y i n t r a c e l l u l a r . More important i s the existence of an e f f e c t i v e i n t r a c e l l u l a r sink of injected radiosodium due to l o n g i t u d i n a l d i f f u s i o n of radiosodium i n i n - jected c e l l s , . a s discussed in section 4. For the c e l l s i n which the sodium content was not r a i s e d by micro i n j e c t i o n of NaCl solutions, t h i s plus radiosodium associated with the c o n t r a c t i l e proteins, i n a r e a l i s t i c estimate, appears to be equivalent to about 1 5 7 D of injected radiosodium i n the experi- ments reported here. The e f f e c t is greater when NaCl is injected. However, the amount l o s t to the sink i s d i f f e r e n t i n d i f f e r e n t c e l l s , and increases from the i n i t i a l value of zero as the experiment progresses. Thus a value Na* for the 'myoplasmic radiosodium' should be used i n place of N a J e l l . A co r r e c t i o n can be applied to the data to account for t h i s . In the d e f i n i t i o n of the e f f l u x density (equation ( 1 ) ) i t i s assumed that the amount of sodium which comes out of the c e l l i n unit time i s r e l a t e d to the amount of radiosodium which comes out i n unit time, Na , v i a the s p e c i f i c a c t i v i t y of radiosodium in a homogeneous i n t r a c e l l u l a r compartment contain- 65 ing only exchangeable sodium. An equivalent statement of this fundamental assumption of the tracer method is that the rate at which radiosodium appears i n the bath, Na*/t, is d i r e c t l y proportional to the amount of exchangeable radiosodium inside the c e l l , Na m. Thus under steady conditions, where the i n t e r i o r of the c e l l i s well-mixed and the s i z e of the e f f l u x doesn't change too much over a c o l l e c t i o n period t = 5 minutes. Note that i t i s assumed i n ad d i t i o n that the continuing loss of radiosodium from the myoplasmic compartment to the i n t r a c e l l u l a r sink is e n t i r e l y independent of the loss of radiosodium across the c e l l membrane. The 'constant' k is then the instantaneous slope of the plot of InNa versus time. I t bears repeating that the approximation w i l l be cl o s e s t to r e a l i t y where the semilog plot (In Na* versus time) i s l i n e a r and not too steep, for then i t is most l i k e l y that the myoplasmic compartment is well-mixed, and that the use of a time r e s o l u t i o n of 5 minutes w i l l not introduce too much error. The e f f e c t of the c o n t r a c t i l e proteins could be corrected for v i a a model for the competition of sodium and potassium for the s i t e s on the protein, i f such a r e l a t i v e l y small c o r r e c t i o n were f e l t to be necessary. Another problem arises with V/A. The membrane i n the c l e f t s presumably is s i m i l a r to that at the surface (the two develop from the same source as the s y n c y t i a l muscle f i b r e is formed, and f u n c t i o n a l l y i t is reasonable that they should have s i m i l a r properties, although the membrane of the TTS probably i s d i f f e r e n t - G i r a r d i e r et a l . 1963). I f the function of the c l e f t system is_ to ensure that a l l parts of the i n t e r i o r of the c e l l are withi n a c e r t a i n distance from some part of the c e l l membrane, the surface- v i a membrane t 66 to-volume r a t i o might well be independent of the diameter of the c e l l . A further complication i s that the volume represented by V should be that of the myoplasmic compartment, as discussed above and in the introduction. It was concluded that reasonable approximations would have to be made in the formulation of a p r a c t i c a l equation. Thus: conductance measurements indicate that the values for the membrane capacitance and resistance for the barnacle muscle c e l l can l a r g e l y be reconciled with those for the membranes of other c e l l s i f the e f f e c t i v e area of homogeneous c e l l membrane is about ten times the apparent surface area of the c y l i n d r i c a l c e l l (Hagiwara et a l . 1964; Brinley 1968). For a c y l i n d e r of radius r, V/A = r/2 so here V/A w i l l be taken to be equal to r/20. Thus: / i / 2 \ i Na* (a„ ) M (moles/cm^sec) = r 1 — — v Na m 20 300sec Na m 0.65 = 2.56 x 10" 1 0 l i t r e r Na*_ cm3 Na* V . ...(4) where r (cm) i s taken to be the average of the c e l l r a d i i measured i n perpendicular d i r e c t i o n s , and ( a ^ a ) m is in m i l l i m o l e s / l i t r e , to y i e l d M^a in moles/cm -sec. Na /Na m is the r a t i o of counts per minute in a 300 second perfusate sample to calculated counts per minute in the c e l l at the s t a r t of the c o l l e c t i o n period for those c e l l s which have a "slope r a t i o " close to unity, i e . i n essence those c e l l s not microinjected and some of those injected with solutions of very low sodium concentration. For other c e l l s , Na*/Na* should be calculated from the slope k of a plot of l n Na* versus time, i d e a l l y only where such a plot i s l i n e a r and not too steep, as Na = k 5 minutes. 67 This l i m i t s the use of microinjected c e l l s to measurements of steady effluxes. Such w i l l occur into normal Ringer's solution, and i n most cases into sodium-free solution, but i n potassium-free or ouabain-containing solutions the c e l l w i l l gain sodium continuously. The e f f l u x increases as ( a ^ a ) m increases, and the slope of the plot of In Na versus time can become zero or even p o s i t i v e . A reasonable estimation of Na*/Na* usually can be made i n these cases, but the r e s u l t i n g calculated value of Mjj a i s more uncertain than i t i s for steady effluxes. Nevertheless, microinjection does have some vi r t u e s . The s e l e c t i v i t y , convenience, and economy of the microinjection technique o f f s e t to some degree the uncertainty due to l o n g i t u d i n a l d i f f u s i o n . A l l 'participatory' i n t r a c e l l u l a r compartments appear to be loaded, just as with passive loading, but the e x t r a c e l l u l a r space is not loaded. Only a small amount of radio- isotope i s required for each experiment. F i n a l l y , c e l l s can be used shortly a f t e r d i s s e c t i o n , when they should be i n a state most l i k e that i n vivo. The determination of Na m would be more accurate i f only the c e n t r a l part of a long segment of injected c e l l were perfused i n microinjection experiments. The strategy adopted i n the present experiments was to perfuse only a long injected segment of the c e l l rather than of perfusing a long c e l l only part of which had been injected. This was only p a r t i a l l y success- f u l i n eliminating the problem of l o n g i t u d i n a l d i f f u s i o n . The problem is eliminated by the use of passive loading, but this method requires a great deal of isotope and long periods of incubation of the c e l l a f t e r dissection, and loads the e x t r a c e l l u l a r space with isotope. The 'time constant for exchange' Na*/Na*e^^ has been taken to r e f l e c t the operation of the transport systems of the membrane most d i r e c t l y (eg. Dick & Lea 1967; B r i n l e y 1968). I t can be asked why the s i z e of the e f f l u x should be calculated. Quite aside from the problems of using Na /Na£ e;Q 68 for microinjected c e l l s , i t can be seen from equation (4) that the sodium content of the myoplasm must be taken into account i f the a c t i v i t y of the transport mechanisms is to be deduced from isotope measurements. I d e n t i c a l values of Na*/Na* i n c e l l s of d i f f e r e n t sodium content r e f l e c t d i f f e r e n t /pumping rates'. As can be seen from the manner i n which equation (2) is derived from the d e f i n i t i o n of flux density (equation (1)), the appearance of the 'time constant' in the e f f l u x equation is i n intimate as s o c i a t i o n with the s p e c i f i c a c t i v i t y of radiosodium inside the c e l l . S i m i l a r i l y , the appearance of (aNa)m e x p l i c i t l y i n equation (4) i s misleading. I t might appear that t h i s imposes a spurious dependence of Mjj a on ( a j j a ) m . Again, examination of equations (1) and (2) reveals that t h i s i s not so. M^a and ( a ^ a ) m mutually dependent i n several ways i n the context of a l i v i n g c e l l during an e f f l u x experiment i n v i t r o . Equation (4) simply separates four measurable quantities from which M^a can be calculated. It must be asked at this point what advance a l l of this is over the method of Keynes and Lewis (1951). Conceptually, i t is c e r t a i n l y a more . r e a l i s t i c model of the c e l l and the ion movements. The main advance is the introduction of the sodium-specific microelectrode. This permits measure- ment of the true s p e c i f i c a c t i v i t y inside the c e l l . The microelectrode also enables one to measure the e f f l u x i n experiments where ( a j j a ) m changes ra p d i l y . Several fundamental problems remain, however. The question of surface- to-volume r a t i o cannot be taken further without the performance of exacting morphological measurements, which themselves are plagued with uncertainties i n the form of changes in c e l l volume during f i x a t i o n . The much more s i g n i f i c a n t and d i f f i c u l t question of the homogeneity of the c e l l membrane with respect to transport properties has not been pursued at a l l . In 69 practice, the best strategy i s to use c e l l s of about the same s i z e , as was done here, so any error due to these factors w i l l be about the same for each c e l l . F i n a l l y , the question of f i l m - c o n t r o l l e d d i f f u s i o n ("unstirred layers") has not been addressed, aside from the e x p l i c i t statement of the assumption that boundary layers are considered to be part of the 'functional membrane'. I f a r e a l l y good estimate of the magnitude of the sodium e f f l u x is to be obtained, these d i f f i c u l t problems must be solved. In the remainder of this thesis, equation (4) i s used to c a l c u l a t e the sodium e f f l u x from barnacle muscle c e l l s , whether loaded with radio- sodium by microinjection or by immersion i n a s o l u t i o n containing radio- sodium. The appropriate c o r r e c t i o n should be applied i n the former case. ( i i ) Steady state d i s t r i b u t i o n of cations. In the experiments described i n section 8 concerning the d i s t r i b u t i o n of hydrogen ions measured during steady conditions (the use of the term 'steady state' i n t h i s context has been c r i t i c i z e d ) , a r e l a t i o n s h i p between the membrane p o t e n t i a l and the transmembrane difference i n pH was found. Such a r e l a t i o n s h i p had been sought by other workers, but had not been found. In the discussion of the r e s u l t s , the r e l a t i o n s h i p derived here from elementary theory w i l l be employed. These considerations apply to sodium ions as well as hydrogen ions. Assume the c e l l membrane i s a lamella uniform i n the y and z directions of a Cartesian coordinate system having the x axis directed perpendicular to the membrane surface. At a point x, i n the membrane, the net flux density j(x) (moles/cm sec) i s assumed to be the sum of a flux density jP(x) due to d i f f u s i o n of hydrogen ions as described by the Nernst-Planck equation, and an ad d i t i o n a l f l u x density j m ( x ) which is not further s p e c i f i e d . Thus 70 j(x) = j m ( x ) - u(x) • c(x) |R • T 9_ In c(x) + F 9 p(x) j ...(5) where u(x) and c(x) are the mobility and concentration (or a c t i v i t y ) of hydrogen ions free to d i f f u s e at x, (6(x) i s the e l e c t r i c a l p o t e n t i a l at x, R i s the gas content, T i s the absolute temperature, and F is the Faraday constant. Note that an e f f l u x is p o s i t i v e i n sign. To obtain a r e l a t i o n between the net transmembrane flux density and the transmembrane differ e n c e of c and p, equation (5) must be integrated across the membrane. Only steady conditions are considered here, so the t o t a l f l u x density j is independent of x in the membrane, although j m need not be. M u l t i p l y i n g both sides by £exp(Fp(x)/RTj)^i(x) and making use of the i d e n t i t y £ — \ c(x) exp(F(6(x)/RT/ = exp(Fp(x)/RT) U_c(x) + c(x)F ? (6(x) ^*  L 1** R-T ^x" - one finds j = _1_ £ M - RT £c(a) - c(0) exp(FE m/RT)J j where * exp[_JL . ( p(x) - p(a) )] Q " I I RT J . dx x=b % j m ( x ) e x p f ^ . ( p( x) -.(6(a) .)] M = j l_RT J_ . dx * u(x) x=o v ' and E m = (6(0) - (6(a) is the membrane p o t e n t i a l , and is generally negative. Also, Q>0 and i f j m ( x ) represents an e f f l u x , M>0. (This r e l a t i o n has been derived by Schwartz (1971) ). If at least one compartment is f i n i t e , the net t o t a l f l u x density must vanish i n the steady state, so M = RT \_ c(a) - c(0) exp F (6/RTj . (Note that this argument could have been applied at equation (6).) I f the i n t e r n a l pH is pH^ = -log^Q c(0) and the external pH i s pH Q = -log-^Q c(a), re- arrangement y i e l d s 71 PH e - p H l - l o g 1 0 f l - — H j - ] - _ I _ E m ( 6 ) Note that the constraint on M i s now 0<M<RT'c(a). Also, x = 0 can be taken to be i n the myoplasm, away from the surface of the membrane, and x = a s i m i l a r i l y to be in the external bathing solution, so surface e f f e c t s need not be considered with respect to (6 or c except i n the evaluation of M, and the values of (pH Q - pH^) and E m i n equation (6) are those measured with microelectrodes. The corresponding r e l a t i o n s h i p for sodium ions i s : logoff.)! - l o g 1 0 ( H a ) o - l o g 1 0 ( l - — M - ^ ) „ 0 I i n f E m . . . ( 7 ) ( i i i ) Electrogenic contribution to the r e s t i n g membrane p o t e n t i a l . A number of physiochemical models of the contribution of electrogenic transport to the r e s t i n g membrane p o t e n t i a l have been made (see review by Thomas 1972a). The usual strategy i s to carry out a d e r i v a t i o n of the membrane po t e n t i a l as a d i f f u s i o n p o t e n t i a l , with the i n c l u s i o n in the flux balance of an extra f l u x of ions due to a c t i v e transport. The d e r i v a t i o n of Moreion (1969) w i l l be adapted for use i n the analysis of the r e s u l t s of the experiments described i n section 7. Moreton (1969) adopted the Goldman-Hodgkin-Katz model, and calculated the passive transmembrane fluxes for sodium ( n ^ ) > potassium (m^), and chloride (m^) from the Nernst-Planck equation i n the constant f i e l d approximation: m N a = - . u d ( } - ( } . d 0 F dx ' N a dx mjr, VOQ-^ s i m i l a r i l y . 72 I f there are no fluxes but these, then i t must happen that "^a + m k = 0 since the c e l l cannot accumulate a net charge. The r e s u l t is the Goldman- Hodgkin-Katz equation, as discussed above. I f there i s an a d d i t i o n a l component M of ion flux due, for example, to an ac t i v e exchange of sodium for potassium which is not one-for-one, then the sum of th i s and the passive fluxes is zero: ^Na + mK " ""Cl + M = 0 and the following expression i s obtained (Moreton 1969) E = E l m In P K ( K ) D + P N a (Na) Q + P c l ( C l ) i + R T M F E„ m E S R_T m -c. In L P K 0 0 i + P N a ( N a > i + PC1 <C1>o + f - | M F Em U + — M F Em W + JL1 M F E m. for convenience. or This is the f a m i l i a r GHK equation, with an ad d i t i o n a l term representing the net pumped cation e f f l u x . When the net e f f l u x increases, the magnitude of the membrane po t e n t i a l increases because the numerical value of the numera- tor i s less than that of the denominator. This is not presented as an exact d e s c r i p t i o n of what occurs i n r e a l i t y , but rather i s developed i n the s p i r i t of the GHK formulation. Some further approximations can be made to f a c i l i t a t e comparison of this r e l a t i o n s h i p with experimental r e s u l t s . Most of the r e s t i n g membrane po t e n t i a l is not due to the pump, ie. U « W, so i t i s a good approximation to neglect the pump term i n the denominator to y i e l d 73 , In U + RJL M W When the c e l l is exposed to ouabain, the pump term changes from M to a new value M ' , and the membrane potential changes from EM to E'. Thus: 4 E M s "m E' m = R T ln 'm U + R T F E' m M' U + R T F E„ M > 0 , where i t is assumed that U and W are unchanged in the time i t takes to complete the measurement of Ê . Upon rewriting this as 4E. R T m In 1 + M'l UFE; j R T ln 1 + R-T U F E Ml m i t is seen that the second term in the argument of each of the logarithmic functions is much less than unity. It is thus a reasonable approximation to expand each of the logarithmic functions in a Taylor series and retain only terms to f i r s t order in the small quantities R T . M' and R T U F E. m M. 4E This yields: m U F E i I F / u E m M where AM = M 1 M < 0 /R T\^ i /R TN̂  I and I } * has been assumed to be equal to / 1 ' U / O - E ; h ( F / U - as jm a matter of convenience. With one further step, the measured change in the sodium efflux on exposure of the c e l l to ouabain can be introduced. If the coupling ratio of sodium to potassium transported by the pump is introduced: 74 (R* - _ ^ a > o then the net pumped cation f l u x is M = ^ a + *K " ( 1 " -P ^ a and A E = /R T\2 . 1 . (1 - 1). 4M . . . (8) . m This approximate expression relates the change in membrane po t e n t i a l 4 E m to the change in sodium e f f l u x AMj^a i n a r e l a t i v e l y simple manner, i n the context of the usual formulation for the o r i g i n of the membrane po t e n t i a l . E i t h e r the coupling r a t i o (R. or the permeabilities i n U can be evaluated from experimental data i f one or the other is already known from independent measurements. The p r o p o r t i o n a l i t y factor between the unbalanced cation e f f l u x and the change i n E is decreased i f the c e l l is hyperpolarized or i f the m concentration gradients are reduced. This feature r e f l e c t s the imposition of the pumped fluxes on the Nernst-Planck equation. The forces which tend to oppose the charge separation which a r i s e s passively likewise oppose the unbalanced flux added phenomenological-ly; because of how the l a t t e r has been imposed i n the current balance. Although mechanistically the unbalanced pumped cation e f f l u x i s l i k e an increased mobility for sodium going out of the c e l l , the existence of this e f f l u x i s f i r s t apparent only as a sort of boundary condition of the steady state passive fluxes. A better and p o t e n t i a l l y very useful model would speci f y some d r i v i n g force for the active e f f l u x , such as the a f f i n i t y of a chemical reaction, and would acknowledge the i n t e r a c t i o n between the a c t i v e and passive fluxes. I t bears repeating that such a model would r e f l e c t the mechanism which brings about a c t i v e cation transport. 75 SECTION 3. THE STATES OF SODIUM IN CELLS The c e l l i s known to be composed of several d i s t i n c t morphological compartments. These were described i n the Introduction. The sodium ions i n the c e l l are d i s t r i b u t e d among these compartments. I f i n an experiment some l a b e l l e d sodium ions are introduced into one compartment, the exchange of sodium ions v i a d i f f u s i o n a l and non-diffusional processes which occurs continuously between communicating compartments w i l l tend to bring about a steady d i s t r i b u t i o n i n which the s p e c i f i c a c t i v i t y of sodium i s the same i n every compartment. The rate at which sodium exchange occurs is not the same for every communicating pair of compartments. One can conceive of a very rapid exchange, as between sodium ions i n the myoplasmic compartment and the sodium ions which are acting as counterions to the fixed negative charges on a macromolecule i n a protein matrix immersed i n the myoplasmic compart- ment. On the other hand, one can conceive of a very slow exchange, as between myoplasmic sodium ions and sodium ions which are counterions i s o l a t e d by the hydrophobic b a r r i e r of a c o i l e d and folded macromolecule. In the l a t t e r case, the exchange rate is probably so slow that, as far as in v i t r o isotope flux experiments are concerned, such sodium ions are non- pa r t i c i p a t o r y . Between these extremes are known to l i e most of the ion transport and exchange processes of the c e l l membrane. In order to interpret flux experiments, one would l i k e to know the exchange rates between communicating i n t r a c e l l u l a r compartments, as well as the f r a c t i o n of the c e l l sodium contained i n each compartment. Ion-specific glass microelectrodes of the type used to make i n t r a - c e l l u l a r measurements are assumed to sample the myoplasmic compartment. 76 This i s the i n t r a c e l l u l a r compartment which behaves very much l i k e a bulk Solution and i s not enclosed by s u b c e l l u l a r membranes, (see, for example, Hinke, C a i l l e , & Gayton 1973 and the discussion following i t , and Edzes & Berendsen 1975). Microelectrode measurements have indicated that only a part of the i n t r a c e l l u l a r sodium, potassium, and chloride measured by chemical analysis of whole c e l l s i s i n free s o l u t i o n i n the bulk water of the myoplasm (McLaughlin & Hinke 1966; Dick & McLaughlin 1969; Lee & Armstrong 1972; Hinke et a l . 1973; Lev & Armstrong 1975). The s i z e of the f r a c t i o n not a c cessible to the microelectrode in each case is not c e r t a i n , because of uncertainty about the volume of the myoplasmic compartment and, e s p e c i a l l y for sodium, because of great uncertainty about the portion of the ions which i s e x t r a c e l l u l a r , i n s o l u t i o n or sequestered (Lev & Armstrong 1975). One extreme estimate is that f u l l y 837, of the i n t r a c e l l u l a r sodium can be inaccessible to the sodium microelectrode (Hinke 1969b). The sodium content of the barnacle muscle c e l l can be increased by immersion of the c e l l i n a potassium-free s o l u t i o n (Beauge & Sjodin 1967), and can be decreased by immersion i n a sodium-free s o l u t i o n ( A l l e n & Hinke 1971). I t was expected that the d i s t r i b u t i o n of sodium inside the c e l l would also change during such manipulations. Thus, changes i n the amount of myoplasmic and 'nonmyoplasmic' i n t r a c e l l u l a r sodium were measured as the t o t a l sodium content of the c e l l was changed. 77 METHODS Specimens. The specimens used i n a l l of the Na experiments were obtained from Georgia S t r a i t . Those used i n the pH experiments were obtained from Puget Sound. The morphology of the d i f f e r e n t species of giant barnacles has been described by P i l s b r y (1916). He states that the o v e r a l l form i s highly v a r i a b l e , and that the shape of the opercular valves, the d e t a i l s of the structure of the plates of the wall, and the structure of the feet are the important c h a r a c t e r i s t i c s . The largest specimens, which are s p e c i f i c a l l y chosen for microinjection work, are Balanus n u b i l i s , and occasionally B. aquila. (These are the largest North American barnacles found i n shallow water.) As noted by P i l s b r y , these larger barnacles are old, so t h e i r s h e l l i s worn and often r i d d l e d by boring animals. This does not appear to a f f e c t the health of the barnacle, but makes the' i d e n t i f i c a t i o n of a species d i f f i c u l t . B. n u b i l i s i s unique i n that as i t grows i t increases i t s i n t e r n a l volume by excavation of the basis, and th i s excavation i s e a s i l y seen during d i s s e c t i o n . Apparently there is no phy s i o l o g i c a l difference between the muscle f i b r e s of these two species. Specimens were obtained by divers, and kept i n a holding tank at the Vancouver Public Aquarium. Seawater drawn from Burrard In l e t was run through the tank continuously. The osmolarity of th i s seawater varied somewhat over the year, (950+50 mOsm on a Fiske osmometer). The residence time i n the tank was not c l o s e l y monitored, but never exceeded three months. Barnacles were moved to a co n t r o l l e d a r t i f i c i a l seawater aquarium (Instant Ocean) at the laboratory 1-3 weeks p r i o r to use. The a r t i f i c i a l seawater was made up to 960 mOsm (the value for normal Ringer's solution) and main- tained at 10-12° C. The barnacles a c t i v e l y extended t h e i r c i r r i i n both 78 aquaria. Dissection. Great care was taken to minimize the trauma experienced by the muscle f i b r e s . The barnacle was quickly k i l l e d by c u t t i n g through the opercular adductor and removing the c i r r i , and the digestive and reproductive organ mass. This l e f t the s i x large depressor muscles attached to the opercular plates and to the basis. In the early experiments the s h e l l was cracked with bone shears to i s o l a t e each muscle bundle i n t a c t (Hoyle 1963), with i t s f i b r e s attached at one end d i r e c t l y (without v i s i b l e tendon) to a fragment of the basis, and at the other end v i a tendons to a fragment of the opercular plate. For most of the experiments, however, a l a p i d a r i s t ' s saw was used. This was far superior, and enabled one to i s o l a t e a muscle bundle very quickly,, with a minimum of manipulation, on a compact fragment of the basis. The i s o l a t e d bundle was immediately suspended by the fragment of operculum, i n a beaker of normal barnacle Ringer's at 5 - 10° C. The muscles used.were the Depressor Scutorum L a t e r a l i s and R o s t r a l i s . The tergal depressor was not used because i t s fibres were heavily invested with connective tissue and were more d i f f i c u l t to i s o l a t e . Single muscle c e l l s were t y p i c a l l y 1.0 - 1 ̂,5 mm i n diameter and 4 - 5 cm i n length. To f a c i l i t a t e the separation of these s i n g l e f i b r e s from one another, a bundle was attached by the opercular and basal fragments to a frame which held the bundle h o r i z o n t a l l y at about the r e s t i n g length, i n a dish of cold Ringer's solution. Fat and connective tissue were c a r e f u l l y removed with jeweller's forceps and iridectomy s c i s s o r s , under a d i s s e c t i o n microscope. To i s o l a t e the f i b r e s , the tendon of an accessible, f i b r e was grasped with the forceps and cut from the operculum. The connections with the other f i b r e s (connective ti s s u e and a few small nerve fibres) were then 79 cut, proceeding from tendon to basis. (Damage to the f i b r e membrane would become apparent immediately as a l o c a l contraction.) This d i s s e c t i o n was c a r r i e d out to as close to the basis as was possible, and the f i b r e was l e f t attached to the basis, since removal from the basis without causing damage to the c e l l membrane is impossible. This procedure was repeated for each of the f i b r e s i n the bundle, and then the bundle was l e f t i n normal Ringer's at 5 - 10° C for 1 - 3 hours before being used. This procedure assured that any damaged fibres would be i d e n t i f i e d , even i f the damage was s l i g h t . Only fibres which were uniform i n contour ( i e . without contractures) and uniform i n translucency were chosen for experiments. Fibres were only removed from the basis at the end of the experiment, when they were taken for weighing and chemical analysis. This use of intact f i b r e s is d i f f e r e n t from the pra c t i c e of other workers (Hagiwara, Chichibu, & Naka 1964; Brinley 1968; B i t t a r , Chen, Danielson, & Tong 1972), who cut the c e l l s o f f at the basis and then cannulated the cut end of the c e l l . Solutions. The a r t i f i c i a l seawater was prepared from Instant Ocean ingredients, to 960 mOsm. The Ringer's solutions were as in Table I. A ouabain (Schwartz-Mann) stock s o l u t i o n (100 mM) was prepared and used to prepare a l l solutions for ouabain experiments, by add i t i o n of the appropriate amount of ouabain stock s o l u t i o n i n making up one of the solutions shown i n Table I. 80 TABLE I COMPOSITION OF SOLUTIONS A l l values are mM. A l l solutions are 960±5 mOsm. Normal Zero-K L i Li-%C1 Choline T r i s * * Sucrose Rinse Na 450 458 -- -- -- K 8 -- 8 16 8 8 8 -- Ca 20 20 20 20 20 20 20 20 Mg 10 10 10 10 10 10 10 10 C l 543 543 522 269 543 543 93 85 T r i s 25 25 25 25 25 475 25 25 L i -- 429 421 -- -- -- -- Choline — -- -- -- 450 -- Sucrose -- -- -- 117 117 675 665 CH„S0. 3 4 -- -- -- 268 -- -- -- -- T r i s is tris-hydroxymethyl aminomethane T r i s C l is not completely dissociated: at pH 7.6 there are 19 mM C l " for 25 mM T r i s C l (Gayton & Hinke 1968). Microelectrodes. Sodium-specific glass microelectrodes were constructed and c a l i b r a t e d by the method of Hinke (1967; 1969a), except that the s e n s i t i v e glass was drawn by hand, and the f i n a l glass-to-glass seal at the t i p was performed with the electrode held h o r i z o n t a l l y i n the microforge and the heater wire brought i n h o r i z o n t a l l y to touch and melt the t i p of the s e n s i t i v e glass. 81 The heat was s u f f i c i e n t to make the glass-to-glass seal, and when the heater wire was then withdrawn h o r i z o n t a l l y the s e n s i t i v e glass was drawn into a fi n e t i p and sealed. The outside diameter of the i n s u l a t i n g glass at the seal was t y p i c a l l y 20-25^, and the length of the s e n s i t i v e t i p was t y p i - c a l l y 75^u. These electrodes were quite durable, and usually broke before they l o s t t h e i r Na s e l e c t i v i t y . With use, the response time lengthened, but an electrode could be 're-activated' by a 10 sec. immersion of the t i p i n 0.1 M hydrof l u o r i c a c i d . * This treatment made.the trips more f r a g i l e . Conventional micropipette electrodes were pulled on a mechanical p u l l e r , e i t h e r from the same lead glass used in the construction of the Na electrode, or from b o r o s i l i c a t e glass (Hinke 1969). The p o t e n t i a l change between 0.2 M NaCl and 0.2 M KC1 was less than 2 mV for electrodes accepted for use (Adrian 1956). New electrodes were constructed the day before or the day of each experiment. Potassium-specific ion exchanger microelectrodes were constructed by the method of Walker (1971), and tested by the method of Hinke (1969a). For most of the experiments, the sodium or potassium electrode was re f e r r e d to an e x t r a c e l l u l a r calomel electrode. The pot e n t i a l difference was measured by a Cary 401 electrometer, and recorded on a chart recorder. The micropipette electrode was also r e f e r r e d to the calomel electrode, and the p o t e n t i a l d i f f e r e n c e was monitored by a Vibron 33B electrometer or by a Kiethley 616 electrometer. For a few experiments the sodium electrode was re f e r r e d d i r e c t l y to the i n t r a c e l l u l a r micropipette electrode. E l e c t r i c a l interference was more of a problem with the l a t t e r method, but the two methods yielded s i m i l a r r e s u l t s . The experimental apparatus was housed in a small metal-walled room, and a l l electrometers, recorders, l i g h t sources, and power syringes were outside the room, except for the pre-amp of the Cary electrometer. The 82 separation of the above equipment from the experimental chamber was less than one metre. Leads, tubing, and f i b r e optic conduit were passed through small ports cut i n the wall of the shielded room. With this arrangement and appropriate grounding, the electrometer readings were very stable throughout. The a x i a l i n s e r t i o n of the i o n - s p e c i f i c electrode into the cannulated c e l l and the r a d i a l i n s e r t i o n of the micropipette electrode are depicted i n F i g . 2. The c a l c u l a t i o n of the i n t r a c e l l u l a r sodium and potassium a c t i v i t i e s from the p o t e n t i a l differences i n the microelectrode c i r c u i t s was by the method of Hinke (1969a). Chemical Analysis. In a p a r t i c u l a r experiment, a c e l l was cut near i t s connection to the basis, rinsed for 30 sec i n sucrose r i n s e solution, and blotted on f i l t e r paper. A short segment of the c e l l was cut from the tendon end and from the other end, and the remaining central part was placed i n a pre-weighed stoppered v i a l . The wet weight was measured, and the dry weight was measured a f t e r drying of the c e l l fragment i n an oven overnight. The c e l l was then wet ashed for analysis for sodium and potassium by atomic absorp- t i o n spectrophotometry. E x t r a c e l l u l a r Space. The volume of the e x t r a c e l l u l a r space was measured for barnacles from the same l o t as those used i n the experiments as the volume of d i s t r i b u t i o n 3 14 of ( H ) i n u l i n or ( C ) s o r b i t o l (New England Nuclear), by standard methods. The composition of the f l u i d i n t h i s volume was assumed to be that of the bathing sol u t i o n , and the t o t a l sodium and potassium contents of the c e l l as determined by chemical analysis were corrected for the contribution of 83 micropipette electrode single muscle c e l l cation - selective microelectrode glass cannula silk tie tendon Figure 2. Configuration of the microelectrodes and cannulated c e l l during an experiment. Not to scale. 84 t h i s e x t r a c e l l u l a r f l u i d i n the usual manner. Myoplasmic and Nonmyoplasmic I n t r a c e l l u l a r Sodium. For a given c e l l , the myoplasmic sodium a c t i v i t y (aj\j a) m i n millimoles/ l i t r e and the i n t r a c e l l u l a r sodium concentration (Na)^ i n (millimoles i n t r a - c e l l u l a r sodium)/(kilogram i n t r a c e l l u l a r water) were measured. The t o t a l amount of i n t r a c e l l u l a r sodium is thus (Na)^ x where is the weight of i n t r a c e l l u l a r water. This amount is uncertain insofar as the f r a c t i o n of the chemically analyzable sodium r e s i d i n g i n the e x t r a c e l l u l a r space i s uncertain. The myoplasmic sodium content i s ( /. ) x (a,T ) x V , where «Y+ J t Na' m m A is taken as 0.65 and V , the volume of the myoplasmic solvent water, is taken as the solvent water f r a c t i o n measured for. the barha'cle (Hirike 1970), (0.73 x V^), rather than the higher figure quoted i n the Introduction as a general figure for c e l l s . The calculated myoplasmic sodium content thus might be an underestimate. A s i m i l a r c a l c u l a t i o n can be done for potassium. The form i n which the sodium content of each compartment w i l l be presented, for the purpose of d i r e c t comparison of the amount,rather than the concentration,in each compartment, is a r r i v e d at as follows. The weight of water i n the c e l l is V = (wet weight) - (dry weight). The e x t r a c e l l u l a r space i s assumed to be 67o of the t o t a l water. The sodium concentration i n normal Ringer's s o l u t i o n i s 450 mM. The sodium content i n s o l u t i o n i n the e x t r a c e l l u l a r space i s thus 0.06 x V t(kg) x 0 . 4 5 0(mole/litre). The tabulated t o t a l i n t r a c e l l u l a r sodium concentration (Na)^ = (mmoles i n t r a - c e l l u l a r Na)/(kg i n t r a c e l l u l a r water ) = [ ( t o t a l analyzed Na) - (e x t r a c e l l u - l a r Na)J/(0.94 x Vfc) . 'Analyzed' refers to flame photometry. The values i n Table II part b and i n Fig. 6 as 'sodium content' have been normalized by d i v i s i o n by V t- Thus the sodium content of the myoplasmic compartment i s (moles Na i n myoplasm)/V t = (a^ a)m0.68/0.65 and the sodiumconteht 85 of the nonmyoplasmic compartment is (moles Na inside c e l l but not i n myoplasm)/Vt = 0.94(Na)^ - (sodium content of the myoplasmic compartment). Two separate experiments w i l l be described i n turn. For c l a r i t y , the Methods, Results, and Discussion for each experiment w i l l be presented separately. A. INCREASE OF CELL SODIUM Methods. C e l l s were loaded with sodium by immersion overnight i n potassium-free Ringer's solution. Four muscle bundles from the same barnacle were dissected as described above. Two were assigned to be experimental and two to be controls. ( aN a)m' ( N a ) i > ( a K ) m , a n d (K)^ were measured on s i x c e l l s from each group. The remaining experimental c e l l s were then placed i n potassium-free Ringer's s o l u t i o n at 2° C for 20 hours. The remaining control c e l l s were kept i n normal Ringer's s o l u t i o n at 2° C. A f t e r 20 hours, the experimental c e l l s were transferred to potassium- free Ringer's s o l u t i o n at room temperature and ( a j j a ) m , (Na) ̂ , ( a j r ) m , and (K)^ were measured for s i x more c e l l s . S i m i l a r measurements were also done for s i x control c e l l s . Half of the remaining experimental c e l l s were then set i n normal Ringer's s o l u t i o n at 10° C for 18 hours, and h a l f were set i n normal Ringer s s o l u t i o n to which had been added ouabain to 10 M, and l e f t at o 10 C for 18 hours. The remaining control c e l l s were kept i n normal o Ringer s s o l u t i o n at 10 C. 86 At the end of 18 hours, measurements of (a.. ) , (Na)., (a„) , and (K). ' x Na'm' v ' 1' v K'm' s ' i were performed on c e l l s from each group. The sodium and potassium content of the two i n t r a c e l l u l a r compartments were calculated for each c e l l as described i n Methods. This procedure was modelled a f t e r a method for measurement of a sodium extrusion dependent on external potassium ( K ) Q and i n h i b i t e d by.ouabain (Steinbach 1940; Beauge & Sjodin 1967). The features relevant to the present problem are the changes i n ion content of the myoplasmic and non- myoplasmic compartments. Results. The r e s u l t s of this experiment i n which c e l l s were 'passively loaded' with sodium by immersion i n potassium-free s o l u t i o n i n the cold and then were allowed to recover i n normal Ringer's solution, are de t a i l e d i n Table I I . The control c e l l s , which were maintained i n normal Ringer's s o l u t i o n throughout the ca. 40 hours of the experiment, underwent a continuous r i s e i n sodium content, amounting to almost 407, o v e r a l l . The potassium content was unchanged over the f i r s t 22 hours, but showed an increase of about 47o over the f i n a l 20 hours. I t had been ant i c i p a t e d that a decline i n the potassium content of the c e l l s would accompany the r i s e i n the sodium content. The membrane p o t e n t i a l was very close to the potassium equilibrium p o t e n t i a l for barnacle muscle c e l l s (Hinke & Gayton 1971), and i t showed no change during t h i s long experiment. The water content of a l l c e l l s i n - creased s l i g h t l y over the f i n a l 20 hours of the experiment. The changes i n the t o t a l amount of sodium and potassium i n the experi- mental c e l l s (where co r r e c t i o n was made for e x t r a c e l l u l a r sodium and potassium on the basis of an e x t r a c e l l u l a r space containing 67, of the c e l l 87 TABLE H a SUMMARY OF MEASUREMENTS ON PASSIVELY-LOADED CELLS Condition ( aNa)m (Na) . l (a K) m (K) i E m % wat :er I n i t i a l : Control 7.08 14. 57 158. 07 195. 75 72.3 74. 5 n = 6 (1.00) (1- 82) (16. 99) (4. 47) (1.2) (0. 3) Experiment 10.12 13. 05 139. 47 193. 88 71.4 74. 9 n = 6 (2.45) (1. 7 8) (21. 58) (2. 25) (2.7) (0. 3) Loaded: Control 7.82 16. 98 149. 68 194. 47 72.5 74. 4 n = 6 (2.30) (2. 56) (24. 28) (6. 21) (4.4) (0. 4) Experiment 15.47 25. 82 113. 68 178. 17 88.6 74. 8 n = 6 (4.18) (4. 44) (37. 41) (1. 91) (9.4) (0. 2) Recovered: Control 9.58 20. 35 127. 85 202. 45 60.6 75. 1 n = 4 (4.09) (3. 45) (34. 74) (5. 88) (15.0) (0. 3) Experiment 5.83 12. 60 136. 30 196. 05 69.1 75. 5 n = 4 (0.79) (1. 21) (10. 28) (5. 11) (2.1) (0. 1) Ouabain 25.48 50. 48 106. 83 159. 98 55.4 75. 5 n = 4 (7.20) (9. 93) (22. 89) (12. 97) (12.0) (0. 4) (a,, ) and (av) are m i l l i m o l e s / l i t r e myoplasmic water. v Na'm x K'm J • (Na)^ and (K)^ are millimoles/kg c e l l water, corrected for e x t r a c e l l u l a r space ions as d e t a i l e d i n Methods. E i s membrane p o t e n t i a l , i n - m i l l i v o l t s . Note that the measurement on the m Loaded-Experiment c e l l s was c a r r i e d out in potassium-free s o l u t i o n . The numbers i n parenthises are the standard deviation of the measured values, and n is the number of c e l l s examined. 88 TABLE l i b ION CONTENT OF THE MYOPLASMIC AND NONMYOPLASMIC COMPARTMENTS Condition Myopl asmic Nonmyoplasmic Total Na K Na K Na K I n i t i a l : Control 7.4 165 6.3 19 13.7 184 Experiment 10.6 146 1.7 36 12.3 182 Loaded: Control 8.2 157 7.8 26 16.0 183 Experiment 16.2 119 8.1 49 24.3 168 Change 4.8 -19 4.9 6 9.7 -14 Recovered: Control 10.0 134 9.1 56 19.1 190 Experiment 6.1 143 5.7 41 11.8 184 Change -11.9 47 -3.7 -38 -15.6 9 Ouabain 26.6 112 20. 8 38 47.4 150 Change 8.6 16 11.4 -41 20.0 -25 A l l values are millimoles/kg t o t a l c e l l water, as d e t a i l e d i n Methods. The change i n the ion content of the experimental c e l l s was corrected by subtraction of the corresponding change i n the control c e l l s . 89 water, as determined by i n u l i n and s o r b i t o l uptake on barnacles from the same l o t using the same b l o t t i n g technique) were s i m i l a r to those found by Beauge and Sjodin (1967) i n a s i m i l a r experiment on barnacle muscle where only chemical analysis was done. When the changes i n ion content of the control c e l l s were subtracted from the changes i n the experimental c e l l s over the corresponding time period, i t was found that the t o t a l sodium gain and potassium loss was about one-for-one on incubation of the c e l l s i n potassium-free sol u t i o n . When c e l l s which had been incubated i n potassium- free s o l u t i o n were allowed to recover i n normal Ringer's solution, sodium was l o s t and potassium gained, again very roughly on a one-for-one basis. When companion c e l l s which also had been immersed i n potassium-free s o l u t i o n were set to 'recover' i n normal Ringer's s o l u t i o n to which ouabain had been -4 added (to 10 M), there was no recovery. Rather, a further gain of sodium and loss of potassium occurred, again on about a one-for-one basis. The changes i n the sodium and potassium content of the myoplasmic compartment, calculated from the measurements made with i o n - s p e c i f i c i n t r a - c e l l u l a r electrodes as described i n Methods, were for the most part just Tike those for the en t i r e c e l l . On immersion of the c e l l s i n a potassium- free solution, sodium was gained and potassium l o s t by the myoplasmic compartment on about a 4Na:IK basis. When c e l l s which had been immersed i n a potassium-free s o l u t i o n were allowed to recover i n normal Ringer's solu- t i o n , sodium was l o s t and potassium gained by the myoplasmic compartment, but i n th i s case on about a lNa:4K basis. For those c e l l s set to 'recover' i n the s o l u t i o n which contained ouabain, sodium was not l o s t , but rather a further gain occurred. The potassium content of the myoplasmic compartment also showed an apparent increase, but i t should be noted that there was a rather large c o r r e c t i o n applied for the behavior of the control c e l l s . This is the only instance i n which the co r r e c t i o n for changes i n the ion 90 content of the control c e l l s caused a change i n the q u a l i t a t i v e r e s u l t for the experimental c e l l s . The uncorrected data show a roughly one-for-one sodium gain and potassium loss by the myoplasmic compartment for c e l l s which were set to recover i n a normal Ringer's s o l u t i o n which contained ouabain. The changes i n the sodium and potassium content of the nonmyoplasmic compartment, calculated as the difference between the change in ion content of the whole c e l l and that of the myoplasmic compartment, were q u a l i t a t i v e l y d i f f e r e n t . On immersion i n potassium-free solution, there was an equal gain of sodium and of potassium by the nonmyoplasmic compartment. On recovery i n normal Ringer's solution, there was a loss both of sodium and potassium by the nonmyoplasmic compartment, on about-a lNa:10K basis. For those c e l l s set to 'recover' i n the s o l u t i o n which contained ouabain, sodium was gained and potassium l o s t by the nonmyoplasmic compartment, on ' roughly a lNa:4K basis. The i n i t i a l sodium d i s t r i b u t i o n i n the experimental group was quite d i f f e r e n t from that in the control group. Large r e c i p r o c a l differences i n the sodium and potassium content of barnacle muscle c e l l s have been noted before (McLaughlin & Hinke 1966). This and the differences i n the t o t a l ion content of barnacles from different, populations (Brinley 1968; Gayton, A l l e n , & Hinke 1969) apparently are normal. I t i s c e r t a i n l y correct to t r y to i s o l a t e changes i n the experimental c e l l s which are due s o l e l y to the experimental manipulation, but the unexpected gain of potassium by the control c e l l s over the f i n a l 20 hours of the experiment suggests the p o s s i b i l i t y of a s i m i l a r caprice by the experimental c e l l s , quite unrelated to the experimental manipulation. For this reason, i t was f e l t that c r e d i - b i l i t y should be assumed only for the q u a l i t a t i v e changes described here. Thus: the myoplasmic compartment behaved, l i k e the c e l l s as a whole. The behavior i s consistent with the model of the myoplasmic compartment as 91 a compartment whose ion content i s governed by a sodium-potassium exchange mechanism which is stimulated by e x t r a c e l l u l a r potassium and i n h i b i t e d by ouabain. The nonmyoplasmic compartment, on the other hand, gains sodium and potassium together i n the absence of external potassium, so there must in e f f e c t be a s h i f t of potassium from the myoplasmic to the nonmyoplasmic compartment. The nonmyoplasmic compartment loses sodium and potassium together when external potassium i s restored. F i n a l l y , the nonmyoplasmic compartment gains sodium but loses potassium on exposure to Ringer's s o l u t i o n which contains ouabain but i s otherwise normal. Discussion. The main problem of concern here is the l o c a t i o n of the sodium not detected by the sodium-specific i n t r a c e l l u l a r electrode. The model employed in the above c a l c u l a t i o n s , i n terms of solvent water and p a r t i t i o n of ions, is due to Hinke (McLaughlin & Hinke 1966; Hinke et a l . 1973). He proposed that the nonmyoplasmic ions were associated with proteins inside the barnacle c e l l . Experiments on intact c e l l s and on membrane-damaged c e l l s indicate that this compartment has a maximum capacity for ions of about 68 m i l l i - equivalents/kilogram dry weight of intact c e l l (Hinke et a l . 1973),^ and a binding preference for sodium ions over potassium ions. In e a r l i e r experi- ments, i t was assumed that a l l of the potassium was free i n the myoplasm (Hinke 1970), but further experiments indicated that some potassium was The membrane-damaged preparation has l o s t soluble organic molecules, which account for about h a l f of the dry weight of the barnacle c e l l . The binding capacity found i n experiments on membrane-damaged c e l l s is roughly twice that found for in t a c t c e l l s , when no account is taken of the d i f f e r - ence i n the dry weight i n the two s i t u a t i o n s . 92 'bound' as well (Hinke et a l . 1973). The actual amount of potassium associated with fixed i n t r a c e l l u l a r anionic s i t e s should be greater than the amount of sodium so bound, though, because the myoplasmic potassium a c t i v i t y is much greater than the myoplasmic sodium a c t i v i t y . However, the t o t a l amount of nonmyoplasmic cation was about twice the capacity of the proteins i n whole c e l l s . Thus there probably is an a d d i t i o n a l component of the nonmyoplasmic compartment, which contains more sodium than potassium. Experiments i n which the water content of the barnacle c e l l was changed by exposure of the c e l l to hypertonic or hypotonic solutions indicated that most of the nonmyoplasmic sodium i n barnacle muscle c e l l s remained immobile despite large changes i n c e l l water (Hinke 1969b). The myoplasmic potas- sium was unchanged when the c e l l water was increased, but decreased when the c e l l water was decreased. Competitive binding of sodium and potassium to i n t r a c e l l u l a r proteins should follow a mass action rul e , so should not be changed by a change i n the amount of water i n the myoplasmic compartment. It i s i n t e r e s t i n g , however, that the potassium behaved s l i g h t l y d i f f e r e n t l y from the sodium i n these experiments, as i t did i n the present experiments. The e f f e c t of potassium-free s o l u t i o n on the myoplasmic sodium and potassium content of frog s k e l e t a l muscle has been reported (Armstrong & Lee 1971; Lee & Armstrong 1974). The r e s u l t s d i f f e r e d from those found here in barnacle muscle, i n that the nonmyoplasmic compartment of frog muscle l o s t potassium on incubation i n potassium-free solution. Thus the behavior of the myoplasmic and nonmyoplasmic compartments in frog muscle was q u a l i - t a t i v e l y the same. However, i t was found that i f the potassium-free s o l u t i o n used for incubation contained much less calcium than frog Ringer's s o l u t i o n does, then a l l of the sodium gained by the c e l l entered the myoplasmic compartment, although potassium was s t i l l l o s t from both the myoplasmic and nonmyoplasmic compartments. Further, exposure to the calcium-poor 93 s o l u t i o n resulted i n a decrease i n the capacity of the nonmyoplasmic compartment. F i n a l l y , when the loading period was extended to 48 hours, the myoplasmic compartment accounted for most of the accumulation of sodium over the f i n a l 24 hours, as i f the nonmyoplasmic compartment had become saturated. Aside from the potassium loss by the nonmyoplasmic compartment, then, the behavior of frog muscle was the same as that of barnacle muscle on exposure to potassium-free sol u t i o n . The e f f e c t s of calcium are p a r t i c u l a r l y relevant to the second l i k e l y s i t e of residence of nonmyoplasmic sodium, the polysaccharides in the e x t r a c e l l u l a r space. The p o s s i b i l i t y that the sodium not detected by the microelectrode might be e x t r a c e l l u l a r was mentioned by Caldwell (1968). Harris and Steinbach (1956) had measured- cation binding by sugars. Brading and Widdicombe (1977) recently published a c a r e f u l study of the capacity of the e x t r a c e l l u l a r cation-exchanging s i t e s i n mammalian smooth muscle. They used the t r i v a l e n t ion lanthanum to displace sodium, potassium, magnesium, and calcium from the tissue, and calculated the contribution of the i n t r a - c e l l u l a r and e x t r a c e l l u l a r space to the e f f e c t . These cations a l l compete for anionic binding s i t e s outside the c e l l . The amount of e x t r a c e l l u l a r c a t i o n displaced from s p e c i f i c s i t e s by lanthanum should be less than the t o t a l amount of cation bound to e x t r a c e l l u l a r s i t e s . I t amounted to about 4 mmole.potassium per kg dry weight, and about 60 mmole sodium per kg dry weight (assuming 807* water) . I t was also found that lanthanum reduced the passive sodium and (to a lesser extent) potassium movement across the c e l l membrane, but did not appear to a f f e c t the ac t i v e ion movements. I t was suggested that the binding of cations to e x t r a c e l l u l a r s i t e s is a stage of passive transmembrane passage of the cations, and that potassium behaves quite d i f f e r e n t l y from sodium i n i t s passive passage of the c e l l membrane. 94 If the e x t r a c e l l u l a r polysaccharide i s s i m i l a r i n barnacle muscle, as seems l i k e l y , there is then a cr e d i b l e second component to the nonmyoplasmic compartment, containing mostly sodium and having an ion-binding capacity s i m i l a r to that of the i n t r a c e l l u l a r proteins. Together, these two compo- nents appear to have a capacity adequate to contain a l l of the nonmyoplasmic sodium (mostly e x t r a c e l l u l a r ) and potassium (mostly i n t r a c e l l u l a r ) . It i s not c l e a r how the behavior of the nonmyoplasmic potassium i n barnacle muscle can be accounted for with binding to these compartments according to the mass action r u l e . I t might be that the e x t r a c e l l u l a r compartment is indeed involved d i r e c t l y i n the transmembrane transport of potassium by a method d i f f e r e n t from that for sodium, but t h i s c e r t a i n l y could not be concluded from the present experiments alone. It seems more l i k e l y that potassium s h i f t s to the TTS from the myoplasm when external potassium i s removed (Birks & Davey 1969), while binding of potassium to fixed charges i s less important. The behavior of the nonmyoplasmic sodium i s accounted for by a model of the nonmyoplasmic compartment as two regions which can bind sodium, one i n t r a c e l l u l a r and containing r e l a t i v e l y l i t t l e sodium i n comparison with potassium, and one e x t r a c e l l u l a r , containing r e l a t i v e l y l i t t l e potassium i n comparison with sodium. At l e a s t some of t h i s e x t r a c e l l u l a r sodium should engage i n rapid exchange with the sodium i n the bathing sol u t i o n , although i n smooth muscle these cations were mobilized only when lanthanum was introduced. Such exchange might be revealed i n an experiment i n which the e f f l u x of sodium into sodium-free solutions is measured. The re s u l t s of such an experiment are described next. 95 B. DECREASE OF CELL SODIUM Methods. In a separate series of experiments, sin g l e barnacle muscle c e l l s were depleted of sodium by immersion i n iso t o n i c sodium-free lithium-substituted Ringer's s o l u t i o n (Table.I). A muscle bundle was dissected as described above, and measurements of ) and (Na). were performed on several c e l l s i n normal Ringer's s o l u t i o n a m 1 at room temperature. The bundle was then immersed i n a sodium-free lithiu m - substituted Ringer's s o l u t i o n for 30 seconds, then transferred to a large volume of th i s s o l u t i o n . Measurements of ( a N a ) m and (Na) i were performed on each of a succession of c e l l s over the next three hours. This procedure was c a r r i e d out on three d i f f e r e n t muscle bundles, from barnacles from the same l o t . In one case, some measurements were made a f t e r 16 hours of immersion i n sodium-free solu t i o n , where the c e l l s were kept at 10° C between the t h i r d and sixteenth hours. The sodium content of the myoplasmic and nonmyoplasmic compartments was calculated, as described above. Results. The r e s u l t s are presented i n Fig. 3 as the change i n the sodium content of the myoplasmic (closed symbols) and nonmyoplasmic (open symbols) com- partments with time, while the c e l l s were immersed i n sodium-free lithiu m - substituted Ringer's sol u t i o n . The zero of time corresponds to the moment of immersion. The l i n e s were drawn by eye as a v i s u a l a i d . Under c e r t a i n conditions, a large rapid f a l l of the myoplasmic sodium a c t i v i t y can occur i n frog s k e l e t a l muscle (White 6c Hinke 1976) and in crab s t r i a t e d muscle (Vaughan-Jones 1977). Such an e f f e c t had been sought 96 o » o E E 14« 12 10 - 8 "E o o o z t o •a • • o O a o • 30 60 90 120 150 f80 time (min.) 210 240 1000 Figure 3. Changes i n the sodium content of c e l l s during incubation i n sodium-free lithium-substituted s o l u t i o n . Correction for e x t r a c e l l u l a r sodium, by standard methods (see t e x t ) , was made for the c e l l s at zero time (normal Ringer's s o l u t i o n ) . Each point represents one c e l l , where the closed symbol at a given time represents the myoplasmic sodium, and the open symbol at that time represents the nonmyoplasmic i n t r a c e l l u l a r sodium. The three d i f f e r e n t symbol shapes represent three d i f f e r e n t experiments. The i r r e g u l a r l y broken l i n e represents the myoplasmic i n t r a c e l l u l a r sodium, and the re g u l a r l y broken l i n e represents the nonmyoplasmic sodium. The l i n e s were drawn by eye to summarize the three experimental runs. 97 and found i n barnacle s t r i a t e d muscle, and is described i n section 6. However, i t is only seen i n barnacle muscle c e l l s which have an elevated sodium content. Two of the muscle bundles used i n t h i s experiment had r e l a t i v e l y low sodium content (tri a n g u l a r and square symbols i n Fig. 3), and i t was not expected that the e f f e c t would be seen i n them. The small i n i t i a l f a l l i n the calculated sodium content of the..myoplasmic compartment, shown i n F i g . 3, might r e f l e c t a rapid e f f l u x from the t h i r d muscle bundle (round symbols), whose i n i t i a l sodium content was higher. Aside from t h i s v a r i a t i o n , the decline of the sodium content of the myoplasmic compartment with time was rather slow. Even a f t e r 16 hours of immersion i n the sodium-free s o l u t i o n (much of which time was spent at 10° C as noted above), the myoplasmic compartment had retained h a l f of i t s i n i t i a l sodium. The decline of the sodium content of the nonmyoplasmic compartment was markedly d i f f e r e n t . There was a large f a l l over the f i r s t 30 to 40 minutes of immersion, but then almost no loss over the next 15 hours. The v a r i a t i o n of the myoplasmic sodium a c t i v i t y ( a j j a ) m i n barnacle muscle c e l l s on b r i e f (less than 60 min) immersion i n sodium-free s o l u t i o n has been measured by other workers (McLaughlin & Hinke 1968; A l l e n & Hinke 1971). They found that ( a ^ a ) m increased i n i t i a l l y , then decreased. However, the l i t h i u m s o l u t i o n they used was prepared by s u b s t i t u t i n g L i C l for NaCl on a one-for-one basis (McLaughlin & Hinke 1968, Table I ) . Such a s o l u t i o n i s hypertonic, so the i n i t i a l behavior of (a.T ) r e f l e c t s the J t r ' v Na'm movement of water out of the myoplasm. This by i t s e l f has been found not to a f f e c t the sodium content of the myoplasmic and nonmyoplasmic compart- ments (Hinke 1969b). A s i m i l a r experiment of 25 minutes' .duration using is o t o n i c sodium-free sucrose-substituted Ringer's s o l u t i o n showed behavior s i m i l a r to that i n F i g . 3 (Hinke 1969b). 98 A rough approximation of the s i z e of the various fractions of c e l l u l a r sodium can be made from F i g . 3. I f the r a p i d l y - l o s t f r a c t i o n of the non- myoplasmic sodium i s assigned to the e x t r a c e l l u l a r space, there remains about 307o of the i n t r a c e l l u l a r sodium not accessible to the microelectrode ( i n the model described e a r l i e r ) . This i s about 10 millimole/kg dry weight. The data for very long time of immersion suggest that i n barnacle muscle perhaps 207o of the c e l l u l a r sodium i s both not accessible to the micro- electrode and so slow to exchange as to be nonparticipatory i n the type of i n v i t r o experiments described i n this thesis. It has often been found that a f r a c t i o n of the c e l l sodium exchanges only very slowly with r a d i o a c t i v e l y - l a b e l l e d sodium i n the bathing s o l u t i o n (Conway & Cary 1955; Troschin 1961; Dunham & Gainer 1968; A l l e n & Hinke 1970). In p a r t i c u l a r , A l l e n and Hinke (1970) found good quantitative agreement i n barnacle muscle for the amount of c e l l u l a r sodium which ex- changes slowly as calculated from isotope f l u x studies with the amount of 'bound' (nonmyoplasmic) sodium calculated from microelectrode studies. Discussion. In the f i r s t experiment, where the sodium content of the c e l l was increased by immersion of the c e l l i n potassium-free solution, i t was found that the behavior of the nonmyoplasmic sodium could be accounted for by a model of the nonmyoplasmic compartment as two regions which can bind sodium: one i n t r a c e l l u l a r , containing a r e l a t i v e l y small amount of sodium; and one e x t r a c e l l u l a r , containing a r e l a t i v e l y large amount of sodium. The second experiment showed that indeed much of the nonmyoplasmic sodium can be washed out very r a p i d l y i n sodium-free solution, while some cannot be washed out even with long immersion i n sodium-free s o l u t i o n . I t seems reasonable to assign the former to the e x t r a c e l l u l a r component of the non- 99 myoplasmic compartment, and the l a t t e r to the i n t r a c e l l u l a r component of the nonmyoplasmic compartment, although some of the e x t r a c e l l u l a r sodium probably is tightly-bound, as i n smooth muscle. To summarize the rough quantitative estimates, about 307> of the sodium which i s t r u l y i n t r a c e l l u l a r i s not accessible to the microelectrode. Most of this (about 207>) cannot be washed out during long immersion of the c e l l i n sodium-free so l u t i o n . I f this 207. is i d e n t i f i e d with the f r a c t i o n of the c e l l sodium which exchanges very slowly with r a d i o i s o t o p i c sodium, then the amount of nonmyoplasmic i n t r a c e l l u l a r sodium which exchanges r a p i d l y with the myoplasmic sodium i s probably less than 107o of the i n t r a - c e l l u l a r sodium. This conclusion i s important because i t allows the model for the measurement of the sodium e f f l u x from barnacle muscle c e l l s to be r e l a t i v e l y simple, as discussed i n section 2.D. The p o s s i b i l i t y that the e x t r a c e l l u l a r nonmyoplasmic sodium and potassium i s within the o v e r a l l mechanism by which these ions pass the c e l l membrane was ra i s e d i n the discussion of the f i r s t experiment. I t i s possible that some of the e x t r a c e l l u l a r nonmyoplasmic sodium can exchange d i r e c t l y with the myoplasmic sodium. However, i t seems prudent to adopt the simpler hypothesis f i r s t , and test i t in prac t i c e . The r e s u l t s of the next two sections support this choice. 100 SECTION 4. MICROINJECTION OF RADIOSODIUM INTO SINGLE MUSCLE CELLS In the preparation of a c e l l for an experiment i n which the e f f l u x of radiosodium i s to be measured, i t is usually necessary to immerse the c e l l for some time i n a s o l u t i o n which contains radiosodium. Both the i n t r a - c e l l u l a r and the e x t r a c e l l u l a r sodium become l a b e l l e d , and in the subsequent experiment both the i n t r a c e l l u l a r and the e x t r a c e l l u l a r sodium contribute to the measured radiosodium e f f l u x . The e x t r a c e l l u l a r radiosodium i s l o s t very rapidly, and a f t e r a short period of time only the i n t r a c e l l u l a r radiosodium contributes appreciably to the observed e f f l u x . However, the behavior of the e f f l u x from the i n t e r i o r of the c e l l immediately a f t e r the experiment has begun is masked. It i s not a simple matter to subtract the estimated contribution of the e x t r a c e l l u l a r sodium to the t o t a l radiosodium e f f l u x , because the e f f l u x from e x t r a c e l l u l a r s i t e s is not always simple (eg. Rogus & Z i e r l e r 1973). In large c e l l s , the i n t e r i o r can be loaded with radiosodium s e l e c t i v e l y by microinjection. As f i r s t described by Hodgkin and Keynes (1956; see also Caldwell & Walster 1963), a fin e c y l i n d r i c a l glass needle was inserted a x i a l l y into a squid axon, and then removed while f l u i d was ejected from the t i p . The injected f l u i d f i l l e d the space vacated by the withdrawing needle. The technique is of in t e r e s t here i n two respects. F i r s t , the d i s t r i - bution of the injected radiosodium among i n t r a c e l l u l a r pools of sodium can provide a test of the model for the c e l l u l a r sodium described in section 3. The i n t e r p r e t a t i o n by B i t t a r and coworkers of sodium microinjection experi- ments i n barnacle muscle seems to be at variance with the model ( B i t t a r , Chen, Danielson, Hartmann, & Tong 1972), as w i l l be described f u l l y below. Second, the technique is convenient i n that the c e l l can be loaded with 101 radiosodium quickly, the e x t r a c e l l u l a r sodium pool can be bypassed, and the sodium content of the c e l l can be rai s e d by i n j e c t i o n of sodium solutions. A l l of th i s can be done with passive techniques as well, but at the expense of long immersion times, often i n nonphysiological solutions. Of course, the ef f e c t s of microinjection on the c e l l and i n p a r t i c u l a r on the sodium transport out of the c e l l must be known before the technique can be adopted as a convenience i n e f f l u x experiments. In the remainder of th i s introductory passage, the r e s u l t s of other workers on the questions of the e f f e c t of microinjection on barnacle muscle c e l l s , and the d i s t r i b u t i o n of injected radiosodium among i n t r a c e l l u l a r sodium pools are discussed. The experimental portion of this section consists of the comparison of the e f f l u x of radiosodium from c e l l s loaded by microinjection, with that from c e l l s loaded passively. M i c r o i n j e c t i o n was f i r s t used i n barnacle muscle by Hagiwara, Chichibu, and Naka (1964). A large needle was used (200 - 500yU o.d.), f l u i d was injected as the needle was advanced down the axis of the c e l l , and enough f l u i d was injected to double the diameter of the c e l l . Even so, the e x c i t a b i l i t y of the c e l l membrane was unimpaired, and the r e s t i n g membrane po t e n t i a l showed a dependence on the transmembrane difference i n the potassium concentration s i m i l a r to that of intact, noninjected barnacle c e l l s (Hagiwara et a l . 1964; Hinke 1970). Brinley (1968) used the technique of Hodgkin and Keynes, but with a microinjector needle which served as an open-tipped i n t r a c e l l u l a r electrode for measurement of the membrane pote n t i a l as the needle was being advanced down the axis of the c e l l (Brinley & Mullins 1965). He found that there was a transient membrane po t e n t i a l depolarization of 2 or 3 m i l l i v o l t s (mV) with each advance of the in j e c t o r needle, with recovery occurring 102 wi t h i n a few seconds a f t e r the advance was halted. He interpreted t h i s as being due to the tearing and rapid r e s e a l i n g of the c l e f t s and transverse tubules, which are open to the bathing s o l u t i o n and penetrate deeply into the c e l l (Hoyle 1973). Br i n l e y found i t d i f f i c u l t to obtain stable membrane po t e n t i a l readings i n his preparation, even when using conventional micro- pipette electrodes. The mean value he found was -68 mV, and i t did not vary with temperature (16 to 22° C). By comparison, Hagiwara et a l . (1964) reported -73.5 mV; Hoyle and Smith (1963) reported -74 to -96 mV on selected intact c e l l s not dissected from the muscle bundle; and McLaughlin and Hinke (1966) reported -71 mV on intact c e l l s . B i t t a r , Chen, Danielson, Hartmann, and Tong (1972) used the o r i g i n a l technique (Caldwell & Walster 1963). Like Hagiwara et al_. and Brinley, they cut s i n g l e barnacle muscle c e l l s from the basis at the point of attachment to the basis, and cannulated the cut end. They found that the membrane p o t e n t i a l was usually unaffected by the process of microinjection, although t h e i r mean value for the r e s t i n g p o t e n t i a l was only -56 mV (range -42 to -72 mV). B i t t a r et: a_l. found that the e f f l u x of injected radiosodium from the c e l l into normal Ringer's s o l u t i o n declined exponentially with time. The f r a c t i o n of the t o t a l i n t r a c e l l u l a r radiosodium lostpper unit time declined slowly over the f i r s t 60 minutes i n the majority of the c e l l s they studied, but was more stable thereafter. They found that the slope of the semilog pl o t versus time of the amount of radiosodium l o s t from the c e l l per unit tiVe ,d_ ln.d „ * . . was greater than the slope of the semilog plot versus M t ' ' W e e l l " time of the amount of radiosodium l e f t i n the c e l l /d « * \, as Mt c e l l did Hodgkin and Keynes.(1956) i n injected squid axon. That i s , the amount of radiosodium i n the c e l l did not decline with time at a rate commensurate to the decline with time of the rate at which radiosodium appeared i n the 103 bath. This was not expected under the conditions of the experiment. I f the rate of u n i d i r e c t i o n a l sodium e f f l u x is constant, and rapid mixing occurs inside the c e l l , then whatever the k i n e t i c r e l a t i o n describing the dependence of this e f f l u x rate on the i n t r a c e l l u l a r concentration of sodium, the f a l l of the t o t a l radiosodium content of the c e l l i s a simple expo- n e n t i a l function of time. That i s , the t o t a l i n t r a c e l l u l a r sodium content and d i s t r i b u t i o n should be constant, while the c e l l is i n normal Ringer's solution, but the pool of radiosodium present at the i n i t i a l time is 22 23 depleted as. Na exits with Na by a random process which is slow r e l a t i v e 22 to d i f f u s i o n i n bulk solutions. D i l u t i o n of the i n t r a c e l l u l a r Na by 23 Na occurs (where mixing inside the c e l l i s assumed to be rapid compared to the e f f l u x r a t e ) , and the rate at which the t o t a l r a d i o a c t i v i t y Na* c e l l due to radiosodium i n the c e l l declines at each instant is proportional to the amount present at that instant: dNa* -I -i ce_L_L = -k-Na* ,, . dt c e l l The rate constant k depends on the rate of u n i d i r e c t i o n a l sodium e f f l u x , which i n turn depends, i n p a r t i c u l a r , on the a c t i v i t y of sodium i n the s o l u t i o n bathing the i n t r a c e l l u l a r s i t e s of the transport mechanisms. Thus: N a * e l l = Na* e l l(t=0) . exp(-kt) and d_ l n N a * e U = _ k = d_ l n d _ N a * e U since dk i s assumed to be zero. B i t t a r et: al. r e f e r to the "slope r a t i o " dt (̂ _ In N a * e l l ) / ( — In — N a ^ e n ) ' w h i c h t h e y found to be less than unity, dt dt dt Hodgkin and Keynes (1956) considered and rejected as a possible explanation that the sodium e f f l u x was not very s e n s i t i v e to changes i n 104 the i n t r a c e l l u l a r sodium concentration (Na)^, since i n j e c t i o n of sodium to r a i s e (Na)± caused an appreciable r i s e i n the sodium e f f l u x . They considered i t possible that t h e i r preparation was slowly deteriorating, so that the u n i d i r e c t i o n a l sodium e f f l u x M^a was d i r e c t l y proportional to (Na)^ at each instant but the proportionality constant slowly declined with time. They demonstrated that at any given time the sodium e f f l u x increased i n s t r i c t proportion to the amount of sodium injected into the axon. B i t t a r et al_. (1972) mentioned the p o s s i b i l i t y that the "sodium pump" was running down i n t h e i r preparation, but argued that the small slope r a t i o was a c t u a l l y due to damage done to the " i n t e r n a l membrane system" by the passage of the microinjector. An examination of injected fibres with the electron microscope had revealed l o c a l d i s r u p t i o n of the sarco- plasmic reticulum and c l e f t s along the i n j e c t i o n track. They hypothesized that sodium and calcium were compartmentalized i n microsome-like v e s i c l e s created by the i n j e c t i o n . This requires that some of the injected radio- sodium be sequestered at the time of i n j e c t i o n and exchange only very slowly with the free i n t r a c e l l u l a r sodium. As Dick and Lea (1967) have pointed out, this would cause the r a t i o of d_ i n N a * e l l t o iL_ l n i l _ Na* n t o e c l u a i dt ' dt dt c e i i the f r a c t i o n of the i n t r a c e l l u l a r radiosodium which i s free i n the myoplasm (assuming that d_ Na* i i ̂ s l i n e a r l y proportional to the amount of free dt c e l 1 l a b e l , as discussed above). From such a calculation,. B i t t a r jet aT. conclude that on average about 30%. of the injected radiosodium i s sequestered, and in some experiments an average of about 75% i s sequestered. In addition, they postulate that the c e l l s i n which the f r a c t i o n of injected radiosodium l o s t per unit time does not f a l l with time, and those i n which i t does f a l l with time, form two d i s t i n c t populations of normal barnacle muscle c e l l s . Some pharmacological experiments were also done by B i t t a r and co- workers. They were interpreted i n terms of bound sodium, and so w i l l be 105 discussed here. B i t t a r and T a l l i t s c h (1975, 1976) showed that exposure to aldosterone of muscles from a barnacle which had been exposed to aldosterone over the previous night r e s u l t s i n a h a l t of the decline of the f r a c t i o n of the injected radiosodium l o s t per unit time. This e f f e c t was r e v e r s i b l e , the f r a c t i o n resuming i t s decline when aldosterone was removed from the bathing s o l u t i o n a f t e r an exposure of less than 30 minutes. They proposed that, a f t e r the pretreatment with aldosterone, acute exposure of the c e l l to aldosterone caused a r e v e r s i b l e release of radiosodium from i n t r a c e l l u l a r binding s i t e s . The act of i n j e c t i n g solutions of NaCl a f t e r radiosodium had been injected (into aldosterone-pretreated c e l l s ) also caused a cessation of the decline of the f r a c t i o n of the injected radiosodium l o s t per unit time, even with solutions of NaCl so d i l u t e that the i n t r a c e l l u l a r sodium concen- t r a t i o n was r a i s e d by only 1 mM. Subsequent acute exposure to aldosterone had no e f f e c t unless the injected NaCl had considerably increased the i n t r a - c e l l u l a r sodium concentration. In the l a t t e r case, the rate of loss of radiosodium rose slowly to a new steady l e v e l . I f i n the overnight pretreatment with aldosterone actinomycin D was included, i t was found that no c e l l showed a decline with time of the f r a c t i o n of injected radiosodium l o s t per unit time. I f a c e l l pretreated only with aldosterone was exposed acutely to spironolactone, subsequent acute exposure to aldosterone was without e f f e c t . It i s i n t e r e s t i n g that i n a l a t e r publication, B i t t a r , Chambers, and Shultz (1976) found that the f r a c t i o n of injected radiosodium l o s t per unit time was constant in almost a l l cases (judging from the data presented in the f i g u r e s ) . Their specimens were obtained from Puget Sound, while for the previous work barnacles both from Puget Sound and from C a l i f o r n i a were used. Differences i n the ion content of these two populations have been 106 reported (Brinley 1968; Gayton, A l l e n , & Hinke 1969). The acute.exposure to aldosterone of aldosterone-pretreated c e l l s also caused a delayed transient stimulation of the radiosodium e f f l u x . This e f f e c t was abolished by acute treatment with actinomycin D, ouabain, DPH (diphenylhydantoin), or injected ethacrynic acid, but was stimulated by maneuvers which would increase the i n t r a c e l l u l a r supply of ATP. To account for these findings, B i t t a r and coworkers suggested that aldosterone induces synthesis of new protein receptors i n the barnacle muscle c e l l , some of which cause the r e v e r s i b l e release of "bound" i n t r a - c e l l u l a r sodium (alleged i n the e a r l i e r paper to be sequestered i n v e s i c l e s created by the microinjector) and some of which cause a delayed stimulation of the ATP-dependent sodium e f f l u x , upon subsequent exposure to aldosterone. A d i f f e r e n t explanation for the observed "slope r a t i o " , i n terms of an e f f e c t i v e i n t r a c e l l u l a r sink for injected radiosodium, w i l l be described i n the discussion of the experimental portion of this section. I t cannot account for the ef f e c t s of aldosterone reported by B i t t a r and coworkers, but i t seems l i k e l y that the main e f f e c t of aldosterone is on the transport systems i n the membrane rather than on the state of the i n t r a c e l l u l a r sodium. The suggestion that over h a l f of the exchangeable i n t r a c e l l u l a r sodium can be r e v e r s i b l y sequestered does not seem reasonable, given our knowledge of the morphology and ion-sequestering properties of the c e l l . I t is possible that i n some c e l l s the supply of metabolic energy i n a su i t a b l e form for u t i l i z a t i o n by the sodium transport systems i s not optimal, so that the system i s indeed 'running down'. Aldosterone s p e c i f i c a l l y promotes the transport of sodium i n some c e l l s by a mechanism which involves the synthesis of new protein by the c e l l . This could cause a c t i v a t i o n of the transport enzymes, provide a d d i t i o n a l energy for the transport enzymes, provide a d d i t i o n a l transport enzymes, or y i e l d a combination of these 107 effects (Feldman, Funder, & Edelman 1 9 7 2 ) . METHODS Dissection of barnacle muscle bundles has been described i n section 3. Previous work on the sodium e f f l u x from barnacle muscle c e l l s was done with c e l l s which had been cut o f f at the basis, as mentioned above. Since th i s cannot be done without damaging the c e l l membrane, sp e c i a l measures were required to prevent the rapid occurrance of d e t e r i o r a t i o n of the c e l l . B r i n l e y ( 1 9 6 8 ) immersed the terminal 5-10 mm of the cut end of the c e l l i n o i l for at l e a s t 3 0 minutes before cannulation and i n j e c t i o n . B i t t a r et a l . apparently adopted a s i m i l a r procedure ( B i t t a r 1 9 6 6 ) . In the present work the c e l l s were kept i n t a c t throughout, with the tendon alone being cannulated. The c e l l membrane was breached only by the i n j e c t i o n needle or the sodium electrode through the tendon end, and by the micropipette electrode r a d i a l l y about 20 mm from the tendon end (Fig. 2 ) . No i n s u l a t - ing o i l was-used, yet only r a r e l y did these manipulations cause v i s i b l e damage to the c e l l (and thereby cause the c e l l to be discarded). ^ N a C l was obtained from New England Nuclear, c a r r i e r - f r e e , i n d i s - t i l l e d water. Before use for i n j e c t i o n , the water was evaporated o f f and 22 23 the NaCl redissolved i n d i s t i l l e d water or i n NaCl sol u t i o n . I n j e c t i o n Apparatus. A Hamilton microsyringe was used throughout. This had a nominal volume of 1.0 m i c r o l i t r e ( A ) , a Chaney adaptor, and a metal c o l l a r protecting the proximal part of the needle (model NCH 7 0 0 1 ) . A fin e needle was drawn from 4 mm o.d. lead glass, on a mechanical micropipette p u l l e r with a long 108 throw (Hinke 1969a). S u f f i c i e n t l y durable needles had c y l i n d r i c a l shaft with o.d. 110-120yU and length from the top of the shoulder to the t i p of 38 mm. The t i p was broken o f f to this length i n such a manner that the t i p was beveled s l i g h t l y . New glass needles were made each day. The glass needle was attached to the syringe needle with s t i c k y wax as follows (Fig. 4). With the plunger withdrawn from the t i p s l i g h t l y , a small c o l l a r of hot s t i c k y wax was put on the metal syringe needle near the t i p , and allowed to harden. D i s t i l l e d water was then drawn up into the syringe to 0 . 9 A , the t i p was dried by b l o t t i n g , and the plunger withdrawn to 0.95^ so no water was at the t i p . The metal needle was then inserted into the glass needle, up to the shoulder. The shoulder (where the wax c o l l a r contacted the inside of the glass) was b r i e f l y passed through a gentle flame so that the s t i c k y wax melted, and the glass needle was then gently pushed further onto the metal needle, u n t i l the metal needle was stopped by the tapering shoulder of the glass. The s t i c k y wax flowed to seal the needles together without bubbles. The stem of the glass needle, which extends inside the metal protective c o l l a r , was then fixed to the metal c o l l a r with dental impression compound. The d i s t i l l e d water was then expelled into the glass needle, and usu a l l y f i l l e d i t without bubbles. When a l l a i r had been expelled from the glass needle, the assembled microsyringe was mounted v e r t i c a l l y on the in j e c t o r (see below) with the t i p of the glass needle submerged i n d i s t i l l e d water. The i n j e c t o r (Fig. 4) consisted of a brace which firmly held the microsyringe by the ba r r e l and by the plunger, and a P r i o r micromanipulator r e b u i l t so that the enti r e microsyringe could be moved in the v e r t i c a l d i r e c t i o n ('positioning'), and so that the bar r e l of the microsyringe could be moved r e l a t i v e to the plunger ('injecting'). The former movement was used to p o s i t i o n the glass needle i n the c e l l , and the l a t t e r to withdraw Figure 4. M i c r o i n j e c t o r . The basic features are shown, not to scale. Inset: d e t a i l of the connection of the glass i n j e c t i o n needle to the Hamilton syringe needle. Not to scale. 110 the needle while e x p e l l i n g a column of i n j e c t i o n f l u i d into the c e l l . The clamp holding the syringe was adjustable i n two h o r i z o n t a l d i r e c t i o n s , so that the glass needle could be aligned with the v e r t i c a l defined by the motion of the micromanipulator. C a l i b r a t i o n of the Microinjector. To test the uniformity of the d e l i v e r y of f l u i d from the syringe, a 22 s o l u t i o n of NaCl i n water was drawn up into the syringe, to 0.75 ^ , and ejected into gamma counter glass counting tubes containing 5 ml of d i s t i l l e d water, in aliquots nominally of 0.1^. Five successive 0.1 "X aliquots could be ejected before the counts in the tube dropped below the t o t a l for each of the preceding tubes. The dropoff presumably was due to mixing of the working f l u i d ( d i s t i l l e d water) with the c a l i b r a t e d f l u i d with which i t was i n contact. In several such tests, and t r i a l ejections of amounts varying from (nominally) 0.05/\ to 0.5^, the ejected volume calculated from the amount of radiosodium ejected agreed with the nominal volume to well within the uncertainty due to counting of the isotope (less than 47o) . Injections into c e l l s during experiments were c a r r i e d out within these l i m i t s of uniform d e l i v e r y . The absolute value of the injected volume was tested by ejections of a s o l u t i o n of known r a d i o a c t i v i t y , and was found to agree with the nominal value to within the uncertainty of counting. C o l l e c t i o n of Isotope. The chamber used during i n j e c t i o n of i n t a c t fibres and c o l l e c t i o n of perfusion f l u i d was s i m i l a r to that described by A l l e n and Hinke (1970) but considerably modified (Fig. 5). The cannulated f i b r e (see below) was held v e r t i c a l l y at i t s rest length, with the fragment of the basis r e s t i n g I l l Figure 5. Apparatus for i s o l a t i o n of a segment of a c e l l for perfusion. The moveable blocks, one containing the inflow channel and the other containing the outflow channel, are shown i n the opened p o s i t i o n . A c e l l attached to a fragment of basis i s outlined. 112 on the f a l s e f l o o r of the lower chamber. The height of the f a l s e f l o o r was adjusted so that the cannulated tendon was at the desired height above the top of the chamber while undue tension was not exerted on the f i b r e . The movable Ple x i g l a s blocks were retracted and the chamber f i l l e d with Ringer's s o l u t i o n for i n j e c t i o n of isotope and placement of microelectrodes. The movable blocks were then brought together by turning the thumb screws, while the displaced f l u i d was withdrawn through the suction tubes. The grooves m i l l e d i n the movable blocks formed a c y l i n d r i c a l chamber around the f i b r e when the blocks were brought together. This was separated from the lower chamber, containing the fragment of basis, by a seal of petroleum j e l l y (Vaseline). Vaseline was also used to seal the movable blocks to the stationary parts of the chamber. The grease seal which separated the upper and lower chambers was r o u t i n e l y tested by r a i s i n g the f l u i d l e v e l i n the lower chamber and observing the f a i l u r e of f l u i d to enter the upper chamber, or by f i l l i n g the upper chamber and observing no leak into the lower chamber. Perfusion f l u i d was delivered from a Braun syringe pump, f i t t e d with a 50 ml syringe (or two 50 ml; syringes i n p a r a l l e l ) , at a constant rate of 1 ml/min. Two such pumps were used a l t e r n a t e l y , so that s o l u t i o n changes were accomplished by disconnecting the deli v e r y tube from one syringe and connecting i t to the other. This switch takes less than two seconds, so the i n t e r r u p t i o n of the perfusion was n e g l i g i b l e . Perfusion f l u i d was drawn o f f through three exhaust ports located near the top of the upper chamber, and c o l l e c t e d d i r e c t l y i n a glass gamma counting tube (Fig. 6). The c o l l e c t i o n period was 5 min. The c o l l e c t i o n tube was changed manually. This interrupted the c o l l e c t i o n for 2 to 3 seconds, but caused no loss of f l u i d . The volume of the upper chamber, which housed the injected portion of 113 suction bleeder from washout chamber rubber stopper glass gamma counter tube Figure 6. Vacuum system. The perfusate from the washout chamber (Fig. 5) i s c o l l e c t e d i n the glass gamma-counter tube. 114 the f i b r e , was 1.0 ml. The washout time of the chamber was tested, with a glass rod placed where the f i b r e would normally be, and was found to be 87% complete wi t h i n 2 minutes, corresponding to a time constant for exponential loss of l a b e l of about 1 min. ^. The e f f e c t of the f i n i t e washout time of the chamber can be estimated •ic ic from a simple model. For the myoplasm (Na^), the washout chamber (Na2), ic and the c o l l e c t i o n tube (Na^) considered as three compartments i n serie s with no backflux, and n _ l = -k x Na* dt d N a 2 = k 1 Na* - k 2 Na* dt d N a 3 = k 2 Na* dt ic ic where at t = 0, Na-̂  — (Na^)^ and Na 2 s Na-j = 0. The general s o l u t i o n for th i s simple l i n e a r system i s Na x(t) = (NaJ) Q e x p C-^t) Na*(t) = ^ ^ f J ^ C 1 " e x P ( " k l t ) ) " k x ( l - exp(-k 2t))] . That i s , ic ^ f i = k ^ N a ^ o exp(-k l t) dt while dNao k.k^Na?),) r ~\ so i f dNa^/dt (which i s measured) i s to approximate -dNa^/dt cl o s e l y , k^ must be much less than k 2. Since k^ is t y p i c a l l y 0.01 min"-1- and k 2 is about 1 min" 1, t h i s condition i s w e l l - s a t i s f i e d , and except for the i n t e r v a l 115 just a f t e r t = 0 the f a l l i n the amount of l a b e l c o l l e c t e d with time c l o s e l y approximates the f a l l i n the amount of l a b e l leaving the muscle c e l l with time. The 5 ml samples of perfusion f l u i d were counted on a well-type gamma counter (Nuclear Chicago). I n i t i a l counting for 1 min. per sample was done during the experiment to monitor the progress of the e f f l u x . Later, the counting was repeated at 20 min. per sample. Sample tubes were re-used a f t e r washing with detergent and then chromic s u l f u r i c acid. Backgrounds for each tube were determined by a 10 min. count and subtracted i n d i v i d u a l l y from the corresponding sample count. This economy was possible because glassware cleaning and unlimited counter time were a v a i l a b l e . Sample counts were almost always more than 10 times background (the exceptions being samples of low a c t i v i t y at the end of a long e f f l u x experiment), and the background of the chemically cleaned tubes was equal to that of new tubes. Micro i n j ec t ion. Cannulation of the tendon of the muscle f i b r e is r e a d i l y accomplished without a f f e c t i n g the c e l l membrane (Fig. 2). The cannulated f i b r e was positioned v e r t i c a l l y i n the P l e x i g l a s chamber as described above, so that the s i l k t i e was about 3 mm above the l e v e l of the f l u i d f i l l i n g the chambers. The cannula was held v e r t i c a l l y , by a P r i o r micromanipulator. The P l e x i g l a s chamber was positioned on the h o r i z o n t a l platform of a Palmer screw stand, and the microinjector was positioned on the hor i z o n t a l p l a t - form of a second Palmer screw stand. The glass needle was then advanced down through the cannula and into the muscle f i b r e , as close as possible to the long axis of the f i b r e . The advance of the needle was viewed through a binocular microscope, from the front and, v i a a small mirror positioned 116 at a 45 degree angle near the muscle f i b r e , from the side. The outside diameters of the f i b r e i n these two views were measured with an eyepiece micrometer. Illumination was provided by a 500 watt lamp (Vo l p i ) , directed obliquely at the muscle f i b r e by a f i b r e optic l i g h t conduit. The needle was quite v i s i b l e i n the opaque muscle f i b r e , and was always kept close to the c e n t r a l axis of the muscle f i b r e . The i n j e c t i n g movement of the microinjector was then used to withdraw the needle while e x p e l l i n g the i n j e c t i o n f l u i d into the c e l l . The i n j e c t i o n track was 22 mm long, corresponding to 0.4^, in most cases, but was shorter when i t proved impossible to advance the needle far enough down the muscle c e l l without having the t i p deviate from the a x i a l l i n e . Injec- t i o n was terminated 5 mm from the point of impalement, to ensure that a l l of the f l u i d was deposited inside the c e l l . With the more concentrated i n j e c t i o n solutions, there would often be a s l i g h t contracture over the injected region, and i f this had not disappeared by the time the microelectrodes were put into place, the experiment was not continued. In most cases, and with the more d i l u t e i n j e c t i o n f l u i d s almost always, the i n j e c t i o n was w e l l t o l e r a t e d by the muscle f i b r e , as far as could be detected by observation through the microscope. The microinjector was removed and the sodium-specific microelectrode was placed in the c e l l a x i a l l y , through the cannula, v i a manipulations s i m i l a r to those used for p o s i t i o n i n g the i n j e c t o r needle. The s e n s i t i v e t i p of the electrode was placed i n about the centre of the injected region of the muscle f i b r e . The micropipette electrode t i p was passed obliquely across the c e l l membrane so i t rested at the l e v e l of the sodium electrode. The movable blocks were then brought together to form the e f f l u x chamber, the grease seal was tested, and the perfusion was started. With a l l of these maneuvers, the f i r s t perfusion f l u i d was c o l l e c t e d 10 to 12 minutes 117 a f t e r the actual i n j e c t i o n . Passive Loading Experiments. From a s i n g l e dissected muscle bundle, three small groups of muscle c e l l s were is o l a t e d , each group on a fragment of the basis. These were 22 placed i n a vessel with 10 ml of normal Ringer's s o l u t i o n to which NaCl had been added, to 20,930 cpm per m i c r o l i t r e , and kept at 5° C overnight. The next day, two of the small groups were used for e f f l u x experiments, as described above for microinjected c e l l s , but they were not injected. The t h i r d group was loaded for 48 hours before being used for e f f l u x measurement. RESULTS Ef f e c t s of microinjection on the c e l l . The e f f l u x of microinjected radiosodium from a s i n g l e barnacle muscle c e l l is shown in Fig. 7. A s i m i l a r plot for a c e l l loaded passively, by immersion i n a s o l u t i o n which contained radiosodium, i s presented as Fig. 9 (page 128). The f a l l of the radiosodium content of the injected c e l l with time and the f a l l of the e f f l u x of radiosodium with time can each be c l o s e l y matched by a simple exponential function. A f t e r the i n i t i a l rapid e f f l u x from the passively-loaded c e l l , ascribed to the e x t r a c e l l u l a r space as discussed below, the plots can each be matched by a simple exponential function during the e f f l u x into normal Ringer's s o l u t i o n (Fig. 9). The. rate constants are s i m i l a r for the two cases. The 'slope r a t i o ' e f f e c t i s discussed below. In F ig. 10 (page 140) i s presented a summary of the raw data and reduced 118 Figure 7. Semilogarithmic plot of the amount of radiosodium c o l l e c t e d i n the perfusate from a c e l l loaded with isotope by m i c r o i n j e c t i o n (upper t r a c e ) , and the amount of radiosodium remaining i n the c e l l at the s t a r t of each c o l l e c t i o n period, calculated by back-addition (lower trace), versus time. The c e l l was perfused with normal Ringer's s o l u t i o n . Lines represent the ^ l i n e a r regression l n y = l n a + bx. Upper: a = 23,112 cpm, b = -0.00928 min -, r ?= 0.97. Lower: a = 548,947 cpm, b = -0.00786 min" 1, r 2 = 1.00. 119 r e s u l t s for a t y p i c a l experiment in the present s e r i e s . Measurements with the sodium-specific microelectrode show that the myoplasmic sodium a c t i v i t y ( a f j a ) m r i s e s a f t e r the i n j e c t i o n i n most c e l l s , sometimes taking 30 minutes to reach a steady value. (The value of ( a ^ a ) m in noninjected c e l l s is t y p i c a l l y about 10 mM - see Table I I ) . This r i s e was less marked in c e l l s of a larger diameter. Further, i t has been reported that immersion of a barnacle muscle c e l l in sodium-free sucrose-substituted Ringer's s o l u t i o n for 5 to 10 minutes washes out most of the e x t r a c e l l u l a r sodium while leaving the t o t a l i n t r a - c e l l u l a r sodium content almost unchanged (McLaughlin & Hinke 1966; Brinley 1968; Brigden, Spira, & Hinke 1971; the 'rapid' sodium-free e f f e c t i n sodium-loaded c e l l s has been mentioned i n section 2 and is discussed f u l l y in section 6). When a c e l l was kept i n sodium-free sucrose-substituted Ringer's s o l u t i o n for 5 to 10 minutes p r i o r to i n j e c t i o n , and i n normal Ringer's s o l u t i o n immediately a f t e r i n j e c t i o n , the r i s e of ( a j j a ) m a f t e r i n j e c t i o n was much less marked. This maneuver was used i n a number of the experiments to obtain data at low sodium l e v e l s ; I t should be mentioned that i n the present series of experiments, no data was used for c a l c u l a t i o n of fluxes u n t i l ( a ^ j a ) m had become steady, except where noted e x p l i c i t l y . The observation by B r i n l e y (1968) of a s l i g h t transient depolarization of the membrane pote n t i a l with each advance of the i n j e c t o r needle was noted i n the Introduction. B i t t a r ejt al_. (1972) reported that the t o t a l sodium content of the c e l l was r a i s e d by i n j e c t i o n but not by cannulation. They suggested that t h i s was due to damage to the membrane of the c l e f t s along the i n j e c t i o n track, causing e x t r a c e l l u l a r sodium to be released into the myoplasm. In Fig. 7 i t can be seen that the f i r s t two points in the plot of the measured amount of radiosodium leaving the c e l l per unit time (upper plot) 120 seem high. In many experiments, the f i r s t few points were lower. This conceivably could be due to a small loss of i n j e c t i o n f l u i d into damaged c l e f t s during the actual i n j e c t i o n . I t a l s o could simply be due to the s l i g h t delay between the i n j e c t i o n i t s e l f and the i n i t i a t i o n of perfusion. B i t t a r et a l . (1972) noted that a second i n j e c t i o n did not a l t e r the course of the e f f l u x of previously injected radiosodium, although B r i n l e y (1968) reported that the c e l l would not t o l e r a t e a second i n j e c t i o n . 3 The e x t r a c e l l u l a r space marker ( H ) i n u l i n does not pass the i n t a c t c e l l 3 membrane. When a s o l u t i o n containing ( H ) i n u l i n was injected into a c e l l , no l a b e l was detected subsequently i n the bathing solution. On the other hand, following i n j e c t i o n of (^CJDMO, (5,5-dimethyl-2,4-oxazolidinedione) , which i s known to cross the c e l l membrane quite r e a d i l y , the l a b e l appeared promptly i n the bathing sol u t i o n . It was concluded that any loss of radio- sodium through damaged membrane during microinjection must be quite small. Altogether, i t seems almost c e r t a i n that the increase i n i n t r a c e l l u l a r sodium (and calcium - B i t t a r et. a_l. 1972) seen a f t e r i n j e c t i o n i s due to a transient i n f l u x of e x t r a c e l l u l a r f l u i d during i n j e c t i o n . The membrane pote n t i a l E i s the most s e n s i t i v e i n d i c a t o r of the r m i n t e g r i t y of the c e l l membrane. As a r u l e , E m was depolarized by 10 to 15 m i l l i v o l t s a f t e r i n j e c t i o n , then recovered slowly (20 to 30 minutes) to assume a steady value close to but generally s l i g h t l y more p o s i t i v e than the p r e - i n j e c t i o n value. Injected c e l l s then showed stable membrane poten- t i a l s for several hours at room temperature i n normal Ringer's solution. Altogether, i t was concluded that while microinjection i s not e n t i r e l y benign, the injected c e l l s recover quickly and appear to behave as non- injected c e l l s do for several hours a f t e r i n j e c t i o n . 121 The 'slope r a t i o ' . For the experiment presented i n Fig. 7, the l i n e a r regressions of In Na and In ̂ a c e n on time indicate that almost a l l of the scatter can be 2 accounted for by random error i n the sampling and counting (r =0.97 and 1.00 r e s p e c t i v e l y ) , and yielded time constants 0.00928 min"-'" and 0.007 86 min-"'" respectively. The slope r a t i o d_ ^ n j d_ in d_ dt dt dt calculated as the r a t i o of the 'slopes' yielded by the l i n e a r regressions, is 0.85. The range of 'slope r a t i o s ' for a number of experiments i s indicated i n F i g . 8. They are plotted versus the myoplasmic sodium a c t i v i t y for the c e l l . Usually the duration of exposure to normal Ringer's s o l u t i o n was o shorter than i n Fig. 7 (50 minutes) and the value for r was s l i g h t l y lower (0.90 to 0.95 i n almost a l l cases). O v e r a l l , these 'slope r a t i o s ' were closer to unity than those reported by B i t t a r et a l . (1972). With respect to the 'slope r a t i o s ' as interpreted by B i t t a r el: a_l., the p r i n c i p a l question is how the injected radiosodium d i s t r i b u t e s inside the c e l l . For convenience, the assumptions adopted in the model of the c e l l employed i n t h i s thesis w i l l be restated. A fundamental assumption of this model i s that the i n t r a c e l l u l a r concentrations which are of d i r e c t importance for transmembrane e f f l u x are those of the myoplasm (given constant e x t r a c e l l u l a r conditions). By "membrane" i s meant the b a r r i e r to d i f f u s i o n , and any boundary.layer of glycocalyx or of water which is strongly influenced by the p r o t e i n - l i p i d lamina can be included as part of the membrane. Ion concentrations can d i f f e r i n the myoplasm and the boundary layer, but electrochemical poten- t i a l cannot, i t being assumed that transmembrane transport is almost always slow r e l a t i v e to mixing inside the c e l l . I t can be demanded formally that ion (sodium) a c t i v i t i e s do not d i f f e r i n the myoplasm and the boundary 122 slope ratio 1.2 1.0 0.8 0.6 0.4 0.2 20 (aNa>m 40 (mM) 60 Figure 8. 'Slope Ratio' is the r a t i o ( — In N a c e l l ) / ( — l n — N a c g l l ) , which i s the r a t i o of the slope of the lower l i n e to that of the upper l i n e i n F i g . 7. Each point represents one e f f l u x experiment i n normal Ringer's so l u t i o n . Not a l l of the experiments done i n the present series are represented. Points omitted for c l a r i t y f e l l i n the lower range of (a ) . 123 layer, as long as this conception of the "membrane" i s kept i n mind. The myoplasm bathes the i n t e r n a l surface of this membrane, and i t i s across t h i s membrane, v i a various mechanisms, that the bulk of the sodium e f f l u x from the c e l l occurs. That i s , for the moment i t i s assumed that sodium e f f l u x into the TTS and the SR i s unimportant under most conditions. Recall that i t was stated above that — In Na i — In — Na dt c e l l dt dt c e l l i f and only i f 4- Na* „ = -k Na* , dt c e l l c e l l ' where d k = 0. The slope r a t i o statements must be made to r e f e r to myo- plasmic parameters: the amount of radiosodium i n the myoplasm Na m is sub- s t i t u t e d for Na^e-Q, and the myoplasmic sodium a c t i v i t y ( a j T a ) m is s u b s t i - tuted for the i n t r a c e l l u l a r sodium concentration as the determinant of the rate of u n i d i r e c t i o n a l sodium e f f l u x . Given previous experimental r e s u l t s , i t was postulated that the rate of u n i d i r e c t i o n a l sodium e f f l u x is an increasing function of the myoplasmic sodium a c t i v i t y over a wide range. Thus as long as the rate of u n i d i r e c t i o n a l sodium e f f l u x i s found to be constant, the net flux of sodium must be zero, given the 'membrane-flux1 model described i n the preceding paragraph. The rate constant k i s , in p a r t i c u l a r , a function of the rate of the u n i d i r e c t i o n a l sodium e f f l u x . This r e l a t i o n s h i p i s usually expressed as a polynomial i n the i n t r a c e l l u l a r sodium concentration. I t has been stated that i f the radiosodium i s well-mixed inside the c e l l at a l l times, and the rate of u n i d i r e c t i o n a l sodium e f f l u x i s constant, the slope r a t i o w i l l be equal to unity. The s i t u a t i o n s i n which these conditions do not obtain can be l i s t e d . - I f there is a net increase i n the sodium content of the myoplasm with 124 time, the s p e c i f i c a c t i v i t y of the myoplasm SA m w i l l f a l l more quickly than would be expected according to the observed e f f l u x of radiosodium. A f a l l of SA m w i l l decrease d wa* , since a smaller f r a c t i o n of the u n i d i r e c t i o n a l M ~T~ m dt sodium e f f l u x w i l l consist of radiosodium. On the other hand, as ( a j j a ) m r i s e s the rate of u n i d i r e c t i o n a l sodium e f f l u x w i l l r i s e , and there w i l l be an increase i n d Na* • The net e f f e c t on the slope r a t i o cannot be — m dt s p e c i f i e d i n the absence of a s p e c i f i c model for the sodium e f f l u x . In any event, the sodium content of injected c e l l s has been monitored with a sodium-specific microelectrode, as noted above. The i n t r a c e l l u l a r sodium content increases upon i n j e c t i o n , due to a transient i n f l u x of e x t r a c e l l u l a r f l u i d , but the sodium content takes at most 30 minutes to s t a b i l i z e , and usu a l l y much less . This cannot account for the observed slope r a t i o s . It i s conceivable that the rate of the u n i d i r e c t i o n a l sodium e f f l u x might change independently. For example, i t might f a l l as the supply of energy from metabolism i n the i s o l a t e d c e l l is depleted. The condition of the 'pump' can be in f e r r e d from observation of the e f f l u x over a long period i n normal Ringer's sol u t i o n . It was found that there was no obvious deviation from l i n e a r i t y "in the semilog plots depicting the loss of radiosodium from injected c e l l s i n experiments of long duration (for example, Fig. 7). This indicates that the 'pump' is not running down. The f i n a l p o s s i b i l i t y i s that the injected radiosodium becomes com- partmentalized inside the c e l l . B i t t a r e_t al_. (1972) adopted the model of Dick and Lea (1967), wherein some of the radiosodium inside the c e l l was sequestered and exchanged with the free i n t r a c e l l u l a r radiosodium only at a n e g l i g i b l y slow rate. As noted above, the slope r a t i o i s equal to the f r a c t i o n of the radiosodium l e f t i n the c e l l which is free. This i s not a 125 constant, but rather declines with time as the free radiosodium i s washed out of the c e l l . Fig. 7 indicates that the slope d_ in j j a * does not decline noticeably dt c e l 1 with time, contrary to the Dick and Lea model i f the si z e of the sequestered f r a c t i o n i s not to be n e g l i g i b l e . Indeed, B i t t a r e_t a l . calculated this f r a c t i o n as up to 707o of the injected radiosodium. The a l t e r n a t i v e is a compartmental model i n which the i n t e r n a l compart- ments can exchange sodium. Many mathematical treatments of such models have been published, but q u a l i t a t i v e considerations can narrow the range of poss i b i l i t i e s . The i n j e c t i o n loads the myoplasmic compartment, s e l e c t i v e l y and very r a p i d l y . I f there were an i n t r a c e l l u l a r compartment of f i n i t e s i z e which exchanged sodium with the myoplasm with a rate constant for exchange comparable to the rate- constant for the transmembrane-flux, then the e f f l u x curve for injected c e l l s would not be so close to a simple exponential for a l l times from near zero. The compartment would load from, then empty into the myoplasm i n the course of the experiment. Consideration of both the s i z e of the hypothetical compartment and the rate at which i t exchanges sodium with the myoplasm is important i n drawing this conclusion. For the range of e f f l u x rate constants found i n the present series of experiments, any such compartment of appreciable but not too large a s i z e (as explained i n the next paragraph) whose rate constant was w i t h i n an order of magnitude of the rate constant for the transmembrane flux would be detectable as a deviation from l i n e a r i t y i n a semilog plot such as Fig. 7. A small compartment which exchanges sodium r a p i d l y with the myoplasm would not be seen as a deviation from l i n e a r i t y , and is of no concern i n the present context. The considerations of section 3 suggest that the 126 sodium associated with i n t r a c e l l u l a r fixed anionic s i t e s is such a compart- ment. A larger compartment which exchanges very slowly with the myoplasm would not be loaded with injected radiosodium i n experiments whose duration was a few hours, although i t might be loaded i n an experiment whose duration was tens of hours. The sodium which exchanges only very slowly, such as that reported for barnacle muscle by A l l e n and Hinke (1971), and the sodium termed "inexchangeable" i n section 3, which probably is the same pool, should be such a compartment. It also i s of no concern in the present context. A very large compartment would load with injected radiosodium from the myoplasmic compartment throughout the duration of the experiment, and might not be seen as a deviation from l i n e a r i t y i n the semilog p l o t i f i t s exchange with the myoplasm can be described by a simple exponential function of time, even i f the exchange were r e l a t i v e l y rapid. It would constitute an i n t r a c e l l u l a r sink for radiosodium, and the t o t a l amount of radiosodium i n the c e l l would not decline with time at a rate commensurate to the decline with time of the rate at which radiosodium appeared i n the bath. That i s , the two slopes would each be constant, but the slope r a t i o would be less than unity. In t h i s connection, comparison of the e f f l u x from injected c e l l s with that from c e l l s loaded with radiosodium by immersion i n a s o l u t i o n which contains radiosodium i s relevant. Three e f f l u x experiments were done on c e l l s from a s i n g l e barnacle which were loaded with radiosodium by immersion at less than 5° C i n normal Ringer's s o l u t i o n which contained some radio- sodium, as described i n Methods. Two experiments were done a f t e r a 24 hour loading period, and one a f t e r a 48 hour loading.period. The e f f l u x determi- nation was c a r r i e d out as for injected c e l l s , but no i n j e c t i o n was done. The semilog plots for the e f f l u x from passively loaded c e l l s d i f f e r e d 127 from those for injected c e l l s . The plots for the second experiment are shown i n Fig. 9. They were l i n e a r for time greater than 50 minutes when perfusion was with normal Ringer's sol u t i o n . A l i n e a r regression was per- formed on the part of the curve from 50 to 135 minutes. The regression l i n e was extrapolated back to zero time, and the difference between the data and this l i n e over the i n t e r v a l from 0 to 50 minutes was re-plotted, as shown i n Fig. 9. This operation yielded a simple exponential. I t i s reasonable to ascribe t h i s rapid e f f l u x to the washout of radiosodium from the e x t r a c e l l u l a r space, as w i l l be discussed below. An e f f l u x of comparable rate from the i n t e r i o r of the c e l l has been implied by measurements i n frog and crab muscle, as noted i n section 2, but only i n sodium-free sol u t i o n . No such rapid e f f l u x into normal Ringer's s o l u t i o n has ever been reported. The slope r a t i o s obtained i n the three experiments were 1.30 and 0.98 for a 24 hour loading period, and 0.99 for a 48 hour loading period. I t was noted at the conclusion of the f i r s t experiment that the c e l l was s l i g h t l y i r r e g u l a r i n appearance. The corresponding slope r a t i o for frog s k e l e t a l muscle loaded passively is also unity (Keynes & Swan 1959). There i s one dif f e r e n c e between the two loading methods which might account for the r e s u l t s . Passive loading loads the ent i r e c e l l , while i n j e c t i o n deposits the radiosodium along a track which does not extend r i g h t to the tendon end of the c e l l and which is acc e s s i b l e to the uninjected portion of the c e l l which extends beyond the grease se a l . D i f f u s i o n of sodium ions i n barnacle muscle c e l l s i s about as rapid as that i n a bulk s o l u t i o n ( C a i l l e & Hinke 1972), but the d i f f u s i o n front s t i l l takes several hours to t r a v e l one centimetre. There i s thus a slow continuous d i l u t i o n of radiosodium throughout even the longest e f f l u x experiment on injected c e l l s . The r e s u l t should be a s l i g h t l y more rapid f a l l o f f of the radio- sodium e f f l u x as the i n t r a c e l l u l a r d i f f u s i o n progresses. Since the t o t a l 128 t i • \' • • i • « « i I I i I l l I I — l I — I — 0 30 60 90 120 150 180 210 time (min.) Figure 9. Semilogarithmic plot of the amount of radiosodium c o l l e c t e d i n the perfusate from a c e l l loaded with isotope by incubation overnight ('passive loading'), and the amount of radiosodium remaining i n the c e l l , versus time. The c e l l was perfused with normal Ringer's so l u t i o n i n i t i a l l y . At ca. 140 minutes perfusion was begun with sodium-free lithium-substituted s o l u t i o n . At ca. 180 minutes t h i s was replaced with potassium-free s o l u t i o n (Table I ) . Resolution of the lower curve into the sum of two exponentials i s i ndicated: y = A exp(-ax) + B exp(-bx) ; A = 22,837 cpm, a = 0.125 min~l, B = 12,830 cpm, b = 0.0072 min~l. Linear regression of upper curve, 55 - 135 minutes, as y = B exp(-bx) i s shown: B = 460 cpm, b = 0.0073 min-"'". 129 i n t r a c e l l u l a r radiosodium at each instant is calculated from back-addition of the radiosodium c o l l e c t e d i n the perfusate, the d i l u t i o n e f f e c t is not taken into account. The measured radiosodium content of the myoplasm w i l l f a l l too slowly. In the present experiments, the slope r a t i o was often somewhat less than unity, e s p e c i a l l y when hypertonic solutions of sodium chloride were injected to r a i s e ( a j r a ) m (see caption to F i g . 8), but o v e r a l l was closer to unity than i n the experiments reported by B i t t a r et a l . (1972). As explained i n Methods, the i n j e c t i o n track in the present experiments was made to ex- tend through the e n t i r e perfused length of the c e l l whenever possible. I n t r a c e l l u l a r d i f f u s i o n would thus occur into the part of the c e l l past the grease s e a l and into the few millimetres at the tendon end which were not injected and which were kept above the l e v e l of the perfusate. B i t t a r £t a l . (1972) injected only a 1 cm column of f l u i d , into c e l l s 3 to 5 cm i n length, and c o l l e c t e d the radiosodium by immersion of the e n t i r e c e l l successively into a series of v i a l s of perfusate. This difference in methods and di f f e r e n c e i n r e s u l t s lend support to the proposal that i n t r a - c e l l u l a r d i f f u s i o n i s responsible for the difference between the r e s u l t s obtained with injected c e l l s and those obtained with passively loaded c e l l s . Comparability of sodium e f f l u x i n passively loaded and injected c e l l s . The radiosodium injected into the c e l l is deposited i n the myoplasm, as noted above. The e f f l u x of t h i s radiosodium represents e f f l u x from the i n t e r i o r of the c e l l . I t exhibits a simple exponential dependence on time for e f f l u x into normal Ringer's s o l u t i o n (Fig. 7). The semilog p l o t for the e f f l u x of sodium from passively loaded c e l l s into normal Ringer's s o l u t i o n i s more complicated (time 0 to 135 minutes in Fig. 9), as noted above. The i n i t i a l rapid e f f l u x was ascribed to the wash- 130 out of radiosodium from the e x t r a c e l l u l a r space, and the slower e f f l u x to the e f f l u x of radiosodium from the i n t e r i o r of the c e l l . The process of separating the two components, known as 'curve peeling', is commonly used, and quite reasonable as long as one can be assured that the components of the net e f f l u x which is measured can each be described by a simple exponen- t i a l , and that the rate constants for the two components d i f f e r by at l e a s t an order of magnitude. Indeed, a p p l i c a t i o n of the procedure to any smooth curve of approxi- mately the shape of a washout curve (no i n f l e c t i o n points, and steady decrease i n the magnitude of the slope) w i l l y i e l d a sum of exponential terms whose rate constants d i f f e r by about an order of magnitude. The process i s i n essence the determination by a process of successive approxi- mations of the power series expansion of the plotted function. Its success i n a p a r t i c u l a r case does not i n i t s e l f constitute proof of the nature of the experimental system, but as a test of a model, e s p e c i a l l y a simple model, i t can be very u s e f u l . The model i n the context of which the e f f l u x curve in Fig. 9 is to be tested i s that the radiosodium i s washed out from two independent pools: the i n t e r i o r of the c e l l , and the e x t r a c e l l u l a r space. A c t u a l l y , there must be some exchange between the i n t e r i o r of the c e l l and the e x t r a c e l l u l a r space, e s p e c i a l l y deep i n the c l e f t system. The well-known 'Huxley c o r r e c t i o n ' takes account of t h i s (A.F. Huxley 1960) but i s an unnecessary refinement here. The questions to. be,addressed are whether the rate of the rapid component i s consistent with e f f l u x from an e x t r a c e l l u l a r s i t e but not with e f f l u x from an i n t r a c e l l u l a r s i t e , and whether the s i z e of the compartment which gives r i s e to the rapid e f f l u x is s i m i l a r to the s i z e of the e x t r a c e l l u l a r space as measured by other means. Very rapid e f f l u x from the i n t e r i o r of the c e l l has been seen in frog 131 s k e l e t a l muscle (White & Hinke 1976) and crab s t r i a t e d muscle (Vaughan-Jones 1977), but only during e f f l u x into sodium-poor solution. A c t u a l l y , what was observed i n these two cases was rapid disappearance of sodium from the myoplasm and the rate constants for t h i s process have a d i f f e r e n t s i g n i f i - cance -- see section 6. The demonstration that, at least i n barnacle muscle, such a rapid disappearance of myoplasmic sodium i s indeed accompanied by a rapid e f f l u x of sodium from the c e l l i s described i n section 6. In normal Ringer's solu t i o n , only effluxes with rate constant of order 0.01 min ^ are seen. Yet th i s figure is the product of p r e c i s e l y the model i t i s desired to t e s t . It can be argued, however, that the assignment of a component of the rapid e f f l u x to the i n t r a c e l l u l a r pool, for e f f l u x into normal Ringer's solut i o n , while the t o t a l sodium content of the c e l l is steady, requires that the i n f l u x and e f f l u x rates change together when the loading bath 22 (normal Ringer's s o l u t i o n containing Na) i s replaced by a bath i d e n t i c a l i n a l l respects except for the absence of radiosodium. Influx experiments exhibit a s i m i l a r i n i t i a l rapid component, so the same argument can be applied there, and the c e l l supposed to sense and respond to the i n c l u s i o n 22 i n or omission from the bathing s o l u t i o n of Na. This does not seem reasonable. The s i z e of the compartment which y i e l d s the rapid e f f l u x of radiosodium into normal Ringer's s o l u t i o n i n F i g . 9 is approximately 5.5% of the volume of the portion of the c e l l which was being perfused. This is an underesti- mate, since a portion of the tendon end of the c e l l was not well perfused, and because the loss of radiosodium from the e x t r a c e l l u l a r space is d i f f u - sive, but agrees w e l l with the values of 6 to. 1% found by various techniques for the s i z e of the e x t r a c e l l u l a r space i n barnacle muscle c e l l s , as noted i n section 3. 132 Altogether, i t seems reasonable to ascribe the rapid component of the radiosodium e f f l u x e n t i r e l y to the e x t r a c e l l u l a r space, and the slow compo- nent to the i n t r a c e l l u l a r compartments. The rate constants for the slow component i n passively loaded c e l l s and the s i n g l e component i n injected c e l l s can thus be compared. In Fig. 7 i t is the upper trace which r e f l e c t s the e f f l u x across the membrane, since the lower trace i s d e c l i n i n g too slowly because of the i n t r a c e l l u l a r sink of radiosodium noted above. The rate constant (0.00928 min i s comparable to but larger than the rate constant for the slow e f f l u x into normal Ringer's s o l u t i o n i n Fig. 9 (0.00729 min . However, the sodium content of the injected c e l l was higher than that of the passively loaded c e l l (ca. 20 mM versus 12 mM), and t h i s alone could account for the difference. The best comparison is of the sodium e f f l u x Mjj a calculated from the radiosodium e f f l u x data. In Fig. 12 (page 143) are presented the caclulated values of Mjr a for a l l injected c e l l s , i n normal Ringer's solution, as a function of the myoplasmic sodium a c t i v i t y ( a j j a ) m . The r e s u l t s from the three passively loaded c e l l s are included as open diamond symbols. They can be seen to l i e i n the region defined by the r e s u l t s for injected c e l l s of comparable sodium content. Brinley (1968) mentioned that the magnitude of the sodium e f f l u x from passively loaded c e l l s was within the range observed with injected c e l l s , but did not report the r e s u l t s on passively loaded c e l l s . A l l e n and Hinke (1970) reported an average rate constant for the slower component of the -1 sodium e f f l u x from passively loaded barnacle muscle c e l l s of 0.0085 min at 15° C. B i t t a r e_t al_. (1972) reported an average rate constant for this -1 o component of about 0.010 to 0.015 min at 23 C for injected barnacle muscle c e l l s . 133 DISCUSSION B i t t a r and coworkers (1972) found that the amount of radiosodium i n barnacle muscle c e l l s loaded by microinjection did not decline with time at a rate commensurate to the decline with time of the rate at which radiosodium appeared i n the bath. They interpreted this i n terms of a model i n which a large portion of the injected radiosodium was sequestered in v e s i c l e s formed from fragments of the c e l l membrane created by the i n s e r t i o n of the micro- i n j e c t o r . This sodium did not exchange at a l l with the free sodium inside the c e l l over the course of the experiment, but could be released by exposure of the c e l l to aldosterone. That so much of the injected sodium could be sequestered at the moment 23 of i n j e c t i o n , that a concomitant amount of Na could also be sequestered (so the apparent f r a c t i o n of 'bound' radiosodium r e f l e c t e d the f r a c t i o n of 'bound' sodium), and that the c e l l membrane fragments which form these v e s i c l e s could become so impermeable to sodium that sodium exchange across them i s n e g l i g i b l y slow compared to sodium exchange across the rest of the c e l l membrane, were not considered to be reasonable hypotheses. On the other hand, i t seems very reasonable that l o n g i t u d i n a l d i f f u s i o n of injected radiosodium i s responsible for the observed slope r a t i o s . The slope r a t i o s were far less than unity i n c e l l s in which the length of the i n j e c t i o n track was much less than the length of the c e l l which was perfused ( B i t t a r et a l . 1972). The slope r a t i o s were closer to but s t i l l less than un i t y when an attempt was made to perfuse only the region of the c e l l which contained the i n j e c t i o n track (present study). The slope r a t i o s were equal to unity i n c e l l s loaded with radiosodium by immersion, i n which no net lon g i t u d i n a l d i f f u s i o n of radiosodium i s expected to occur (present study and r e s u l t s of other workers for frog s k e l e t a l muscle). F i n a l l y , the slope 134 of the semilog plots of sodium e f f l u x versus time are constant over the e n t i r e duration of long experiments i n injected c e l l s , contrary to the expectation that the magnitude of the slope should decline i f a fixed amount of the injected radiosodium is sequestered. The e f f e c t s of aldosterone remain to be explained, but probably r e f l e c t an a c t i o n on the transport systems i n the c e l l membrane. The behavior of the sodium e f f l u x into normal Ringer's s o l u t i o n from microinjected c e l l s i s indistinguishable from that from passively loaded c e l l s (Fig. 12, page 143). A d e t a i l e d study of the response of the slow component i n passively loaded c e l l s to ouabain and to changes i n the composition of the bathing s o l u t i o n was not c a r r i e d out, but i n the experi- ments which were done the behavior of the passively loaded c e l l s was q u a l i t a t i v e l y and q u a n t i t a t i v e l y the same as for injected c e l l s . The use of microinjection leads to a complication i n the measurement of the sodium e f f l u x , however, due to the l o n g i t u d i n a l d i f f u s i o n . The quantity which appears i n the e f f l u x equation, equation (4) of section 2.F, is Na*/Na* the r a t i o of the amount of radiosodium which leaves the c e l l m during a c o l l e c t i o n i n t e r v a l to the amount of radiosodium i n the myoplasmic compartment at the s t a r t of that i n t e r v a l . The myoplasm is continuously l o s i n g radiosodium to l o n g i t u d i n a l d i f f u s i o n as well as to the bathing s o l u t i o n . i The r a t i o Na*/Na* would be equal to the slope of the plot of In Na* versus time i f the loss of radiosodium by l o n g i t u d i n a l d i f f u s i o n from the myoplasm being perfused were e n t i r e l y independent of the loss across the c e l l membrane, and the rate of the loss across the c e l l membrane was proportional to the amount of radiosodium i n the myoplasm at each instant. Then the slope of a l i n e drawn through the data points of the semilog plot could be assumed to be equal to Na*/Na*!, and used in equation (4) to 135 c a l c u l a t e Mjj a, but only over time i n t e r v a l s during which the slope changed very l i t t l e ( i e . dk = 0). dt This has been done for the e f f l u x into normal Ringer's s o l u t i o n for the experiment depicted in Fig. 10 (page 140). The drawing of a smooth curve averages out small var i a t i o n s i n the raw data. The net e f f e c t i s a value for Mjj a which i s about 107o higher than the average of the values c a l c u l a t e d d i r e c t l y from equation (4). The slope r a t i o for t h i s experiment was 0.95. The e f f e c t of ignoring the i n t r a c e l l u l a r sink due to l o n g i t u d i n a l d i f f u s i o n w i l l be greater in c e l l s loaded with sodium, for which the slope r a t i o tends to be lower (Fig. 8). This c o r r e c t i o n to the calculated s i z e of the sodium e f f l u x from i n - jected c e l l s i s systematic, and involves a severe averaging of fluctuations i n the raw data before M^a can be calculated. I t would be preferable to employ equation (4) as written, and then consider the effects of the uncertainty i n Na* and i n V /A during discussions to which the absolute J m m ° s i z e of the sodium e f f l u x i s important, rather than to apply corrections to the raw data. However, comparison of the uncorrected and the corrected r e s u l t s of most experiments revealed a profound e f f e c t of the above effects on the data, as w i l l be discussed i n d e t a i l l a t e r . F i n a l l y , the apparent s i z e of the e x t r a c e l l u l a r pool of sodium yielded by the e f f l u x curves for the passively loaded c e l l s must be mentioned. The value obtained (about 5.57») was s i m i l a r to the i n u l i n space. However, the pool of r a p i d l y exchanging e x t r a c e l l u l a r sodium might be expected to appear much larger than t h i s . The s i z e of the r a p i d l y exchanging e x t r a c e l l u l a r sodium f r a c t i o n proposed i n section 3 i s about 12 millimoles Na/kg c e l l water (Fig. 3). The amount of sodium i n s o l u t i o n i n the e x t r a c e l l u l a r space, i f the l a t t e r i s taken as 67, of the c e l l volume, is approximately 450 mM x 0.06 or 27 millimoles Na/kg c e l l water. Thus one might expect the s i z e of the e x t r a c e l l u l a r space deduced from the radiosodium washout to be somewhat larger than the i n u l i n space. There are two main reasons why this does not occur. F i r s t , the loss of radiosodium from the e x t r a c e l l u l a r space i s by free d i f f u s i o n , the same process which mixes the e x t r a c e l l u l a r sodium which is free i n s o l u t i o n . This causes the y-intercept i n F i g . 9 to be low, and the s i z e of the e x t r a c e l l u l a r space to be underestimated. Second, the e x t r a c e l l u l a r nonmyoplasmic cations are not a l l highly mobile. In the experiments on smooth muscle, lanthanum was used to free them. In addition, the zero of time i s not p r e c i s e l y definable. In a l l , the value obtained for the e x t r a c e l l u l a r space i s not unreasonable. 137 SECTION 5. SURVEY OF THE SODIUM EFFLUX FROM SINGLE MUSCLE CELLS In t h i s section, the dependence of the sodium e f f l u x from s i n g l e whole barnacle muscle c e l l s on the myoplasmic sodium a c t i v i t y is surveyed. This serves f i r s t o f . a l l as a tes t of the techniques described i n section 2.F for measuring the sodium e f f l u x , since the re s u l t s of s i m i l a r experiments on barnacle muscle c e l l s loaded with radiosodium by microinjection are a v a i l - able for comparison (Brinley 1968; B i t t a r £t aT. 1972). The r e s u l t s of section 3 suggest that the a p p l i c a t i o n of the new technique w i l l not y i e l d r e s u l t s markedly d i f f e r e n t from those found with the usual techniques, because most of the nonmyoplasmic sodium does not exchange r a p i d l y with the myoplasmic sodium. This means that the use of the sodium-specific i n t r a c e l l u l a r electrode w i l l only improve the estimate of the s i z e of the i n t e r n a l sodium concentration on which the e f f l u x depends. The nature of the dependence should be the same with either method. However, the use of microinjection has been shown to give r i s e to a s i g n i f i c a n t uncertainty, i n that most but not a l l of the radiosodium injected into a c e l l is a v a i l a b l e for exchange with e x t r a c e l l u l a r sodium when the usual techniques are employed. There w i l l be an underestimate of the s i z e of the sodium e f f l u x , as explained i n sections 2.F and 4. Further, this underestimate w i l l be greater i n microinjected c e l l s with an elevated sodium content, as indicated by Fig. 8. Therefore, the nature of the dependence of the sodium e f f l u x on the sodium content of the c e l l found by other workers using microinjected c e l l s might be incorrect. The dependence of the sodium e f f l u x Mjj a on the myoplasmic sodium a c t i v i t y ( a j j a ) m r e f l e c t s the contribution of more than one transport system i n muscle c e l l s . The prominent systems are thought to be the (Na+K)ATPase, 138 and a system which mediates sodium-sodium exchange. The behavior of the e f f l u x can reasonably be expected to be d i f f e r e n t during conditions which favour one or another transport mode. The dependence of M ^ a on ( a j * a ) m was measured i n normal Ringer's solu- tion, which should correspond c l o s e l y to the normal conditions of the c e l l i n vivo; i n potassium-free solution, where the major mode of the (Na+K)ATPase should be disabled; i n sodium-free solutions, where the sodium-sodium exchange mode reported i n muscle should be disabled; and in the presence of ouabain, where almost a l l of the reactions of the (Na+K)ATPase should be disabled. I t was found that a p p l i c a t i o n of the co r r e c t i o n to Na£ e-Q for c e l l s loaded with radiosodium by microinjection changed the re s u l t s appreciably. METHODS The method of preparing c e l l s and the use of the sodium-specific microelectrode to measure (a,T ) were described i n section 3. The method Na m of i n j e c t i n g , c o l l e c t i n g , and counting the radiosodium was described i n section 4. The c a l c u l a t i o n of the sodium e f f l u x Mjr a was ca r r i e d out v i a equation (4) of section 2.F, both with and without the cor r e c t i o n to Na*e-Q described in sections 2.F and 4. Steady conditions are of inte r e s t here for two reasons. F i r s t , the response time of the transport mechanisms to changes in the myoplasmic sodium a c t i v i t y is not known, so the most r e l i a b l e data should be obtained during steady conditions. Second, the cor r e c t i o n to ic N a c e l l can only be made with confidence during steady conditions, as ex- 139 plained i n sections 2.F and 4. L i n e a r i t y of the semilog plot and ste a d i - ness of (^a),^ over at least four c o l l e c t i o n periods was the c r i t e r i o n for the s e l e c t i o n of data. Experiments i n which the sodium e f f l u x is impaired, as by removal of e x t r a c e l l u l a r potassium or by exposure to ouabain, r e s u l t i n a steady r i s e i n the myoplasmic sodium a c t i v i t y as the sodium i n f l u x is no longer adequ- a t e l y countered by sodium extrusion (for example, F i g . 10 of th i s section). This u s u a l l y caused no problem but i n a few cases the e f f l u x f e l l to a minimum, then rose slowly as ( a j j a ) m rose. A subjective judgment then had to be made about the value of M^a to extract for analysis. The value judged to r e f l e c t the maximum e f f e c t of the experimental manipulation was extracted, along with the value of ( a ^ a ) m at that time. The uncertainty due to this was quite small, but the e f f e c t i s of inte r e s t , as w i l l be discussed below in connection with the dose-response curve for ouabain. Use of Day-old C e l l s . For some experiments, such as loading with radiosodium by incubation i n radiosodium-containing solutions, measurements on the dissected c e l l cannot be performed u n t i l 24 to 48 hours a f t e r the di s s e c t i o n . Dissected c e l l s kept i n normal Ringer's s o l u t i o n at less than 5° C are found to main- t a i n t h e i r ion gradients and membrane p o t e n t i a l : f o r several days (for example, Table I I ) . In p i l o t experiments, the behavior of the e f f l u x of injected radiosodium from barnacle muscle c e l l s was not noticeably d i f f e r e n t i n c e l l s which were injected 24 hours a f t e r d i s s e c t i o n from that i n c e l l s which were injected a few hours a f t e r d i s s e c t i o n . Some differe n c e (enhanced M^a) was noted i n a few c e l l s tested at 48 hours a f t e r d i s s e c t i o n . The use of c e l l s from a barnacle dissected the preceding day makes much more e f f i c i e n t use of the a v a i l a b l e specimens, and saves a considerable amount of time. 140 E 30 Q. 20 O •o 0> o 0> tt> o o 10 1 0.5 I normol | K-free | normal | I0"4M. ouabain • • • •••• 0 . 2 H 26 18 10 2 H | 24 r I I I •••••• «E 2 0 1 - o 3 16 I I. I 1 1 0 30 60 90 120 150 180 210 240 time (min.) Figure 10. Summary of the raw data and reduced r e s u l t s for a t y p i c a l experiment. Upper trace: logarithm of the amount of radiosodium c o l l e c t e d i n each 5 minute c o l l e c t i o n period, i n counts per minute; lower trace: myoplasmic sodium a c t i v i t y as measured by a sodium-specific glass micro- electrode, i n mM; middle trace: sodium e f f l u x deduced from the data v i a equation (4), plotted i n picomoles/cm sec (pes). For the i n t e r v a l i n normal Ringer's s o l u t i o n , the corrected value of M^ i s indicated as a dashed l i n e . 141 About h a l f of the experiments reported on here were done on such 'day-old' c e l l s . The data obtained from such c e l l s is indicated in the figures. The s i m i l a r behavior of fresh and day-old c e l l s (eg. Fig. 11) is. discussed below. RESULTS In Fig.- 10 a summary of the raw data and reduced r e s u l t for a t y p i c a l experiment is presented. Reference w i l l be made to t h i s figure l a t e r . (a) Sodium e f f l u x into normal Ringer's s o l u t i o n . The r e s u l t s from 58 experiments are presented in Fig. 11 and F i g . 12 as plots of M N a versus ( a - N a ) m . In Fig. 11, M^a was calculated according to ic equation (4) using ^ a c e n ' In F i g . 12, the same data was employed but the ic i *** corrected value of Na /Na m was used i n equation (4). In F i g . 11, the r e l a t i o n s h i p between Mjj a and ( a j j a ) m appears to be s l i g h t l y sigmoidal: saturation appears to occur at higher values of ( a j j a ) m . B r i n l e y (1968) used (Na)^ rather than (Na) m i n c a l c u l a t i n g Mj^, and con- ducted h i s experiments at 0° C. I t had been a n t i c i p a t e d that the present r e s u l t s would be s i m i l a r to his ( a f t e r c o r r e c t i o n for surface area by a factor of 10), but s h i f t e d to lower values on the abscissa since ( a j j a ) m was used instead of (Na) , and with a larger e f f l u x at a given sodium content due to the higher temperature. This was found. Brinley's empirical r e l a t i o n s h i p i s shown as a broken l i n e i n Fig. 11. In Fig. 12, the r e l a t i o n s h i p between and ( a j T a ) m i s quite d i f f e r e n t . It appears to be a f f i n e , as was found i n s n a i l neurone by Thomas (1972). 142 Figure 11. Sodium e f f l u x into normal Ringer's sol u t i o n , calculated from equation (4), without correction for Na* , ., . S o l i d c i r c l e s : c e l l s dissected ——— c e l l on the day of the experiment. S o l i d diamonds; c e l l s dissected on the day before that of the experiment. Open t r i a n g l e s : c e l l s loaded with radiosodium by immersion overnight i n l a b e l l e d s o l u t i o n . S o l i d curve: model c a l c u l a t i o n for three sodium ions binding successively to equivalent independent s i t e s per cycle of the transport enzyme (k = 15.75 mM, = 45 pes). Dashed curve: experimental data of Brinley (1968) as M„ versus (Na)., where (Na). i s on the same numerical scale as (a„ ) . Ma i x ' i Na m 143 Figure 12. Sodium e f f l u x into normal Ringer's s o l u t i o n , calculated from equation (4), with correction for N a* e-Q- Symbols as i n F i g . 11. S o l i d l i n e : curve to which k i n e t i c models were f i t t e d by t r i a l and error. Dashed l i n e : experimental data of Brinley (1968), as i n F i g . 11. Elevation of (a„ ) above the normal range (ca. 10 mM) was accomplished by i n j e c t i o n of Na m NaCl into the myoplasm. 144 No saturation i s evident. Acceptable data at higher sodium content was d i f f i c u l t to obtain. The value of (ajyj a) m usually did not become steady when large amounts of 5 M NaCl were injected, so i t was concluded that the permeability of the c e l l membrane had been compromised. Very rapid effluxes were seen in such cases. High values of (Na)^ (about 70 mM) have been alleged to unmask pre-formed transport enzymes i n frog s k e l e t a l muscle ( E r l i j & G r i n s t e i n 1976a,b), although i t is hard to imagine that such a challenge would ever occur i n a l i v i n g animal. In one acceptable experiment at ( a N a ) m = 70 mM, a r e l a t i v e l y high e f f l u x was found (Fig. 11). The use of prolonged immersion of the c e l l s i n potassium-free s o l u t i o n as a means of passively r a i s i n g the sodium content was not investigated, but should be a better method for loading the c e l l with sodium. In this connection, note, however, that while Fig. 10 suggests that ( a j ^ a ) m can be r a i s e d e a s i l y i n potassium-free solution, Table II shows that the increase in ( a ^ a ) m over 20 hours i n potassium-free s o l u t i o n i s not very great. No diff e r e n c e i s seen between the r e s u l t s for fresh ( s o l i d c i r c l e s ) and day-old ( s o l i d diamonds) c e l l s i n Fig. 12. I t has been reported that the dependence of the response of frog s k e l e t a l muscle to ouabain on the sodium content of the c e l l d i f f e r s i n fresh and 'aged' c e l l s (Horowicz, Taylor, & Waggoner 1970), as does the response to removal of external sodium (Keynes 6c Swan 1959; Keynes 6c Steinhardt 1968) . Fig. 12 does not reveal any d e f i n i t i v e information about the k i n e t i c s of the extrusion of sodium from the c e l l , because of the scatter of the data but more importantly because of the u n a v a i l a b i l i t y of data for low values of ( a ^ a ) m with the present techniques. I t i s clear, however, that the behavior revealed with the new method d i f f e r s from that obtained with the usual method. 145 (b) Sodium e f f l u x into potassium-free sol u t i o n. The r e s u l t s of 20 experiments i n which the sodium e f f l u x into potassium- free s o l u t i o n was measured are presented i n Figs. 13 and 14 as plots of M^a versus C 3 - ^ ) ^ I n Fig- 13, was calculated from equation (4) without the c o r r e c t i o n to Na* e^, while i n Fig. 14, Mjj a was calculated with this c o r r e c t i o n . The behavior of M^a i s s i m i l a r i n the two plots, aside from the cor r e c t i o n i n F i g . 14 of the underestimate of the s i z e of M^a i n Fig. 13. The d e f i n i t e plateau is markedly d i f f e r e n t from the behavior found i n normal Ringer's solution, Fig. 12. The sodium extrusion mechanism which does not require external potassium appears to have a li m i t e d capacity, although over the 'physiological range' of ( a ^ a ) m i t responds to an increase i n (a.T ) by increasing i t s rate. v Na'm 3 ° Further, i t appears that the sodium extrusion mechanism which does require external potassium does not saturate at myoplasmic sodium a c t i v i t i e s up to 70 mM. By comparison, Keynes and Swan (1959) found i n frog s t r i a t e d muscle that the reduction i n the sodium e f f l u x caused by removal of external potassium was greater as (Na)^ was raised. Two other observations on.the e f f e c t of removal of external potassium can be made. As i l l u s t r a t e d by Fig. 10, the e f f e c t of removal of external potassium i s reversed by r e s t o r a t i o n of external potassium. I t should be noted that B i t t a r ejt al_. (1972) describe a r i s e of the sodium e f f l u x to a l e v e l above that obtained before external potassium was removed i f external potassium i s subsequently restored. This occurred only for c e r t a i n c e l l s , those for which they calculated a large "sequestered f r a c t i o n " of sodium by the slope r a t i o method. Such c e l l s are found to have high myoplasmic sodium a c t i v i t y , as discussed i n section 4. B i t t a r et al_. reported no appreciable change i n the ion content of c e l l s incubated i n potassium-free 146 50k ^ i J m ( m M ) Figure 13. Sodium e f f l u x from the c e l l into a potassium-free bathing s o l u t i o n , calculated from equation (4) without c o r r e c t i o n for Na*g^^, versus myoplasmic sodium a c t i v i t y at the time (>of the change from potassium-containing to potassium-free s o l u t i o n . C i r c l e s : c e l l s dissected the day of the experiment. Diamonds: c e l l s dissected the day before that of the experiment. S o l i d l i n e drawn by eye. Dashed l i n e represents the sodium e f f l u x into normal Ringer's s o l u t i o n calculated i n a s i m i l a r manner (Fig. 11). Note: the ordinate i s d i f f e r e n t from that i n F i g . 11 and F i g . 12. 147 Figure 14. Sodium e f f l u x from the c e l l into a potassium-free bathing s o l u t i o n c a l culated from equation (4) with c o r r e c t i o n for Na* e^, versus myoplasmic sodium a c t i v i t y at the time of the change from potassium-containing to potassium-free s o l u t i o n . Symbols as i n F i g . 13. S o l i d l i n e : k i n e t i c model for three sodium ions binding successively to equivalent independent s i t e s per cycle of the transport enzyme (k = 15 mM, ^ m a x = ^0 pes). Dashed l i n e : e f f l u x into normal Ringer's sol u t i o n , from F i g . 12. 148 s o l u t i o n for 50 to 70 minutes, although i t is clear from Table I I that (Na)^ must r i s e , and from F i g . 10 that ( a j j a ) m w i l l be increased by such treatment. B i t t a r et a l . assert that i n these c e l l s the behavior of the sodium e f f l u x is altered, but i t seems cl e a r that the "extra e f f l u x " can reasonably be a t t r i b u t e d to the raised myoplasmic sodium a c t i v i t y . They have not demonstrated that changes i n the sodium e f f l u x caused by removal of external potassium are not r e v e r s i b l e . As i l l u s t r a t e d by F i g . 24 (page 190), the membrane p o t e n t i a l does change when external potassium i s removed. The e f f e c t when E m becomes steady i s a depolarization i n this p a r t i c u l a r c e l l , although immediately a f t e r the s o l u t i o n change there is a transient hyperpolarization. In most of the c e l l s tested, the net e f f e c t was found to be a hyperpolarization. For c e l l s i n which ( a ^ a ) m w a s less than 40 mM p r i o r to the s o l u t i o n change, the r a t i o of the membrane po t e n t i a l i n potassium-free s o l u t i o n to that i n normal Ringer's s o l u t i o n p r i o r to the change was 1.05 (n = 11, SD = 0.02) while for ( a j j a ) m greater than or equal to 40 mM the r a t i o was 1.10 (n = 11, SD = 0.06). (Note: there are fewer than 22 points i n F i g . 13 because E m some- times was stable when M^a was not.) (c) Sodium e f f l u x into sodium-free s o l u t i o n . Sodium-free solutions substituted with lithium, t r i s , choline, or sucrose were employed (Table I ) . The e f f e c t s on the sodium e f f l u x of re- placement of the normal Ringer's s o l u t i o n bathing a c e l l by one of the above solutions are shown i n Fig. 15. M^a was calculated from equation (4) with- out c o r r e c t i o n for Na*e-Q, since the c o r r e c t i o n cannot be applied with confidence when the e f f l u x i s not steady. To make comparison easier, the value plotted i s the r a t i o of M^a at each time to the steady value of H^a found before the change from normal Ringer's sol u t i o n . Both i n h i b i t o r y and 149 0.50r • i i 1 i i • • 0 20 40 60 time after solution change (min.) Figure 15. The e f f e c t on the sodium e f f l u x of removal of sodium from the e x t r a c e l l u l a r medium. At time zero minutes (arrow), the e x t r a c e l l u l a r s o l u t i o n was changed from normal Ringer's s o l u t i o n to a sodium-free solu t i o n , substituted as indicated. Sodium e f f l u x has been normalized to 1.0, so each c e l l serves as i t s own co n t r o l . It can be seen from F i g . 16 that the c e l l - t o - c e l l v a r i a - t i o n i n the s i z e of the e f f l u x into normal Ringer's s o l u t i o n and into the various sodium-free solutions i s so great that the d i f f e r e n t response to d i f f e r e n t sodium-free solutions is obscured. 150 stimulatory e f f e c t s can be seen, and the transient e f f e c t s appear to be d i f f e r e n t for the d i f f e r e n t substitute ions. They can be considered i n turn. Lithium. The replacement of external sodium by l i t h i u m caused a f a l l i n the value of M^a i n 17 of 22 experiments. In the time immediately following the s o l u t i o n change, M^a changed e r r a t i c a l l y , yet there was always an i n i t i a l abrupt f a l l . The r e l a t i v e reduction was by 0.30 (SD = 0.17, n = 17). This almost always s e t t l e d into a slow decline as ( a ^ ^ f e l l . I t seemed l i k e l y that both a transient stimulatory and a sustained i n h i b i t o r y e f f e c t resulted from the replacement of the external sodium by lithium. The portions of F i g . 15 which deal with the time period immediately a f t e r the change to sodium-free s o l u t i o n are only presented as q u a l i t a t i v e r e s u l t s . The transients i n the sodium e f f l u x w i l l be described separately i n s e c tion 6. Only the sustained i n h i b i t o r y e f f e c t w i l l be considered here. In one experiment, at very high ( a j r a ) m , the s o l u t i o n change was followed by an abrupt drop and then a marked increase i n M^, plus a con- t r a c t i o n of the c e l l , as was found by B r i n l e y (1968). Return of the c e l l to normal Ringer's s o l u t i o n seemed to reduce the e f f l u x of radiosodium, but M^ja could not be calculated because the contraction dislodged the electrodes. Baker, Blaustein, Hodgkin ejt al. (1969) have suggested that contractions i n this s i t u a t i o n might be due to an increased entry of calcium into the c e l l v i a the sodium-calcium exchange mechanism operating opposite to i t s usual manner due to the absence of external sodium. They observed such an e f f e c t i n squid axon. Choline. In f i v e experiments where choline-substituted sodium-free s o l u t i o n was used, the i n i t i a l behavior was again e r r a t i c . There was an abrupt drop in M^, followed by a r i s e to a l e v e l above the i n i t i a l l e v e l ( r e l a t i v e increase by 0.17, SD = 0.14, n = 5). Again, the sustained e f f e c t 151 was a slow decline of the e f f l u x . T r i s . In two experiments where t r i s - s u b s t i t u t e d sodium-free s o l u t i o n was used, there was an abrupt r i s e i n Mj-a ( r e l a t i v e increase by 0.36 on average), while again the sustained e f f e c t was a slow decline of the e f f l u x . Sucrose. In two experiments where sucrose-substituted sodium-free s o l u t i o n was used, the radiosodium e f f l u x rose a f t e r the so l u t i o n change and then declined slowly. Measurements of ( a ^ a ) m were not done i n these two experiments. The q u a l i t a t i v e behavior of the sodium e f f l u x probably can be deduced from examination of the radiosodium e f f l u x alone i n this case. As indicated i n Fi g . 15, M^a seldom attained a low steady value a f t e r the removal of external sodium. I t seems reasonable to a t t r i b u t e t h i s slow decline to the f a l l of ( a j j a ) m as c e l l u l a r sodium is l o s t to the bathing s o l u t i o n . The s i z e of the sodium e f f l u x during t h i s slow decline was estimated from equation (4) using the co r r e c t i o n for Na c e-Q, and the res u l t s plotted i n Fig. 16. The r e s t r i c t i o n s of this corrected c a l c u l a t i o n make i t impossible to estimate Mjj a at high values of ( a j r a ) m , as explained i n sections 2.F and 4. The values at 30 and 35 mM are less c e r t a i n than the others. Note, however, that the behavior is the same for lithium, choline, and t r i s solutions. This i s the behavior a f t e r long immersion i n sodium- free s o l u t i o n (beyond 30 minutes) while F i g . 15 shows i n addition the behavior immediately a f t e r the change to sodium-free sol u t i o n . The v a r i a t i o n of M^a with ( a ^ a ) m can be presented over a s l i g h t l y wider range of ( a j r a ) m i f equation (4) i s employed without correction. In Fig. 17, the e f f l u x of sodium into sodium-free lithium-substituted s o l u t i o n i s presented as the change i n M^a of i n d i v i d u a l c e l l s as sodium i s l o s t from them into the sodium-free bathing sol u t i o n . This is an approximate c a l c u l a - 152 50 (pes) 25 A** 9 o n l A •,.<> 0 I 10 20 ( ° N a ) m (mM) 30 0 "40 Figure 16. Sodium e f f l u x from the c e l l i n t o a sodium-free solution, calculated from equation (4) with co r r e c t i o n for Na*e-^, versus myoplasmic sodium a c t i v i t y at the time the e f f l u x was calculated. Diamonds: lithium-substituted s o l u t i o n . C i r c l e s : T r i s - s u b s t i t u t e d s o l u t i o n . Open symbols: c e l l s dissected on the day of the experiment. Closed symbols: c e l l s dissected on the day before that of the experiment. Dashed l i n e : e f f l u x into normal Ringer's solu t i o n , from F i g . 12. Note: scale is d i f f e r e n t from F i g . 12. 153 t i o n because of the time constants involved and the use of Na£ £-Q, as has been discussed i n section 2.F. Ap p l i c a t i o n of the cor r e c t i o n for Na c e-Q would r a i s e the estimate of M^a, e s p e c i a l l y at higher sodium content. The range of C 3 ^ ^ remains just short of the region of most int e r e s t , about 40 mM, at which the e f f l u x into potassium-free s o l u t i o n exhibits a shoulder. The e f f l u x into sodium-free s o l u t i o n seems not to have a shoulder, and so to be s i m i l a r to the e f f l u x into normal Ringer's solution, F ig. 12, but unfortunately t h i s cannot be alleged with cer t a i n t y on the basis of thi s data over the f u l l range i n which the e f f l u x into normal Ringer's s o l u t i o n has been measured. A s i m i l a r i t y between the r e l a t i o n s h i p of ^ a N a ^ i to (a£[a) i ^n sodium-free and sodium-containing s o l u t i o n has been dt found i n s n a i l neurone by Thomas (1972b). The behavior found here for barnacle muscle is quite s i m i l a r to that found i n s n a i l neurone. (d) Sodium e f f l u x into solutions containing ouabain. In the present series of experiments, only one concentration of ouabain was tested on a p a r t i c u l a r c e l l . Exposure of the c e l l to ouabain causes a continuing r i s e i n ( a ^ ) ^ , as shown i n F i g . 10, and i t was not known how the ouabain-insensitive sodium e f f l u x depended on ( a ^ a ) m - l n the case of submaximal i n h i b i t i o n , M^a should increase as ( a ^ a ) m increases (Fig. .12). As explained i n Methods, a value for M^& could be calculated only a f t e r the i n i t i a l rapid decline i n M^a was completed, so data at low concentrations of (a ) could not. he obtained. Na m In F i g . 18, the dependence of the sodium e f f l u x on the myoplasmic sodium a c t i v i t y i n the presence of ouabain i s presented. Mjj a was calculated from equation (4) with the co r r e c t i o n for Na c e-Q. In the presence of 10 M ouabain (,- .r c i r c l e s ) , the sodium e f f l u x into normal Ringer's s o l u t i o n is s i m i l a r to but smaller than that into normal Ringer's s o l u t i o n which 154 Figure 17. Sodium e f f l u x from the c e l l into sodium-free lithium-substituted s o l u t i o n , calculated from equation (4) without c o r r e c t i o n for Na* e^, versus myoplasmic sodium a c t i v i t y . Each l i n e represents a sin g l e c e l l , and the change in the sodium e f f l u x as the myoplasmic sodium a c t i v i t y declined during immersion of the c e l l i n the sodium-free s o l u t i o n . This is an approximate c a l c u l a t i o n , as explained i n the text. The dashed l i n e i s the corresponding approximate c a l c u l a t i o n for e f f l u x into normal Ringer's s o l u t i o n ( F ig. 11). 155 Figure 18. Sodium e f f l u x from the c e l l into normal Ringer's s o l u t i o n to -6 -4 which had been added 10 M ( c i r c l e s ) or 10 M (diamonds) ouabain, versus myoplasmic sodium a c t i v i t y . Open symbols represent c e l l s dissected on the day of the experiment. Closed symbols represent c e l l s dissected the day before that of the experiment. S o l i d l i n e : k i n e t i c model for three sodium ions binding successively to equivalent independent s i t e s per cycle of the transport enzyme (k = 15 mM, M =55 pes). Dashed l i n e : e f f l u x into normal max * Ringer s solution, from Figure 12. Correction for Na used i n this instance is explained i n the text. 156 contains no ouabain (Fig. 12 and broken l i n e i n Fig. 18). In the presence -4 of 10 M ouabain (' diamonds ), the sodium e f f l u x d i f f e r s markedly, being much reduced at higher sodium concentrations. -4 The sodium e f f l u x into normal Ringer's s o l u t i o n i n the presence of 10 M ouabain shows only a weak dependence on ( a j j a ) m . I t i s very s i m i l a r to -4 the e f f l u x into potassium-free s o l u t i o n ( F ig. 14). I f 10 M ouabain y i e l d s almost t o t a l i n h i b i t i o n of the (Na+K)ATPase, F i g . 18 shows the sodium e f f l u x which i s mediated by other transport mechanisms. The construction of a dose-response curve i s made d i f f i c u l t by the fact that the e f f l u x depends quite strongly on ( a£j a) m at ouabain concentra- tions which y i e l d submaximal i n h i b i t i o n , while even p a r t i a l i n h i b i t i o n of the sodium transport system can r e s u l t i n appreciable increases i n ( a j j a ) m . A dose-response curve could be constructed by comparing the e f f l u x measured for a given concentration of ouabain with the e f f l u x into normal Ringer's s o l u t i o n at the same value of (a-j|a)m> v i a Fig. 12. The e n t i r e dose-response curve should represent c e l l s of s i m i l a r sodium content. Because there is so much scatte r i n the data, this endeavour was thwarted by the absence of a large number of experimental points i n a small range of (ajja^m" Q u a l i t a t i v e observations can be made. There was no e f f e c t of solutions containing 10"^, 10"**, or 10"^ M ouabain. The e f f e c t of 10"^ M ouabain i s seen i n F i g . 18 to be s l i g h t , while the e f f e c t of 10"^ M ouabain was marked. The binding of ouabain to an enzyme renders i t unable to transport sodium, but the many enzymes which do not have ouabain bound to them constitute a very large functional reserve of sodium extrusion. Yet i f the enzyme responded only to the myoplasmic sodium a c t i v i t y , the e f f l u x at a given value of ( a ^ a ) m i n the presence of enough ouabain to bind to the enzymes appreci- ably should r e s u l t i n a lower t o t a l sodium e f f l u x . This appears to be the case i n Fig. 18. In order to characterize the dose-response to ouabain 157 properly, more data for 10 ° M and 10 3 M ouabain must be obtained. B r i n l e y (1968) used the technique of exposing the c e l l to a series of solutions each with a greater concentration of strophanthidin than the preceding one. He also did experiments in which only a s i n g l e concentration of strophanthidin was used on a c e l l , at the concentration judged to y i e l d maximal i n h i b i t i o n (about 5 x 10 ~* M) . He found the f r a c t i o n a l i n h i b i t i o n of the sodium e f f l u x to be greater i n c e l l s of lower estimated sodium content (Na) „ This is confirmed by F i g . 18. Brinley found that the dose for half-maximal i n h i b i t i o n of the sodium e f f l u x by strophanthidin varied from about 1 x 10 ^ M for c e l l s of low sodium content to about 5 x 10 ^ M for c e l l s of higher sodium content. B i t t a r at al. (1973) reported a dose for half-maximal i n h i b i t i o n by ouabain of about 5 x 10 ^ for barnacle muscle. Fig. 18 indicates that the actual dose of ouabain for half-maximal i n h i b i t i o n -6 is greater than 10 M except at r e l a t i v e l y low sodium content. DISCUSSION Micro i n j e c t i o n. It was noted i n section 4 that microinjection causes only transient changes i n the permeability, ion content, and transport properties of the barnacle muscle c e l l . An exception might be when concentrated solutions of NaCl are injected, as t h i s often resulted in a sustained r i s e i n ( a ^ a ) m . A s i g n i f i c a n t technical problem has been i d e n t i f i e d , i n what appears to be l o n g i t u d i n a l d i f f u s i o n of injected radiosodium i n t o non injected?;-, regions of the c e l l . Mixing in the r a d i a l d i r e c t i o n s along the injected portion of the c e l l appears to be quite rapid. 158 An attempt was made to i n j e c t a long segment of the c e l l , and to c o l l e c t radiosodium only along this injected region. This reduced the e f f e c t of lon g i t u d i n a l d i f f u s i o n , but did not eliminate i t . A correction for the e f f e c t of lon g i t u d i n a l d i f f u s i o n has been described. This involves no assumptions beyond those i m p l i c i t i n the statement of the 23 r e l a t i o n between the flux of radiosodium and the flux of Na, that i s , the fundamental assumption of the tracer technique. It can only be applied i n the form presented when the rate at which sodium i s expelled from the c e l l i s constant. Correction at other times requires a c a l c u l a t i o n of the rate of change of the sodium e f f l u x . C a l c u l a t i o n of the sodium e f f l u x was avoided during the i n t e r v a l s i n which the sodium content of the c e l l was changing rapidly, such as immed- i a t e l y a f t e r c e r t a i n changes i n the composition of the external solution, because i t was not cl e a r whether the i n t e r i o r of the c e l l could be assumed to be well-mixed at such times. S i m i l a r l y , the a p p l i c a b i l i t y of the fl u x model when the sodium e f f l u x was very rapid was not known. I t i s thought that an 'unstirred layer' at the i n t e r n a l surface of the c e l l membrane could l i m i t the sodium e f f l u x i n such circumstances. In this connection, the data of Figs. 16 and 17 are of in t e r e s t . The flu x calculated at each instant during r e l a t i v e l y rapid changes i n ( a ] ; j a ) m (Fig. 17) appears i f anything to exceed the corresponding flux calculated during steadier conditions (Fig. 16). I f there were appreciable l i m i t a t i o n of the e f f l u x by an un s t i r r e d layer at the in t e r n a l surface of the c e l l membrane, the former should be less than the l a t t e r . Of course, the measured value of ( a j g a ) m should lag as well during f i l m - c o n t r o l l e d d i f f u s i o n , and l i t h i u m is thought to be able to stimulate the sodium e f f l u x as potassium does, but altogether i t appears that r a d i a l mixing of ions inside the c e l l , which is d i f f u s i v e , is not a great problem with the time.resolution a t t a i n - 159 able i n the present flux studies. The implications of t h i s , to continue this speculation further, is that the distance from any point i n the i n t e r i o r of the c e l l to the c e l l membrane is small. It is known that the c l e f t system i n barnacle muscle makes this so. Further, i t is implied that the transport properties of the membrane l i n i n g the deep c l e f t s are s i m i l a r to those of the rest of the c e l l membrane. This seems reasonable from a functional point of view, but of course cannot be concluded with c e r t a i n t y from these considerations. In p r a c t i c a l terms, one could attempt to reduce the ef f e c t s of longitu- d i n a l d i f f u s i o n further by i n j e c t i n g a longer region of the c e l l and c o l l e c t - ing isotope only at the centre of the injected region. This would make the technique of microinjection more d i f f i c u l t , and might not make the e f f e c t of l o n g i t u d i n a l d i f f u s i o n n e g l i g i b l e . Overall, passive loading seems preferable for sodium e f f l u x studies, even though i t requires prolonged immersion of the dissected c e l l s p r i o r to the performance of the experiment. The c o r r e c t i o n devised here for the microinjection technique is as f u l l y j u s t i f i e d as the use of the tracer technique i t s e l f . However, as a general rule, i t seems desirable to design experiments so that the le a s t manipula- t i o n of the raw data must be done before an answer to the question under i n v e s t i g a t i o n can be obtained. Aged c e l l s . The maintenance of dissected barnacle muscle c e l l s i n normal Ringer's s o l u t i o n has been shown to a f f e c t the c e l l s but l i t t l e . The c e l l s tend to gain sodium and lose potassium, but the membrane p o t e n t i a l and water content remain constant (eg. Table I I ) . In the e f f l u x experiments i t has been found that the behavior of fresh and day-old c e l l s i s the same (Figs. 11; 14; 16 with less certainty; and 18). The difference i n the response of 160 'fresh' and 'aged' frog s k e l e t a l muscle c e l l s noted above i s probably due to differences i n the sodium content, as noted for example by Keynes and Steinhardt (1968). . Modes of sodium extrusion. The dependence of the sodium e f f l u x on the sodium content of the barnacle muscle, for e f f l u x into normal Ringer's solution, i s s i m i l a r to that reported i n s n a i l neurone (Thomas 1972b) and i n squid axon (Hodgkin & Keynes 1956; Sjodin & Beauge 1967; Brinley & Mullins 1968). It i s d i f f e r e n t from that reported i n red blood c e l l s (Garay & Garrahan 1973) and in frog s k e l e t a l muscle (Harris 1965) i n the f a i l u r e to detect saturation even at r e l a t i v e l y high l e v e l s of i n t r a c e l l u l a r sodium content. I t also d i f f e r s from the r e s u l t s of Br i n l e y (1968) and of B i t t a r et al. (1972) for barnacle muscle c e l l s , almost c e r t a i n l y because of the problems involved i n working with microinjection, as discussed at length above and i n section 4. The s t r i k i n g feature of the dependence is the apparent absence of saturation at myoplasmic sodium a c t i v i t i e s up to 50 mM and perhaps up to 70 mM. I t is not clear-why the barnacle muscle c e l l should have such a large functional reserve for sodium extrusion. Action potentials do not propagate i n the barnacle muscle c e l l membrane under ordinary conditions, so t his cannot be a large source of sodium i n f l u x _in vivo. Perhaps a sodium-calcium exchange across the c e l l membrane i s required for re l a x a t i o n of the muscle, since i t s sarcoplasmic reticulum i s so small. Frog s t r i a t e d muscle, by comparison, shows saturation at r e l a t i v e l y low sodium content (shoulder at about 10 millimole sodium per kg tissue - Harris 1965). The e f f l u x into normal Ringer's s o l u t i o n i s thought to be composed of several components. The dominant ones are thought to be sodium-potassium exchange v i a the (Na+K)ATPase and sodium-sodium exchange v i a some other 161 mechanism, as noted i n the Introduction. A small contribution appears to be made by the sodium-sodium exchange mode of the (Na+K)ATPase i n potassium- free s o l u t i o n and in energy-depleted c e l l s , since a ouabain-sensitive sodium- sodium exchange has been reported i n frog s k e l e t a l muscle under these conditions (Keynes & Steinhardt 1968; Kennedy & De Weer 1976). Thus the in t e r p r e t a t i o n of Fig. 12 must be c a r r i e d out by comparison with the corres- ponding plots when one or another of the sodium extrusion mechanisms is disabled. For t h i s purpose, the curves f i t t e d by eye to Figs. 12, 14, 16, and 18 are c o l l e c t e d i n Fig. 19. The dependence of M^a on ( a ^ a ) m i n t n e absence of external potassium and that i n the.presence of 10"^ M ouabain are c l o s e l y correlated, but d i f f e r from the dependence i n normal Ringer's s o l u t i o n i n that they show a d e f i n i t e saturation. This is consistent with the hypothesis that a dominant mode of sodium extrusion i s the sodium-potassium exchange mode of the (Na+K)ATPase. The e f f l u x which remains i n the presence of ouabain or i n the absence of external potassium r e f l e c t s the maximum contribution that other mechan- isms can make to the t o t a l sodium e f f l u x . This appears to be almost as great- as. the e f f l u x into normal Ringer's s o l u t i o n over the 'physiological range'. However, i t is widely held that this is mostly t i g h t l y - l i n k e d sodium-sodium exchange, and so i n e f f e c t u a l as far as sodium regulation is concerned. Further, even i f t h i s were a l l e f f e c t i v e sodium extrusion, i t seems to f a l l short of what is needed under normal conditions to balance the i n f l u x . Thus exposure to ouabain or removal of external potassium w i l l cause the sodium e f f l u x to f a l l below the sodium i n f l u x . (aNa^m w :'- i i r i s e s t e a d i l y , and the s p e c i f i c a c t i v i t y of the myoplasm w i l l be reduced. This w i l l cause a reduction i n the e f f l u x of radiosodium. This reduction i n the e f f l u x of radiosodium could be interpreted as representative of a f a l l i n the sodium e f f l u x , i f the myoplasmic sodium 162 Figure 19. Summary of sodium e f f l u x from the c e l l into various solutions, extracted from Figures 12 (normal Ringer's s o l u t i o n - s o l i d curve), 14 (potassium-free s o l u t i o n - lower dashed curve), 16 (sodium-free s o l u t i o n - upper dashed curve, representing choline), and 18 (ouabain i n normal Ringer's s o l u t i o n at 10 ^ M - l i n e as for potassium-free s o l u t i o n ) . The r e l a t i v e p o s i t i o n of the d i f f e r e n t curves over the p h y s i o l o g i c a l range, (approx. 10 - 20 mM) was deduced form the behavior of a c e l l compared to i t s e l f as control, for a given test s o l u t i o n . The sc a t t e r of the grouped data does not permit the r e l a t i v e positions to be distinguished over this range. Thus; the e f f l u x at a given value of (a., ) increases when the b ° Na m external s o l u t i o n is changed from normal Ringer's s o l u t i o n to sodium-free choline-substituted s o l u t i o n , but decreases when i t i s changed to a potassium-free s o l u t i o n or to one containing ouabain. The curves for the -4 l a t t e r two cases (with 10 M ouabain) are almost i n d i s t i n g u i s h a b l e i n the present series of experiments, and the e f f l u x into potassium-free ouabain- containing s o l u t i o n was not examined. The curve for sodium-free lithiu m - substituted s o l u t i o n would be p a r a l l e l to the choline curve, but below the normal curve. 163 a c t i v i t y i s not monitored for the purpose of c a l c u l a t i n g the sodium e f f l u x from radioisotope movement. That i s , the f a l l i n the sodium e f f l u x due to i n h i b i t i o n of the pump might have been overestimated i n the past. If the mechanism which remains operational i n the absence of external potassium and i n the presence of ouabain does indeed represent sodium-sodium exchange, then i t should be markedly reduced, i f not eliminated, by the removal of sodium from the bathing s o l u t i o n . It has been found by other workers, however, that removal of external sodium and potassium, even when combined with exposure to ouabain, does not reduce the sodium e f f l u x to the l e v e l expected i f only passive fluxes are present (eg. Brinley 1968). Only when ATP i s almost completely removed from the c e l l and i n h i b i t o r s of metabolism are applied are the fluxes reduced to the passive rates, as discussed i n section 2. It appears from F i g . 19 that for barnacle muscle c e l l s the e f f l u x of sodium into sodium-free s o l u t i o n is very s i m i l a r to the e f f l u x into normal Ringer's s o l u t i o n . Further, this appears to be independent of the cation used i n place of sodium i n the bathing s o l u t i o n (Fig. 16). Is is conceivable but hardly l i k e l y that these cations could each substitute for sodium i n a sodium-sodium exchange, or enter the c e l l by some other path, to y i e l d the behavior observed. I f i t i s assumed that the e f f l u x into normal Ringer's s o l u t i o n r e f l e c t s the normal operation of the c e l l , i t appears that a sodium e f f l u x which depends on external sodium contributes l i t t l e to the normal flux i n barnacle muscle c e l l s . Through the use of the i n t r a c e l l u l a r sodium-specific microelectrode, the net reduction i n the sodium e f f l u x seen when the bathing s o l u t i o n i s changed from one containing sodium to one not containing sodium has been revealed to be due to the consequent f a l l i n ( a ^ a ) m ' ° v e r the 'physiological range' of (a„ ) and somewhat beyond. That the e f f l u x into sodium-free s o l u t i o n v Na'm J seems to be s l i g h t l y greater i n magnitude than the e f f l u x into sodium- containing solutions i s consistent with the fi n d i n g of other workers 164 that external sodium can i n h i b i t the sodium e f f l u x . The r e s u l t s with l i t h i u m probably r e f l e c t i n addition the a b i l i t y of l i t h i u m to stimulate l i k e potassium (eg. Beauge 1975). The existence of sodium-sodium exchange was f i r s t suggested by Ussing (1949). The f i r s t evidence i n favour of i t s existence i n muscle c e l l s was presented by Keynes and Swan (1959) and t h e i r r e s u l t s demand closer considera- ti o n . In t h e i r experiments, whole muscles of frogs were loaded with radio- 24 sodium ( Na) by immersion for about 3.5 hours i n normal frog Ringer's s o l u t i o n which contained radiosodium. The muscle was then transferred at 5 or 10 minute in t e r v a l s through a succession of test-tubes containing i n a c t i v e Ringer's or other s o l u t i o n . The amount of r a d i o a c t i v i t y leaving the muscle during each i n t e r v a l ( c a l l e d Na* i n this thesis) was measured, corrected for decay of this s h o r t - l i v e d isotope, and plotted l o g a r i t h m i c a l l y against time. Their estimate of changes in the.rate of loss of sodium, 23 that i s , of Na, from the muscle c e l l s were calculated from displacements of t his curve caused by changes i n the ioni c composition of the medium. They found that the slope "k£" of the plot of l n Na versus time to be constant during e f f l u x into normal frog Ringer's sol u t i o n . Likewise, the slope "k|" of the pl o t of l n N a * e l l versus time was constant, where N a * e l l was calculated by back-addition as was done i n the present series of experiments on barnacle muscle (see Methods, section 4). The slope r a t i o — l / — 1 W a S v e r y c l ° s e to unity, as expected for passively-loaded c e l l s . When the external s o l u t i o n was changed from normal frog Ringer's solu- t i o n to sodium-free lithium-substituted s o l u t i o n , the value of Na decreased. The plot of In Na* versus time assumed a steadier, almost l i n e a r slope ii "k^" a f t e r about 20 minutes. The slope of the pl o t of l n N a c e i i versus time exhibited a break, to a new smaller s l o p e " ^ " for sodium-free lithium- substituted s o l u t i o n . Keynes and Swan found that k^/loj was close to 3. 165 It was th i s discrepancy that gave r i s e to t h e i r enquiry into d i f f e r e n t models for the nature of the sodium e f f l u x . They considered the plo t of Na* versus Na*e-Q for e f f l u x into sodium-free sol u t i o n , which i s l i k e a power function with power three, and f i n a l l y s e t t l e d on a model i n which sodium i s transported out of the c e l l from a sing l e homogeneous compartment, at a rate proportional to the cube of the sodium content of the c e l l . In fact, the reason for the value of k^/k^ being greater than unity i s quite simple. The fundamental assumption of the tracer technique i s that the tracer ( 2 2Na or 2 4 N a) i s i n a well-mixed compartment, and behaves just as the 2 - % a does. Then no matter what the mechanism by which sodium i s transported out of the compartment, as long as the rate of sodium transport is steady the rate of e f f l u x of tracer w i l l be proportional only to the amount of tracer l e f t i n the compartment (assuming no backflux). Thus, dNa = -k x Na* where k is a p o s i t i v e constant. This equation defines - z — c e l l c e l l ^ ^ at the exponential function : Na*e-Q = Na* e^^(t=0).exp(-kt). The 'rate constant 1 k for radiosodium e f f l u x is determined by the rate of the sodium e f f l u x . When this rate is steady, k is constant and the plo t of In Na c e;Q versus time i s l i n e a r . This occurs for e f f l u x into normal Ringer's s o l u t i o n for frog,as i t does for barnacle muscle, but not for sodium-free so l u t i o n . I f the rate of mixing i n the compartment is always rapid compared to the rate of transport out of the compartment (which i s an assumption of the tracer method), then a s i m i l a r s i t u a t i o n should occur at each instant of time even when the rate of sodium transport is changing: N a * e l l ( t ) = Na* e U ( t = 0 ) - e x p ( - k ( t ) - t ) where now the 'rate constant' k is a function of time. •k The rate of change of Na c e-Q must r e f l e c t both the rate of sodium 166 transport and the r a t e . of change of the sodium transport, and t h i s accounts for the value of ^ 2 ^ 2 f° u nd i n frog. More formally, i f the t o t a l amount of radiosodium inside the c e l l is N ^ c e l l ' and i n i n t e r v a l At of time an amount of radiosodium Na* i s c o l l e c t e d i n the bathing sol u t i o n , then experimentally dNa* e l l = - Na*/dt dt where again the shorthand of 'pseudo-calculus' is employed. A plot of l n ic • ic Na versus time is a p l o t of In d N a C e l l versus time, and i t s slope is dt d i n dNa*e-Q; th i s is "k"' of Keynes and Swan. A pl o t of l n N a * e l l versus dt dt ic time has slope d_ In ^a'cenl this is "k" of Keynes and Swan. Straight- dt forward d i f f e r e n t i a t i o n of the above expression for Na c e-^^(t) reveals: ( k(t) ..+ t ^ , ) 2 " ( 2 ^ + ) dt dt ( k(t) + t ^ ) and k = ( k(t) + t ^ ) . dt When k(t) i s constant, as i n normal Ringer's solution, where (Na)^ is steady, then k' = k = k and k'/k = 1, as has been noted before. However, when the muscle i s i n sodium-free sol u t i o n , (Na)^ w i l l f a l l roughly as an exponential function of time i n frog muscle (White & Hinke 1976), and so k(t) w i l l f a l l . Then ( ? dk d 2k k'/v = 1 " ( k ( t ) + t f ) 2 * It is not unreasonable to assume s o l e l y for the purposes of this discussion that the decline of k(t) i s also describable by an exponential function: 167 k(t) k Q exp(-bt) where b i s a p o s i t i v e constant, roughly equal to 0.01 min"''" a f t e r the f i r s t 20 minutes for frog muscle (White & Hinke 1976). Then k'/ k ~ 1 - b ( b-t - 2 ) k ( 1 - b-t ) 2 and k'/k. w i l l exceed unity while b-1< 2, which i n t h i s very rough, semiquanti- t a t i v e example is during the f i r s t 200 minutes. The conclusion to be drawn, then, is that the "discrepancy" which led Keynes and Swan (1959) and Keynes and Steinhardt (1968) to postulate the existence of more than one i n t r a c e l l u l a r compartment was a misinterpretation of the tracer data. S i m i l a r i l y , the "plateaus" found by Mullins and Frumento (1963) for radiosodium e f f l u x from frog s k e l e t a l muscle into sodium-free s o l u t i o n can be accounted for by the above expression for k', and the e l e c t r i c a l coupling they proposed need not be invoked. Further, a l l of the r e s u l t s on the sodium-free e f f e c t i n frog muscle can be accounted for in terms of the changes in the rate of sodium e f f l u x which occur as (Na) ̂  changes. I t i s i n t e r e s t i n g that Keynes and Swan (1959) and Keynes and Steinhardt (1968) t r i e d to account for t h e i r r e s u l t s by postulating that sodium-sodium exchange occurred at low values of (Na)^ but not at high values. That i s , they acknowledged the e f f e c t of t h e i r manipu- la t i o n s of the muscle c e l l on (Na)^, and the dependence of the rate of sodium expulsion on (Na)^, but did not take these features into account in t h e i r analysis of t h e i r tracer data. Apparently they did not f e e l that (Na). could decline so p r e c i p i t o u s l y i n sodium-free s o l u t i o n (Keynes 1965). Recognition of t h i s e f f e c t has come from measurements with sodium-specific i n t r a c e l l u l a r microelectrodes (Thomas 1972b- White & Hinke 1976; Vaughan- Jones 1977). 168 The r e s u l t s imply that the behavior of the sodium transport i n muscle is much more straightforward than previously had been thought. The linked sodium-sodium exchange which was supposed to be ouabain-insensitive and to comprise almost h a l f of the sodium transport measured i n muscle does not occur i n barnacle muscle. Evidence previously presented for i t s existence i n frog muscle has been shown to be incorrect. In this connection, i t i s i n t e r e s t i n g that when (Na)^ was maintained by i n t e r n a l d i a l y s i s i n squid axon, sodium-free solutions caused no reduction i n the sodium e f f l u x as long as ATP was included i n the d i a l y s i s s o l u t i o n (Mullins & Brinley 1967). The various e f f e c t s on the sodium e f f l u x of ouabain or changes i n the i o n i c composition of the external solution, reported here for barnacle and by others for frog muscle, appear almost a l l to be explained by the curves of Fig. 19. Most of the r e s u l t s i n nerve can be explained i n l i k e manner. It . almost-seems that the overwhelming dominant mode of sodium transport in muscle and nerve, and perhaps i n most c e l l s , under normal conditions, is the sodium-potassium exchange mode of the (Na+K)ATPase. However, i t would have to be proposed in add i t i o n that exposure of the c e l l s to ouabain cannot i n h i b i t a l l of the transport enzymes i n the c e l l membrane, as suggested, for.example, by Brinley (1968), and that 'recycling' of potassium which leaks out of the c e l l into potassium-free s o l u t i o n can occur, as suggested,Jfor example, by. Beauge (1975). The contribution of sodium trans- port modes involving calcium, amino acids, etc., should be r e l a t i v e l y small normally. C e r t a i n l y no proof of this hypothesis is claimed here, but these considerations seem to be a worthwhile basis on which to plan further experiments to f i n d the mechanism of sodium expulsion into potassium-free and into ouabain-containing solutions. 169 K i n e t i c s . Much of the contribution of the (Na+K)ATPase to the sodium e f f l u x can be eliminated s e l e c t i v e l y by exposure of the c e l l s to ouabain. The remaining e f f l u x can be assumed to be due mostly to one mechanism as a f i r s t approxi- mation, and i t s k i n e t i c c h a r a c t e r i s t i c s examined. Of course, this might represent more than one mechanism, but there is no good reason to assume this now. I f a simple two-parameter model of the type discussed i n section 2 is applied to the experimental data, a reasonable f i t can be found for some values of the parameters. Se l e c t i o n of the most applicable model then depends on a knowledge of the values these parameters can assume, since each has a physical i n t e r p r e t a t i o n . MJJ^JJ is the maximum value the e f f l u x can a t t a i n , and can be estimated quite well from the data at higher values of ( a ^ a ) m i f saturation occurs. I f saturation cannot be detected in the data, M m a x cannot be estimated with any degree of assurance. The value of k, the apparent d i s s o c i a t i o n constant of the enzyme- sodium complex when used i n this context, r e f l e c t s the binding energy. This energy can be quite high since the binding r e s u l t s i n a s l i g h t change i n the conformation of the enzyme, as discussed i n section 2. For a c y c l i c a l c a r r i e r system, the apparent d i s s o c i a t i o n constants are complicated functions of the rate constants which describe the various reactions in the c y c l i c sequence. They can be very d i f f e r e n t from the actual a f f i n i t i e s (Caldwell 1969). I t appears that the apparent d i s s o c i a - t i o n constants at one surface of the membrane are independent of changes in the composition of the s o l u t i o n bathing the opposite surface under most conditions, even though such changes would a l t e r some of the reaction rates i n the c y c l i c sequence (Hoffman & Tosteson 1971; Garay & Garrahan 1973; 170 C h i p p e r f i e l d & Whittan 1976). It is l i k e l y , then, that the apparent a f f i n i t i e s obtained v i a a good model w i l l r e f l e c t the true a f f i n i t i e s of the sodium s i t e s . From studies of the action of sodium i n protecting the (NaH-K)ATPase from i n a c t i v a t i o n by DCCD (dicyclohexylcarbodi-imide), Robinson (1974) was able to estimate the d i s s o c i a t i o n constant for the enzyme-sodium complex. In the absence of other ligands, the value was 2.3 mM. In the c e l l , potas- sium w i l l compete with sodium for the sodium s i t e s , and so the apparent d i s s o c i a t i o n constant w i l l be too large. However, since the a f f i n i t y of the sodium s i t e for potassium i s much less than that for sodium, the apparent value w i l l probably be of the correct order of magnitude. With respect to the models discussed i n section 2, for "n" ions binding before transport occurs, the value of k which gives a good f i t to the data i s lower for higher values of n. The data for ouabain (Fig. 18) are almost i d e n t i c a l to the data for potassium-free s o l u t i o n (Fig. 14). A l i n e a r regression of l / ( a ^ a ) m on 1/M^ y i e l d s k = 105 mM, M^^ = 116 pes for the e f f l u x i n the presence of 10"^ M oubain, and k = 116 mM, \ i a K = 124 pes for the e f f l u x into potassium- free s o l u t i o n . These values for k are too large, however. For two ions binding successively, the best f i t to the data, by t r i a l and error, was with k = 20 mM, M =60 pes. For three ions i t was k = 15 mM, M „„ = 60 ' max r max pes. For four ions i t was k = 15 mM, = 55 pes. On the basis of the above discussion, as a f i r s t approximation only, the data are f i t t e d best by a model with at l e a s t three sodium ions binding successively to equivalent independent s i t e s . The key to a more precise conclusion i s good data at low l e v e l s of ( a f j a ) m j a s discussed previously, plus extension of the data i n normal Ringer's s o l u t i o n and sodium-free s o l u t i o n to the region of saturation. 171 Continuing i n th i s rather speculative vein, i t can be noted again that the e f f l u x into sodium-free s o l u t i o n i s very s i m i l a r to the e f f l u x into normal Ringer's s o l u t i o n (Fig. 19). I f i t i s assumed that the dominant mode of sodium transport normally is sodium-potassium exchange, the two- parameter models can be applied. There is no good i n d i c a t i o n of M ^ ^ but a good f i t can be achieved for one ion binding with k = 200 mM, M^.^ = 400 pes; for two ions with k = 50 mM, M^^j = 225 pes; for three ions binding with k = 30 mM, = 175 pes; and for four ions with k = 27.5 mM, M^^ = 200 pes. That i s , the f i t is better for higher numbers of ions binding successively. Thus the two modes defined operationally here appear to d i f f e r i n t h e i r k i n e t i c c h a r a c t e r i s t i c s i n the context of the two-parameter models. For no value of n is i t found that k is the same but M i s lower for c e l l s max in which the (Na+K)ATPase i s i n h i b i t e d compared to c e l l s i n normal Ringer's s o l u t i o n . This suggests that the e f f l u x observed i n the presence of ouabain and i n the absence of external potassium i s not simply due to the operation of fewer transport enzymes. When more data at very low and very high levels of (Na) m can be obtained, i t w i l l be i n t e r e s t i n g to perform a more de t a i l e d k i n e t i c analysis i n terms of i n h i b i t i o n of d i f f e r e n t types. 172 SECTION 6. COMPARISON OF SODIUM ELECTRODE AND RADIOSODIUM MEASUREMENTS Two 'sodium-free' e f f e c t s have been reported i n sodium e f f l u x studies i n muscle, as summarized in section 2.C. They d i f f e r i n t h e i r time course. The better-known e f f e c t i s a sustained reduction i n the sodium e f f l u x when sodium i s removed from the bathing s o l u t i o n . In the l a s t section, i t was reported that t h i s e f f e c t appears to be due to the f a l l i n the myoplasmic sodium a c t i v i t y alone, at least i n barnacle muscle c e l l s . The second 'sodium-free e f f e c t ' is a large rapid f a l l i n the i n t r a - c e l l u l a r sodium a c t i v i t y measured with an i n t r a c e l l u l a r sodium-specific microelectrode. This e f f e c t has been observed i n frog s k e l e t a l muscle (White & Hinke 1976) and i n crab s t r i a t e d muscle (Vaughan-Jones 1977). A s l i g h t l y d i f f e r e n t rapid f a l l was found i n s n a i l neurone (Thomas 1972b). This rapid f a l l i n ( a j q a ) m coincides roughly with the transient stimula- t i o n of the sodium e f f l u x seen in Fig. 15 with choline and t r i s , and with the 'biphasic transient' with lithium. The change from sodium-containing to sodium-free s o l u t i o n i s not instantaneous, and as noted i n section 4, the experimental apparatus was not designed to measure transient phenomena. However, i t was f e l t to be worthwhile to compare the information from the i n t r a c e l l u l a r microelectrode and the radioisotope, p a r t i c u l a r l y during the period where the transients occur. In the experiments on frog muscle, chemical analysis indicated that the f a l l i n the myoplasmic sodium a c t i v i t y is due to movement of sodium ions out of the c e l l (White & Hinke 1976). Studies of the e f f l u x of radiosodium from frog muscle into sodium-free s o l u t i o n have revealed some transient rapid fluxes, but these were us u a l l y ascribed to the e x t r a c e l l u l a r space and ignored (eg. Hodgkin & Horowicz 1959). Measurements with the i n t r a c e l l u l a r microelectrode alone can only 173 reveal a loss of sodium from the major i n t r a c e l l u l a r compartment, the myoplasm. They cannot reveal the fate of the l o s t sodium. A treatment of the data was thus devised i n which microelectrode and radioisotope r e s u l t s are combined, so that changes i n the sodium content of the myoplasm could be compared with the simultaneous loss of sodium from the c e l l . The experi- ments were conducted p r i o r to the p u b l i c a t i o n of the re s u l t s for crab muscle. It was found that a rapid f a l l i n the myoplasmic sodium a c t i v i t y s i m i l a r to that seen i n s n a i l neurone and i n frog and crab muscle occurs in barnacle muscle under c e r t a i n conditions, and that i t i s accompanied by a commensu- rate loss of sodium from the c e l l . The transient changes i n the sodium e f f l u x which occur immediately a f t e r the-change to sodium-free s o l u t i o n appear to r e f l e c t a d i r e c t e f f e c t on the transport mechanism, as well as an e f f e c t due to rapid changes i n the sodium content, of the c e l l s . METHODS Dissection of s i n g l e muscle c e l l s , i n j e c t i o n and c o l l e c t i o n of radio- sodium, and measurements with microelectrodes have been described i n previous 22 23 sections. The f l u i d injected contained NaCl and, sometimes, NaCl, the l a t t e r i n order to r a i s e the myoplasmic sodium a c t i v i t y . 174 RESULTS In F ig. 20 i s presented the time course of the decline i n ( a ^ a ) m for three c e l l s of d i f f e r e n t i n i t i a l sodium content, during immersion i n a sodium-free lithium-substituted s o l u t i o n . The behavior i s q u a l i t a t i v e l y d i f f e r e n t for c e l l s of d i f f e r e n t sodium content. The plot i s semilogarith- mic. A simple exponential f a l l of ( a j j a ) m with time was seen i n c e l l s having a lower i n i t i a l value of ( . ^ a ) m - A rapid i n i t i a l f a l l , of (aji a) m> s i m i l a r to that seen i n s n a i l neurone (Thomas 1972b) but not unlike that seen i n frog s k e l e t a l muscle (White & Hinke 1976) and i n crab s t r i a t e d muscle (Vaughan- Jones 1977), was seen i n c e l l s having a higher i n i t i a l value of ( % a ) m - In frog and crab muscle, a large i n i t i a l f a l l of ( a M ) was seen even i n IN a , Ul c e l l s having low i n i t i a l values of (a„ ) . Na m This behavior of barnacle muscle c e l l s i s d i f f e r e n t from that reported by McLaughlin and Hirtke (1968) and by A l l e n and Hinke (1971). However, they had used a hypertonic sodium-free sol u t i o n , and the i n i t i a l behavior of (a„ ) i n t h e i r experiments included the e f f e c t s of water movement, as v Na'm ^ ' discussed i n section 3.B. The time course of the f a l l i n ( a j j a ) m i n the c e l l s having a high i n i t i a l sodium content could be f i t t e d quite well by the sum of two exponentials, but for the c e l l s having a lower i n i t i a l sodium content a s i n g l e exponential or occasionally even a l i n e a r function s u f f i c e d . The hazards of 'curve peeling' were mentioned i n section 4. There i s no reason to propose that the behavior of the c e l l s of high i n i t i a l sodium content r e f l e c t s the sum of two independent processes. I t seems equally l i k e l y that a s i n g l e mechanism is operating but the rate constant is d i s - playing a dependence on the gradient of the chemical p o t e n t i a l for sodium. When rate constants for the rapid e f f l u x were calculated by 'curve peeling', 175 0 20 40 60 time (min.) Figure 20. F a l l of the myoplasmic sodium a c t i v i t y upon exposure of the c e l l to sodium-free lithium-substituted s o l u t i o n . Solution change from normal Ringer's s o l u t i o n occurred at time 5 minutes. Prior to that time, the steady value of the myoplasmic sodium a c t i v i t y of each c e l l i n normal Ringer's s o l u t i o n i s shown. The plot i s semilogarithmic. For the c e l l which st a r t e d with the highest sodium content (upper curve), i t i s shown how the 'size of the i n i t i a l rapid f a l l 1 was calculated for F i g . 21, by extrapolation back to zero time of the l i n e a r t a i l of the curve. 176 the values were found to show considerable scatter and to be poorly corre- la t e d with the i n i t i a l value of ( a ^ a ) m - I t appeared that above a c e r t a i n threshold value of (a„T ) of about 15 mM the fast rate was present, while ^ Naym below this threshold i t was not. The mean value of the larger rate constant was comparable to the value which describes the washout of the e x t r a c e l l u l a r space (0.15 min"-'-, SD = 0.06 min"''' for 9 c e l l s f i t t e d by two exponentials; a s i m i l a r value was reported by White & Hinke 1976 for frog muscle). The d i f f e r e n t s i g n i f i c a n c e of the rate constant i n the two cases w i l l be con- sidered i n the Discussion. Vaughan-Jones (1977) noted that the change of ( a j j a ) m during 15 minutes of immersion of crab muscle i n sodium-poor s o l u t i o n correlated well with the i n i t i a l value of (a^T ) i n the c e l l s . A s i m i l a r r e s u l t was found i n barnacle muscle, as shown i n Fig. 21. The / s i z e of the rapid f a l l ' i n mM was calculated by the method indicated i n Fig. 20 top tracing. This r e a l l y j u s t confirms the impression gained from Fig. 20 that, whatever the i n i t i a l value of (ajjg^jjj above about 15 mM, the rapid f a l l is 'switched o f f when the value of (a ) f a l l s to ca. 15 mM during immersion of the c e l l i n the x Na'm sodium-free s o l u t i o n . The 'threshold' value of ( a - ^ a ) m i n crab muscle was about 2 mM (Vaughan-Jones 1977). A d i f f e r e n t and better approach i s to measure the i n i t i a l slope of the tracings of ( a j j a ) m versus time, to y i e l d a s i n g l e rate describing the rapid f a l l . When th i s rate is plotted versus the value of (a,T ) at the s t a r t of r Na m the f a l l , a c o r r e l a t i o n i s seen (Fig. 22). The dependence is very s i m i l a r , even i n the s l i g h t d i f f e r e n c e i n slope for d i f f e r e n t c e l l s at a given value of ( a j j a ) m J to that displayed i n Fig. 17 for the e f f l u x M^a (calculated from equation (4) without c o r r e c t i o n for Na* ,,) versus (a„ ) . This i n turn n * c e l l Na m is s i m i l a r to the corrected value F i g . 16, as noted i n section 5. In theory, the r e l a t i o n s h i p between the two is V m*^^ a^m = A-M^a, 177 ( o N o ) m (mM.) Figure 21. Size of the rapid f a l l i n the myoplasmic sodium a c t i v i t y upon exposure of the c e l l to sodium-free lithium-substituted s o l u t i o n (see F i g . 20 and text), versus the steady value of the myoplasmic sodium a c t i v i t y i n the c e l l p r i o r to the change from normal Ringer's to sodium-free s o l u t i o n . One data point was excluded from the l i n e a r regression represented by the l i n e , and is shown i n parentheses. 178 c E 2 E o 0.7 0.6 0.5 0.4 0.3 3 0.2 0.1 i_ 10 20 Na'm 30 (mM.) 40 Figure 22. Rate of f a l l of the myoplasmic sodium a c t i v i t y immediately a f t e r exposure of the c e l l to sodium-free lithium-substituted s o l u t i o n , versus the steady value of the myoplasmic sodium a c t i v i t y i n normal Ringer's s o l u t i o n p r i o r to the change to sodium-free s o l u t i o n . The point i n parentheses is from the c e l l excepted i n F i g . 21. C i r c l e s ; c e l l s dissected on the day of the experiment. Diamonds; c e l l s dissected on the day before that of the experiment. Triangles: c e l l s dissected on the day of the experiment and subjected only to microelectrode experiments, not m i c r o i n j e c t i o n . 179 where A i s the area of the surface enclosing V , and across which the e f f l u x m of sodium occurs. The two are not completely independent, since measured values of ( a N a ) m appear i n both. However, M^a i s determined i n addition by the isotope e f f l u x . A check of consistency is to eliminate ( a ^ a ) m between Figs. 17 and 22. The c e l l s used were of about the same s i z e . A plot of Mjj a vs. d _ ( a M a ) m i s l i n e a r , and the slope Vm/J(.A i s 0.004 cm. This dt m m should equal 0.68 r , so the mean value of the c e l l r a d i i should be 0.0765 0.65 20 cm. The actual mean value of the c e l l r a d i i for the experiments using sodium-free s o l u t i o n is 0.066 cm (SD = 0.013 cm, 19 c e l l s ) . This suggests that the approximations used i n a r r i v i n g at equation (4) are quite good. It also suggests that the rapid f a l l i n ( a j ^ a ) m is r e a l l y due to transport across the c e l l membrane, and further that the mechanism behaves j u s t l i k e the mechanism which expels sodium from the c e l l into normal Ringer's s o l u t i o n . The r e l a t i o n s h i p between the microelectrode measurements and the radioisotope measurements of the e f f l u x into sodium-free s o l u t i o n can be examined In more d e t a i l , i n l i g h t of the re s u l t s of the preceding sections. The injected radiosodium i s deposited i n s o l u t i o n in the myoplasm. I t very quickly e q u i l i b r a t e s with the small pool of bound but rapidly-exchang- ing sodium associated with the c o n t r a c t i l e proteins. Exchange between the myoplasmic sodium and th i s loosely-bound sodium i s much more rapid than transport of sodium across the c e l l membrane, so this bound radiosodium plus the free radiosodium constitute one compartment as far as the membrane transport i s concerned. Radiosodium i s continuously l o s t across the c e l l membrane to the bathing sol u t i o n , and v i a lo n g i t u d i n a l d i f f u s i o n to an e f f e c t i v e i n t r a - c e l l u l a r sink. These processes have been assumed to be independent. The myoplasmic sodium a c t i v i t y ( a ^ a ) m i s re l a t e d to the sodium content 180 of the myoplasmic compartment Na m as X-Nara/Vm = ( aN a) m> by the d e f i n i t i o n s of this model. A change in the sodium content of the myoplasmic compart- ment i n a given time is then ANa = V • A (a ) ... (9) m -m v Na'm . v ' *± I f i t i s assumed that the change i n the sodium content of the myoplasm which occurs when the c e l l is immersed in sodium-free s o l u t i o n i s e n t i r e l y due to passage of sodium across the c e l l membrane and into the bath, then ANa m . 4|££ i S£. ta.. - S . <a S a) m . ...(10) S Am Na* Na* h SA a c t u a l l y includes free and loosely-bound sodium and radiosodium. The m electrode measures only free sodium. The exchangeable radiosodium (free and loosely-bound) i s given by the slope of the plot of In Na versus time, as explained in sections 2.F and 4. For t h i s reason, (10) as written is a s l i g h t underestimate o f ^ N a . I f N a „ n 1 l is used in place of Na , then ° m c e l l r m' (10) underestimates ANa m by a larger amount. The amount of the underesti- mate due to t h i s factor . w i l l increase with time, but should amount at most to about 257c here, given the sodium content of these c e l l s (Fig. 8). This must be considered because the use of the instantaneous slope of the plot of In Na versus time to c a l c u l a t e Na /Na m can only be done when a l i n e can be drawn through the data points, as explained i n sections 2.F and 4, while this plot is quite e r r a t i c just a f t e r the change to a sodium-free s o l u t i o n (Fig. 15), since the rate constant for sodium transport i s changing. The point of a l l of this i s that (9) i s what the electrode measures and (10) i s what the radioisotope measures. I f the f a l l i n ( a j ( j a ) m i n sodium- free s o l u t i o n i s s o l e l y or mostly due to passage of sodium out of the c e l l , then the r a t i o of (9) to (10) should be close to unity. Since the only 181 value for (10) obtainable from the data under these circumstances is an underestimate, the r a t i o of (9) to (10) w i l l be greater than unity by at most about 2570. I f the f a l l i n (a ) i s due to an appreciable amount of Na m sodium going elsewhere i n the c e l l , then the r a t i o of (9) to (10) w i l l be lower. What is 'appreciable' here i s about 257« of the f a l l i n ( a j j a ) m - Of the 22 experiments with sodium-free lithium-substituted s o l u t i o n i n which Mjr a was measured, ( a j r a ) m f a i l e d to f a l l i n three,. and a contracture interrupted one. For the remaining 18 experiments, the r a t i o of (9) to (10) was calculated: M£Na>m At RATIO = I * 1 Na . . . 5 min N a * *-aNa'm " d c e l l for each c o l l e c t i o n period from the moment of immersion i n sodium-free so l u t i o n . The r e s u l t s are shown i n Fig. 23. The value of the RATIO ri s e s to about 1.4, then declines to a value near 1.0 over the f i r s t 40 minutes in sodium-free sol u t i o n . Given the above considerations, i t appears that almost a l l of the f a l l i n ( a j r a ) m seen i n sodium-free s o l u t i o n i s due to movement of sodium out of the c e l l , and not to a r e d i s t r i b u t i o n of sodium among i n t r a c e l l u l a r compart- ments. The i n i t i a l transient v a r i a t i o n s of K j r a for lithium-substituted s o l u t i o n might be an a r t i f a c t of the so l u t i o n change, but the stimulation i n t r i s - and choline-containing s o l u t i o n i s more d i f f i c u l t to explain. There was no transient i n (Na) m a f t e r the change to sodium-free solution, j u s t the onset of a well-behaved decline l i k e those i n F i g . 20. That i s , the response of the transport mechanism to the substrate concentrations is d i f f e r e n t when t r i s or choline is present while external sodium i s absent. At a given value of (Na) m, compared to the e f f l u x into normal Ringer's 182 Figure 23. Value of the RATIO which represents the e f f l u x of sodium from the myoplasm a f t e r exposure to sodium-free s o l u t i o n (time zero) as measured by the microelectrode r e l a t i v e to that measured by the e f f l u x of radiosodium, as explained i n the text. S o l i d l i n e simply connects the data points. Each point represents the mean of the r e s u l t s f or 17 - 18 c e l l s (time 5 min to 40 min) or for 6 - 1 6 c e l l s (time beyond 40 min), and the bars represent two standard deviations. V 183 s o l u t i o n the sodium e f f l u x appears to be s l i g h t l y greater i n the absence of external sodium with:lithium as a substitute, and to be greater s t i l l with t r i s or choline as the substitute. An hypothesis which could account for a l l of these cases i s that external sodium i n h i b i t s sodium transport, that l i t h i u m removes some of this i n h i b i t i o n but as a small cation s t i l l acts much as external sodium does i n the i n h i b i t i o n of sodium transport, and that the large cations choline and t r i s are much less able to act as external sodium does to i n h i b i t sodium transport. Then the M$ja versus ( a ^ a ) m c h a r a c t e r i s t i c would r i s e more steeply with lithium-substituted sodium-free s o l u t i o n than with normal Ringer's solu t i o n , and more steeply s t i l l for choline- or t r i s - s u b s t i t u t e d sodium- free s o l u t i o n . The c h a r a c t e r i s t i c curves for the three s i t u a t i o n s would be close together at low values of ( a ^ ) , ^ a s found i n Fig. 16, but farther apart at higher values of (a„ ) . Then at a higher value of (a„ ) , when ^ & v Na'm ^ Na'm' the external s o l u t i o n i s changed to one containing t r i s rather than sodium, the e f f l u x w i l l jump to the higher c h a r a c t e r i s t i c curve, and decline along this curve as (a,T ) f a l l s . The i n i t i a l e f f e c t w i l l then be a stimulation v Na'm of M^a for c e l l s of higher i n i t i a l sodium content, with the decline of M^a always occuring as ( a ^ a ) m f e l l , as found i n Fig. 15. The biphasic response with l i t h i u m presumably r e f l e c t s this summation of stimulatory and i n h i b i t o r y e f f e c t s , but cannot be explained i n d e t a i l . Membrane P o t e n t i a l . In almost a l l experiments with lithium-substituted sodium-free solution, there was a depolarization of a few m i l l i v o l t s s h o r t l y a f t e r the change from normal Ringer's s o l u t i o n to sodium-free sol u t i o n . No good c o r r e l a t i o n between the amount of depolarization and the rate of the rapid e f f l u x could 184 be demonstrated. In the experiments with choline-substituted sodium-free solutions, there always was a hyperpolarization following the change to sodium-free sol u t i o n . In the experiments with t r i s - s u b s t i t u t e d sodium-free solut i o n , there was a less dramatic hyperpolarization following the s o l u t i o n change. These features w i l l be discussed i n section 7, which is concerned with the membrane po t e n t i a l and e l e c t r o g e n i c i t y . DISCUSSION A rapid f a l l i n ( a ^ a ) m i n barnacle muscle c e l l s occurs on immersion of the c e l l s i n sodium-free sol u t i o n . The e f f e c t d i f f e r s from that observed i n frog muscle and crab muscle. F i r s t , i t only occurs in barnacle muscle c e l l s whose sodium content i s elevated. The e f f e c t is seen even in frog and crab muscle c e l l s of low sodium content. Second, the time course is d i f f e r e n t . The rapid e f f l u x is complete a f t e r about 3 minutes i n crab muscle, and a f t e r 5 to 20 minutes i n frog muscle, but takes about 20 minutes i n barnacle muscle. I t seems l i k e l y that both features are due to d i f f e r - ences i n the M ^ a versus ( a j r a ) m c h a r a c t e r i s t i c curves for frog compared to barnacle ( r e c a l l : i n frog there i s a steep r i s e of M ^ a and saturation at low (a ) - Harris 1965). But crab is a crustacean l i k e barnacle and should v Na<m ' not behave so d i f f e r e n t l y . Other features of the electrode measurements i n crab muscle are unusual. There is almost no change in ( a j r a ) m during a 100 minute period of exposure of the c e l l to crab Ringer's s o l u t i o n to which had been added ouabain to 2 x 10"^ M (Vaughan-Jones 1977 - F i g . 5 ) , or during a 55 minute period of exposure of the c e l l to potassium-free s o l u t i o n (Vaughan-Jones 1977 - Fig. 4 ) . 185 This d i f f e r s from the immediate and sustained r i s e of ( a - ) seen when N Na'm s n a i l neurone, frog s k e l e t a l muscle, or barnacle muscle c e l l s are exposed to ouabain or potassium-free s o l u t i o n (Thomas 1972b; White & Hinke 1976; Fig. 10 of this t h e s i s ) . Yet the re s u l t s reported here for the response of barnacle muscle to sodium-free solutions show i n general the same behavior as was seen i n s n a i l neurone and frog and crab muscle. For now, i t w i l l be assumed that a s i m i l a r process y i e l d s the rapid f a l l i n a l l four preparar t ions. The rapid f a l l of ( a j j a ) m i n barnacle muscle i s associated with a commensurate e f f l u x of sodium from the c e l l . Thomas (1972b) noted that the rate of f a l l of (Na) was appropriate for the s i z e of the u n i d i r e c t i o n a l sodium e f f l u x i n the absence of sodium i n f l u x i n s n a i l neurone. Vaughan- Jones (1977) f e l t that i n t r a c e l l u l a r : r e d i s t r i b u t i o n of sodium could not be ruled out by h i s r e s u l t s . White and Hinke (1976) concluded on the basis of chemical analysis of the frog muscle that the f a l l of (a„ ) was J ° N Na'm associated with an e f f l u x of sodium from the c e l l . They were mainly concerned with the microelectrode measurements but presented some radio- sodium measurements as we l l . The radiosodium measurements were r e l a t i v e l y unsophisticated i n that a passive loading period of four hours was used, on muscle bundles rather than sin g l e c e l l s , and the large e x t r a c e l l u l a r space was not cleared by immersion of the bundle i n normal frog Ringer's so l u t i o n before measurements i n sodium-free s o l u t i o n were made. Any ra p i d e f f l u x of sodium was thus masked by e f f l u x from the e x t r a c e l l u l a r space. The rate constant describing the i n i t i a l rapid f a l l of ( a j j a ) m j deduced by the curve peeling technique, is comparable i n magnitude to the rate constant describing the e f f l u x of radiosodium from the e x t r a c e l l u l a r space. The comparison is deceptive, however, since i n the case of the e f f l u x of radiosodium from the e x t r a c e l l u l a r space, the rate is the constant rate 186 determined by the d i f f u s i o n of sodium i n bulk so l u t i o n . (In fact, the fundamental assumption of the tracer technique, that the rate of loss of tracer i s proportional to the amount of tracer present, is not s a t i s f i e d i n the case of the loss of radiosodium from the e x t r a c e l l u l a r space, since the process by which radiosodium leaves this compartment i s the same as the process which mixes the compartment - d i f f u s i o n i n bulk solution.) In the case of the plo t of In Na versus time for radiosodium leaving the i n t e r i o r of the c e l l , the slope r e f l e c t s not only the rate constant k but i n addition, i t s rate of change dk. dt The point i s , Na*/Na* never approaches the value of the slope of the semilog plot when that p l o t is not close to l i n e a r , and the s i z e of the e f f l u x never approaches the s i z e of the e f f l u x from the e x t r a c e l l u l a r space, even though s i m i l a r values for rate constants can be obtained by the imposi- t i o n of the tracer analysis on the data. In summary, the radioisotope and microelectrode measurements are con- s i s t e n t , and this indicates that the model for the states of the i n t r a - c e l l u l a r solvent and ions is consistent insofar as i t was used i n the in t e r p r e t a t i o n of these measurements. The sodium-free e f f e c t as i t i s generally regarded, as the i n h i b i t i o n of a t i g h t l y - l i n k e d sodium-sodium exchange, has been found to be something quite d i f f e r e n t . The res u l t s i n barnacle and most of the r e s u l t s i n other muscles and i n nerve concerning the stimulation of sodium e f f l u x when the external sodium i s removed can be viewed as r e f l e c t i n g an i n h i b i t i o n of sodium transport by external ions. The f a l l i n the sodium e f f l u x which follows removal of external sodium appears to be due to the consequent f a l l i n the myoplasmic sodium a c t i v i t y . 187 SECTION 7. ELECTROGENIC SODIUM TRANSPORT IN BARNACLE MUSCLE The p r i n c i p a l mode of a c t i v e transport of sodium i n barnacle muscle c e l l s appears to be the sodium-potassium exchange mode of the (Na+K)ATPase. I f sodium ions are expelled from the c e l l s at a rate greater than the rate at which potassium ions are taken up v i a this mechanism, the net e f f l u x of cations which r e s u l t s w i l l give r i s e to an e l e c t r i c a l p o t e n t i a l d i f f e r e n c e across the c e l l membrane. The mechanism by which t h i s p o t e n t i a l d i f f e r e n c e a r i s e s , as discussed in section 2.D, probably is s i m i l a r to that by which the larger p o t e n t i a l difference due to the passive movement of potassium ions out of the c e l l a r i s e s , as discussed i n section 2.D. However, the small hyperpolarization of the c e l l d i r e c t l y due to the sodium pump w i l l only be present when the sodium-potassium exchange is not one-for-one, while the larger potassium ' d i f f u s i o n p o t e n t i a l ' w i l l p e r s i s t as long as the potassium ion gradient across the c e l l membrane is maintained. An electrogenic e f f e c t i s most e a s i l y detected i n c e l l s which have been loaded with sodium, as then the rate of a c t i v e sodium extrusion i s more rapid, but the e f f e c t can also be seen i n c e l l s with normal or low sodium content (Kerkut & Thomas 1965; Thomas 1972b). However, i t has been detected in the course of the normal functioning of several c e l l types as well: post-tetanic hyperpolarization i n nerve (eg. Rang & R i t c h i e 1968); post- synaptic potentials i n nerve (eg. L i b e t & Kobayashi 1968); regulation of tension i n vascular and g a s t r o i n t e s t i n a l smooth muscle (eg. Prosser 1977); the response of pancreatic acinar c e l l s to pancreozymin (Kanno 1975); and the response of pancreatic i s l e t c e l l s to glucose (Matthews & Sakamoto 1975; Atwater & Meissner 1975). The degree to which e l e c t r o g e n i c i t y i s necessary i n the normal functioning of c e l l s i s not yet known. An electrogenic contribution by the (Na+K)ATPase can be detected by 188 observation of the e f f e c t on E m of a l t e r a t i o n s i n the pump rate. Most s p e c i f i c is the observation of a depolarization of the c e l l when i t i s exposed to ouabain. An a l t e r n a t i v e method is to observe a hyperpolariza- t i o n of the c e l l when the pump is stimulated by a sodium load, although t h i s is not as s p e c i f i c as the use of ouabain (eg. DeWeer & Geduldig 1973). A v a r i a t i o n of these methods is to voltage clamp the c e l l , that i s , to measure E m with an i n t r a c e l l u l a r electrode and pass a current across the membrane v i a a d i f f e r e n t electrode i n order to keep E m steady. Then changes in the amount of current which must be passed to maintain E m i n the face of some challenge such as a sodium load indicate the changes i n the contribu- t i o n of the pump to E m which the challenge can e l i c i t . In fact, this method bypasses the manifestation of electrogenic transport, the changes i n E m, and measures the unbalanced cation e f f l u x by exactly opposing i t (eg. Thomas 1969; Kostyuk et a l . 1972). None of the methods is id e a l . A p p l i c a t i o n of ouabain can i n h i b i t much of the act i v e sodium transport, but i t is not known how the ouabain-insensi- t i v e transport changes i n the face of th i s challenge. Voltage clamping involves the passage of a poly i o n i c current across the membrane, and the composition of this current is not completely c l e a r . The experiments described i n this section involve the measurement i n a sin g l e c e l l of two quantities simultaneously: the membrane po t e n t i a l and the u n i d i r e c t i o n a l sodium e f f l u x . The response of these quantities to exposure of the c e l l to ouabain i s compared. It is shown that an e l e c t r o - genic e f f e c t i s associated with the sodium e f f l u x i n barnacle muscle, and the c o r r e l a t i o n of the changes i n E and M N is examined. 189 METHODS The measurement of the sodium e f f l u x has been described in sections 4 and 5. In this series of experiments, a s i n g l e muscle c e l l was dissected, injected with radiosodium solution, and mounted i n the perfusion chamber i n the usual manner. The c e l l was perfused with normal Ringer's s o l u t i o n u n t i l a steady e f f l u x was established. Then the perfusing s o l u t i o n was changed to normal Ringer's to which ouabain had been added, to 10" to 10" M i n d i f f e r e n t experiments. ' The sodium e f f l u x M^a was calculated from equation (4), with c o r r e c t i o n for Na* e-^ since the best quantitative estimate was sought. RESULTS The change i n the membrane po t e n t i a l E , recorded with a micropipette electrode, assumed a new steady value a f t e r exposure of the c e l l to ouabain, -4 as indicated i n Fig. 24. S i m i l a r i l y , the sodium e f f l u x f e l l and, with 10 M ouabain, remained quite steady for.30 to 60 minutes despite a large increase in ( a j j a ) m . In order to apply the c o r r e c t i o n for l o n g i t u d i n a l d i f f u s i o n , the 'slope r a t i o ' for e f f l u x into normal Ringer's s o l u t i o n before the exposure of the c e l l to ouabain was used. This was necessary because the c o r r e c t i o n for Na*e-Q described previously cannot be applied when the e f f l u x rate is changing. Fig. 24 is a summary of more data from the experiment depicted in Fig. 10. Of p a r t i c u l a r i n t e r e s t here are the changes in the membrane p o t e n t i a l V I t can be seen that while the c e l l is being perfused with normal 190 M N ( J (p/cs) -E m (mV) 26 18- I Oi 70- 66- 62- NORMAL • K-FREE NORMAL OUABAIN • • • • • • • • • A A A A ..1 A A (aN«An ( m M ) 24j 20- 16' • * • • 50 loo 150 TIME (min.) 200 Figure 24. Change i n the membrane p o t e n t i a l which occurs on exposure of the -4 c e l l to potassium-free or ouabain (10 M) containing solutions. A d d i t i o n a l data from the same experiment i s presented i n F i g . 10. Top: sodium e f f l u x c a l c ulated from equation (4) without c o r r e c t i o n for Na* ,,. Middle: membrane c e l l p o t e n t i a l measured with micropipette electrode. Bottom: myoplasmic sodium a c t i v i t y measured with a sodium-specific microelectrode. 191 Ringer's sol u t i o n , (a,T ) was steady at about 18 mM, E became steady at & v Na'm J ' m J about -68 mV, and M^a was reasonably steady at about 18 pes. As presented in t h i s figure, M^a i s calculated d i r e c t l y from equation (4) without 7 ̂ correction, to i l l u s t r a t e the de t a i l e d q u a l i t a t i v e changes. NaCl had been injected to r a i s e (a ) , and E recovered r e l a t i v e l y slowly i n th i s experi- Na m M ment, but attained a steady value. Exposure to potassium-free s o l u t i o n i s expected to slow the sodium- potassium exchange, and the membrane po t e n t i a l does f a l l s l i g h t l y . The i n i t i a l e f f e c t on E m, a transient r i s e , probably r e f l e c t s the e f f e c t of removal of external potassium on the membrane p o t e n t i a l as expressed by the Goldman-Hodgkin-Katz equation, i e . the 'potassium d i f f u s i o n p o t e n t i a l ' . The net e f f e c t seen should be the sum of a hyperpolarization due to the l a t t e r , and a depo l a r i z a t i o n due to i n h i b i t i o n of act i v e sodium-potassium exchange. The decline of M^a i n potassium-free s o l u t i o n i s seen i n Fig. 24, as i s the accompanying r i s e i n ( a j j a ) m - Restoration of the external potassium causes the sodium e f f l u x to r i s e to i t s former l e v e l , and ( a j j a ) m becomes steady. I t would have been expected from Fig. 19 that M ^ would be s l i g h t l y higher at the higher value of (a ) . Apparently the e f f e c t i s too small to be revealed i n this Na m uncorrected c a l c u l a t i o n of M^. The membrane p o t e n t i a l r i s e s , to a value somewhat higher than before. This cannot convincingly be ascribed to increased sodium pumping at the higher concentration of i n t r a c e l l u l a r sodium, since the expected r i s e i n the sodium e f f l u x has not been demonstrated by comparison with i t s previous value i n normal Ringer's sol u t i o n . The extra r i s e i n E m might r e f l e c t a change in the sodium-potassium exchange r a t i o due to the sodium load brought about by the b r i e f removal of external potassium, as w i l l be discussed l a t e r i n th i s section. Exposure of the c e l l to normal Ringer's s o l u t i o n to which ouabain had 192 been added to 10" 4 M i s expected to slow the sodium extrusion considerably. Fig. 24 shows that Mjra f a l l s quite abruptly, while ( a j T a ) m commences a steady r i s e , and f a l l s abruptly with M^^. This demonstrates that by the accepted c r i t e r i o n , there i s an electrogenic contribution to the r e s t i n g membrane p o t e n t i a l i n barnacle s t r i a t e d muscle, at least when i t is loaded with sodium. This had not been shown before. Fig. 25 is a summary of a number of experiments i n which c e l l s were exposed to ouabain. The change A E m i n the membrane po t e n t i a l upon exposure to ouabain i s plotted versus the corresponding simultaneous change i n the sodium e f f l u x AM^a q u a n t i t a t i v e l y . The value of AE^ i s expected to be accurate, since any uncertainties involved i n the use of the micropipette electrode do not influence such a measured differ e n c e change in E m < The value of ^M^a requires more attention. The approximations and uncertainties involved i n the measurement of Mjj a have been discussed at length i n previous sections, and the approach taken is f e l t to y i e l d the best possible estimate. The consistency of M^a and ~ (a^ ) presented in section 6 supports this assertion. Thus i t seems j u s t i f i e d to seek a quantitative d e s c r i p t i o n of the r e l a t i o n s h i p between 4 E m and The q u a l i t a t i v e r e l a t i o n s h i p between ̂ E ^ and AM^ a is clear from Fig. 25. The l i n e drawn i s r e a l l y j u s t a v i s u a l aid. A quantitative expression of the r e l a t i o n s h i p between A E m and ^M^a i n the context of the Goldman- Hodgkin-Katz theory has been developed i n section 2.F. The passive trans- membrane fluxes of ions are treated i n the constant f i e l d approximation, and the net a c t i v e cation e f f l u x of electrogenic transport i s included phenomenologically. That i s , the passive fluxes are assumed to a r i s e from a s p e c i f i e d d r i v i n g force, while the net a c t i v e c a t i o n e f f l u x i s j u s t added into the current balance equation. The r e s u l t i s 193 Figure 25. Change i n the membrane p o t e n t i a l AE m which occurs upon exposure of the c e l l to ouabain, plotted versus the corresponding simultaneous change in the sodium e f f l u x -AM calculated from equation (4), with c o r r e c t i o n for * -4 N a -5 T6 Na ... C i r c l e s : 10 M. Diamonds: 10 M. Triangles; 10 M. Open c e l l symbols represent c e l l s dissected on the day of the experiment. Closed symbols represent c e l l s dissected on the day before that of the experiment. 19.4 where R, T, and F have t h e i r usual s i g n i f i c a n c e , U = P (K) + P (Na) + K o Na o P ^ ^ ( C l ) ^ . Pj is the permeability of the membrane to species j , ( K ) q and (Na) Q are the external concentrations, ( C l ) ^ i s the i n t r a c e l l u l a r concentra- tion, and (J*\= -M â/Mj, > 0 is the coupling r a t i o for sodium-potassium exchange, assumed here to be almost e n t i r e l y a ctive. Fig. 25 exhibits a great deal of scatter, but i f i t i s interpreted in terms of (8), i t seems that (R. is greater than unity at smaller values of AM.̂  , but c l o s e r to unity at higher values. Interpretation of (8) and Fig. 25 w i l l be discussed i n d e t a i l l a t e r i n this section. In F i g . 26 are presented the measured steady values of the membrane po t e n t i a l E for c e l l s of d i f f e r e n t sodium content. The sodium content of r m the c e l l s was r a i s e d by i n j e c t i o n of NaCl. Several e f f e c t s were anticipated. The sodium load should stimulate a c t i v e sodium expulsion, and any e l e c t r o - genic e f f e c t should become more prominent. On the other hand, the i n j e c t i o n of chloride w i l l y i e l d a depolarization, as can be seen from the Goldman- Hodgkin-Katz equation. The i n j e c t i o n of sodium tends to y i e l d a hyperpolari- zation i n a s i m i l a r manner, but P i s small compared to P so the e f f e c t Na C l w i l l also be small. Also, the i n j e c t i o n of concentrated solutions of NaCl might change the permeability of the c e l l membrane. The net e f f e c t i s seen i n Fig. 26 to be s l i g h t . 195 8 0 6 0 > E i 4 0 2 0 O^lo 20 30 40 50 60 ( a N Q ) m (mM.) Figure 26. Resting membrane p o t e n t i a l for c e l l s i n normal Ringer's s o l u t i at approximately 23°C, a f t e r microinjection, versus the steady value of th myoplasmic sodium a c t i v i t y . 196 DISCUSSION Nonelectrogenic membrane p o t e n t i a l . In the GHK equation for the r e s t i n g membrane, pot e n t i a l , the sodium, potassium, and chloride concentrations appear. T y p i c a l l y , P^ a i s small compared to P^ and P^- I t was noted that shows l i t t l e dependence on (a ) o v e r a l l (Fig. 26), and that t h i s could be due to the c a n c e l l a t i o n of Na m several d i f f e r e n t effects of i n j e c t i o n of NaCl. In section 6, it.was noted that i n experiments with lithium-substituted sodium-free s o l u t i o n there was a depolarization of a few m i l l i v o l t s s h o r t l y a f t e r the change from normal Ringer's s o l u t i o n to sodium-free solution, while there was a hyperpolariza- t i o n a f t e r the change from normal Ringer's s o l u t i o n to choline- or t r i s - substituted sodium-free s o l u t i o n . It was noted above that there was a transient hyperpolarization and a sustained depolarization a f t e r the change from normal Ringer's s o l u t i o n to potassium-free s o l u t i o n . Choline and t r i s w i l l pass the c e l l membrane at a much slower rate than w i l l the small inorganic cations. The GHK equation predicts that removal of external sodium should cause a s l i g h t hyperpolarization, s l i g h t because P„ is small r e l a t i v e to P„ and P„_. The rate of the sodium e f f l u x into Na K C l sodium-free s o l u t i o n is not very d i f f e r e n t from that into normal Ringer's s o l u t i o n (Fig. 19), but the i n i t i a l stimulation of the sodium e f f l u x i n choline- and t r i s - s u b s t i t u t e d solutions might cause an increase i n the electrogenic hyperpolarization of the c e l l . The coupling r a t i o (R. of the (Na+K)ATPase might be changed by these manipulations, but there i s no basis on which to speculate about such an e f f e c t . It might be that the permea- b i l i t y of the membrane to l i t h i u m i s greater than that to sodium, so that the switch from normal Ringer's s o l u t i o n to lithium-substituted s o l u t i o n y i e l d s a depolarization as l i t h i u m ions enter the c e l l . 197 Thus the net depolarization i n sodium-free lithium-substituted, s o l u t i o n can be accounted for as the sum of two passive e f f e c t s : small hyperpolarization due to the rever s a l of the sodium concentration gradient, and larger depolarization due to the creation of a large cation (lithium) gradient opposing the potassium gradient. On the other hand, the net depol a r i z a t i o n i n potassium-free s o l u t i o n can be accounted for as the sum of a passive and an electrogenic e f f e c t : a hyperpolarization due to an increase i n the potassium concentration gradient, and, i n the case of Fig. 25, a larger depolarization due to i n h i b i t i o n of the electrogenic sodium- potassium exchange when external potassium i s removed. The size of the l a t t e r e f f e c t is indicated by the larger depolarization which occurs when the electrogenic transport i s i n h i b i t e d with ouabain. In this case, the potassium concentration gradient does not change abruptly. Electrogenic sodium transport. The r e l a t i o n between AE • and AM.T,, w i l l be discussed i n terms of in iNci equation (8) of's e c t i o n 2.F, which was reproduced above. The unbalanced ca t i o n e f f l u x i s (1 - l/fo_)- A M j r a d e f i n i t i o n , and as i t increases the s i z e of the membrane pot e n t i a l should increase. The p r o p o r t i o n a l i t y factor between A E m and (1 - l/<ft)-Al% a was discussed i n section 2.F. It has been assumed i m p l i c i t l y i n the de r i v a t i o n of this expression that the coupling r a t i o (R. is unchanged by the a p p l i c a t i o n of ouabain. That i s , i t has been assumed that a c e r t a i n number of the transport enzymes are transporting more sodium than potassium, a l l with the same r a t i o . Exposure to ouabain has been assumed to h a l t the operation of some of these enzymes, but to leave the others unchanged, since i n i s o l a t e d (Na+K)ATPase preparations a l l but a few p a r t i a l reactions are halted by the binding of ouabain. This would be so i f the transport enzymes were independent, and 198 i f the rate for each enzyme were governed only by the concentration of substrates, i n p a r t i c u l a r by (Na)^. Such an assumption was avoided i n the examination of the k i n e t i c c h a r a c t e r i s t i c s of sodium transport i n section 5, but w i l l be made here as the simplest case for the purposes of discussion. Thus i t i s assumed that for each data point, (R. was unchanged by the manipulation which brought about AEM and AM̂ . Differences from l i n e a r i t y i n Fig. 26 are then due to d i f f e r e n t values of (R for d i f f e r e n t experiments, although some of the scatter i s probably due to differences i n the value of E for d i f f e r e n t experiments. The l a t t e r can cause vari a t i o n s of 10 to 15% m i n the p r o p o r t i o n a l i t y factor i n equation (8) (see Fig. 26). Attempts to c a l c u l a t e the value of (R. for each experiment were compli- cated by the fact that the c a l c u l a t i o n involves the r a t i o of small d i f f e r - ences between large quantities which are subject to experimental uncertainty. A consistent d e s c r i p t i o n can be formulated, however. The slope of the best l i n e drawn by eye i n Fig. 25 i s 0.52 mV/pcs. The value of U can be estimated from published data to be 10 pes ( (Na)^ = 450 mM, ( K ) Q = 8 mM, where )ft = 0.7; ( a c l ) m = 23 mM (Hinke, C a i l l e , & Gayton 1973); P C 1 / P K = 0.04, (Hagiwara et a l . 1968); i t i s assumed that P = 1 x 10"^ cm/sec and K P /P = 0.01 by analogy with nerve and vertebrate s t r i a t e d muscle). Then Na K J (R, = 2.1, or somewhat greater than the value of 1.5 found i n red blood c e l l s . For squid axon the value of OK. has been reported to vary with the sodium content (Mullins & B r i n l e y 1969). No claim can be made here beyond consistency of the model and data, but consistency is found. The assumption that (j\. i s constant .is consistent with the r e s u l t s of Thomas (1969) for s n a i l neurone. He found that the feedback current in a voltage clamp was d i r e c t l y proportional to the sodium e f f l u x stimulated by a sodium load, and speculated that the ATP:Na:K r a t i o of 1:3:2 might be u n i v e r s a l . The r e s u l t i n squid axon indicates otherwise. 199 Further, Kostyuk e£ a l . (1972) found that the feedback current for a voltage clamped s n a i l neurone was e x q u i s i t e l y s e n s i t i v e to the value of the membrane p o t e n t i a l . I t appeared that Mjj a was r e l a t i v e l y unaffected by small hyperpolarizations, while was greatly increased. The net e f f e c t was suggested to be a decrease i n the pump r a t i o (R toward unity as the c e l l was hyperpolarized. The experiment on which t h i s conclusion i s based appears to show that the pump r a t i o responds almost instantaneously to changes i n the membrane p o t e n t i a l . It should be mentioned here that G l i t s c h (1972) presented for cardiac muscle a p l o t s i m i l a r to F i g . 25. He found almost perfect c o r r e l a t i o n between and during recovery of the cardiac muscle from incubation i n the cold. However, i n his c a l c u l a t i o n of the u n i d i r e c t i o n a l sodium e f f l u x as the differe n c e between the net change i n sodium content of the tiss u e as measured by flame photometry, and the estimated change i n the passive sodium i n f l u x i n the constant f i e l d theory, he has imposed on his data exactly the functional dependence he claims to f i n d . It seems l i k e l y that electrogenic sodium transport does occur i n cardiac muscle, but his methods cannot be used to obtain accurate measurements of th i s phenomenon. In summary, i t has been demonstrated that there i s a depolarization of the barnacle muscle c e l l when the (Na+K)ATPase is i n h i b i t e d by ouabain. The s i z e of th i s d epolarization i s correlated i n s i z e and in time course with the accompanying decrease i n the u n i d i r e c t i o n a l sodium e f f l u x measured with radiosodium and a sodium-specific microelectrode. These r e s u l t s e s t a b l i s h the existence of electrogenic sodium transport i n barnacle muscle. The response of the membrane p o t e n t i a l to potassium-free and to sodium-free solutions can be explained as the sum of an e f f e c t on the passive and the act i v e contributions. It was not possible to ca l c u l a t e the coupling r a t i o for a c t i v e transport 200 for the i n d i v i d u a l experiments, because the c a l c u l a t i o n involves the r a t i o of small differences i n large quantities and the scatter of the data i s quite large. In addition, the permeability of the barnacle muscle c e l l membrane to the major inorganic cations has not been measured accurately. The r e s u l t s of the present experiments can be interpreted c o n s i s t e n t l y i n terms of a pheriomenological extension of the constant f i e l d model, with a coupling r a t i o of about two sodium ions transported out per potassium ion transported in, presumably v i a the (Na+K)ATPase. It was not possible to detect a dependence of the coupling r a t i o on the membrane po t e n t i a l or on the rate of sodium transport, but such a dependence cannot be ruled out. This approach to the measurement of the electrogenic p o t e n t i a l could be quite f r u i t f u l . A s p e c i f i c mechanism for the act i v e cation e f f l u x , such as the assumption of a d r i v i n g force involving the a f f i n i t y of the hydrolysis of ATP and the transmembrane differ e n c e i n the electrochemical p o t e n t i a l for sodium, potassium, and chloride, could be used i n the de r i v a t i o n of an expression for the t o t a l membrane p o t e n t i a l . A great deal of preliminary study of the passive permeabilities and the composition of the transmembrane i o n i c currents w i l l have to be c a r r i e d out before d e t a i l e d conclusions can be drawn from radioisotope and voltage clamp experiments, but the findings of such study would be of in t e r e s t i n other contexts as well. 201 SECTION 8. DISTRIBUTION OF HYDROGEN IONS DURING STEADY CONDITIONS It has been known for some time that the normal i n t r a c e l l u l a r pH is higher than would be the case i f hydrogen ions were d i s t r i b u t e d passively across the c e l l membrane (eg. Caldwell 1956; Waddell & Bates 1969). I t has been concluded that some process dependent on metabolism and equivalent to the expulsion of hydrogen ions from the c e l l must ex i s t , but u n t i l very recently nothing more could be said. As summarized i n section 2.A, evidence has been found for the involve- ment of several processes i n the act i v e control of the i n t r a c e l l u l a r pH. The electron transport system of aerobic metabolism appears to be able to cont r o l the destination of the hydrogen ions i t produces. There appears to be i n the c e l l membrane an ATPase which i s able to transport hydrogen ions. The e x t r a c e l l u l a r sodium concentration has been found to influence the in t e r n a l pH and this has been interpreted as sodium-hydrogen exchange. An ass o c i a t i o n between anion fluxes and the i n t e r n a l pH has been found and interpreted as chloride-bicarbonate exchange. F i n a l l y , a b a c t e r i a l membrane protein which can translocate hydrogen ions when exposed to l i g h t has been is o l a t e d . The use of tracer techniques l i k e those described i n the preceding sections i n connection with the sodium flux cannot be used to study hydrogen fluxes. The experimental study of hydrogen ion transport can only be ca r r i e d out by i n d i r e c t methods, a l l of which involve the measurement of changes i n the i n t r a c e l l u l a r pH. It thus is very important to asce r t a i n the accuracy and r e l i a b i l i t y of the methods cur r e n t l y used to measure the i n t r a c e l l u l a r pH. A d i r e c t comparison of the two most commonly used methods for measuring the i n t r a c e l l u l a r pH thus was c a r r i e d out, and the res u l t s are presented i n section 9 of th i s t h e s i s . 202 During these experiments, i t was noticed that the transmembrane dif f e r e n c e i n pH measured with a pH-specific i n t r a c e l l u l a r microelectrode (which agreed with the res u l t s of an indicator method), showed a c o r r e l a t i o n with the s i z e of the membrane p o t e n t i a l E^. Such a c o r r e l a t i o n i s predicted by the current theory of the passive movement of ions across the c e l l membrane, and has been sought by other workers, but has not been found (Caldwell 1954, 1958; Kostyuk & Sorokina 1961; A i c k i n & Thomas 1975; Thomas 1976b). One worker did f i n d a c o r r e l a t i o n , but of a d i f f e r e n t sort from that predicted by theory (Carter et a l . 1967; Carter 1972). It appears that these r e s u l t s were due to some problem with the measuring techniques, however ( P a i l l a r d 1972; Lev & Armstrong 1975). In t h i s s ection are presented new experimental r e s u l t s pertaining to the r e l a t i o n s h i p between the membrane po t e n t i a l and the transmembrane differe n c e i n pH. These r e s u l t s are analyzed i n terms of the transmembrane cation d i s t r i b u t i o n predicted by current theory. METHODS Single muscle c e l l s of the giant barnacle were dissected as described in section 3. The composition of the sp e c i a l solutions used to cause changes i n the i n t r a c e l l u l a r pH was as follows. To r a i s e the i n t r a c e l l u l a r pH: normal barnacle Ringer's s o l u t i o n without t r i s c h l o r i d e but with 5mM (NH^SO^, pH = 8.2 ( referred to as "NH^ Ringer's s o l u t i o n " ) . To lower the i n t r a c e l l u l a r pH: normal barnacle Ringer's s o l u t i o n without t r i s c h l o r i d e but with 1 mM NaHC03, through which is bubbled continuously at a 203 constant rate a 957, 0 2 — . 57, CO2 § a s mixture to maintain a pH as close as possible to 6.2 (referred to as " C O 2 Ringer's s o l u t i o n " ) . The composition of normal Ringer's s o l u t i o n without t r i s c h l o r i d e is Na 450 mM, K 8 mM, Ca 20 mM, Mg 10 mM, Cl 518 mM. Normal barnacle Ringer's s o l u t i o n contains, in addition, 25 mM t r i s c h l o r i d e and is t i t r a t e d to pH 7.6. The pH of the bathing solutions was monitored throughout the exposure of the c e l l s to them, with a conventional pH meter (Instrumentation Labora- tories) . Standard buffer solutions for the c a l i b r a t i o n of the pH meter and the pH-specific glass microelectrodes were prepared accoring to Robinson (1967). These agreed exactly with commercially made buffer solutions, so the two sets were combined to give f i v e c a l i b r a t i o n points over the pH range investigated here. pH-specific glass microelectrodes were constructed by the method of Hinke (1969). Micropipette electrodes for the measurement of membrane po t e n t i a l were constructed by conventional techniques (eg. Hinke 1969), and tested for small t i p p o t e n t i a l as described i n section 3. The technique for making measurements with these microelectrodes was as described i n section 3 for sodium-specific electrodes. Occasionally the pH-specific microelectrode was referred d i r e c t l y to the i n t r a c e l l u l a r micropipette electrode as a test of consistency. (The use of an indicator to measure the i n t r a c e l l u l a r pH of identically-prepared companion c e l l s i s described in section 9.) At the end of each microelectrode measurement, the impaled c e l l and a companion c e l l were removed for analysis for sodium, potassium, and water content. The e l e c t r o l y t e and water contents of the impaled and non-impaled c e l l s were not s i g n i f i c a n t l y d i f f e r e n t . Furthermore, t h e i r mean values (not corrected for the e x t r a c e l l u l a r space) were not d i f f e r e n t from the mean values of c e l l s used i n the experiments involving the pH indicato r DMO 204 (section 9). RESULTS V i a b i l i t y of is o l a t e d c e l l s . As i n a l l of the work i n barnacle muscle c e l l s reported i n this thesis, i f an impaled c e l l showed any sign of damage the data from i t was discarded. The impaled c e l l s , judged to be damage-free a f t e r prolonged immersion i n a test s o l u t i o n , showed r e s t i n g potentials and e l e c t r o l y t e composition s i m i l a r to those reported by others (Tables I I I and IV). No dependencies were detected between the sodium, potassium, or water content of each c e l l , on the one hand, and the i n t r a c e l l u l a r pH or the transmembrane pH gradient, on the other hand. Mean values of c e l l sodium, potassium, and water content are presented i n Table IV i n order to provide an i n d i c a t i o n of the ph y s i o l o g i c a l state of the c e l l s under the three experimental conditions. Most of the v a r i a t i o n demonstrated here probably is due to animal v a r i a t i o n s (related to si z e , age, season, and nu t r i t i o n ) rather than to experimental conditions. However, the c e l l s e q u i l i b r a t e d i n CO Ringer's s o l u t i o n had a larger i n u l i n space (1470 of c e l l water) than did the c e l l s e q u i l i b r a t e d i n normal Ringer's s o l u t i o n (8.27o) or i n NH^ Ringer's s o l u t i o n (7.97o) and this would account for the larger t o t a l sodium content of these c e l l s (Table IV). The r e s t i n g membrane p o t e n t i a l of the c e l l s during the various steady conditions ranged from -45 to -90 mV (as indicated i n F i g . 29), although the mean value was -72 mV (Table I I I ) . Usually, the membrane pote n t i a l is used as an indicator of the v i a b i l i t y of the c e l l , as noted i n previous 205 TABLE I I I INTRACELLULAR pH AND MEMBRANE POTENTIAL IN CRUSTACEAN MUSCLE Species Temp. Mean E m P H e Mean pR\ Reference A) Maia squinado 20°C -62 mV 7.55 7.15 Caldwell (1958) B) Care inus maenus 20 -40 7.8 7.20 Caldwell (1958) C) C. maenus 23 -60 7.65 6.91 P a i l l a r d (1972) D) C. maenus 20 -65 7.5 7.27 A i c k i n & Thomas (1975) E) Balanus nubilus 23 -72 7.6 7.32 McLaughlin & Hinke (1968) F) B. nubilus 15 -50 7.8 7.34 Boron & Roos (1976) G) B. nubilus 22 -59 7.5 7.31 Boron (1977) H) B. nubilus 23 -72 7.8 7.23 This study TABLE IV MEAN WATER AND ELECTROLYTE CONTENT OF TEST CELLS E q u i l i b r a t i o n No. of C e l l C e l l (Na) C e l l (K) bath c e l l s water (%) (mmole/kg c e l l water) Normal Ringer's 73 74.56 42.9 172.4 0.11* 1.3 1.6 C0 0 Ringer's 55 75.10 50.4 182.3 0.15 2.3 2.3 NH. Ringer's 16 73.90 36.6 181.0 4 0.28 3.4 2.1 E x t r a c e l l u l a r space contents are included i n the analyses. C e l l s had been immersed for more than. 2 hours i n the s p e c i f i e d solution. S.E. of mean. 206 sections. Here, c e l l s with membrane potentials which were r e l a t i v e l y low (yet as i t turned out, s t i l l w ithin the normal range reported by other workers) were accepted i f no damage was apparent on v i s u a l inspection, a f t e r immersion for 2 hours i n the chosen solu t i o n , and the sodium, potassium, and water content was within the normal range established by previous work. This was done to see i f the r e l a t i o n between membrane p o t e n t i a l and pH found i n the higher range extended into the lower range. I n t r a c e l l u l a r pH from microelectrodes. Figures 27 and 28 provide examples of changes i n the i n t r a c e l l u l a r pH value (pH^), e x t r a c e l l u l a r pH value (pH e), and membrane p o t e n t i a l E m with time following a change in the bathing s o l u t i o n . These plots were con- structed from data taken every 5 to 15 minutes (where necessary) from o r i g i n a l continuous recordings. In Fig. 27, c e l l A was allowed to e q u i l i - brate for 105 minutes a f t e r impalement. During this period, the i n t r a - c e l l u l a r pH was very steady even though the membrane p o t e n t i a l increased from -75 to -81.5 mV and the external pH decreased from 7.69 to 7.62. The 'acid i n j u r y ' and the slow recovery following electrode penetration as described by Thomas (1974) and A i c k i n and Thomas (1975) using the recessed- t i p pH-specific microelectrode on smaller c e l l s ( s n a i l neurone) was not observed here with the spear-type pH-specific microelectrode and barnacle muscle c e l l s . Following the bath change to C0 2 Ringer's s o l u t i o n the i n t r a c e l l u l a r pH of c e l l A decreased from 7.31 to 6.94 in about 30 minutes, remained r e l a t i v e l y stable for the next 30 minutes, then began to decrease for no apparent reason. Such behavior by a c e l l has not been explained but was considered undesirable and hence, the few c e l l s showing i t were not used i n any analysis r e l a t e d to the membrane pote n t i a l (or to DM0 - next section). 207 78 74 64 6 Ok 7 4 1 7 0 6 6 Normal Ringer ( solution C O , Ringer solution Fibre A | impaled ^ i i i i > i i i i i Fibre B j impaled Fibre C impaled 30 60 90 0 30 60 90 120 150 Time (min) 180 210 240 270 Figure 27. Re-constructed tracings of i n t r a c e l l u l a r pH, pR\, and membrane po t e n t i a l , E , from three c e l l s of a muscle which was subjected to a bath change from Sormal Ringer's s o l u t i o n to C0 2 Ringer's s o l u t i o n . V e r t i c a l arrows indicate when each c e l l was impaled. pH Q i s the e x t r a c e l l u l a r pH. 208 N H „ + Ringer solution 8-4 (- ! p H 0 J 1 1 1 1 1 I 0 3 0 6 0 9 0 1 2 0 1 S 0 1 8 0 Time (min) Figure 28. Re-constructed tracings o f pH. and membrane p o t e n t i a l E from one c e l l of a muscle which was subjected t o a bath change from normal Ringer's s o l u t i o n to NH^ Ringer's s o l u t i o n . Not shown are the complete tracings following electrode impalement which occurred 90 minutes before zero time. pH is the e x t r a c e l l u l a r pH. 209 Also shown i n Fig. 27 are the r e s u l t s from two companion c e l l s with a h i s t o r y of exposure to test solutions i d e n t i c a l to that of c e l l A. C e l l B was impaled a f t e r 180 minutes and c e l l C was impaled a f t e r 240 minutes of e q u i l i b r a t i o n i n CO2 Ringer's solu t i o n . Fig. 28 shows a t y p i c a l t r a c i n g from a c e l l which was allowed to e q u i l i b r a t e for more than 60 minutes in normal Ringer's s o l u t i o n (not shown) then exposed to NH^ Ringer's s o l u t i o n for about 200 minutes. It was noted that no transients i n the i n t r a c e l l u l a r pH occurred when the c e l l was exposed to CO2 Ringer's or NH4 Ringer's solutions. Transients were reported during s i m i l a r experiments on s n a i l neurone (Thomas 1974). The transients consisted of a slow decline of the i n t r a c e l l u l a r pH during immersion of the c e l l s i n NH^ s o l u t i o n and comparable to the NH^ Ringer's s o l u t i o n used i n the present work, and a slow r i s e of the i n t r a c e l l u l a r pH during immersion of the c e l l s i n HCO3 s o l u t i o n comparable to CO2 Ringer's s o l u t i o n . Such ef f e c t s had not been seen in the preparation used i n the studies described here, which were performed p r i o r to the p u b l i c a t i o n of the r e s u l t s i n s n a i l neurone. A f t e r the r e s u l t s i n s n a i l neurone were published, measurements of the type shown i n Figs. 27 and 28 were repeated on barnacle muscle c e l l s from a d i f f e r e n t batch, and i t was confirmed that such transients do not occur in the preparation used in these studies. Subsequently, pH transients s i m i l a r to those seen i n s n a i l neurone were reported i n squid "axon (Boron & DeWeer 1976), and i n barnacle muscle c e l l s (Boron 1977). The preparation used by Boron (1977) was a barnacle muscle c e l l cut from the basis and cannulated at both ends. The "barnacle sea water" used by Boron was s l i g h t l y d i f f e r e n t from but, on the whole, quite s i m i l a r to barnacle Ringer's solution, aside from the use of 5 mM (N-((2-hydroxy-ethyl)piperazine)-N'-2-ethyl s u l f o n i c a c i d (HEPES) as buffer rather than t r i s . However, the c e l l s had to be immersed i n calcium-free 210 s o l u t i o n during the cannulation procedure to prevent the occurrence of serious damage. This might have changed the permeability properties of the membrane. I t was noted that the membrane po t e n t i a l of this preparation was r e l a t i v e l y low (Table I I I ) . The sodium, potassium, and water content of the c e l l s were not reported. Membrane po t e n t i a l and the transmembrane gradient of pH. In Fig. 29 is plotted the transmembrane differ e n c e i n pH, (pH - pH.), measured by pH-specific electrodes, versus the corresponding membrane pot e n t i a l measured by a conventional micropipette electrode. Only one value of pH. and E were taken from each c e l l . The c e l l s had been immersed I m i n one of the three test solutions for at least 2 hours before measurements were made. Acceptable values were those which varied no more than 0.04 pH units and +lmV over a 30 minute period. Almost a l l c e l l s tested were acceptable by t h i s c r i t e r i o n . The mean values of (pH e - pHj) and E m for the d i f f e r e n t c e l l types l i s t e d i n Table I I I are plotted as l e t t e r s A to G for comparison. In Fig. 29, from above downward, the continuous l i n e represents the Gibbs-Donnan r e l a t ion for hydrogen: pHg - pH-̂  — -(FE^)/(2.3RI) ; the upper dashed l i n e is the l i n e a r regression of the data for c e l l s i n normal Ringer's solution; and the lower dashed l i n e i s the l i n e a r regression for c e l l s i n CO2 Ringer's sol u t i o n . The l i n e a r regression for c e l l s i n NH^ Ringer's s o l u t i o n was s i m i l a r to that for c e l l s i n normal Ringer's so l u t i o n and i s not shown. The slope and y-intercept for each regression plot are l i s t e d i n Table V. Fig. 29 shows that the steady conditions for each t e s t s o l u t i o n do not conform to a Donnan equilibrium for hydrogen ions across the c e l l membrane, but they do show a dependency of (pH £ - pH^) on E m with a Donnan-type 211 Figure 29. Relationship between the l°g^Q °f the transmembrane hydrogen ion a c t i v i t y gradient and the r e s t i n g membrane p o t e n t i a l from s i n g l e c e l l s e q u i l i b r a t e d for more than 2 hours i n one of three s o l u t i o n s . F i l l e d c i r c l e s : normal Ringer's s o l u t i o n . Open t r i a n g l e s : NH^ Ringer's s o l u t i o n . Open squares: CO2 Ringer's s o l u t i o n . The two dashed li n e s are the regression li n e s for the normal Ringer's data and the C 0 2 Ringer's data. The l i n e for the NH^ Ringer's data is not shown. The upper continuous l i n e represents the Gibbs- Donnan r e l a t i o n . Note that a c e l l i s only represented once in this plot and no c e l l was included i f i t s i n t e r n a l pH was found to vary by more than 0.04 i n 30 minutes a f t e r a 2 hour incubation. C e l l membrane p o t e n t i a l and bath pH always varied less than pH.. Letters A - F represent data from other . workers summarized in Table I I I . 212 slope (-F/RT) TABLE V CALCULATION OF FLUX j m FROM DATA OF FIG. 29 Solution NH, Normal CO, No. of experiments Mean pH e S.E. 12 8.18 0.007 21 7.84 0.03 20 6.19 0.016 Slope (mV ) S.E. y - i n t e r c e p t * S.E. j m / P (mole-cm ^ H at E „ = -70mV) m ' y-intercept = log-^Q .m Vs1. •0.0140 0.004 -0.37 0.30 -0.0155 0.002 -0.52 0.14 1.1 x 10" L 1 3.0 x 10" 1 - exp((FE m)/(RT)) 11 (FE m)/(RT) -0.0165 0.005 -1.55 0.32 1.8 x 10"! DISCUSSION Absence of pH transients. Transient changes of pH^ have been found i n preparations of s n a i l neurone (Thomas 1974), squid axon (Boron & DeWeer 1976b), and barnacle muscle (Boron 1977) upon exposure of these c e l l s to NH^ and C O 2 solutions s i m i l a r to NH^ Ringer's and C O 2 Ringer's sol u t i o n . On the other hand, no such transient changes of pH^ were found i n the barnacle muscle c e l l preparation used here. The properties of the c e l l membrane in nerve might 213 be d i f f e r e n t from those i n muscle. The barnacle muscle c e l l preparation used by Boron (1977) was cut from the basis, exposed to calcium-free sol u t i o n , and cannulated at both ends. I t seems l i k e l y that t h i s treatment and perhaps the s l i g h t l y d i f f e r e n t solutions used have a l t e r e d the permea- b i l i t y properties of the c e l l membrane. For example, the membrane po t e n t i a l is rather low i n Boron's preparation. The technique of 'pH t r a n s i e n t s ' has revealed several new properties of the mechanism which regulates pH , notably the influence of i anion fluxes and e x t r a c e l l u l a r sodium. Because of the disagreement of the r e s u l t s i n intact c e l l s exposed only to p h y s i o l o g i c a l solutions before t e s t i n g , and the preparation of Boron (1977), i t i s not c e r t a i n to what extent the pH transients detected r e f l e c t the normal functioning of the c e l l . I t seems imperative that an attempt be made to c l a r i f y t his s i t u a t i o n , so the r e s u l t s obtained with t h i s p o t e n t i a l l y very useful new technique can be interpreted with confidence i n muscle c e l l s . (pH e - pHj) versus E m . The steady value of (pH e - pH^) from c e l l s i n three d i f f e r e n t condi- tions of a c i d i t y show a l i n e a r dependence on the membrane po t e n t i a l with a slope near F/(2.3RT), (Table V), the slope of the Donnan l i n e . Both Caldwell (1954, 1958) and Kostyuk and Sorokina (1961) searched for a r e l a - t i o n between pH. and E„ but were unable to e s t a b l i s h one. r l m Carter and co-workers (1967) observed a l i n e a r r e l a t i o n between (pH e - pH.j) and E m but t h e i r r e l a t i o n was d i f f e r e n t i n that a l l t h e i r data points followed the Donnan l i n e . Furthermore, Carter reported that whenever the membrane p o t e n t i a l was a l t e r e d the i n t r a c e l l u l a r pH changed almost instan- taneously to an appropriate value along the Donnan l i n e . Although no 214 deliberate attempt to change E m was made in the present experiments, these experiments provide numerous examples (eg. Figs. 27 & 28) which indicate that pH^ and E m often varied independently on a short time scale. A i c k i n and Thomas (1975) observed l i t t l e change i n pE^ for s n a i l neurone when the membrane p o t e n t i a l was increased for 10 minutes following modest changes i n the potassium content of the external s o l u t i o n . S i m i l a r l y , examination of the r e s u l t s on the sodium d i s t r i b u t i o n i n the c e l l s on which sodium flux studies reported in the preceding sections were done revealed l i t t l e depen- dence of (Na) m on E m- Recall that the sodium content of the c e l l s had been changed a r t i f i c i a l l y to obtain a range of values of (Na) m. The reason that d i f f e r e n t workers have sought a r e l a t i o n s h i p between (pH e - v R j ) and E m is that a dependency between the two i s predicted by the accepted theory for ion fluxes across membranes. Active ion transport also occurs, however, so the dependency might not be apparent i n the whole c e l l . A formal de r i v a t i o n of the r e l a t i o n s h i p between the steady transmembrane d i s t r i b u t i o n of cations and the membrane p o t e n t i a l i s presented i n section 2 . F ( i i ) . The r e s u l t for hydrogen ions i s where F, R, and T have t h e i r usual s i g n i f i c a n c e , ( H ) e = 10 e, and M i s a d e f i n i t e i n t e g r a l which contains the a c t i v e hydrogen ion transport (or i t s equivalent). No assumption has been made except that the d r i v i n g force for passive cation flux i s given by the Nernst-Planck equation and is indepen- dent of the a c t i v e flux. The r e s u l t (6) has been cast i n a form which corresponds to the equation for the l i n e s i n Fig. 29 to emphasize that quite elementary p r i n c i p l e s seem to predict such a r e l a t i o n s h i p . I f now the constant f i e l d assumption i s adopted and the a c t i v e flux . .. (6) -pH 215 j i s assumed to be constant, the i n t e g r a l M can be evaluated and (6) becomes PH -PH, = l o g i n [ l - - J - ! - / l - e x p ( F E m / ( R T ) ) \ ] - — 1 ! — E ...(11) 1 0 L ¥ H ) e \ FE m/(RT) 7j 2" 3 R T m where i s the permeability of the membrane to hydrogen ions. The term in square brackets corresponds to the y-intercept l i s t e d i n Table V. The term in square brackets in (11) cannot be expected to assume the same value during a l l steady conditions i n a given test solution, since i t contains E m and because J m/Pjj might vary with E m < However, since the slope of the p l o t for each test condition appears to be r e l a t i v e l y constant, and nearly equal to F/(2.3RT), (Fig. 29), i t follows that the y-intercept for a given test condition must be r e l a t i v e l y constant, at l e a s t for the range of membrane po t e n t i a l i n question (-45 to -90 mV). I t also- follows that j m / P j j must vary with E m at about an equal and opposite rate as the round bracket term varies, i f the constant f i e l d approximation i s taken at face value for the purposes of discussion. The c a l c u l a t i o n of parameters v i a the approximate equation (11) is semiquantitative, but i t provides a means of estimating the s i z e of j m i n the three test s i t u a t i o n s . In Table V are shown the values of j m / P j j for each s i t u a t i o n when E m i s -70 mV. An estimate for PJJ of 5 x 10"^ cm/sec has been provided by Woodbury (1971). The values i n Table V indicate that the hydrogen ion transport system i s required to function about two orders of magnitude faster when the i n t r a c e l l u l a r pH i s reduced from 7.2 to about 6.7 by immersion of the c e l l i n Ringer's s o l u t i o n . They also indicate that the hydrogen ion transport system i s not challenged when the i n t r a - c e l l u l a r pH i s raised to 7.6 by immersion of the c e l l i n NH^ Ringer's s o l u t i o n . In summary, i t has been found that following prolonged immersion of 216 the c e l l s i n the various test solutions, the transmembrane hydrogen ion d i s t r i b u t i o n i s r e l a t e d to the membrane po t e n t i a l but not i n accordance with a simple Gibbs-Donnan d i s t r i b u t i o n . A model which recognizes the existence of two independent net fluxes of hydrogen ions across the c e l l membrane was developed to account for the r e s u l t s . One of the fluxes represents passive movement and the other represents a c t i v e hydrogen ion transport or an equivalent process. Semiquantitative estimates from the model y i e l d an increase i n the a c t i v e hydrogen transport by two orders of magnitude when pH^ i s reduced from 7.2 to 6.7. In the absence of more information about the actual mechanisms which regulate the i n t r a c e l l u l a r pH, and about the passive' permeability of the membrane to hydrogen ions, nothing further can be gained from these r e s u l t s . 217 SECTION 9. COMPARISON OF THE INTRACELLULAR pH MEASURED BY DMO AND BY MICROELECTRODES The experimental study of hydrogen ion transport can only be c a r r i e d out by i n d i r e c t methods, a l l of which involve the measurement of changes i n the i n t r a c e l l u l a r pH, (pH^ (eg. Waddell & Bates 1969). Use of an i n t r a - c e l l u l a r pH-specific microelectrode i s probably the method of choice, e s p e c i a l l y for the measurement of the recently-discovered transient changes in pH^ which seem to be due to an act i v e process which regulates pH^ (Thomas 1974; Boron & DeWeer 1976b; Boron 1977). Of the several methods employed or proposed for measuring pH^ (Waddell & Bates 1969; Rose 1968; Moon & Richards 1973), the one most often used i s the 'weak e l e c t r o l y t e d i s t r i b u t i o n ' method, with the weak a c i d 5,5-dimethyl- oxazolidine-2,4-dione (DMO) as the indicat o r . Indicator methods y i e l d an estimate for pH^ which r e f l e c t s the pH of a l l compartments in the c e l l which are accessible to the indicator, and not a l l of these have the same pH. For example, mitochondria behave as i f they have a r e l a t i v e l y high pH (Garthwaite 1977). An i n t r a c e l l u l a r pH-specific microelectrode w i l l measure the pH i n the i n t r a c e l l u l a r s o l u t i o n which surrounds the s e n s i t i v e t i p , but i t i s t e c h n i c a l l y very d i f f i c u l t to construct such electrodes with s e n s i t i v e t i p s s u f f i c i e n t l y small for use i n most c e l l u l a r systems of in t e r e s t . Waddell and Bates (1969) noted that some of the uncertainties of the weak e l e c t r o l y t e method could be c l a r i f i e d by a study which combines the weak e l e c t r o l y t e and microelectrode methods i n the same c e l l u l a r system. The r e s u l t s of such a study (accounts of which have already been published - Menard, Nee, & Hinke 1975; Hinke & Menard 1976, 1978) are presented i n th i s section. A s i m i l a r study using barnacle muscle c e l l s was c a r r i e d out indepen- 218 dently by Boron and Roos (1976), and w i l l be discussed. A comparison of the DMO and e l e c t r o l y t e methods as applied to c e l l s of the plant N i t e l l a translucens has also been published (Spanswich & M i l l e r 1977). I t suffers from problems with ensuring that the s e n s i t i v e t i p of the electrode i s in t a c t and e n t i r e l y w i t h i n the i n t r a c e l l u l a r region of in t e r e s t , and with determining the cytoplasmic volume. I t w i l l not be discussed further. The r e s u l t s from the studies using barnacle c e l l s indicate that the DMO method can y i e l d a value for the pH inside the c e l l during steady condi- tions which is not too d i f f e r e n t from the microelectrode r e s u l t . However, some new d i f f i c u l t i e s with the DMO method were found. METHODS Two muscle bundles from a sin g l e barnacle were dissected as described i n s e ction 3. Both were then immersed in one of three test solutions (normal Ringers, CO2 Ringer's, or NH4 Ringer's solutions) for 2 hours at 23° C before measurements were begun. One bundle was used for microelec- trode measurements and one for a DMO uptake experiment. At the s t a r t of the DMO experiment, the bathing s o l u t i o n was a l t e r e d only by the addition of radioisotopes. Measurements with pH-specific microelectrodes and chemical analysis of the c e l l s were described i n section 8. The DMO technique w i l l be described in d e t a i l here. The base plate of the muscle bundle was anchored to a glass bar and the short tendon of each c e l l was anchored to another glass bar by means of s i l k threads during incubation. The c e l l s were more or less p a r a l l e l to 219 one another and approximately at t h e i r i n s i t u length. This assembly was then immersed in a continuously s t i r r e d 100 ml bath of Ringer's s o l u t i o n (one of the three above) to which was added (^C)DMO (to 300 disintegrations per minute (dpm)/yul, SA = 8.8 mc/mole) and ei t h e r (^H)inulin (to 1000 dpm/yul, SA = 122 mc/g) or ( 3 H ) s o r b i t o l (to 1000 dpm/yul, SA = 200 mc/mmole) . At various times over the next 3 hours, sin g l e c e l l s were cut away from the assembly, rinsed for 30 seconds i n i s o t o n i c sucrose wash s o l u t i o n (Table I ) , blo t t e d on f i l t e r paper and placed i n pre-weighed stoppered l i q u i d s c i n t i l l a - t i o n v i a l s . A f t e r recording the wet weight and constant dry weight, the amounts of and i n each c e l l were determined on two channels of the Mark II L i q u i d S c i n t i l l a t i o n Counting System (Nuclear-Chicago, Searle). A manually adjustable window was used for the H channel and a pre-set double-label window was used for the ^ C channel to maximize sin g l e channel counting e f f i c i e n c i e s and to minimize channels overlap. E f f i c i e n c i e s were determined by the external standards r a t i o method. The sixteen quenched standards (Amersham-Searle) contained H or X^C diss olved in the same s c i n t i l l a t i o n medium as the unknown samples. Every sample was counted for at least 20 minutes and a l l sample counts were stable. Some c e l l s were wet ashed and analysed for sodium and potassium content by flame photometry to check on the p h y s i o l o g i c a l state of the c e l l s . The bath was r o u t i n e l y analysed for 14-c, 3JI, sodium, and potassium at the beginning and end of each experiment. The pH of the bath was monitored continuously using commercial electrodes and a conventional pH meter. Calculations. F i g . 30 summarizes how DMO i s expected to cross the membrane and, once across, how i t is expected to behave. I t i s assumed that the non-ionized form of the weak acid (HDM0) crosses the membrane r e a d i l y and passively, 220 H + + DMO' tl HDMO bath cell H+ + DMO tl HDMO Figure 30. Diagram i l l u s t r a t i n g the c e l l u l a r permeation, e q u i l i b r a t i o n , and d i s s o c i a t i o n of a weak acid (HDMO), assuming only the non-ionized form of, the aci d passes the membrane. 221 and that a steady state i s eventually achieved when (HDMO) equals (HDMO)_. i e In fact, of course, the ionized form of DMO w i l l cross the membrane but only at a r e l a t i v e l y very slow rate. I t i s also assumed that the two aqueous phases are homogeneous and that HDMO i s able to d i s t r i b u t e i t s e l f uniformly i n each phase. F i n a l l y , i t must also be assumed that the d i s s o c i a t i o n constant, K', for HDMO is s i m i l a r i n the two aqueous phases. With these assumptions, Waddell and Butler (1959) proposed the existence of two d i s t i n c t i o n i z a t i o n e q u i l i b r i a f o r HDMO, one inside and the other outside the c e l l (Fig. 30). They combined the two mass law equations de f i n i n g the two e q u i l i b r i a to obtain the following expression for P H i s " P W : pR± = pK1 + l o g 1 0 . f£t f i + M - l Ce V V V i i 1 0 ( P H -PK') + J ! ...(12) where pH e i s the pH of the bath, i s the analysed c e l l water, V e i s the ex t r a c e l l u l a r water of the c e l l , = V t - V e, C t i s the t o t a l DMO content of the c e l l , and C £ i s the t o t a l e x t r a c e l l u l a r DMO of the c e l l . Waddell and Butler examined the p a r t i a l d e r i v a t i v e of the r a t i o C t/C e with respect to pH £ as an index of p r e c i s i o n when c a l c u l a t i n g pH^ from the weak e l e c t r o l y t e d i s t r i b u t i o n method. They recommended the use of DMO pa r t l y because of i t s inertness i n t r a c e l l u l a r l y and p a r t l y because the pK' of DMO was such as to y i e l d a high value for the p a r t i a l d e r i v a t i v e b (C t/ Ce> ' * P He In the present experiments where two radioactive labels were used simultaneously, C t and C g are of less i n t e r e s t than the seven parameters which were a c t u a l l y measured to obtain P H J J MQ. The Waddell and Butler equation was therefore rewritten as follows: 222 pH-DMO = pK1 + l o g 1 0 ^ A-i/ L B c l D : H 1 0(pH e-pK') + l (13) where = ( ( 3 H ) i n u l i n ) e i n dpm per unit bath volume, B c = ((^C)DMO) e i n 3 dpm per unit bath volume, D H i s t o t a l H (dpm) i n a blotted f i b r e , and D c i s t o t a l 1 4 C (dpm) i n a blotted f i b r e . This was the equation used to c a l c u l a t e a pH D MQ value for every c e l l exposed to the two isotopes regard- less of how long the c e l l was i n the e q u i l i b r a t i n g medium. pK' for DMO. The ( 1 4C)DM0, ( 3 H ) i n u l i n , and ( 3 H ) s o r b i t o l were obtained from New England Nuclear at the s p e c i f i c a c t i v i t i e s stated above. Non-radioactive DMO was obtained from Eastman Organic Chemicals. The l a t t e r was used to determine a pK1 value for DMO at 23° C and i n the presence of half-molar NaCl, corresponding to the uptake experiment. NaOH (C02 _free) was used to t i t r a t e the acid form of DMO at two concentrations (0.5 and 1.0: mM), both in the absence and i n the presence of 0.573 M NaCl. The NaOH (C02 _free) was obtained by t r e a t i n g a f r e s h l y prepared AgOH p r e c i p i t a t e with a purged NaCl solution: the f i n a l concentration was tested by t i t r a t i o n of potassium hydrogen phthalate. The conductimetric end point of the DMO-NaOH t i t r a t i o n i n the absence of NaCl was determined f i r s t and then the t i t r a t i o n curve for the same volume of solu t i o n , containing the same amount of DMO, plus NaCl to 0.573 M was obtained, using a Radiometer T i t r i g r a p h . The r e s u l t i n g mean pK1 values are 6.170 (SD of an observation = 0.032, n = 4) i n the presence of 0.573 M NaCl and 6.443 (SD of an observation = 0.051, n = 4) i n the absence of NaCl. The former value was used i n equation (13) for a l l pH(DMO) ca l c u l a t i o n s . For comparison, Boron and Roos (1976) give a pK1 value of 6.18 at 25° C for DMO i n "barnacle sea water 1 and a pK' value of 6.33 at 25° C for DMO i n water. 223 RESULTS . Course of the uptake of DMO and i n u l i n . The time course of penetration for the e x t r a c e l l u l a r space marker 3 ( ( H)inulin) into the sin g l e c e l l can be expressed as the r a t i o (Vg/V t) of the calculated volume of the e x t r a c e l l u l a r space to the t o t a l c e l l water content (assuming water density is 1 g/cm i n both cases). This quantity is s u i t a b l e for comparison between d i f f e r e n t c e l l s . The same c a l c u l a t i o n 14 can be done for the ( C)DMO marker, to y i e l d a space which has no s i g n i f i - cance as a volume but which is useful as a c e l l - s p e c i f i c index for compari- son of the DMO content i n d i f f e r e n t c e l l s . Ideally, when the uptake of (^C)DMO or ( " % ) i n u l i n i s calculated i n th i s manner and plotted against time of exposure to the isotopes, the r e s u l t i n g curve for each isotope should show a fast i n i t i a l r i s e from zero to a value which remains constant with time. Actually, what one finds is a fast i n i t i a l r i s e followed by a slow sustained r i s e which i s nearly l i n e a r with time. In Fig. 31, for example, are shown the curves for the uptake of (^CJDMO and (̂ H) i n u l i n by the c e l l s of one bundle (after 2 hours of immersion i n non-radioactive normal Ringer's solution) placed at time zero into the normal Ringer's s o l u t i o n containing the two isotopes. The slow l i n e a r r i s e i n both these curves is usually interpreted as the r e s u l t of some non-saturable non-specific adsorption and not as slow entry of the isotopes into regions to which access i s d i f f i c u l t . This i n t e r p r e t a t i o n is supported by the fin d i n g that the add i t i o n of 1 mM unlabelled DMO (more than 65 times the concentration of (^C)DMO) to the Ringer's solution, both before and during uptake, did not produce any change i n the slopes of the 'plateau' of the (•'-̂ C)DMO or ( 3H) i n u l i n uptake curves. The slow uptake for ( C)DMO and ( H ) i n u l i n , as i n Fig. 31, was rather s i m i l a r i n a l l ten 224 Figure 31. Uptake of the i n d i c a t o r compounds with time by c e l l s i n normal Ringer's s o l u t i o n a f t e r 2 hours of pre-incubation i n non-radioactive Ringer's s o l u t i o n . The data are from one muscle and each entry i s from a separate c e l l . Triangles: i n u l i n . Open c i r c l e s : DMO. F i l l e d c i r c l e s : calculated pH. for each c e l l , from equation (13). Continuous l i n e s are plotted from a 1 l i n e a r regression and dashed l i n e s are drawn by eye. 225 experiments but an inspection of Figs. 32 and 33 shows that the 'plateau' slopes for ( C)DMO and ( H ) i n u l i n can be opposite to one another. This indicates that the adsorptions of DMO and i n u l i n are quite independent of one another. I n t r a c e l l u l a r pH from DMO. The double isotope technique permitted the c a l c u l a t i o n of a pH(DMO) for every c e l l removed from the bath (using equation (13)). For example, the i n t r a c e l l u l a r pH(DMO) points i n the upper curve of Fig. 31 were obtained from the (l^C)DMO and (^H)inulin points of the two uptake curves of the same figure. I t can be noted that a l l of the pH(DMO) values are rather s i m i l a r , i.e. they change very l i t t l e with uptake time. Even during the f i r s t 10 minutes when (14-c)DM0 and (̂ H) i n u l i n are taken up rather r a p i d l y , the calculated pH(DMO) i s s i m i l a r to the pH(DMO) values for the slow r i s e period (times greater than 45 minutes). The pH of the bath for this experiment remained constant at 7.79. An example of double isotope uptake during low pH conditions is shown in Fig. 32, and an example during high pH conditions is shown i n Fi g . 33. For a l l three conditions the pH(DMO) curve c o n s i s t e n t l y declined l i n e a r l y with time and i t does not appear to be r e l a t e d to the uptake behavior of either one of the two isotopes. A l l the (^C)DMO uptake points i n Fig. 32 were divided by a factor of 5.5 i n order to present the data of the low pH experiment i n a more compact form. The fact that i t was necessary to do th i s i l l u s t r a t e s that a great deal more neutral DMO i s a v a i l a b l e for i n t r a c e l l u l a r penetration under low pH conditions than under normal or high pH bath conditions. For example, the concentration of neutral DMO (pK = 6.17) should increase sixteen times when the bath pH i s lowered from 7.66 to 6.20. 226 Figure 32. Uptake of i n d i c a t o r compounds with time by c e l l s i n C0 2 Ringer's s o l u t i o n a f t e r 2 hours of pre-incubation i n non-radioactive CC>2 Ringer's s o l u t i o n . Symbols are as i n F i g . 31. Note: a l l of the DMO uptake values (open c i r c l e s ) were divided by 5.5 to f a c i l i t a t e p l o t t i n g . 227 78r Ql I I 1 : 1 1 1 0 60 120 180 Time (min) Figure 33. Uptake of i n d i c a t o r compounds with time by c e l l s i n NH^ Ringer's s o l u t i o n a f t e r 2 hours of pre-incubation i n non-radioactive NH^ Ringer's s o l u t i o n . Symbols are as i n F i g . 31. 228 I n t r a c e l l u l a r pH from microelectrodes. It was shown i n the preceding section that the i n t r a c e l l u l a r pH measured with the microelectrode was very steady a f t e r two hours of immer- sion i n any of the three test solutions (Figs. 28 & 29). Comparison of pH(DMO) and pHi r e s u l t s . Two methods were adopted to compare the pH(DMO) values with the pR\ (microelectrode) values i n the three t e s t conditions. In one (Fig. 34a), the pH(DMO) value at zero time (obtained by extrapolation to zero time of the regression l i n e through the points a f t e r 45 minutes of the pH(DMO) . curve) i s compared to the mean pH^ for microelectrode measurements on companion c e l l s selected as described above. In the second method (Fig. 34b), the mean of a l l pH(DMO) values a f t e r 45 minutes is compared to the same mean pH^. The 'extrapolated' pH(DMO) value i n Fig. 34a should be independent of a r t i f a c t s due to non-specific adsorption of the two indica- t o r s . The second method is s i m i l a r to the common pract i c e of assuming that DMO uptake reaches a sing l e constant value a f t e r 1 hour. Only complete experiments are represented i n Fig. 34a and 34b, i. e . a f u l l double isotope uptake experiment combined with successful microelectrode impalements of at le a s t three and usu a l l y f i v e c e l l s . I t can be noted that the 'extrapolated' pH(DMO) value i s nearly always higher than the mean pR\ value. For example, the regression l i n e (continuous) through the points predicts a pH(DMO) of 7.18 when pHL i s 7.00. Since the regression l i n e is nearly p a r a l l e l to the i d e n t i t y l i n e (dashed), one can expect the 'extrapolated' pH(DMO) values to y i e l d c o r r e c t l y the change i n i n t r a c e l l u l a r pH. I t can be noted from Fig. 34b, however, that the regres- sion l i n e for the 'plateau' pH(DMO) values crosses the i d e n t i t y l i n e . I t is u n l i k e l y , therefore, that measured changes i n 'plateau' pH(DMO) would 2 2 9 8.0I- 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 A - P H ( D M O ) C A L C U L A T E D BY E X T R A P O L A T I N G TO t=o B - PH(DMO) C A L C U L A T E D F R O M D A T A AFTER 45 M I N . 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 p H ( E L E C T R O D E ) Figure 34. Corr e l a t i o n l i n e s between pH(DMO) and pH(electrode) from c e l l s on which pH. was measured by the DMO and the pH microelectrode methods. For l i n e A, the1pH(DM0) was calculated by extrapolation to zero time of data taken a f t e r 45 minutes. For l i n e B, the pH(DMO) was calculated as the mean of a l l points a f t e r 45 minutes. The dashed l i n e indicates equality. 230 c o r r e c t l y r e f l e c t the r e a l changes i n i n t r a c e l l u l a r pH. DISCUSSION DMO technique. Fig. 31 revealed that the calculated values of pH(DMO) are s i m i l a r on magnitude even though the amount of l a b e l l e d indicator varied considerably with time. In equation (13) (or (12)), there are seven parameters each of which i s measured with a c e r t a i n degree of uncertainty, yet the calculated P%)M0 values do not r e f l e c t the degrees of uncertainty of the seven parameters i n an obvious way. To obtain a rough i n d i c a t i o n of how a change i n each of the seven parameters might a f f e c t p H D M 0 > the p a r t i a l d e r i v a t i v e of pH D MQ with respect to each parameter X^ at a t y p i c a l point i n the 'plateau' region (time greater than 45 minutes) for c e l l s under normal, acid, and a l k a l i n e conditions was evaluated. The change i n PHJ^Q, SPHQMQ, due to changes i n the X^ parameters, &X^, can be estimated to f i r s t order from the expression: SpHDM0 = ^ ^PHDM0 CX- -..(14) The s e n s i t i v i t y of pH^Q to a change i n X^ is indicated by £ (PH^Q)/«) X^, but i t i s more informative to consider the f r a c t i o n a l change, 5X./ X., of each parameter which would produce a small but important change i n P%)M0' i , e - a n increase of 0.05. The r e s u l t s of t h i s exercise are summar- ized i n Table VI. Also included, as the f i r s t column of this table, are the estimations of maximum uncertainty i n the measurement of the seven 231 TABLE VI SENSITIVITY OF pH(DMO) TO ERRORS IN MEASUREMENT Normal A c i d i f i e d A l k a l i n i z e d Estd. ?X± P X - L P X ± 2X± 2X± ^ uncertainty ^ ~^7 ~x"7 Vt(mg) 0. 1% -0. 95 87o -0. 52 57, -0. 87 87o PHE 0. TL 0. 04 0.57o 0. 07 17o 0. 04 0.57o pK 0. 5% 0. ,35 67o -0. ,20 37o 0. 53 970 DH(dpm) 1. 51 -354 247o 919 367o -168 147o D^(dpm) 1. 5% 77 6 7 o 272 67o 38 57» BH(dpm/A) 2-•3% -175 197o -200 257» -147 157o Bc(dpm/A) 2-•3% -17 67o -16 67o -14 57o Notes: (a) The density of water was taken to be 1 g/ml. Therefore, 1 mg = U (b) The values of the seven parameters i n a given c a l c u l a t i o n were taken from the 15th f i b r e of each experiment. For example, the values of the parameters for the normal f i b r e were: V^ = 12.035 mg, pH e = 7.73, pK' = 6.17, D R = 1456 cpm, D c = 1293 cpm, Bjj = 937 cpm/* , B c = 285 cpm/A . parameters. The most r e l i a b l e parameter is pH £. Notice, however, that pH g need only change by 0.57, to e f f e c t an increase of 0.05 i n P H J J ^ Q . I n contrast, there can be a 247, change i n D^., which i s a measure of the e x t r a c e l l u l a r space of the c e l l , before pBpMQ changes by 0.05. This fin d i n g suggests that r e l i a b l e extinctions of P H J ^ Q can be expected i n experiments were mean e x t r a c e l l u l a r space determinations are made, rather than ' i n d i v i d u a l ' determinations as i n th i s study. Since pH^^Q i s so sen s i t i v e to pH £, the l a t t e r should be monitored continuously i n an experi- 232 merit to enable the experimenter to maintain a chosen value or to match the value of pH e at a given time to the other parameters at that time for a given c a l c u l a t i o n . The data i n Table VI also show that pK' can increase by 67o i n normal Ringer's s o l u t i o n before an increase i n pH D MQ of 0.05 occurs. This estimate i s , of course, based on the assumption that the two pK' entries i n equation (13) are i d e n t i c a l , or, put another way, that the i n t e r n a l and external pK' values for DMO are i d e n t i c a l . I f pK' (DMO) were 6.5 or greater, i t s precise value would have to be taken more s e r i o u s l y (although the other parameters would be d i f f e r e n t as w e l l ) . DMO was recommended as an ind i c a t o r for pRi p a r t l y because of i t s pK' (Waddell & Butler 1959). The more d i f f i c u l t question, whether pK'^ = pK' e remains unanswered although there i s some evidence which suggests that pK^ i s very close to pK'e- F i r s t , Boron and Roos (1976) have shown that pK(DMO) i s not s e r i o u s l y a f f e c t e d by ionic strength or by a change i n e l e c t r o l y t e from NaCl to KCL. Second, Hinke (1970) has shown that -most of the i n t r a c e l l u l a r water in muscle behaves l i k e e x t r a c e l l u l a r bulk water in i t s solvent properties and that the a c t i v i t y c o e f f i c i e n t for cations i n the i n t r a c e l l u l a r aqueous phase is s i m i l a r i n magnitude to the ioni c a c t i v i t y c o e f f i c i e n t i n barnacle Ringer's s o l u t i o n . The barnacle c e l l l i k e other muscle c e l l s does, of course, contain c o n t r a c t i l e protein filaments with a number of fixed negative charge s i t e s which can act as a proton sink, but i t is d i f f i c u l t to imagine how such negative charge s i t e s could a l t e r the DMO d i s s o c i a t i o n process i n a way other than how one might imagine an a l t e r a t i o n i n DMO di s s o c i a t i o n to be caused by a buffer with a reasonable capacity, i n the external medium. F i n a l l y , i t is inconsistent to worry about possible f a l s e a c t i v i t i e s of HDMO and DMO i n myoplasm which might produce a d i f f e r e n t pK!̂ without also worrying about the true value of V for DMO which should be 233 used i n equations (12) and (13) rather than V t (Hinke 1970). Two more points can be made from the data i n Table VI. F i r s t , the r e l a t i v e weight of each parameter for the c a l c u l a t i o n of PHJ^Q does not change when the c e l l i s under acid or a l k a l i n e conditions. Second, the p a r t i a l derivatives of pH^Q with respect to some of the parameters have negative signs. Of p a r t i c u l a r i n t e r e s t are the opposite signs of the p a r t i a l d erivatives with respect to D„ and By, because these are most relevant to the uptake curves of Fig. 31. During the f i r s t 30 minutes of uptake, both and are increasing appreciably, yet the calculated values of pH^Q in thi s period are a l l s i m i l a r i n magnitude and close to pH^Q values obtained a f t e r 45 minutes, when the changes i n D R and are smaller. I t appears that the parameters D^ and i n equation (13) are so placed as to s t a b i l i z e the PHDMO c a l c u l a t i o n during the double-isotope experiments. I f this reasoning i s v a l i d , then i t may be used as a good reason to advocate the use of the double-isotope technique even though, as was pointed out e a r l i e r , the technique does not seem warranted i f one looks only at the r e l a t i v e l y weak dependence of pHjj M 0 o n °H (Table VI). An id e a l volume marker should produce an uptake curve which r i s e s r a p i d l y at f i r s t and then quickly bends to a hori z o n t a l plateau. In Fig. 31 14- 3 i t i s shown that neither ( C)DM0 nor ( H ) i n u l i n behaved i n such an ide a l manner. Instead, both markers produced s l i g h t l y i n c l i n e d plateaus a f t e r 45 minutes and up to 180 minutes, which suggests that e i t h e r an equilibrium i s not attained, or that each marker i s involved i n a non-saturable non- s p e c i f i c adsorption process. To obtain an independent measure of the rate of passage of ("*"̂ C)DM0 across the c e l l membrane, (^C)DMO was microinjected into the myoplasm of an int a c t c e l l . The e f f l u x of the radioisotope was observed to occur with a rate constant of 0.09 min"''". With such a rapid transmembrane fl u x rate, i t can r e a d i l y be calculated that DMO should reach 234 a steady state across the membrane within minutes. Thus, the slowly r i s i n g l i n e a r plateau of the (•*"̂ C)DMO uptake curve cannot be a t t r i b u t e d to a f a i l u r e to achieve a steady state. ( H ) i n u l i n was also injected into a c e l l and i t was observed that the radioisotope did not cross the membrane. From t h i s r e s u l t one can conclude (a) that the rapid e f f l u x of ( 1 4C)DM0 was not due to injury to the membrane following microinjection, and (b) that the slowly r i s i n g l i n e a r plateau of the ( H ) i h u l i n uptake curve could not be explained by. a slow penetration of ( 3 H ) i n u l i n into the c e l l . To i n v e s t i - gate the slow r i s e further, several muscle c e l l s were preincubated i n a bath containing a r e l a t i v e l y high concentration of non-radioactive DMO (more than 65 times that of (•*"̂ C)DMO). In these experiments, no change i n the slope of the slowly, r i s i n g plateau was observed on either the (^C)DMO 3 curve or on the ( H ) i n u l i n curve. In other experiments, the uptake curves 3 of ( H ) s o r b i t o l with and without preincubation i n unlabelled s o r b i t o l (5 mM) were examined and i n both cases a slow uptake was ^observed, just as with i n u l i n . I t has not been possible to explain the uptake curves for c e l l s which were treated with CO2 or NH^ Ringer's solution. They do not r e f l e c t actual changes i n pH^, since the microelectrode measurements reveal that pH^ was steady under the conditions of the DMO experiment. I t is not l i k e l y that the DMO was transported or metabolized by the c e l l . It bears repeating that the use of CO2 and NH^ solutions to a l t e r pR\ i s a commonly used technique. pH(DMO) versus microelectrode measurements. The value for the i n t r a c e l l u l a r pH obtained with the pH-specific glass microelectrode is assumed/to be the best measurement of this quantity. I t r e f l e c t s the pH i n the i n t r a c e l l u l a r s o l u t i o n which surrounds the s e n s i t i v e 235 t i p of the microelectrode. In the barnacle muscle c e l l , there are few i n t r a c e l l u l a r compartments, as discussed i n section 2.B. The d i s t r i b u t i o n of the indicator DMO can reasonably be expected to be dominated by the properties of the large myoplasmic compartment. Since i t i s the properties of t h i s compartment that the microelectrode measures, the accuracy of pH(DMO) i n barnacle muscle c e l l s is revealed by the closeness of i t s value to the value measured with the microelectrode. I t has been found that the best value obtained from the DMO d i s t r i b u - t i o n , the 'extrapolated' pH(DMO), i s co n s i s t e n t l y 0.1 to 0.2 units higher than pIL ( i . e . pH(microelectrode)). As discussed i n section 2, this might at f i r s t glance be taken to r e f l e c t the existence of r e l a t i v e l y a l k a l i n e i n t r a c e l l u l a r compartments. By comparison, Boron and Roos (1976), using s i m i l a r electrodes but the 'plate'au' value of pH(DMO), reported that pH(DMO) was smaller by about 0.05 than the values of 7.26 to 7.35 measured with the microelectrode for the cells'when i n 'barnacle sea water'. (In the present study, mean pH^ for the c e l l s when i n normal Ringer's s o l u t i o n was 7.23 - Table III.) They suggested three explanations for the small values of pH(DMO): ( i ) the existence of a low i n t r a c e l l u l a r pK' value for DMO; ( i i ) an appreciable permeability of the c e l l membrane to ionized DMO; ( i i i ) the presence of an a c i d i c i n t r a c e l l u l a r compartment. Actually, the use of the 'plateau' pH(DMO) could by i t s e l f account for the s l i g h t discrepancy between t h e i r r e s u l t s and those reported here. In l i g h t of the preceding discussion, however, i t is d i f f i c u l t to ascribe much s i g n i f i c a n c e to the small difference between the best values for pH(DMO) and pH(microelectrode). Uncertainties i n the DMO method i n - volving the neglect of 'nonsolvent water 1, the assumption of equality of pK^ and pK^, the possible permeability of the c e l l membrane to ionized DMO, 236 and the non-ideal uptake behavior of the indicators would have to be resolved before such a discrepancy could p r o f i t a b l y be examined i n d e t a i l . The most important conclusion to be drawn from these studies is a p r a c t i c a l one. pH(DMO) can give an estimate of the i n t r a c e l l u l a r pH accurate to about 0.2 units, and a rather better estimate of the difference i n the value of pR\ during d i f f e r e n t steady conditions, i f 'extrapolated' pH(DMO) i s evaluated. The DMO technique should be of l i t t l e use i n the measurement of transient changes i n pH, although i t s use for such a purpose has been reported (Roos 6c Boron 1978). 237 SECTION 10. SIGNIFICANCE OF THE RESULTS AND SUGGESTIONS FOR FUTURE WORK The work on sodium fluxes reported in t h i s thesis f a l l s into three parts: the formulation of a model for measuring sodium e f f l u x , the t e s t i n g of the model, and the a p p l i c a t i o n of the model to two s p e c i f i c problems. A p r a c t i c a l model for the c a l c u l a t i o n of the u n i d i r e c t i o n a l e f f l u x of sodium from the myoplasm was devised. The myoplasm bathes the inner surface of the c e l l membrane, and the sodium a c t i v i t y i n i t can be measured d i r e c t l y with a sodium-specific glass microelectrode. The new r e s u l t which made a simple formulation of the model possible was that the nonmyoplasmic sodium does not appear to exchange appreciably with the myoplasmic sodium, at l e a s t during the washout of c e l l u l a r sodium into a lithium-substituted sodium-free s o l u t i o n . I t i s not easy to reconcile this r e s u l t with the existence of a great deal of 'loosely bound 1 sodium associated with the c e l l u l a r proteins, although there i s good experimental evidence that such sodium e x i s t s . Further work should be done on the s i z e and the l o c a t i o n of the mobile and immobile fractions of c e l l u l a r sodium. In any case the r e s u l t s of the present experiments can be interpreted consistently i n the model that: some c e l l u l a r sodium is free i n s o l u t i o n i n the myoplasm, as envisaged i n the early models; a r e l a t i v e l y small amount i s 'bound loosely' to f i x e d charges inside the c e l l , as envisaged i n more recent models; some i s highly mobile and outside the c e l l membrane, a f r a c t i o n acknowledged but not q u a n t i f i e d previously; and f i n a l l y some i s nonparticipatory as far as these experiments are concerned, and probably i s purely ' s t r u c t u r a l ' i n the sense that i t has l i t t l e to do with the ion movements which are so v i t a l to the moment-to-moment l i f e of the c e l l . Most of the l a t t e r f r a c t i o n i s probably located outside the c e l l membrane, i n the glycocalyx. Each of the fractions w i l l probably be found to be heterogeneous when characterized i n terms other than j u s t mobility. 238 The r a t i o n a l e for the new technique (the combination of sodium-specific microelectrode and radiosodium measurements) was straightforward. The f r u i t s of i t are better measurements of the 'true' f l u x . Just as the use of a ro t a t i n g frame of reference i n mechanics gives r i s e to complicated f i c t i t i o u s forces, so errors such as incor r e c t estimation of the myoplasmic a c t i v i t i e s or neglect of the lo n g i t u d i n a l d i f f u s i o n of an in j e c t e d marker substance give r i s e to ' f i c t i t i o u s fluxes' and complicated models. Once the f i c t i t i o u s fluxes were eliminated from the sodium e f f l u x measurements for barnacle muscle c e l l s , i t was found that the notion of compartments which exchange ions i n a complicated fashion v i a many d i f f e r e n t transport systems need not be invoked yet. There i s s t i l l progress to be made by using simple models c o r r e c t l y . The k i n e t i c c h a r a c t e r i s t i c s of the sodium e f f l u x were interpreted i n the usual manner, as r e f l e c t i n g a chemical r e a c t i o n mediated by a membrane-bound p r o t e i n - l i p i d enzyme complex. Some of the e f f l u x can be ascribed to the (Na+K)ATPase, on the basis of the response to ouabain. Further, the s i m i l a r i t y of the response to ouabain and to potassium-free s o l u t i o n implies that i t i s only v i a the sodium-potassium exchange mode that the (Na+K)ATPase makes an appreciable contribution to the active sodium e f f l u x i n barnacle muscle c e l l s . I f this were so, a d i f f e r e n t mechanism for active sodium transport, i n s e n s i t i v e to ouabain and (perhaps) not re q u i r i n g external potassium, would have to be postulated. What seem better working hypotheses are that not a l l of the i n s i t u (Na+K)ATPase can be affe c t e d by ouabain, or that the binding of ouabain to an enzyme i n s i t u does not stop i t from transporting ions, contrary to the case with the i s o l a t e d enzyme. Thus, the k i n e t i c c h a r a c t e r i s t i c s of the sodium e f f l u x i n the presence of d i f f e r e n t concentrations of ouabain could be analyzed i n the context of the established models for the various possible actions of i n h i b i t o r s of enzyme-mediated reactions. The results obtained with ouabain are at once i n t r i g u i n g and f r u s t r a t i n g . 239 Because so much ground had to be covered i n order to make the complicated new technique for flux measurement p r a c t i c a l , because the time a v a i l a b l e to do the experiments was l i m i t e d by the constraints of the M.D.-Ph.D. program, and because the thesis supervisor moved to a d i f f e r e n t u n i v e r s i t y , i t was not possible to do as many experiments as were desired. A major part of future work w i l l be on the cha r a c t e r i z a t i o n of the ouabain-sensitive and - i n s e n s i t i v e components of the e f f l u x . A complication which appears to have been resolved i s the e f f e c t of e x t r a c e l l u l a r sodium on the sodium e f f l u x . I t had been known that replacement of the sodium i n the e x t r a c e l l u l a r s o l u t i o n with some other species could cause an increase i n the sodium e f f l u x from nerve or muscle c e l l s of high sodium content. A sodium-sodium exchange not mediated by the (Na+K)ATPase (or at least, not s e n s i t i v e to ouabain) and prominent only when the i n t r a - c e l l u l a r sodium concentration was low, was postulated. The res u l t s reported here for barnacle muscle c e l l s are consistent with a much simpler explanation. The rate of active sodium transport i s governed almost e n t i r e l y by the i n t r a - c e l l u l a r sodium concentration under normal conditions. Removal of external sodium causes a decline i n the i n t r a c e l l u l a r sodium concentration, and consequently a decline i n the sodium e f f l u x . In addition, e x t r a c e l l u l a r sodium appears to be able to modify s l i g h t l y the response of the sodium transport mechanism to the i n t r a c e l l u l a r sodium concentration. At a given i n t r a c e l l u l a r sodium concentration the sodium e f f l u x i s greater when the external sodium i s replaced by a large cation. This i s a synthesis of hypotheses put forward by other workers i n a s i m i l a r context. This explanation i s consistent with the res u l t s reported by other workers for nerve and muscle c e l l s . An inco r r e c t i n t e r p r e t a t i o n of radio- sodium flux experiments has been shown here to be the cause of what appears to be a longstanding misconception concerning "sodium-sodium exchange". The use of the new technique reported here, i n which care i s taken to determine 240 the actual magnitude of the sodium flux, revealed the true behavior of the sodium e f f l u x , although i n retrospect the r e s u l t could have been deduced from the microelectrode experiments published by other workers. I t i s not claimed that sodium-sodium exchange does not occur, but rather that the contribution i t makes to the sodium e f f l u x i n muscle c e l l s i s much smaller than had been thought. The new r e s u l t s lead to a refinement of the current models concerning the sodium d i s t r i b u t i o n and movements i n c e l l s . The new technique also y i e l d e d a nice demonstration that electrogenic sodium transport occurs i n barnacle muscle c e l l s . This c e r t a i n l y i s not an unexpected finding, but i t had not been shown convincingly before. More important are the quantitative r e s u l t s of these experiments. Measurement of the absolute value of the sodium e f f l u x i s e s s e n t i a l i f a quantitative c o r r e l a - t i o n between the changes i n sodium e f f l u x and membrane p o t e n t i a l i s to be obtained. Such a c o r r e l a t i o n i s important for the evaluation of mechanistic models of ion movement, as i t can provide an independent measurement of parameters. A model was derived here, by making the simplest possible extension from the current successful model for the r e s t i n g membrane p o t e n t i a l . The d e r i v a t i o n presented was intended to express the basic physical process which appears to give r i s e to the electrogenic p o t e n t i a l . Approximations were employed i n order to obtain a s i m p l i c i t y of form, because the data obtained so far do not admit to d e t a i l e d a n a l y s i s . (As i t turned out, however, the quantitative r e s u l t s were quite good.) The way i s indicated for an i n v e s t i g a - t i o n of the d e t a i l e d c o r r e l a t i o n between the flux and the membrane p o t e n t i a l . This w i l l permit modelling of the actual mechanism of the transport process to be done and tested. The work on the hydrogen ion reported here, which a c t u a l l y was done as a b r i e f project before the work on sodium, y i e l d s a p r a c t i c a l r e s u l t and presents two puzzles. One puzzle concerns the DMO technique. How can i t occur that when the c e l l i s a c i d i f i e d with C0 o or a l k a l i n i z e d with NH_, the DMO i s f i r s t 241 taken up but then i s expelled? The i n t r a c e l l u l a r pH measured by the micro- electrode remains quite stable i n these s i t u a t i o n s . The very slow a c i d i f i c a t i o n following the a l k a l i n i z a t i o n by NH^ i s i n agreement with Roos' model of NH^ penetration (Roos 1965), and the absence of a l k a l i n i z a t i o n following the a c i d i f i c a t i o n by CO^ i s probably due to the low concentration of HCO^ used. The c e l l s do not gain or lose water. The i n d i c a t o r i s not metabolized. That the e f f e c t occurs i n both a c i d and a l k a l i n e conditions implies that i t i s due to the techniques rather than to some physicochemical process, yet only the standard techniques were employed. This question demands further i n v e s t i g a t i o n . The second puzzle concerns the r e l a t i o n s h i p between the transmembrane pH difference and the membrane p o t e n t i a l . As was discussed i n section 8, an elementary model predicts the e f f e c t seen, and analysis of the techniques used revealed no source of er r o r . The fact remains, however, that other workers have sought and f a i l e d to f i n d this e f f e c t for pH. Further i n v e s t i g a - t i o n of t h i s e f f e c t , for other ions as w e l l as for hydrogen, i s c e r t a i n l y indicated. I t might be that the pH-sensitive p o t e n t i a l difference measurable i n glycerinated c e l l s i s involved i n this e f f e c t . The p r a c t i c a l r e s u l t i s the demonstration that c a r e f u l use of the weak-electrolyte (DMO) method can y i e l d accurate measurements of changes i n the i n t r a c e l l u l a r pH between states of the c e l l i n each of which conditions are steady. 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