Passive and active calcium fluxes across plasma membranes

ProO. Biophys. molec. Biol., Vol. 35, pp. 135-195 © Pergamon Press Ltd. 1980. Printed in Great Britain

0079-6107/80/05014)135505.00/0

PASSIVE A N D ACTIVE CALCIUM FLUXES ACROSS PLASMA MEMBRANES P. V. SULAKHEand P. J. ST. LOUIS Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO

CONTENTS

I. INTRODUCTION

136

II. SPECIFIC TISSUE STUDIES

138 138 138 139 139

1. Axons (a) Na+-dependent Ca 2+ effiux (b) ATP-indaced aOinity changes (f) Ca2+-dependent Ca 2+ e ~ u x (d) A TP-dependent Ca 2 + e.~ux (e) Summary 2. Brain (a) .Na +-dependent Ca 2 + efllux (b) Studies on isolated membranefractions (i) Isolation of synaptic plasma membranes (SPM) (ii) Ca 2 +-stimulated A TPase (iii) Ca 2+ -transport activity (ATP-dependent) (iv) lntrasynaptosomal ATP-dependent Ca2+-storaoe systems (v) Microsomal Ca 2 +-transport system (vi) Modulation of Ca 2+ transport and Ca 2 +-A TPase by calmodulin in brain membrane fractions (vii) Summary of results from isolated membrane fractions 3. Cardiac Muscle (a) Tissue studies (i) Na + effects on Ca 2 + fluxes (ii) Na+-dependent Ca 2+ efl/ux Off) Effects of cardiac glycosides (iv) Active (ATP-dependent) Ca 2+ transport (b) Isolated membrane studies (i) Isolation of cardiac sarcolemma (ii) Ca2 +-stimulated A TPases in cardiac sarcolemma (iii) Calcium bindino to sarcolemma (iv) Implications of the type of membrane preparation used in the study of A T P-dependent Ca 2+ transport (v) Na+--Ca 2+ exchange in isolated membrane vesicles (vi) Monovalent cation effects on the Ca 2+ pump of sarcoplasmic reticulum (vii) Phosphorylation of cardiac sarcolemma (c) Does N a + - C a 2+ exchange carrier mediate Ca 2+ influx into the myocardial cell? 4. Smooth Muscle (a) Na+-dependent Ca 2+ eOtux (b) A TP-dependent Ca 2+ efflux (c) Studies on isolated plasma membrane-enrichedfractions 5. Skeletal Muscle (a) Ca 2 + efltux studies with isolated skeletal muscle preparations (b) Calcium transport studies with isolated surface membranefractions (c) Ca2+-ATPase/Mg~+-ATPaseofskeletalmusclesarcolemma (d) M g 2 +, Ca 2 +-ATPase and A TP-dependent Ca 2 +-transport system of skeletal muscle surface membrane (e) Comparative biochemical analysis of membranesfrom skeletal muscle involved in the excitationcontraction coupling (f) Phosphorylation of skeletal muscle sarcolemma and effect on Ca 2+ binding and accumulation by the membranefraction 6. Liver III. CONCLUSIONS

140 141

142 142 142 143 144 146 148 148 149 149 150 150 151

151 152 153 153 154 155 157 158 161 162 170 171 171 171 172 173 174 175 175 176 176 177 181 182

ACKNOWLEDGEMENTS

185

REFERENCES

185 135

J.p.n. 35/3--^

140

136

P.V. SULAKHEand P. J. ST. Louis I. I N T R O D U C T I O N

CALCIUM ion is critically involved in numerous cellular, subcellular and molecular regulatory processes including excitation--contraction coupling (in all types of muscle), excitation-secretion coupling, neuronal excitability, intermediary metabolism of carbohydrate and fats, ionic permeability of membranes in general, and cell growth and differentiation. A unique feature of Ca 2 +-regulatable processes is that regulation is generally a power function of Ca 2 ÷ and in some instances the rate at which a given process responds to Ca 2 ÷ varies as the fourth power of the cation concentration. Also, regulation by Ca 2 + is both of short-term and long-term duration and in many instances the response to Ca 2+ is biphasic--a marked stimulation at lower and inhibition at higher concentration. It is thus not surprising that tissues are endowed with systems, both intracellular and plasma membrane-associated, that maintain and regulate the intracellular concentration of calcium ion within narrow limits. In some situations such as the beat-to-beat contraction of cardiac muscle and synaptic transmission in the central nervous system, it is conceivable that these regulatory systems must operate with extreme rapidity and display high affinity towards this cation. In the present discussion, we have directed our attention to Ca 2 + fluxes across the cell plasma membrane and the likely mechanisms by which membrane-associated processes mediate (or assist) bidirectional cation movement with particular emphasis on the efflux activity. Cell surface membrane is not impermeable to C a 2 + and yet is under the continuous constraint imposed by the large electrochemical gradient that exists between the extracellular and intracellular Ca 2 + concentration. Although the exact intracellular Ca 2 ÷ concentration of a "resting" (i.e. unstimulated) tissue is still a matter of speculation, a number of estimates indicate it to be no greater than 0.1 #M. With extracellular Ca 2 ÷ concentration of 1 mM (and this would be the lower limit), a concentration gradient of about four orders of magnitude therefore probably exists across the cell plasma membrane. The "passive" influx of Ca 2 + into the cellular cytoplasm is generally determined by two main factors: (i) the existing transmembrane concentration gradient and (ii) the permeability of the membrane. Further, a number of studies indicate that there are at least three separate "gates" or "channels" by which Ca 2 ÷ enters the cellular cytoplasm. One of these is independent of the membrane potential and displays a high cation selectivity (Ca 2 + being the preferred cation). Perhaps it is this channel that is activated by certain external stimuli (e.g. hormones) which do not affect or induce membrane depolarization. The other gate is membrane potential (or voltage)dependent and is regulated by those external stimuli that cause membrane depolarization. Many studies suggest that this gate represents the so called "slow" (or "late") calcium channel. Finally, following membrane depolarization, some Ca 2 + can also enter the cell via the "early" or "fast" calcium channel; since C a 2 ÷ entry via this route can be blocked by tetrodotoxin it is likely that it is identical to "Na ÷ gate". Depending on the tissue, C a 2 ÷ entry into the intracellular matrix occurs via single or multiple gates with each route utilized to a varying degree. Exactly how a given tissue relies on a single or multiple channel for C a 2 + influx is not understood and still represents a challenge for future investigations. In contrast to the "passive" entry of C a 2+ into the cells, C a 2 + efflux is an "active" movement or process since the cation has to be translocated against its own concentration gradient. Numerous studies have provided evidence that suggests at least two separate mechanisms are involved in Ca 2 ÷ efflux. According to one of these, the plasma membrane possesses a specific C a 2 + - A T P a s e whose activity is regulated by C a 2 + and which catalyzes ATP breakdown to provide the energy necessary for the uphill movement of this cation. Such a mechanism implies that the catalytic site as well as Ca 2 ÷ binding-site(s) of this protein complex are accessible from the intracellular face of the membrane and also that Ca 2 ÷ is translocated by the enzyme. In other words, it is a Ca 2 ÷ pump protein. A vast amount of evidence supports this mechanism in the case of the red blood cell and comparable suggestive evidence has recently been provided for other tissues as well. An interesting recent observation, which has attracted many investigators and which may turn out to be of fundamental significance, suggests that calmodulin or calmodulin-like proteins are in-

Passive and active calciumfluxesacross plasma membranes

137

timately involved in the mechanism by which Ca 2+ regulates the ATP-dependent C a 2 + pump of cell plasma membranes in general and red cell membranes in particular. The other mechanism proposed for Ca 2 + efflux describes the involvement of a N a + - C a z + exchange system. The exchange system, which is probably mediated by a carrier molecule (or complex), is presumed to have no enzymic activity (at least as ATPase) and is capable of binding either Na + or Ca 2 + at cation binding sites present on both the extracellularly and intracellularly exposed regions of the presumptive carrier. The mobility of the carrier is suggested to be dependent on the simultaneous occupancy of both internal and external binding sites. Theoretically, the exchange system is thus capable of exchanging internal Na + for external Ca 2+ or Na + as well as exchanging internal Ca 2+ for external Na + (or Ca2+). For an electroneutral exchange, one Ca 2 + is exchanged for two Na +. In other words, n Ca 2 + for 2n Na + and, depending on the charge ( - 2 or - 4 ) on the carrier, one could envisage exchange of one Ca 2 + for two Na +, or two Ca 2 + for four Na +. The carrier, according to some investigators, is believed to be capable of electrogenic cation movement as well--for example, one Na + with two Ca 2 +, or three Na ÷ for one Ca 2 +. Under the conditions that prevail physiologically, the most important exchange that the carrier mediates is believed to be that of extracellular Na ÷ for intraceUular Ca 2 +, i.e. efflux of Ca 2 +. Even though the carrier would depend on the supply of energy for an uphill Ca 2 + movement, it is believed to be derived from the Na + gradient which in turn is maintained by the classical Na ÷ pump (Na +, K +-ATPase). In addition, the Na +-Ca 2 ÷ exchange system has been implicated by some to mediate Ca 2 + influx as well. Since Ca 2 ÷ is known to influence the Na + pump activity whereas Na ÷ or K ÷ in turn can modulate the membrane-bound Ca 2 + pump (such as that of fast skeletal muscle or cardiac sarcoplasmic reticulum), it is not entirely clear how Na ÷ pump and Ca 2 ÷ pump as well as the Na +-Ca 2 ÷ exchange carrier will participate in the Ca 2 ÷ efflux across the cell plasma membrane. As is the case for Ca 2 ÷ influx, a single or multiple efflux mechanism may be operational depending on the tissue examined. The red blood cell appears to rely almost exclusively on the Ca 2 ÷ pump whilst the frog ventricle relies on the Na +:-Ca 2 ÷ exchange system for Ca 2 ÷ efflux. Many other tissues fall in between these two extreme situations. However, if one considers the expression of the Na ÷ pump and Ca 2÷ pump activities are due to a single protein (except in red blood cell where the evidence suggests that these are separate proteins), which has been postulated from time to time, then the situation becomes even more complex. Nevertheless, there exists (fortunately) an agreement in the literature that Ca 2 + efflux does occur and it represents a process of critical importance in cellular regulation. Irrespective of the basic mechanisms involved in Ca 2 ÷ influx and efflux across the cell plasma membrane, it is increasingly documented that a variety of stimuli increase or decrease the turnover of C a 2+ a c r o s s the cell. In a number of instances, hormones (such as catecholamines, acetylcholine, glucagon) that stimulate or inhibit tissue functions (contractility, secretion, metabolism) elicit rapid changes in the concentrations of cyclic nucleotides (cyclic A M P and cyclic G M P ) as well. A considerable number of studies have dealt with a myriad of interactions between Ca 2+ and the so-called "second messengers" (i.e. cyclic nucleotides), some of which are undoubtedly of significance in the hormonal effects on the target tissues. For the present discussion, we have directed our attention to the likely role played by cyclic AMP-dependent phosphorylation of plasma membrane proteins in the C a 2 + fluxes (influx and efflux) wherever appropriate. At the outset, it should be emphasized that the review is not intended to be exhaustive and hence the references are limited to those which we felt, amongst those that we are aware of, are most relevant to the topic of this article. We would also like to clarify that by no means do we consider the roles played by mitochondria or sarcoplasmic reticulum (or endoplasmic reticulum) less important in the cellular Ca 2 + homeostasis but nevertheless these aspects have been ably reviewed (perhaps rather frequently) in recent years. However, whenever appropriate, attention has been drawn to these organdies as well.

138

P.V. SULAKHEand P. J. ST, Louis

II. S P E C I F I C

TISSUE STUDIES

1. A x o n s

The fluxes of Ca 2+ (and Na ÷) across the axolemma (axonal plasma membrane) of cephalopods, particularly, the giant squid, have been extensively studied by several groups. These axons can be quite large (up to 1 mm diameter) and are relatively robust lending themselves to various experimental manipulations (see for example Mullins and Brinley, 1975; Di Polo et al., 1976). While it is hoped that results obtained from studies using these preparations may be extrapolated to mammalian nervous tissue there is, so far, no evidence that this is indeed so (Baker, 1976). Despite this limitation, the studies on squid axon have provided very useful information on Ca 2 + fluxes as well as the likely mechanisms involved in such fluxes. It is known that the axolemma is not impermeable to Ca 2 ÷ and Ca 2 + influx into the axoplasm, probably by movement down a concentration gradient, has been documented by Blaustein and Hodgkin (1969) and Baker et al. (1969). In addition, Ca 2 + enters the axon during the (nerve) action potential. There is an early phase of entry probably occurring through Na+-specific channels, and a late phase which appears to involve Ca 2 +-specific channels (Baker, 1972). The ionic composition of the extracellular fluid bathing the squid axon (blood or hemolymph) closely resembles that of sea water, containing Ca 2 + at 10-11 mM and Na + at 640 mM (Hodgkin and Keynes, 1956). The total axoplasmic concentrations of Ca 2 + and Na ÷ are of the order of 0.2--0.5 mM and 50 mM respectively (Keynes and Lewis, 1956; Blaustein and Hodgkin, 1969; Di Polo, 1974); however, the ionized (free) Ca 2 + in the axoplasm has been estimated to be only 30--100 nM (Baker et al., 1971; Di Polo et al., 1976). It should be clarified at this point that the following discussion in no way denies the importance of the axoplasmic buffers and intra-axonal organelles, such as mitochondria, in the regulation of the levels of ionized Ca 2 ÷ within the axon (see for example Blaustein and Goldring, 1975; Baker and Schlaepfer, 1978). (a) Na ÷-dependent Ca 2 + e ~ u x The marked difference in intracellular and extracellular concentrations of Ca 2 + would suggest that the axon possesses some mechanism for the extrusion of Ca 2 ÷ against its concentration gradient. Experimental observations of several workers have in fact shown that Ca 2 ÷ movements across the axolemma, and hence Ca 2 + homeostasis, are markedly influenced by intra- and extracellular levels of Na + and of Ca 2 +. Recent studies in this area have utilized internally dialyzed axon preparations which thus allow the control of the intracellular as well as the extracellular ionic composition. For example, Brinley et al. (1975) have shown, using internally dialyzed squid axon, that at 62/AM [Ca 2 +]i, C a 2 + efflux was 0.25 pmol/cm 2 in the presence of 80 mM [Na +] ~and increased 3-fold when [-Na + ]~ was raised from 1 mM to 80 mM. Also, when [Ca 2 +]~ was 1/AMthe replacement of external N a + with Li + reduced Ca 2 ÷ efflux by 50~o; at [Ca 2 +]~ of less than 0.1/AM efflux, however, was not sensitive to Na ÷ levels. Another report by Mullins and Brinley (1975) showed that, in the absence of metabolic substrates (following treatment with cyanide), at [Ca 2 +]~ of 1/.tM or greater most of the Ca 2+ efflux is dependent on [Na+]o and [Ca2+]o. Blaustein and Russell (1975) reported that, in intact axons loaded with 45Ca2+, replacement of [Na+]o with Tris or choline caused efflux to decrease by about 30~ although in both cases the residual efflux is apparently sufficient to maintain normal steady-state values for [Ca 2 +]~. Recently, Baker and McNaughton (1976) also showed that Ca 2 + efflux was decreased by 33~o when axons were perfused with Na+-free solutions. As early as 1969, Baker et al. had proposed, on the basis of the observed effects of Na + concentrations on Ca z + fluxes, that Ca 2 + efflux was mediated by a N a + - C a 2 + exchange mechanism. Hence studies showing the ionic dependence of Ca 2 ÷ fluxes, such as those cited above, have been interpreted as supporting the concept that a N a + - C a 2+ exchange mechanism is involved in maintaining axoplasmic Ca 2 ÷ levels. In addition, studies using metabolically poisoned (ATP depleted) axons seemed to suggest that the utilization of ATP is not directly involved in (the Na+-concentration dependence of) Ca 2 + efflux (see review by

Passive and active calcium fluxes across plasma membranes

139

Baker, 1972; also Blaustein, 1974; Brinley et al., 1975; Baker and McNaughton, 1976; Requena et al., 1977). Indeed Baker and co-workers (Baker et al., 1969; Baker, 1972) had postulated that a significant portion of the Ca 2+ efltux from axons is not directly dependent on metabolic energy. Assuming the validity of the proposed Na+-Ca 2+ exchange mechanisms, theoretical considerations based on the known physiological concentrations of Ca 2 ÷ and Na ÷ outside and within the axon argue against electroneutral exchange (two Na ÷ for one Ca ~+). It was thus suggested (Baker, 1972) that a simple exchange of three (or more) Na ÷ for one Ca 2 ÷ could maintain the observed [Ca 2 +]i: [ Ca2 +]o ratios. Such a model would probably involve a carrier (molecule or complex) and would be electrogenic, involving a net charge transfer. Alternatively it has been postulated that the three for one stoichiometry could involve a complementary Na +-K ÷ exchange involving the third Na ÷. This particular model was supported by Blaustein (Blaustein et al., 1974; Blaustein, 1976) who has noted that the Na ÷dependent Ca 2÷ effiux is influenced by membrane potential. Blaustein (1976) has also proposed that the carrier (either unloaded or fully loaded) may well be capable of undergoing site translocation, thereby promoting the ion fluxes. More recently Mullins (1977) has described a model, based on studies by Requena et al. (1977) and Brinley et al. (1977), where the binding of four Na ÷ to a carrier site causes induction of a Ca 2÷ binding-site on the opposite side of the membrane. ATP, which appears to influence Ca 2 ÷ influx as well as efflux but seems not to affect net transfer of Ca 2 +, was suggested to be responsible for altering the affinity of the various sites for Na ÷ and Ca 2 + without providing direct energy input into the translocation reaction. (b) A T P-induced affinity chanoes Baker and McNaughton (1976) observed that the [Na+]o-aCtivated component of the Ca 2 + efflux is affected primarily by the levels ofATP; this was true both at (constant) low and high magnitudes of Ca 2 ÷ efflux. Additionally, changes in the Ca 2 ÷ effiux kinetics, from high apparent affinity to low affinity were influenced by ATP levels. ATP-dependent changes in the affinity of the Ca 2 ÷-transport system for external cations have been shown (Baker and Glitsch, 1973 ; Di Polo, 1974) and Di Polo (1976) has further reported the occurrence of ATPinduced changes in the affinity of internal sites for Ca 2+. It was thus suggested by Baker and McNaughton (1976) that ATP-binding to the carrier (transport system) may well be essential before the binding of external Na ÷ can occur. This binding of Na ÷ would then facilitate the binding of Ca 2 ÷ to specific internally-located sites. The question of whether or not ATP is consumed (hydrolyzed) as a result of or even during such an ATP-dependent step is still unanswered. The observations of Baker and McNaughton (1976) and of Di Polo (1977) that non-hydrolyzable analogues of ATP are not capable of activating Ca 2 ÷ efflux may well be construed as evidence in favour of the degradative involvement of ATP in mediating the affinity changes in the carrier system. However, whether this could be interpreted in terms of an ATP-linked Ca 2 ÷ pump activity is an open question. (c) Ca 2 +-dependent Ca 2 + eJ~ux A detailed analysis of the ion-dependent effiux systems thought to be operational in (squid) axons, including Na+-dependent Ca 2+ efflux, has been presented by Baker and McNaughton (1976). It appears that a Ca 2 +-dependent Ca 2 + efflux activity also exists in axons. Requena et al. (1977) showed that the Ca 2 + load of an axon could be varied by altering [ Ca2 +]o. When Ca 2÷ was 8 mM steady state Ca 2 ÷ flux was observed, i.e. [Ca 2 +]i remained constant. Beyond this level of [Ca 2+]o the rate of effiux appeared to be constant although Ca 2 + influx increased, thereby altering the net Ca 2 + flux. As Baker (1976) has further pointed out, about half the Ca 2 + effiux observed from loaded axons is dependent on [Ca2+]o. In unpoisoned axons Ca 2 +-dependent Ca 2 + efflux requires 2 #M [Ca2+]o for half-maximal activation. However, Baker and McNaughton (1976) indicated that the data on Ca 2+dependent Ca 2 + efflux are not conclusive but do strongly suggest the existence of a Ca 2+Ca 2+ exchange reaction. Further, the evidence suggested that the Ca 2+-dependent Ca 2+ efflux system observed in unpoisoned (control) axons is different from that in metabolically

140

P.V. SULAKHEand P. J. ST. Louis

poisoned axons. A significant point that was raised is the possibility that in unpoisoned axons the Ca 2 +-dependent (Na+-independent) Ca 2 ÷ efflux may reflect an uncoupled exchange of Ca 2 + from a Ca 2 +-binding matrix. The probable existence of such a matrix has also been put forward in a subsequent report by Baker and McNaughton (1978) and will be discussed later. (d) ATP-dependent Ca z + efftux Evidence from recent studies has indicated that, contrary to earlier observations, ATP does influence Ca 2 + efflux. Thus, Mullins and Brinley (1975) reported that, using internally dialyzed squid axon preparations, the addition of ATP (up to 5 mM) to the dialysate caused a 3-fold enhancement of Ca 2 + etttux ; however, efflux declined to about one-third of the new level when the preparation was superfused with N a ÷-free medium. Baker and McNaughton (1976) have also reported that small amounts of ATP (up to 60 #M final) injected into an intact axon greatly enhanced Ca 2 ÷ efflux. In a subsequent report (Baker and McNaughton, 1978) it was shown that in unpoisoned axons 50-909/0 of the Ca2÷-activated efflux can continue in the absence of external Ca 2 ÷ and may reflect uncoupled extrusion of Ca 2 +. These studies are supported by reports from Requena et al. (1977) and Di Polo (1974, 1977, 1978) which show that a significant portion of the Ca 2 ÷ efflux from squid axons is indeed ATPdependent. Further, the results of Di Polo (1977) and Baker and McNaughton (1978) show that only hydrolyzable analogs of ATP (such as 2-deoxyATP and ~t-~, methylene ATP) can activate Ca 2 ÷ efflux. The ATP requirement of the Ca 2+ efflux appeared to be critical at concentrations of internal ionized Ca 2 + near the physiological level (0.02-0.06 #M); also the ATP-dependent Ca 2 + extrusion depended on external Na ÷ while a large fraction (about 60~o) was apparently not coupled to any external ion (Di Polo, 1978). Additionally, at higher levels of ionized [ C a 2 + t (up to 100/./M) the ATP-dependent component of Ca 2 ÷ efflux, though less important, still represented 50-609/0 of the total ettlux (Di Polo, 1977). At these levels, Na +-dependent and [Ca 2 ÷ ]o-dependent Ca 2 ÷ efflux assumed more significance and it has been suggested (Di Polo and B e a u # , 1979) that Na ÷-Ca 2 ÷ exchange becomes effective only when internal Ca 2 + levels are increased beyond the physiological range. These observations therefore strongly suggest that at least part of the net efflux of Ca 2 ÷ from the axon is directly dependent on ATP. The question of whether or not ATP hydrolysis per se contributes to (active) Ca 2 ÷ efflux by a system analogous to that described for red blood cell membranes remains to be established. The specific demonstration of Ca 2 +stimulated ATPase activity in squid axonal membranes will obviously be the next goal of researchers in this area. In fact, Di Polo (1977) has indicated that they have some evidence for the presence of such an activity in a highly purified membrane fraction from lobster leg nerve. Recent work in our laboratory showed that a membrane fraction enriched in axolemmal membrane fragments isolated from rat brain white matter contained ouabain-sensitive Na 4, K ÷-ATPase (so called Na + pump) of very high specific activity (200/~mol P J m g protein/hr). Based on the formation of Na ÷-dependent phosphoenzyme intermediate as well as its (K + sensitive) dephosphorylation, the membrane fraction showed an enrichment of about 15- to 20-fold over the starting homogenate. Interestingly, this membrane contained Mg 2 +, Ca 2 +ATPase and the Ca 2 +-dependent formation of the phosphoenzyme intermediate of Mg 2 +, Ca 2 ÷-ATPase was also noted (P. V. Sulakhe and E. Petrali, unpublished work). Clearly, more detailed work on the biochemical properties of axolemma will provide additional useful information concerning the likely mechanism(s) involved in the axolemmal Ca 2 ÷ fluxes.

(e) Summary The results of the studies on Ca 2 + fluxes in squid axon may be described by a schematic diagram (Fig. 1) that incorporates the major hypotheses put forward by numerous investigators in this area. Model I in essence depicts the idea from Baker and others that the N a + - C a 2 + exchange carrier (C) mediates Ca 2 + efflux in a non-electrogenic fashion by exchanging [Ca 2 +]i with [Na+]o. Further, it is believed that the efflux is energized by the Na + gradient, which in turn is maintained by the N a + - K + pump. Model II takes into account the likely role of ATP in altering the apparent affinity of the carrier but without

Passive and active calciumfluxesacross plasma membranes Na K

K

Na

Na

Ca Ca

141

ECF

C

NaK

ICF ATP

Na K

ADP Pi

K

Na

Na

ATP

ADP Pi

ATP

K

Na

Na K

Na K

Ca Ca

a Ca

Ca Ca

~ ADP Pi

ATP

ICF

ADP Pi

Na ATp

ECF

ECF

ICF

ADP P~

FIG. 1. ThreemodelsofCaz+ et]fluxfromaxonalplasmamembranes.Brokenlinesshow the direction of passive cation movement; C, the presumptiveNa+-Ca 2+ exchangecarder; ECF, extracellular fluid ; ICF, intracellular fluid. necessarily being hydrolyzed by the carrier. Model III, on the other hand, shows two separate cation pumps, N a + - K + pump and Ca 2+ pump, which derive energy direcdy from the hydrolytic cleavage of the terminal phosphate bond of ATP. It'is implied that both pumps are capable of functioning simultaneously and independently of each other. It is also possible that models II and III can be amalgamated to provide the fourth model describing N a + - K + pump, Ca 2 + pump and N a + - C a 2 + exchange system, all functioning in the axonal plasma membrane. The scheme (Fig. 1) also shows passive influx of Na + and Ca z + and eiflux of K + (broken lines) across the membrane in the direction of their respective concentration gradients. It will become obvious as we describe the results obtained with other tissues in the following sections that minor modifications in these basic models are adequate to explain most of the results on Ca 2 + fluxes across other (mammalian) tissues as well. We are therefore of the opinion that the results on axonal Ca z + fluxes, despite some uncertainties in the exact mechanisms involved, have contributed significantly towards understanding of the Ca 2 + fluxes across plasma membranes of other tissues as well. It is this belief that prompted us to introduce these models before describing the results from other systems. However, we should emphasize here that investigations on other tissues have indeed been carded out simultaneous with and independent from those on the squid axon and these studies are by no means of lesser significance.

2. Brain As mentioned earlier in the case of squid axons, mammalian neurons are not impermeable to Ca 2+ ions (Blaustein, 1974). Further, it is well documented that Ca 2+ is accumulated within the presynaptic nerve terminals during depolarization and in fact, influx of Ca 2 + into these terminals is absolutely necessary for release of stored neurotransmitters (see Blaustein, 1974, 1975; Miledi, 1973). In order that neurons return to their "resting state", Ca 2 + that is accumulated during neuronal activity must be extruded. If one were to consider the membrane potential oftbe order of - 70 mV and extracellular Ca z + concentration of 1 mM, the intracellular Ca 2 + at equilibrium would be expected to be at least 10 mM (calculations give a value around 100 mM), However, the total Ca 2+ content of mammalian brain or

142

P.V. SULAKHEand P. J. ST.LouIs

synaptosomes is of the order of 10- 3 mol/kg (Tower, 1969) and the free Ca 2+ is estimated to be lower than #M. It is obvious that much of the Ca z + is sequestered by intracellular organeUes (mitochondria, endoplasmic reticulum) and, in addition, there must be a process within the synaptosomal plasma membrane which aids in Ca 2 + efflux and which depends on energy supply for translocation of Ca 2+ against its concentration gradient. Much of the earlier work on the mammalian central nervous system has been carried out using brain slices and synaptosomal preparations. However, in recent years, microsomal fractions (that contain fragments of endoplasmic reticulum) and synaptic vesicles as well as coated vesicles have been shown to possess Ca 2+-sequestering mechanism(s). Interestingly, in contrast to the earlier belief that the synaptosomal Ca 2 + level was regulated exclusively by intrasynaptosomal mitochondria, there is some recent evidence that intrasynaptosomal organelles, besides mitochondria, are involved in regulating synaptosomal Ca 2 + concentration. In the following sections, the results from studies on Ca 2+ efflux (slice or synaptosomal preparations) as well as on ATPase and Ca 2÷ transport activities of isolated membranes are presented. Some discussion concerning the methods of isolation of these membrane fractions and their characterization, which is relevant to the studies of Ca 2 ÷ transport in isolated membrane vesicles, is also included. (a) Na+-dependent Ca 2 + efflux Data obtained by Blaustein and Weisman (1970) suggested that at least some of the energy required for Ca 2+ extrusion from brain presynaptic terminals may come from the electrochemical Na + gradient across the plasma membranes. Reports from studies using brain slices (Cooke and Robinson, 1971 ; Bull and Trevor, 1972; Stahl and Swanson, 1972) indeed supported the suggestion that a [Na+]o-dependent Ca z + efflux mechanism may be operative at the level of the synaptic terminals. Thus, Swanson et al. (1974) have shown *SCa2 + uptake by isolated synaptosomes increased when the preparations were transferred to medium low in Na ÷ ; this effect was also demonstrable in the presence of rotenone which is known to inhibit ATP-dependent Ca 2 ÷ uptake by mitochondria. These results thus support the concept of a Na +-Ca 2+ exchange system operating to regulate Ca 2 + fluxes across the synaptic membrane. Blaustein and Osborn (1975) have now shown, using isolated synaptosomes, that Ca 2 + etttux into Ca 2+-free medium is largely dependent on [Na +]o and that [Na+]o-dependent Ca 2+ etttux proceeds quite readily in metabolically poisoned (cyanide treated) synaptosomes. Similarly, Ichida et al. (1976a) have interpreted their results with isolated rat brain synaptosomes in support of a Na+-dependent Ca 2 + efflux system at nerve endings particles. The available data thus implies that a carrier-mediated Na +~Ca 2 + exchange system may be involved in Ca 2 + efflux (in brain) although the evidence is not conclusive. In fact a series of experiments by several other groups have suggested that ATPdependent Ca 2 + transport by synaptic membranes (including plasma membrane) may mediate the active efflux of Ca 2+ from synaptosomes. (b) Studies on isolated membrane fractions (i) Isolation of synaptie plasma membranes (SPM). Various groups have prepared synaptosomal fractions (synaptosomes) from homogenates of brain tissue. These fractions comprised pinched off synaptic terminals containing subeellular membranes such as mitochondria and synaptic vesicles (Whittaker et al., 1964; Robinson and Lust, 1968; Nakamaru and Schwartz, 1971). Synaptosomes have been further fractionated, usually by procedures involving density gradient centrifugation, to give fractions specifically enriched in synaptic plasma membranes (SPM; see for example de Robertis, 1964; Whittaker et al., 1964; Cotman and Matthews, 1971 ; Morgan et al., 1971 ; Saito et al., 1972; Gurd et al., 1974; Krishnan and Balaram, 1976; also see Mahler, 1977). The specific nature and relative purity of SPM have usually been estimated using putative marker enzyme activities (see Mahler, 1977). Thus early studies relied almost solely on determination of the relative enrichment compared to starting material, of Na +, K +-ATPase activity in the SPM (Nakamaru and Schwartz, 1971 ; Saito et al., 1972). Ohashi et al. (1970) have also used acetylcholine content and K +-sensitive phosphatase activity as criteria of purity. In addition to Na +, K+-ATPase, Sulakhe and Jan (1975) have given (comparative)

Passiveand activecalciumfluxesacrossplasmamembranes

143

values for adenylate cyclase in synaptic plasma membranes and other membrane fractions from rat and rabbit cerebral cortex while Krishnan and Balaram (1976) have compared the 5'-nucleotidase, acetylcholinesteraseand adenylate cyclase activities in monkey brain plasma membranes and in homogenate. Some of the values reported for membrane-associated Na +, K+-ATPase, as measured by Pi release from ATP, in brain plasma membranes are 9 /zmol/mg protein/hr (Nakamaru and Schwartz, 1971), 24-30/~mol/mg protein/hr (Ohashi et al., 1970; Saito et al., 1972; Sulakhe and Jan, 1975) and greater than 100/zmol/mg protein/hr (see Mahler, 1977). Interestingly, the values for SPM adenylate cyelase reported by Sulakhe and Jan (1975) and Krishnan and Balaram (1976) are similar, being both of the order of 400 pmol/mg/min. On the basis of the various enzyme activities measured by Krishnan and Balaram (1976) their plasma membranes were purified 2.5-5-fold relative to homogenate while the SPM fraction of Sulakhe and Jan (1975) showed 9-fold enrichment of Na ÷, K ÷ATPase and 6-fold enrichment of adenylate cyclase. In addition to estimation of marker enzyme activity to determine the relative purification of preparations of brain plasma membranes, electron microscopy has also been used to determine the nature and homogeneity of some of these preparations (de Robertis, 1964; Whittaker, 1965; Ohashi et al., 1970; Nakamaru and Schwartz, 1971; Cotman and Matthews, 1971 ; Morgan et al., 1971 ; Gurd et al., 1974). Results indicate that the fractions designated as plasma membranes consist primarily ofmembraneous material, some of which appears as (re)sealed vesicles. Electron microscopy does further indicate, however, that these preparations are relatively, but not completely, homogenous. For example, the fraction examined by Ohashi et aL (1970) contains membrane fragments as well as small vesicles bearing attached ribosomal-like material which thus probably represents microsomal contamination. Examination of results obtained by some workers (Nakamaru and Schwartz, 1971; Ohashi et al., 1970; Sulakhe and Jan, 1975) indicates that some Na ÷, K+-ATPase activity is detectable in brain microsomal fractions as well as in SPM, although the reported specific activities are lower in microsomes than in SPM. Another critical point is that the absence of specific enzyme markers for microsomes limits the use of this approach to determine microsomal contamination of SPM. In view of this, and the heterogeneity of those SPM preparations which have been examined morphologically as well as the low degree of relative purification (as indicated by marker enzyme activity) of some preparations, there must be some doubt about the purity of SPM fractions so far described. It follows that there will be some reservations about the interpreting of results obtained using these preparations. (ii) Ca 2 +-stimulated A TPase. Mg 2+, Ca 2 +-ATPase (Ca 2 +-stimulated Mg 2 +-dependent ATPase) activity has been reported in brain plasma membrane fractions prepared by various groups and it has been unanimously suggested that this activity may, at least in part, represent a Ca 2 ÷ "pump" activity. Thus Yoshida and co-workers (Ohashi et al., 1970) have shown that "extra" ATPase [(Mg 2 ÷ + Ca 2 +) - Mg 2 +] is present in SPM from rat cerebral cortex and the activity (15.4 #mol/mg/hr) is maximal at 8 x 10 -5 M CaCI2 in the assay. Nakamaru and Schwartz (1971), Sulakhe and Jan (1975), Robinson (1976), and O'Driscoll and Duggan (1977) have also shown that Mg 2 ÷, Ca 2+-ATPase is present in brain membrane fractions enriched in Na ÷, K+-ATPase activity (presumed thus to represent partially purified plasma membranes). The specific activities where given lie in the range 6--18 #mol/mg/hr. Nakamaru and Schwartz (1971) and Robinson (1976) have both shown that of various nucleotides tested only ATP was significantly hydrolyzed by the plasma membrane Mg 2+, Ca 2 +-ATPase. Robinson (1978) has also shown that Ca 2+ stimulated the (hydroxylamine sensitive) phosphorylation of a Mg 2 +, Ca 2÷-ATPase preparation from rat brain microsomes. The preparation which also exhibited Na +, K +-ATPase activity, was probably of heterogeneous subceUular origin. Duncan (1976) showed that the Mg 2 +-ATPase activity in a SPM preparation from rat brain (30 #mol/mg/hr) was maximally stimulated to 150% by 2 x 10 - 6 M C a 2+. Duncan (1976) specifically stated that the properties of the Ca 2+stimulated ATPase, including Km (4 x 10- 7 M C a 2 +) and activation range (1 unit ofpCa 2 ÷), suggested its involvement in mediating Ca 2 + efflux from (intact) synaptic terminals rather than an involvement in the process of exocytosis. The preparation described by O'Driscoll

144

P.V. SULAKHEand P. J. ST. LOUIS

and Duggan (1977) was obtained from a crude mitochondrial fraction whereas the other procedures referred to involved fractionation of a crude synaptosomal (microsomal) preparation. It is interesting that the reported specific activities of the Mg 2 +, Ca 2 +-ATPase in the various preparations cited above are generally similar as are the values of K,4ca2+ ), these latter being in the //M range. These observations suggest that the various SPM preparations are, in fact, comparable. Further, the observation that the distribution of the Mg24, Ca 2 +-ATPase is similar to that of the Na +, K+-ATPase, in these membranes from brain, is consistent with the hypothesis that this Ca 2 +-stimulated enzyme reflects the activity of a system which could mediate the active (ATP dependent) efflux of Ca z + across the synaptic plasma membrane. (iii) Ca2+-transport activity (ATP-dependent). Saito et al. (1972) have showed that the binding of Ca 2 + to SPM was ATP- and temperature-dependent and was maximal (as was the Mg 2+, Ca 2 +-ATPase activity of the preparation) at 9 x 10-s M CaC12 in the medium. The K m for ATP was 30 #M and Mg 2 + was required for binding; maximal binding occurred at 2 mM Mg 2+ (Ichida et al., 1976a). ATP-dependent binding (8-10 nmol/mg protein) was stimulated by oxalate with maximal stimulation at 60 mM oxalate (up to 52 nmol/mg protein). The Ca 2 +-binding activity exhibited properties, such as temperature dependence, which indicated that it was distinct from the Ca 2 + binding activity of brain mitochondrial and microsomal fractions (Saito et al., 1972; Ichida et al., 1976a). Since the SPM preparations also contained Mg 2+, Ca2+-ATPase (Ohashi et al., 1970; Ichida et al., 1976a) it was considered that the membranes did possess a Ca 2 + pump activity. However, examination of the effects of La 3 +, Mn 2 + and ruthenium red on the Ca 2 +-binding and Mg 2 +, Ca 2 +-ATPase activities led these workers to suggest that the Ca 2 +-binding activity of the isolated SPM is not closely coupled with Mg 2 +, Ca 2 +-ATPase (Ichida et al., 1976b). Direct evidence for the binding of Ca 2 + to brain plasma membranes has also been provided by other groups. Thus Robinson and Lust (1968) had earlier shown the presence of an ATP-dependent Ca 2 +-binding activity in a membrane fraction, considered to represent enriched SPM, from rat brain. Oxalate, however, did not augment the Ca 2 +-accumulation activity. Sulakhe and Jan (1975) have reported Ca 2 + uptake by enzymically characterized SPM from rat cerebral cortex displayed a Vmaxof 20-30 nmole/mg and K,,(Ca 2+) of 5-10/~M and O'Driscoll and Duggan (1977) have found that Ca 2 + uptake and Mg 2 +, Ca z +-ATPase activities were present (and enriched) in brain membranes enriched in acetylcholinesterase and Na +, K+-ATPase. Interestingly, Nakamaru and Schwartz (1971) have previously reported their inability to detect significant (ATP-supported) Ca 2 + binding to a brain plasma membrane enriched fraction which exhibited Mg 2+, Ca 2 +-ATPase activity. These workers suggested, nonetheless, that the plasma membrane Mg 2 +, Ca 2 +-ATPase may be involved in the active extrusion of intracellular Ca 2 +. It is evident that although attention has been directed for several years to the question of the existence of a mechanism for the (active) efflux of Ca 2 + from synaptic terminals, only limited success has been achieved. The definition of Ca z +-stimulated ATPase in preparations enriched in brain synaptic membranes has led to the suggestion that this activity, by analogy with the red blood cell, reflects the presence of an ATP-dependent Ca 2 + "pump" which mediates the active efflux of Ca 2 +. In one case where ATP-supported Ca z + binding has been described in a preparation of synaptic membranes which exhibit Ca z +-stimulated ATPase (Ichida et al., 1976a, b) there is some doubt as to the relationship between the two activities (Ca z+ binding and ATP hydrolysis). The subsequent observation that Na + decreased Ca 2 + binding and promoted Ca z + release prompted the suggestion that a Na +Ca a + exchange mechanism may also be operative in these membranes (Ichida et al., 1976b). However, K + was found to stimulate Ca 2+ binding both in the absence and presence of oxalate (Ichida et al., 1976a). It would have been informative if Yoshida and co-workers had also examined the effects of K + on the Mg 2 +, Ca z +-ATPase. Nonetheless, the reports of Robinson and Lust (1968), Sulakhe and Jan (1975) and O'Driscoll and Duggan (1977) on the presence of both Mg 2+, Ca 2 +-ATPase and ATP-dependent Ca 2 +-binding activities in preparations enriched in SPM do provide evidence in support of the proposal that an

Passive and activecalciumfluxesacross plasma membranes

145

ATPase linked Ca 2 +-transport system exists in brain plasma membranes. Further critical examination of the relationship between the Ca 2 +-stimulated ATPase and Ca 2 +-transport activities of SPM is obviously required. The lack of specific Ca 2 +-binding (ATP-dependent) activity in some of the synaptic plasma membrane preparations referred to above may be a reflection of the orientation (sidedness) of the membrane vesicles in the preparations. That is, since the activity is supposed to mediate efflux of Ca 2 +, specific binding sites for the cation should be located on the internal (cytoplasmic) aspect of the membrane in vivo. Thus rightside-out (RSO) membrane preparations would not be expected to exhibit (ATP-dependent) Ca 2 +-binding activity. For such preparations to exhibit Mg 2+, Ca2+-ATPase activity, however, it is necessary to postulate that the vesicles are not permeability intact in order to account for the accessibility of the substrate (ATP), as well as the Mg 2 + and Ca 2 + ions, to the enzyme sites. In this case it should really be possible to document some specific Ca 2+ binding to the membrane preparation. It appears more likely that isolated synaptic plasma membranes are oriented inside-out (IO). This proposal is supported by a recent report which indicated that 80% of the nerve endings became inverted when synaptosomes were isolated from rat cerebral cortex (Logan and Waters, 1976). Of relevance to the present discussion is the demonstration of the specific interaction of Ca 2 + with isolated brain plasma membranes. Madeira and Madeira (1973), and Krishnan and Balaram (1976), using anionic sulfonate fluorescent probes, have shown that synaptic membranes specifically bind Ca 2 + and other ions probably at non-lipid membrane sites. The activity described was not an ATP-dependent process. Madeira and Madeira (1973) suggested that the interaction of Ca 2+, which results in conformational changes in the membrane, may reflect a phase transition effect of importance in the formation of an "active release state". The implication of these two studies is that the isolated membranes are probably oriented RSO, in contrast to the proposal above, or they are leaky or exist as fragments rather than vesicles. Examination of the various reports cited above on the isolation of brain plasma membranes (where electron microscope analysis is reported) shows that there is variability and preparations such as that used by Ohashi et al. (1970) comprises both vesicles and membrane fragments. This obviously renders interpretation of any results more difficult. It must be emphasized, nonetheless, that the existence of specific sites for Ca 2 + on both the external and internal aspects of the plasma membrane is a distinct possibility. Such a possibility has in fact been considered by Ichida et al. (1976c). With IO membrane vesicles the question of accessibility of substrates and specific ligands to the active site(s) of the Ca 2 + pump (efflux) does not arise. However, it should be possible to demonstrate ATP-dependent Ca 2 ÷ binding, such as described by Yoshida and co-workers (Saito et al., 1972; Ichida et al., 1976a). The observation of Ichida et al. (1976b) that Ca 2+ uptake by synaptic plasma membranes is not closely coupled with Mg 2 +, Ca 2 +-ATPase activity could probably be due to a partial uncoupling of the two activities as a result of the membrane isolation procedure. Complete uncoupling could well be the reason why workers such as Nakamaru and Schwartz (1971) have noticed Mg 2 +, Ca 2+-ATPase, but not Ca 2 +binding activity, in brain plasma membrane preparations. The results presented by Sulakhe and Jan (1975) and by O'Driscoll and Duggan (1977) do not allow us to speculate on the extent of coupling of the ATPase and Ca 2 +-uptake activities exhibited by their SPM preparations. Enhancement of ATP-supported Ca 2 + binding by the addition of a permanent anion such as oxalate has been used as an indicator of the transmembrane movement of Ca 2 + ions, the assumption being that the intravesicular formation of Ca 2 +-oxalate complex maintains low free Ca 2+ ions within the vesicles and thus promotes the translocation of Ca 2 + from the extravesicular space. Oxalate at high concentrations was found to enhance the net Ca 2 +binding activity of synaptic plasma membranes (Ichida et al., 1976b) the activities were 8 nmole Ca2+/mg protein and 51.5 nmol Ca2+/mg protein in the absence and presence respectively of 60 mM oxalate. This observation, of course, reinforces the suggestion that there exists in synaptic plasma membranes a system which mediates the ATP (energy)dependent efflux of Ca 2 + in oioo. In another report (Ichida et al., 1976c), these investigators

146

P.v. SULAKHEand P. J. ST. Louis

suggest that there are in fact three mechanisms of Ca 2 + efflux from brain synaptic terminals: (i) Ca 2 + efflux coupled to Mg 2 ÷, Ca 2 ÷-ATPase (ii) efflux due to Ca 2 +-Ca 2 + exchange and (iii) Na +-dependent Ca 2 + efflux. Interestingly, these workers also implied that there is some doubt that the ATP-dependent Ca 2 ÷ binding with synaptic plasma membranes represents a partial reaction of Ca 2 + ettlux. In summary, therefore, it has been shown that partially enriched brain plasma membrane fractions exhibit Ca 2 +-stimulated ATPase activity and ATP-dependent Ca 2 +-binding or -uptake activity. However, it is still not certain whether the two activities are indeed part of a linked system. Further, the significance of this ATPase activity is still not clear. (iv) Intrasynaptosomal A T P - d e p e n d e n t Ca 2+ storage systems. Earlier studies (see for example Alnaes and Rahamimoff, 1975; Sulakhe and Jan, 1975; Vickers and Dowdall, 1976) indicated that the observed Ca 2 +-sequestering activity of synaptosomes was due primarily to the mitochondria present in the preparation. Sulakhe and Jan (1975) in fact estimated that about 80~o of homogenate (rat brain cortex) Ca 2÷ uptake was associated with a crude mitochondrial fraction and 10~ with a crude microsomal fraction. When the crude mitochondrial fraction was fractionated on sucrose gradients, the evidence suggested that the bulk of synaptosomal Ca 2 +-uptake activity can be accounted for by the activity of intrasynaptosomal mitochondria. More recent studies, however, offer alternate suggestions. For example, Kendrick et al. (1977) compared Ca 2 + uptake by mitochondria and disrupted synaptosomes and suggested that within presynaptic terminals there are vesicular bodies, besides mitochondria, which store Ca 2 + in the presence of MgATP 2 -. In fact, Kendrick et al. (1977) believe that, since these vesicular bodies exhibit a high affinity towards Ca 2 + (1/./M or less) whereas mitochondria show much lower affinity (10-20#M), these membrane vesicles are critical in maintaining low Ca 2 ÷ within the presynaptic nerve particles under physiological conditions. Further, Kendrick et al. (1977) suggested that the mitochondria may aid in buffering intraterminal Ca 2+ primarily by supplying ATP to the transport and storage system of the membrane vesicle. The facts that oxalate promoted ATP-dependent Ca 2 + uptake, and a divalent cation specific ionophore, A23187, prevented ATP-dependent uptake and released previously accumulated Ca 2÷ indicated a net gain of Ca 2 + against its electrochemical gradient. In the subsequent work (Blaustein et al., 1978a, b) the details of the non-mitochondrial, intrasynaptosomal membrane-associated Ca 2 ÷-transport system were described. Blaustein et al. (1978a,b) provided some suggestive evidence that the membrane vesicles studied were separate from synaptic plasma membrane, intrasynaptosomal mitochondria or brain mitochondria, synaptic vesicles (plain and/or coated) as well as microsomes. Since, if proven, it will represent a potentially vital mechanism for maintaining low Ca 2 ÷ in the presynaptic nerve ending particle, the evidence that led to the conclusion by Blaustein et al. (1978a,b) deserves mention at this stage. (1) ATP-dependent Ca 2 + uptake was determined in the presence of azide, dinitrophenol and oligomycin, which effectively block the mitochondrial uptake system. (2) Trypsin inactivated the non-mitochondrial process whereas it has no effect on the mitochondria under the conditions tested. (3) When Ca 2 ÷ is low, such as < 0.3 #M, mitochondria took up little Ca 2 + whereas the non-mitochondrial system was capable of sequestering Ca 2+. (4) Ruthenium red at lower concentrations inhibited mitochondrial Ca 2 ÷ uptake and FCCP, a mitochondrial uncoupling agent, caused the release of stored Ca 2 ÷ from mitochondria; neither of these effects were seen with the nonmitochondrial system. (5) The lack of the effect of either saponin or digitonin (at low concentrations) as well as of monovalent cations (especially that of Na ÷) suggested that plasma membrane (everted) vesicles did not contribute to the measured Ca 2 ÷ uptake by the membrane fraction. (6) Synaptic vesicles isolated by the classical De Robertis method did not show ATP-dependent Ca2÷-uptake activity. Further, since synaptic vesicles have a cholesterol content similar to that of synaptic plasma membrane and saponin, which disrupts cholesterol-rich membranes, did not influence the Ca 2 ÷ uptake, they suggested that the membrane fraction is not contaminated with synaptic vesicles either. In addition, subfractionation of the synaptosomal fraction indicated that the Ca2+-uptake activity did not sediment with the synaptic vesicle fraction. (7) Although Blitz et al. (1977) observed an ATPdependent Ca2+-uptake activity in the purified coated vesicle, Blaustein et al. (1978a,b)

Passiveand activecalciumfluxesacrossplasma membranes

147

indicated that contamination of the coated vesicle fraction by other organclles was not ruled out in the study by Blitz et al. (1977). Blaustein et al. (1978a,b) implied that perhaps the intraterminal smooth endoplasmic reticulum, which their work suggested to possess the active Ca 2 +-transport system, could have been present in the preparation of coated vesicle used in the study by Blitz et al. (1977). Further, if coated vesicles were to be involved in the synaptic membrane recycling phenomenon, as postulated by Blitz et al. (1977), then it is conceivable that components of the smooth ("uncoated") synaptic vesicles are also involved. In other words, it appears that whether or not coated vesicles have the ATP-requiring Ca 2 +uptake system is not yet established. In fact, recent studies carried out by us (P. V. Sulakhe, E. H. Petrali and B. J. Thiessen, unpublished work) indicated that distributions of the coated vesicles (based on the content of clathrin, a marker for coated vesicles) and ATP-dependent Ca 2 +-uptake activity showed dissimilar patterns within the density gradient subfractions analyzed. Although we observed that MgATP 2--promoted Ca 2+ uptake (compared to that seen without MgATP 2-), oxalate failed to further increase it and Ca 2 +-stimulated Mg z +dependent ATPase could not reliably be detected in these gradient subfractions. Irrespective of the question of the identity of the Blaustein membrane preparation, the evidence, nevertheless, shows considerable similarity between the so-called intrasynaptosomal non-mitochondrial system and the muscle sarcoplasmic reticulum. For example, both display Kca 2+ (apparent half-saturation constant) around 0.3-0.5 #M, Ks for ATP around 10 /~M, a stoichiometric relation of 2 mole of Ca 2+ transported per mole ofATP hydrolyzed and requirement for Mg 2 + for Ca 2 + uptake as well as ATP hydrolysis. However, the ATP-dependent C a 2+ uptake and Mg2+-dependent Ca2+-stimulated ATPase activities of the Blaustein's membrane fraction did not appear to be tightly coupled. For example, the maximal rate of ATP hydrolysis was about 50--100-fold greater compared to the Ca 2 +-uptake activity. Several possibilities such as non-linear reaction rates for Ca 2 + uptake, leaky vesicles in the preparation as well as contamination by the contractile proteins (like actomyosin) present in the presynaptic nerve terminals were considered by Blaustein et al. (1978a,b) to account for these differences. Future work should hopefully clarify these aspects. Rahamimoff and Abramovitz (1978a,b), on the other hand, have reported ATP-supported Ca 2 +-uptake and Mg z +, Ca 2 +-ATPase activities in the synaptosomal vesicle; however, these showed some interesting differences from those of the sarcoplasmic reticulum (for example, weak effect of Mersalyl and the lack of Ca 2 + stimulation of Mg 2+-ATPase). These studies, nevertheless, are only preliminary and a more detailed analysis is warranted before any firm conclusions can be made. At the same time, the possibility that synaptosomal vesicles participate in the regulation of Ca z + within the nerve terminals, as suggested by Rahamimoff and Abramovitz (1978a, b), cannot be overlooked. Recently, Rahamimoff and Spanier (1979) provided preliminary data that suggested the presence of a Na+-Ca 2+ exchange system in their synaptosomal vesicle preparation. Briefly, the vesicles accumulated significant amounts of Ca 2+ when there existed an outwardly directed Na + gradient but not K + gradient. The vesicles also showed Ca 2 +dependent Na + uptake. However, in view of the crude preparation utilized, the exact stoichiometry of the Na +-Ca 2 + exchange system could not be estimated and further, it is not yet clear whether the exchange system is due to the synaptic plasma membranes or other (intra)cellular membrane fragments present in the vesicular preparation. Nonetheless, the fact that Ca 2 + is indeed accumulated by the vesicular fraction in the presence of a Na + gradient appears to indicate that IO vesicles are present in the preparation. Unfortunately, however, Rahamimoff and Spanier (1979) have not tested the RSO and IO content of the preparation. In another study, Papazian et al. (1979) reported reconstitution of the ATPsupported Ca 2 +-uptake system from synaptosome into artificial lipid vesicles. The transport system was also purified by the sucrose density gradient centrifugation technique. Interestingly, the purified, reconstituted vesicles showed the presence of two major polypeptides, one of Mr 94,000 and another of M, 140,000, following electrophoresis in the presence of SDS. It is worth mentioning here that the molecular weights of Ca 2 +-ATPase from muscle sarcoplasmic reticulum and RBC respectively are about 100,000 and 140,000 (MacLennan and Holland, 1975; Drickamer, 1975).

148

P.V. SULAKHEand P. J. ST.Louis

Despite a general agreement amongst various investigators that the Na+-Ca z ÷ exchange system is of significance, its exact subcellular location remains unclear. Michaelis et al. (1979), in their recent abstract, suggest that it is present in the synaptic plasma membrane. Similar suggestion has also been put forward by other investigators in the earlier studies. However, further detailed analysis is warranted before any firm conclusions can be made. (v) Microsomal Ca z ÷-transport system. As early as 1965, Otsuka et al. (1965) reported the presence of ATP-dependent Ca 2 +-uptake activity in the microsomal fraction from rat brain. Since then, a number of studies have confirmed this activity as well as Mg 2 +, Ca 2+-ATPase in microsomal preparations from brain (Robinson and Lust, 1968; Otsuki, 1969 ; De Meis et al., 1970; Nakamaru and Schwartz, 1971 ; Trotta and De Meis, 1975 ; Sulakhe and Jan, 1975 ; Robinson, 1976, 1978; Trotta and De Meis, 1978). Even though the microsomal fraction utilized in most of these studies contains membrane vesicles derived from endoplasmic reticulum and from other sources such as plasma membranes, synaptic vesicles, some of the results, especially those dealing with phosphorylation of the ATPase, deserve mention at this point. Robinson, who earlier observed Mg 2+, Ca2+-ATPase in rat brain microsomes (Robinson, 1976), recently noted a Ca 2+-dependent phosphorylation of the Lubrol-treated microsomal fraction. This phosphorylation was considered due to the formation of an acylphosphoenzyme intermediate and the monomeric ATPase gave a molecular weight of 100,000. Trotta and De Meis (1978) have recently reported some interesting results on brain microsomes. Their work showed that microsomes catalyze ATP-P~ exchange as well as ATP hydrolysis, depending on the concentration of Ca 2 +. Increasing Ca 2 + promoted ATP-PI exchange reaction (half maximal inhibition at 2 mM); low Ca 2+ (10--100 #M), in fact, promoted ATP hydrolysis. De Meis's group suggests that Ca 2+ binding to a low affinity site (KCa2+ of 2 mM), which resides at the intravesicular surface, favors ATP synthesis whereas Ca 2+ binding to a high affinity site (KCa2÷ of 50 #M) located at the external surface favors ATP hydrolysis. In other words, there exists a 40-fold difference in the affinities of internal and external Ca 2+ binding-sites in the case of brain microsomes; this is considerably less than that noted for muscle sarcoplasmic reticulum where nearly 3 to 4 orders of magnitude difference exists between the internal and external Ca 2+ binding-sites. An interesting suggestion that Trotta and De Meis put forward was that cerebral ATPase is maximally activated by a much smaller transmembrane gradient, which might also account for the lower efficiency of this system for calcium storage or accumulation, compared to fast muscle sarcoplasmic reticulum. However, it should be noted that the apparent affinity of the high affinity site in brain microsomes is at least 50 times less than that of the Blaustein's membrane fraction; the latter fraction, however, accumulates significantly less quantity of calcium compared to microsomal preparations. (vi) Modulation of Ca 2+ transport and Ca 2 +-ATPase by calmodulin in brain membrane fractions. The observations that a calcium dependent modulatory protein (calmodulin), which is required for the stimulatory effect of Ca 2÷ on brain cyclic nucleotide phosphodiesterase and adenylate cyclase (see review by Wang and Waisman, 1979), stimulated Mg 2+, Ca 2 +-ATPase activity in red blood cell membrane (Jarret and Penniston, 1977; Gopinath and Vincenzi, 1977) as well as Ca 2+ uptake in IO vesicles of human red blood cell membranes (Maclntyre and Green, 1977; Hinds et al., 1978) have now been extended to the Ca 2+-transport systems in membranes from a variety of tissues including brain, heart and skeletal muscle (see also later sections of this article). Yoshida's group (Kuo et al., 1979; Sobue et al., 1979) observed that purified calmodulin restored the Mg 2 +, Ca 2 ÷-ATPase and Ca 2+-binding activities of the EGTA-pretreated synaptic plasma membrane preparation. The EGTA-treatment, which reduced these activities (40-60%), was found to remove a protein factor functionally equivalent to calmodulin. These results support a view that calmodulin regulates translocation of Ca 2 + across synaptic plasma membranes. Iqbal et al. (1979) also observed a similar stimulatory effect on Mg 2+, Ca 2 ÷-ATPase activity of neural microsomal membrane fraction. In the study by Sorensen and Mahler (1979) and by Kuo et al. (1979), about 3-6 #g of calmodulin per mg of membrane protein gave the half-maximal stimulatory effect on ATPase activity and the apparent affinity for Ca 2 ÷ was estimated to be

Passiveand activecalciumfluxesacrossplasma membranes

149

about 3-10/~M. Interestingly, not all of the membrane-bound calmodulin or calmodulin-like protein(s) can be extracted from the membrane by EGTA treatment or other treatments (such as hypotonic shock treatment). However, Hano and Penniston (1979) reported the lack of stimulatory effect of calmodulin on brain membrane Ca 2 +-ATPase. In this study, a partially purified Mg 2+, Ca2÷-ATPase preparation isolated according to the Robinson method (Robinson, 1978) as well as synaptosomal plasma membrane isolated according to Jones and Matus (1974), were tested. Whether or not the methods of preparations and/or assay conditions may explain the differences in the action of calmodulin cannot be determined at present. In the electron microscopic study by Wood et al. (1979), calmodulin and its binding protein (of M, 80,000) were localized in the post-synaptic densities and also along the post-synaptic microtubules. Siekevitz and co-workers (Grab et al., 1979) have indeed also shown that calmodulin as well as calmodulin-dependent protein kinase are present in the post-synaptic density. Further studies on these aspects will no doubt provide very useful and interesting information and these appear to have been now initiated in numerous laboratories including ours. The mechanism(s) by which calmodulin stimulated synaptic membrane Ca2÷-uptake and Mg 2+, Ca2÷-ATPase activities have not been investigated. One likely possibility is that a specific membrane protein(s) is phosphorylated by the calmodulin dependent protein kinase present in the membrane fraction and such phosphorylation mediates the stimulatory effect on the "pump". We observed that in the membrane fraction, which is essentially similar to those used in the study by Kuo et al. (1979) and Sorensen and Mahler (1979), several polypeptides are phosphorylated by Ca 2÷dependent protein kinase in a calmodulin-dependent manner (Petrali et al., 1978). Recent work also indicated a relation between the phosphorylation of membrane by Ca 2+calmodulin dependent kinase and Mg 2÷, Ca2÷-ATPase activity (Sulakhe et al., unpublished). This is quite interesting since the results provide a clue of a likely mode of in vivo regulation of synaptosomal Ca 2 ÷ fluxes. (vii) Summary of results from isolated membrane fractions. The above discussion clearly reveals the complexity in the mechanisms involved in the regulation ofintracellular Ca 2 ÷ in nervous tissue. Although we have not reviewed it here, considerable evidence shows brain mitochondria capable of sequestering large amounts of Ca 2+, although there is still some doubt whether these organelles have sufficiently high affinity to be operational under physiologically relevant conditions (i.e. when lCa 2 +]~ is well below/aM). Synaptic vesicles, synaptosomal vesicles, coated vesicles, smooth endoplasmic reticulum as well as plasma membranes reportedly contain ATP-requiring Ca 2 ÷-uptake systems as well. In the case of plasma membrane, a Na +-Ca 2 ÷ exchange system for Ca 2 ÷ effiux has also been considered by many investigators. Besides these membrane-associated transport processes, cytoplasmic proteins that display high affinity towards Ca 2+ are also capable of buffeting [Ca2+]/. Considerable future work is required to understand how I-Ca2 +]i is maintained by these systems and whether these act in a coordinated, independent or interdependent fashion (see Fig. 2). In future studies, investigators must address themselves to the question of relative importance of apparent affinity versus capacity of these systems in addition to the rapidity with which a given transport system responds to altered Ca 2÷ levels that accompany or result from enhanced neuronal activity. The role, if any, played by non-neuronal cells (such as glia) in the regulation of [Ca 2 +]o is still a matter of speculation and represents a challenge for future studies. 3. Cardiac Muscle The central role played by Ca 2 + ions in the excitation-contraction coupling process in all types of muscle is well established. In cardiac muscle, following depolarization, Ca 2 + enters the sacroplasm via the fast Na ÷ channels as well as through slow (Ca 2 ÷-sensitive) channels. It is believed that Ca 2 ÷ influx into the myocardial cell is somehow critically involved in the contractile response of cardiac muscle. In addition there is probably an inward "leak" of extracellular Ca 2 ÷, comparable to that occurring in other cells and tissue types, into the myoplasm. Under normal conditions the myoplasmie levels of Ca 2 + are maintained within

150

P.V. SULAKHEand P. J. ST. Louis

II

O

tI

vesicles

\\

~-ai -

-

~

extrusio~n

J.dY//

II "'°"'"'e

Ca 0

/

\

FIG. 2. A schematic diagram of various organelles, plasma membranes and cytoplasmic proteins associated with cellular Ca2+ homeostasis in the case of pre-synaptic neuron. strict limits during the contraction and the relaxation phases of the cardiac cycle. Hence this Ca 2 +, which leaks in, must be effluxed and, more importantly, the Ca 2 ÷ which influxes with each beat must be returned to the exterior of the cardiac cell also on a beat-to-beat basis. The efflux of Ca 2 + across the cardiac sarcolemma has been documented by a number of laboratories and evidence is available which suggests that a N a + - C a 2 + exchange carrier can mediate Ca 2+ efflux. In addition, however, some biochemical studies have raised the possibility of the existence in the sarcolemma of a Ca 2 ÷-transport activity which may be linked to a Ca 2 +-stimulated ATPase activity, i.e. an ATP-dependent Ca 2 + pump. In the following sections we shall therefore discuss the various published findings and how they in fact relate to the overall question of the mechanism of Ca 2 + etttux across the cardiac plasma membrane (sarcolemma). Recent studies suggest that/~-adrenergic amines accelerate Ca 2 ÷ influx by increasing the number of functional Ca 2 + channels but apparently without any detectable effect on the characteristics of such channels. There is also some preliminary evidence that a N a + - C a 2 + exchange system m a y mediate an increased Ca 2 ÷ influx, at least under certain experimental situations. Interestingly, m a n y studies implicate a likely role for cyclic nucleotides in the Ca 2 + influx. Increase or decrease in Ca 2 + influx due to interactions of sympathetic amines and cholinergic agents respectively with the specific sites (receptors) present at the external aspect of myocardial sarcolemma, and the mechanism(s) through which cyclic nucleotides regulate the Ca 2÷ influx are under intense investigations in m a n y laboratories. The possibility that cyclic A M P modulates the "active" efflux of Ca 2 ÷ has also been considered by some investigators. Thus, some discussion about the likely mechanisms by which cyclic nucleotides influence sarcolemmal Ca 2+ fluxes is included. Isolation methods for sarcolemma as well as their biochemical characteristics are also discussed. The findings on sarcoplasmic reticulum or other intracellular systems are included only whenever these are relevant to the sarcolemmal Ca 2 ÷-transport systems. (a) Tissue studies (i) Na+effects on Ca z ÷ fluxes. Several early reports had suggested that the levels of N a ÷ both outside and within the cardiac muscle cell somehow could influence myocardial levels of Ca 2+. Thus Niedergerke (1959), Niedergerke et al. (1969) and Langer and Brady (1963)

Passiveand activecalciumfluxesacrossplasmamembranes

151

showed that the external concentration ofNa + ions ([Na+]o) influenced force development in cardiac muscle, and proposed that this was due to effects on trans-sarcolemmal Ca 2 + movements. Langer (1964) further showed that [Na +]o did influence Ca 2 + influx into cardiac muscle preparations. On the other hand Gfitsch et al. (1970) found that increasing [Na+]~ caused an enhanced influx of aSCa2 + into guinea-pig auricles suggesting that [Na+]i also influenced bidirectional Ca 2 + fluxes across cardiac muscle cells. Other studies by Nayler and co-workers (Nayler, 1967; Nayler and Fassold, 1977) and by Langer and co-workers (see Langer, 1973) have provided evidence in support of the concept that [Na+]o and [Na+]i could mediate calcium movements across the myocardium and so regulate myocardial Ca e + concentration.

(ii) Na+-dependent Ca 2+ efflux. A critical study by Reuter and Seitz (1969) showed that Ca 2 + effiux from guinea pig ventricular trabeculae and auricles was dependent on both [C ae +]o and [Na+]o . In particular Ca 2 + effiux decreased to 70% in Ca 2+-free solution, to 20% in Ca2+-free, Na+-free solution and to 65% in Na+-free solution. These studies suggested that Ca 2 + efflux depended to a large extent on the ratio [Ca 2+]o: [Na+]o and the Na +-activated fraction of the Ca 2 + efflux was found to exhibit high specificity for Na +. On the basis of their results Reuter and Seitz (1969) proposed that a modified exchange diffusion mechanism (see Ussing, 1960), involving a carrier system with at least four binding-sites, was involved in a Na+-mediated Ca 2 + efflux system. The various sites would be expected to exhibit specific affinities for the cations and the results obtained showed that the activation site for Ca 2+ efflux (presumably located internally) exhibited a much lower affinity for Na + than for Ca e +. In a more detailed presentation of the proposed mechanism Reuter (1974) suggested that under normal conditions the bulk of the Ca 2+ efflux from the myocardium is an exchange with external Na +, i.e. a Na+-Ca e + exchange. This carrier scheme for Na +Ca 2+ exchange involved the exchange of two Na + (from outside the cell) for one Ca 2 + (to outside the cell). That is, quantitatively two Na + ions and one Ca 2+ ion would compete for any one transport (carrier) site on the membrane and the exchange should be electroneutral. Studies using agents, such as caffeine and cyanide, which cause release of Ca 2 + from intracellular stores, have provided support for the proposal and for a pump ratio of two Na + for each Ca e + transported (Jundt et al., 1975). The studies of Tillisch and Langer (1974) on the effects of [Na +]o on ion fluxes in cardiac muscle are also consistent with this proposed Na +Ca 2 + exchange mechanism. The energy for the operation of the proposed carrier is supposedly derived from the electrochemical gradient for Na + across the membrane (see discussion on axons). Thus the downhill movement ofNa + along its concentration gradient would provide the driving force to promote the uphill movement of Ca 2+ (via the suggested carrier). The Na + gradient itself is, of course, maintained by the Na+-K + pump probably represented by the plasma membrane Na +, K +-ATPase (see Glynn and Karlish, 1975). Thus the Ca 2 + effiux system is only indirectly dependent on ATP hydrolysis. (iii) Effects of cardiac glycosides. One area of experimentation which also appears to support Na +-Ca 2 + exchange as a significant mechanism in the regulation of Ca 2 + effiux (and influx) in cardiac muscle is studies on the effects of cardiac glycosides on cardiac muscle contractility. It is well documented that these agents (such as ouabain) depress (myocardial) Na +, K+-ATPase, increase transmembrane Na + influx and also enhance force of contraction (see review by Schwartz et al., 1975; Ku et al., 1977). The suggested mechanism which links these two effects is as follows. By inhibiting Na ÷, K ÷-ATPase the glycoside will cause enhanced [Na +]i. This in turn results in greater competition between Na + ions and Ca 2 + ions for the internally located carrier sites, thereby effectively reducing Ca 2+ efflux and so raising [Ca 2 +]i. This of course will result in enhanced force of contraction. A recent report by Wood and Schwartz (1978) showed that the Qlo for the effect of ouabain on Ca 2+ efflux from fragments of guinea-pig heart (Qlo = 1.64) was comparable to that found by Reuter and Seitz (1969) in their studies on Ca 2+ efltux from guinea-pig heart auricles (Q~o = 1.35). Overall the results of Wood and Schwartz (1978) appear to be consistent with the hypothesis that ouabain, by specifically effecting a change in the Na + gradient, facilitates decreased J.F'.a. 35/3--a

152

P.V. SULAKnEand P. J. ST. Louis

Ca 2 + efflux. This would therefore tend to support the proposal that Na +-Ca 2 + exchange is the (principal) mechanism involved in Ca 2 ÷ efflux from the myocardium. Significantly, workers in this area have failed to document conclusively that [Na+]i is in fact elevated at concentrations of glycoside which induce an inotropic response (see Lee and Klaus, 1971 ; Akera and Brody, 1976) although correlation has been observed between the inhibition of myocardial N a +, K ÷-ATPase and enhancement of contractility (see review by Schwartz et al., 1975 ; Akera et al., 1977). For these reasons the opinion is held that glycosideinduced inotropy results from an indirect action of these agents rather than from an ionconcentration effect on a Na ÷-Ca 2 ÷ exchange system. The suggestion is that the interaction of the glycoside with the (membrane-associated) Na +, K ÷-ATPase perturbs membrane siteCa 2 + ion interactions, probably by altering the affinity of Ca 2 ÷ for lipids (see Lullman and Peters, 1976; Gervais et al., 1977). Recently, however, Akera and co-workers (Akera et al., 1976; Akera, 1977) have indicated that at normal heart rates inotropic concentrations of ouabain do in fact cause elevation of intracellular Na ÷ transients (that is a transient increase in [Na+]~ associated with each membrane excitation) in guinea-pig hearts. On the other hand, myocardial Na ÷ accumulation was observed only when the heart rate was increased (rate of stimulation raised to 240/rain from 120/min). These results in fact confirmed their computer-based predictions and are significant in that they answer the objections indicated above. That is, they indicate that specific alterations in [Na +]i can occur at levels of ouabain which induce an inotropic response. The idea of alterations ofintracellular N a ÷ transients has further implications with respect to ion fluxes and the regulation of cardiac muscle function. The probability must now be considered that a specific Na ÷ space exists in the region of the intracellular aspect of the cardiac plasma membrane. The concentration of Na ÷ in this space could be varied independently of the bulk myoplasmic N a +, thereby facilitating the occurrence of Na ÷ transients. Sodium ions in the two pools (sub-sarcolemmal matrix and myoplasm) would, of course, be in equilibrium. This Na ÷ matrix may well be closely associated with, or even identical with, the sub-sarcolemmal pool of activating Ca 2 ÷ proposed by Langer and others (see Langer, 1976 ; Nayler et al., 1976) to be involved in the excitation-induced release of Ca 2 ÷ for the activation of contraction. It is then conceivable that competition between Na ÷ and Ca 2 ÷ for the membrane-located sites would influence the effective internal concentrations of Na ÷ ions and Ca 2÷ ions. Such a system could thus be involved in the regulation of the activities of these ions (as opposed to the concentrations; see Fozzard, 1977). It must be emphasized that the foregoing discussion relates to the internal membrane-associated events rather than specifically to trans-membrane movements of ions. The manner in which the system suggested here may be involved in these ion fluxes will be considered later. (iv) Active (ATP-dependent) Ca 2 ÷ transport. A major problem with the exchange diffusion system as proposed by Reuter (1974) is that, on the basis of the known distribution ratio for Ca 2÷ and Na ÷ across the myocardium the proposed mechanism dictates that the intracellular Ca 2 ÷ concentration would be 10- 5 M. However, the free myoplasmic Ca 2 ÷ ion concentration during relaxation must be of the order of 10-7 M (see Langer, 1973; Reuter, 1973). Energetically the 2 : 1 pump ratio is sufficient to maintain [Ca 2 ÷]~ levels of 10- 7 M only if [Na+]~ is 1-2 mM, suggesting that either [ N a + l is lower than usually estimated or some other factors must be involved in the removal of Ca 2 ÷ from the cell. In this regard Fozzard (1977) has indicated, as a result of the determination of the activity coefficient of N a ÷ in heart muscle, that the use of values for the chemical concentration of Na ÷ in the interpretation of experimental results may be misleading. However, the possibility must be considered as suggested by Reuter (1974) that there is another energy source, in addition to the Na ÷ gradient, which mediates the efflux of Ca 2 ÷ from the myocardial cell. Such a source, probably an energy-requiring system, was originally proposed by Weber (1966) who suggested that a Ca 2 + pump system, comparable to the so-called N a ÷ pump, may operate to transport Ca z ÷ out of the cardiac muscle cell against its concentration gradient. Numerous workers have, subsequently, attempted to identify Ca 2 ÷-dependent ATPase and Ca 2 ÷-transport activities, comparable to those identified in red blood cells and in muscle sarcoplasmic reticulum, in plasma membrane fractions from cardiac muscle.

Passive and activecaldumfluxesacrossplasmamembranes

153

(b) Isolated membrane studies (i) Isolation of cardiac sarcolemma. Several procedures for the isolation of cardiac sarcolemma have been reported (Anand et al., 1977; Feldman and Weinhold, 1977a,b; see St. Louis and Sulakhe, 1976a, for earlier references). Some of these procedures are in fact modifications of methods described for the isolation of sarcolemmal membranes from skeletal muscle. Essentially the methods reported involved the treatment of homogenates or crude membrane fractions with salts such as KCI or LiBr, to effect solubilization of contractile proteins, in combination with differential centrifugation and/or gradient centrifugation to separate various subcellular particles. As is usually the case purity was estimated by analysis for specific marker enzymes, including Na +, K+-ATPase, adenylate cyclase and 5'-nucleotidase, and contamination by other subcellular fractions was estimated using putative marker enzymes. The following brief discussion will serve to indicate the diverse nature of the various sarcolemmal preparations. It should be clarified that not every aspect considered is applicable to all the preparations. On a protein basis the yield of sarcolemma (where given) relative to homogenate was generally of the order of 5% (Stare et al., 1970; Williamson et al., 1975; Hui et al., 1976; St. Louis and Sulakhe, 1976a). Values as low as 0.5% were reported (Barr et al., 1974; Wollenberger et al., 1975) while particularly high recoveries were reported by Tada et al. (1972; 8%) and Jarrot and Picken (1975; 15%). Na +, K+-ATPase activity in most preparations was in the range 4.5-10.5 #mol/mg/hr for sarcolemmal membranes from rat and pig heart respectively. In the latter reports there is unfortunately no comment on the homogenate activity and hence on the relative purification of the fractions. In addition sarcolemmal membrane fraction isolated by Wollenberger et al. (1975) exhibited particularly low fluoride-stimulated adenylate cyclase activity (122 pmol/mg/min) when compared to values quoted for other preparations (up to 3600 pmol/mg/min; Williamson et al., 1975 ; Hui et al., 1976; St. Louis and Sulakhe, 1976a). On the basis of these two enzyme activities sarcolemmal membranes obtained by various groups was purified, relative to homogenate, 4-15-fold. It should be noted that variations in specific activity of the same enzyme in different sarcolemmal preparations may reflect both species differences as well as variations in the isolation procedures. However, in none of the reports examined was the relative purification of Na +, K+-ATPase comparable to that of adenylate cyclase. This apparent discrepancy, as well as the relatively low degree of purification of sarcolemma (on the basis of marker enzyme activity) should be viewed in the light of the known complexity of the sarcolemma. Thus as McNutt (1975) has shown the cardiac plasma membrane is physically complex, comprising several distinct layers of regions. Further, it is (considered to be) the locus of numerous enzyme activities, specific ligand receptors, etc. Finally the various preparations described have been useful in contributing to our knowledge of the functions of cardiac plasma membranes. Recent studies by Besch et al. (1976) and Jones et al. (1977) showed an apparent latency of Na +, K +-ATPase in microsomal (sarcoplasmic reticulum) membranes from cardiac muscle. This of course has particular implications concerning the use of this enzyme as a marker for plasma membranes. Indeed, St. Louis and Sulakhe (1976a) have shown that variations of the conditions used in the preparation of membranes can cause marked activation, or loss of, both Na +, K ÷-ATPase and adenylate cyclase in membrane fractions isolated from cardiac muscle. Further, reports by Entman et al. (1969), Katz et al. (1974), Sulakhe and Dhalla (1973) and Sulakhe and Narayanan (1978) suggest that cardiac microsomal preparations possess adenylate cyclase activity. In order therefore to partly answer the objection that the Na +, K +-ATpase and adenylate cydase activities observed in cardiac sarcolemmal fractions may well be, in part, due to contamination by sarcoplasmic reticulum membranes, St. Louis and Sulakhe (1976a) examined specific [3HI ouabain binding to membranes. Sarcoplasmic reticulum displayed negligible binding activity (0.01-0.03 pmol/mg) which was unaffected by various ligand conditions. Binding to sarcolemma on the other hand exhibited response characteristics similar to those reported for purified, Na +, K +-ATPase from cardiac muscle and on this basis (specific ouabain binding) sarcolemma was purified about 7-fold (activity in the presence of ATP + N a + was 10.56 pmol/mg). Interestingly, these sarcolemmal

154

P.V. SULAKHEand P. J. ST.Louis

membranes also exhibited approximately 7-fold increase (over homogenate) in specific activity of Na +, K +-ATPase. It would appear, from a more recent report by Besch et al. (1977), that Na ÷, K+-ATPase and adenylate cyclase activities displayed by (classically) isolated cardiac sarcoplasmic reticulum membranes may in fact be due at least in part to the presence of sarcolemmal vesicles in these membranes. The foregoing observations underline the problems associated with the isolation of purified subcellular membranes and emphasize the need for critical evaluation of such preparations as are obtained. The morphology of cardiac sarcolemma from several species is well defined as a result of electron microscope examination of gross preparations (see McNutt, 1975). Examination of isolated cardiac sarcolemma from rat (Kidwai et al., 1971), frog (Barr et al., 1974) and guineapig (Tada et al., 1972, Hui et al., 1976; St. Louis and Sulakhe, 1976a; Anand et al., 1977) showed that the preparations comprised vesicles of varying size having a characteristic triplelayered appearance as well as sheets and fragments of extracted myofibrils. In all these preparations contamination by other subcellular fractions was found to be absent or negligible. In summary, therefore, preparations of isolated cardiac sarcolemmal membranes do represent fractions of varying degrees of purity. However, they have proved to be valuable tools in studies on the possible existence of an active Ca2+-transport system in the sarcolemma which may contribute to the regulation of myocardial levels of Ca 2+ and hence influence contractility. (ii) Ca 2+-stimulated A T P a s e s in cardiac sarcolemma. Ca 2 +-activated A T P a s e . Several groups have earlier described the isolation of cardiac sarcolemmal membrane preparations which exhibited Ca 2 +-dependent ATPase (Ca 2 +ATPase) activity (Boldyrev, 1971; Dietz and Hepp, 1971; Sulakhe and Dhalla, 1971; McNamara et al., 1974). Of interest was the observation by Sulakhe and Dhalla (1971) that the activity was maximal when the concentrations of Ca 2+ and ATP were equimolar suggesting that CaATP 2- was the substrate for the enzyme. More recently, reports from Hui et al. (1976) and Sulakhe et al. (1976a) state that values of the order of 12 #mol/mg/hr and 36 #mol/mg/hr respectively were obtained for Ca 2 +-ATPase ( C a 2 + = 5 mM) present in well characterized guinea-pig cardiac sarcolemmal membrane, while Anand et al. (1977) have shown that rat heart sarcolemma exhibits Ca 2 ÷-ATPase activity which was maximally stimulated at 5 mM Ca 2÷ (40 #mol/mg/hr). This activity exhibited some differences in properties, such as pH optimum, when compared to Ca 2 +ATPase in cardiac microsomal and mitochondrial fractions. Stam et al. (1970) have reported the presence of Ca 2 +-ATPase in a dog heart sarcolemmal fraction (3 #mol/mg/hr). Interestingly, ouabain had no effect on the enzyme at pCa 2 + of 8.2 but was inhibitory at pCa 2 ÷ of 6. It was thus suggested that this Ca 2 +-ATPase may be involved in a Ca 2÷ efflux reaction and further that the observed effect of ouabain could be related to its inotropic action on heart muscle. It was suggested by the groups cited above that this Ca 2 +-ATPase activity could be involved in the active efflux of Ca 2+ across the cardiac plasma membrane. It is interesting to note, at the same time, that these preparations all displayed a separate Mg 2+-ATPase activity which was not stimulable by Ca 2 +. In fact Mg 2+ was found to inhibit (up to 30~o) the sarcolemmal Ca2+-ATPase activities described by Dhalla and co-workers in various sarcolemmal preparations (McNamara et al., 1974; Dhalla et al., 1977; Anand et al., 1977). Ca 2 +-stimulated Mg 2 + - A T P a s e (Mg 2+, Ca 2 +-ATPase). It was reported by Stam et al. (1973) that their sarcolemmal membrane fraction from dog heart possessed, in addition to Ca2+-ATPase, a Mg2+-ATPase activity which could be stimulated by Ca 2÷. The stimulation of the ATPase at #M levels of Ca 2 + (pCa 2+ = 6), as well as the inhibition of this stimulation by so-called "interstitial" levels of Na + (120 raM) was interpreted to suggest that the activity was localized at the intracellular sarcolemmal surface and probably involved in Ca 2+ efflux. More recently, Mg 2+-ATPase which could be stimulated by #M levels of Ca 2 + has been identified in well characterized plasma membrane preparations from guinea-pig cardiac muscle (Hui et al., 1976; Sulakhe et al., 1976a). The Mg 2+, Ca 2 +-ATPase activity described by Hui et al. (1976) was of the order of 9 #mol/mg/hr while the extra-ATPase

Passiveand activecalciumfluxesacrossplasmamembranes

155

[-(Mg2+ -+- Ca 2+) - Mg 2+] was approximately 3/zmol/mg/hr. The corresponding values from the data of Sulakhe et al. (1976a) were Mg 2+, CaZ+-ATPase = 42/zmol/mg/hr and extra-ATPase = 6/zmol/mg/hr. These values may be compared with those reported for guinea-pig cardiac microsomes by Nayler and Fassold (1977): Mg 2+, Ca2+-ATPase = (approx) 80/zmol/mg/hr and extra-ATPase = approx) 48/zmol/mg/hr. It is now fairly well accepted that the sarcoplasmic reticulum extra-ATPase activity reflects the energy liberating component of the membrane Ca 2 ÷ pump. The relatively lower magnitude of the cardiac sarcolemmal ATPase (compared to that in sarcoplasmic reticulum) does not however preclude its involvement in a similar efflux system. This rather suggests that the (postulated) sarcolemmal system may well have (kinetic) characteristics distinct from those of the sarcoplasmic reticulum system. The activation of the Mg 2 +-ATPase by/.tM levels of Ca 2 + may well be significant in that the system would be expected to be functional at lower levels of intracellular ionized Ca 2 +. This observation is based on the operation of the sarcoplasmic reticular system as the principal mechanism for lowering the "effective" concentration of myoplasmic Ca 2 +.

(iii) Calcium binding to sarcolemma. ATP-independent binding. Cardiac sarcolemma membrane preparations isolated by Williamson et al. (1975) and by Wollenberger et al. (1975) have been shown to exhibit ATPindependent Ca 2 +-binding activity. Williamson et al. (1975), using Scatchard plot analysis, determined the presence of high affinity (Kin 16/zM) and low affinity (Kin 800/~M) sites. They suggested that the low affinity sites may represent specific externally-oriented sites for Ca 2 + entry into the myocardium while the high affinity (low K ~ sites may represent Ca 2 + bindingsites on the inside surface of the sarcolemma related specifically to a Ca 2 +-effhix system. Wollenberger et al. (1975), on the other hand, determined the presence of two classes of high affinity (low Kin)binding-sites in their membrane preparation. These workers have proposed that these sites may be concerned with a system for the extrusion of Ca 2+ from the cell via either an ATP-associated Ca 2+ pump or a Na+--Ca 2+ exchange mechanism. ATPindependent Ca 2 +-binding activity has also been described in rat heart sarcolemma by Dhalla et al. (1976) who indicated that they had no evidence for energy-linked Ca 2+ binding to their membranes. The data presented also showed that the Ca 2 +-binding activities at 50 /zM and 1.25 mM Ca 2 + (approx. 29 and 260 nmol/mg/5 rain) were inhibited up to 50% by ATP at concentrations up to 4 mM. Addition of Mg 2+ in the assay also markedly inhibited this ATP-independent Ca 2÷ binding. It may be noted that this sarcolemmal preparation exhibited Mg 2 +-ATPase and Ca 2+-ATPase and that the Ca 2 +-ATPase was also markedly inhibited by Mg 2÷ (see also Anand et al., 1977). ATP-independent Ca 2 + binding was exhibited by a sarcolemmal preparation obtained from rat heart by Feldman and Weinhold (1977a); oxalate markedly enhanced (3-fold) this Ca 2 +-binding activity. At the same time, it was found that Ca 2 + (at 10/tM) stimulated a Mg 2 +-ATPase activity present in the fraction raising the possibility that this activity may be involved in a Ca 2 + pump system. A subsequent report by Feldman and Weinhold (1977b) has described the isolation of a calcium binding lipoprotein component from their preparation of rat heart plasma membrane. This lipoprotein exhibited a high Ca 2 + capacity (52 moles Ca +/mole lipoprotein). In view of its high Ca 2 + affinity (74/zM) it was suggested, however, that the lipoprotein is in fact located at the extracellular side of the membrane and functions as a membrane storage site for Ca 2 +. Limas (1977) reported two classes of calcium binding sites, one of high affinity(Kinaround 30/zM) and the other of low affinity (K, around 2 mM), in the rat heart sarcolemmal preparation. Calcium binding to the membrane fraction was mostly due to binding of Ca 2 + to protein rather than lipids or sialic acids of the preparation. He also indicated that carboxyl residues are involved in Ca 2 + binding whereas sulphydryl, amino or thiol groups are not required. Lanthanum and hydroxylamine affected (reduced) Ca 2 ÷ binding to the high affinity site. Ruthenium red, on the other hand, inhibited both binding sites. Morcos and Jacobson (1979) have also observed that ATPindependent calcium binding to guinea-pig heart sarcolemma involves two classes of binding

156

P.V. SULAKHEand P. J. ST.Louis

sites. In the same study ATP-dependent calcium binding was shown to involve only one class of binding site (Kin around 74/AM). Very recently, we have found that ATP-independent calcium binding (measured at 10 s_ 10-2 M CaC12) to rat or guinea-pig sarcolemmal preparations occurs at more than one class of binding sites. The three sites detected, I, II and III, showed apparent affinity of 0.5-1/AM, 10-30/AM and 1.0-2 mM respectively. Suggestive evidence was obtained that indicated site I binding probably represents the high affinity binding to the Ca 2 + pump (Mg 2+, Ca 2+ ATPase) protein whereas site II represents the binding site of another protein (likely to be the calmodulin protein); both these sites are of inside-out vesicles present in the preparation. Site III, on the other hand, was that of the rightside-out vesicles and probably represents the external site on the presumptive Na+--Ca 2+ exchange carrier or glycoprotein(s) of sarcolemma. A T P - d e p e n d e n t Ca 2+ bindin#. Williamson et al. (1975), Wollenberger et al. (1975) and Limas (1977) have indicated that ATP could stimulate the Ca 2 +-binding activity of their respective sarcolemmal membrane preparations. However, no in-depth investigation of this activity was performed. Hui et al. (1976) and St. Louis and Sulakhe (1976a) have described (separate) procedures for the isolation of sarcolemmal membranes, from guinea-pig cardiac muscle, in a high degree of purity. Interestingly, these preparations exhibited ATP-dependent Ca 2 + binding and oxalate enhanced this activity. Guinea-pig heart sarcolemma isolated by Hui et al. (1976) bound 0.7 and 3.5 nmol CaZ+/mg/min in the absence and presence respectively of 2 mM ATP. The binding activity was rapid and reached a maximum within 1 minute. In the presence of 5 mM oxalate the Ca 2 ÷-transport (uptake) activity was increased to 8.8 nmol/mg/min. The specific activities of the Ca2+-transport activity present in sarcolemma isolated by St. Louis and Sulakhe (1976b) were comparable 0.5 and 4.5 nmol/mg/min in the absence and presence respectfully of 2.5 mM ATP, and 8-12 nmol/mg/min in the presence of ATP + 5 mM oxalate (Sulakhe et al., 1976a). On the other hand, Porsius and Van Zwieten (1976) using guinea-pig cardiac sarcolemma isolated by the procedure of Lullman and Peters (1976) have reported Ca2+-uptake activity (+ ATP + oxalate) of the order of 25 nmol/mg/min. Recently, Morcos and Jacobson (1979) observed about 2 and 12 nmole of Ca 2+ bound/mg protein in the absence and presence of ATP in guinea-pig sarcolemma prepared essentially by the method of Hui et al. (1976). An interesting finding in this study was that sodium ions selectively reduced ATP-dependent, but not ATPindependent, calcium binding in the membrane fraction. The effect of sodium showed both time and concentration dependence. These studies are all significant in that they provide more direct evidence for the presence of a plasma membrane localized Ca 2 + pump in cardiac muscle. The detailed studies of the cardiac sarcolemmal Ca 2 +-transport activity were reported by St. Louis and Sulakhe (1976b), and Sulakhe and St. Louis (1978). Thus it was shown that sarcolemmal Ca 2 + transport was stimulated by Ca 2+ up to 10 - 4 M (added in the assay); higher concentrations of Ca 2 + were inhibitory (St. Louis and Sulakhe, 1976b). The Km for Ca 2÷ was calculated to be in the range 10-20/AM both in the absence and the presence of oxalate. In this regard it is interesting to note that Mas-Oliva et al. (1979) have independently shown that cardiac sarcolemmal membranes isolated following the procedure of St. Louis and Sulakhe (1976a) displayed ATP-dependent Ca 2 +-binding activity with an apparent Ks for Ca 2 + of 25/AM. It should also be noted here that a direct comparison of some of the properties of the Ca 2 +-transport activities present in cardiac sarcolemmal and sarcoplasmic reticulum membranes has been reported (St. Louis and Sulakhe, 1976b; Sulakhe and St. Louis, 1978). The results presented showed that the properties of the ATP-dependent Ca 2 +transport activity of guinea-pig cardiac sarcolemma were clearly different from those of sarcoplasmic reticulum from the same tissue. While ATP was shown to enhance Ca 2÷ binding by sarcolemma in a concentration dependent manner, the bound Ca 2 + could be released upon depletion of ATP in the medium (St. Louis and Sulakhe, 1976b). This statement is based on the observation that in the presence of ATP at 0.1 and 0.25 mM sarcolemmal bound Ca 2 + increased up to 30 sec and release of Ca 2 + occurred thereafter; increasing the concentration of ATP delayed the release reaction and no release was evident after 20 min incubation in the presence of 2.5 mM ATP. This observation is important since

Passiveand activecalciumfluxesacrossplasma membranes

157

for meaningful participation ofthe sarcolemmal system in the contraction-relaxation cycle it is necessary that Ca 2+ binding be reversible. (iv) Implications of the type of membrane preparation used in the study of A TP-dependent Ca 2+ transport. Guinea-pig cardiac sarcolemma isolated by Hui et al. (2976) and by us (Sulakhe et al., 1976a) exhibited both Ca 2 +-ATPase and Mg 2 +, Ca 2 +-ATPase (see earlier section) as well as the ATP-dependent Ca 2 +-transport activity discussed above. Likewise Ca2+ATPase and ATP-dependent Ca2+-transport activities were described in a rat heart sarcolemmal fraction prepared by Funcke and Rattenhuber (1976) and in the sarcolemmal membrane preparation used by Mas-Oliva et al. (1979). Presence of these enzymes was also reported by Morcos and Jacobsen (1979) in rat and guinea-pig sarcolemmal preparations. It would thus appear possible that ATPase (either Ca 2 +-activated and/or the extra ATPase) could be associated with the ATP-dependent Ca 2 +-transport activity, forming part of an energy-linked Ca 2 +-pump system. The results so far presented by various laboratories do not allow us to conclusively answer this question. The stoichiometric relationship between Pi liberation (reflected by extra ATPase) and Ca 2+ uptake (oxalate present) in terms of moles ATP hydrolyzed per mole Ca 2 + transported was calculated to be in the range 2-10 (St. Louis and Sulakhe, unpublished data, also see St. Louis and Sulakhe, 1978). This apparently low efficiency may well be a reflection of the various factors described below. Nature of membrane preparations. Electron microscopic observation has indicated that the sarcolemmal membranes isolated by Hui et al. (1976), Funcke and Rattenhuber (1976), and St. Louis and Sulakhe (1976a) are to a large degree, but not completely, vesicular. By definition Ca 2 + uptake (or accumulation) implies the occurrence of a translocation reaction across a vesicular membrane into the intravesicular space. Hence non-vesicular membrane fragments in a preparation would not be expected to contribute to the total uptake capacity of the preparation but would still display Ca 2 +-activated ATPase, thereby lowering the observed efficiency. " t eaky" vesicles. It is quite likely that of the membrane vesicles present in isolated sarcolemma, some portions have lost the selective permeability characteristic of the intact membrane. That is, the procedures used for isolation including salt extraction, may have rendered these vesicles more permeable, i.e. "leaky". In such a case the continued loss of Ca 2 + from the intravesicular space would be expected to reduce the observed efficiency for the ATP-coupled activity. Uncoupling. The techniques used for isolation of (sarcolemma) membrane fragments, including mechanical disruption of the tissue and extraction in the presence of high salt concentrations, may well effect, to a variable degree, the uncoupling of the ATPase and Ca 2 +-transport activities. In fact this could be one reason why some groups have failed to observe ATP-dependent Ca 2 +-transport activity in membrane preparations which exhibit Ca 2 +-dependent ATPase activity. The suggestion that the Ca 2 +-dependent ATPase and ATP-dependent Ca 2 +-transport activities exhibited by isolated cardiac sarcolemma reflects the existence of a sarcolemmal Ca 2 + pump implies that the membrane preparations comprise or contain vesicles which are oriented inside out. The degree of stimulation of enzyme activities, such as ouabain-sensitive Na +, K +-ATPase, present in sarcolemmal membranes induced by treatment with detergents can be used to estimate the relative contribution of IO and RSO vesicles in a membrane preparation. Using this approach we have examined the effects of deoxycholate on the Na +, K+-ATPase, K+-sensitive p-nitrophenyl phosphatase and specific [3H]-ouabain binding activities of isolated sarcolemmal membranes. Analysis of the results obtained indicate that our preparation comprises approximately 50% IO and 50% RSO vesicles (St. Louis and Sulakhe, 1978a). It is quite likely therefore that the preparations described by Hui et al. (1976) and Funcke and Rattenhuber (2976) may also be heterogeneous with respect to orientation. It now becomes necessary to attempt to resolve such membrane preparations into fractions with homogeneous populations of vesicles. This will greatly facilitate the investigation and characterization of the properties of the (proposed) cardiac sarcolemmal active Ca 2 +transport system.

158

P.V. SULAKHEand P. J. ST. LOUIS

In summary, therefore, the evidence discussed here strengthens the proposal that a unique plasma membrane-localized Ca 2 +-transport system exists in heart muscle and that this system mediates, in vivo, the active efflux of Ca 2 + across the myocardium. More conclusive proof will depend on critical analysis of the observed Ca 2 ÷-stimulated ATPases and ATPdependent Ca 2 +-transport activities and in particular on the isolation of pure preparations which exhibit a more reasonable stoichiometry between ATP hydrolysis and Ca 2 ÷ transport. (v) N a + - C a 2 + exchange in isolated membrane vesicles. Sarcolemma. All the studies so far discussed which deal with the possible presence of a Na +-Ca 2 + exchange mechanism in the plasma membranes of cardiac muscle cells have been performed using perfused muscle preparations. There are two recent reports however which are of significance in that they provide data, from studies using isolated (fragmented) muscle membrane preparations which support the existence of a sarcolemmal-located Na +-Ca 2 + exchange mechanism in cardiac muscle. Reeves and Sutko (1979) and Pitts (1979) have both shown that isolated membrane vesicles previously loaded with Na + can accumulate Ca 2 + (in the absence of ATP) and this uptake activity was dependent on the transmembrane Na + gradient. Indeed external Na + [Na +]o markedly inhibited Ca 2 + uptake (1/2 maximal inhibition at 16 mM Na +) and La 3 + could displace only part (20%) of the pre-accumulated Ca 2 + suggesting that the bulk of this Ca 2 + had been sequestered within the membrane vesicles (Reeves and Sutko, 1979). In addition the data obtained by these latter workers also suggested that the vesicles had effected the accumulation of Ca 2 + against a concentration gradient. Another interesting point that emerged from the work of both groups is the apparent requirement of the uptake activity for K + ions. This will be discussed later in this section. The observations just mentioned, in themselves, are not striking in that essentially they document (ATP-independent) Ca 2÷ uptake by (plasma) membrane vesicle fractions. Assuming that the vesicles are inside-out with respect to the intact membrane in situ, then the observed activity may well reflect the existence of a Ca: +-efflux activity. However, in order to document the counter-transport of Na ÷ and Ca 2÷ (i.e. N a + - C a 2÷ exchange)in these membrane preparations both Reeves and Sutko (1979) and Pitts (1979) have studied the Ca 2 +-efflux-Na ÷-influx activity. Briefly, membranes were pre-loaded with Ca z ÷ (or 45Ca2 ÷) and the efflux of Ca 2 + and/or the uptake of Na ÷ by loaded vesicles was investigated. Reeves and Sutko (1979) observed that when KCI or LiC1 was present in the medium Ca 2 + efflux exhibited a rapid initial phase, which accounted for 10-15~ of the total ettlux, and a subsequent slow phase with a t 1/2 of 9.4 min. In the presence o f K C l + NaCI, however, there was a dramatic increase in the total rate of Ca 2 ÷ efflux (t 1/2 = 0.31 min) indicating a marked dependence on [Na+]o. This approach was taken further by Pitts (1979) who showed, by following the 22Na + influx into C a 2 +-lOaded vesicles and the Na +-induced efflux of 45Ca2 + from loaded vesicles that the apparent N a + - C a 2 ÷ exchange exhibited a stoichiometry of three Na + for one Ca 2 +. It may be noted that such a stoichiometry has indeed been suggested for the proposed Na +-Ca 2 ÷ exchange in squid axon (Blaustein, 1976) and, further, this ratio would render the system electrogenic. Faced with this problem, proponents of the Na +Ca 2 + exchange mechanism including Pitts have implied that another ion, probably K +, may also be transported by the carrier in such a manner as to render the Na +-Ca 2 ÷ exchange system electroneutral. It is interesting to note that while Ca 2 ÷ uptake by the membranes could reflect the in vivo presence of a Ca 2 +-efflux activity, experiments involving Ca 2 ÷ release-Na + uptake by isolated IO membrane vesicles would really represent, in vivo, a Na ÷-Ca 2 + exchange activity mediating Ca 2 ÷ influx into the myocardium as suggested by Langer and co-workers (Langer, 1977). The more rigorous experiment necessary to document a Na ÷-Ca 2 ÷ exchange activity mediating Ca 2 + etttux would involve the examination of the Ca2+-uptake-Na+-release activity of(the) membranes. Pitts (1979) does, in fact, briefly examine this using 1 mM Ca 2 ÷ in the external assay medium; the other experiments involving 45Ca 2 + uptake and Na ÷ release had employed 40 #M C a 2+. The results obtained in this experiment showed that Ca 2÷ markedly activated 22Na+ efflux from previously loaded vesicles. Of significant interest,

Passive and activecalciumfluxesacrossplasmamembranes

159

however, is the fact that ATP (2 mM) was added in the assay medium, It is thus difficult to preclude the possibility of ATP involvement in the efflux ofNa + from the membrane vesicles in this particular experiment. It is evident that more detailed experiments in this direction are needed. One point of concern in the studies just described is the purity of the membrane preparations used. As Reeves and Sutko (1979) themselves stated, their membrane fraction indeed contained fragments of mitochondria and sarcoplasmic reticulum. However, based on preliminary observations, they suggested that the observed Na +-Ca 2+ exchange activity is due to the sarcolemmal fragments in their preparation. The cardiac sarcolemmal membrane preparation used by Pitts (1979) was more purified than that of Reeves and Sutko (1979) but probably also contained some intracellular membrane contaminants. Pitts (1979) in his report indicated that, in his hands, isolated cardiac mitochondria did not display significant Ca 2+ uptake into Na+-loaded membranes and proposed that the Na+-Ca 2+ exchange activity displayed by his membranes was probably not due to mitochondrial contamination. However, it has been shown in a series of elegant experiments by Carafoli and Crompton (1978) that mitochondria isolated from rat heart exhibited a Na +-induced Ca 2 +efflux activity. In fact Crompton et al. (1979) suggested that mitochondria possess two distinct mechanisms for the extrusion of Ca 2+. Further, it appears that in heart mitochondria the Na +-Ca 2 ÷ antiporter system is very active while the other system (Na ÷-insensitive Ca 2 ÷ efflux) is much less active. Further experimentation should resolve the apparent discrepancy in the results from these two laboratories. To summarize, therefore, the in vitro studies of Reeves and Sutko (1979) and Pitts (1979) indeed document that isolated cardiac plasma membranes can accumulate Ca 2÷ in the absence of ATP but in the presence ofa Na ÷ gradient. This activity is stimulated by K ÷ and is purported to reflect the operation of a Na ÷-Ca 2÷ exchange activity. However, as is evident from our discussion, some questions do remain to be answered, such as the actual in vivo direction of the observed activity and the specific membrane fraction(s) responsible for the observed activity. Another point worthy of note is the apparent involvement of the other major cellular cation, K ÷, in regulation of the exchange-carrier mediated Na ÷ and Ca 2 ÷ fluxes. Significantly, K ÷ markedly stimulated the ATP-dependent Ca z ÷-uptake activity (St. Louis and Sulakhe, 1976b) and Ca 2÷-stimulated ATPase activities (St. Louis, 1979) exhibited by isolated cardiac sarcolemmal membranes. It is thus likely that K ÷ indeed plays a regulatory role in the activity of membrane-associated Ca 2 ÷-transport systems. In any case investigations of the actions ofK ÷ as well as Na ÷ on membrane Ca 2 ÷ pumps are of potential significance and may provide clues concerning the possible interactions between the Na ÷ pump (Na + K +ATPase) and the cardiac sarcolemmal Ca 2+-efflux activity whether it be a Na +-Ca z ÷ exchange carrier system or a "Ca 2 ÷ pump" activity. Na+-Ca 2÷ exchange system in mitochondria and its role in Ca 2+ efflux from mitochondria. Since the initial report by Vasington and Murphy (1962), a vast number of studies have shown that mitochondria isolated from virtually all mammalian tissues are capable of in vitro accumulation of Ca 2 ÷ (see recent reviews by Carafoli and Crompton, 1976, 1978; Bygrave, 1977). Much recent work indicates that inward transport of Ca z ÷ by mitochondria occurs by an electrophoretic process driven by the proton motive force generated by coupled respiration and results in a transfer of two charges. The postulated mechanisms of charge neutralization by Ca 2+-H ÷ antiport (Reed and Bygrave, 1975; Akerman, 1978) or Ca 2 +P O ] - symport systems have now been questioned by the findings of Crompton et al. (1978). The reported apparent affinity of the uptake system towards Ca 2÷ has varied from one study to another, and apparently depends on the tissue or the method of determination. For heart mitochondria, the apparent Km of about 10 gM has been found by Crompton et al. (1976a,b), which interestingly shifts to 30 #M in the presence of Mg 2 +. Mitochondria from heart and liver differ in several regards including the effect of Mg 2 +, which is an effective inhibitor of the cardiac uptake system but produces only a weak inhibition of liver mitochondrial Ca 2 ÷ uptake. Mg 2+ also influences the kinetic behaviour in that hyperbolic dependence on Ca 2+ of the uptake system observed without Mg 2÷ changes to a sigmoidal (or co-operative) dependence in the presence of Mg 2÷ (see Crompton et al., 1976a,b; Vinogradov and Scarpa,

160

P.V. SULAKHEand P. J. ST. LOUIS

1973; Akerman et al., 1977). With the phosphate present, a maximal rate of 3-10 nmol Ca 2 +/sec/mg protein has been observed under in vitro assay. Mitchell (1966) and Mitchell and Moyle (1969) have estimated a membrane potential difference of 180 mV (negative inside) across mitochondria. In view of transfer of two charges per cycle by the mitochondrial Ca 2 ÷ carrier (as an electrophoretic uniporter), a gradient of Ca 2 + activities across the inner membrane of 106 will be expected based on the Nerst equilibrium. Indirect measurements, however, suggest a much lesser gradient, being no greater than 103. For example, Denton et al. (1972, 1978) showed that the matrix Ca 2 ÷ oscillates from 10- 5 to 10- 6 M and the generally accepted cytosolic Ca 2 + concentration appears to be around 0.1 ~M in "resting" state thus providing an estimate of Ca 2 ÷ gradient to be about 102-103. Such observations imply that an equilibrium is usually not attained between the mitochondria and the medium (cytosol), since if attained against - 180 mV potential difference, the cytosolic Ca 2 ÷ level will be extremely low (10-11 or lower), which is not the case. The work by Drahota et al. (1965) indicated that following its accumulation, Ca 2 ÷ can be released from the mitochondria. Since then a number of substances, such as phosphate (Rossi and Lehninger, 1964), prostaglandins (Malmstr6m and Carafoli, 1975), phosphoenol pyruvate (Chudapongse, 1976), pyridine nucleotides (Lehninger et al., 1978), and monovalent cations, especially Na + (Carafoli and Crompton, 1976) have been found to promote the release of Ca 2 ÷ from mitochondria. In the present context, emphasis is given to the mechanism by which Na ÷ stimulates mitochondriai Ca 2÷ efflux in order to see if any similarities are found between the mitochondrial and plasma membrane systems. Significant progress in the study of the Na +-dependent Ca 2 + efflux was made following the observation of Moore (1971) that ruthenium red completely inhibited the Ca 2÷ influx into the mitochondria via the electrophoretic uniporter (Carafoli et al., 1974). Using cardiac mitochondria, the Carafoli group suggested a mechanism for the Na ÷ promotion of Ca 2 ÷ efflux (noted in the presence of ruthenium red) which did not appear to be a simple reversal of the influx mechanism. Instead, their study suggested a separate mechanism for Ca 2 ÷ efftux. Since then, extensive work by Crompton and Carafoli has favored this postulate (see Crompton et al., 1976a,b; 1979; Crompton and Heid, 1978). The results of Stuckie and Ineichen (1974), however, suggest that ruthenium red binding to mitochondria is influenced by the potential difference across inner membrane and imply that some Ca 2 ÷ release is likely to occur in the presence of ruthenium red via the electrophoretic uniporter. Caroni et al. (1978) found that the Ca 2 +-Ca 2÷ exchanger is indeed inhibited by ruthenium red in deenergized mitochondria and their results apparently ruled out the likelihood of the electrophoretic uniporter mediating the efflux of Ca 2÷. The possibility of N a + - H + antiporter operating in association with a fast H +-Ca 2 ÷ antiporter and thus accounting for the Na +-promoted Ca 2 ÷ release is not supported by the findings of Nicholl (1978). Nicholl's results imply that Na + requirement of Ca 2 + release is not the result of dissipation of excess H ÷ in the matrix. Thus, although further work is needed to establish unequivocally the presence of a highly specific N a + - C a 2 + exchange carrier in mitochondria mediating Ca 2 + efflux, it is safe to conclude that the available data do not exclude this interesting mechanism at least in certain tissues. There still remains a puzzle why mitochondria from tissues such as liver, kidney and lung fail to show the Na +-promoting effect on Ca 2 + efflux (Crompton et al., 1978 ; Nicholl, 1978 ; Nedergard et al., 1979), although these mitochondria do release Ca 2 +. Comparative analysis of the heart and liver mitochondria by Crompton et al. (1979) indicates that the contributions by the Na ÷-sensitive and -insensitive mechanisms vary in the two tissues examined; in the cardiac mitochondria, the Na ÷-sensitive antiporter is much more active whereas the reverse is the case of liver mitochondria. Nevertheless, the results from the Carafoli laboratory do indicate that the mechanism for Ca 2 ÷ efflux is separate from the influx mechanism and do not support the postulates of Bygrave (1978) and Pozan et al. (1977) that implicate a single carrier mediating bidirectional flux of Ca 2 + across mitochondrial inner membrane. Carafoli suggests that, since the influx and etttux rates for Ca 2÷ are approximately similar (even though mediated by different systems), under in vivo situations mitochondria from "non-pathological" tissues do not store substantial amounts of Ca 2 ÷ but instead are critical in short term regulation of intracellular Ca 2 +. This view is indeed very

Passiveand activecalciumfluxesacrossplasmamembranes

161

interesting and offers an explanation how calcification of mitochondria can be prevented under normal in rive situation. Further, if one accepts the estimation that up to 90% of the total Ca 2 + transport membrane area in tissues (except red blood cell) is accounted by mitochondria, it will indeed be difficult to overlook the important role for this organdie in the intracellular regulation of ionized Ca 2 + irrespective of the questions the affinity of both the influx and efflux mechanisms towards Ca 2 + and the likely factors that initiate or regulate Ca z + efflux and influx. Further, the activity of the Na+-Ca 2 + exchange system of cardiac mitochondria (10-20 nmol/gm wet weight/sec, 25°C) apparently is much greater than that of the heart sarcolemma (0.1-1 nmol/gm wet weight/sec). For the latter membrane, very recently a stoichiometric ratio of 3 Na + : 1 Ca 2+ in the Na +-Ca 2+ exchange cycle has been obtained (Pitts, 1979), which supports the concept of an electrogenic exchange. Similar stoichiometric data, however, are not as yet available for the cardiac mitochondrial exchange system. Carafoli and Crompton (1978), however, have interpreted the sigmoidal nature of the Na+-promoted Ca z+ release from mitochondria to mean that at least two Na + ions are exchanged per Ca 2+. It would be interesting to know whether Ca 2+ efflux from the inside-out vesicles ofmitochondrial inner membrane is accompanied by Na + uptake, which apparently has been observed now for heart sarcolemmal vesicles (Reeves and Sutko, 1978; Pitts, 1979) and synaptosomal vesicles (Rahamimoff and Spanier, 1979). (vi) Monovalent cation effects on the Ca 2 + pump of sarcoplasmic reticulum. Sarcoplasmic reticulum from fast skeletal muscle and other muscle types has been established to contain a highly active Ca 2 +-transport system (see reviews by MacLennan and Holland, 1976; Tada et al., 1978). In recent years, considerable attention has been given to the possible regulation of this pump by monovalent cations, especially Na + and K +. These studies have provided very interesting results and are of considerable relevance to the present article. Many years back, Carvalho and Leo (1967) noted that the total cation binding capacity of isolated sarcoplasmic reticulum was similar when Ca 2 + uptake was determined with and without ATP present in the assay. ATP, which promoted the net Ca 2 + uptake by these membranes, appeared to cause two main effects--it changed the apparent affinity for Ca 2 + of the uptake system and the net gain of Ca 2+ was accompanied by a proportionate loss of either Mg 2 + or K +. Duggan (1966), Katz and associates (Rubin and Katz, 1967; Katz and Repke, 1967), Sulakhe (1971), and Sulakhe and Dhalla (1973) reported an increase by monovalent cations in the ATP dependent, oxalate-facilitated storage of Ca 2+ into the sarcoplasmic reticulum vesicles. De Meis (1970), on the other hand, found a marked inhibitory effect ofNa + or K +. However, in these studies the mechanisms involved in the monovalent cation effect were not analyzed in detail. Recently, Shigekawa and associates (Shigekawa and Pearl, 1976; Shigekawa et al., 1976) and Duggan (1977), using skeletal muscle sarcoplasmic reticulum, and Jones et al. (1977, 1978), using cardiac sarcoplasmic reticulum, have provided some details concerning the likely mechanisms involved in the effect of alkali metal salts on the Ca 2+ pump. Briefly, the findings of these investigations demonstrated that K+-(or Na +-) stimulated Mg 2 +, Ca2 +-ATPase and the rate of ATP-dependent, oxalate-facilitated storage by accelerating the rate of breakdown of phosphoprotein intermediate of the Mg 2+, Ca 2 +ATPase. Jones et al. (1978) also showed that for a maximal effect of K +, mM concentrations of ATP are required and proposed an allosteric control by both monovalent cation and ATP of the rate-limiting dephosphorylation step of the cycle. Ribeiro and Vianna (1978) suggested the presence of two K + sites, only one of which interacted with Mg 2 +. However, since in the cell sarcoplasmic reticulum bathes in the cytosolic milieu containing 100-150 mM K + and 510 mM Na +, it is not clear whether indeed K + (or monovalent cations) truly "regulate" the Ca 2 + pump as their concentrations do not change appreciably. Further, K + or monovalent cations also promote the release of previously accumulated Ca 2+ in the sarcoplasmic reticulum (Sulakbe and Kowalsky, 1978; and unpublished observations), and this efflux did not necessarily reflect a reversal of the "pump" (Kirchberger and Wong, 1978 ; Katz et al., 1977). It is conceivable that monovalent ions may influence the membrane structure in general or the pump protein, in particular. In other words, monovalent cations maintain a configuration of the pump protein in the membrane such that it displays its maximal

162

P.v. SULAKHEand P. J. ST. Louis

function, i.e. Ca 2 + influx coupled to ATP hydrolysis. Supportive evidence in this regard is that by Louis et al. (1974) which showed an orderly proteolytic degradation of the ATPase protein only when K + (or other monovalent cations) were present during tryptic exposure. Further evidence favoring the conformational and stabilization roles of monovalent cations was obtained in our laboratory using rat fast skeletal muscle sarcoplasmic reticulum (Sulakhe and Kowalsky, 1978). Briefly, in agreement with the finding by Louis et al. (1974), trypsinization of the sarcoplasmic reticulum or partially purified ATPase led to orderly fragmentation of the ATPase protein only when K + or Na + (100 mM) were present. We interpreted this observation to indicate that trypsin-sensitive portions in K+-induced conformation of the ATPase protein were limited whereas in the absence of monovalent cations, the ATPase protein had many more trypsin-sensitive portions made accessible due to its "unnatural" configuration. Thus, K+-induced conformation, which our data also showed to display the maximal Ca2+-transport ability or Mg 2÷, Ca 2 +-ATPase activity, represents the "native" state of this protein in the membrane. Experiments in which sarcoplasmic reticulum was isolated in the "K +-free" or "K + -containing '' states and was stored, following isolation, without and with K +-containing media, clearly indicated that K + (or Na +) conferred the stability to the "pump" as well. In fact, it seemed that certain minimal amount of K + surrounding the pump protein must be present in order for it to remain in its active state, since a "monovalent cation-free" sarcoplasmic reticulum was irreversibly inactivated. Calcium ions are also capable of imparting the stability to the ATPase protein (see Mclntosh and Berman, 1978). Such observations indicate that monovalent cations may effectively maintain the stability and integrity of sarcoplasmic reticulum membrane and of the ATPase protein, in particular and this is reflected in the reported increase in the energydependent Ca 2 + uptake, Mg 2 +, Ca 2 +-ATPase and Ca 2 +-efflux activities of the membrane. One important point to note is that, while K + or Na ÷ are equieffective on the sarcoplasmic reticular Ca 2 + pump (even though the apparent affinity for K + is slightly higher than for Na +) and produce similar action, the Na +-Ca 2 ÷ exchange system shows a much greater specificity for Na + compared to K +. Whether or not monovalent cations also influence the plasma membrane, the internal and external sides of which are exposed to high K ÷ and high Na + respectively, remains yet to be investigated. It is nevertheless recognized that external Ca 2 + can regulate Na +-permeability (Van Breemen et al., 1979) and the internal Ca 2 +-, K +permeability (Lew and Ferriera, 1978). The reverse, that is whether asymmetric distribution of Na + and K + across cellular plasma membrane controls Ca 2 +-permeability of this membrane, is an interesting topic for future study. (vii) Phosphorylation of cardiac sarcolemma. It is well established that fl-adrenergic catecholamines and cholinergic agents have opposing effects on myocardial function including contractility as well as myocardial metabolism. Considerable evidence is also available that rapid and marked changes occur in the levels of cyclic nucleotides in the myocardial cell following interaction of adrenergic and cholinergic agents with their specific sites (receptors). In a number of instances, an increase in cyclic A M P has now been shown to precede a fl-adrenergic amine-induced increase in the force of contraction. Electrophysiological studies also suggested that epinephrine accelerated Ca 2 + influx via voltage-dependent slow Ca 2 + channels, an effect that was probably mediated by cyclic AMP. There are also reports that describe a stimulatory effect of cyclic AMP on the active uptake of Ca 2 + by cardiac sarcoplasmic reticulum and which appears to involve phosphorylation of specific protein (phospholamban) of these membranes. This latter observation has been interpreted to be of significance in the fl-adrenergic amine-induced increase in the rate of myocardial relaxation. Several interesting schematic models have been published that link cyclic nucleotides and Ca 2 + as mediators in the cardiac effects of a variety of agents including epinephrine. The basic view in all these postulates centers around the likelihood of cyclic AMP-promoted phosphorylation of proteins or enzymes regulating a number of Ca 2 +-regulatable processes including actomyosin shortening, Ca 2+ pump of sarcoplasmic reticulum as well as glycogenolysis. In the following section, we describe the results from the studies of

Passiveand activecalciumfluxesacrossplasma membranes

163

myocardial sarcolemmal phosphorylation. Most of these have utilized isolated sarcolemmal membrane-enriched preparations but there is some evidence that under in vivo conditions, sarcolemmal proteins are indeed phosphorylated. Phosphorylation of isolated sarcolemmal fractions. Independently, Hui et al. (1976) and Sulakhe et al. (1976a) observed that a well-characterized preparation of guinea-pig ventricular sarcolemma contained protein kinase activity that effected phosphorylation of sarcolemma. Krause et al. (1975) and Dowd et al. (1976), on the other hand, used sodium deoxycholate-extracted microsomal preparation enriched in the Na +, K+-ATPase, and found that exogenous protein kinase catalyzed phosphorylation of this preparation. Dowd et al. (1976) noted the phosphorylation of two low molecular weight polypeptide regions--one of about 16,000 and the other of about 12,000 molecular weight. Krause et al. (1975), on the other hand, observed the phosphorylation of a 24,000 molecular weight polypeptide band. Several speculative interpretations were put forward from these initial attempts. (1) Sarcolemmal phosphorylation in general and phosphorylation of a specific protein in particular may mediate the increased Ca z+ influx that reportedly occurs following interaction of fl-adrenergic amine with the cardiac muscle. This then would have significance in initiation of myocardial contraction. It was of course assumed that phosphorylation is dependent on or stimulated by cyclic AMP and catalyzed by the membrane-associated or cytosolic enzyme (protein kinase). (2) Phosphorylation may have a direct action on the sarcolemmal ATP-dependent Ca 2÷ pump which mediates the "active" efflux of this cation since Mg 2 +, Ca2+-ATPase and oxalate-supported calcium uptake activities were proportionately increased by protein kinase in the presence of cyclic AMP. This pump would thus assist the sarcoplasmic ventricular pump in bringing about the increased rate of relaxation. (3) Phosphorylation may influence either the activity or efficiency of the classical Na ÷ pump, which may have a capability to act like a Ca 2÷ pump and thus mediate Ca 2 ÷ efflux. (4) Phosphorylation could also influence the Na+--Ca 2÷ exchange carrier that presumably mediates Ca 2 ÷ efflux as well. In order to establish whether or not each one of these postulates represent a likely event in the myocardial cell, additional work is undoubtedly required and has been undertaken in many laboratories. Recently, several publications have been addressed to these critical issues and the findings obtained so far are clearly divergent, although certain similarities can also be found. In our laboratory we have been examining phosphorylation of sarcolemmal preparations from guinea-pig and rat heart ventricles (Sulakhe and St. Louis, 1976, 1977a,b, 1978; Sulakhe et al. 1976a, 1977, 1978; St. Louis and Sulakhe, 1978a, 1979). The results obtained are summarized below. Sarcolemmal preparations contained endogenous protein kinase activity and protein substrates for this enzyme. Endogenous kinase showed many characteristics similar to those observed with the cytosolic (purified) cyclic AMP dependent protein kinase; these included half maximal stimulation by 0.5 /tM cyclic AMP, apparent Km of 50 pM towards ATP, dependence ofMg 2 ÷ (half-maximal activity at 1-2 mM) as well as histone (type II)-induced dissociation of the holoenzyme. A weak but consistent stimulatory effect of added cyclic AMP was considered the result of the formation of sufficient cyclic AMP by sarcolemmal highly active adenylate cyclase during incubation (see Fig. 3). When following phosphorylation, sarcolemmal proteins were fractionated by SDS-polyacrylamide disc gel electrophoresis, a number of polypeptides were phosphorylated (Fig. 4); peak e (M,, 48,000) was the major phosphorylated band. Sarcolemmal proteins were also phosphorylated by exogenous protein kinase(s)--purified from heart or skeletal muscle of beef and rabbit. Exogenous kinase(s) increased phosphorylation of most of the bands seen with endogenous protein kinase; however, there was a marked increase in phosphorylation of bands f (Mr, 22,000) and g (Mr, 16,000). Purified phosphoprotein phosphatase effected dephosphorylation of all phosphorylated bands. Exposure to trypsin, but not to phospholipase C, of the membrane fraction reduced phosphorylation ofsarcolemma and of peaks c (Mr, 86,000), fand #. The above mentioned observations were obtained with guinea-pig heart membrane and with a short (6 cm) electrophoretic separation of the sarcolemmal proteins by the WeberOsborn technique. A significant limitation in this technique was that the exact identity of the

164

P. V, SULAKHEand P. J. ST. Louis 300

E ~ 200

/

o~ ,_c

/

~

~11"

cAMP. Histone

100

Ill

-cAMP

1

.cAMP

2

3 MINUTES

4

5

FIG. 3. Demonstration of endogenous protein kinase in isolated cardiac sarcolemma. Note that cyclic AMP produced only a weak stimulatory effect in the absence of histone and a marked stimulatory effect in the presence of histone IP. J. St. Louis and P. V. Sulakhe, unpublished data, 1976).

0,2 10(

0.4

Relative mobility 0.6

0.8

1.0

autophosphorylation of PK

5

10

15

20

25

30

35

40

45

50

20 jug purified PK(RC) ÷ SL 20 pg purified PK(C) + SL

40(

SL

.~_ E

g 3O(

c

e

20¢

10(

, , , ,

. . . . . . . . . . . . . . . 5

10

15

,

20

. . . .

L . . . . . . . . . . . . . . 25

slice

30

35

,

40

. . . . . . . . .

45

, 50

number

FIG. 4. Identification of phosphorylated proteins of cardiac sarcolemma. Following its isolation, the membrane fraction (from guinea-pig heart ventricle) was incubated without and with exogenous protein kinase (purified from bovine skeletal muscle; RC, holoenzyme; C, catalytic subunit) and [? - 3 2 p ] A T P . Membranes were then solubilized in SDS and phosphoproteins identified by electrophoresis, followed by counting of radioactivity in gel slices (each 1.13 mm thick). Upper panel (A) shows autophosphorylation of exogenous kinase (membrane fraction absent) and lower panel (B) shows the incorporation of radioactivity into various polypcptide bands under three different assay conditions (P. J. St. Louis and P. V. Sulakhe, unpublished data, 1976).

Passiveand activecalciumfluxesacrossplasmamembranes

165

phosphorylated band with the corresponding polypeptide band of the stained gel could not be achieved. In the subsequent study, we utilized the slab gel electrophoretic method of Laemmli and phosphorylated proteins were identified by autoradiography. Some rather unexpected and interesting observations were made. Because the separation of phosphorylated bands (high and low molecular weight) was considerably better (15 cm total distance from the origin to the dye front) using either low (6-7.5~o) or high (12.5-15~) acrylamide contents of the separating gel, many more phosphorylated bands were readily detected. Further, a direct comparison between heart sarcolemmal and sarcoplasmic reticulum proteins could be made. The following results were obtained. Heart sarcolemma--with endogenous kinase, phosphorylation of peptides of Mr, 94,000, 63,000, 60,000, 46,000 and 10,000 was detected and 46,000 peptide incorporated the maximum 32p_ phosphate. With exogenous kinase, many additional phosphorylated bands were detected, especially of high molecular weights; the most prominent of these were of Mr, 20,000, 140,000, 115,000 and 94,000. Exogenous kinase also promoted phosphorylation of 63,000, 60,000, 46,000, 16,500, 14,500, i1,000 and 9,500, with very high incorporation in M, of 9,500 and 46,000 (Figs. 5 and 6). Heart sarcoplasmic reticulum--endogenous kinase promoted phosphorylation of 60,000, 49,000, 46,000 and 9,500 molecular weight peptides while exogenous kinase promoted phosphorylation of 90,000, 60,000, 14,500 and 9,500 molecular weight peptides. These results hence showed some similarities and differences in the phosphorylatable peptides of guinea-pig cardiac sarcolemma and sarcoplasmic reticulum. However, the rather striking observation made was the absence of significant phosphorylation in the region of 20,000-22,000, which is a major phosphorylated band of dog heart sarcoplasmic reticulum (Kirchberger et ai., 1975; Tada et al., 1975; Tada et al., 1979; Jones et al., 1979). Recently, using dog heart sarcolemmal vesicles isolated from the microsomal fraction by sucrose density gradient centrifugation, phosphorylation of 165,000, 90,000, 56,000, 24,000 and 11,000 molecular weight peptides was observed in the presence of cyclic AMP and endogenous kinase (Jones et al., 1979). The electrophoretic technique used was that of Laemmli or Porzio and Pearson, and autoradiographic detection of the phosphorylated peptides was carried out. Their results are quite comparable to those described above. This is very interesting since the methods for isolation of sarcolemma used by us and Jones et al. (1979) are quite different. Jones et al. (1979) suggest that sarcolemma contains a distinct protein kinase, which is not present in sarcoplasmic reticulum. More work is, however, required to establish this with certainty. Despite the methodological differences for isolation of sarcolemma, general agreement exists that fl-adrenergic receptors and catecholamine-stimulable adenylate cyclase are mainly present in the cardiac sarcolemma. The work from our laboratory has, in addition, described the presence of g-adrenergic receptors (Wei and Sulakhe, 1979), muscarinic cholinergic receptors (Ma et al., 1978 ; Wei and Sulakhe, 1978; Wei and Sulakhe, 1979), digitalis receptors (St. Louis and Sulakhe, 1976b; Sulakhe et al., 1978), guanylate cyclase (Sulakhe and St. Louis, 1975; Sulakhe, P. V. et al., 1976b; Sulakhe, S. J. et al., 1976; Sulakhe and Sulakhe, 1978; St. Louis and Sulakhe, 1976c), cyclic nucleotide phosphodiesterases (St. Louis and Sulakhe, 1976c) and ATPase(s) (Sulakhe et al., 1976a, 1977, 1978 ; St. Louis and Sulakhe, 1976a,b, 1978a). Phosphorylation of low molecular weight polypeptides of cardiac sarcolemmal preparations.

Dowd et al. (1976) reported an interesting finding that the Na +, K+-ATPase-enriched preparation from beef heart contained two low molecular weight polypeptides (16,000 and 11,500) which were phosphorylated by exogenous cyclic AMP-dependent protein kinase. However, phosphorylation failed to show any action on Na +, K+-ATPase activity of the preparation, an observation that was also reported by us with neural Na +, K+-ATPase (Sulakhe et al., 1976c). One interesting speculation put forward by Dowd et al. (1976) was that phosphorylation increases the efficiency of the Na + pump in the cell. Using a similar type of preparation (that is, the membrane fraction stripped of extrinsic membrane proteins by detergents and high salts), the WoUenberger group (Krause et al., 1975), however, observed phosphorylation of a 24,000 molecular weight peptide. In the subsequent study from this laboratory, phosphorylation of a 11,500 molecular weight peptide was observed using purified pigeon heart sarcolemma. As described above, we, as well as Jones et al. (1979) have

166

P.V. SULAKHEand P. J. ST. Louis

cA

+cA+PK

- -- -I- 1 2 3 4 5 6 (xl~r ~ ...........

cA -

+

+cA+PK

PK alone

1 2 3 4 5 6 6

230 -i 150 -~

Mr (x4n-3) I |

63_! 60 ..................

46-

43-!

26-i 17r~

- 14.5 : 10.5 sarcolemma

sarcoplasmic reticulum

FIG. 5. Wells numbered I through 6 show phosphorylation with increasing concentrations of protein kinase (PK) in the presence of cyclic AMP (cA, 10-6M).

Mr

0

12

34567

0 1 2 3 4 5

(x 10-3)

67

Mr (x 10-3)

T -86

T 928262-

_49 -46

46-

16.514.511.0-

-14.5 -11.0 - 9.5

9.5sarcolemma

sarcop lasmic reticulum

FIG. 6. 0, means cA or PK absent during phosphorylation; 1, cA present; 2 through 7, cA present with protein kinase concentration increasing from 2 to 7.

FIGS. 5 and 6. Autoradiographic detection of phosphorylated polypeptides of heart sarcolemma and sarcoplasmic retieulum. Following phosphorylation, electrophoresis was carried out by the Laemmli procedure and the dried (stained) gel was exposed to Kodak X-ray film (St. Louis and Sulakbe, unpublished data, 1977). Electrophoretic separation was carried out on the gel containing 10% acrylamide (Fig. 5) or 12.5~o aerylamide (Fig. 6) in order to achieve better resolutions of the high and low molecular weight polypeptide regions.

Passiveand activecalciumfluxesacrossplasma membranes

167

found phosphorylation of peptide of very similar molecular weight in guinea-pig and dog heart sarcolemmal preparations. Feldman and Weinhold (1977b) have isolated a lipoprotein (molecular weight 12,300) from rat heart sarcolemma and have shown that it has a very high calcium binding capacity (52 mole/mole). However, location of the lipoprotein is not yet established, although Feldman and Weinhold suggest that it may be located externally (i.e. facing extracellular fluid). Identity of the Feldman-Weinhold peptide with the phosphorylated low molecular weight peptide reported by Dowd et al. (1976), Will et al. (1978), Jones et al. (1979) and by us (see above) is of great interest. Wollenbcrger and Will (1978) have indeed offered an interesting suggestion that the phosphorylated low molecular weight protein of pigeon sarcolemma could conceivably form the wall of the membrane channel presumably identical to the "slow" Ca 2 + channel. A similar view is also expressed by Jones et al. (1979). Further support to this postulate comes from the observations by Reuter (1979), Tsien (1978), Sperelakis and Schneider (1976) as well as Watanabe and Besch (1974) that imply a role of cyclic AMP (presumably via phosphorylation of a specific protein) in the fladrenergic amine-induced increase in the slow Ca 2 + current (or influx) in cardiac muscle. However, whether or not in vitro phosphorylation of a 10,000-12,000 molecular weight peptide is markedly enhanced by cyclic AMP is still not clear. Will et al. (1978) and ourselves observe a weak but consistent stimulatory effect of cyclic AMP whereas Jones et al. (1979) show a marked stimulation by cyclic AMP of its phosphorylation catalyzed by endogenous kinase. On the other hand, Will et al. (1978) and ourselves observe a marked increase in the phosphorylation with exogenous kinase ( _ cyclic AMP) whereas Jones et al. (1979)) do not observe this. In fact, they could not detect phosphorylation in this region in their autoradiogram obtained with a short exposure of the X-ray film. Not only are the species used (guinea-pig, dog, pigeon) different but so it is true for the methods of isolation of membrane fractions as well as phosphorylation assay conditions. One hopes that future investigations will clarify these discrepancies. Phosphorylation of sarcolemmal preparations in relation to Ca 2 + binding and storage by the membrane preparations. In 1976, Hui et al., and Sulakhe et al. (1976a) described an increase in Ca 2 + uptake (ATP dependent, oxalate-facilitated) following phosphorylation of guinea-pig sarcolemma by endogenous or exogenous protein kinase; calcium binding (ATP dependent or independent, but assayed without oxalate), however, was not influenced. The increase in Ca 2 + uptake was not due simply to an increase in negative charge added to the membrane preparation due to its phosphorylation. Hui et al. (1976) noted that for every nmole of phosphate incorporated, there were extra 15 nmole of Ca 2+ taken up by the membrane. As shown in Fig. 7, we observe a stoichiometric relationship of 230 (increase in Ca 2+ taken up/increase in phosphate incorporated), which argues strongly against an involvement of a simple electrostatic or charge interaction in the observed increase in Ca 2 +

c E .c_

20 ol • Exp.2

i

7

/ 10

0

.~5

.1;

~Phosphorylation(nmole/mgprotein/5 min)

FIG. 7. Stoichiometric relation between the increase in Ca2+ uptake (ATP-dependent, oxalatefacilitated) and in phosphorylation of heart sarcolemma(St. Louisand Sulakhe,unpublished data, 1977). J.P.a. 35/3---C

168

P. V, SULAKI-IEand P. J. ST. LOUIS 10( B

40

0.01 m M Cacl 2 1.0 m M

3o

/

#

o ~ ....

8(

.....

::.-2

ec

20 /

tO

.e ........

J

2~ catalytic subunit i 2

I 5 Minutes

2 I

ILO

2

5

30 L

10 Minutes

FIG.8. Effectof purifiedmuscleprotein kinaseon calciumuptake by heart sarcolemmalvesicles.The results clearly show that purified bovine skeletal muscle kinase catalytic subunit increases ATPdependent, oxalate-facilitatedCa2+ uptake by heart sarcolemma under various assay conditions (P. J. St. Louis and P. V. Sulakhe, unpublished data, 1977).

uptake. In our initial studies (Sulakhe et al., 1976a; Sulakhe and St. Louis, 1978) and those by Hui et al. (1976), Will et al. (1976) and Funcke and Rattenhuber (1976) partially purified protein kinase (bovine heart) preparations were used. Recent studies from our laboratory indicated a similar effect using a fully purified protein kinase--either from heart or skeletal muscle (see Fig. 8). Will et al. (1976) found that sarcolemmal vesicles prepared according to the method of Lullman and Peters (1976) contained phosphorylatable substrate proteins, whose phosphorylation was effected by endogenous or exogenous kinase. The increase in Ca 2 + uptake per unit time by phosphorylation was dependent on the concentration of Ca 2 +, with an almost 7-fold increase seen at 0.14 #M Ca 2 +. ATP-dependent Ca 2 ÷ binding was also reported by the Nayler's group (Mas-Oliva et al., 1979) using sarcolemmal vesicles essentially isolated by the method of Sulakhe et al. (1976a; also see St. Louis and Sulakhe, 1976a). The above mentioned observations support the view of modulation of sarcolemmal Ca 2 ÷ fluxes by phosphorylation. However, whether or not these are related to increase in Ca 2 ÷ influx or efflux or both is not yet clear. There are two major problems that require discussion at this point. The first deals with the purity of the sarcolemmal preparations used at least in terms o f a likely contamination by the fragments of sarcoplasmic reticulum which possesses an active Ca 2 + pump. This is a very complex issue to be resolved conclusively at this time. We have provided and discussed in detail the evidence that the observed sarcolemmal fluxes are probably not the result of such contamination (Sulakhe et al., 1976a; St. Louis and Sulakhe, 1976a,b,c, 1979; Sulakhe and St. Louis, 1978; Sulakhe and Narayanan, 1978). A number of other studies (Hui et al., 1976; Mas-Oliva et al., 1979) tend to support this claim. On the other hand, the Besch group (Besch et al., 1976; Jones et al., 1979) report that the sarcoplasmic reticulum isolated by the Harigaya-Schwartz method (1969) contains sarcolemmal membrane fragments and hence, imply that a comparison between the Harigaya-Schwartz type sarcoplasmic reticulum preparation and sarcolemma-enriched preparations (isolated by our method or others) may not give clear-cut results or differences between these membranes. However, the main objection from such studies centers around whether or not sarcolemma contains an ATP-requiring Ca 2 ÷ pump that is being modulated by cyclic AMP-dependent phosphorylation. At the same time, Besch's group, as well as other investigators, suggest that sarcolemma binds Ca 2÷ in ATP-independent manner and that phosphorylation may modulate Ca 2 ÷ binding which presumably reflects modulation of the slow Ca 2 ÷ current (or influx) channel of sarcolemma. The other issue concerns the sidedness of the membrane vesicles comprising the sarcolemmal fraction. Recent work from our laboratory (St. Louis and Sulakhe, 1978a) described that sarcolemmal fraction contains both rightside-out (RSO) and inside-out (IO) vesicles of almost similar proportion (509/0 each). Such findings imply

Passive and activecalciumfluxesacrossplasmamembranes

169

that, provided the vesicles (RSO and IO) are permeability intact, ATP-supported, oxalate facilitated Ca2+-uptake activity is expressed mainly by the IO vesicles while ATPindependent Ca 2 +-binding activity is that of the RSO vesicles. Since phosphorylation influenced Ca 2 + uptake (Sulakhe et al., 1976a; Hui et al., 1976; Will et al., 1976; Sulakhe and St. Louis, 1978; also see Figs. 7 and 8), these observations may well support the role of phosphorylation in the Ca 2 + effiux via modulation of the Ca 2 + pump, the effect that is of significance in the accelerated rate of relaxation of the myocardium due to cyclic AMPpromoting fl-adrenergic catecholamines. On the other hand, passive (ATP-independent) Ca 2 +-binding activity has not been reported so far to be influenced by phosphorylation (Sulakhe et al., 1976a; Hui et al., 1976; Funcke and Rattenhuber, 1976) in well-characterized sarcolemmal preparations. However, in these studies, Ca 2 + used in the assay was in the #M range and hence it is likely that the assay conditions used were inappropriate. This is based on the assumption that the affinity of the Ca 2 + binding-site of the externally facing side of the membrane fraction towards Ca 2 + is low (say > 100 #M or even in the mM range). In more recent work, we observed a slight increase (20-30%) in the "passive" Ca 2 + binding assayed at 1 mM Ca 2+ following the kinase-catalyzed phosphorylation of sarcolemma. Further detailed study of this important aspect is needed to establish this. In situ phosphorylation of sarcolemmal proteins. It is generally agreed that in vitro phosphorylation of membranes, following their isolation, is of limited significance, especially if a correlation between the phosphorylation of a specific protein(s) and the change in membrane functions (ion transport, permeability, enzyme activities, etc.) cannot be tested. Protein kinase(s) are not a very specific enzyme(s) in that they tend to display a broad substrate specificity. In some cases, a non-phosphorylatable protein may become a "good" phosphate acceptor following alterations such as freeze-thawing of the membrane fraction, denaturation, etc. It is thus inevitable that considerable caution is required to interpret the results of in vitro phosphorylation studies of isolated membrane fractions. An approach of investigating the phosphorylation of membrane proteins under the conditions that likely prevail physiologically is, therefore, of great value. Perfusing rat heart with radioactive tracers (inorganic 32p-phosphate or ATP) in order to label the intracellular ATP pool and then isolating membrane fractions from control and stimulated (e.g. with catecholamine) hearts has been the approach recently utilized in the study by Walsh et al. (1979). Their results indicated that two phosphoproteins are present in a putative sarcolemmal fraction--protein A, of molecular weight 36,000 and protein B, of molecular weight 27,000. Increased phosphorylation of protein B occurred in response to epinephrine. Walsh et al. (1979) also observed phosphorylation of proteins of similar molecular weight in sarcolemma isolated by the method of Sulakhe et al. (1976a; also see St. Louis and Suiakhe, 1976a) and incubating it in the presence of exogenous protein kinase. Both in vitro and in vivo results indicated that protein A can be phosphorylated by cyclic AMP-independent and cyclic AMP-dependent protein kinases whereas that of protein B required cyclic AMP-dependent kinase. Interestingly, these phosphoproteins were not present in the rat heart sarcoplasmic reticulum preparation isolated according to the Harigaya-Schwartz method. Amongst the other findings, Walsh et al. (1979) observed that half-maximal stimulation by epinephrine of protein B phosphorylation occurred at 0.2 #M, was a result of the fl-adrenergic receptor stimulation (although glucagon, which also increases cyclic AMP in rat heart, promoted phosphorylation of protein B as well), and was maximal at 45 sec following epinephrine stimulation. The latter observation (i.e. the time course of stimulation of phosphorylation of protein B) raises an important question of its relevance in the epinephrine-induced stimulation of myocardial contraction. As Walsh et al. (1979) stated, under their experimental conditions, a maximum inotropic response was attained at 10-15 sec at which time there was only barely detectable increase in the phosphorylation of protein B. Although it is still possible that the sarcolemmal protein B is involved in the stimulatory action of epinephrine, along with other phosphorylatable (regulatory) proteins such as phosphorylase kinase, troponin and sarcoplasmic reticulum-bound protein(s), a detailed study is warranted to establish this. Nevertheless, Walsh et al. (1979) have provided significant direction in this area and their findings are indeed interesting and valuable.

170

P.V. NULAKHEand P. J. ST. Louis

(c) Does N a + - C a 2 + exchange carrier mediate Ca 2 + influx into the myocardial cell? In the preceding sections, evidence in support of an involvement of a Na +-Ca 2 + exchange carrier system in Ca 2 + efflux was discussed. There is, however, some evidence that the carrier may also assist or mediate Ca 2 + influx as well. This view has primarily been supported by Langer and associates (see Langer, 1976, 1977). Tillisch et al. (1979) observed that the contractile response of rabbit papillary muscle to altered external Na + consists of a brief initial transient change in tension development (with a t 1/2 of 1.1 min) followed by recovery in tension development similar to that obtained prior to changes in external Na +. Further analysis of the contractile response indicated that the fast initial component was dependent on the l-Ca 2÷ ]o :l-Na ÷]o ratio but the steady-state tension development was independent of external Na ÷. From these observations, they proposed a model that describes how the exchange carrier assists or mediates Ca 2 + influx. The carrier is considered to bind either Ca 2 + or Na ÷ in the ratio of n Ca 2 + to 2n N a +. A transient change in the tension response of rabbit papillary muscle due to alterations in [Na÷]o is thus reflected in this ratio (i.e. Ca 2 + :Na +) and further changes in [Na÷]i in response to [Na÷]o affect the rate of carrier movement. Since sarcoplasmic reticulum maintains a low I-Ca2+]i, competition between [Na + ]i and [Ca 2 ÷ ]i is minimal and therefore [Na +]i binding to the internal site of the carrier predominates in carrier regulation. It is also suggested that under physiological conditions, the net direction of exchange is [Ca2+]o-[Na+]v Hence, when [ N a + t is increased, the carrier movement is increased and this brings about increased [Ca2+], entry. Also, this would counteract the inhibitory effect of higher [Na ÷]o on Ca 2 ÷ influx. The reverse is then expected when [Na÷]i is decreased in response to lowering of [Na+]o. Further support for this view comes from the observation that Ca 2 + efflux is not affected when [Na +]o is changed from 36 to 200 mmol/1 (Wendt and Langer, 1977). Although Ca 2 + efflux is reduced when [Na+]~ is below 36 mmol/1, it is unlikely that increase in [Na+]o above this value exerts an effect on tension development by affecting Ca 2 + efflux (but without any action on Ca 2÷ influx). Thus two factors mainly appear to regulate Ca 2 + influx : (1) Na +-Ca 2 + competition at the external site on the carrier that assists in Ca 2 + entry by exchanging [Ca2+]o with [Na+]~, (2) rate of carrier movement that is primarily determined by [Na+]v The carrier hence can maintain steady state Ca 2 + influx at a constant level despite large changes in [Na+]o concentrations. Recently, Anderson et al. (1977) have provided the evidence in frog ventricle of a net Ca 2 + influx via the Na +-Ca 2 + exchange system. Ca 2 + influx was increased by increase in [Ca 2÷ ]o or decrease in [Na +]o as well as by positive electrical polarization of the membrane. For the latter intervention, it was suggested that the electrical field may cause a change in the relative dissociation constants of the carrier for the two competing cations such that stoichiometry or driving force remain unaffected and selective modification of the permeability brings about net influx of Ca 2 +. Interestingly, for the frog ventricle, Kavaler et al. (1978) suggest that relaxation (beat to beat) can be solely brought about by Ca 2 + efflux (mediated by the N a + - C a 2 + exchange carrier system) without any participation of the Ca 2 + pump in sarcoplasmic reticulum. Requirement for [Na +]o in the positive inotropic effect of fladrenergic catecholamine, isoproterenol, on guinea-pig atria was recently suggested by Linden and Brooker (1978). In this study, ability of isoproterenol to elevate intracellular cyclic A M P was reduced at low [Na +]o or when [Na +]o was absent but was not abolished. It was argued that during membrane depolarization, the presumptive N a + - C a 2 + exchange system favors Ca 2 + influx that precedes each beat (Benninger et al., 1976). When [Na+]o was low, diastolic tension was increased, an indication of raised [Ca 2 +]~ and this gain in [Ca 2 +]i could be a reflection of an inhibition of the rate of Ca 2 + efflux seen under low [Na +]o (Wendt and Langer, 1977). Also [Ca2+]i is believed to inhibit myocardial adenylate cyclase and increase cyclic A M P phosphodiesterase and this may account for the reduced cyclic A M P concentrations with or without isoproterenol in [Na+]o-free situations. Since time to peak tension or half relaxation-time were increased in [Na +]o-free medium, the likelihood of an impairment of sarcoplasmic reticular Ca / + pump was ruled out. Instead, it was suggested that isoproterenol-induced positive inotropism and its dependence on [Na +]o are initiated by sarcolemmal events. One such event could very well be that under normal [Na+]o isoproterenol influences the trans-sarcolemmal equilibrium between Na + and Ca 2+ such

Passive and activecalciumfluxesacross plasmamembranes

171

that the rate of Ca 2 + influx via the presumptive carrier is increased leading to the positive inotropic action. In summary, although the evidence favoring the likely role of the N a + - C a 2÷ exchange carrier in Ca 2 + influx in heart is not extensive and further the concept has been challenged by many, it is premature to overlook such a possibility. It would be equally interesting to know whether or not dependence of the isoproterenol inotropy on external Na + is reflected in the subtle requirement of this cation or external Ca 2+ in regulating the affinity of the fladrenoceptor, whose activation leads to increased synthesis of myocardial cyclic AMP. Further, whether or not cyclic A M P via sarcolemmal phosphorylation alters Ca 2 + influx via the presumptive carrier remains to be tested. 4. Smooth Muscle Various studies on smooth muscle indicate that activator Ca 2 + might originate from both extracellular and intracellular Ca z+ stores. Further, excitation-contraction coupling depends mainly on extracellular Ca 2÷ (Godfraind and Kaba, 1969; Bohr, 1973; Van Brcemen, 1977) and it appears that large tonic responses of (arterial) smooth muscle depend on the presence of extracellular Ca 2 ÷ ions while small phasic contractions can still be produced in CaZ+-poor solutions (Godfraind, 1976). Efflux of Ca z÷ across the plasma membrane is thus thought to be one mechanism whereby intraceUular Ca 2 ÷ levels are regulated in smooth muscle (see for example Bohr, 1973) and several groups have been seeking to identify the mechanism by which this efflux activity occurs. (a) Na+-dependent Ca z + eJ~ux Evidence is available which suggests that the transmembrane Na ÷ gradient is involved in the extrusion of Ca 2 ÷ from smooth muscle. Thus Katase and Tomita (1972) showed that, under specified conditions, Na + ions could effect the relaxation of taenia muscle and Reuter et al. (1973) have demonstrated the existence of a Na+-sensitive component of Ca a ÷ efflux from rabbit aortic muscle preparations. Goodford (1970) and more recently, Brading and Widdicombe (1977) have shown that superficial sites specific for Na ÷ and for Ca z÷ are present in taenia coli and have suggested that these may be involved in a N a + - C a a+ exchange reaction mechanism. Similar observations have been made by Raeymaekers et al. (1974) who also noted that varying external Na ÷ levels caused alterations in Ca 2 ÷ efflux from guinea-pig taenia coli. Van Brcemen et al. (1975) observed, also using taenia coli, that metabolic depletion induced a release of cellular 45Ca 2 ÷ from loaded preparations. Other reports by Brading (Brading, 1973, 1978; Brading and Widdicombe, 1976) also strongly suggest that Na ÷-dependent Ca z ÷ extrusion is, at least in part, responsible for mediating the net efflux of Ca 2÷ from smooth muscle. For example, Brading and Widdicombe (1976) showed that Ca 2 ÷ influx increased when [Na +]i was elevated in taenia coli. Also, depletion of intracellular ATP caused enhanced Ca 2 ÷ influx, probably due to increased membrane 'leakiness' and the rate of change in leakiness was affected by the Na ÷ gradient. Nonetheless, as discussed by Casteels and Van Breemen (1975) and by Brading (1978), calculations based on the (probable) distribution ofNa ÷ and Ca z ÷ across the smooth muscle plasma membrane indicate that the energy potential represented by the Na + gradient is not sufficient to account for the observed Ca z ÷ fluxes. (b) A TP-dependent Ca 2 + e~ux Van Brcemen and co-workers have shown that ATP is somehow involved in Ca 2 + effiux from taenia coli and have suggested that this outward movement is mediated by an ATPdependent Ca 2+ pump. Thus Van Brcemen et al. (1973, 1975) showed that, following metabolic (ATP) depletion, the small early change in the Na + gradient is insufficient to account for the observed change in Ca 2+ influx. In another report (Castcels and Van Breemen, 1975) several observations were reported. In preparations with normal ATP levels, when the Na + gradient was altered using K +-free perfusate, normal efflux rates and hence normal Ca 2 + levels were still maintained. Further, when tissue was loaded with 45Ca 2 + at 0°C (to minimize the energy dependent uptake of Ca 2 + by intracellular pools) extrusion

172

P.V. SULAKHEand P. J. ST, Louis

proceeded at relatively normal rates in the presence of lowered Na ÷ when the preparations were rewarmed to 37°C. Finally, when the Na ÷ gradient was very much reduced or reversed (by using Li 2 ÷ and/or choline solutions) net extrusion was still observed. Hence, Casteels and Van Breemen (1975) have concluded that Ca 2 ÷ extrusion in smooth muscle cells is a process which depends on metabolic energy rather than the energy represented by the transmembrane Na ÷ gradient. A recent excellent review by Van Breemen et al. (1979) discusses in critical detail, in relation to the molecular mechanism(s) of Ca 2 ÷ efflux, a large number of studies dealing with Ca 2 ÷ (and Na ÷ and K ÷) fluxes across the plasma membrane of various smooth muscle types. The studies briefly cited above are also included in their review. The overall conclusion which has emerged from this review is that while Na ÷ may indeed influence Ca 2÷ homeostasis, including both Ca 2÷ efflux and influx, yet a N a + - C a 2+ exchange mechanism does not appear to represent the sole or major mechanism for Ca 2 ÷ efflux from smooth muscle. For example, Katase and Tomita (1972) concluded from their studies of the effects of Na ÷ and Ca 2 ÷ on the recovery of taenia coli muscle preparations from K ÷ contracture that Ca / ÷ extrusion was mediated by an active Ca 2 ÷ pump involving a mechanism other than Na +-Ca 2 ÷ exchange. The studies by Van Breemen and co-workers (1979) involving the effects of metabolic inhibitors and of cooling on Ca 2 ÷ fluxes in taenia coli suggested that an inwardly directed Na ÷ gradient was not involved in Ca 2 ÷ extrusion and indeed studies by Raeymaekers et al. (1974) suggested that a large component of the Ca 2 ÷ efflux, which appears to be influenced by Na~+xt levels in fact involves the washout of Ca 2÷ from extracellular binding sites. Recent data from Van Breeman et al. (1979) also showed that the active extrusion of Ca 2 ÷ from taenia coli preparations can occur in the total absence of Na ÷ and in their review Van Breemen et al. (1979) further showed that Ca 2+ etttux from uterine smooth muscle is not affected by alterations in external Na ÷ concentrations. It is interesting to note that Van Breemen et al. (1979) suggested that a N a + - C a 2÷ exchange mechanism may be operating in series with the Ca 2 ÷ uptake pump present in the sarcoplasmic reticulum to promote net Ca 2 ÷ efflux via a sarcoplasmic reticulum uptake plasma membrane efflux pathway. This implies a linkage of the sarcoplasmic reticulum with the plasma membrane probably via a regulated pore (channel) system. The evidence for this, however, is at best tenuous. The studies so far cited have all involved the examination of ion fluxes using perfused smooth muscle preparations. Recently, however, there have been studies on the Ca 2÷transport and ATPase activities of broken cell preparations derived from smooth muscle which appear to provide data in support of the existence of a Ca 2 ÷ pump mechanism in the plasma membrane. These are discussed below. (c) Studies on isolated plasma membrane-enriched fractions ATP-dependent Ca 2 + binding and uptake, as well as Ca z ÷-stimulated ATPase activities, have been described in non-mitochondrial ("microsomal') membrane preparations (see Ford, 1976, for a brief review). However, the extent to which surface-derived membranes contribute to the total membrane composition of these preparations is still a matter of some debate. For example, ATP-dependent Ca 2 + incorporation was reported in a vesicular fraction from bovine aorta (Ford and Hess, 1975) which also exhibited Ca 2 +-stimulated ATPase activity (Hess and Ford, 1974). Although this preparation was enriched in Na 4, K ÷ATPase and 5'-nucleotidase data from enzymic analysis indicated a good amount of heterogeneity, indicating significant sarcoplasmic reticulum content. On the other hand, Wei et al. (1976) have reported the isolation and characterization of plasma membranes from rat mesenteric arteries. The preparation which was vesicular in appearance exhibited ATPdependent Ca 2 ÷-binding activity which was only slightly enhanced by oxalate and was little affected by Na ÷ or K 4. The properties of this Ca 2 ÷-binding activity were shown to be different from those of endoplasmic reticulum from the same tissue. The lack of any significant effect of oxalate implies that the vesicles were either relatively impermeable (to oxalate) or very leaky. The possible involvement of the observed Ca 2 ÷-binding activity in transplasma membrane Ca 2 ÷ movements was, however, not discussed by the authors and

Passiveand activecalciumfluxesacrossplasma membranes

173

further no mention was made concerning the presence or absence of Ca2+-stimulated ATPase in the membrane preparations. It is interesting that the same group of workers have reported the isolation and characterization of a sarcolemmal membrane fraction from rat and from human myometrium (Janis et al., 1977). These preparations, which appeared vesicular on electron microscopy, also exhibited ATP dependent Ca2+-binding activity and this activity was enhanced by oxalate. Further, the sarcolemmal membranes displayed Ca2+-stimulated ATPase activities and the results have been interpreted as providing direct evidence for a role for the plasma membrane in the regulation of Ca 2 + concentrations in smooth muscle (Janis et al., 1977). Ca 2 +-ATPase activity has also been described in a sarcolemmal membrane preparation from intestinal smooth muscle (Oliviera and Holzhacker, 1974). One important point noted by Janis et al. (1977), however, was the probable contribution of sarcolemmal membranes to Ca 2 ÷-transport activity exhibited by the endoplasmic reticulum (microsomal) fraction prepared from the myometrium. In this regard it could be noted that Ca 2+ incorporation by a microsomal fraction from guinea-pig ileum was reported by Godfraind et al. (1976) who suggested that the preparation was comprised of fragments of plasma membrane and of sarcoplasmic reticulum. In fact the "microsomal" fraction exhibited both Na +, K ÷-ATPase and Ca 2 +-ATPase. A similar situation was reported by Hurwitz et al. (1973) who found that the specific activity of Ca 2 + uptake in a "microsomal" membrane fraction and from guinea-pig ileum paralleled the specific activity of plasma membrane marker enzymes. Kutsky and Goodman (1978) have also shown that a microsomal preparation from canine aorta exhibited ATP-dependent Ca 2÷ binding and oxalate stimulated this activity. Interestingly, the preparation was enriched about 3-fold in both 5'nucleotidase (a plasma membrane marker) and NADPH-cytochrome c reductase (a microsomal marker). These observations reflect one problem inherent in attempts to purify subcellular membranes and that is the separation of homogeneous fractions. This problem is greater in the case of smooth muscle since, as described by Devine et al. (1972) and by Godfraind et al. (1973), electron micrographs show the presence of sarcoplasmic reticulum located close to the plasma membrane in association with mitochondria and caveolae. It follows therefore that so-called microsomal and plasma membrane fractions isolated from smooth muscle are, probably, heterogeneous. This raises obvious difficulties in deciding the significance of the Ca 2 +-transport activities, detected in these membrane preparations, in the Ca 2 ÷ homeostasis of the cell. The purity, as well as the orientation (sidedness), of membrane fractions isolated from smooth muscle will have to be determined before a meaningful answer to this question can be obtained. 5. Skeletal M u s c l e

In contrast to cardiac muscle, skeletal muscle contractility can be observed for several hours in the absence of extracellular Ca 2 +. This observation has generally been interpreted to indicate a lack of critical role of extracellular Ca 2+ or Ca 2 + fluxes across sarcolemma in regulating skeletal muscle contraction and/or relaxation. However, it is known, since the early study by Bianchi and Shanes (1960), that Ca 2÷ influx does occur following muscle activation, although a number of additional factors such as the quantity of Ca 2 + that enters and the speed at which it will diffuse and reach the myofibril to activate contraction, have raised a serious doubt about its direct role in initiation or maintainance of contraction. Instead, most investigators suggest that "activator" Ca 2+ is released from the internal storage sites such as those present in the extensively developed network of sarcoplasmic reticulum following muscle cell membrane depolarization by a process that is still not completely understood. Amongst the various possibilities, C a 2 + - i n d u c e d C a 2 + release or a simple change in the permeability of the terminal cisternae have received support from biochemical and physiological investigations (see reviews by Endo, 1977; Tada et al., 1978). There is no doubt that sarcoplasmic reticulum from fast skeletal muscle has an extremely active and high affinity Ca z + pump and considerable Ca 2 + storage capability such that both contraction and relaxation can be regulated in rive by the amount of Ca 2 + release from and

174

P.v. SULAKHEand P. J. ST. Louis

taken up by these membranes. This fact raises a serious question whether or not fast skeletal muscle indeed has any requirement of sarcolemmal transport system for Ca 24. In fact, such a view has hampered, in the previous years, detailed studies of sarcolemma from this tissue, either biochemically or physiologically, in terms of its role in Ca 2 + fluxes. However, in recent years, there is increasing evidence that Ca 2 + transport systems are present in plasma membranes from most tissues and there is no a priori reason to believe that it is absent in the surface membrane of skeletal muscle--fast or slow. The evidence that a concentration gradient for Ca 2 + across muscle cell exists is rather overwhelming. The question, therefore, is how this is brought about irrespective of whether Ca z ÷ flux across sarcolemma has a direct role in the mechanical events in skeletal muscle. Weber (1966) speculated that there must be an active efflux process for Ca z ÷ in skeletal muscle but failed to indicate the nature of such a process. Investigations of the presence of a Na +-Ca 2 ÷ exchange carrier in other excitable tissues mediating Ca z ÷ efflux provided one likely clue. This led Blaustein and associates (1971) and Di Polo and Caputo (1977) to test whether such a system is present in skeletal muscle. It is interesting that despite its existence in skeletal muscle noted in these studies, investigations of the Na +-Ca z ÷ exchange system have been very limited. On the other hand, the presence of Ca z +-ATPase and Ca 2 ÷-binding ability of isolated sarcolemma has now been documented in numerous biochemical investigations. Even though neither the radiolabelled Ca 2 ÷ efflux studies from the tissue preparations (i.e. slices or individual fibers) nor biochemical reports of Ca 2÷ binding and storage--energy dependent and independent--by isolated sarcolemma have established any physiologically relevant role of the sarcolemmal Ca 2 +-transport system in regulating skeletal muscle contraction and relaxation, it is well recognized that muscle sarcolemma are intimately involved in the excitation-contraction coupling mechanism. We have thus elected to present a brief review of the literature pertaining to these studies. Some recent investigations of the transverse tubular system have provided interesting information about the similarities and differences between this membrane and sarcolemma and thus these studies have also been included. As is the case for the sections on brain, heart and smooth muscle, isolation of putative surface membrane fractions and more recently of transverse tubular membranes is a complex subject and thus currently there are a number of controversies in this area. We are, however, optimistic that a continued and systematic study of this topical and critical subject will eventually provide useful and pertinent information. (a) Ca2 +efflux studies with isolated skeletal muscle preparations Nearly 20 years ago, Bianchi and Shanes (1960), and Cosmos and Harris (1961) observed Ca 2+ efflux from frog sartorious muscle and showed that it depended on the ionic composition of the external medium. In single frog muscle fibers, Ca 2 ÷ efflux occurs in three phases characterized by time constants of 18, 300 and 882 min (Curtis, 1970). This observation thus indicated a multicompartmental distribution of Ca 2 + in skeletal muscle. Following stimulation of the fiber, the compartment with a time constant of 300 min showed an increased turnover of Ca 2 + and this was interpreted to suggest that it was involved in the response of the fiber to electrical (or external) stimulus. Extensor longus digitii from frog were later found to contain four compartments involved in Ca 2 + exchange (Kirby et al., 1975). In more recent work, Caputo and Bolanos (1978) report that calcium washout from frog muscle after 1 hr has a time constant of 165 min and can be described by a single exponential curve. In fact, the rate coefficient remained constant after first 90 min of washout. In the same study, the fractional rate coefficients of Ca 2+ efflux showed dependence on external Na +. For example, it was reduced from 10.3 × 10 -3 min -1 to 4.1 × 10 -3 min -1 when external Na + was absent. It was estimated that about 60~ of the total C a 2 + efflux was dependent on external Na + and C a 2 +, a finding in agreement with that earlier observed by Bianchi and Shanes (1960), Cosmos and Harris (1961), and Watson and Winegrad (1973). The results from the study by Caputo and Bolanos (1978) suggested that C a 2 + efflux in frog muscle consists of three components, one sensitive to external N a ÷, one sensitive to external C a 2+ and one insensitive (or independent) to external Na ÷ and C a 2 +. A similar finding was earlier noted by Blaustein et al. (1971) with barnacle muscle fiber. An increased Ca 2 + influx into frog (Cosmos

Passiveand activecalciumfluxesacrossplasmamembranes

175

and Harris, 1961) or barnacle (Di Polo, 1971) muscle fibers has been observed when external Na + was absent. However, since Ca 2+ efflux of similar magnitude occurs when external Na + is absent, irrespective of the presence of external Ca 2+, it was suggested that the effect of reducing external Na + on Ca 2 + efflux is due to inhibition of the Na +--Ca2 + exchange system. Similarly, the inhibitory effect of reducing external Ca 2 + on Ca 2 + efflux could be explained by the lower activity of this exchange process. However, Curtis (1963) noted that reducing external Ca 2+ to less than 100/zM causes depolarization of muscle membrane and thus this could conceivably affect the membrane conductance to different ions including Ca 2+. Even though much more detailed work is required to establish the Na +-Ca 2 + exchange system in skeletal muscle, it is clear that considerable efflux (30--40%), which is insensitive to external Na + and Ca 2 +, must occur by mechanism(s) other than the Na +-Ca 2 + exchange process. Di Polo and Caputo (1977) indeed have observed the dependence on ATP of the exchange system. However, it is still not clear whether the effect of ATP is simply via alterations in the affinities of the carrier towards Na + and Ca 2 + or it is in fact hydrolyzed to liberate energy, which is utilized in the efflux process. (b) Calcium transport studies with isolated surface membrane fractions Sacrolemma isolated from frog (Koketsu et al., 1964; Sato et al., 1971), rabbit (Hotta and Usami, 1967; Madeira and Carvalho, 1972; Severson et al., 1972; Sulakhe et al., 1973a), rat (Schapira et al., 1974) and guinea-pig (Thorpe and Seeman, 1971) skeletal muscle is known to bind Ca 2+. In fact, Sulakhe et al. (1973a,b), showed that Ca 2+ binding to sarcolemmal membrane is enhanced in the presence of MgATP 2- and evidence was provided that this was not due likely to the presence of contaminating sarcoplasmic reticulum fragments. Numerous investigators have found the presence of an ATPase in isolated muscle sarcolemma whose activity can further be increased in the presence of Mg 2+ or Ca 2 + (Boegman et al., 1970; Sulakhe et al., 1971, 1973b; Severson et al., 1972; Andrew and Appel, 1974; Sulakhe and Drummond, 1974; Ferdman et al., 1974; Schapira et al., 1974; Heffron and Duggan, 1975; Barchi et al., 1977). In rabbit muscle sarcolemma, Mg 2 +-ATPase was found to be increased by micromolar Ca 2+ (Sulakhe et al., 1973a,b,c; Sulakhe and Drummond, 1974) and this activity was suggested to be of relevance in the ATP-dependent Ca2+-uptake activity. Following its uptake, Ca 2+ was shown to be released from the sarcolemma and La a +, in a concentration dependent manner, inhibited Ca 2 + efflux (Sulakhe et al., 1973a). These observations thus demonstrate that isolated sarcolemma from skeletal muscle possesses Ca 2 +-binding and -storage functions as well as ATPase(s) activities. An important implication from these biochemical investigations is that sarcolemma may contain an active Ca 2+-transport system that mediates active Ca 2 + efflux. (c) Ca2 +-A TPase-Mg2 +-A TPase of skeletal muscle sarcolemma It has been a puzzling feature that a highly active ATPase, which utilizes either Mg 2 ÷ or Ca 2+, is present in sarcolemmal preparations, even in those preparations that fail to show ATP-dependent Ca 2 +-binding and -storage abilities. There are at least three independent observations which suggest this activity to be that of an ecto-enzyme with its properties similar in many regards to myosin (or actomyosin) ATPase. As early as 1957, Marsh and Haugaard noted an ecto-ATPase on the surface of rat diaphragm. However, this was firmly established by the work of Manery and co-workers on the frog skeletal muscle (Dunkley et al., 1966; Manery et al., 1968). Carvalho et al. (1971) made an interesting observation that isolated rabbit muscle sarcolemma displayed a radial contraction in the presence of ATP. These workers concluded that the contraction was an intrinsic activity of sarcolemma. Such an observation also indirectly suggested the presence of contractile proteins in isolated sarcolemma. In many recent studies, the presence of myosin in cell plasma membrane from numerous tissues has been documented (see Brandon, 1975). The effects of a variety of salts of monovalent and divalent cations, - S H group reagents and other ATPase inhibitors observed on sarcolemmal Ca 2 +-ATPase appear very similar to those known from myosin ATPase or actomyosin ATPase (Sulakhe et al., 1971 ; McNamara et al., 1971 ; Sulakhe et al., 1973b,c; Heffron and Duggan, 1975; Gimmel'reikh et al., 1978). In fact, Gimmel'reikh et al.

176

P.V. SULAKHEand P. J. ST.LOUIS

(1978) have purified Ca 2+-ATPase from sarcolemma and have shown it to resemble myosin ATPase. Electrophoretic analysis of rabbit muscle sarcolemma carried out in our laboratory has further indicated the presence of myosin and actin even when the membrane fraction was isolated after extensive salt extraction that removes all myofibrillar contaminants (Sulakhe et al., unpublished data). The data, in fact, suggest that membrane-bound myosin and actin are resistant to salt extraction procedures (Sulakhe et al., 1973b,c). The fact that removal of nearly 80~ of the membrane phospholipid did not cause any inactivation of Mg / +-ATPase or Ca2+-ATPase (Ferdman et al., 1974) also distinguishes this enzyme from the Ca 2+transport ATPase, which is a lipoprotein at least in the case of sarcoplasmic reticulum (MacLennan and Holland, 1975). Myosin ATPase survives a mild Triton X-100 treatment of the myofibrills and Triton X-100 is able to remove virtually all phospholipid from membrane such as sarcolemma or sarcoplasmic reticulum. Nevertheless, the above discussion suggests that Ca 2+-ATPase (ATPase assayed with Ca z + as a sole divalent cation) is not likely to be of relevance in the active efflux of Ca 2+ but instead may participate in other functions such as cell contact, deformability and membrane motility. (d) Mg 2+, Ca2 +-A TPase and A TP-dependent Ca2 +-transport system of skeletal muscle surface membrane Thorpe and Seeman (1971) reported that ATP increased Ca 2 + binding to isolated guineapig sarcolemma. However, this observation was interpreted to suggest the presence of sarcoplasmic reticulum fragments. The suggestion was based on the assumption that sarcolemmal membranes are exclusively devoid of an active Ca2+-transport system. Severson et al. (1972) were the first to report that a highly purified rabbit muscle sarcolemma possessed an ATP dependent Ca z +-accumulation ability. This was further supported by detailed studies by Sulakhe et al. (1973a,b,c) and Sulakhe and Drummond (1974). Mg 2 +dependent Ca 2 +-stimulated ATPase activity was also present. In many regards, this enzyme and the sarcolemmal ATP-dependent Ca 2+ uptake process resembled the extensively characterized sarcoplasmic reticulum Ca 2 +-transport system (see Tada et al., 1978). These findings thus provided evidence for the first time that supported a view of the presence of an active Ca 2 + pump in muscle surface membrane capable of mediating active Ca 2 + efflux. Perhaps it can now be suggested that about 30--40~ of Ca 2+ elttux, which occurs in the absence of external Ca 2 + and Na + (Caputo and Bolanos, 1978) and which thus is not likely to be mediated by the Na +-Ca 2+ exchange carrier, relies on this mechanism, i.e. Ca 2+ pump. However, on a quantitative basis, the sarcolemmal Ca2+-efflux activity only assists the sarcoplasmic reticular pump in lowering the cytosolic Ca 2 + during relaxation of muscle and by itself will be incapable of bringing about relaxation. It is not surprising that muscle sarcolemma contains a Ca 2 + pump, since virtually all cell plasma membranes examined so far, including from those tissues discussed in the earlier sections of this review article, show the presence of an active Ca 2 +-transport system. The question of its physiological role in skeletal muscle contractile function still remains unanswered and represents a challenging topic of future studies.

(e) Comparative biochemical analysis of membranes from skeletal muscle involved in the excitation-contraction coupling In recent years, since the observation by Meissner (1975), there have been increasing investigations of the subfractions of sarcoplasmic reticulum-enriched microsomal fraction obtained by sucrose density gradient centrifugation. Meissner's study showed that light, intermediate and heavy subfractions differed from each other in the content of calsequestrin, a protein that MacLennan and Wong (1971) purified from sarcoplasmic reticulum and assigned it a role in Ca 2 + storage within the lumen of sarcoplasmic reticulum. That one of these subfractions might contain transverse tubules and the others longitudinal and/or terminal cisternae was considered by many, including us. Lau et al. (1976, 1977) extended these observations and suggested that the heavy subfraction contains terminal cisternae along with transverse tubule attached to it either in diadic or triadic junctions. On the other hand, Sarazala and Michalak (1978) and Zubryska et al. (1978) considered that light and

Passiveand activecalciumfluxesacrossplasma membranes

177

heavy fractions represented inside-out and rightside-out vesicles, a view that is not supported by the subsequent study by Michalak et al. (1979). Lau et al. (1977) presented the evidence that electron microscopic appearance of vesicles in the putative transverse tubule fraction was suggestive of triadic junctions with vesicles being of 2000°A length and 200°A width. Also, protein composition of the tubular fraction was quite distinct from that of the terminal cisternae or sarcoplasmic reticulum. While the heavy fraction of Meissner showed enrichment in calsequestrin in agreement with the later studies by Sarazala and Michalak (1978) and Zubryska et aL (1978), Lau et al. (1977) indicated an enrichment in the 55,000 protein band. That this band consists of multiple proteins has become evident from the study by Michalak et aL (1979), who show that high affinity calcium binding protein (M, = 55,000) is common to both transverse tubule and sarcoplasmic reticulum while a glycoprotein of a similar molecular mass is present exclusively in the sarcoplasmic reticulum but absent in the transverse tubule fraction. Barchi et al. (1979) indicate that the membrane vesicles in their LiBr-KCI fraction are of sarcolemmal origin since it showed an enrichment in the Na + channels, acetylcholine receptor sites and p-nitrophenyl phosphatase, whereas the low saltsucrose fraction contained T-tubular membranes. In this study, both sarcolemma- and Ttubule membrane-enriched fractions showed quantitative but not qualitative differences amongst the proteins detected by electrophoresis. Smith and Appel (1977), on the other hand, distinguished the transverse tubular membranes based on the presence of a protein kinase that specifically phosphorylated 28,000 molecular weight polypeptide band. Ouabainsensitive Na ÷, K ÷ -ATPaseis reportedly present in the putative T-tubule fraction of Lau et al. (1977) and Rosenblatt et aL (1979) but not of Scales and Sabbadini (1979). Ca 2 ÷-ATPase, on the other hand, is reportedly present in the T-tubule fraction (Brandt et al., 1979; Rosenblatt et al., 1979). Do transverse tubule membranes have an energized Ca 2 ÷ pump activity? Brandt et al. (1979) provide preliminary evidence that this is indeed present. This activity was correlated with the [3H]-ouabain binding activity amongst the gradient fractions. The Caswell group (Caswell et al., 1979) also believes that p-adrenoceptor-coupled adenylate cyclase is present in the transverse tubular membranes. This is quite interesting in view of the previous studies by Sulakhe et al. (1973a,b,c, 1974; also see Severson et al., 1972) using a putative sarcolemmal fraction that have identified many of these biochemical activities, some of which are now being considered also present in the T-tubule membrane. Sulakhe et al. (1973a) stated that, since so little was known then of the biochemistry of the T-tubule, some of the biochemical functions of the sarcolemmal fraction may indeed be those of the T-tubules present in the surface membrane fraction used. In fact, an enrichment of the 55,000 protein band (Lau et al., 1977) as well as the presence of protein kinase capable of phosphorylating 50,000 (Pinkett and Perlman, 1976) and 32,000 (Smith and Appel, 1977) protein bands were also found by Sulakhe et al. (1977). It is difficult to imagine that T-tubules, which in essence represent a continuation of the surface membrane, would have different biochemical functions than sarcolemma. So far, the findings of Sulakhe et al. have shown an enrichment of ]/-adrenergic receptors, adenylate cyclase and guanylate cyclase, cyclic nucleotide phosphodiesterases and ouabain-sensitive Na ÷, K ÷-ATPase and the presence of energized Ca 2 + pump including (Ca 2 +-ATPase), p-nitrophenyl phosphatase, protein kinases (cyclic AMP dependent and independent) and phosphatase in the sarcolemma-T-tubule membrane fraction. Recent studies from many laboratories have confirmed these basic observations. However, it is important that studies along these lines be continued and these will provide information of potentially high significance. For the present article, it is important to recognize that ATP-dependent Ca 2 ÷ pump, whether present in T-tubule or sarcolemma or both, is receiving increasing support and this makes the possible mediation by such a pump in the active Ca 2 + ettlux from the skeletal muscle a likely alternate route to the classical Na +Ca 2 + exchange system. (f) Phosphorylation o f skeletal muscle sarcolemma and effect on Ca z + binding and accumulation by the membrane fraction

Irrespective of the question of whether or not sarcolemmal Ca 2 + fluxes are directly linked to skeletal muscle contractile function, there exists a possibility that sarcolemmal Ca 2+

178

P.V. SULAKHEand P. J. ST. Louis

TABLE 1. EFFECT OF CYCLICAMP-DEPENDENT PROTEIN KINASEON CALCIUMTRANSPORTAND ATPASE ACTIVlTY OF SARCOPLASMICRETICULUMAND ON ATPAsE ACTIVITYOF PURIFIED ATPAsE PREPARATION

Sarcoplasmic Reticulum Calcium binding (nmol/mg protein/2 rain) Calcium accumulation (#mol/mg protein/10 min) Mg-ATPase (#mol PJmg protein/min) Mg, Ca-ATPase (/zmol/PJmg protein/min) ATPase Preparation Mg-ATPase (#mol PJmg protein/min) Mg, Ca-ATPase (#mol Pt/mg protein/min)

Control

Protein kinase + Cyclic AMP

Significance

80.50 + 2.50

81.80 ± 3.20

N.S.

2.28 ± 0.14

2.97 ± 0.12

p < 0.05

0.30 + 0.11

0.41 ± 0.05

N.S.

4.72 ± 0.75

5.88 5:0.71

p < 0.05

0.30 5:_0.05

0.45 5: 0.10

N.S.

15.40 +_ 0.30

19.69 + 1.20

p < 0.05

The results are means + S.E. of the means of four experiments. N.S. indicates not significant. The statistical significance was determined by the Student's paired t-test (P, V. Sulakhe, P. J. St, Louis, J. W, Wei and S. H. Jan, unpublished results, 1977).

stores participate in such key metabolic reactions in the skeletal muscle as glycogenolysis. For example, Yeaman and Cohen (1975) provided the evidence that phosphorylase kinase is totally dependent on calcium ions for its activity and the reversible activation by Ca 2 ÷ occurred at the concentration range of Ca 2 ÷ believed to exist during muscle contraction. Whether or not a portion of the Ca 2 ÷ pool, which activates this kinase, comes from the sarcolemmal pool has not been ruled out. Further, it is conceivable that epinephrine, which activates skeletal muscle sarcolemmal cyclase in a fl-adrenergic fashion (Severson et al., 1972; Wei et al., 1977; Wei and Sulakhe, 1979), regulates the availability of the sarcolemmal and/or sarcoplasmic reticular Ca 2+ store via cyclic AMP-stimulated phosphorylation of the membrane protein(s). Initially Schwartz et al. (1976) and subsequently Bornet et al. (1977) and Fabiato and Fabiato (1978) provided the evidence that cyclic AMP promotes Ca 2 ÷ accumulation in isolated fast skeletal muscle sarcoplasmic reticulum (but also see

TABLE 2. PROTEIN KINASE-CATALYZEDPHOSPHORYLATION OF PROTEINS OF THE ATPAsE PREPARATION AND OF SARCOPLASMICRETICULUM ATPase Average molecular weight (range) 70,000 (65,000-72,000) 50,000 (48,000-53,000) 35,000 (34,000-38,000) Unfractionated

Expt 1

Expt 2

Sarcoplasmic reticulum Expt 3 Expt 1 pmol 32p/mg protein

Expt 2

9.02

8.87

41.24

39.50

523.20

59.74

167.40

26.37

115.21

235.20

9.64

6.62

14.46

50.63

54.00

4.42

14.33

8.78

32.50

201.20

Sarcoplasmic reticulum (50-100 #g protein) or the ATPase preparation (85/~g protein) was incubated for 5 min at 24°C in the reaction mixture containing 40/zg bovine muscle kinase (Expt 1) or 48 #g bovine heart kinase (Expt 2) or 6 #g catalytic subunit of bovine muscle kinase (Expt 3), 1 ktM cyclic AMP and 40 p ~ [), - 32P]ATP. Phosphorylated proteins were separated by electrophoresis. The amount of protein present in each Coomassie blue-stained band was estimated from the densitometric scans. Results shown for sarcoplasmic reticulum are taken from a representative experiment ; similar results were obtained with three separate membrane preparations. For the ATPase preparation, Expts 1, 2 and 3 were carried out using the same preparation but with different protein kinascs as indicated; the values are average of duplicate determinations (P. V. Sulakhe, P. J. St. Louis, J. W. Wei and S. H. Jan, unpublished results, 1977).

Passive and active calcium fluxes across plasma membranes

//[ /

/

200

-=~ o

"

~

.

4": / ,

3 too-

o m

E_

/

. -

#

i

,i

/

.; /

,£ t / /,ji..•

Assay condition ~,~& ATP+oxalate | O , I ATP+Pi [ o, • ATP ~?eqlI passive

&~i,•~V Phosphorylated ~O~Ot~ Control . I --''11

r,~

,,

to I

/

•

~" l l i 0

.OOt

179

-

.....¢.01

.1

i

_ ~ -5 ,J, 0

Ca2+, mM

FIG. 9. Dependence of calcium binding and storage of skeletal muscle sarcolemmal vesicles on varying CaCI 2 concentrations in the assay. Rabbit skeletal muscle membranes were incubated with "5CaC12 under various assay conditions as shown. Membrane fraction prephosphorylated by [~ - 32P[ATP] in the presence of exogenous purified protein kinase catalytic subunit was also used simultaneously. Note that phosphorylated membrane vesicles accumulated Ca 2+ greater than the corresponding control membrane vesicles. Fig. also shows that oxalate-faeilitated Ca2+-uptake process is not saturated at Ca 2 + concentrations which saturate this process measured in the presence of phosphate. ATP-dependent Ca 2 + binding is a saturable process, however, phosphorylation did not affect this process. Passive Ca 2 + binding to the low affinity site of the membrane fraction is shown and is increased by phosphorylation (P. V. Sulakhe, J. W. Wet, P. J. St. Louis and S. H. Jail, unpublished data, 1977).

Kirchberger and Tada, 1976). Similar results were also obtained in our laboratory (see Table 1). Interestingly, however, in contrast to the observation on cardiac sarcoplasmic reticulum, cyclic AMP promoted phosphorylation of non-phospholamban-type proteins (Table 2) in fast muscle sarcoplasmic reticulum. That calcium uptake into skeletal muscle sarcolemmal fraction is enhanced by cyclic AMP stimulated membrane phosphorylation was also obtained (Sulakhe and Drummond, 1974). A detailed subsequent study in this laboratory indicated that muscle sarcolemma contains a cyclic AMP-dependent protein kinase that phosphorylates mainly a 50,000 molecular weight polypeptide band. Phosphorylation of this [ ATP-dependent~ 8t binding [

200

if +l

160

$ rain-bindinQ

U

-~ o E_

8o

SL-pretreltment

Assay Addition

['7 None

None

None cAMP+PI<

40

PTaso None PTase cAMP*PK 2-rain accumulation 10-rainaccumulation

ATP-dependent, Oxalate- facilitated FIG. 10. Increa~ in Ca z+ uptake by phosphorylation and its reversal by dephosphorylafion. Note that ATP-independent calcium binding (passive binding, left inset) and ATP-dependent, oxalatefacilitated Ca 2 + storage, but not ATP-dependent calcium binding (right inset), are increased by phosphorylation. Also shown is an interesting observation that phosphatase (PTase)-treatment of control membrane vesicles reduced both passive and oxalate-facilitated calcium uptake (36-37%) indicating that isolated membranes contain phosphorylated sites. It was also then predictable that the phosphatase-treated membranes would show greater degrees of stimulation by the added protein kinase (PK) and this was found to be the ease (see values on top of each bar) (P. V. Sulakhe, J. W. Wet and P. J. St. Louis, unpublished data, 1978).

180

P.V. SULAKHEand P. J. ST. Louis

"7

ATP -dependent

ATP -independent

2.0

I

1,6

.20 .16

+ pCMB J

~ 1.2

.12

~ .s

.08

~

.4

,04 .

.

.

20

.

o..s,

.

40

S0

80

20 100 (CaCI2)-1 mM -1

40

60

80

100

0

FIG. 11. Passive and ATP-dependent calcium binding sites of isolated rabbit skeletal muscle sarcolemma. The results shown in the left panel and inset are those for passive calcium binding sites. The data indicated the presence of high and low (inset) affinity binding sites ; the affinity of the latter sites is affected by p-chloromercuribenzoate (pCMB), which does not influence passive calcium binding to the high affinity sites, pCMB, however, influences both Vmx and the Km of the ATPdependent calcium binding system (J. W. Wei and P. V. Sulakhe, unpublished data, 1977).

band was related to the stimulatory effect of protein kinase on Ca 2 ÷ uptake (ATPdependent) (Fig. 9) and the effect of phosphorylation was reversible (Fig. 10). Interestingly, besides 50,000 molecular weight protein, a protein of about 30,000 molecular weight was also phosphorylated. The latter has now been shown by Smith and Appel (1977) in the putative Ttubule fraction while the former, by Pinkett and Perlman (1974) in the sarcolemmal membrane vesicle fraction. In the T-tubule fraction, the kinase appears to depend on divalent cation, especially Ca 2÷ (Smith and Appel, 1977). Walaas et al. (1977) have shown an endogenous phosphorylation of multiple proteins in rat muscle sarcolemma with the highest incorporation into a low molecular weight peptide band (M, = 15,000). However, with the exception of the studies by Sulakhe and co-workers (Sulakhe and Drummond, 1974; Sulakhe et al., 1977), the others have not as yet examined the effects of phosphorylation on membrane Ca z÷ fluxes. That the skeletal muscle sarcolemma passively binds Ca 2÷ at two sites characterized by high and low affinities towards Ca 2÷ has also been observed (Fig. 11). TABLE 3. EFFECT OF PHOSPHORYLATION AND DEPHOSPHORYLATION ON CALCIUM BINDING BY RABBIT SKELETAL MUSCLE SARCOLEMMA

Preincubation conditions Experiment - cAMP + cAMP - cAMP + cAMP

Additions to the incubation

Calcium binding (nmol/mg protein)

Significance

I

+ +

PK PK PK PK

Experiment II + cAMP + P K - PPase + cAMP + P K + PPase

-ATP -ATP + ATP + ATP

0.85 2.10 7.50 8.00

+ + + _

0.10 0.20 0.40 0.30

-ATP

2.80 _ 0.30

-ATP

0.50 ___0.10

p < 0.05 N.S.

p < 0.05

Experiment I: Isolated rabbit skeletal muscle sarcolemma (2 mg protein were preincubated in the mixture (1 ml) containing 20 mM Histide-HC1 (pH 6.8), 0.5 mM MgCI2, 0.5 mM EGTA and 20 #M ATP in the absence or presence of cyclic A M P (cAMP) and protein kinase (PK) for 15 min at 22°C. Membranes were centrifuged, washed and then assayed for calcium binding in the presence or absence of ATP in the standard binding assay. Calcium binding values were obtained by 5 rain incubation and are means _+ S.E. of three preparations. Experiment II : Following preincubation in the presence of cAMP and P K (see Expt I), the sedimented and washed membranes were further incubated in the presence and absence of phospho-protein phosphatase (PPase). The membranes were then centrifuged, washed and assayed for calcium binding (5 min-incubation, ATP absent). Binding values are means of duplicate determinations. Note that while phosphorylation promoted passive (i.e. when ATP was absent in the Ca 2 + binding assay) Ca 2+ binding, dephosphorylation reduced it; ATP-dependent Ca 2 ÷ binding was not influenced (P. V. Sulakhe, P. J. St. Louis and J. W. Wei, unpublished results, 1977). N.S. indicates not significant. The statistical significance was determined by the Student's paired t-test.

Passiveand activecalciumfluxesacrossplasmamembranes

181

Further, it seemed that phosphorylation promoted passive binding to the low affinity site (Fig. 10), again in a reversible manner (Table 3). Phosphorylase b kinase also promoted phosphorylation of rabbit muscle sarcolemma as well as Ca 2 + uptake (ATP-dependen0 into the membrane vesicles (J. W. Wei and P. V. Sulakhe, unpublished). These findings thus imply that sarcolemmal Ca 2 + fluxes are likely to be involved in the action of fl-adrenergic catecholamines or other hormones such as insulin on skeletal muscle. The observed phosphorylation of sarcolemmal and/or T-tubular proteins may be of significance in altering Ca 2 + permeability and/or active Ca 2 + flux across these membranes. However, the studies bearing on this question have really just begun and it is difficult at present to know the precise significance of the sarcolemmal Ca 2+ flux in the metabolic responses of skeletal muscle to external stimuli including epinephrine. Further, the relation between the sarcoplasmic reticular and the surface membrane (including T-tubular) fluxes of Ca 2+ remains an enigma and presents a challenging topic to future muscle biologists. 6. Liver

Experiments using perfused liver and liver slices suggest that the intracellular Ca 2+ concentration could be regulated by an outwardly directed transport mechanism (see Van Rossum, 1970). Earlier Judah and Ahmed (1964) using liver slices reported that Ca 2+ efflux increased when [Na +]o was decreased and efflux was also increased in slices that had been depleted of ATP. They therefore suggested that Ca 2 + efflux was in fact linked to the Na + gradient and ATP was indirectly required to maintain the Na + gradient (via the Na + pump). Later Van Rossum (1970), also using liver slices, provided data showing that Ca 2+ efflux was not dependent on the Na + gradient but rather was directly dependent on high-energy compounds. It was thus suggested that a system for the active efflux of Ca 2 +, as opposed to a Na+-Ca 2÷ exchange system, was present in the liver plasma membrane (see also Van Rossum et al., 1976). Ca 2+-stimulated Mg 2 +-ATPase activities (exhibiting Km for ATP of 0.35 mM and 0.88 mM) have now been demonstrated in a well-characterized plasma membrane fraction obtained from mouse liver (Garnett and Kemp, 1975). In addition to the Mg 2+, Ca 2÷ATPases, distinct Na+-activated and K+-activated Mg 2+, Ca2+-ATPases were also identified suggesting the operation of a complex enzyme system, at least in the mouse liver plasma membrane. It would thus appear that the Mg 2+, Ca 2 +-ATPase may be involved in the active efflux of Ca 2+. The suggested involvement of K + or Na + ions in the regulation of sarcoplasmic reticulum Ca 2 +-transport ATPase (see sections on heart and skeletal muscle) would further imply that the various activities described in the mouse liver membrane may well reflect monovalent-cation induced regulation of the (putative) Ca 2 + transport ATPase. Additional support for the suggestion that liver plasma membranes possess an "active" Ca 2 + extrusion mechanism has been derived, by implication, from reports that Ca 2+ efflux from the liver of different species is modulated by various hormones (Friedman and Park, 1968; Chausmer et al., 1972; Haylett, 1976). In contrast to the results of Garnett and Kemp (1975), Chambaut et al. (1974) were unable to detect Ca 2 +-stimulated Mg 2 +-ATPase activity in plasma membranes isolated from rat liver; Ca 2 + in the range 0.1/~M-0.5 mM failed to enhance ATPase activity measured in the presence of 5mM Mg 2+-ATP 2 -. A Ca 2+-dependent ATPase activity (27.6/~mole/mg/hr in the presence of 3 mM CaCI 2 + 5 mM ATP) was, however, detectable. Further examination of the Mg 2 +-ATPase and Ca 2+-ATPase activities failed to reveal any distinguishing properties. These observations indicated that the rat liver plasma membrane does not possess ATPdependent Ca 2+ efflux system. However, in the same study, the plasma membrane preparation possessed Ca 2 +-binding activity which exhibited both low and high affinity binding sites as determined by Scatchard plot analysis and ATP (and Mg 2+) markedly inhibited the Ca 2 + binding. As is the case for other tissues, the experimental evidence still does not allow us to conclusively state which of the two suggested mechanisms is operative in mediating Ca 2 + efflux from the hepatic cell. Claret-Berthon et al. (1977) contend that an extracellular Ca 2 + pool accounts for 63~ of the total exchangable Ca 2 + in the liver cell and suggest that an

182

P.v.

SULAKRE and P. J. ST. LOUIS

active efflux system may well exist in the plasma membrane. The lack of stronger supportive evidence may be a function of the relative "intactness" of the isolated membrane preparations so far studied. Wisher and Evans (1977) have described the isolation from rat liver, of plasma membrane subfractions which retain their functional polarity. Such preparations may prove to be useful tools in resolving the question of the existence in liver plasma membranes of an ATP-dependent Ca z +-transport system as well as of a N a + - C a 2 ÷ exchange mechanism. II1. C O N C L U S I O N S It is apparent from the foregoing presentation that there are two lines of evidence which bear on the question of the mechanism of cellular Ca 2 ÷ efflux. In broad terms one of these lines favour a Na ÷-Ca 2 ÷ exchange hypothesis while the other provides evidence supporting the existence of an ATP-linked Ca 2 ÷-pump activity. There are, however, valid reservations about the interpretation and/or significance of the available data, and indeed a certain degree of convergence can now be found in these two proposals. The various results on the influence of external Na + on Ca 2 ÷ etttux activity, derived from elegant studies on axons, cardiac muscle, and to some extent, on smooth muscle, do strongly suggest that a N a + - C a 2 ÷ exchange mechanism mediates Ca 2 + efflux. Further, if we accept this hypothesis, then [Na÷]i will likely influence the amount of Ca z÷ bound to specific transport sites on the postulated carrier and hence will also modulate Ca z ÷ efflux. Indeed, it has been shown for cardiac muscle that increasing [Na ÷]i caused decreased Ca z ÷ etflux and also increased Ca 2 ÷ influx. Support for the Na ÷-Ca 2 + exchange hypothesis has also come from a recent study by Malaisse and Couturier (1978). Using the cationic ionophore BrX537A incorporated into a carbon tetrachloride organic phase in a Pressman cell, these workers showed that a Na ÷ gradient ([Na+]o > [Na+]i) could be sufficient to maintain a Ca z + gradient and to further insure the transport of Ca 2 ÷ against such a gradient. One major objection to the N a + - C a 2 + exchange system hypothesis is the fact that, given the known transmembrane distribution o f N a ÷ in vivo both in axons and cardiac muscle, the equilibrium conditions predicted by the equation defining the hypothesis cannot account for the low [Ca 2 +]i levels observed in the "resting" state (see Jundt et al., 1975; Mullins, 1979; Van Breemen et al., 1979). In fact, this has led to the proposal that a complex 4 N a + - I Ca 2 + exchange occurs which thus renders the system electrogenic (Reuter, 1974; Mullins, 1979). Another possible rebuttal to this objection lies in the suggestion that the intracellular activities of Na ÷ and Ca 2+ may well be different from the estimated intracellular concentration (Lee and Fozzard, 1975; Fozzard, 1976). In addition to the objection cited above it has been reported that ATP (even at ~/M levels) does influence the [Na+]o-dependent Ca z÷ efflux from axons (Di Polo, 1973; Baker and McNaughton, 1976, 1978) and cardiac muscle (Jundt and Reuter, 1977). ATP-induced changes in the affinity of the exchange-carrier for Ca z ÷ and for Na + have also been reported by Baker and co-workers using axonal preparations (Baker and Glitsch, 1973; Baker and McNaughton, 1976). These studies, and others by Di Polo (e.g. see Di Polo, 1976), have now led to the proposal that the N a + - C a e+ exchange carrier operates as an ATP-dependent system. It has also been found that non-hydrolyzable analogs of ATP do not influence Ca z +efflux activities (Di Polo, 1977; Baker and McNaughton, 1978) leading one to infer that hydrolysis of ATP could be involved in the activation of Ca z ÷ efflux. Indeed, the suggestion had earlier been made from the studies of the dependence of Ca 2+ efflux on [Na+]o in metabolically (ATP)-depleted preparations (of heart and axon) that there may well have been sufficient residual ATP to modulate or mediate the observed Ca 2 ÷ efflux (Di Polo, 1974; Jundt et al., 1975). Hence, those reports which indicate the involvement of ATP in Ca 2 ÷efflux activities in axons (Di Polo, 1978, 1979), guinea-pig auricles (Jundt and Reuter, 1977) and goldfish ventricles (Busselen and Van Kerkhove, 1978) are in support of this view. In general, earlier investigations on Ca 2 +-el'flux activities used non-physiological Ca 2 ÷ loads in experimental preparations (e.g. studies involving the use of caffeine and dinitrophenol). Further, some of these studies involved the disruption of the normal functioning of endogenous Ca2+-buffering systems such as the mitochondria and endoplasmic (or sacroplasmic) reticulum. It should be noted here that the generally accepted Ca 2 ÷ capacities

Passive and activecalcium fluxesacrossplasmamembranes

183

and rates of Ca 2 +-transpOrt activities of these systems are sufficient to accommodate (i.e. buffer) Ca 2 ÷ loads to which the cell is normally exposed. It is thus significant that recent studies have shown that at physiological concentrations of Ca 2+, there appears to be a component of Ca 2 + efflux which is independent of [Na +]o and [Ca 2 +]o (see for example, Busselen and Van Kerkhove, 1978; Di Polo, 1978 ; Di Polo and Beaugt, 1979). Further, while it is apparent that, in axons in particular, a Na+--Ca 2 + exchange system may be of greater significance at elevated intracellular levels of Ca 2+, the evidence shows that at least part of the ATP-dependent Ca 2÷ efflux is modulated by external levels of Na +. The entire situation is nonetheless not clear-cut and indeed as indicated by Mullins (1978) the effect of ATP on Ca 2+ fluxes could well be a catalytic one rather than that of a substrate on the Ca 2 + pump. Numerous biochemical investigations, particularly those on isolated plasma membrane preparations, provide substantial evidence in support for the existence of an ATP-requiring Ca 2 ÷ pump whose activity is manifested as Mg 2 +, Ca 2 +-ATPase involved in the (active) uptake of Ca 2 ÷ by the plasma membrane vesicles (see above sections). Assuming that a major proportion of the membrane vesicles used in these studies represent everted (i.e. inside-out) vesicles, for which there is some evidence, the observed active storage of Ca 2 ÷ into these vesicles in essence represents on active efflux of Ca 2 ÷ by the RSO vesicle. Red blood cell membrane provides an excellent example of the ATP-dependent Ca 2+-pump mediated Ca 2+-efflux process; however, it fails to show a significant Ca 2+ efflux via the Na +-Ca 2 + exchange process. In other tissues, especially those discussed in this article, the situation is complex. For example, as yet there is no proof that in isolated plasma membrane preparations active Ca 2 ÷ uptake and Mg 2÷, Ca 2 +-ATPase are indeed tightly coupled. Numerous factors, including the sidedness of the membrane fraction, permeability properties, the type of treatments given prior to isolation of the membrane fraction, could all contribute to the lack of successful stoichiometry (i.e. moles Ca 2÷ transported per mole ATP utilized) and thus considerable further work is needed to establish this conclusively. Another major criticism has been the purity (or the lack of it) of the membrane fraction used. Since intracellular organdies (mitochondria, endoplasmic or sarcoplasmic reticulum) are known to contain highly active (ATP-requiring) Ca 2 ÷ pumps, the evidence that these organelles are totally absent from the isolated plasma membrane fraction is prerequisite to acceptance of a qualitatively similar pump in the plasma membrane. Perhaps, as the techniques of isolation and characterization of the plasma membranes improve, which certainly appears to be the case in recent years, even more rigorous examination of this question than what is already provided in published reports will be expected and this will hopefully provide compelling and conclusive evidence in support for an active Ca 2 ÷ pump at the cell surface membrane. Two exciting recent developments in the area of modulation of the membrane-associated Ca2+-pump activity deal with the likely involvement of calmodulin- and cyclic AMPdependent protein kinase(s) in the control of Ca 2 ÷ fluxes across cellular membranes in general and plasma membranes in particular. The former kinase, in fact, requires Ca 2÷ for its optimal activity. With either kinase, specific membrane proteins appear to be phosphorylated and these appear to control both passive and active fluxes of Ca 2 +. Although most of the available evidence implicating the roles for calmodulin and cyclic nucleotides in membrane Ca2+-transport process is quite recent and still far from complete, it has nevertheless provided significant clues for future investigations. Perhaps, the most appealing feature in this type of regulation is that the "active" Ca 2 ÷ efflux process appears tightly linked to the key cellular metabolic processes that are regulated by the intracellular "second messengers". Ca 2 + fluxes across cell plasma membranes and their modulation by cyclic AMP- and calmodulin-dependent processes are schematically presented in Figs. 12 and 13 using cardiac sarcolemma as one specific example of the plasma membrane, since for this membrane both biochemical and physiological evidences are now available. The diagram (Fig. 12) indicates the presence of Ca 2 + gradients across and in the external vicinity of the sarcolemma. The membrane is shown to contain the presumptive Na+-Ca 2 + exchange carrier, Ca 2 + pump, (Na +-K +) pump, as well as the processes that appear to regulate membrane Ca 2 ÷ fluxes, i.e. enzymatic systems responsible for the synthesis (cyclases), degradation (phosphodiesterases) ,r.p.B, 35/3--D

184

P.V. SULAKHEand P. J, ST Louis

Extracellular side

Ca

Ca(2-3mM)

Ca

~a - (~-

Ca Ca

.L ,L I~I~X ~. L - ~ Cytoplasmic side

I'

".. ,

Cail0mM) ~'

~

-

.Ca,

p"mp

~.....o-

.p

."

pp.

_

Lamina

Ca

Ca

AC~i.l R~C~ , L

ATP ADP ATP ADP Pi Pi Ca2+(0 IjJM)

AMP PPi

:.,cyclic GMP PPi

J~ Surface

-~J-

j~ Plasma ymemb . . . .

GTP

FIG. 12. Schematic diagram of the structure of heart sarcolemma and of biochemical activities of the plasma membrane relevant to passive and active Ca 2+ fluxes. GP, glycoprotein ; C, the presumptive Na+-Ca 2+ exchange carrier; MP, a membrane protein that spans the entire width of plasma membrane or that is present in the cytoplasmic monolayer of the plasma membrane; LP, lipoprotein ; CaM, calmodulin or calmodulin-like proteins; PPase, phosphoprotein phosphatase; HR, hormone receptor; AC, adenylate cyclase; PK, protein kinase with its regulatory (R) and catalytic (C) subunits ; PDE, phosphodiesterase; GC, guanylate cyclase. Ca2+ binding-sites on lipoprotein, lipids and glycoproteins of the plasma membrane as well as in the external aspect (surfacecoat, lamina) are also shown. Concentrations of Ca z ÷ in cytoplasmic and extracellular fluids are shown; Ca 2+ concentrations in the membrane itself, in the area between the external half of the plasma membrane and the surface coat, and in the external lamina represent indirect estimations. and actions (kinases) of cyclic nucleotides as well as calmodulin (Fig. 12)• The nature of passive binding sites for Ca 2 ÷ on and external to the plasma m e m b r a n e proper are also depicted. It was also intentional on our part to show in the diagram (Fig. 12) that complex structures (in this particular case surface coat and external lamina) "protect" the plasma m e m b r a n e from the external environment. Serious considerations to these structures should be given in future studies of Ca 2 ÷ fluxes, especially in view of the C a 2 + binding-sites that these "external" structures provide. In fact, the presence of an "external matrix" has been increasingly recognized for plasma m e m b r a n e s from a variety of tissues and it can be anticipated that detailed studies on this will provide some additional clues not as yet k n o w n a b o u t the ways by which extracellular Ca 2 + is held in the vicinity of the plasma membrane• It is this region or "pool" from which the required Ca 2 ÷ is derived during the cell activation and into which an active Ca 2 + efflux occurs during the recovery of the activated cell• Figure 13 incorporates various postulates concerning the m o d u l a t i o n of passive Ca z ÷ influx as well as active Ca 2 ÷ efflux by m e m b r a n e phosphorylation. M o d u l a t i o n of the former is considered by m a n y to be of significance in the fl-adrenergic activation of the myocardial contractility while that of the latter, in the accelerated relaxation of the cardiac muscle under the influence of fl-adrenergic catecholamines. N o t shown in Fig. 13 is the likely influence of

Ca [ ~c~

I

Ca I

/

l~-Adrenergic Catecholamine

Ca A

~V--~

Ca A

| :,-"~, ~ , r - ' - - - ' - / §_AR i ~ - ~ H x r t II II ili !/ ~ Sarcolemma I1o~.11 iii l ~+R RC ~ RC ReC.. ? II II . . . . . .

mr

jr,.'

~

.,' ;J<• X._/'

#

/

X ~ '~,.ll cyc.c 1AC [c~,c:'lc

After \ /Before AMP ~ . _ _ ~ A M ' phosphorylation/ ATP kip • ~/ . - z mow inwara .

II

f l / ~ I / ATP ADP \ After { Pi ~ S e f o r e phosphorylation ] ATP-dependent Ca2+ efflux

FIG. 13. Schematic presentation of the modulation of the passive and active Ca 2+ fluxes by cyclic AMP-dependent phosphorylation of heart plasma membrane, fl-AR, fl-adrenergic receptor ; AC, adenylate cyclase; RC, protein kinase; ECF and ICF, extracellular and intrac~llular fluids respectively.

Passive and active calcium fluxes across plasma membranes

185

membrane phosphorylation on the presumptive Na+-Ca 2+ exchange carrier-mediated Ca 2 + fluxes, since no data is as yet available on this aspect. Whether or not it is also subjected to regulation by cyclic AMP- or calmodulin-dependent protein kinases remains an interesting topic for future work. ACKNOWLEDGEMENTS The published a n d u n p u b l i s h e d results of studies from o u r l a b o r a t o r y described in this article were s u p p o r t e d by grants received from the Medical Research C o u n c i l of C a n a d a , the Saskatchewan H e a r t F o u n d a t i o n a n d the M u s c u l a r D y s t r o p h y Association of C a n a d a (PVS). P. J. St. Louis was a Research Fellow of the C a n a d i a n H e a r t F o u n d a t i o n during the p r e p a r a t i o n of this review. His c u r r e n t address is D e p a r t m e n t of Biology, Cave Hill C a m p u s of the University of West Indies, Barbados.

REFERENCES AGAPITO,M. T. and CABEZAS,J. A. (1977) Isolation and chemical composition of sarcolemmal membranes from rabbit and frog skeletal muscle,lnt, 2. Biochem. & 811-817. Ageg~ T. (1977)Membrane adenosine triphosphatase: a digitalis receptor? Science 198, 569-574. AKERA,T. and BRODY,T. M. (1976) Inotropic action of digitalis and ion transport. Life Sci, 18, 135-142. AKERA,T. and BRODY,T. M. (1978)The role of Na +, K +-ATPasein the inotropic action of digitalis.Pharmacol. Rev. 29, 187-220. AKERA,T., BENNETT,R. T., OLGAARD,M. K. arid BRODY,T. M. (1976)Cardiac Na +, K +-adenosinetriphosphatase inhibition by ouabain and myocardial sodium: A computer simulation. J. Pharmac. exp. Ther. 199, 287-297. AKEgA,T., OLGAARD,M. K., TEMMA,K. and BRODY,T. M. (1977) Development of the positive inotropic action of ouabain: Effect of transmembrane sodium movement. J. Pharmac. exp. Ther. 203, 675-684. AKERMAN,K. E. O. (1978) Changes in membrane potential during calcium ion influx and effiux across the mitochondrial membrane. Biochim. biophys. Acta 502, 359-366. AKERMAN,K. E. O., WIKSTROM,M. K. F. and SARIS,N. E. (1977) Effects of inhibitors on the sigmoidicityof the calcium ion transport kinetics in rat liver mitochondria. Biochim. biophys. Acta 464, 287-294. ALNAeS, E. and RAHAMIMO~,R. (1975) On the role of mitochondria in transmitter release from motor nerve terminals. J. Physiol. (Lond.)248, 285-306. ANAND,M. B., CHAUHAN,M. S. and DHALLA,N. S. (1977) Ca2+/Mg2+ ATPase activities of heart sarcolemma, microsomes, and mitochondria. J. Biochem. 82, 1731-1739. ANDERSON,T. W., HIRSCH,C. and KAVALER,F. (1977)Mechanismof activation in frog ventricular muscle.Circ. Res. 41, 472-480. ANDREW,C. G. and APPEL,S. H. (1973) Macromolecuiar characterization of muscle membranes. I. proteins and sialic acid of normal and denervated muscle, g. biol. Chem. 248, 5156-5163. ANDREW,C. G., ROSES,A. D., ALMON,R. R. and APPEL,S. H. (1973) Phosphorylation of muscle membranes: Identification of a membrane-bound protein kinase. Science 182, 927-929. BAKER,P. F. (1972) Transport and metabolism of calcium ions in nerve. Proo. Biophys. molec. Biol. 24, 177-223. BAKER,P. F. (1976) Regulation of intracellular Ca and Mg in squid axons. Fed. Proc. 35, 2589-2595. BAKER,P. F. and GLrrSCH,H. G. (1973)Does metabolic energyparticipate directlyin the Na-dependent extrusion of Ca45 ions from squid giant axons? J. Physiol. (Lond.)223, 44p-46p. BAKER,P. F. and McNAUGHTON,P. A. (1976)Kinetics and energeticsof calciumeffluxfrom intact squid giant axons. J. Physiol. (Lond.) 2~9, 103-144. BAKER,P. F. and MCNAUGHTON,P. A. (1978) The influenceof extracellular calcium binding on the calcium effiux from squid axons. J. Physiol. (Lond.) 276, 12%150. BAKER,P. F. and SCHLAEPFER,W. W. (1978)Uptake and binding of calcium by axoplasm isolated from giant axons of Loligo and myxicola. J. Physiol. (Lond.)276, 103-125. BAKER,P. F., BLAUSTEIN,M. P., HODGKIN,A. L. and STEINHARDT,R. A. (1969)The influenceof calcium on sodium efflux in squid axons. J. Physiol. (Lond.)200, 431-458. BAKER,P. F., HODGKIN,A. L. and RIDGEWAY,E. G. (1971) Depolarization and calcium entry in squid axons. J. Physiol. (Lond.)218, 709-755. BARCHI,R. L., BONILLA,E. and WONG,M. (1977)Isolation and characterization of musclemembranes using surfacespecific labels. Proc. nat. Acad. Sci. U.S.A. 74, 34-38. BARCHI,R. L., WEIGELE,J. B., CHALIKIAN,D. M. and MURPHY,L. E. (1979)Musclesurfacemembranes. Preparative methods affect apparent chemical properties and neurotoxin binding. Biochira. biophys. Acta 550, 59-76. BARR,L., CONNOR,J. A., DEWEY,M. M., APRILLE,J. and JOHNSTON,P. V. (1974)The isolation of plasma membrane from frog cardiac muscle cells. Biochira. biophys. Acta 345, 336-347. BENNINGER,C. J., EINWAeHTER,H. M., HAAS,H. G. and KERN,R. (1976)Calcium-sodium antagonism on the frog's heart : a voltage clamp study. J. Physiol. (Lond.)259, 617-645. BESC8,H. R., JR, JONES,L. R. and WATANASE,A. M. (1976) Intact vesiclesof canine cardiac sarcolemma: Evidence from vectorial properties of Na +, K+-ATPas¢. Circ. Res. 39, 586-595. BESCH,H. R., JR, JONES,L. R., FLEMING,J. W. and WATANABE,A. M. (1977)Parallel unmasking of latent adenylate cyclase and (Na +, K+)-ATPase activities in cardiac sarcolemmal vesicles. A new use of the channel-forming ionophore alamethicin. J. biol. Chem. 252, 7905-7908. BXANCm,C. P. and SHAt~S, A. M. (1959) Calcium influx in skeletal muscle at rest, during activity, and during potassium contracture. J. gen. Physiol. 42, 803-811.

186

P.V. SULAKHEand P. J. ST. LoLqs

BLAUSTEIN, M. P. (1974) The interrelationship between sodium and calcium fluxes across cell membranes. Rev. Physiol. Biochem. exp. Pharmac. 70, 33-82. BLAUSTEIN, M, P. (1975) Effects of Potassium, Veratridine and Scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Physiol. (Lond.) 247, 617-655. BLAUSTEIN,M. P. (1976) The ins and outs of calcium transport in squid axons: internal and external ion activation of calcium efflux. Fed. Proc. 35, 2574-2578. BLAUSTEIN, M~ P. and GOLDRING, J. M. (1975) Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials. J. Physiol. (Lond.) 247, 589-615. BLAUSTEIN, M. P. and HODGKIN, A. L. (1969) The effect of cyanide on the efflux of calcium from squid axons. J. Physiol. (Lond.) 200, 497-527. BLAUSTEIN,M. P. and O~ORN, C. J. (1975) The influence of sodium on calcium fluxes in pinched-off nerve terminals in vitro. J. Physiol. (Lond.) 247, 657-686. BLAUSTEIN,M. P. and RUSSELL,J. M. (1975) Sodium-calcium exchange and calcium--calcium exchange in internally dialyzed squid giant axons. J. Mere. biol. 22, 285-312. BLAUSTEIN, M. P. and WIESMANN, W. P. (1970) Effect of sodium ions on calcium movements in isolated synaptic terminals. Proc. natn. Acad. Sci. U.S.A. 66, 664-671. BLAUSTE1N,M. P., SHIELD,C. E. and SANTIAGO,F. (1971) Sodium ions and calcium fluxes in giant barnacle muscle fibers. Biophys. Soc. 15th Ann. Meet. Abstrs. 141a. BLAUSTEIN, M. P., RUSSELL,J. M. and DEWEER, P. (1974) Calcium efflux from internally-dialyzed squid axons. The influence of external and internal cations. J. Supramol. Struct. 2, 558-581. BLAUSTEIN, M. P., RATZLAFF, R. W., KENDRICK, N. C. and CnWE~TZER, E. S. (1978a) Calcium buffering in presynaptic nerve terminals I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism. J. gen. Physiol. 72, 15-41. BLAUSTEIN,M. P., RATZLAFF,R. W. and SCHWEITZER,E. S. (1978b) Calcium buffering in presynaptic nerve terminals II. Kinetic properties of the nonmitochondrial Ca sequestration mechanism. J. gen. Physiol. 72, 43-66. BUTZ, A. L., FINE, R. E. and TOSELLI, P. A. (1977) Evidence that coated vesicles from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum. J. Cell Biol. 75, 135-147. BOEGMAN,R. J., MANERY,J. F. and PlYrERIC, L. (1970) The separation and partial purification of membrane-bound (Na ÷ + K+)-dependent Mg2+-ATPase and (Na ÷ + K+)-independent Mg2+-ATPase from frog skeletal muscle. Biochim. biophys. Acta 203, 506-530. BOHR, D. F. (1973) Vascular smooth muscle updated. Cir. Res. 32, 665-672. BOLDYREV, A. A. (1971) N a +, K ÷-dependent ATPase of the sarcotemma. Biokhimiya 36, 826-833. BORNET, E. P., ENTMAN, M. L., VAN WINKLE, W. B., SCHWARTZ,A., LEHOTAY,D. C. and LEVEY,G. S. (1977) Cyclic AMP modulation of calcium accumulation by sarcoplasmic reticulum from fast skeletal muscle. Biochim. biophys. Acta 468, 188-193. BRADING, A. F. (1973) Ion distribution and the role of calcium in cellular function. Ion distribution and ion movement in smooth muscle. Phil. Trans. R. Soc. London Ser. B. Biol. Sci. 265, 35-46. BRADING, A. F. (1975) Sodium/sodium exchange in the smooth muscle of guinea-pig taenia coli. J. Physiol. (Lond.) 251, 79-105. BRADING, A. F. (1978) Calcium-induced increase in membrane permeability in the guinea-pig taenia coli : Evidence for involvement of a sodium-calcium exchange mechanism. J. Physiol. (Lond.) 275, 65-84. BRADING, A. F. and WIDDICOMBE,J. H. (1976) Interaction between sodium and calcium movements in smooth muscle. In : Colloquium on Smooth Muscle, Pharmacology and Physiology, pp. 235-245 (Eds. M. WORCEL and G. VASSORT)Paris INSERM. BRADING,A. F. and WIDDICOMBE,J. H. (1977) The use of lanthanum to estimate the number of extracellular cationexchanging sites in the guinea-pigs taenia coli and its effects on transmembrane monovalent ion movements. J. Physiol. (Lond.) 266, 255-273. BRANDON, D. L. (1975) Myosin-like polypeptides in plasma membrane preparations. FEBS Lett. 58, 349-352. BRANDT, N. R., CASWELL, A. H. and BRUNSCHWlG,J-P. (1979) ATP-energized Ca 2 + pump in transverse tubules isolated from skeletal muscle. Xlth Int. Congress Biochem. Abst. 454. BRIGGS,F. N., WLSE,R. M. and HEARN, J. A. (1978) The effect of lithium and potassium on the transient state kinetics of the (Ca + Mg)-ATPase of cardiac sarcoplasmic reticulum. J. biol. Chem. 253, 5884-5885. BRINLEV, F. J. JR., SPANGLER,S. G. and MULLINS, L. J. (1975) Calcium and EDTA fluxes in dialyzed squid axons. J. .qen. Physiol. 66, 223-250. BRINLEY, F. J. JR., TIFFERT, T., SCARPA, A. and MULLINS, L. J. (1977) lntracellular calcium buffering capacity in isolated squid axons. J. 9en. Physiol. 70, 355-384. BULL, R. J. and TREVOR, A. J. (1972) Sodium and the flux of calcium ions in electrically-stimulated cerebral tissue. J. Neurochem. 19, 1011-1022. BUSSELEN, P. and VAN KERKHOVE, E. (1978a) Study of the Na-Ca exchange hypothesis in the goldfish ventricle. Archs Int. Physiol. Biochim. 86, 186-188. BUSSELEN, P. and VAN KERKHOVE, E. (1978b) The effect of sodium, calcium and metabolic inhibitors on calcium efflux from goldfish heart ventricles. J. Physiol. (Lond.) 282, 263-283. BYGRAVE,F. L. (1977) Mitochondrial calcium transport. In : Current Topics in Bioenergetics, Vol. 6, pp. 259-318 (Ed. D. R. SANADI) Academic Press, New York. BYGRAVE, F. L. (1978) Calcium movements in cells. Trends Biochem. Sci. 3, 175-178. CAPUTO,C. and BOLANOS,P. (1978) Effect of external sodium and calcium on calcium efflux in frog striated muscle. J. Mere. biol. 41, 1-14. CARAFOLI, E. (1979) The calcium cycle of mitochondria. FEBS Lett. 104, 1-5. CARAFOLI, E. and CROMPTON, M~ (1976) Calcium ions and mitochondria. Syrup. Soc. exp. Biol. 30, 89-115. CARAFOLI, E. and CROMPTON, M. (1978) The regulation of intracellular calcium. In: Current Topics in Membranes and Transport, Vol. 10, pp. 151-216 (Eds. F. BRONNER and A. KLEINZELLER) Academic Press, New York.

Passive and active calcium fluxes across plasma membranes

187

CARAFOLI, E., TIOZZO, R., LUGLI, G., CROV~YrI, F. and KRATZING, C. (1974) The release of Ca 2+ from heart mitochondria by sodium. J. Mol. Cell. Cardiol. 6, 361-371. CAROm, P., S C B W ~ A ~ , K. and CARAFOIa,E. (1978) Separate pathways for Ca 2÷ uptake and release in liver mitochondria. FEBS Lett. 96, 339-342. CARVAtaO, C. A. M. (1978) Chtorotetracycline as an indicator of the interaction of calcium with brain membrane fractions. J. Neurochem. 30, 1149-1155. CARVALHO,C. A. M. 0979) Fluxes of Ca 2+, Sr 2+ and Mg 2÷ in synaptosomes. Life Sci. 25, 73-82. CARVALHO,A. P. and LEo, B. (1967) Effects of ATP on the interaction of Ca 2*, Mg 2+ and K + with fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle. J. oen. Physiol. 50, 1327-1352. CARVALHO,A. P., MADEIRA,V. M. C. and ANTU~ES-MADE]~, M. C. (1971) Effect of potentiators of muscular contraction on contractile and enzymatic activities of sarcolemma. Biochim. biophys. Acta 234, 210-221. CAS~,~LS, R. and VANBREEMEN,C. (1975) Active and passive Ca 2+ fluxes across cell membranes of the guinea-pig taenia coli. Pflfioers Arch. 359, 197-207. CASWELL,A. H., LAU, Y. H. and BRUNSCHWlG,J-P. (1976) Ouabain-binding vesicles from skeletal muscle. Archs B iochem. Biophys. 176, 417-430. CHAMBAUT,A. M., LERAY-PEcKER,F., F~LOMAN,G. and HANOUNE,J. (1974) Calcium binding properties and ATPase activities of rat liver plasma membranes. J. gen. Physiol. 64, 104-126. CHAUSMER,A. B., SHERMAN,B. S. and WALLAeH,S. (1972) The effect of parathyroid hormone on hepatic cell transport of calcium. Endocrinology 90, 663-672. CHUDAPONGSE,P. (1976) Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria. Biochim. biophys. Acta 423, 196-202. CLARET-BERTHON,B., CLARET,M. and MAZET, J. L. (1977) Fluxes and distribution of calcium in rat liver cells: Kinetic analysis and identification of pools. J. Physiol. (Lend.) 272, 529-552. COOKE,W. J. and ROmNSO~,J. D. (1971) Factors influencing calcium movements in rat brain slices. Am. J. Physiol. 221, 218-225. COSMOS,E. and HARRIS,E. J. (1961) In vitro studies of the gain and exchange of calcium from frog skeletal muscle. J. gen. Physiol. 44, 1121-1130. COTMAN,C. W. and MATTHEWS,D. W. (1971) Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim. biophys. Acta 249, 380-394. CROMPTON, M. and HEID, I. (1978) The cycling of calcium, sodium, and protons across the inner membrane of cardiac mitochondria. Eur. J. Biochem. 91, 599-608. CROMPTON,M., SIGEL,E., SALZMANN,M. and CARAFOLI,E. (1976a) A kinetic study of the energy-linked influx of Ca 2+ into heart mitochondria. Eur. J. Biochem. 69, 429-434. CROMPTON, M., CAPANO, M. and CARAFOLI, E. (1976b) The sodium-induced effiux of calcium from heart mitochondria. A possible mechanism for the regulation of mitochondrial calcium. Ear. J. Biochem. 69, 453--462. CROMPTON,M., HEDIGER,M. and CARAFOLI,E. (1978) The effect of inorganic phosphate on calcium influx into rat heart mitochondria. Biochem. biophys. Res. Commun. 80, 540-546. CROMPTON,M., HEID, ]., BASCHERA,C. and CARAFOLI,E. (1979) The resolution of calcium fluxes in heart and liver mitochondria using the lanthanide series. FEBS Lett. 104, 352-354. CURTIS,B. A. (1963) Some effects of Ca-free choline-Ringer solution on frog skeletal muscle. J. Physwl. (Lend.) 166, 75-86. CURTIS,B. A. (1970) Calcium effiux from frog striated muscle fibers. J. gen. Physiol. 55, 243-253. DE ME]S, L. (1971) Allosteric inhibition by alkali ions of the Ca 2+ uptake and adenosine triphosphatase activity of skeletal muscle microsomes. J. biol. Chem. 246, 4764-4773. DE MEIS,L., RUmN-ATTSCHUL,B. M. and MACHADO,R. D. (1970) Comparative data of Ca 2+ transport in brain and skeletal muscle microsomes. J. biol. Chem. 245, 1183-1189. DEm'ON, g. M., RANDLE,P. J. and MARTIN,B. R. (1972) Stimulation by calcium ions of pyruvate dehydrogenase phosphate phosphatase. Biochem. J. 128, 161-163. DENTON,R. M., RICHARDS,D. A. and CHIN,J. G. (1978) Calcium ions and the regulation of NAD +-linked isocitrate dehydrogenase from the mitochondria of rat heart and other tissues. Biochem. J. 176, 899-906. DE ROmmTIS, E. (1964) Electron microscope and chemical study of binding sites of brain biogenic amines. In: Progress in Brain Research, Vol. 8, pp. 118-136 (Eds. H. E. HIMWlCHand W. A. H]MWICH)Elsevier, New York. DEVINE,C. E., SOMLYO,A. V. and SOMLYO,A. P. (1972) Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles. J. Cell Biol. 52, 690-718. DHALLA, N. S., ZIEGELHOFFER,A. and HARROW,J. A. C. (1977) Regulatory role of membrane systems in heart function. Can. J. Physiol. Pharmac. 55, 1211-1234. DIETZE, G. and HEPP, K. D. (1971) Calcium stimulated ATP-ase of cardiac sarcolemma. Biochem. biophys. Res. Commun. 44, 1041-1049. DIETZE, G. and HEPP, K. D. (1972) Effect of 3'5'-AMP on calcium-activated ATPase in rat heart sarcolemma. Biochem. biophys. Res. Commun. 46, 269-278. DI POLO,R. (1973) Sodium-dependent calcium influx in dialysed barnacle muscle fibres. Biochim. biophys. Acta 298, 279-283. DI POLO, R. (1974) Effect of ATP on the calcium efliux in dialyzed squid giant axons. J. gen. Physiol. 64, 503-517. D1 POLO, R. (1976) The influence of nucleotides on calcium fluxes. Fed. Proc. 35, 2579-2582. D] POLO, R. (1977) Characterization of the ATP-dependent calcium efltux in dialyzed squid giant axons, d. oen. Physiol. 69, 795-813. D] POLO, R. (1978) Ca pump driven by ATP in squid axons. Nature 274, 390-392. D] POLO,R. and BEAUGE',L. (1979) Physiological role of ATP-driven calcium pump in squid axon. Nature 278, 271273. D] POLO, R. and CAPUTO,C. (1977) The effect of ATP on calcium efliux in dialyzed barnacle muscle fibers. BiocMm. biophys. Acta 470, 389-394. D] POLO, R., REQUENA,J., BRINLEY,F. J. JR., MULL1NS,L. J., SCARPA,A. and TWFERT,T. (1976) Ionized calcium concentrations in squid axons. J. gen. Physiol. 67, 433-467.

188

P.V. SULAKHEand P. 5. ST. LooIs

DOWD, F. J. JR., PITTS,B. J. R. and SCHWARTZ,A. (1976) Phosphorylation of a low molecular weight polypeptide in beef heart Na +, K+-ATPase preparations. Archs Biochem. Biophys. 175, 321-331. DRAHOTA,Z., CARAFOLI,E., ROSSI,C. S., GAMBLE,J. L. and LEHNINGER,A. L. (1965) The steady state maintenance of accumulated Ca + + in rat liver mitochondria. J. biol. Chem. 240, 2712-2720. DRICKAMER,L. K. (1975) The red cell membrane contains three different adenosine triphosphatases. J. biol. Chem. 250, 1952-1954. DUGGAN,P. F. (1968) The monovalent cation-stimulated calcium pump in frog skeletal muscle. Life Sci. 7, 913-919. DUGGAN,P. F. (1977) Calcium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. J. biol. Chem. 252, 1620-1627, DUNCAN,C. J. (1976) Properties of the Ca 2 +-ATPase activity of mammalian synaptic membrane preparations. J. Neurochem. 27, 1277-1279. DUNKLEY, C. R., MANERY,J. F. and DRYDEN, E. E. (1966) The conversion of ATP to IMP by muscle surface enzymes. J. Cell. Physiol. 68, 241-247. ENDO, M. (1977) Calcium release from the endoplasmic reticulum. Physiol. Rev. 57, 71-108. ENTMAN,M. L., LEVEY,G. J. and EPSTEIN,S. E. (1969) Demonstration of adenyl cyclase activity in canine cardiac sarcoplasmic reticulum. Biochem. biophys. Res. Commun. 35, 728-733. ENTMAN, M. L., BORNET,E. P., VAN WINKLE, W. B., GOLDSTEIN,M. A. and SCHWARTZ,A. (1977) Association of glycogenolysis with cardiac sarcoplasmic reticulum: II. Effect of glycogen depletion, deoxycholate solubilization and cardiac ischemia : Evidence for a phosphorylase kinase membrane complex. J. Mol. Cell Cardiol. 9, 515-528. FAmATO, A. and FABIATO, F. (1978) Cyclic AMP-induced enhancement of calcium accumulation by the sarcoplasmic reticulum with no modification of the sensitivity of the myofilamcnts to calcium in skinned fibres from a fast skeletal muscle. Biochim. biophys. Acta 539, 253-260. FABIATO,A. and FABIATO,F. (1979) Calcium and cardiac excitation-contraction coupling. A. Rev. Physiol. 41,473484. FELDMAN, D. A. and WEINHOLD,P. A. (1977a) Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-Propranolol. Biochem. Pharmac. 26, 2283-2289. FELDMAN,D. A. and WEINHOLD,P. A. (1977b) Calcium binding to rat heart plasma membranes: Isolation and purification of a lipoprotein component with a high calcium binding capacity. Biochemistry 16, 3470-3475. FERDMAN,D. L., HIMMELREICH,N. G. and DYADYUSHA,G. P. (1970) The enzyme activity in a detergent-treated sarcolemma of skeletal muscles. Biochim. biophys. Acta 219, 372-378. FESTOFF, B. W. and ENGLL, W. K. (1974) In vitro analysis of general properties and junctional receptor characteristics of skeletal muscle membranes. Isolation, purification, and partial characterization of sarcolemmal fragments. Proc. natn. Acad. Sci. U.S.A. 71, 2435-2439. FIEHN, W., PETER, J. B., MEAD, J. F. and GAN-ELEPANO,M. (1971) Lipids and fatty acids of sarcolemma, sarcoplasmic reticulum, and mitochondria from rat skeletal muscle. J. biol. Chem. 246, 5617-5620. FORD, G. D. (1976) Subcellular fractions of vascular smooth muscle exhibiting calcium transport properties. Fed. Proc. 35, 1298-1301. FORD, G. D. and HESS, M. L. (1975) Calcium-accumulating properties of subcellular fractions of bovine vascular smooth muscle. Circ. Res. 37, 580-587. FOZZARD,H. A. (1977) Heart: excitation-contraction coupling. A. Rev. Physiol. 39, 201-220. FRANK, J. S., LANGER, G. A., NUDD, L. M. and SERAYDARIAN,K. (1977) The myocardial cell surface, its histochemistry, and the effect of sialic acid and calcium removal on its structure and cellular ionic exchange. Circ. Res. 41, 702-714. FRIEDMANN,N. and PARK,C. R. (1968) Early effects of 3'-5' adenosine monophosphate on the fluxes of calcium and potassium in the perfused liver of normal and adrenalectomized rats. Proc. natn. Acad. Sci., U.S.A. 61, 504-508. FUNCKE, H. J. V. and RATTENHUBER, E. (1976) Stimulierung des aktiven calcium-transportes am Herzmuskelsarkolemm durch CAMP-abhangige Proteinkinase. Verh. Dtsch. Ges Kreislaufforschg. 42, 120126. GARNET'r, H. M. and KEMP, R. B. (1975) (Ca 2+ + Mg2+)-Activated ATPases in the plasma membrane of mouse liver cells. Biochim. biophys. Acta 382, 526-533. GERVAIS, A., LANE, L. K., ANNER, B. M., LINDENMAYER,G. E. and SCHWARTZ,A. (1977) A possible molecular mechanism of the action of digitalis. Ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney. Circ. Res. 40, 8-14. GIMMEL'REIKH,N. G., KRAVETS,L. G., POPOVA,G. N. and KURSKn,M. D. (1978) Purification and certain properties of Ca-ATPase of skeletal muscle sarcolemma. Biokhimiya 43, 491-497. GLITSCH,H. G., REUTER,H. and SCHOLZ,H. (1970) The effect of the internal sodium concentration on calcium fluxes in isolated guinea-pig auricles. J. Physiol. (Lond.) 209, 25-43. GLYNN, I. M. and KARLISH,S. J. D. (1975) The sodium pump. A. Rev, Physiol. 37, 13-55. GODFRAIND,T. (1976) Calcium exchange in vascular smooth muscle, action of noradrenaline and lanthanum. J. Physiol. (Lond.) 260, 21-35. GODFRAIND,T. and KABA, A. (1969) Blockade or reversal of contraction induced by calcium and adrenaline in depolarized arterial smooth muscle. Br. J. Pharmac. 36, 549-560. GODERAIND,T., STURBOIS,X. and VERBEKE,N. (1973) Ultrastructure du muscle longitudinal de l'il6on de cobaye et de ses microsomes. Archs Int. Physiol. Biochem. 81, 786-791. GODFRAIND, T., STURBOIS,X. and VERBEKE,N. (1976) Calcium incorporation by smooth muscle microsomes. Biochim. biophys. Acta, 455, 254-268. GOODFORD,P. J. (1970) Ionic interactions in smooth muscle. In : Smooth Muscle, pp. 100-121 (Eds. E. BLtLBRING, A. F. BRADING,A. W. JONESand T. TOMITA)Edward Arnold, London. GOPINATH, R. M. and VINCENZI, F. F. (1977) Phosphodiesterase protein activation mimics red blood cell cytoplasmic activation of (Ca 2 +-Mg 2÷)ATPase. Biochem. biophys. Res. Commun. 77, 1203-1209. GRAB, D. J., BERZINS,K., COHEN, R. S. and SIEKEVlTZ,P. (1979) Presence of calmodulin in post-synaptic densities isolated from canine cerebral cortex. J. biol. Chem. 254, 8690-8696.

Passive and active calcium fluxes across plasma membranes

189

GURD,J. W., JONES,L. R., MAHLER,H. R; and MOORE,W. J. (1974) Isolation and partial characterization of rat brain synaptic plasma membranes. J. Neurochem. 22, 281-290. HA~D~, S. and LF.ONAaD,E. J. (1976) A calcium transport system for mammal/an cells. L/fe So/. 17, 1527-1533. HARIGAYA,S. and SCHWARTZ,A. (1969) Rate of calcium binding and uptake in normal animal and failin$ human cardiac muscle. Circ, Res, 25, 781-794. HARROW,J. A. C., DAS, P. K. and DHALLA,N. S. (1978) Influence of some divalent cations on heart sarcolenamal bound enzymes and calcium binding. Biochem. Pharmac. 27, 2605--2609. HAYLETT,D. G. (1976) Effects of sympathomimetic amines on 45Ca effiux from liver slices. Br. J. Pharmac. 57, 158160. HEFFRON,J. J. A. and DUGGAN,P. F. (1975) Adenosinetriphosphatase activity of isolated sarcolemmal tubules of frog skeletal muscle. Int. J. Biochem. 6, 219-229. HEMMINKI,K. (1974) Calcium binding to brain plasma membranes. Biochim. biophys. Acta 363, 202-210. HESS, M. L. and FORD,G. D. (1974) Calcium accumulation by subcellular fractions from vascular smooth muscle. J. Mol. Cell. Cardiol. 6, 275--282. HICKS, M. J., SHIGEKAWA,M. and KATZ,A. M. (1979) Mechanism by which cyclic adenosine 3' : 5'-monophosphatedependent protein kinase stimulates calcium transport in cardiac sarcoplasmic reticulum. Circ. Res. 44, 384391. HINDS, T. R., LARSEN,F. L. and V~CeNZa, F. F. (1978) Plasma membrane Ca 2+ transport: stimulation by soluble proteins. Biochem. biophys. Res. Commun. 81, 455-461. HODGrdN, A. L. and KEYN~S,R. D. (1956) Experiments on the injection of substances into squid giant axons by means of a micro-syringe. J. Physiol. (Lond.) 131, 592-616. HOTTA,K. and USAMI,Y. (1967) Enzymatic activity ofisulated skeletal muscle sarcolemma. J. B/ochem. 61, 407-410. HuI, C-W., DRUMMOND, and DRUMMOND, G. I. (1976) Calcium accumulation and cyclic AMP-stimulated phosphorylation in plasma membrane-enriched preparations of myocardium. Archs Biochem. Biophys. 173, 415-427. HURWITZ,L., FITZPATRICK,D. F., DEBBAS,G. and I_ANt)ON,E. J. (1973) Localization of calcium pump activity in smooth muscle. Science 179, 384-386. ICHIDA,S., Kuo, C-H., UCHIDA,S., NAGAI,K. and YOSHIDA,H. (1976a) Bound forms of Ca taken up by the synaptic plasma membrane. Jap. J. Pharmac. 26, 551-558. ICHIDA, S., KUO, C-H., MATSUDA,T. and YOSHIDA,H. (1976b) Effects of La + + +, Mn + + and Ruthenium Red on Mg-Ca-ATPase activity and ATP-dependent Ca-binding of the synaptic plasma membrane. Jap. J. Pharmac. 26, 39-43. ICI-nDA,S., HATA, F., MATSUDA,T. and YOSHIDA,H. (1976c) Effects of Na + and other monovalent cations on Caefflux from synaptosomes. Jap. J. Pharmac. 26, 31-37. IKEMOTO,N., MORGAN,J. F. and YAMADA,S. (1978) Ca z +-controlled eonformational states of the Ca 2+ transport enzyme of sarcoplasmie reticulum. J. biol. Chem. 253, 8027-8033, IQBAL,Z., GARG, B. P. and OCHS, S. (1979) Caimodulin activation of brain and nerve Ca 2 +-Mg 2+-ATPase Soc. Neurosci. Abst. 5, 60. ITANO, T. and PE~ISTON, J. T. (1979) Ca2+ATPases from brain membranes and calmoduiin. Xlth Int. Cono. Biochem. Abst. 455. JAN/S,R. A., CRANKSHAW,D. J. and DANIEL,E. E. (1977) Control of intraceilular Ca 2+ activity in rat myometrium. Am. J. Physiol. 232, C50-C58. JARREYr,H. W. and PEN~ISTON,J. T. (1977) Partial purification of the Ca 2 +-Mg 2+ATPase activation from human erythrocytes : Its similarity to the activator of Y: Y-cyclic nucleotide phospliodiesterase. Biochem. biophys. Res. Commun. 77, 1210-1216. JAgRO'rT, B. and PICKEN, G. M. (1975) Cardiac adenylate cyciase. I. Preparation and characterization of a subcellular fraction containing catecholamine-sensitive adenylate cyclase. J. Mol. Cell. Cardiol. 7, 685--695. JONES, D. H. and MATUS,A. I. (1974) Isolation of synaptic plasma membrane from brain by combined flotationsedimentation density gradient centrifugation. Biochem. biophys. Acta. 356, 276-287. JONES, L. R., BESCH,H. R. JR., and WATANABE,A. M. (1977) Monovalent cation stimulation of Ca 2+ uptake by cardiac membrane vesicles. Correlation with stimulation of Ca 2+-ATPase activity. J. biol. Chem. 252, 33153323. JONES,L. R., BESCH,H. R. JR. and WATANABE,A. M. (1978) Regulation of the calcium pump of cardiac sarcoplasmic reticulum. Interactive roles of potassium and ATP on the phosphoprotein intermediate of the (K +, CaZ+)ATPase. J. biol. Chem. 253, 1643-1653. Joints, L. R., BESCrt,H. R. JR., FLEMIr~G,J. W., MCCONNAOCrIEY,M. M. and WATANABE,A. M. (1979) Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic retieulum. J. biol. Chem. 254, 530-539. JUDAH,J. D. and AHMED,K. (1964) The biochemistry of sodium transport. Biol. Rev. 39, 160--193. JUNDT,H., PORZIG,H., REUTER,H. and STUCKI,J. W. (1975) The effect of substances releasing intracellular calcium ions on sodium-dependent calcium effiux from guinea-pig auricles. J. Physiol. (Lond.) 246, 229-253. KATASE,T. and TOMITA,T. (1972) Influences of sodium efflux and calcium on the recovery process from potassium contracture in the guinea-pig taenia coli. J. Physiol. (Lond.) 224, 489-500. KATZ, A. M. and REPKE,D. I. (1967) Sodium and potassium sensitivity of calcium uptake and calcium binding by dog cardiac microsomes. Circ. Res. 21, 767-775. KATZ, A. M., REPKE, D. 1., TADA, M. and CORKEDALE,S. (1974) Propranolol-induced inhibition of cardiac mierosomal calcium-uptake, calcium-binding, and epinephrine-stimulated adenylate cyclase. Cardiovase. Res. 8, 541-549. KATZ, A. M., REPKE, D. I., FUDYMA,G. and SHIGEKAWA,M. (1977) Control of calcium effiux from sareoplasmic reticulam vesicles by external calcium. J. biol. Chem. 252, 4210--4214. KAVALER, F., ANDERSON,T. W. and FISHER, V. J. (1978) Sarcolemmal site of caffeine's inotropic action on ventricular muscle of the frog. Circ. Res. 42, 285-290. KENDRIeK, N. C., BLAUST~rN,M. P., FRIED, R. C. and RATZt.AFF,R. W. (1977) ATP-dependent calcium storage in presynaptic nerve terminals. Nature 265, 246-248.

190

P.V. SULAKHEand P. J. ST. LouIS

KEYNES, R. D. and LEWIS, P. R. (1956) The intraceUular calcium contents of some invertebrate nerves. J. Physiol. (Lond.) 134, 399-407. KIDWAI, A. M., RADCLIFFE, M. A., DUCHON, G. and DANIEL, E. E. (1971) Isolation of plasma membrane from cardiac muscle. Biochim. biophys. Res. Commun. 45, 901-910. KIDWAI, A. M., RADCLIFFE, M. A., LEE, E. Y. and DANIEL, E. E. (1973) Isolation and properties of skeletal muscle plasma membrane. Biochim. biophys. Acta 298, 593-607. KIRSY, A. C., LINDLEY, B. D. and PICKEN, J. R. (1975) Calcium content and exchange in frog skeletal muscle. J. Physiol. (Lond.) 253, 37-52. KIRCHBERGER, M. A. and TADA, M. (1976) Effects of adenosine 3': 5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting muscle. J. biol. Chem. 251, 725-729. KIRCHBERGER, M. A. and WONG, D. (1978) Calcium efftux from isolated cardiac sarcoplasmic reticulum. J. biol. Chem. 253, 6941-6945. KIRCHBERGER, M. A., TADA, M. and KATZ, A. M. (1974) Adenosine 3': 5'-monophosphate-dependent protein kinase catalysed phosphorylation reaction and its relationship to calcium transport in cardiac sarcoplasmic reticulum. J. biol. Chem. 249, 6166-6173. KOKETSU, K., KITAMURA,R. and TANAKA,R. (1964) Binding of calcium ions to cell membrane isolated from bullfrog skeletal muscle Am. J. Physiol. 207, 509-512. KRAUSE, E. G., WILL, H. SCHIRPKE, B. and WOLLENBERGER, A. (1975) Cyclic AMP-enhanced protein phosphorylation and calcium binding in a cell membrane-enriched fraction from myocardium. In : Advances in Cyclic Nucleotide Research, Vol. 5, pp. 473-490 (Eds. G. I. DRUMMOND, P. GREENGARD and G. A. ROBISON) Raven Press, Hewlett, New York. KRISNAN, K. S. and BALARAM,P. (1976) Cation binding to brain plasma membranes: An evaluation of the use of anionic fluorescent probes. Archs Biochem. Biophys. 174, 420--430. Ku, D. D., AKERA, T., FRANK, M., BRODY, T. M. and IWASA, J. (1977) The effects of grayanotoxin 1 and ctdihydrograyanotoxin II on guinea-pig myocardium. J. Pharmac. exp. Ther. 200, 363-372. Kuo, C-H., ICHIDA, S., MATSUDA, T., KAKIUCHI, S. and YOSHIDA, H. (1979) Regulation of ATP-dependent Cauptake of synaptic plasma membranes by Ca-dependent modulator protein. Life Sci. 25, 235-240. KUTSKY, P. and GOODMAN, F. R. (1978) Calcium incorporation by canine aortic smooth muscle microsomes. Archs Int. Pharmac. Ther. 231, 4-20. LANGER, G. A. (1964) Kinetic studies of calcium distribution in ventricular muscle of the dog. Circ. Res. 15, 393-405. LANGER, G. A. (1971) The intrinsic control of myocardial contraction-ionic factors. Physiol. Med. 285, 1065-t071. LANGER, G. A. (1973) Heart: Excitation-contraction coupling. A. Rev. Physiol. 35, 55-86. LANGER, G. A. (1976) Events at the cardiac sarcolemma: localization and movement of contractile-dependent calcium. Fed. Proc. 35, 1274-1278. LANGER, G. A. (1977) Ionic basis of myocardial contractility. A. Rev. Med. 28, 13-20. LANGER, G. A. and BRADY,A. J. (1963) Calcium flux in the mammalian ventricular myocardium. J. gen. Physiol. 46, 703-720. LAU, Y. H., CASWELL,A. H. and BRUNSCHWIG,J. P. (1976) Fractionation of the triad junction from rabbit skeletal muscle. Fed. Proc. 35, 847. LAU, Y. H., CASWELL,A. H. and BRUNSCHWIG,J. P. (1977) Isolation of transverse tubules by fractionation of triad junctions of skeletal muscle. J. biol. Chem. 252, 5565-5574. LAU, Y. H., CASWELL,A. H., BRUNSCHWIG,J. P., BAERWALD,R. J. and GARCIA, M. (1979) Lipid analysis and freezefracture studies on isolated transverse tubules and sarcoplasmic reticulum subfractions of skeletal muscle. J. biol. Chem. 254, 540-546. LAZAREWICZ, J. W., HALJAMAE, H. and HAMBERGER, A. (1974) Calcium metabolism in isolated brain cells and subcellular fractions. J. Neurochem. 22, 33-45. LEE, K. S. and KLAUS, W. (1971) The subcellular basis for the mechanism of inotropic action of cardiac glycosides. Pharmacol. Rev. 23, 193-261. LEHNINGER, A. L., VERCESI,A. and BABABUNMI,E. A. (1978) Regulation of Ca 2 + release from mitochondria by the oxidation-reduction state of pyridine nucleotides. Proc. natn. Acad. Sci. U.S.A. 75, 1690-1694. LEW, V. L. and FERREIRA, H. G. (1978) Calcium transport and the properties of a calcium-activated potassium channel in red cell membranes. In: Current Topics in Membranes and Transport, Vol. 10, pp. 217-277 (Eds. F. BRONNER and A. KLEINZELLER) Academic Press, New York. LIMAS, C. J. (1977) Calcium-binding sites in rat myocardial sarcolemma. Archs Biochem. Biophys. 179, 302-309. LINDEN, J. and BROOKER,G. (1978) Sodium requirement for the positive inotropic action ofisoproterenol on guinea pig atria. Science 199, 539-541. LOGAN, J. G. and WATERS,G. A. (1976) The hydrolysis of exogenous ATP by rat cerebral synaptosomes. J. Physiol. (Lond.) 256, 30P-31P. Louis, C. F., BUONAFFINA,R. and BINKS, B. (1974) Effect of trypsin on the proteins of skeletal muscle sarcoplasmic reticulum. Archs Biochem. Biophys. 161, 83-92. LI~ILLMANN,H. and PETERS,T. (1976) On the sarcolemmal site of action of cardiac glycosides. In : Recent Advances in Studies on Cardiac Structure and Metabolism: The Sarcolemma, Vol. 9, pp. 311-328 (Eds. P. E. RoY and N. S. DHALLA) University Park Press, Baltimore. MA, S. K., SULAKHE, P. V. and LEUNG, N. L. K. (1978) Binding of [ZH] atropine by cardiac plasma membraneenriched fractions. In : Recent Advances in Studies on Cardiac Structure and Metabolism: Heart Function and Metabolism, Vol. 11, pp. 249-256 (Eds. T. KOBAYASHI,T. SANO and N. S. DHALLA) University Park Press, Baltimore. MACINTYRE, J. D. and GREEN, J. W. (1977) Further studies on the red cell calcium pump using inside-out vesicles as a model. Fed. Proc. (Abst,) 36, 271. MACLENNAN, D. H. and HOLLAND, P. C. (1975) Calcium transport in sarcoplasmic reticulum. A. Rev. Biophys. Bioenq. 4, 377-404. MACLENNAN, D. H. and HOLLAND, P. C. (1976) The calcium transport ATPase of sarcoplasmic reticulum. In : The Enzymes of Biological Membranes, Vol. 3, pp. 221-259 (Ed. A. MARTONOS1)Plenum Publishing Co., New York.

Passive and active calcium fluxes across plasma membranes

191

MACLENNAN,D. H. and WONG, P. T. S. (1971) Isolation of a calcium-sequestering protein from the sarcoplasmic reticulum. Proc. natn. Acad. Sci. U.S.A. 68, 1231-1235. MADEIRA,V. M. C. and ANTUNES-MADEIRA,M. C. (1973) Interaction of Ca 2+ and Mg 2+ with synaptic plasma membranes. Biochim. biophys. Acta 323, 396--407. MADEIRA, V. M. C. and CARVALHO,A. P. (1972) Interaction of cations and local anesthetics with isolated sarcolemma. Biochim. biophys. Acta 266, 670--683. MAHLER,H. R. (1977) Proteins of the synaptic membrane. Neurochem. Res. 2, 119-147. MALAISSE,W. J. and COUTURIER,E. (1978) Ionophoretic model for Na-Ca counter transport. Nature 275, 664665. MALSTRtM,K. and CARAFOta,E. (1975) Effects of prostaglandins on the interaction of Ca 2+ with mitochondria. Archs Biochem. Biophys. 171, 418-423. MANERY,J. F., RIORDAN,R. J, and DRYDEN,E. E. (1968) Characteristics of nucleotide-converting enzymes at muscle surfaces with special reference to ion sensitivity. Can. J. Physiol. Pharmac. 46, 537-547. MARSH,J. B. and HAUGAARD,N. (1957) Adenosine triphosphatase activity of intact muscle cells. Biochim. biophys. Acta 23, 204-205. MAS-OLIVA,J., WILLIAMS,A. J. and NAYLER,W. G. (1979) ATP-induced stimulation of calcium binding to cardiac sarcolemma. Biochem. biophys. Res. Commun. 87, 441-447. MCINToSH, D. B. and BERMAN, M. C. (1978) Calcium ion stabilization of the calcium transport system of sarcoplasmic reticulum. J. biol. Chem. 253, 5140-5146. MCNAMARA,D. B., SULAKHE,P. V. and DHALLA,N. S. (1971) Properties of the sarcolemmal calcium ion stimulated adenosine triphosphatase of hamster skeletal muscle. Biochem. J. 125, 525-530. MCNAMARA,D. B., SINGH,J. N. and DHALLA,N. S. (1974) Properties of some heart sarcolemmal bound enzymes. J. Biochem. 76, 603--609. MCNUTT, N. S. (1975) Ultrastructure of the myocardial sarcolemma. Circ. Res. 37, 1-13. MEISSNER, G. (1975) Isolation and characterization of two types of sarcoplasmic reticulum vesicles. Biochim. biophys. Acta 389, 51-68. MICHAELIS,M. L., MICHAELIS,E. K. and MYERS,S. L. (1979) Characterization of the Na-Ca exchange system in synaptosomes and synaptic membrane ghosts. Soc. Neurosci. (Abst.) 5, 306. MICHALAK,M., CAMPBELL,K. P. and MACLENNAN,D. H. (1980) Localization of the high affinity calcium binding protein and an intrinsic glycoprotein in sarcoplasmic reticulum membranes. J. biol. Chem., in press. MILEDI,R. (1973) Transmitter release induced by the injection of calcium ions in nerve terminals. Prec. R. Soc. Lend. B. Biol. Sci. 183, 421-425. MITCHELL, P. (1966) Chemiosmotic coupling in Oxidative and Photosynthetic Phosphorylation. Glynn Res. Ltd., Bodmin, England. MITCHELL, P. and MOYLE, J. (1969) Estimation of membrane potential and pH difference across the cristal membrane of rat liver mitochondria. Eur. J. Biochem. 7, 471-484. MOORE, C. L. (1971) Specific inhibition of mitochondrial Ca 2+ transport by ruthenium red. Biochem. biophys. Res. Commun. 42, 298-305. MORCOS, N. C. and JACOaSOlq,A. L. (1978) Interaction of sodium with the sarcolemmal calcium system. Can. J. Biochem. 56, 1-6. MORCOS,N. C. and JACOBSON,A. L. (1979) Calcium binding in cardiac sarcolemma. Can. J. Physiol. Pharmac. 57, 529-534. MORGAN,I. G., WOLFE, L. S., MANDELL,P. and GOMBOS,G. (1971) Isolation of plasma membranes from rat brain. Biochim. biophys. Acta 241, 737-751. MULLINS,L. J. (1977) A mechanism for Na/Ca transport. J. oen. Physiol. 70, 681-695. MULLINS,L. J. (1979) The generation of electric currents in cardiac fibers by Na/Ca exchange. Am. J. Physiol. 236, C103-C110. MULLINS,L. J. and BRINLEY,F. J., JR. (1975) Sensitivity of calcium efflux from squid axons to changes in membrane potential. J. gen. Physiol. 65, 135-152. NAGATOMO,T. and PETER, J. B. (1975) (Na + + K+)-ATPase in subcellular fractions of rat skeletal muscle. Exp. Neurol. 49, 345-355. NAKAMARU,Y. and SCHWARTZ,A. (1971) Adenosine triphosphate dependent calcium binding vesicles; magnesium, calcium adenosine triphosphatase and sodium, potassium adenosine triphosphatase: Distributions in dog brain. Archs Biochem. Biophys. 144, 16-29. NAYLER,W. G. (1967) Some factors involved in the maintenance and regulation of cardiac contractility. Circ. Res. 20, 21, Suppl. 3 : 213-217. NAYLER, W. G. and FASSOLD,E. (1977) Calcium accumulating and ATPase activity of cardiac sarcoplasmic reticulum before and after birth. Cardiovas. Res. 11, 231-237. NAYLER,W. G., SULLIVAN,A. T., DUNNETI',J., SLADE,A. M. and TRETHEWIE,E. R. (1976) The effect of cardiotoxic component of the venom of the Indian Cobra (Naja nigricollis) on the subeelluiar structure and function of heart muscle. J. Mol. Cell. Cardiol. 8, 341-360. NEDERGARD, J., AL-SHAIKHALY,M. H. M. and CANNON, B. (1979) In: Function and Molecular Aspects of Biomembrane Transport, Vol. 3, pp. 175-178 (Ed. E. QUAGLIARIELLOet al.) Elsevier/North-Holland, Amsterdam, New York. NICHOLLS,D. G. (1978) Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart. Biochem. J. 170, 511-522. NIEDERGERKE,R. (1959) Calcium and activation of contraction. Experientia 15, 128-130. NIEDERGERKE,R., PAGE, S. and TALBOT,M. S. (1969) Calcium fluxes in frog heart ventricles. Pflfigers Arch. ges Physiol. 306, 35%360. O'DPdSCOLL,G. and DUGGAN,P. F. (1977) Distribution of the calcium-ion-transport system in subeellular fractions from rabbit brain. Biochem. Soc. Trans. 571st Meeting, Dublin pp. 1708-1710. OHASHI, T., UCHIDA,S., NAGAI, K. and YOSHIDA,H, (1970) Studies on phosphate hydrolyzing activities in the synaptic membrane. J. Biochem. 67, 635-641.

192

P.V. SULAKHEand P. J. ST. Louis

OLIVEIRA, M. M. and HOLZHACKER, S. (1974) Isolation and characterization of smooth muscle cell membranes. Biochim. biophys. Acta 332, 221-232. OTSUKA, M., OTSUKI,I. and EBASHI,S. (1965) ATP-dependent Ca binding of brain microsomes. J. Biochem. 58, 188190. OTSUKI, I. (1969) ATP-dependent Ca uptake of brain microsomes. J. Biochem. 66, 64~650. PAPAZIAN,D., RAHAMIMOFF,H. and GOLDIN, S. i . (1979) Reconstitution and purification by "transport specificity fractionation" of an ATP-dependent calcium transport component from synaptosome-derived vesicles. Proc. hath. Acad. Sci. U.S.A. 76, 3708-3712. PETRALI, E. H., THIESSEN,B, J., DESSENS,P. N. and SULAKHE,P. V. (1978) Polypeptide compositions and Ca 2 + - and cAMP-promoted phosphorylation of membrane proteins of synaptic subfractions. Soc. Neurosci. (Abst.) 4, 247. PINKETT, M. O. and PERLMAN,R. L. (1974) Phosphorylation of muscle plasma membrane protein by a membranebound protein kinase. Biochim. biophys. Acta. 372, 379-387. PITTS, B. J. R. (1979) Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the Sodium pump. J. biol. Chem. 254, 6232-6235. PORSlUS, A. J. and VAN ZWIETEN, P. A. (1975) The negative ionotropic action of propanidid. Influence of calcium movements in atrial tissue. Eur. J. Pharmac. 34, 283-292. POZZAN,T., BRAGADIN,M. and AZZONE,G. F. (1977) Disequilibrium between steady-state Ca 2 + accumulation ratio and membrane potential in mitochondria. Pathway and role of Ca 2 +. Biochemistry 16, 5618-5625. RAEYMAEKERS,L., WUYTACK,F. and CASTEELS,R. (1974) N a S a exchange in taenia coli of the guinea-pig. Pfliigers Arch. 347, 329-340. RAHAMIMOFF, H. and ABRAMOVITZ,E. (1978a) Calcium transport in a vesicular membrane preparation from rat brain synaptosomes. FEBS Lett. 89, 223-226. RAHAMIMOFF,H. and ABRAMOVITZ,E. (1978b) Ca transport and ATPase activity of synaptosomal vesicles from rat brain. FEBS Lett. 92, 163-167, RAHAMIMOFF,H. and SPANIER,R. (1979) Sodium-dependent calcium uptake in membrane vesicles derived from rat brain synaptosomes. FEBS Lett. 104, 111-114. REED, K. C. and BYGRAVE,F. L. (1975) A kinetic study ofmitochondrial calcium transport. Eur. J. Biochem. 55, 497504. REEVES,J. P. and SOTKO,J. L. (1979) Sodium-calcium ion exchange in cardiac membrane vesicles. Proc. narD. Acad. Sci. U.S.A. 76, 590-594. REQUENA, J., DI POLO, R., BR1NLEY, F. J., JR. and MULLINS, L. J. (1977) The control of ionized calcium in squid axons. J. gen. Physiol. 70, 329-353. REUTER, H. (1973) Divalent cations as charge carriers in excitable tissues. Prog. Biophys. Mol. Biol. 26, 1-43. REUTER, H. (1974) Exchange of calcium ions in the mammalian myocardiaum. Mechanisms and Physiological significance. Circ. Res. 34, 599-605. REUTER, H. and SEITZ,N. (1968) Dependence of calcium effiux from cardiac muscle on temperature and external ion composition. J. Physiol. (Lond.) 195, 451-470. REUTER, H., BLAUSTEIN, M. P. and HAEUSLER, G. (1973) Na-Ca exchange and tension development in arterial smooth muscle. Phil. Trans. R. Soc. B 265, 87-94. RIBEIRO,J. M. C. and VIANNA,A. L. (1978) Allosteric modification by K ÷ of the (Ca 2 + + Mg 2 +)-dependent ATPase of sarcoplasmic reticulum. J. biol. Chem. 253, 3153-3157. ROBINSON, J. n . (1976) (Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation. Archs Biochem. Biophys. 176, 366-374. ROBINSON, J. D. (1978) Calcium-stimulated phosphorylation of a brain (Ca + Mg)-ATPase preparation. FEBS Letts 87, 261-264. ROBINSON, J. D. and LUST, W. D. (1968) Adenosine triphosphate-dependent calcium accumulation by brain microsomes. Archs Biochem. Biophys. 125, 286-294. ROSENBLATT, C., HIDALGO, C., VERGARA,C. and IKEMOTO, N. (1979) Isolation and characterization of a purified t-tubule membrane fraction from rabbit skeletal muscle. Xlth Int. Conoress ofBiochem. (Abst.) 358. RossI, C. S. and LEHNINGER, A. L. (1964) Stoichiometry of respiratory stimulation, accumulation of Ca ÷ + and phosphate, and oxidative phosphorylation in rat liver mitochondria. J. biol. Chem. 239, 3971-3980. RUBIN, B. B. and KATZ, A. M. (1967) Sodium and potassium effects on skeletal muscle microsomal adenosine triphosphatase and calcium uptake. Science 158, 1189-1190. SAERMARK, T. and VILHARDT, n. (1979) Isolation and partial characterization of magnesium ion- and calcium iondependent adenosine triphosphatase activity from bovine brain microsomal fraction. Biochem. J. 181, 321-330. SAITO, K., UCrtlDA, S. and YOSHIDA, H. (1972) Calcium binding of isolated synaptic membranes from rat cerebral cortex. Jap. J. Pharmac. 22, 787-798. SARAZALA, M. G. and MICHALAK, M. (1978) Studies on the heterogeneity of sarcoplasmic reticulum vesicles. Biochim. biophys. Acta. 513, 221-235. SATO, T., SAKAI, R. and SAKAI, T. (197t) The membrane-bound enzyme activities and their localization in toad skeletal muscles. Jikei. Med. J. 18, 53-66. SCALES,D. J. and SABBADINI,R. A. (1979) Microsomal T System. A Stereological analysis of purified microsomes derived from normal and dystrophic skeletal muscle. J. Cell Biol. 83, 33-46. SHAPIRA,G., DOBOCZ,I., PIAU, J. P. and DELAIN,E. (1974) An improved technique for preparation of skeletal muscle cell plasma membrane. Biochim. biophys. Acta 345, 348-358. SCHIMMEL,S. D., KENT, C., BISCHOEF,R. and VAGELOS,P. R. (1973) Plasma membranes from cultured muscle cells : Isolation procedure and separation of putative plasma-membrane marker enzymes. Proc. hath. Acad. Sci. U.S.A. 40, 3195-3199. SCHWARTZ,A., LINDENMAYER,G. E. and ALLEN,J. C. (1975) The sodium-potassium adenosine triphosphatase; pharmacological, physiological and biochemical aspects. Pharmac. Rev. 27, 3-134. SCHWARTZ,A., ENTMAN, M. L., KANOKE, K., LANE, L., VAN WINKLE,B. and BORNET, E. P. (1976) The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Effects of cyclic AMPdependent protein kinase and phosphorylase b kinase. Biochim. biophys. Acta 426, 57-72.

Passive and active calcium fluxes across plasma membranes

193

SeVErN, S. E., BOLDYREV,A. A. and TICACUK,V. A. (1974) Some properties of Na +, K+-stimulated ATPase from normal and dencrvated muscles of rabbit with a spedal r~erence to its sensitivity to acetylcholine. Comp. gen. Pharmac, 5, 181-185. SEVERSON,D. L., DRUMMOND,G. I. and SULAKM~P. V. (1972) Adenylate cyclase in skeletal muscle. J. biol. Chem. 247, 2949:-2958, SHIGEKAWA,M, and PEARL,L. J. (1976) Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations. J. biol. Chem. 251, 6947-6952. SHIGEKAWA,M., FINEGAN,J. M. and KATZ,A. M. (1976) Calcium transport ATPase of canine cardiac sarcoplasmic reticolum. J. biol. Chem. 7,51, 6894-6900. SMITH, P. B. and APPEL, S. H. (1977) Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle. Biochim. biophys. Acta 466, 109-122. SOBUE,K., ICHIDA,S., YOSHIDA,H., YAMAZAKI,R. and KAK]UCm,S. (1979) Occurrence of a Ca 2+- and modulator protein-activatable ATPase in the synoptic plasma membranes of brain. FEBS Left. 99, 199-202. SORE~Sl~N,R. and MAHLER,H. R. (1979) Stimulation by calmodulin of ECa2+-Mg 2+]ATPase and Ca 2+- dependent calmodulin-requiring protein kinase in rat brain synaptic plasma membranes. Soc. Neurosci. (abst.) 5, 309. SPERELAKIS,N. and SCHNEIDER,J. A. (1976) A metabolic control mechanism for calcium ion influx that may protect the ventricular myocardial cell. Am. J. Cardiol. 37, 1079-1085. STAHL,W. L. and SWANSON,P. D. (1972) Calcium movements in brain slices in low sodiumor calcium media. J. Neurochem. 19, 2395-2407. STAM, A. C., JR., WEGLICK], W. B., FF.LDMAN,D., SHELBOURNE,J. C. and SO~NBLICK, E. H. (1970) Canine myocardial sarcolemma: Its preparation and enzymic activity. J. Mol. Cell. Cardiol. 1, 117-130. STAM,A. C., JR., WEGLICK],W. B., GERTZ, E. W. and SONNENBLICK,E. H. (1973) A calcium-stimulated, ouabaininhibited ATPase in a myocardial fraction enriched with sarcolemma. Biochim. biophys. Acta. 298, 927-931. ST. Louis, P. J. (1979) Ca2+-activated ATPase activities of cardiac sar¢olemmal membranes. Xlth Int. Congr. Biochem. (abst.) 454. ST. LOLaS, P. J. and SULAKHE,P. V. (1976a) Isolation of sarcolemmal membranes from cardiac muscle. Int. J. Biochem. 7, 547-558. ST. LOUIS,P. J. and SULAKHE,P. V. (1976b) Adenosine triphosphate-dependent calcium binding and accumulation by guinea-pig cardiac sarcolemma. Can. J. Biochem. 54, 946-956. ST. Louis, P. J. and SffLAKHE, P. V. (1976c) Adenylate cyclase, guanylate cyolase, and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma. Biochem. J. 158, 535-541. ST. Louis, P. J. and SffLAKHE,P. V. (1978a) Protein analysis of cardiac sarcolemma: Effects of membrane-pert urbing agents on membrane proteins and calcium transport Biochemistry 17, 454(1-4550. fir. Louis, P. J, and SULAKHE,P. V. (1978b) Protein (PR) and phosphorylation (PN) profiles of sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes from guinea-pig heart ventricles. J. Moll. Cell. Cardiol. (abst.) 10,117. ST. Louts, P. J. and SULAKHE,P. V. (1979) Phosphorylation of cardiac sarcolemma by endogenous and exogenous protein kinase. Archs Biochem. Biophys. 198, 227-240. STUCK], J. W. and INEICHEN,E. A. (1974) Energy dissipation by calcium recycling and the efficiency of calcium transport in rat liver mitochondria. Eur. J. Biochem. 48, 365-375. SULAKI-IE,P. V. (1971) Ph.D. Thesis, University of Manitoba, Winnipeg. SULAg.HE,P. V. and DHALLA,N. S. (1971) Excitation contraction coupling in heart VI. Demonstration of Ca 2+ activated ATPase in dog heart saroolemma. Life Sci. 10, 185-192. SULAKHE,P. V. and DHALLA,N. S. (1973a) Adenylate cyclase of heart sarcotubular membranes. Biochim. biophys. Acta. 293, 379-396. SULAgHE, P. V. and DHALLA,N. S. (1973b) Further studies on energy linked calcium transport by subcellular particles in the failing heart of myopathic hamsters. Biochem. Med. 8, 18-27. SULAIC~E,P. V. and DRtrMMOND,G. I. (1974) Protein kinase catalyzed phosphorylation of muscle sarcolemma. Archs Biochem. Biophys. 161, 448-455. SULAKHE,P. V. and JAN, S-H. (1975) Characterization of active calcium transport systems in isolated membranes from cerebral cortex. Can. Physiol. (abst.) 6, 58. SULAKHE,P. V. and KOWALSKY,D. D. (1978) Role of potassium ions in sarcoplasmic reticulum (SR) calcium pump. J. Mol. Cell. Cardiol. (abst.) 10, 117. SULAgSE, P. V. and NARAYANAN,N. (1978) Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle. B/ochem. J. 175, 171-180. SULAKHE,P. V. and ST. LoUIs, P. J. (1976) Membrane phosphorylation and calcium transport in cardiac and skeletal muscle membranes. Gen. Pharmac. 7, 313-319. SULAKHE, P. V. and ST. LOUIS, P. J. (1977a) Stimulation of calcium accumulation in cardiac sarcolemma by phosphorylase kinase. Biochem. J. 164, 457-459. SULAK]-IE,P. V. and ST. Louis, P. J. (197To) Phospho-dephosphorylation of cardiac sarcolemma. J. Mol. Cell. Cardiol. (abst) 9, 54. SULAKHE,P. V. and ST. Louis, P. J. (1978) Characteristics of heart and sarcolemmal calcium transport system and effect of protein kinase on sarcolemmal calcium accumulation. In : Recent Advances in Studies on Cardiac Structure and Metabolism: Heart Function and Metabolism, Vol. 11, pp. 241-247 (Eds. T. KOEAYASm,T. SANO and N. S. DHALLA)University Park Press, Baltimore. SULAKHE, P. V., FEDELOSOVA,M., MCNAMARA,D. B. and DHALLA, N. S. (1971) Isolation of skeletal muscle membrane fragments containing active Na +-K + stimulated ATPase: Comparison of normal and dystrophic muscle sarcolemma. Biochem. biophys. Res. Commun. 42, 793-800. SULAKHE,P. V., DRUMMOND,G. I. and NG, D. C. (1973a) Calcium binding by skeletal muscle sarcolcmma. J. biol. Chem. 248, 4150-4157. SULAKHE,P. V., NG, D. C. and DRUMMOND,G. I. (1973b) Biochemical characterization of muscle sarcolemma membrane. In : Myocardial Biology, Vol. 4, pp. 195-208. University Park Press, Baltimore. SULAKI-IE,P. V., DRUMMOND,G. I. and NG, D. C. (1973c) Adenosine triphosphatase activities of muscle sarcolemma. J. biol. Chem. 248, 4158-4162.

194

P.V. SULAKHEand P. J. ST. LOUIS

SULAKHE, P. V., LEUNG, N. L-K. and ST. LOUIS, P. J. (1976a) Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase. Can. J. Biochem. 54, 438-445. SULAKHE, P. V., SULAKHE, S. J., LEUNG, N. L-K., ST. LOUIS, P. J. and H1CKIE, R. A. (1976b) Guanylate cyclase : Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver. Biochem. J. 157, 705-712. SULAKHE,P. V., SULAKHE,S. J. and JAN, S-H. (1974) Modulation of calcium transport and ATPase activity in membranes of excitable tissues by cyclic AMP-dependent protein kinase (C-PK) Proc. Int. Union Physiol. Sci. (abst) XI, p. 12. SULAKHE, P. V., ST. LOUIS, P. J. and WEI, J. W. (1977) Sarcolemmal calcium transport and its regulation by cyclic AMP- and calcium-dependent phosphorylation of membrane proteins. Proc. Can. Fed. Biol. Soc. (abst) 20, 114. SULAKHE, P. V., ST. LOUIS, P. J., NARAYANAN, N. and WEI, J. W. (1978) Biochemical characterization of heart sarcolemma (SL). J. Mol. Cell. Cardiol. (abst) 10, 118. SULAKHE, S. J. and ST. Louis, P. J. (1975) Guanylate cyclase in cardiac muscle. Can. Physiol. (abst) 6, 59. SULAKHE, S. J. and SULAKHE, P. V. (1978) Properties of membrane-bound and soluble guanylate cyclase of cardiac and skeletal muscles. In : Recent Advances in Studies on Cardiac Structure and Metabolism: Heart Function and Metabolism, Vol. 11, pp. 257-273 (Eds. T. KOBAYASHI, T. SAND and N. S. DHALLA) University Park Press, Baltimore. SULAKHE, S. J., LEUNG, N. L-K., and SULAKHE, P. V. (1976) Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver. Biochem. J. 157, 713719. SWANSON, P. n., ANDERSON, L. and STAHL, W. L. (1974) Uptake of calcium ions by synaptosomes from rat brain. Biochim. biophys. Aeta 356, 174-183. TADA, M., FINNEY, J. O., JR., SWARTZ, M. n . and KATZ, A. M. (1972) Preparation and properties of plasma membranes from guinea-pig hearts. J. Mol. Cell. Cardiol. 4, 417-426. TADA, M., KIRCHBERGER, M. A. and KATZ, A. M. (1975) Phosphorylation of a 22,000 dalton component of the cardiac sarcoplasmic reticulum by adenosine 3'-5'-monophosphate-dependent protein kinase. J. biol. Chem. 250, 2640-2646. TADA, M., YAMAMOTO, T. and TONOMURO, Y. (1978) Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol. Rev. 58, 1-79. TADA, M., OHMORI, F., YAMADA,M. and ABE, H. (1979) Mechanism of the stimulation of Ca 2 ÷-dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3' : 5'-monophosphate-dependent protein kinase. Role of the 22,000-dalton protein. J. biol. Chem. 254, 319-326. THORPE, W. R. and SEEMAN, P. (1971) Effect of denervating skeletal muscle on calcium binding by isolated sarcolemma. Exp. Neurol. 30, 277-290. TILLISCH, J. H. and LANGER, G. A. (1974) Myocardial mechanical responses and ionic exchange in high sodium perfusate. Circ. Res. 34, 40-50. TILLISCH, J. H., FUNG, L. K., HUM, P. M. and LANGER,G. A. (1979) Transient and steady-state effects of sodium and calcium on myocardial contractile response. J. Mol. Cell. Cardiol. 11, 137-148. TOWER, D. B. (1969) Inorganic constituents. In: Handbook of Neurochemistry, Vol. 1, pp. 1-24 (Ed. H. LAJaa-L~) Plenum Press, New York. TROTTA, E. E. and DE MEIS, L. (1975) ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate. Biochim. biophys Acta 394, 239-247. TROTTA, E. E. and DE MEIS, U (1978) Adenosine 5'-triphosphate -orthophosphate exchange catalyzed by the Ca 2 ÷ transport ATPase of brain. J. biol. Chem. 253, 7821-7825. TSUDZUKI,T. (1979) Adenosine triphosphate-dependent calcium uptake of synaptic vesicle fraction is largely due to contaminating microsomes. J. Biochem. 86, 777-782. USSING, H. H. (1960) The alkali metal ions in isolated systems and tissues. In: Handbuch der experimentellen Pharmakologie, Band 13, p. 50, Berlin, G6ttingen, Heidelberg: Springer. VAN BREMEN,C. (1977) Calcium requirement for activation of intact aortic smooth muscle. J. Physiol. (Lurid.) 272, 317-329. VAN BREEMEN,C., FARINAS, B. R., CASTEELS,C., GERBA, P., WUYTACK,F. and DETH, R. (1973) Factors controlling cytoplasmic Ca 2 ÷ concentration. Phil. Trans. R. Soc. B 265, 57-71. VAN BREEMEN,C., WUYTACK, F. and CASTEELS, R. (1975) Stimulation of 4SCa efflux from smooth muscle cells by metabolic inhibition and high K depolarization, Pflaoers Arch. 359, 183-196. VAN BREEMEN, C., AARONSON, P. and LOUTZEmIISER, R. (1979) Sodium--calcium interactions in mammalian smooth muscle. Pharmac. Rev. 30, 167-208. VANDENBURGH, H. H., SHEFE, M. F. and ZACKS, S. I. (1974) Chemical composition of isolated rat skeletal sarcolemma. J. Mem. Biol. 17, 1-12. VAN ROSSUM,G. D. V. (1970) Net movements of calcium and magnesium in slices of rat liver. J. teen. Physiol. 55, 1832. VAN ROSSUM, G. D. V., SMITH,K. P. and BEETON, P. (1976) Role of mitochondria in control of calcium content of liver slices. Nature 260, 335-337. VERJOVSKI-ALMEIDA, S. and INESI, G. (1979) Fast-kinetic evidence for an activating effect of ATP on the Ca 2÷ transport of sarcoplasmic reticulum ATPase. J. biol. Chem. 254, 18-21. VICKERS, G. R. and DOWDALL, M. J. (1976) Calcium uptake in preterminal central synapses: Importance of Mitochondria : Exp. Brain. Res. 25, 429--445. VINOGRADOV, A. and SCARPA, A. (1973) The initial velocities of calcium uptake by rat liver mitochondria, d. biol. Chem. 248, 5527-5531. WALAAS,O., WALAAS,E., LYSTAD,E., ALERTSEN,AA. R., HORN, R. S. and FOSSOM,S. (1977) A stimulatory effect of insulin on phosphorylation of a peptide in sarcolemma-enriched membrane preparation from rat skeletal muscle. F EBS Lett. 80, 417-422. WALLICK, E. T., LANE, L. K. and SCHWARTZ,A. (1979) Biochemical mechanism of the sodium pump. A. Rev. Physiol. 41, 397-411. WALSH, D. A., CLIPPINGER, M. S., SIVARAMAKRISHNAN,S. and McCULLOUGH, T. E. (1979) Cyclic Adenosine

Passive and active calcium fluxes across plasma membranes

195

monophosphate dependent and independent phosphorylation of sarcolemma membrane proteins in perfused rat heart. Biochemistry 18, 871-877. WATANABE,A. M. and BESCR,H. R., JR. (1974) Myocardial adenylate cyclase; studies on the relationship of activity to purity of sarcolemmal preparation. J. Mol. Cell. Cardiol. 7, 563-565. WATSON,P. T. and WINEGRAD,S. (1973) A possible sodium-calciumexchange in skeletal muscle. Fed. Prec. (abst) 32, 374. WEBER,A. (1966) Energized Calcium transport and relaxing factors. In: Current Topics in Bioenergetics, Vol. 1,203254 (Ed. D. R. SANADI)Academic Press Inc., New York. WEI, J. W. and SULAKHE,P. V. (1978) Regional and subcelluiar distribution of myocardial muscarinic cholinergic receptors. Fur. J. Pharmac. 52, 235-238. WEI, J. W. and SULAKHE,P. V. (1979) Properties of the muscarinic eholinergic receptors in rat atrium. NaunynSchmiedeberg' s Arch. Pharmac. 309, 259-269. WEI,J. W., JANIS,R. A. and DANIEL,E. E. (1976) Isolation and characterization of plasma membrane from rat mesenteric arteries. In: Blood Vessels, Vol. 13, 27%292 (Ed. J. A. BEVAN,M. COMELand L. LASZT)S. Karger, Basel. WEI, J. W., NARA'CANAN,N. and SuI.Ard-m,P.. V. (1977) Characterization of a//-adrenoceptor-coupled adenylate cyclase system of sarcolemma. Prec. Can. Fed. Biol. Soc. (abst) 20, 176. WENDT,I. R. and LANGER,G. A. (1977) Sodium--calcium relationship in mammalian myocardium: effect of sodium deficient perfusion on calcium fluxes. J. Mol. Cell. Cardiol. 9, 551-564. WHITTAKER,V. P. (1965) The application of subcellularfractionation techniques to the study of brain function. Pro0. Biophys. Mol. Biol. 15, 3%96. WI-nTTAKER,V. P., MICHELSON,I. A. and KIP,KLAND,J. A. (1964) The separation of synaptic vesicles from nerveending particles ('synaptosomes'). Biochent J. 90, 293--303. WILL,H., BLANCK,J., SMELTON,G. and WOLLENBERGER,A. (1976) A quench flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence ofimtial velocity on free calcium ion concentration and influence of preincubation with protein kinase MgATP and cyclic AMP. Biochim. biophys. Acta 449, 295-303. WILL, H., LEVCHENKO, T. S., LEVITSKY, D. O., SMIRNOV, V. N. and WOLLENBERGER,A. (1978) Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum. Biochim. biophys. Acta 543, 175-193. WILLIAMSON,J. R., WOODROW,M. L. and SCARPA,A. (1975) Calcium binding to cardiac sarcolemma. In : Recent Advances in Studies on Cardiac Structure and Metabolism: Basic Functions of Cations in Myocardial Activity,

Vol. 5, pp. 61-71 (Eds. A. FLECKENSTEINand N. S. DHALLA)University Park Press, Baltimore. WISHER, M. H. and EVANS, W. H. (1977) Preparation of plasma-membrane subfractious from isolated rat hepatocytes. Biochem. J. 164, 415--422. WOLLENBERGER,A. and WILL, H. (1978) Protein kinase-catalyzed membrane phosphorylation and its possible relationship to the role of calcium in the adrenergic regulation of cardiac contraction. Life Sci. 22, 1159-1178. WOLLENBERGER,A., WILL,H. and KRAUSE,E. G. (1975) Adenosine Y,5'-monophosphate, the myocardial cell membrane, and calcium. In: Recent Advances in Studies on Cardiac Structure and Metabolism: Basic Functions of Cations in Myocardial Activity, Vol. 5, pp. 81-93 (Eds. A. FLECKENSTEINand N. S. D~tALLA)University Park Press, Baltimore. WOOD,J. G., WALLACE,R., WHlTAKER,J. and CHEtnqG,W. J. (1979) Studies of the localization ofcalmodulin and its binding protein in brain. Soc. Neurosci (abst) 5, 310. WOOD,J. M. and SCHWARTZ,A. (1978) Effects ofouabain on calcium-45 flux in guinea pig cardiac tissue. J. Mol. Cell. Cardiol. 10, 137-144. WRAY,H. L. and GRAY,R. R. (1977) Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated, Mg 2+-dependent ATPase in cardiac sarcoplasmic reticulum. Biochira. biophys. Acta 461, 441-459. WLrVTACg,F., LANOON,E., FLEISHER,S. and HAgDMAN,J. G. (1978) The calcium accumulation in a mierosomal fraction from porcine coronary artery smooth muscle. A study of the heterogeneity of the fraction. Biochim. biophys. Acta 540, 253-269. YEAMAN,S. J. and COHEN, P. (1975) The hormonal control of activity of skeletal muscle phosphorylase kinase. Phosphorylation of the enzyme at two sites in rive in response to adrenalin. Eur. J. Biochent 51, 93-104. ZUBRZYCgA,E., KORCZAK,B., SARAZALA,M. G. and DRAaitowsrd, W. (1978) Location of the peripheral proteins in sarcoplasmic reticulum vesicles. F EBS Lett. 90, 215-217.