Cdl caldm (lMJ2) 13, 027-634 0 LongmanGroupUK Ltd 1982

Cellular origin of the rapidly exchangeable calcium pool in the cultured neonatal rat heart cell J.A. POST* and G.A. LANGER Departments of Physiology and Medicine, Cardiovascular Research Laboratories, UCLA School of Medicine, Los Angeles, California, USA Abstract - Calcium in the myocardial cell is highly compartmentalized and in the cultured neonatal rat heart cells over 66% of the exchangeable calcium exchanges extremely fast (Q/z < I s). The goal of the present study was to investigate, in the intact cell, the locus of thls pool. By comparing myoblasts and fibroblasts and their respective plasma membranes, it is concluded that in the intact myocyte a significant fraction of the large lanthanum displaceable calcium pool is attributable to intracellular components, not present in the fibroblast. At least 36% of the lanthanum displaceable pool resides intracellularly, as is shown with the use of the drugs nifedipine, ryanodine and thapslgargin. It is proposed that the diadic subsarcolemmal junctional region represents a significant locus for the pool.

Calcium in the myocaxdial cell is highly compartmentalized and in two recent papers these kinetically distinct compartments have been described in isolated adult rat heart cells [l] and in cultured neonatal rat heart cells [2]. In the cultured cells 4 compartments were described: (i) a non-exchangeable compartment; (ii) a slowly exchangeable compartment, with a ttn of 19 min; (iii) 2 intermediate compartments with tla of 19 and IO3 s; and (iv) a fast compartment with tin < 1.5 s. A similar qualitative compartmentation was found for adult cells and it was shown that the slow phase resides in the mitochondria and the intermediate phases in the samoplasmic reticulum [ll. The fast compartment could not be positively localized to a cellular organ* Present address: Dr JA Post, Institute of Biomembranes, Padualaan 8.3584 CH Utrecht, The Netherlands

elle, but its rapid exchange characteristic tentatively placed it at, or in virtually instantaneous equilibrium with, the sarcolemma. In on-line experiments, in which the myocardial cells are equilibrated with 45Ca, exposure to lanthanum results in a displacement of cell-associated calcium [l-31. The amount of calcium displaced by lanthanum, 3.1 mmoleskg dry weight, represents over 66% of the total exchangeable calcium When a washout is initiated by rapidly washing cells with a 45Ca-free buffer, after pre-labelling with 45Ca, and lanthanum is added to the washout medium after 2 s, no increase in the 45Ca content of the effluent is observed [ll. The absence of any lanthanum displaceable calcium at this time means that the lanthanum displaceable calcium had already exchanged and is in a very rapidly exchangeable compartment, with a ttn of < 1 s. As stated above, the lanthanum 627

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displaceable compartment contains over 66% of the total cellular exchangeable calcium in the cultured neonatal cells [2] aad is likely to play a role in the excitation-contraction coupling process. Even though several calcium compartments have been identified within the intact myocaniial cell aad related to function and orgaaelle of origin [l], this large, rapidly exchangeable lanthanum displaceable pool of calcium has not been localized. We recently studied the lanthanum displaceable calcium binding to isolated sarcoleauna of cultured neonatal rat heart cells [4, 51 and this study showed that the majority of these sarcolemmal calcium binding sites in these isolated merabraaes are within the cytoplasmic leaflet of the sarcolemmal bilayer. This contribution of the inner rather than the outer leatlet had not been predicted aad prompted further investigation, in the intact cell, of the locus of this largest of the cell’s exchangeable calcium pools.

Materials and Methods Cell culture Culturing and isolation of the myocardial cells was done according to a modification of the method of Harary aad Farley [6]. Neonatal rats (1-2 days old) were decapitated, the hearts were excised aad minced. The miace was incubated in a spinner flask at 37°C with 0.05-0.1% trypsia (in 137 mM NaCI, 5 mM KCl, 4 HIM NaHCO3, 5 mM glucose and penicillin (100 000 units/l)/streptomycla (100 mg/l). The incubation fluid was decanted and new medium was added. The supematant from the first 3 incubations (15 min each) was discarded. The cell pellets were spun (8 min. 430 g) and resuspended in growth medium (Gibco. Ham FlO, supplemented with 10% fetal calf serum, 10% horse serum, penicillin (100 000 uaits/l)/streptomycia (100 mg/l), Arabinose C (IO @I, to inhibit fibroblast growth) and CaClz (tiaal concentration 1 mM). The cells were plated on Falcon 3000 dishes for 2-3 h during which time fibroblasts adhere aad myocytes xrmaia freely suspended [7]. Finally, the myocytes were plated on Primaria treated disks (Falcon Plastics) which contain sciatillant material (Bicroa) and within 3 days a confluent monolayer of spoataae-

CELL CALCIUM

ously beating, virtually pun?. myocytes was formed. Fibroblasts were obtained by culturing the cells which had been stack to the Falcon 3000 dishes, in the same growth medium, without Arabinose C. After the cells reached confluency, they were removed from the substrate by Qypsinatioa and were plated on the same, sciatillaat containing plastic, until they reached confluency. Isolationof the sarcolemma The plasraa membraae of the myocytes and the fibroblasts were isolated by the so-called ‘gasdissection’ technique which has been previously described ia detail [8,9]. In short, the disk with cell monolayer attached is placed at the ceatre of a platform in a chamber, which is closed. The platform is then elevated so that a valve extending into the chamber makes firm contact with the ceatre of the disk. Upon rapid (< 1 s) opening of the inlet valve, N2 (2000 psi) exits in a stream parallel to the surface of the monolayer, shearing open the upper surface of the cells, blowing out the cellular material and leaving the sarcolemrna, of high purity, attached to the disk. Purity and recovery of this sarcolemma preparation has been described before 191 and is summarized as follows: a 50-fold purification of Nat/K+-ATPase activity (over homogenate), an increase of the cholesterol/phospholipid ratio from 0.35 to 0.49 (raol/mol), an increase of the phospholipid to protein ratio of 0.24 to 1.4 (pm01 Pilmg protein), absence of sarcoplasmic reticulum, a small mitochoadrial contamination, and a sarcolemma recovery of 43%. 45Ca monitoring The 45Ca binding and uptake by the cells on the ‘gas-dissected’ membranes is monitored by a scintillation flow cell technique. described in detail by Frank et al. [lo]. In short, the plastic disks on which the cells are grown, contain a scintillant material. The disks are mounted in a flow cell, which then is placed in the well of a specially The following designed scintillation counter. perfusate was used: (in mM) 133 NaCl, 3.6 KCl, 0.3 MgClz, 16 glucose and 5 Tris(hydroxymethy1) aminomethane rnaleate (pH 7.20MaOH) and various

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The insert shows the lower lanthanum concentrations

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in

Closed circles represent the cells, the open circles

the ‘gas-dissected’ membranes

CaClz concentrations. To this solution 45Ca2t (ICN) was added at 1 pCi/ml. The 45Ca2tsignal of the cells or the membranes, in close proximity of the scintillation disk, counts with a much higher efficiency (39%) than the 4sCa2t in the bulk solution (< 5%). Since the signal of the bulk solution does not change, the effect of the interventions can be determined.

In the first series of experiments, we studied the effect of increasing amounts of lanthanum on the amount of calcium displaced from ‘gas-dissected* membranes and intact cells at a constant calcium concentration of 1 mh4. The lanthanum displacement at 1 mM was maximal and was, in order to directly compare the ‘gas-dissected’ membranes with the intact cells, set at 100% for both the ‘gasdissected’ membranes and the cells (for the intact cells 100% represented 3.1 mmol/kg dry weight and for the ‘gas-dissected’membranes 100% represented 45 nmolhq sarcolemmal protein). Figure 1 shows that the lanthanum dependency of the calcium displacement for the ‘gas-dissected’membranes and the cells are essentially superimposable. Figure 2 shows the calcium dependency of the lanthanum displaceable calcium pool. We varied the extracellular calcium concentration from 30 @I to 1000 JLMand obtained the maximal amounts of calcium displaced by lanthanum Again, to compare the ‘gas-dissected’ membranes and the cells, we set the amount of calcium displaced at a calcium concentration of 1 mM to 100% (at 1 mM extracellular calcium the 100% values were the same as above). Figure 2 clearly shows that there is a significant difference between the results obtained using the cells and the ‘gas-dissected’ membranes. At

Drugs used

A stock solution of nifedipine (Sigma) was made up in ethanol, as was thapsigargin (LC Services Corporation). Both were used at 1 pM concentration and the amount of ethanol (0.1%) alone added to the cells has no effect on function or 45Ca characterRyanodine (Agrisystems) was directly istics. dissolved in the buffer used. Statistics

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calcium concentrations.

All data are presented as mean + standard deviation. Data are analyzed and compared using the unpaired Student’s t-test.

100

membranes 100%.

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at various

For both the cells and the ‘gas-dissected’

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The solid bars represent the. cells, the open ones the

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displaceable calcium pool ‘saturates’ at much lower calcium cooceotratioos io the intact cell. Subsequently we compared the data obtained for the myocytes with non-muscle cells, fibroblasts. isolated from the same hearts. Myocytes showed a lanthanum displaceable calcium pool (at 1 mM extracellular calcium) of 3.1 + 0.3 mmol/kg dry weight (o = 6). At the same calcium concentration the fibroblasts showed a laotbaoum displaceable calcium pool of only 0.7 + 0.1 mmol/kg dry weight (o = 9). a highly significant difference (P < 0.001). The ‘gas-dissected* membranes of the myocytes showed a laothaoum displaceable calcium pool, at 1 o&l [Cal of 45 f 10 mmol/mg protein (0=9), whereas the ‘gas-dissected’ membranes of the fibroblasts showed a lanthanum displaceable calcium pool of 79 * 50 oolovmg protein (0 = 9). Thus, even though the isolated plasma membrane of the fibroblast binds the same amount of calcium as the sarcolemon of the myocyte, the displacement from intact cells is the opposite, i.e. the fibroblasts show a much smaller lanthanum displaceable calcium pool. 10 order to investigate whether part of tbe lanthanum displaceable calcium pool resides iotracellularly, the neonatal rat heart cells or the ‘gasdissected’ membranes were labelled for a period with4?a (extracellular calcium concentration is 1 mM) until a steady state was reached. Subsequently, 1 mM laotbaoum was added to the perfusate and, after stabilization of the signal, we switched to the original buffer aod studied the reversibility of the lanthanum displacement (Fig. 3A). The signal obtained befonz the addition of lanthanum was set at 100%. the one obtaioed at the end of the lanthanum exposure at 0%. Figure 3B shows that the signal of the ‘gas-dissected’ membranes returns to 100% within 30 tin, whereas the signal of the intact cells requires a much longer period to return to 100%. Blockage of the L-type calcium channel, by the addition of 1 @VI oifedipioe, just before aod during the removal of lanthanum, showed that the recovery of the signal in the intact cells was slowed down aod reached only 60% of the original signal. The same was observed with the use of ao ioorgaoic blocker of the channel. 50 @VIZo [ll]. Blockage of the T-type calcium channels by 50 J.LMNi [12] did not affect the degree of recovery of the signal.

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Ng. 3 (A) The 45Ca signal from cells labelled with “Ca (1 @@x101) at a calcium concentration of 1 mM. After equilibration, at the upper arrow. 1 mM lanthanum is added to the supetperfusate (no change in 45Ca specific activity). After another 30 min, lanthanum is removed from the superperfusate at the lower arrow and the recovery of the original signal is studied. (B) Return to the original signal after removal of the lanthanum for cells (closed circles), ‘gas-dissected’ membranes (open circles) and cells in the presence of 1 pM nifedipine (closed triangles). The original signal (before the addition of lanthanum) is set at 100%; the signal after the displacement is set at 0%

100 pM calcium the lanthanum displacement io the intact cells is already 75% of that at 1000 pM, whereas it is only 15% io the case of the ‘gasdissected’ membranes. These results show sigoificaotly different behaviour between the neonatal rat heart cells aod their sarcolemrna, i.e. the lanthanum

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To further study the possible role of intracellular calcium in the lanthanum displaceable pool, the neonatal rat heart cells were preincubated with 1 @I thapsigargin, an inhibitor of the calcium ATPase of the sarcoplasmic reticulum [13, 141. This resulted in a reduction of the lanthanum displaceable calcium pool from 3.2 -I- 0.4(n = 10) to 2.3+ 0.2(n = 5) mmol/kg dry weight (P c 0.001). Pmincubation with 1 p.M nifedipine, to block the L-type calcium channel, also resulted in a reduction of this pool, to 2.4 + 0.7 mmovkg dry weight (n = 6; P < 0.02).

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Ryanodine (1 ph4) reduced the pool to 1.8 + 0.5 mmol/kg dry weight (P < 0.001) and 1 mM ryanodine reduced the pool to 2.65f 0.73mmofig dry weight (n = 5; P < 0.08). Since the above results, as will be discussed, indicate that part of the lanthanum displaceable calcium pool depends upon an intracellular source, we attempted to determine the route by which the intracellular calcium moves out of the cell when displaced by lanthanum. The Na/Ca exchanger is a possible candidate [2]. In a recent study [15], a calcium pool in adult rat cells was described, whose exchange is dependent on the NaKa exchanger. This pool is also present in the neonatal cells (Post, Kuwata and Langer, unpublished observations) and can be used to study the time course of inhibition of the Na/Ca exchanger by lanthanum. When isotopically labelled cells are perfused with a calcium and sodium free buffer, this pool is retained in the cells, but, as soon as sodium and calcium are returned this pool, is released. This is shown in Figure 4A. The curve in Figure 4B (typical of 3 experiments) shows that when sodium and calcium are reintroduced in the presence of 1 mM lanthanum, the release of 45Ca is eliminated. This indicates an immediate inhibition of the Na/Ca exchanger and eliminates the exchanger as the route from the cell. The possible role of the Na/Ca exchanger in the lanthanum displaceable calcium pool was also tested by analyzing this pool in the absence of extracellular sodium (choline chloride substituted). Under those conditions, the size of the lanthanum displaceable calcium pool was 3.2 + 0.4 mmobkg dry weight (n = 9), thus identical to the size in the presence of sodium.

Discussion The goal of this study was to gain further insight into the lanthanum displaceable calcium pool in intact myocardial cells. This pool contains over 66% of the total exchangeable calcium in the cells used in this study [2] and it is, therefore, likely to represent an important calcium pool in excitationcontraction coupling. One of the approaches compared the ‘gasdissected’ sarcolemma of the cultured neonatal cells

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Ccal(PM) Fig. 5 Curve fitting of the obtained lanthanum displaceable calcium pools using Michaelis-Menton equation. The intact cells (closed circles) show a Kd of 55 pM whereas the ‘gas-dissected’ membranes (open circles) show a fi of 1 n&l. The left Y-axis is for the intact cells, the right axis for the ‘gas-dissected’ membranes (SL - sarcolemma)

with the intact cells. Since the samolemmal recovery during the ‘gas-dissectioning’ is known (43%) [9], as well as the absolute amount of sarcolemma obtained per gram cells (19.1 mg/g cell dry weight) [18], we can calculate the amount of calcium displaced from the ‘gas-dissected’ membranes in terms of its contribution in the intact cell. In this study, the ‘gas-dissected’ membranes of the heart cells showed a lanthanum displaceable calcium pool of 45 nmol/mg sarcolemmaI protein. Using the factors described above, 1 kg of dry weight of cells contains (19.1/O-43 =) 44g sarcolemmaI protein, which would lead in the intact cells to 2.0 mmohkg dry weight. This is somewhat less than previous values obtained, being 3.2 mmol/kg dry weight [18]. The reason for this discrepancy is unexplained, but given improvements in technique, it is likely that present values are more reliable. It has to be kept in mind that in the ‘gasdissected’ membranes both sides of the membrane are accessible to several chemical probes 191and, therefore, most likely also to lanthanum and also to the calcium concentration in the buffer. In the intact cell, only the extracellular leaflet of the sarcolemma is exposed to the buffer system used. Therefore, the amount of lanthanum displaceable calcium from

intact cells, derived from the ‘gas-dissected’membranes, is most likely an overestimation. This then indicates that the sarcolemmal lanthanum displaceable calcium pool cannot explain the total cellular lanthanum displaceable calcium pool. Despite the above mentioned complications, a qualitative comparison of the two systems, to gain further insights into the locus of the pool, is certainly possible. Firstly the fact that both systems show the same lanthanum dependency (Fig. 1) indicates that in the intact cells either the total lanthanum displaceable calcium pool resides at the extracellular samolemmal surface or that a fraction resides intracellularly, but is displaced by lanthanum in the intact cell with the same characteristics as lanthanum displaces it from the inner leaflet of the ‘gas-dissected’membranes. Figure 2 shows that the calcium dependency of the lanthanum displaceable calcium pool is dramatically different when the intact myocardial cells are compared with their ‘gas-dissected’ sarcolemma. The intact cells maintain their lanthanum displaceable calcium pool at low calcium concentrations to a much greater extent than the membranes. Treating both curves as if they were binding curves one obtains clearly different results for the two preparations (Fig. 5), with a Kd of 55 pM for the cells and a IQ of 1 mM for the UXmbraW. This SUggeStS that, in order to maintain the total cellular lanthanum displaceable pool, the intact myocyte is lE@HI.

This hypothesis was further tested by comparing the results obtained from the myocytes with those obtained from fibroblasts. It was found that intact fibroblasts showed a much Smaller lanthanum displaceable calcium pool, only 22% of that of the myocytes (expressed per kg dry weight). However, the ‘gas-dissected*plasma membrane of the fibroblast plasma membrane showed a lanthanum displaceable calcium pool which is comparable in size to that of the isolated myocyte sarcolemma. This indicates that, in the tibroblast plasma memb- rane, the majority of the calcium binding sites are in the cytoplasmic leaflet. The comparable size of the lanthanum displaceable calcium pool on the plasma membrane of both cell types and the difference in the size of the pool in the intact cells indicate that, in the intact myocyte, a significant portion of the

NEONATAL RAT HEART CELLS: RAPIDLY EXCHANGEABLE Ca POOL

large lanthanum displaceable calcium pool is atbibutable to intracellular components, not present in the fibroblasts. Interestingly, vascular smooth muscle cells also show a large lanthanum displaceable calcium pool [16] and these cells have, in common with the myocytes, a well-developed sarcoplasmic reticulum. Figure 3 gives further evidence that at least part of the cellular lanthanum displaceable calcium pool is dependent upon intracellular calcium. This part is, directly or indirectly, supplied via the L-type calcium channel, because blockage of this channel prevents repletion of the pool. Furthermore, preincubation of the myocytes with nifedipine results in a reduction of the lanthanum displaceable calcium pool, again indicating that calcium entering the cell through the L-type calcium channel plays an important role in this pool. The data obtained with thapsigargin and ryanodine provide further insight into the origin of the intracellular component of the lanthanum displaceable calcium pool. Thapsigargin is an inhibitor of the Ca-ATPase of the sarcoplasmic reticulum [13, 141 and results in a depletion of the caffeine releasable calcium in the sarcoplasmic reticulum (Post and Langer, unpublished observations). Preincubation of the neonatal cells with this agent significantly reduces the lanthanum displaceable calcium pool by 30%. This could indicate that 30% of the lanthanum displaceable calcium pool resides directly in the sarcoplasmic reticulum. However, a more likely explanation is that upon thapsigargin treatment a new steady state redistribution of cellular calcium occurs, which then results in a decrease in the lanthanum displaceable calcium pool. In any event, the data clearly show that at least a part of the lanthanum displaceable calcium pool depends upon a source of intracellular calcium. The data obtained with ryanodine support these results. The large difference in the size of the lanthanum displaceable calcium pool in myocytes and fibroblasts, despite similar calcium binding to their isolated plasma membranes, points in the direction of a specialized cell organelle or configuration of cell components, which is required to obtain a large lanthanum calcium displacement, In detailed studies on the lanthanum displaceable calcium pool from ‘gas-dissected’ sarcolemma we used proteases,

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neuraminidase and phospholipases to elucidate the origin and topological distribution of the sarcolemmal calcium binding sites [4, 51. Two classes of calcium binding sites were found: low affinity binding sites of lipidic origin and high affinity binding sites, most likely of proteinaceous origin. It was shown that the majority of the binding sites, constituting the sarcolemmal lanthanum displaceable calcium pool, are located in the cytoplasmic leaflet of the sarcolemmal bilayer [4, 51. These binding sites dramatically alter the subsarcolemmal calcium concentration profile in the so-called ‘cleft-area’, the space between the junctional sarcoplasmic reticulum and the cytoplasmic leaflet of the sarcolemma, and significant calcium binding to the inner sarcolemmal leaflet can occur [17]. The sites could accommodate more than 4 mm01 calcium per kg dry weight of cells, more than sufficient to account for the lanthanum displaceable compartment. The amount bound is conceived to be in equilibrium with that contained in the junctional sarcoplasmic reticulum. If this decreases (e.g. with tbapsigargin, ryanodine or nifedipine), the amount bound which is putatively accessible to lanthanum, will decline. Although the sarcoplasmic reticulum of the neonatal cells is less well developed than in adult rat heart cells, it is clearly well developed and specialized subsarcolemmal regions are present [18]. Fibroblast endoplasmic reticulum will certainly not play a role in cellular calcium regulation to the extent found in the cardiac myocyte sarcoplasmic reticulum and the fibroblast does not have the sarcoplasmic reticulum junctional structure found in the myoblasts. Interestingly, vascular smo-oth muscle cells do have a well developed sarcoplasmic reticulum, which is clearly engaged in control of cellular calcium and this cell type also demonstrates a large lanthanum displaceable calcium pool [16]. Thus it might well be that a specialized region in the cell, such as the diadic subsarcolemmal junctional region (‘cleft’) represents a significant locus for the rapidly exchangeable lanthanum displaceable calcium pool. If binding sites at the inner sarcolemmal leaflet represent a portion of the lanthanum displaceable compartment, as seems likely, two problems arise: (i) such binding requires that calcium concentrations in the sub-sarcolemmal ‘cleft’ (see above) be in the 50-100 p&I range and such concentrations are,

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indeed, predicted by a recently developed model [18]. Such high concentration in the region from which calcium induced calcium release [17] is assumed to occur, however, tend to keep the sarcoplasmic reticulum release channels permanently closed. (ii) Displacement by lanthanum from the inner leaflet requires that the displaced calcium move across the membrane to the extracellular space. Since lanthanum blocks the two known calcium efflnx pathways, the Na/Ca exchanger (Fig. 4) and the sarcolemmal calcium pump [19], the route via which calcium is removed is not presently apparent. It is clear that further study is required.

Acknowledgements We thank Mrs Eloise Andrew-Farley for skilful isolation and culturing of the neonatal rat heart cells. This study was supported by USPHS grants HL 28539-07. 08, the Laubisch Fund, the Castera Foundation and the American Heart Association, Greater Los Angeles Affiliate.

References 1. Langer GA. Rich TL. Omer Ff3. (1990) Ca exchange under

2.

3. 4. 5.

6.

7.

non-perfusion-limited conditions in rat ventricular cells: identification of subcellular compaameuts. Am. J. Physiol., 259, HS92-H602. Kuwata JH. Langer GA. (1989) Rapid, non-perfusion-limited calcium exchange in cultured neonatal myocardial cells. J. Mol. Cell Cardiol., 21, 1195-1208. Langer GA. Frank JS. (1972) Lanthanum in heart cell culture. J. Cell Biol., 54, 441-445. Post JA. Langer GA. (1991) Molecular origin of samolemmal calcium binding sites. FASEB J., 5, Al051. Post JA. Langer GA. (1992) Sarcolemmal calcium binding sites in heart: I. Molecular origin in ‘gas-dissected’ sarcolemma. J. Membr. Biol., 129, 49-57. Harary L. Farley B. (1963) In vitro studies of single tat heart cells. I. Growth and organization. Exp. Cell l&s., 29, 451-465. Blonde1 B. Roijeir T. Clrzneval JP. (1971) Heart cells in culture: a simple method for increasing the proportion of myoblasts. Experientia, 27,356-358.

8. Langer GA. Frank JS. Philipson KD. (1978) Pmpamtion of sarcolemmal membrane from myocardial tissue culture monolayer by high velocity gas dissection. Science, 200, 1388-1391. 9. Post JA. Langer GA. Op den Kamp JAF. Verkleij AJ. (1988) Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and ‘gas-dissected’ membranes. Biochim. Biophys. Acta, 943,256-266. 10. Frank JS. Langer GA. Nudd LM. 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. 11. Hille B. (1984) Ionic Channels of Excitable Membranes. Sunderland, MA, Sinauer Associates Inc. 12. Tytgat J. Vemecke J. Carmeliet J. (1990) A combined study of sodium current and T-type calcium current in isolated cardiac cells. Ptliigers Arch., 417, 142-148. 13. Thapstrup 0. Cullen PJ. Drobak BK. Hartley MR. Dawson AP. (1990) Thapsigargin, a tumor promoter discharges intracellular Ca2’ stores b specific inhibition of the $+ endoplasmic reticulum Ca ATPase. Proc. Natl. Acad. Sci. USA, 87,2466-2470. 14. Kijima Y. Ogunbunmi E. Fleischer S. (1991) Drug action of thapsigargin on the Ca2+pump protein of sarcoplasmic reticulum. J. Biol. Claem., 266.22912-22918. 15. Langer GA. Rich TL. (1992) Identification and characterization of a discrete Na-Ca exchange-dependent calcium compartment in rat ventricular cells. Am. J. Physiol., 262, Cl 149C1153. 16. Pierce ON. Langer GA. Wright GW. Kutryk MJB. (1989) Calcium is rapidly exchangeable in cultured vascular smooth muscle cells from rabbit aorta. J. Mol. Cell Cardiol.. 21, 889-899. 17. Peskoff A. Post JA. Langer GA. (1992) Sarcolemmal calcium binding sites in heart: II. Mathematical model for diffusion of calcium released from the sarcoplasmic reticulum into the diadic region, J. Membr. Biol., 129, 59-69. 18. Langer GA. Frank JS. Nudd LM. (1979) Correlation of calcium exchange, structure and function in myocardial tissue. Am. J. Physiol.. 237, H239-H246. 19. Carafoli E. (1991) Calcium pump of the plasma membrane. Physiol. Rev., 71, 129-153. Please send reprint requests to : Dr Glenn A. Langer, Department of Medicine, Cardiovascular Research Laboratories, UCLA School of Medicine, McDonald Research Laboratories Building, Room 3-645, 675 Circle Drive South, Los Angeles CA QOU24-1760,USA Received : 6 February 1992 Revised : 25 March 1992 Accepted : 11 May 1992