©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Sodium-Calcium Exchange and Store-dependent Calcium Influx in Transfected Chinese Hamster Ovary Cells Expressing the Bovine Cardiac Sodium-Calcium Exchanger
ACCELERATION OF EXCHANGE ACTIVITY IN THAPSIGARGIN-TREATED CELLS (*)

(Received for publication, August 7, 1995; and in revised form, December 20, 1995)

Galina Chernaya (§) Melissa Vázquez John P. Reeves (¶)

From the Department of Physiology, University of Medicine and Dentistry of New Jersey, The New Jersey Medical School, Newark, New Jersey 07103

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effects of extracellular Na on store-dependent Ca influx were compared for transfected Chinese hamster ovary cells expressing the bovine cardiac Na-Ca exchanger (CK1.4 cells) and vector-transfected control cells. Store-dependent Ca influx was elicited by depletion of intracellular Ca stores with ionomycin, thapsigargin, or extracellular ATP, a purinergic agonist. In each case, the rise in [Ca] upon the addition of extracellular Ca was reduced in CK1.4 cells compared with control cells at physiological [Na]. When Li or NMDG was substituted for Na, the CK1.4 cells showed a greater rise in [Ca] than control cells over the subsequent 3 min after the addition of Ca. Under Na-free conditions, SK& 96365 (50 µM), a blocker of store-operated Ca channels, nearly abolished the thapsigargin-induced rise in [Ca] in the control cells but only partially inhibited this response in the CK1.4 cells. We conclude that in the CK1.4 cells, Ca entry through store-operated channels was counteracted by Na-dependent Ca efflux at physiological [Na], whereas Ca entry was enhanced through Na-dependent Ca influx in the Na-free medium. We examined the effects of thapsigargin on Ba entry in the CK1.4 cells because Ba is transported by the Na-Ca exchanger, but it enters these cells only poorly through store-operated channels, and it is not sequestered by intracellular organelles. Thapsigargin treatment stimulated Ba influx in a Na-free medium, consistent with an acceleration of Ba entry through the Na-Ca exchanger. We conclude that organellar Ca release induces a regulatory activation of Na-Ca exchange activity.


INTRODUCTION

Agents that promote the production of 1,4,5-inositol trisphosphate (InsP(3)) (^1)give rise to a biphasic increase in cytosolic Ca. The initial, transient phase is primarily due to release of Ca from intracellular stores, whereas the more prolonged plateau phase involves an accelerated influx of extracellular Ca. The Ca influx pathway involves low conductance Ca channels and is activated, through a poorly understood mechanism, by the loss of Ca from the InsP(3)-sensitive stores(1, 2, 3, 4) . This process is designated as capacitative Ca entry (3) or store-dependent Ca influx (SDCI)(4) . When cells are exposed to a Ca-mobilizing agent in the absence of extracellular Ca, the SDCI pathway remains activated (even after removal of the Ca-mobilizing agent) until Ca is restored and the InsP(3)-sensitive stores refill with Ca. Inhibitors of sarco(endo)plasmic reticulum Ca ATPase such as thapsigargin (Tg) prevent Ca reaccumulation by the InsP(3)-sensitive stores, resulting in prolonged activation of SDCI(5) .

The Na-Ca exchanger is a carrier-mediated transport process that couples the transmembrane movement of 3 Na ions to the movement of a single Ca in the opposite direction. In cardiac cells, it is widely accepted that the exchanger transports a portion of the Ca released from the sarcoplasmic reticulum out of the cells by Na-dependent Ca efflux and in this way regulates the amount of stored Ca available for release during subsequent beats (reviewed in (6) ). However, in many other types of cells the downstream response to Ca-mobilizing agents depends more on the sustained influx of Ca following agonist addition than on the magnitude of the Ca transient itself. For noncardiac cells, there is a wealth of evidence supporting the generalized activity of Na-Ca exchange in mediating Ca efflux and in modulating the Ca content of intracellular stores(7) . However, the precise physiological role of the exchanger and its interactions with other Ca hemeostatic mechanisms is not as clearly defined in noncardiac cells as in myocardial cells.

A major difficulty in investigations of exchanger function in intact cells is the absence of a selective inhibitor for exchange activity. In this report, we utilize transfected CHO cells to bypass this limitation. CHO cells do not normally express Na-Ca exchange activity, but after transfection with an expression vector coding for the bovine cardiac Na-Ca exchanger, high levels of activity are observed(8, 9) . Comparing the effects of Na on Ca mobilization between transfected and vector-transfected control cells provides a means of identifying functional roles of exchange activity in relation to other cellular mechanisms for intracellular Ca handling. The results of this study indicate that Ca efflux via the Na-Ca exchanger limits the rise in [Ca] during sustained Ca entry, suggesting a potential modulatory function for Na-Ca exchange in the Ca signaling process. Additional evidence suggests that the exchanger itself undergoes a regulatory activation during Ca release from intracellular stores.


EXPERIMENTAL PROCEDURES

Cells

CK1.4 cells were prepared by transfection of dhfr Chinese hamster ovary cells with a mammalian expression vector (pcDNA-NEO; Invitrogen) containing a cDNA insert coding for the bovine cardiac Na-Ca exchanger(8) . Control cells were prepared by transfection of the CHO cells with the vector alone (i.e. no insert). The cells were grown in Iscove's modified Dulbecco's medium containing 10% fetal calf serum and 500 µg/ml geneticin (G418) as described(8) . Unless specified otherwise, biochemicals were obtained from Sigma.

Fura-2 Assays

Cells grown in 75-cm^2 plastic culture flasks were washed three times with Na-PSS, which contains (in mM) 140 NaCl, 5 KCl, 1 MgCl(2), 1 CaCl(2), 10 glucose, and 20 mM Mops, buffered to pH 7.4 (37 °C) with Tris. PSS prepared by substituting 140 mM NaCl with 140 mM LiCl is designated as Li-PSS, and nominally Ca-free PSS refers to PSS in which 1 mM CaCl(2) has been omitted. The cells were released from the flask by the addition of 5 mM EDTA to Ca-free Na-PSS, centrifuged, and resuspended twice in Na-PSS. Aliquots (300 µl) of cells were loaded for 30 min in Na-PSS containing 3 µM fura-2 AM (Molecular Probes) and 0.25 mM sulfinpyrazone (to retard transport of fura-2 out of the cells; (8) ). After loading, the cells were centrifuged and resuspended in 100 µl of Ca-free Na-PSS containing 0.3 mM EGTA and, as indicated, either 10 µM ionomycin or 200 nM thapsigargin. After 1 min, the cells were centrifuged, resuspended in the desired medium (specified in the individual experiments), and placed in a fluorescence cuvette; 0.25 mM sulfinpyrazone was included in the cuvette solutions. Occasional variations in this protocol are designated in individual experiments. All experiments were conducted at 37 °C. Fluorescence was monitored at 510 nm emission with excitation at 340 and 380 nm in a Photon Technology International RF-M 2001 fluorometer. Calibration of the 340/380 ratios was accomplished by adding 10 µM digitonin to the cuvettes in the presence of excess EGTA or Ca for determination of R(min) and R(max), using the formula of Grynkiewicz et al.(10) to calculate the corresponding values of [Ca]. No differences in calibration between CK1.4 and control cells were observed. All fluorescence values were corrected for autofluorescence determined for each set of experiments using unloaded cells. For experiments involving Ba entry, the excitation wavelengths were 350 and 390 nm(11) ; calibrations were not conducted for the Ba experiments. The results are presented for multiple experiments as mean values ± S.E. (error bars shown in figures). Significance testing was carried out using Student's t test (two-tailed) for unpaired samples.

Ca Uptake

Cells were grown in 24-well plastic dishes. The medium was replaced with 1 ml of nominally Ca-free Na-PSS, and the cells were preincubated for 30 min at 37 °C. Midway through the preincubation period, Tg (50 nM) or vehicle (0.1% dimethyl sulfoxide) was added to the desired wells. The preincubation medium was replaced with the 200 µl of assay medium (either Na-PSS or NMDG-PSS, pH 8.0, containing 1 mMCaCl(2)), and after the desired intervals, the wells were washed 4 times with 1 ml of termination medium (100 mM MgCl(2) + 10 mM LaCl(3) + 5 mM Mops/Tris, pH 7.4). The contents of the wells were extracted with 0.1 N HNO(3) and counted.

Protocols

Refilling of Ca Stores

Ca stores were depleted by treating 0.1 ml of fura-2-loaded cells for 1 min with 10 µM ionomycin in Ca-free Na-PSS containing 0.3 mM EGTA (37 °C). The cells were centrifuged and resuspended in nominally Ca-free PSS (with substitutions for Na as indicated) containing 0.3% fatty acid free bovine serum albumin to scavenge residual ionomycin. After 30 s, 1 mM CaCl(2) was added to the medium, and [Ca](i) was monitored. After the desired interval, 3 mM EGTA and, 10 or 30 s later, 0.3 mM ATP were added to bring about Ca release from the InsP(3)-sensitive stores(8) . The peak of the Ca transient was taken as a measure of the amount of Ca in the InsP(3)-sensitive pool.

Tg Treatment

Cell suspensions (0.1 ml) were loaded with fura-2 and treated with 200 nM Tg in Ca-free Na-PSS containing 0.3 mM EGTA for 1 min at 37 °C. The cells were centrifuged and suspended in nominally Ca-free PSS containing 0.3 mM EGTA (with or without Na substitutes as indicated) in the cuvette. CaCl(2) (1 mM) was added as indicated, and [Ca](i) was monitored.


RESULTS

Refilling of Intracellular Ca Stores

Intracellular Ca stores in fura-2-loaded cells were depleted by treatment with ionomycin in a Ca-free medium. The cells were then re-exposed to Ca, and the state of filling of the InsP(3)-sensitive Ca stores was assessed after the desired intervals by the addition of 3 mM EGTA followed by 0.3 mM ATP. ATP elicits the formation of InsP(3) in these cells (8, 12) through its interaction with a P purinergic receptor(12) . As shown in Fig. 1(top trace, left panel) when 0.3 mM ATP was added to CK1.4 cells after pretreatment with ionomycin, no [Ca](i) transient was observed, indicating that the InsP(3)-sensitive stores had been completely depleted; similar results were obtained with control cells (data not shown). In each of the other traces in Fig. 1, CaCl(2) (1 mM) was added at 30 s (arrows), and then 3 mM EGTA was added after various intervals, followed by 0.3 mM ATP 30 s later. For the control cells (right panel, Fig. 1), [Ca](i) increased markedly upon the addition of 1 mM CaCl(2), and the stores refilled rapidly with Ca, attaining a maximal level within 2.5 min. For the CK1.4 cells (left panel, Fig. 1), the increase in [Ca](i) upon the addition of 1 mM CaCl(2) was smaller than for the control cells, and the rate of store refilling was slower, requiring 5 min of exposure to Ca to achieve a maximal level.


Figure 1: Rate of store refilling in CK1.4 cells (left panel) and vector-transfected controls (right panel). Intracellular Ca stores were depleted by treatment with ionomycin and placed in nominally Ca-free Na-PSS containing 0.3% bovine serum albumin (cf. ``Experimental Procedures''). CaCl(2) (1 mM) was added at 30 s (arrows), and after the desired interval, 3 mM EGTA was added () followed by 0.3 mM ATP 30 s later (). Individual traces are displaced vertically by one 340/380 ratio unit for clarity; note the different ordinate scales for the left and right panels. For the control cells, the [Ca] values at the peak of the Ca transients were 95, 110, 220, and 190 nM after exposure to 1 mM CaCl(2) for 0.5, 1, 2.5, and 5 min, respectively. For the CK1.4 cells, the peak [Ca] values were 40, 113, and 160 after 0.5, 2.5, and 5 min of exposure, respectively.



For the CK1.4 cells, the rate of store refilling was markedly increased in a Na-free medium (Li substitution). As shown in Fig. 2(left panels), the increase in [Ca](i) upon the addition of CaCl(2) in Li-PSS was larger than in Na-PSS, and the [Ca](i) transients elicited by ATP were markedly increased; in contrast, there was no significant difference between the sodium- and lithium-based media for the control cells (right panels, Fig. 2). The peaks of the Ca transients in the CK1.4 cells after 30 and 150 s of exposure to Ca were 48 ± 2 nM (n = 5) and 140 ± 13 nM (n = 3) in Na-PSS as compared with 140 ± 22 nM (n = 3) and 427 ± 83 nM (n = 5) in Li-PSS, respectively. The differences between the sodium- and lithium-based media were highly significant (p < 0.01). The comparable values for the control cells were 112 ± 31 nM (n = 5) and 327 ± 46 nM (n = 8) in Na-PSS versus 109 ± 25 nM (n = 3) and 312 ± 64 nM (n = 5) in Li-PSS. The difference between the CK1.4 cells and the control cells in Na-PSS (150 s) was significant (p < 0.01); other comparisons showed no significant differences. The larger size of the [Ca](i) transient for the CK1.4 cells in the Na-free medium was primarily a reflection of the increased amount of Ca in the InsP(3)-sensitive stores. As shown below (see Fig. 8), the ATP-evoked [Ca](i) transient in cells containing equivalent amounts of stored Ca was smaller in Na-PSS than in Li-PSS, but the effect of Na was small compared with that shown in Fig. 2. The results in Fig. 1and Fig. 2indicate that extracellular Na reduces the rate of store refilling in CHO cells expressing the Na-Ca exchanger.


Figure 2: Effect of Na-PSS and Li-PSS on store refilling in CK1.4 cells (A and B) and vector-transfected controls (C and D). Fura-2-loaded cells were treated with ionomycin and placed in nominally Ca-free Na-PSS (bold traces; error bars down) or Li-PSS (light traces; error bars up). CaCl(2) (1 mM) was added at 30 s (arrows), and after either 30 (A and C) or 150 s (B and D), 3 mM EGTA was added () followed by 0.3 mM ATP 10 s later (). The results are the mean values for three to five determinations in two (control) and three (CK1.4) independent experiments.




Figure 8: Effect of Na on [Ca] transients elicited by ATP or ionomycin. CK1.4 cells were loaded with fura-2, preincubated in 100 µl of Na-PSS + 1 mM CaCl(2), and added to a cuvette containing 3 ml of Na- or Li-PSS containing 0.3 mM EGTA. At the times indicated, 0.3 mM ATP (left panel) or 2 µM ionomycin (right panel) were added to the cuvette. The data in each panel are the mean values from six to eight determinations in three experiments.



Tg-induced Ca Entry in CK1.4 Cells

Another method of depleting intracellular Ca stores is by treating the cells with Tg, an irreversible inhibitor of sarco(endo)plasmic reticulum Ca ATPases(13, 14) . In cells pretreated with 200 nM Tg, ATP does not induce a [Ca](i) transient either before or after the addition of extracellular Ca (data not shown). This indicates that the InsP(3)-sensitive Ca stores were fully depleted by the Tg treatment and that refilling of the stores does not occur, as expected from the blockade of sarco(endo)plasmic reticulum Ca ATPase activity. As in other cell types, Tg treatment increases the influx of Mn, a Ca surrogate, as measured by the decline in fluorescence due to quenching of fura-2 by intracellular Mn. As shown in Fig. 3, Mn entry was stimulated by Tg to the same extent in vector-transfected and CK1.4 cells, indicating that intracellular store depletion is equally effective in activating store-operated Ca channels in the two types of cells. No difference was observed in the rate of Mn entry between Na- and Li-PSS for either type of cell (data not shown). Mn is not transported by the Na-Ca exchanger in these cells (data not shown).


Figure 3: Effect of Tg on Mn entry in CK1.4 cells (left panel) and control cells (right panel). Fura-2-loaded cells were pretreated with Tg and placed in Na-PSS + 1 mM CaCl(2). MnCl(2) (0.2 mM) was added at 30 s, and the fluorescence (emission, 510 nm; excitation, 360 nm) was monitored. The results are the mean values of four to six determinations from three independent experiments with each cell type.



The right panel of Fig. 4shows that the addition of 1 mM CaCl(2) to Tg-treated control cells produced a marked increase in [Ca](i), which was essentially identical in Na-PSS or Li-PSS. For CK1.4 cells, however (left panel, Fig. 4), the rise in [Ca](i) was greatly reduced in Na-PSS (trace c) compared with Li-PSS (trace a). Traces b and d depict the rise in [Ca](i) in Li-PSS and Na-PSS, respectively, for CK1.4 cells that had not been treated with Tg. For these cells, the rise in [Ca](i) in Li-PSS (trace b) was significantly greater than in Na-PSS (trace d) but was still much less than that shown by Tg-treated cells in Li-PSS (trace a). Similar results were obtained when NMDG was used as the Na substitute instead of Li (data not shown). The average values of [Ca](i) observed between 90 and 120 s after the addition of CaCl(2) for the data shown in Fig. 4are summarized in Table 1. Compared with the vector-transfected control cells, Tg-treated CK1.4 cells exhibited a reduced [Ca](i) in Na-PSS but an elevated [Ca](i) in Li-PSS (p < 0.01 in each case). The initial portions of the traces from the CK1.4 cells and control cells in Na-PSS are directly compared in the inset to the right panel of Fig. 4. The increases in [Ca](i) are identical initially but as [Ca](i) approaches 100 nM, the rise in [Ca](i) slows dramatically in the CK1.4 cells; the difference between the two types of cells becomes statistically significant (p < 0.05) at all times after the asterisk shown in the inset.


Figure 4: Effect of Na on Ca entry in Tg-treated CHO cells. Fura-2-loaded CK1.4 cells (left panel) or control cells (right panel) were pretreated with or without 200 nM Tg and placed in nominally Ca-free Na- or Li-PSS. CaCl(2) (1 mM) and EGTA (3 mM) were added as indicated. For the CK1.4 cells, the traces labeled a and c correspond to Tg-treated cells in Li-PSS and Na-PSS, respectively; the traces labeled b and d correspond to untreated cells in Li-PSS and Na-PSS. For the control cells (right panel) the data are presented with error bars down for Li-PSS and error bars up for Na-PSS. The results are the mean values of five determinations from four independent experiments except for trace d, which represents nine determinations from five experiments. Inset, direct comparison of data for Tg-treated CK1.4 cells and control cells. The differences between the two types of cells are significant (p < 0.05 or less) for all points following the asterisk.





A similar pattern of results was obtained in Ca flux experiments. The data in Fig. 5depict Ca uptake by vector-transfected control and CK1.4 cells in Na-PSS and in Na-free PSS (NMDG substitution). Ca accumulation under these conditions is markedly enhanced at alkaline pH, and so these experiments were conducted at an external pH of 8.0. As shown in the right panel of Fig. 5, Tg treatment (filled symbols) stimulated Ca uptake in the control cells in both Na-PSS and in NMDG-PSS. For the CK1.4 cells, however (left panel, Fig. 5), Tg had practically no effect on Ca uptake in Na-PSS, although in NMDG-PSS it stimulated Ca uptake as well as in the control cells. The results in NMDG-PSS were similar to those obtained with Li-PSS (data not shown). Thus, the conclusions of the Ca experiments are consistent with those obtained in the fura-2 measurements: Tg-induced Ca entry was markedly reduced in the CK1.4 cells by the presence of extracellular Na.


Figure 5: Effect of Tg on Ca uptake in Na-free media. CK1.4 cells (left panel) or control cells (right panel) were treated with Tg (filled symbols) in Ca-free Na-PSS and assayed for Ca uptake in either Na-PSS (squares) or NMDG-PSS (circles). The unfilled symbols represent data for cells not treated with Tg. The transport assays were conducted at pH 8.0. The results are the means of two separate experiments for each cell type.



In many types of cells, Ca entry via the SDCI pathway is blocked by SK& 96365(15, 16) . As shown in the right panel of Fig. 6, 50 µM SK& 96365 sharply reduced the rise in [Ca](i) in vector-transfected control cells that had been treated with Tg prior to adding 1 mM CaCl(2); SK& was equally effective in Na- or in Li-PSS. In contrast, SK& only partially inhibited the rise in [Ca](i) for Tg-treated CK1.4 cells in Li-PSS (left panel). Thus, in the CK1.4 cells a substantial fraction of Ca enters the cell via a pathway that is insensitive to inhibition by SK& 96365; because this pathway is absent in the control cells, it probably reflects Ca influx via reverse Na-Ca exchange. Incubation of the Tg-treated cells under Na-free conditions for 5 min prior to adding CaCl(2) reduced the increase in [Ca](i) and increased its sensitivity to SK& 96365, consistent with a reduced [Na](i) and decreased contribution of Ca influx via the exchanger (data not shown). Note also that the decline in [Ca](i) after addition of EGTA was slowed by the presence of SK& 96365 (left panel, Fig. 6), consistent with previous reports of an inhibitory effect of this agent on Ca efflux(16, 17) .


Figure 6: Effect of SK& 96365 on Ca entry in Tg-treated CK1.4 cells and vector-transfected controls. Control cells (right panel) were pretreated with 200 nM Tg and placed in nominally Ca-free Na-PSS or Li-PSS with or without 50 µM SK& 96365. CaCl(2) (1 mM) and EGTA (3 mM) were added as indicated. For the CK1.4 cells (left panel), only results with Li-PSS are shown, and the data with SK& 96365 are depicted with downward error bars; note the slower decline in [Ca] after the addition of EGTA in the presence of SK& 96365. For the control cells, the data in Li-PSS are shown with downward error bars. The results are the means of three to six determinations in three experiments for the control cells and seven or eight determinations for five or six experiments for the CK1.4 cells.



Ca Efflux from CK1.4 Cells

The results presented above suggest that Na(o)-dependent Ca efflux via the Na-Ca exchanger is responsible for the attenuation of Tg-induced Ca entry in the presence of Na. To assess the magnitude of Na(o)-dependent Ca efflux from CK1.4 cells, we adopted the following protocol. After loading with fura-2, [Ca](i) was elevated by preincubating a concentrated suspension of the cells for 1 min with Li-PSS containing 200 nM Tg and 1 mM CaCl(2). The cell suspension was then diluted 30-fold into a cuvette containing 0.3 mM EGTA in either Li- or Na-PSS, and the decline in [Ca](i) was monitored. As shown in Fig. 7, the rate of decline of [Ca](i) was more rapid in Na-PSS than in Li-PSS, consistent with a contribution of Na-Ca exchange to Ca efflux. We cannot be certain that the decline in [Ca](i) under these conditions is entirely due to Ca efflux from the cell because Ca sequestration by Tg-resistant compartments could also play a role. However, when control cells were used instead of the CK1.4 cells in similar experiments, there was no difference between Na- or Li-PSS, and the rate of decline in [Ca](i) was similar to that observed in Li-PSS for the CK1.4 cells (data not shown). Thus, the Na-dependent component of the decline in [Ca](i) probably represents Ca efflux via the Na-Ca exchanger.


Figure 7: Effect of Na on Ca efflux in CK1.4 cells. CK1.4 cells were loaded with fura-2 and preincubated for 1 min in 100 µl of Li-PSS containing 1 mM CaCl(2) plus 200 nM Tg. The cells were then diluted into a cuvette containing 3 ml of Na-PSS or Li-PSS, and [Ca] was monitored continuously thereafter. The inset depicts the initial declines in [Ca] in Li-PSS (filled symbols) and Na-PSS (open symbols) as first order plots; the data points were fit to third order polynomials in each case. The data are the mean values from six to eight determinations in four experiments.



The data in the inset of Fig. 7depict the decline in [Ca](i) during the first 40 s of the experiment, expressed as a first order plot. Nonlinear first order plots were observed in both Na- and Li-PSS. At equivalent concentrations of cytosolic Ca, the rate of decline in [Ca](i) was approximately 2.5-fold greater in Na-PSS than in Li-PSS throughout the concentration range examined.

Effects of ATP on [Ca](i) in the Presence of Extracellular Ca

It is important to know if the exchanger modulates Ca movements elicited by physiological agonists such as ATP. Therefore, we examined the effects of Na on the shape of the [Ca](i) transient elicited by ATP in CK1.4 cells in the absence of extracellular Ca. As shown in Fig. 8(left panel), the peak of the [Ca](i) transient was smaller in Na-PSS than in Li-PSS, and the transient decayed to prestimulation levels more quickly. A similar pattern was observed when a [Ca](i) transient was evoked with 2 µM ionomycin instead of ATP (right panel, Fig. 8). With ionomycin, organellar Ca sequestration would be blocked, and the decay of the [Ca](i) transient should reflect Ca efflux from the cell; the acceleration of Ca efflux in Na-PSS compared with Li-PSS therefore provides an index of Na-Ca exchange activity. For control cells, no difference between Na- and Li-PSS was observed in comparable experiments (cf. Fig. 9for data with ATP). The results confirm the importance of Na(o)-dependent Ca efflux in regulating [Ca](i) in the CK1.4 cells.


Figure 9: Effect of ATP in presence of extracellular Ca in CK1.4 cells (left panel) and control cells (right panel). For these experiments, cells were preincubated in Na-PSS containing 1 mM CaCl(2) prior to adding them to the cuvette. The cuvette contained nominally Ca-free PSS plus 0.3 mM EGTA with either 140 mM NaCl (traces labeled c), 140 mM LiCl (a), or 40 mM NaCl + 100 mM KCl (b) as the principal cations. After 30 s of incubation, the following additions were made: 0.3 mM ATP plus 1 mM CaCl(2) (top panel), 0.3 mM ATP alone (center panel), or 1 mM CaCl(2) alone (bottom panel). The stock mixture of 15 mM ATP + 50 mM CaCl(2) added to the cuvette formed a precipitate that dissolved immediately upon dilution in the cuvette. The results with error bars are the mean values from three or four determinations from three experiments; where error bars are not shown, mean values from two separate experiments are presented.



The effects of ATP in the presence extracellular Ca are compared for vector-transfected control cells and the CK1.4 cells in Fig. 9. The cuvette solutions in these experiments initially contained 0.3 mM EGTA and PSS with 140 mM NaCl, 140 mM LiCl, or 40 mM NaCl + 100 mM KCl as the principal salts. The effects of adding 0.3 mM ATP plus 1 mM CaCl(2) (upper traces, Fig. 9), ATP alone (center traces, Fig. 9), or CaCl(2) alone (lower traces, Fig. 9) were examined for each cell type. For the control cells (right panels, Fig. 9), the addition of ATP alone produced a transient rise in [Ca](i), whereas the addition of ATP + 1 mM CaCl(2) elicited a sustained increase in [Ca](i), which was higher than that evoked by the addition of Ca alone. There were no major differences in the behavior of the control cells among the various media, although there was a tendency for the sustained phase of Ca entry to be reduced in the 40/100 sodium/potassium medium (trace b, upper right panel, Fig. 9); the latter effect is probably due to the reduced driving force for Ca entry in cells depolarized by the high potassium concentration.

For the CK1.4 cells (left panel, Fig. 9), dramatic differences were observed among the different media when ATP and Ca were added together. In 140 mM Na (trace c; upper left panel, Fig. 9), the initial [Ca](i) transient was followed by a sustained phase that was slightly but significantly reduced (p < 0.05) compared with that observed in the control cells. The levels of [Ca](i) attained in the Na-free or 40 mM Na media (traces a and b; upper left panel, Fig. 9) were much higher than those produced by the addition of Ca alone (lower left panel, Fig. 9) and were markedly elevated compared with the sustained [Ca](i) levels in the vector-transfected control cells, particularly in the case of Li-PSS. Ca entry was similarly enhanced when NMDG was used as the Na substitute (data not shown). As in the case of Tg-treated cells, the sustained increases in [Ca](i) in the low [Na] media were only partially inhibited by SK& 96365 (data not shown), suggesting that a portion of the Ca entry under these conditions was conducted by the Na-Ca exchange system. Intracellular Ca stores refilled rapidly after ATP addition (assessed by ionomycin-induced Ca release; data not shown) indicating that Ca sequestration was not blocked under these experimental conditions. The pronounced increase in [Ca](i) in the low [Na] media suggests that exchange activity might have been accelerated during the release of Ca from InsP(3)-sensitive stores. This possibility is explored further in the experiments described below.

Barium Influx in Tg-treated CK1.4 Cells

Ba is transported by the cardiac-type Na-Ca exchanger (18) and reportedly enters several types of cells via the SDCI pathway(11, 20, 21, 22) , although in either pathway it is less effectively transported than Ca. Importantly, however, Ba is not sequestered by intracellular organelles such as the endoplasmic reticulum or the mitochondria(20, 23) . Experiments to be reported elsewhere confirm the exchanger's ability to transport Ba and the absence of organellar Ba sequestration in the CK1.4 cells. (^2)Because Ba is not accumulated by intracellular organelles, the fura-2 signal should provide a relatively direct assessment of Ba influx.

The data in Fig. 10(right panel) show the effects of adding 1 mM BaCl(2) to control cells with or without prior treatment with Tg. Ba entry produces an initial abrupt rise in the 350/390 ratio in these experiments, which is probably due to small amounts of extracellular fura-2. The subsequent gradual rise in the 350/390 ratio reflects Ba entry into the cells and is only slightly enhanced in Tg-treated cells (trace a, Fig. 10) compared with untreated cells (trace b, Fig. 10). No difference between Li-PSS and Na-PSS was observed, and so the results with both media have been combined in each trace. Ba influx therefore occurs only weakly through the SDCI pathway in these cells. Note that after the addition of EGTA at 180 s, only a small decline in the fura-2 signal was observed, indicating that Ba is not readily transported out of the cell under these conditions.


Figure 10: Effect of Tg on Ba influx in CHO cells. Control (right panel) and CK1.4 (left panel) cells were loaded with fura-2, treated with or without 200 nM Tg for 1 min in Ca-free Na-PSS + 0.3 mM EGTA, and placed in a cuvette containing Ca-free Na- or Li-PSS with 0.3 mM EGTA. BaCl(2) (1 mM) was added at 30 s, and EGTA (10 mM) was added at 180 s. No difference was observed between Na- or Li-PSS for the control cells, and the combined results are shown. For the CK1.4 cells, no effect of Tg was observed in Na-PSS, and the combined results with and without Tg are shown. Control cells, trace a, Tg-treated cells in Na- or Li-PSS; trace b, nontreated cells in Na- or Li-PSS. CK1.4 cells: trace a, Tg-treated cells in Li-PSS; trace b, nontreated cells in Li-PSS; trace c, Tg-treated and nontreated cells in Na-PSS. The results are the mean values of four determinations from three experiments for the CK1.4 cells and six or seven determinations from three experiments for the control cells.



In CK1.4 cells (left panel, Fig. 10), Ba entry in Li-PSS is substantially increased by Tg (trace a, Fig. 10) compared with untreated cells (trace b, Fig. 10); the fura-2 ratios attained in trace a (Fig. 10) are significantly higher than observed in the vector-transfected control cells. SK& 96365 (50 µM) had no effect on Ba entry in the CK1.4 cells (data not shown), consistent with the absence of significant barium entry via store-operated channels. In Na-PSS, Ba influx (trace c, Fig. 10) was only slightly less than in the nontreated cells in Li-PSS (trace b, Fig. 10); no difference was observed between Tg-treated and untreated cells in Na-PSS, and both conditions have been combined in trace c (Fig. 10). Because a significant acceleration of Ba influx is observed only in Tg-treated CK1.4 cells under Na-free conditions, we conclude that Tg treatment accelerates Na-Ca exchange activity.

In the experiments described above, the cells had been pretreated with Tg in Ca-free Na-PSS, and it is conceivable that increased Na entry might be responsible for the subsequent acceleration of Ba entry by the Na-Ca exchanger. Therefore, we determined whether Tg could accelerate Ba entry when added to the cells in a Na-free medium. CK1.4 cells were loaded with fura-2 and placed in a cuvette containing either Ca-free Na-PSS or Li-PSS and 0.3 mM EGTA. After 30 s, either 200 nM Tg or vehicle (dimethyl sulfoxide) was added to the cuvette, and 1 mM BaCl(2) was added 2 min later. As shown in Fig. 11, in the absence of Tg treatment, there was little or no difference between Na- or Li-PSS for barium entry in the CK1.4 cells (A) or the control cells (C). For both types of cells (Fig. 11, B and D), the addition of Tg resulted in a slowly developing transient rise in the 350/390 fluorescence ratio, which undoubtedly reflects the gradual release of Ca from internal stores. (The choice of 350/390 excitation wavelengths optimizes the Ba signal but still allows detection of increases in [Ca](i).) For the Tg-treated control cells (Fig. 11D), no difference was observed in Ba entry between the Na-PSS and the Li-PSS; the ratios were not significantly higher than those observed in the absence of Tg, again indicating that Ba does not enter these cells efficiently by the SDCI pathway. However, the Tg-treated CK1.4 cells (Fig. 11B) exhibited an accelerated Ba entry in Li-PSS (trace a) compared with Na-PSS (trace b). We conclude that the Tg-induced acceleration of Ba entry via Na-Ca exchange is independent of a possible effect of Tg on Na entry. When the CK1.4 cells were treated with 200 nM ionomycin (which does not transport Ba; (24) ) in otherwise identical experiments, similar results were obtained, i.e. ionomycin stimulated Ba uptake in Li-PSS but not in Na-PSS (data not shown).


Figure 11: Effect of Tg on Ba entry in the presence and the absence of extracellular Na. CK1.4 cells (left panel) and control cells (right panel) were loaded with fura-2, washed once in Ca-free Na-PSS containing 0.3 mM EGTA, and resuspended in cuvettes containing 3 ml of either Na-PSS or Li-PSS (nominally Ca-free plus 0.3 mM EGTA). Tg (200 nM; B and D) or dimethyl sulfoxide (6 µl; A and C) was added at 30 s, and 1 mM BaCl(2) was added at 150 s. For B, trace a, Tg-treated CK1.4 cells in Li-PSS; trace b, Tg-treated CK1.4 cells in Na-PSS. In all experiments, data obtained in Li-PSS are shown with error bars up. The results are the mean values of ten or eleven determinations from six experiments for the CK1.4 cells and four to eight determinations from four experiments for the control cells.




DISCUSSION

The expression of the cardiac Na-Ca exchanger in a cell type that does not normally exhibit this activity confers several new attributes to cellular Ca homeostasis. In the CK1.4 cells, physiological concentrations of extracellular Na retard store refilling and attenuate Tg-induced Ca entry compared with control cells. Upon the addition of Ca(o) to Tg-treated cells, the initial rise in [Ca](i) is identical in CK1.4 cells and in control cells until [Ca](i) reaches 100 nM, when the increase slackens markedly in the CK1.4 cells (inset, Fig. 4). Na(o)-dependent Ca efflux provides the simplest explanation for these results. We suggest that a portion of the Ca entering the cell through store-operated Ca channels is transported back out of the cell by the exchanger, thereby reducing net Ca entry and attenuating the rise in [Ca](i) during SDCI. In this view, the exchanger generates circulatory movements of Ca across the plasma membrane during Ca channel activity; this circulation could play an important role in Ca signaling processes, as suggested by Alkon and Rasmussen(27) .

The effects of Na(o) in stimulating Ca efflux (Fig. 7) and reducing the [Ca](i) transients elicited by ATP or ionomycin (Fig. 8) lend support to this interpretation. However, these data were obtained in Ca-free media and do not necessarily reflect the exchanger's activity in the presence of physiological [Ca](o). On one hand, the efficiency of the exchanger as a Ca pump could be substantially reduced by Ca entry via Na(i)-Ca(o) or Ca(i)-Ca(o) exchanges. On the other hand, local gradients due to Ca channel activity might elevate [Ca](i) beneath the plasma membrane compared with that of the bulk cytosol. The exchanger's rapid turnover (>2,000 s; (25) and (26) ) and high K(m) for Ca (4 µM; ref. 26) would ensure a high level of efficiency in transporting Ca from a region with locally elevated [Ca](i).

In the absence of Na(o), store refilling and Tg-induced Ca influx are accelerated in CK1.4 cells compared with control cells (Fig. 1, Fig. 2, Fig. 4, and Fig. 5and Table 1). Under these conditions, Ca efflux via the exchanger is blocked and Ca enters the cell both by SK&-sensitive channels (Fig. 6) and by ``reverse mode'' Na-Ca exchange (Na(i)-dependent Ca influx), a process that is insensitive to the SK& compound. An unexpected feature of our results is that exchange activity is accelerated during Ca release from internal stores. In Na-free or low [Na] media, the plateau levels of [Ca](i) following the simultaneous addition of ATP and Ca(o) to CK1.4 cells were greatly enhanced compared with either the addition of Ca alone or the responses of the control cells (Fig. 9). Although these observations are consistent with an increase in exchange activity, alterations in other Ca handling pathways could also contribute to the results.

The Ba experiments provide firm support for activation of the exchanger during Ca release. The fura-2 signal for Ba provides a better index of divalent cation influx than that for Ca because Ba is not sequestered by intracellular organelles, and its cytosolic concentration should therefore be unaffected by blockade of sarco(endo)plasmic reticulum Ca ATPase activity. Moreover, Ba entry via store operated Ca channels appears to be minimal in CHO cells ( Fig. 10and Fig. 11), allowing a better assessment of alterations in exchange activity. With CK1.4 cells, Tg differentially stimulates Ba entry in Li-PSS compared with Na-PSS but has no such effect with the control cells (Fig. 10, 11). Similar results were obtained using ionomycin to release Ca from internal stores (data not shown). Recent experiments indicate that ATP also evokes a transient increase in exchange-mediated Ba influx in the CK1.4 cells. (^3)

It is important to distinguish between regulatory activation of exchange activity and increased activity that is simply due to a rise in the concentration of Na or Ca as a transport substrate. Although increased Ca efflux via the exchanger following organellar Ca release has been reported on numerous occasions, to our knowledge a linkage between Ca release and regulatory activation of exchange activity has not been shown previously in any cell type. In our experiments, enhanced exchange activity was measured as Na(i)-dependent Ba influx and could be observed in a Na-free medium (Fig. 11). We therefore conclude that exchange activity is activated by a regulatory process during Ca release from internal stores.

The mechanism of activation is uncertain. Two modes of regulation of the cardiac exchanger have been described: ATP-dependent regulation and secondary Ca activation(28, 29, 30, 31) . A phosphorylation mechanism has been suggested for ATP-dependent activation of exchange activity in squid giant axons(28) , but experiments with sarcolemmal membrane patches (29, 30, 31) and our previously published results with CK1.4 cells (9) raise doubts as to the relevance of this mechanism for the cardiac exchanger. Moreover, preliminary experiments indicate that the Tg-induced increase in Ba entry is not inhibited by the nonspecific protein kinase inhibitor staurosporine (1 µM; data not shown). Thus, it seems unlikely that exchange activity is accelerated by a protein kinase-dependent mechanism. Other suggested modalities of ATP-dependent regulation, such as aminophospholipid translocase activity (30) or cytoskeletal alterations (9) , have not yet been tested.

Secondary activation by Ca would seem to be a plausible mechanism, because acceleration of exchange activity is linked to Ca release from internal stores. This mode of regulation involves the interaction of Ca with regulatory sites distinct from the transport sites for Ca that lie within the central hydrophilic domain of the exchanger. The molecular identity of these sites has recently been described by Philipson and his colleagues(32, 33) . Secondary Ca activation has been extensively studied in squid giant axons(28, 34) , barnacle muscle (35) , myocardial cells(36) , and cardiac sarcolemmal membrane patches (29, 31, 37) , but its physiological importance is not well understood.

The data in Fig. 9(ATP) and Fig. 11(Tg) are consistent with secondary Ca activation as the mechanism accelerating exchange activity, because in both cases the increased Ca or Ba influx was associated with an elevation in [Ca](i). However, under conditions where the cells were pretreated with Tg, there does not appear to be an elevation of [Ca](i) compared with untreated cells prior to adding extracellular Ca ( Fig. 4and Fig. 6) or Ba (Fig. 10). Thus, it appears that accelerated exchange activity does not necessarily correlate with increased cytosolic Ca. Experiments currently under way suggest that Ca(i)-dependent changes in exchange activity involve complex interactions between cytosolic Ca and the Ca content of internal stores. Additional studies will be required to resolve these issues.

Regardless of the precise mechanism(s) involved, our findings indicate that Ca release from intracellular stores is coupled to regulatory activation of Na-Ca exchange activity. The exchanger is potentially a focal point for a variety of regulatory influences, as suggested by reports that the sensitivity of the exchanger to secondary activation by Ca can itself be modulated by an ATP-dependent mechanism(19) . Activation of exchange activity during Ca release could therefore provide an adjustable negative feedback mechanism for controlling the amount of Ca that is resequestered by the sarco(endo)plasmic reticulum and, as demonstrated in this report, for limiting net Ca entry into the cell during Ca channel activity. Na-Ca exchange activity thus provides a potentially rich source of regulatory control for cellular Ca traffic.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL49932. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral Research Fellow of the American Heart Association (New Jersey affiliate).

To whom correspondence should be addressed. Tel.: 201-982-3890; Fax: 201-982-7950; reeves{at}umdnj.edu.

(^1)
The abbreviations used are: InsP(3), inositol 1,4,5-trisphosphate; CHO, Chinese hamster ovary; NMDG, N-methyl-D-glucamine; PSS, physiological salts solution; SDCI, store-dependent Ca influx; Tg, thapsigargin; Mops, 3-(N-morpholino)propanesulfonic acid.

(^2)
M. Condrescu, G. Chernaya, J. G. Patel, and John P. Reeves, manuscript in preparation.

(^3)
M. Vázquez and J. P. Reeves, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Vijay Patel for assistance with the ionomycin experiment in Fig. 8and Drs. Abraham Aviv, Madalina Condrescu, and Masayuki Kimura for advice and helpful discussions during the course of this work.


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