Store-operated Ca2+ entry and coupling to Ca2+ pool depletion in thapsigargin-resistant cells.

The release of Ca2+ from intracellular Ca2+ pumping pools and the entry of extracellular Ca2+ are tightly coupled events. The potent and specific intracellular Ca2+ pump inhibitor, thapsigargin, blocks Ca2+ accumulation and allows Ca2+ release from pools within mammalian cells, inducing major changes in endoplasmic reticulum function and cell growth. Recent studies characterized the pools of Ca2+ within permeabilized DC-3F/TG2 cells (a thapsigargin-resistant variant form of the DC-3F Chinese hamster lung fibroblast line, able to grow in 2 μM thapsigargin), revealing highly thapsigargin-resistant intracellular Ca2+ pumping activity capable of accumulating Ca2+ within an inositol 1,4,5-trisphosphate-releasable Ca2+ pool (Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961). Using intact fura-2-loaded thapsigargin-resistant DC-3F/TG2 cells, the present study investigated the role of this unusual Ca2+ pumping activity in maintaining cytosolic Ca2+, generating Ca2+ signals, and mediating Ca2+ entry. The thapsigargin-resistant Ca2+ pumping pool was capable of generating rapid cytosolic Ca2+ signals in response to the phospholipase C-coupled agonist, oleoyl lysophosphatidic acid. The resting level of cytosolic Ca2+ in DC-3F/TG2 cells was 2-fold elevated compared with control cells (the parent DC-3F line), and transient extracellular Ca2+ removal induced a large “overshoot” in cytosolic Ca2+. The overshoot response was blocked by the Ca2+ influx inhibitor, SKF96365, and was kinetically identical to that induced in parent DC-3F cells after thapsigargin-induced Ca2+ pool emptying, indicating that the thapsigargin-resistant DC-3F/TG2 cells had “constitutively” opened Ca2+ entry channels coupled to an emptied or partially emptied thapsigargin-sensitive Ca2+ pumping pool. Even though oleoyl lysophosphatidic acid-mediated Ca2+ release induced little Ca2+ entry, complete ionomycin-activated emptying of the thapsigargin-resistant Ca2+ pool in DC-3F/TG2 cells induced a large, sustained entry of Ca2+ that was also completely blocked by SKF96365. The results revealed that the thapsigargin-resistant Ca2+ pump does maintain physiological Ca2+ levels, is able to fill an agonist-responsive Ca2+ pool in DC-3F/TG2 cells, and is likely responsible for the ability of these cells to function and grow in the presence of thapsigargin. In addition, Ca2+ influx in the resistant DC-3F/TG2 cells reflects emptying of pools that accumulate Ca2+ by both thapsigargin-sensitive and -resistant Ca2+ pumps; since these pumps accumulate Ca2+ in distinct pools in parent DC-3F cells, it is possible that more than one pool is coupled to Ca2+ influx in the resistant DC-3F/TG2 cells.

The release of Ca 2؉ from intracellular Ca 2؉ pumping pools and the entry of extracellular Ca 2؉ are tightly coupled events. The potent and specific intracellular Ca 2؉ pump inhibitor, thapsigargin, blocks Ca 2؉ accumulation and allows Ca 2؉ release from pools within mammalian cells, inducing major changes in endoplasmic reticulum function and cell growth. Recent studies characterized the pools of Ca 2؉ within permeabilized DC-3F/ TG2 cells (a thapsigargin-resistant variant form of the DC-3F Chinese hamster lung fibroblast line, able to grow in 2 M thapsigargin), revealing highly thapsigargin-resistant intracellular Ca 2؉ pumping activity capable of accumulating Ca 2؉ within an inositol 1,4,5trisphosphate-releasable Ca 2؉ pool (Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961). Using intact fura-2-loaded thapsigarginresistant DC-3F/TG2 cells, the present study investigated the role of this unusual Ca 2؉ pumping activity in maintaining cytosolic Ca 2؉ , generating Ca 2؉ signals, and mediating Ca 2؉ entry. The thapsigargin-resistant Ca 2؉ pumping pool was capable of generating rapid cytosolic Ca 2؉ signals in response to the phospholipase C-coupled agonist, oleoyl lysophosphatidic acid. The resting level of cytosolic Ca 2؉ in DC-3F/TG2 cells was 2-fold elevated compared with control cells (the parent DC-3F line), and transient extracellular Ca 2؉ removal induced a large "overshoot" in cytosolic Ca 2؉ . The overshoot response was blocked by the Ca 2؉ influx inhibitor, SKF96365, and was kinetically identical to that induced in parent DC-3F cells after thapsigargin-induced Ca 2؉ pool emptying, indicating that the thapsigargin-resistant DC-3F/TG2 cells had "constitutively" opened Ca 2؉ entry channels coupled to an emptied or partially emptied thapsigargin-sensitive Ca 2؉ pumping pool. Even though oleoyl lysophosphatidic acid-mediated Ca 2؉ release induced little Ca 2؉ entry, complete ionomycin-activated emptying of the thapsigargin-resistant Ca 2؉ pool in DC-3F/TG2 cells induced a large, sustained entry of Ca 2؉ that was also completely blocked by SKF96365. The results revealed that the thapsigargin-resistant Ca 2؉ pump does maintain physiological Ca 2؉ levels, is able to fill an agonist-responsive Ca 2؉ pool in DC-3F/TG2 cells, and is likely responsible for the ability of these cells to function and grow in the presence of thapsigargin. In addition, Ca 2؉ influx in the resistant DC-3F/TG2 cells reflects emptying of pools that accumulate Ca 2؉ by both thapsigargin-sensitive and -resistant Ca 2؉ pumps; since these pumps accumulate Ca 2؉ in distinct pools in parent DC-3F cells, it is possible that more than one pool is coupled to Ca 2؉ influx in the resistant DC-3F/TG2 cells.
Ca 2ϩ signals in many cells comprise both release of Ca 2ϩ from intracellular pools and entry of Ca 2ϩ across the plasma membrane. These two events are closely coupled, entry of Ca 2ϩ being triggered by emptying of Ca 2ϩ from pools in response to InsP 3 1 (1,2). Whereas the precise localization of Ca 2ϩ pools involved in activating Ca 2ϩ entry is uncertain (3,4), in general it appears that the ER or subcompartments thereof are the site of InsP 3 -mediated Ca 2ϩ release (5)(6)(7). Quite substantial changes in intraluminal Ca 2ϩ levels occur in response to InsP 3induced Ca 2ϩ release from the ER (8). Such changes in ER Ca 2ϩ are responsible for a number of different cellular responses. Thus, the decrease in Ca 2ϩ stored within the ER appears to be the primary determinant for activating the opening of store-operated channels (SOCs) 2 in the plasma membrane, allowing the entry of Ca 2ϩ , which serves to enhance cytosolic Ca 2ϩ signals as well as allow refilling of intracellular pools (1)(2)(3). The level of Ca 2ϩ in pools also appears to control the function of InsP 3 -sensitive Ca 2ϩ release channels (5,6). In addition to controlling Ca 2ϩ signal generation within the cytosol, the level of Ca 2ϩ within pools mediates important control over intraluminal events including the essential ER functions of translation, folding, processing, and assembly of proteins (9 -13); such effects may be mediated by the large array of intraluminal Ca 2ϩ -binding proteins, several of which function as molecular chaperones (14,15). Last, it has become clear that the Ca 2ϩ content of intracellular Ca 2ϩ pools exerts profound control over cell proliferation and progression of cells through the cell cycle (16 -19).
The ER accumulates Ca 2ϩ via the function of intracellular sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) Ca 2ϩ pump proteins (20 -22); these SERCA pumps have been shown to be highly sensitive to the Ca 2ϩ pump blocker, thap-sigargin (23,24). Thapsigargin binds with extremely high affinity to intracellular Ca 2ϩ pumps resulting in a virtually irreversible inhibition of Ca 2ϩ accumulation within the ER (16, 24 -26). Emptying of ER Ca 2ϩ with either thapsigargin or other Ca 2ϩ pump blockers, including 2,5-di-tert-butylhydroquinone and cyclopiazonic acid, causes cells to undergo one of two different growth responses. DDT 1 MF-2 smooth muscle cells progress through S-phase and become arrested in a quiescent G 0 -like state in which they remain viable and stable for as long as 7 days (16,17). Other cells such as prostatic cancer cells enter an irreversible apoptotic state in which endonucleases are activated, DNA becomes fragmented, and cells undergo morphological degeneration and death (27). One criterion that may determine these growth responses to Ca 2ϩ pump blockade is the extent of Ca 2ϩ influx activated by pool emptying. Prolonged Ca 2ϩ influx via SOCs and hence sustained elevated cytosolic Ca 2ϩ appears to trigger apoptosis (4,7,27), whereas survival in a growth-arrested state is observed in cells in which Ca 2ϩ influx is rapidly deactivated, such as DDT 1 MF-2 smooth muscle cells (7,17,28). In the latter line, application of high serum or arachidonic acid induces the pool-depleted quiescent cells to synthesize new pump protein, to develop new functional Ca 2ϩ pools, and to reenter the cell cycle (17)(18)(19).
Even though thapsigargin-induced Ca 2ϩ pool emptying has such profound effects on cell function and growth, a variant of the DC-3F Chinese hamster lung fibroblast cell line was recently developed that is resistant to thapsigargin (29). The resistant variant line, DC-3F/TG2, was generated by exposure of DC-3F cells to gradually increasing levels of thapsigargin over a 10-month period. Unlike the parent DC-3F cells that have a high sensitivity, cytotoxic response to thapsigargin, DC-3F/TG2 cells retain normal appearance and grow and divide in culture medium containing 2 M thapsigargin (29). This remarkable resistance to thapsigargin did not appear to stem from expression of high levels of the multidrug resistance factor, P-glycoprotein, which can confer resistance to toxic hydrophobic molecules; nor did it appear to reflect substantially increased levels of SERCA pump expression (29). Instead, the resistance is attributable to expression of a novel intracellular Ca 2ϩ pump activity with 20,000-fold lower sensitivity to thapsigargin (30). In spite of this vast difference in thapsigargin sensitivity, the resistant pump has similar high affinity for Ca 2ϩ (K m 0.1 M), ATP dependence, and sensitivity to vanadate as normal SERCA pumping activity (30). A small amount of what appears to be the same thapsigargin-resistant pump activity is detectable in parent DC-3F cells; however, it does not mediate any Ca 2ϩ accumulation within InsP 3 -sensitive Ca 2ϩ pools; for this reason, it was considered that in parent DC-3F cells the resistant Ca 2ϩ pump functions to accumulate Ca 2ϩ within a pool that is distinct from the InsP 3 -sensitive Ca 2ϩ pool (30). In the resistant DC-3F/TG2 cells, InsP 3 -sensitive Ca 2ϩ pools clearly exist, but in contrast with parent DC-3F cells, their uptake of Ca 2ϩ occurs exclusively via the thapsigarginresistant Ca 2ϩ pump (30). In addition to this important Ca 2ϩ pump distinction, other changes in Ca 2ϩ pool function within resistant cells were noted, including differences in pool heterogeneity, anion permeability, and the translocation of Ca 2ϩ between pools. From these results it was concluded that the cells contain two different Ca 2ϩ pools distinguished by sensitivity to thapsigargin and that in the resistant DC-3F/TG2 cells, InsP 3 receptors are expressed within the pool accumulating Ca 2ϩ via the thapsigargin-insensitive Ca 2ϩ pump (30).
Whereas these analyses using non-intact cells revealed that the resistant DC-3F/TG2 cells contained thapsigargin-resistant Ca 2ϩ pumps capable of accumulating Ca 2ϩ within InsP 3sensitive pools, no information was available on the physiolog-ical function of these pools in intact cells. Two important questions remained to be addressed. The first was whether the Ca 2ϩ pumping activity operating within the thapsigargin-resistant cells was able to fill agonist-releasable Ca 2ϩ pools. The second was to determine whether release of Ca 2ϩ from such pools was coupled to SOC-mediated Ca 2ϩ influx. The results indicate that Ca 2ϩ pools inside the resistant DC-3F/TG2 cells are indeed functional, that the resistant pump accumulates Ca 2ϩ within a pool that can be mobilized by agonists, and that emptying of this pool within the intact cells is coupled to activation of Ca 2ϩ influx through authentic store-operated channels. The results also reveal that the activation of Ca 2ϩ influx in the resistant DC-3F/TG2 cells reflects operation of both normal and resistant Ca 2ϩ pumping activities possibly residing in distinct pools.

Culture of Parent DC-3F and Thapsigargin-resistant DC-3F/TG2
Cells-DC-3F Chinese hamster lung fibroblasts were cultured in ␣-modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal bovine serum (Life Technologies, Inc.) as described previously (29,30). Cells received a change of medium after 2 days and were passaged the following day. Selection of the thapsigargin-resistant DC-3F/TG2 cell line by successive culturing in the presence of increasing thapsigargin concentrations was described previously (29). DC-3F/TG2 cells were cultured in ␣-modified Eagle's medium with 5% heat-inactivated fetal bovine serum together with 2 M thapsigargin; as for the parent line, the cells also received a change of medium (still containing 2 M thapsigargin) on the second day after passaging and were passaged the following day. Both cell types were also cultured on glass coverslips for use in intracellular Ca 2ϩ measurements.
Intracellular Free Ca 2ϩ Measurements-The methodology for measurement of intracellular free Ca 2ϩ within DC-3F and DC-3F/TG2 cells using fura-2 was as described previously (8,17,28,30). Cells were grown on glass coverslips as described above and fura-2-loaded by incubation with 2 M fura-2/AM ester for 10 min at 20°C in Hepesbuffered Krebs medium (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO 4 , 1 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 11.5 mM glucose, 20 mM Hepes-KOH, pH 7.4, 0.1% bovine serum albumin). Under these conditions approximately 95% of dye was restricted to the cytosol as judged by the signal remaining after permeabilization with saponin. Fluorescence emission at 505 nm was monitored at 25°C using a PTI dual wavelength spectrofluorometer system with excitation at 340 and 380 nm. Calculations of free intracellular Ca 2ϩ concentrations were as described by Grynkiewicz et al. (31) using a K d of 135 nM. Dye was considered saturated upon addition of 40 M ionomycin, whereas minimum fluorescence ratio was determined in the presence of 10 mM EGTA together with ionomycin. For each of the traces shown, groups of 10 -15 cells were analyzed. Unless indicated otherwise, all results are representative of a minimum of three experiments.
Materials and Miscellaneous-Parent DC-3F and the thapsigarginresistant DC-3F/TG2 cell lines were kindly provided by Dr. Arif Hussain (University of Maryland Cancer Center). Thapsigargin was purchased from LC Services, Worcester, MA. Fura-2 was from Molecular Probes, Inc., Eugene OR. Miscellaneous procedures and materials were as described previously (16 -18).

Cytosolic Ca 2ϩ Responses to Thapsigargin in Intact Parent DC-3F and Thapsigargin-resistant DC-3F/TG2 Cells-
Whereas almost all mammalian cells accumulate Ca 2ϩ within pools via intracellular Ca 2ϩ pumps with extreme sensitivity to thapsigargin (24 -26), the DC-3F/TG2 thapsigargin-resistant cell line contains highly resistant Ca 2ϩ pump activity (30). Unknown was whether this distinct Ca 2ϩ pumping could function to sustain physiologically operational Ca 2ϩ pools within intact thapsigargin-resistant cells. Initial experiments examined Ca 2ϩ levels in parent DC-3F and resistant DC-3F/TG2 cells and cytosolic Ca 2ϩ changes in response to exogenously added thapsigargin. Using fura-2-loaded parent DC-3F cells grown on coverslips, application of a high dose of thapsigargin (2 M) induced a rapid and large increase in cytosolic Ca 2ϩ (Fig.  1A). Although the peak Ca 2ϩ level partially subsided with time, the increase in Ca 2ϩ was persistent with time as a result of continued influx of Ca 2ϩ through store-operated Ca 2ϩ channels (see below). As shown in Fig. 1A, 30 min after thapsigargin addition, the level of Ca 2ϩ was still above 120 nM. From many experiments, the mean resting level of Ca 2ϩ in these cells (i.e. without thapsigargin) was measured as 27.7 Ϯ 7.0 nM (n ϭ 94), and the mean level of Ca 2ϩ after 30 min of thapsigargin treatment was 148.4 Ϯ 33.1 nM (n ϭ 52). Even several hours following thapsigargin treatment this increased level of Ca 2ϩ was maintained. We noted that in another cell type, the DDT 1 MF-2 smooth muscle line, although thapsigargin induced a similarly rapid and large Ca 2ϩ increase due to pool release, the ensuing SOC-mediated influx of Ca 2ϩ was short-lived due to rapid deactivation (7,(17)(18)(19). Indeed, it was considered that the efficient deactivation of SOCs may explain why DDT 1 MF-2 cells remain viable (albeit in a growth-arrested state) and able to undergo growth recovery with high serum treatment for up to 7 days following thapsigargin treatment (7). In contrast, once treated with thapsigargin, DC-3F cells cannot be induced to reenter the growth cycle and undergo necrosis (30). This may result from the cytotoxic action of the prolonged increase in cytosolic Ca 2ϩ that follows thapsigargin-induced pool release.
In marked distinction from the parent DC-3F cells, the thapsigargin-resistant DC-3F/TG2 cells showed no response to exogenously added thapsigargin. Thus, as shown in Fig. 1B, no change in cytosolic Ca 2ϩ was observed after addition of either 100 nM or 2 M thapsigargin. Although there was no effect of thapsigargin, it was clear from the data in Fig. 1B that the resting Ca 2ϩ level in these cells was higher than in the parent DC-3F line. Indeed, from a large number of experiments, the mean resting level of cytosolic Ca 2ϩ in resistant DC-3F/TG2 cells was 70.4 Ϯ 15.5 nM (n ϭ 154), that is approximately double that in the parent line. This suggested that either there was less Ca 2ϩ pumping activity in these cells or that persistent entry of Ca 2ϩ was occurring perhaps as a result of some degree of pool emptying. Although levels of plasma membrane Ca 2ϩ pumps are unknown, the level of SERCA pump protein in resistant DC-3F/TG2 cells is slightly increased relative to parent DC-3F cells (29). Experiments described below indicate that there is an endogenously activated increased basal level of Ca 2ϩ influx in resistant DC-3F/TG2 cells.
Agonist-mediated Ca 2ϩ Pool Release in Parent DC-3F and Resistant DC-3F/TG2 Cells-The absence of a thapsigarginsensitive Ca 2ϩ pool in intact DC-3F/TG2 cells was consistent with observations that these cells lack thapsigargin-sensitive Ca 2ϩ pumping activity. It was also clear from previous experiments with permeabilized DC-3F/TG2 cells that they express a highly thapsigargin-insensitive Ca 2ϩ pump (30). This activity has an IC 50 for thapsigargin of approximately 4 M, about 20,000-fold less sensitive than most of the Ca 2ϩ pumping activity inside parent DC-3F cells, which has an IC 50 for thapsigargin of approximately 200 pM (30). Interesting was the finding that a small fraction (approximately 20%) of the total pump activity in parent DC-3F cells has an IC 50 for thapsigargin (4 M) identical to that of pumping in resistant DC-3F/TG2 cells. From this it was inferred that both pump types are coexpressed in normal cells. However, each pump appears to function in a distinct Ca 2ϩ pool; thus, from experiments with permeabilized DC-3F cells, the resistant pump did not function to accumulate Ca 2ϩ within an InsP 3 -sensitive pool. In contrast, in permeabilized resistant DC-3F/TG2 cells, the resistant pump clearly did accumulate Ca 2ϩ within an InsP 3 -releasable pool (30). The important question was therefore whether in intact DC-3F/ TG2 cells this pool was coupled to agonist-induced Ca 2ϩ signal generation.
As shown in Fig. 2A, release of agonist-sensitive Ca 2ϩ pools in parent DC-3F cells is activated by LPA receptors coupled to phospholipase C stimulation and InsP 3 production, as in many other cell types (32). Upon addition of a maximally effective dose of LPA (100 M), there was a rapid increase in cytosolic Ca 2ϩ , reaching a peak within 30 s and thereafter returning to resting levels between 100 and 150 s later. The peak Ca 2ϩ release from pools was not as high as with thapsigargin and did not include a significant influx component. Reapplication of LPA a second time after return to basal Ca 2ϩ levels caused no further Ca 2ϩ response (data not shown), likely reflecting desensitization of the LPA receptor. Importantly, the resistant DC-3F/TG2 cells also responded to LPA (Fig. 2B). The response in these cells was very similar to that in the parent line. The rate of onset was rapid and there was a fast termination of the response; the decrease in Ca 2ϩ back to basal levels was consistently faster and often dipped below the starting Ca 2ϩ level. Although the peak level of Ca 2ϩ attained in resistant DC-3F/ TG2 cells was a little higher, the peak height above resting Ca 2ϩ was very similar with both cell types, approximately 100 nM. Thus, the size of the response did not appear to be affected by the increased level of basal Ca 2ϩ within DC-3F/TG2 cells. Whereas the LPA response in parent DC-3F cells was completely abolished by prior treatment with 3 M thapsigargin, the Ca 2ϩ pump blocker had no effect on LPA-induced Ca 2ϩ release in resistant DC-3F/TG2 cells (data not shown). These results are significant since they provide direct proof that the thapsigargin-resistant Ca 2ϩ pumping within DC-3F/TG2 cells causes Ca 2ϩ accumulation within an agonist-sensitive Ca 2ϩ pool.
Coupling of Ca 2ϩ Pools to Ca 2ϩ Influx in Parent DC-3F and Resistant DC-3F/TG2 Cells-An important further question to address was the relationship between Ca 2ϩ pool release and the activation of Ca 2ϩ entry in both cell types. In the parent DC-3F cells, emptying of pools with thapsigargin appeared to induce quite substantial and long lasting Ca 2ϩ influx (Fig. 1A). The smaller degree of Ca 2ϩ influx observed following agonistinduced Ca 2ϩ release ( Fig. 2A) could reflect less than complete pool emptying by LPA, resulting from desensitization of the LPA receptor, as mentioned above. In apparent contrast, neither LPA nor thapsigargin induced Ca 2ϩ entry in the resistant DC-3F/TG2 cells. To investigate activation of Ca 2ϩ influx further, we took advantage of the fact that SOCs can be induced to open by transient removal of extracellular Ca 2ϩ . As described above, in many cells, SOC-mediated Ca 2ϩ influx becomes partially or completely deactivated with time (7,28), and brief exposure to low extracellular Ca 2ϩ causes temporary reactivation of SOCs (28,33). As shown in Fig. 3A, treatment of parent DC-3F cells with nominally Ca 2ϩ -free medium for 3 min had virtually no effect upon cytosolic Ca 2ϩ levels. However, if the cells were first treated with thapsigargin for 30 min, the removal of Ca 2ϩ caused a fast and very substantial decrease in cytosolic Ca 2ϩ (Fig. 3B). Under this condition, pools have been emptied and SOCs remain at least partially activated, as shown in Fig. 1A. The removal of Ca 2ϩ from the external medium has two effects; first, the driving force for Ca 2ϩ entry through channels is greatly diminished and second, the lowered external Ca 2ϩ level may increase the coupling efficiency of the plasma membrane Ca 2ϩ pump as has been observed for operation of intracellular Ca 2ϩ pumps with decreased luminal Ca 2ϩ levels (34). The net result is a rapid decrease in cytosolic Ca 2ϩ , reaching a new equilibrium within 30 s. As discussed below, the lowered cytosolic Ca 2ϩ level and possibly also the lowered extracellular Ca 2ϩ level enhance opening of Ca 2ϩ influx channels. When Ca 2ϩ was added back, the open Ca 2ϩ channels allowed Ca 2ϩ to rapidly enter the cells and a transient overshoot of Ca 2ϩ was observed (Fig. 3A). As channels returned to a partially deactivated state, the cytosolic Ca 2ϩ level re-turned to almost the same level as before Ca 2ϩ removal. The decrease in Ca 2ϩ entry likely reflects an inhibitory effect of cytosolic Ca 2ϩ on Ca 2ϩ entry through SOCs, Ca 2ϩ interacting with a site possibly close to the channel itself (2,28,33,35). There is also evidence that Ca 2ϩ may associate with an extracellular site (or possibly a site within the channel) and exert an inhibitory effect on SOC opening (36).
Significantly, as shown in Fig. 3C, the same brief extracellular Ca 2ϩ removal applied to DC-3F/TG2 cells caused a cytosolic Ca 2ϩ decrease followed by a substantial overshoot, both responses being similar to the responses seen with thapsigargin-treated parent DC-3F cells. This is important because it indicates that in the DC-3F/TG2 cells, Ca 2ϩ influx channels are at least partially activated. The interpretation of this observation is that even though these cells have agonist-releasable pools that are not emptied, their state of SOC activity closely resembles that of the parent cells after thapsigargin-induced emptying of pools. Therefore, it appears that a thapsigarginsensitive pool that operates to activate Ca 2ϩ entry exists in the resistant DC-3F/TG2 cells and remains in an empty or partially empty state. Since the DC-3F/TG2 cells are continually exposed to thapsigargin during growth and since the action of thapsigargin is irreversible (16) it is reasonable that such a pool would be empty.
Even though the signal for SOC-mediated Ca 2ϩ entry appears to be turned on in DC-3F/TG2 cells, the channels remain in a mostly deactivated state, as judged by the relatively low resting Ca 2ϩ levels in these cells. A question arising is how reactivation of channels by transient external Ca 2ϩ removal occurs and whether this process is the same in both DC-3F and DC-3F/TG2 cells. As shown in Fig. 4, experiments assessed the time dependence of Ca 2ϩ removal on subsequent reactivation of Ca 2ϩ influx in the two cell types. Using parent DC-3F cells treated for 30 min with 2 M thapsigargin, extracellular Ca 2ϩ was removed for 30 s to allow a new steady state to be attained. Normal extracellular Ca 2ϩ was returned either immediately following this time or at 30-s intervals up to 2 min later (Fig.  4A). The immediate return of Ca 2ϩ following the 30-s removal resulted in a rapid return of cytosolic Ca 2ϩ to the original equilibrium level; under this condition there was no overshoot. Overshoots were observed following removal of Ca 2ϩ for 1 min or longer; maximal overshoots were observed after removal of Ca 2ϩ for 1.5 min or longer. Results from a series of experiments that analyzed the kinetics of the activation of overshoots showed that the peak level of Ca 2ϩ influx resulted in a doubling of the basal Ca 2ϩ level and that half-maximal peak height was induced between 0.5 and 1 min after the Ca 2ϩ removal period (Fig. 4A, inset). This period of time is very similar to that shown by Zweifach and Lewis (35) for the current mediated by SOCs to recover in Jurkat T lymphocytes following Ca 2ϩ removal. Most likely, the reactivation of Ca 2ϩ influx reflects dissociation of Ca 2ϩ from a Ca 2ϩ binding site that mediates inhibitory control over SOC activity, either within cells (2,35) or possibly on the outer surface (36). Clearly, the reactivation process that occurs in the DC-3F/TG2 cells following Ca 2ϩ removal is virtually identical (Fig. 4B). In this case, even though the peak reactivation following Ca 2ϩ readdition was slightly less than with parent DC-3F cells, it represented a 3-fold enhancement over the basal level of Ca 2ϩ influx occurring; the time dependence for reactivation was very similar to parent cells. Therefore, it appears that the state of activation of SOCs and the mechanisms for deactivating and reactivating Ca 2ϩ influx remain the same for both resistant DC-3F/TG2 cells and the thapsigargin-treated parent DC-3F cells.
Steady State Ca 2ϩ Influx and Ca 2ϩ Overshoots Are Both Inhibited by the Ca 2ϩ Influx Blocker, SKF96365-From the above studies, we considered that the increased resting levels of Ca 2ϩ within the resistant DC-3F/TG2 cells were due to continual influx through Ca 2ϩ entry channels. We sought to investigate this further by analyzing pharmacological modification of Ca 2ϩ entry channels using the imidazole derivative, SKF96365. This compound was originally shown to inhibit SOC-mediated Ca 2ϩ entry by Merritt et al. (37). Even though the selectivity of SKF96365 is not confined to SOC activity (for example, it has effects on voltage-sensitive Ca 2ϩ influx as well as other channel activities), its effects on blocking SOC-mediated influx have been widely described (2,37,38). Clearly, SKF96365 does terminate the influx of Ca 2ϩ in DC-3F cells induced by thapsigargin-mediated Ca 2ϩ pool depletion. As shown in Fig. 5A, the application of 100 M SKF96365 to parent DC-3F cells treated with 2 M thapsigargin for 30 min caused a large decrease in cytosolic Ca 2ϩ levels, reducing the level from more than 150 M down toward 50 M within 100 s. Subsequent removal of external Ca 2ϩ made little further difference, and readdition of Ca 2ϩ , even after a maximally effective 3-min delay (see Fig. 4), caused only a slight change in Ca 2ϩ . Thus, under this emptied pool condition, clearly, channels are open, and addition of SKF96365 causes a rundown of cytosolic Ca 2ϩ to a new steady state as a result of channel closure. Once blocked, the overshoot response almost completely disappeared (Fig. 5A), indicating that very little reopening of channels occurred as a result of transient Ca 2ϩ removal. SKF96365 applied to normal DC-3F cells with filled pools was without any effect on cytosolic Ca 2ϩ levels (data not shown), indicating that there is no measurable effect on other channel or pump activities in these cells. In Fig. 5B, thapsigargintreated parent DC-3F cells were treated with SKF96365 but, in this case, after Ca 2ϩ removal. In this experiment the rate at which the new steady state is reached is significantly faster, presumably, as described above, due to more efficient pumping across the plasma membrane as a result of the decreased Ca 2ϩ gradient. The new equilibrium level of Ca 2ϩ (approximately 25 M) was unaffected by addition of SKF96365, and the overshoot response following readdition of Ca 2ϩ was completely abolished. From this, it is clear that the blocking action of SKF96365 is not restricted to channels actively conducting Ca 2ϩ and is not dependent on the presence of external Ca 2ϩ . These experiments show that the action of SKF96365 is to effectively block SOC activity. Moreover, they suggest that both Medium was changed to nominally Ca 2ϩ -free medium for 30 s, followed by continuation in this Ca 2ϩ -free condition for either 0, 0.5, 1.0, 1.5, or 2.0 min, as shown. The times shown represent times from the moment of return to normal (1 mM) external Ca 2ϩ . Insets show analyses of the kinetics of overshoot activation (expressed as -fold increase above starting Ca 2ϩ levels) over a range of Ca 2ϩ removal times from 0 to 5 min; these are means Ϯ S.D. of measurements taken from three separate experiments. Measurements of Ca 2ϩ were as described under "Experimental Procedures." the steady state increases in Ca 2ϩ as well as the overshoot responses are due to Ca 2ϩ entry through the same channel. When applied to resistant DC-3F/TG2 cells (Fig. 5C), although the resting level of Ca 2ϩ was not as high, the effect of SKF96365 in reducing Ca 2ϩ is unmistakable and closely resembles its action on thapsigargin-treated parent cells. Again, the Ca 2ϩ overshoot after Ca 2ϩ readdition was completely prevented. These experiments provide strong evidence that the increased levels of Ca 2ϩ in resistant DC-3F/TG2 cells result from continued entry of Ca 2ϩ through SOCs.
Ionomycin-induced Emptying of the Thapsigargin-resistant Ca 2ϩ Pool Activates Additional Ca 2ϩ Entry in Resistant DC-3F/TG2 Cells-An intriguing question to address was the relationship between possible different Ca 2ϩ pools and the activation of Ca 2ϩ entry in the resistant DC-3F/TG2 cells. Thus, whereas a thapsigargin-resistant Ca 2ϩ pumping pool exists in these cells that is agonist-responsive, these cells also have some constitutively activated Ca 2ϩ influx that appears to result from the continuously empty (or partly empty) state of a thapsigargin-sensitive Ca 2ϩ pumping pool. Although agonistmediated release of the thapsigargin-insensitive Ca 2ϩ pool did not appear to induce significant Ca 2ϩ influx (Fig. 2B), it was possible that agonist-induced Ca 2ϩ release was incomplete in emptying this pool as a result of receptor desensitization, as described above; certainly, the finding that agonist-sensitive Ca 2ϩ pools are a distinct entity from Ca 2ϩ pools coupled to SOC activation would be curious. We therefore sought to determine whether more complete emptying of pools could be effected using the Ca 2ϩ ionophore, ionomycin, and whether such pool emptying was coupled to Ca 2ϩ entry. Initial experiments shown in Fig. 6 assessed the relative extent of ionomycininduced Ca 2ϩ pool release in parent DC-3F cells and resistant DC-3F/TG2 cells. These experiments were undertaken in the absence of external Ca 2ϩ so that release could be measured without any Ca 2ϩ entry. Using parent DC-3F cells, removal of Ca 2ϩ had virtually no effect on resting Ca 2ϩ levels (approximately 25 M), and thapsigargin caused a large release of Ca 2ϩ , reaching a peak within 50 s and then declining back to the resting level within a further 5 min (Fig. 6A). After this emptying of thapsigargin-sensitive Ca 2ϩ pools (and the subsequent pumping of released Ca 2ϩ out of the cells), the application of 10 M ionomycin caused a further release of Ca 2ϩ , accounting for approximately 20% of the Ca 2ϩ released by thapsigargin. Although we cannot be sure all thapsigargin-sensitive Ca 2ϩ pools were emptied, the ionomycin-induced Ca 2ϩ release is believed to be derived predominantly from pools filled via thapsigargininsensitive Ca 2ϩ pumps. Indeed, the size of this pool relative to the thapsigargin-sensitive pool corresponds very well with the relative sizes of thapsigargin-sensitive and -insensitive Ca 2ϩ pumping pools determined from 45 Ca 2ϩ flux experiments using permeabilized parent DC-3F cells (30). Thapsigargin treatment

FIG. 5. SKF96365-induced inhibition of Ca 2؉ influx in parent DC-3F cells and in thapsigargin-resistant DC-3F/TG2 cells.
Changes in cytosolic Ca 2ϩ measured as described under "Experimental Procedures" were followed in DC-3F cells pretreated for 30 min with 2 M thapsigargin (A and B) or in DC-3F/TG2 cells that were not treated with thapsigargin after removal from dishes (C). At the times shown, cells were treated with 100 M SKF96365 or nominally Ca 2ϩ -free medium. periods longer than that in Fig. 6A resulted in similar ionomycin-induced Ca 2ϩ release from DC-3F cells; however, prolonged Ca 2ϩ -free treatment does eventually result in some rundown of all pools. In the resistant DC-3F/TG2 cells, as shown above, removal of Ca 2ϩ caused the higher resting level of Ca 2ϩ (approximately 70 M) to decrease as the endogenously active Ca 2ϩ entry was prevented. Thapsigargin addition had no effect, whereas addition of ionomycin released the whole thapsigargin-insensitive Ca 2ϩ pumping pool (Fig. 6B). Generally the amount of Ca 2ϩ released from this pool was less than the total Ca 2ϩ released from pools in the parent cells, and this again is in agreement with total size of pools measured in flux experiments (30).
Pool release experiments were then conducted in the presence of external Ca 2ϩ to assess whether ionophore-induced Ca 2ϩ release from thapsigargin-resistant pools was linked to Ca 2ϩ entry. In these experiments, a limiting concentration of ionomycin (2 M) was used which, although still effectively releasing Ca 2ϩ from intracellular stores, did not increase the permeability of the plasma membrane to Ca 2ϩ . Parent DC-3F cells were treated with thapsigargin for 30 min to completely release thapsigargin-sensitive Ca 2ϩ pools. As shown in Fig. 7A, after steady state Ca 2ϩ influx had been achieved as a result of opening of SOCs (see Fig. 1), the addition of ionomycin caused only a slight change in cytosolic Ca 2ϩ , suggesting that emptying of a residual Ca 2ϩ pool was not inducing any significant Ca 2ϩ influx. This result was in contrast to the actions of ionomycin on resistant DC-3F/TG2 cells shown in Fig. 7B. In this case, ionomycin induced a rapid peak of cytosolic Ca 2ϩ due to pool release, followed by a sustained increase in Ca 2ϩ lasting for many minutes (trace a). This sustained increase was likely due to activation of SOCs since the application of SKF96365 at any time (for example, 200 s in trace b) caused an immediate and rapid decrease in cytosolic Ca 2ϩ as a result of inhibiting entry through SOCs. If SKF96365 was added together with ionomycin (trace c) then only the ionophore-induced peak of Ca 2ϩ release was observed. As shown in the latter two traces, the level of Ca 2ϩ reached after addition of SKF96365 was below the starting level, which was consistent with the action of SKF96365 in blocking both the constitutively activated Ca 2ϩ entry (see Fig. 5C) and the additional Ca 2ϩ entry resulting from ionophore-induced emptying of the thapsigargin-insensitive Ca 2ϩ pool.
Concluding Remarks-Previous 45 Ca 2ϩ flux studies (30) using permeabilized cells of the parent DC-3F fibroblast line revealed that InsP 3 -sensitive Ca 2ϩ pools are exclusively filled via an intracellular Ca 2ϩ pump with high sensitivity to thapsigargin (IC 50 200 pM). A small amount of highly thapsigarginresistant pumping activity (IC 50 4 M) is detectable in these cells but is not involved in filling InsP 3 -releasable pools. However, similar flux studies with the thapsigargin-resistant DC-3F/TG2 cell line revealed that an identical thapsigargin-resistant Ca 2ϩ pump is the only apparent operational pump in these cells and pumps Ca 2ϩ into InsP 3 -sensitive Ca 2ϩ pools (30).
Thus, it appears that the adaptive change that permits these cells to continuously grow in the presence of 2 M thapsigargin (29) is the coexpression of InsP 3 receptors and thapsigarginresistant pumps within a single pool (30). The present studies reveal that in intact resistant DC-3F/TG2 cells, this same pool is capable of generating agonist-responsive cytosolic Ca 2ϩ signals. Moreover, it is most likely that the same pumping activity serves to sustain the normal intraluminal Ca 2ϩ levels within the ER that appear necessary to maintain the many essential functions of ER (4, 7, 9 -15) and normal cell growth (7, 16 -19).
The resting level of Ca 2ϩ in the resistant DC-3F/TG2 cells is higher and appears to reflect a significant amount of constitutively activated SOC-mediated Ca 2ϩ influx. This view is confirmed by the effects of brief removal of external Ca 2ϩ , which causes a characteristic transient reactivation of the Ca 2ϩ influx channels; in contrast, in parent DC-3F cells, transient removal of Ca 2ϩ has no effect and SOCs are clearly closed under resting conditions. From these experiments, SOCs appear to exist in three different states: either closed, maximally open, or in a partially deactivated state. Only closed SOCs are present in the parent DC-3F cells under resting conditions. The maximally open state of SOCs appears to be a transient event and occurs only after complete emptying of at least one Ca 2ϩ pool; the maximally open state can be transiently reactivated in cells after brief removal and readdition of external Ca 2ϩ . Apart from this transient maximally open state, SOCs return to a partially deactivated state in which a small amount of Ca 2ϩ enters and the level of cytosolic Ca 2ϩ is modestly raised. Most likely, the deactivation of Ca 2ϩ channels occurs as a result of the interaction of Ca 2ϩ with a site that negatively controls SOCs and is responsive to levels of Ca 2ϩ significantly above resting levels. When Ca 2ϩ is removed from the outside, decreased entry of Ca 2ϩ and efficient clearing of cytosolic Ca 2ϩ by the plasma membrane Ca 2ϩ pump result in Ca 2ϩ being rapidly lost from the vicinity of the influx channel and hence dissociating from a site that inhibits Ca 2ϩ influx through SOCs. Clearly, there is ample precedent for such a negative controlling action of Ca 2ϩ on SOC activity (2,28,33); indeed, evidence from Zweifach and Lewis (35) indicates that a Ca 2ϩ -dependent deactivation site is located close, perhaps within 3-4 nm of the cytosolic mouth of the entry channel. In addition, Putney and co-workers (36) revealed that in NIH 3T3 cells an additional slower mechanism of control over SOCs occurs as a result of Ca 2ϩ acting at an extracellular site; at present, our results do not really distinguish between these possibilities.
Whereas the resting level of cytosolic Ca 2ϩ in resistant DC-3F/TG2 cells is higher than in parent DC-3F cells, it is less than half of the level that persists within parent DC-3F cells following treatment with thapsigargin. As described earlier, thapsigargin treatment of parent DC-3F cells induces an irreversible cytotoxic state that may be a consequence of the persistently elevated cytosolic Ca 2ϩ level (30). In contrast, other cells such as DDT 1 MF-2 smooth muscle cells have more complete deactivation of influx and, following thapsigargin-induced pool emptying, enter a growth-arrested state from which recovery can be induced (17)(18)(19). Whereas the resistant DC-3F/TG2 cells do have some persistent Ca 2ϩ entry likely due to an empty thapsigargin-sensitive pool, the level of resting Ca 2ϩ (70 nM) may be below the cytotoxic threshold; thus, in these cells, either there are less channels opened by emptying of the pool or the deactivation of the open channels is more efficient. The results in Figs. 3 and 4 do not really distinguish between these possibilities.
The additional Ca 2ϩ influx resulting from ionophore-induced emptying of the thapsigargin-resistant pool in resistant DC-3F/ TG2 cells is an intriguing observation. Our previous evidence indicated that the two distinct intracellular Ca 2ϩ pump activities expressed in parent DC-3F cells are clearly located in distinct pools, with only the thapsigargin-sensitive Ca 2ϩ pump functioning to accumulate Ca 2ϩ within InsP 3 -sensitive pools (30). Although only thapsigargin-resistant pumping can be observed in DC-3F/TG2 cells, the constitutive influx of Ca 2ϩ suggests that a thapsigargin-sensitive pool does exist in these cells but, obviously, in a persistently and irreversibly empty state as a result of the 2 M thapsigargin present in the culture medium. Even though we do not know the molecular identity of the thapsigargin-resistant pump, Western analysis reveals the presence of at least the same or even increased levels of SERCA pump protein in the DC-3F/TG2 cells (29). We suggest that in the resistant DC-3F/TG2 cells both types of Ca 2ϩ pumping pool exist and that Ca 2ϩ influx is activated by the empty or partially empty state of either pool. Although the two components of Ca 2ϩ influx are additive and both are blocked by SKF96365, at this stage we are unable to definitively conclude that the same single entry channel is activated by emptying of the different pools. Indeed, whereas the evidence is strong for two distinct pools within the parent DC-3F cells, the evidence for two separate pools in the resistant DC-3F/TG2 cells is less compelling; hence we cannot rule out the possibility that the two components of influx could represent different degrees of emptying of a single pool. In the parent DC-3F cells, emptying of the separate thapsigargin-resistant Ca 2ϩ pool with ionophore does not result in any significant influx of Ca 2ϩ . Our previous studies indicated that in resistant DC-3F/TG2 cells, the InsP 3 receptor becomes redirected to be expressed within the thapsigargininsensitive Ca 2ϩ pumping pool (30). If this is the case and assuming the equivalent separate Ca 2ϩ pools exist in both DC-3F and DC-3F/TG2 cells, then it appears that the ability of the pool to signal Ca 2ϩ entry is conferred upon the pool by the presence of InsP 3 receptors and not merely by emptying of Ca 2ϩ . This presents an interesting scenario wherein both pool emptying and the presence of InsP 3 receptors are required for SOC activation. Such a scheme may be in keeping with earlier (39) and more current hypotheses (2) that suggest InsP 3 receptors are closely associated with the Ca 2ϩ influx machinery.