©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thapsigargin-resistant Intracellular Calcium Pumps
ROLE IN CALCIUM POOL FUNCTION AND GROWTH OF THAPSIGARGIN-RESISTANT CELLS (*)

Richard T. Waldron , Alison D. Short , Donald L. Gill (§)

From the (1) Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
REFERENCES

ABSTRACT

Exposure of cells to the intracellular Ca pump blocker, thapsigargin (TG), results in emptying of Ca pools and termination of cell proliferation (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4986-4990). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A.(1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 µM TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of Ca uptake into permeabilized parent DC-3F cells revealed two distinct Ca pump activities with 20,000-fold different sensitivities to TG; the IC values for TG were 200 pM and 4 µM, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 ± 0.10 and 0.18 ± 0.08 nmol of Ca/10 cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca pumping; however, almost all pumping was blocked at higher TG concentrations with an IC of 5 µM. In both cell types, each Ca pump activity (regardless of TG sensitivity) had high Ca affinity (Kvalues 0.1 µM) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca pool was releasable with inositol 1,4,5-trisphosphate (InsP) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP-releasable. GTP-induced Ca uptake in the presence of oxalate indicated Ca transfer between distinct pools in the DC-3F cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca accumulation was releasable with InsP; unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca transfer between pools in the presence of oxalate. Thus, whereas InsP releases Ca only from the high TG sensitivity Ca pumping pool in parent DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant Ca pumping pool now contains functional InsP receptors. It is concluded that the ability of TG-resistant DC-3F/TG2 cells to grow in the presence of 2 µM TG results from expression of a distinct TG-resistant Ca pump which serves to accumulate Ca within an InsP-releasable Ca pool; this provides further evidence for the essential role of functional Ca pools in progression of cells through the cell cycle and cell division.


INTRODUCTION

Intracellular Ca pools play an essential role in signaling events within cells. Although the precise identity and distribution of Ca pools remain elusive, it is clear that the endoplasmic reticulum (ER)() or subdomains thereof constitute at least a major component of Ca pools (1, 2, 3) . The Ca within ER not only provides an important source of cytosolic Ca signals, but also appears to be the primary determinant in the opening of agonist-dependent ``capacitative'' Ca entry channels mediating sustained Ca signaling events (4, 5) . However, generation and modulation of Ca signals is not the only role of Ca accumulated within ER. Thus, intraluminal Ca levels appear to exert control over essential ER functions including folding, processing, and assembly of proteins (6, 7, 8) ; such control may be effected through a range of luminal Ca-binding and chaperone proteins (9, 10) . In addition, it has become clear that the Ca content of intracellular Ca pools exerts profound control over cell proliferation and progression of cells through the cell cycle (11, 12, 13, 14) . Therefore, modification of Ca within the ER exerts substantial effects on the viability, growth, and function of cells.

Ca is accumulated within pools via members of the intracellular sarcoplasmic/endoplasmic reticulum CaATPase (SERCA) family of Ca pump proteins which appear widely distributed in the endoplasmic reticulum of most cells (15, 16) . These pumps have been shown to be highly sensitive to blockade by the plant sesquiterpene lactone, thapsigargin (17, 18) . Thapsigargin binds with high affinity to intracellular Ca pumps resulting in virtually irreversible inhibition of Ca accumulation within ER (11, 18, 19) . The selective blockade of Ca accumulation within ER using thapsigargin has made it possible to assess the significance of intraluminal Ca in cell function and growth. Thus, using cultured DDTMF-2 smooth muscle cells, brief treatment with nanomolar thapsigargin levels causes a long-lasting emptying of Ca pools and the entry of cells into a growth-arrested G-like state in which the cells remain stable for several days (11) . Treatment of these arrested cells with 20% serum induces synthesis of new Ca pump protein, the refilling of Ca pools, and the return of cells into the cell cycle (12, 13) . Not all cells undergo this thapsigargin-induced cytostatic response; instead, other cells can undergo cytotoxic responses to thapsigargin. For example, nanomolar thapsigargin levels induce an irreversible cessation of proliferation of prostatic cancer cells; in these cells, endonucleases are activated, genomic DNA becomes fragmented, and cells undergo apoptosis resulting in a generalized breakdown of cell morphology (14) . Such a cytotoxic response may be attributable to more substantial thapsigargin-induced capacitative entry of Ca in the latter cells; in contrast, Ca entry in DDTMF-2 cells due to thapsigargin is comparatively small and short-lived (11, 12, 13) .

In spite of the profound influence of thapsigargin-induced Ca pool emptying on cell growth, it was recently shown that cells of the DC-3F Chinese hamster lung fibroblast line could be made resistant to thapsigargin (20) . This was accomplished by gradual exposure of DC-3F cells in culture to increasing concentrations of thapsigargin over a period of 10 months. The resulting cell line, DC-3F/TG2, exhibits neither the cytostatic effects of thapsigargin in DDTMF-2 cells nor the cytotoxicity observed in the parent DC-3F cell line; indeed, cells retain normal morphology and continue to divide while growing in 2 µM thapsigargin. The basis of this remarkable resistance is an important question. Toxic hydrophobic molecules can be efficiently transported out of cells by overexpression of the multidrug resistance factor, P-glycoprotein (Pgp). However, although overexpression and amplification of high levels of Pgp can reduce TG sensitivity, the thapsigargin-resistant DC-3F/TG2 cells express only modestly increased levels of Pgp (3.7-fold), enough to only slightly decrease sensitivity to members of the multidrug resistance class of compounds (20) . Resistance to thapsigargin might also have resulted from overexpression of Ca pumps; however, the slight increase (3-fold) in SERCA pump expression in DC-3F/TG2 cells (20) was insufficient to explain the massive decrease in thapsigargin sensitivity.

The basis of the current study was therefore to assess the intriguing question of how DC-3F/TG2 cells survive in high levels of thapsigargin, and whether any Ca pools actually exist in these cells. To address this, experiments sought to directly assess Ca pool function within permeabilized cells. The results reveal function of a novel and remarkably thapsigargin-insensitive Ca pumping activity. This pump accumulates Ca within a pool that has characteristics similar to Ca signaling pools in normal DC-3F cells but which also has certain different properties. The results, in addition to providing evidence for a novel Ca pump, are consistent with the hypothesis that intracellular Ca pumping activity is essential for cells to be able to grow and proliferate.


EXPERIMENTAL PROCEDURES

Culture of Parent DC-3F and Thapsigargin-resistant DC-3F/TG2 Cells

Cells of the DC-3F Chinese hamster lung fibroblast line were cultured in -modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% heat-inactivated fetal bovine serum (Life Technologies, Inc.) as described previously (20) . Cells received a change of medium after 2 days and were used in Ca flux experiments the following day. Selection of the thapsigargin-resistant DC-3F/TG2 cell line by successive culturing in the presence of increasing thapsigargin concentrations was as described previously (20). DC-3F/TG2 cells were cultured in -modified Eagle's medium with 5% heat-inactivated fetal bovine serum together with 2 µM thapsigargin; they received a change of medium (still containing 2 µM thapsigargin) on the 3rd day after passaging and were used in Ca flux experiments the following day. For cell proliferation experiments, DC-3F or DC-3F/TG2 cells were passaged directly into 4-well plates and counted over a period of 4 days. Both cell types were also cultured on glass coverslips for use in intracellular Ca measurements as described below.

Permeabilization of Cells and CaFlux Measurements

Cells were permeabilized with 0.005% saponin and used to measure Ca fluxes into and out of intracellular Ca pools as described previously (21, 22) . Uptake was measured in the presence of an intracellular-like medium (ICM: 140 mM KCl, 10 mM NaCl, 2.5 mM MgCl, 10 mM Hepes-KOH, pH 7.0) containing 1 mM ATP, 50 µM CaCl (with 150 Ci/mol Ca) buffered to 0.1 µM with EGTA, 3% polyethylene glycol, and 10 µM ruthenium red, at 37 °C for the times shown. The low free Ca level together with the inclusion of ruthenium red ensured no mitochondrial component of Ca accumulation. Uptake was quenched with ICM containing 1 mM LaCl, and cells were rapidly filtered on glass fiber filters and accumulated Ca was counted. Results are presented as ATP-dependent Ca accumulation with that component of Ca associated with permeabilized cells in the absence of ATP subtracted. For both cell lines, ATP-independent passive influx of Ca into pools was typically 1-2% of total ATP-dependent Ca pumping (between 2 and 5 pmol/10 cells/min).

Intracellular Free CaMeasurements

Procedures for measurement of intracellular free Ca within DC-3F and DC-3F/TG2 cells using fura-2 were as described previously (11, 12, 13, 23) . Cells were grown on glass coverslips as described above and fura-2-loaded by incubation with 2 µM fura-2/AM ester for 10 min at 20 °C in Hepes-buffered Krebs medium (107 mM NaCl, 6 mM KCl, 1.2 mM MgSO, 1 mM CaCl, 1.2 mM KHPO, 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 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 concentrations were as described by Grynkiewicz et al.(24) , using a Kof 135 nM. Dye was considered saturated upon addition of 10 µM ionomycin, while minimum fluorescence ratio was determined in the presence of both 10 µM ionomycin and 5 mM EGTA.

Materials and Miscellaneous Procedures

The DC-3F and DC-3F/TG2 cell lines were provided by Dr. Arif Hussain (University of Maryland Cancer Center). Thapsigargin was from LC Services (Woburn, MA). Fura-2 was from Molecular Probes. Other materials and miscellaneous procedures were as described previously (11-13).

RESULTS AND DISCUSSION

Effects of Thapsigargin on Growth Rates of DC-3F and DC-3F/TG2 Cells

The ability of cells to grow and proliferate has been found to closely correlate with their ability to express functional intracellular Ca-pumping pools (11, 12, 13) . That Ca pools are essential to cell proliferation has been shown by blocking Ca uptake into Ca pools using three different intracellular Ca pump inhibitors: thapsigargin, 2,5-di-tert-butylhydroquinone, and cyclopiazonic acid (11, 12) . The finding that cells could be obtained, albeit through a lengthy and rigorous selection procedure (20) , that survive and grow in a supermaximally effective concentration of thapsigargin (2 µM), was therefore difficult to reconcile. Growth of normal DC-3F Chinese hamster lung fibroblasts is blocked by thapsigargin with an EC of less than 10 nM(20) . An initial question concerning the selected thapsigargin-resistant DC-3F/TG2 cells was whether the rate of cell division was appreciably different from that of the parent cell line. The rates of cell division for both cell types are compared in Fig. 1. In the absence of thapsigargin, both cell types had doubling times of approximately 24 h. In the presence of 2 µM thapsigargin, the rate of DC-3F/TG2 cells was virtually unaltered, whereas, for the parent DC-3F cells, 2 µM thapsigargin totally prevented cell division. Indeed, after 3-4 days in culture with thapsigargin, cell viability decreased considerably; by this time, the majority of cells were rounded, detached from dishes, and nonviable, indicating a cytotoxic effect of thapsigargin on the parent DC-3F cells. This contrasts with the cytostatic effect of thapsigargin observed using the DDTMF-2 smooth muscle cell line in which cell viability is maintained for up to 7 days even though cell division is completely arrested (11) . This is an interesting difference which may relate to the coupling of Ca pool emptying with Ca influx, as described below.


Figure 1: Effects of thapsigargin on growth rate of parent DC-3F and resistant DC-3F/TG2 cells. DC-3F cells (, ) or DC-3F/TG2 cells (, ) were cultured under identical conditions either in the absence of thapsigargin (, ) or in the presence of 2 µM thapsigargin (, ). Results are the means ± S.D. of measurements from four wells, and are typical of three separate experiments.



Distinct Intracellular CaPumps within DC-3F and DC-3F/TG2 Cells Exhibit a 20,000-fold Difference in Thapsigargin Sensitivity

A major question concerning cells of the thapsigargin-resistant DC-3F/TG2 line was whether they contained any functional intracellular Ca pools. If they did not, then our premise that Ca pools are essential for progression of cells through the cell cycle (11, 12, 13) would be in question. As reported previously, the possibility that thapsigargin resistance was arising from export of thapsigargin mediated by Pgp overexpression, was not supported by measurements on Pgp levels (20) . Therefore, unless a different resistance factor was being expressed, the levels of thapsigargin would be sufficiently high within the cells to render any known intracellular Ca pumps nonfunctional. With regard to which pumps do exist within these cells, previous Western analyses revealed that the major pump protein of normal DC-3F cells was the SERCA-2 subtype; the same pump was also expressed in the resistant DC-3F/TG2 cells at a slightly increased level, but not sufficient to give a significant change in thapsigargin sensitivity (20) . It should be noted that Western analysis also revealed SERCA-2 pump protein within thapsigargin-treated quiescent DDTMF-2 cells; however, measurements of phosphorylated pump intermediate formation revealed that none of this pump protein was functional due to irreversible inhibition by thapsigargin (13) .

Considering these facts, it was essential to examine Ca pumping activity directly within DC-3F/TG2 cells. This was undertaken using saponin-permeabilized cells in which Ca pool function could be measured without the possibility of any interference from export of molecules across the plasma membrane. The results of studies comparing the action of thapsigargin on Ca pump activity within permeabilized normal DC-3F and resistant DC-3F/TG2 cells are shown in Fig. 2 . In DC-3F cells grown under standard conditions, most ATP-dependent Ca pumping was highly sensitive to thapsigargin (Fig. 2A). Thus, 1 nM thapsigargin blocked almost 75% of uptake. The range of thapsigargin concentrations from 3 nM up to 1 µM all induced virtually the same effect, inhibiting approximately 80% of Ca accumulation. A further reduction of the remaining 20% of Ca uptake was observed with 3 µM thapsigargin, and 10 µM caused maximal inhibition.


Figure 2: Inhibition of Ca accumulation within DC-3F and DC-3F/TG2 cells by thapsigargin. A, uptake was measured using permeabilized DC-3F cells (grown under standard conditions without thapsigargin) in the presence of 0 (), 1 (), 3 (), 10 (), 30 (), 1,000 (), 3,000 (), or 10,000 () nM thapsigargin. B, uptake was measured using permeabilized DC-3F cells (grown in the presence of 2 µM thapsigargin for 16 h before use) in the presence of either 0 () or 3,000 () nM thapsigargin. C, uptake was measured using permeabilized DC-3F/TG2 cells (grown continuously in the presence of 2 µM thapsigargin) in the presence of either 0 (), 300 (), 1,000 (), 3,000 (), or 10,000 () nM thapsigargin. Details of the Ca uptake conditions are given under ``Experimental Procedures.'' Results are typical of three similar experiments.



If DC-3F cells had been treated overnight with 2 µM thapsigargin, there was essentially no thapsigargin-inhibitable Ca accumulation (Fig. 2B). Significantly, and in contrast to the parent DC-3F cells, thapsigargin-resistant DC-3F/TG2 cells grown continuously in the presence of 2 µM thapsigargin (the standard condition for these cells), clearly displayed ATP-dependent Ca pumping activity (Fig. 2C). This uptake could be inhibited by thapsigargin. However, inhibition was observed only at the higher concentrations of thapsigargin; that is, in the range of 300 nM to 10 µM.

The fact that there is pumping activity within the DC-3F/TG2 cells grown in the presence of 2 µM thapsigargin was unexpected. Indeed, as shown in , the maximal level of Ca uptake attainable in these cells was not significantly less than in parent DC-3F cells; however, substantial blockade of Ca pumping using 100 nM thapsigargin could only be observed with the latter. The differences in thapsigargin sensitivity of Ca pumping in the two cell lines suggested that distinct Ca pumps were operating. In order to investigate this, a detailed analysis of the thapsigargin concentration dependence in the two cell lines was undertaken, as shown in Fig. 3 . For DC-3F cells, there were clearly two widely separated regions of thapsigargin sensitivity. To more clearly illustrate this, the effects of including thapsigargin in the 30 pM to 1 nM range are depicted separately from those resulting from inclusion of thapsigargin in the 100 nM to 10 µM range. The IC values for thapsigargin in these regions were 0.2 nM and 4 µM, respectively. The former value is very close to that reported by Sagara and Inesi (19) for inhibition of ATP-dependent Ca pumping into sarcoplasmic reticulum vesicles. Interestingly, the concentration dependence of the low sensitivity region for these cells is almost superimposable upon that of the thapsigargin sensitivity of Ca pumping in the resistant DC-3F/TG2 cells (Fig. 3), which in these cells comprises all of the pumping activity. In the latter cells, the IC was approximately 5 µM. Thus, it appears that the parent DC-3F cells contain a mixture of two distinct Ca pumps with widely differing (20,000-fold) sensitivity to thapsigargin; in these cells, the majority of Ca accumulation occurs through the high thapsigargin sensitivity Ca pump. In DC-3F/TG2 cells, it appears that all of the Ca pumping occurs through the low thapsigargin sensitivity pump type (referred to subsequently as the thapsigargin-insensitive pump) and that this is the only Ca pump activity functioning within these cells. Selection of this type of Ca pump likely represents the major adaptation within this cell line that permits growth in thapsigargin.


Figure 3: Thapsigargin sensitivity of intracellular Ca pump activity in DC-3F and DC-3F/TG2 cells. Uptake was undertaken using either DC-3F cells () or DC-3F/TG2 cells () under similar conditions as in Fig. 2 except accumulation was for a standard 9-min period. For DC-3F cells, 100% of Ca uptake was defined as either that observed in the absence of thapsigargin (left curve) or that observed in the presence of 100 nM thapsigargin (right curve). For DC-3F/TG2 cells, 100% uptake was defined as that observed in the absence of added thapsigargin. Each point represents the mean ± S.D. of Ca uptake measured in three separate experiments.



Even though the thapsigargin concentration dependence of both types of pumping activity differs widely, other parameters of Ca pump function were remarkably similar. Thus the Kfor Ca of pump activity in DC-3F cells in the absence of thapsigargin and that for Ca pumping in DC-3F/TG2 cells were almost identical, both measured as approximately 0.1 µM (data not shown) using standard Ca flux procedures under EGTA-buffered conditions, as described previously (21) . This value is very similar to the Kmeasured for intracellular Ca pumps in a variety of other cell types (2, 21, 25) . We also investigated the sensitivity of thapsigargin-sensitive and -insensitive Ca pumps in both cell types toward the other Ca pump inhibitors, 2,5-di-tert-butylhydroquinone and cyclopiazonic acid. In other cell types, the IC for these reagents is approximately 5 and 10 µM, respectively (11, 12) . Although resistant DC-3F/TG2 cells showed a slightly lower sensitivity to 2,5-di-tert-butylhydroquinone (approximately 30 µM), this difference was small; moreover, the nonspecific effects of these agents at concentrations approaching or beyond 100 µM made determination of the absolute sensitivity of pumping or growth to these reagents difficult to measure. Both thapsigargin-sensitive and -insensitive Ca pump activities present in DC-3F and DC-3F/TG2 cells displayed similar dependence on ATP, and both were completely inhibited by 1 mM vanadate (data not shown). Therefore, it appears that the two pump activities are functionally equivalent, each being capable of mediating accumulation of Ca into pools from normal resting cytosolic free Ca levels. Since measured Ca accumulation reflects both pumping and passive leak rates, we cannot make assertions as to the actual or relative amounts of Ca pumping activity measured in these cells and, hence, the relative sizes of the pools involved. However, that intracellular Ca pump activity exists within DC-3F/TG2 cells continuously exposed to thapsigargin is clear; therefore, these data do not challenge our previous assertion that actively pumping intracellular Ca pools are a prerequisite for cell growth and division (11, 12, 13) .

It has been suggested that Ca accumulation into InsP-sensitive Ca pools within pancreatic acinar cells may be mediated by a mechanism distinct from direct ATP-dependent Ca pumping; this may involve a multistep process of ATP-dependent proton transport and Ca/H exchange (26, 27) . It was also shown that the proton pump inhibitor, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, blocks this Ca accumulation within InsP-sensitive pools (28) . However, experiments we have undertaken reveal no effect of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole on Ca accumulation within either DC-3F or DC-3F/TG2 cells (data not shown). This suggests that the thapsigargin-insensitive Ca uptake activity observed in these cells is unlikely to involve proton pumping and is instead more likely to reflect function of a pump directly utilizing ATP hydrolysis to transport Ca.

Thapsigargin-insensitive CaPumps Fill an InsP-sensitive CaSignaling Pool in DC-3F/TG2 Cells

The important question arising from discovery of a thapsigargin-insensitive Ca pumping pool in the DC-3F/TG2 cells was whether this pool is functionally significant, in particular, whether Ca could be released from the pool in response to InsP. We therefore examined the actions of InsP on Ca accumulated within both DC-3F and DC-3F/TG2 cells as shown in Fig. 4. The data in Fig. 4A reveal that Ca accumulated within nonmitochondrial stores in saponin-permeabilized parent DC-3F cells could be directly released by addition of InsP or GTP. In these cells, approximately 70% of ionophore A23187-releasable Ca was rapidly released upon addition of 10 µM InsP. As with other cell types, addition of 20 µM GTP resulted in an apparently similar release of Ca; however, this action of GTP reflects function of a very different mechanism believed to reflect G protein-mediated translocation through junctions between closely apposed membranes of distinct pools (29, 30, 31) . Such junctions may be important in maintaining and/or controlling luminal continuity of Ca pools in the ER (32) . Although not observed in all cells, GTP-mediated Ca release into the medium likely results from a small number of leaky or nonsealed pools through which Ca is lost to the medium after junctions are formed between pools (2, 33, 34) .


Figure 4: InsP and GTP-mediated Ca release from Ca pools within DC-3F and DC-3F/TG2 cells. Ca uptake experiments were performed as described under ``Experimental Procedures'' using either permeabilized parent DC-3F cells (A) or permeabilized DC-3F/TG2 cells (B). For DC-3F/TG2 cells only, the entire uptake and release experiments were undertaken in the presence of 100 nM thapsigargin. Release of Ca was monitored after addition of either 10 µM InsP (), 20 µM GTP (), 10 µM InsP together with 20 µM GTP (), 5 µM A23187 (), or control buffer (), each added at the arrow. Arrows show the time of reagent additions. Data are from single experiments representative of at least three experiments with each cell type. Details of uptake conditions are given under ``Experimental Procedures.''



Most importantly, InsP-mediated Ca release was also clearly observable using the thapsigargin-resistant DC-3F/TG2 cells (Fig. 4B). Although the extent of release was somewhat less (approximately 45% of ionophore-releasable Ca), the actions of InsP and GTP were qualitatively similar. In this experiment, Ca uptake and Ca release were undertaken throughout in the presence of 100 nM thapsigargin. Using the parent DC-3F cells under the same condition (that is, with 100 nM thapsigargin), the small component of Ca accumulation measurable (see Fig. 2A) was entirely unaffected by addition of InsP or GTP (data not shown). These results demonstrate therefore that Ca pool function within DC-3F/TG2 cells has undergone a clearly defined modification in which the thapsigargin-insensitive Ca pump, which formerly functioned to accumulate Ca only within a pool unresponsive to InsP, now actively accumulates Ca within a functional, InsP-releasable Ca pool.

Pool function within the parent DC-3F cells closely resembles that which we described in other cells, for example, DDTMF-2 smooth muscle cells (2, 18, 29, 30, 31) . Thus, we noted previously that InsP-sensitive Ca pools in DDTMF-2 cells accumulate Ca exclusively through thapsigargin-sensitive Ca pumps (18) . In those cells, although a distinct thapsigargin-insensitive Ca pool was measurable, that pool was InsP-insensitive and persisted within thapsigargin-treated growth-arrested cells (18) . These observations contributed to the view that thapsigargin-insensitive pumps lacked any physiological role in mediating signaling events. Indeed, the inability of thapsigargin-treated cells to grow was directly attributable to the lack of a functional thapsigargin-sensitive, InsP-releasable Ca pool (11, 12) ; moreover, the re-entry of thapsigargin-arrested cells back into the growth cycle was dependent upon expression of new SERCA pump protein and the return of a functional InsP-sensitive Ca pool (13) . With DC-3F/TG2 cells, we conclude that the selection procedure has been successful in selecting cells in which either (a) thapsigargin-insensitive Ca pumps have now been recruited to function within InsP-releasable pools or (b) InsP receptors are now expressed within the pool accumulating Ca via thapsigargin-insensitive Ca pumps. Some limited information on distinguishing between these possibilities is given below. In addition, these results provide a substantial reinforcement of our previous conclusion that functional InsP-sensitive Ca pools are necessary for cells to be able to proliferate (2, 11, 12, 13) .

Distinctions in CaPool Function and CaTranslocation between Pools in DC-3F and DC-3F/TG2 Cells

We have previously shown that a further distinguishing characteristic of InsP-sensitive Ca pools within cells is their permeability to anions, in particular, oxalate (29, 30, 35, 36) . The entry of oxalate appears to be mediated by nonselective anion channels existing within specific organelles including sarcoplasmic reticulum of muscle and InsP-releasable elements in ER (2, 36, 37) ; the role of such channels may be to assist rapid Ca release events by dissipation of charge build-up (36, 37) . In Ca flux experiments, entry of oxalate into Ca pumping pools results in greatly enhanced Ca accumulation due to formation of the insoluble Ca-oxalate complex. In previous studies with other cell types, we demonstrated that oxalate permeability is a property specific to InsP-sensitive Ca pools within cells (29, 30, 31, 33, 36) . As shown in Fig. 5A using DC-3F cells, oxalate above 5 mM results in a large increase in Ca accumulation; at 10 mM oxalate, Ca uptake becomes approximately linear with time indicating substantial entry of oxalate into Ca-accumulating pools within DC-3F cells. As was shown for other cell types (29) , this large increase in Ca uptake with oxalate was completely prevented by InsP (data not shown), indicating that oxalate was functioning exclusively upon the InsP-releasable pool of Ca within these cells. In contrast, with DC-3F/TG2 cells, addition of oxalate induced almost no increase in Ca accumulation (Fig. 5B) indicating the absence of an oxalate-permeable pool in these cells. It is concluded therefore that in DC-3F/TG2 cells, the InsP-sensitive Ca pool, which comprises at least a substantial fraction of the Ca pumping pools present in these cells (see Fig. 4B), is clearly different from that functioning in parent DC-3F cells with respect to oxalate permeability.


Figure 5: Oxalate dependence of Ca uptake into DC-3F and DC-3F/TG2 cells. Ca uptake was measured in either permeabilized DC-3F cells (A) or DC-3F/TG2 cells (B) in the presence of either 0 (), 3 (), 5 (), 8 (), 10 (), or 20 () mM oxalate, as shown. Data are from a single experiment representative of four experiments with each cell type. Details of Ca uptake conditions are given under ``Experimental Procedures.''



The difference in Ca pools within the two cell types is further substantiated by examining the actions of GTP in the presence of oxalate (Fig. 6). We noted previously that GTP can induce a profound increase in Ca accumulation in the presence of submaximally effective oxalate concentrations due to GTP-activated Ca translocation between oxalate-permeable and -impermeable compartments (29, 33, 36) . Thus, GTP appears to activate junctional processes between the two compartments allowing Ca pumped into oxalate-impermeable pools to access oxalate-permeable pools, effectively increasing the size of the oxalate-permeable pool (2, 29, 36) . It appears that oxalate-precipitated Ca cannot pass through the GTP-activated junctions between pools, hence Ca is not released via connections with nonsealed pools and instead remains trapped within the oxalate-permeable pool (29, 36) . As shown in Fig. 6A, whereas the presence of 5 mM oxalate caused only a modest increase in Ca accumulation in DC-3F cells, when 20 µM GTP was also present, there was a very large increase in Ca accumulation. In contrast, with DC-3F/TG2 cells, the slight increase in Ca accumulation in the presence of 5 mM oxalate was definitely not enhanced by GTP (Fig. 6B); instead, in these cells GTP activates only release of Ca resulting in decreased Ca accumulation just as in the absence of oxalate, as shown in Fig. 4B. Thus, it is clear that whereas connections between membranes can be activated by GTP in both cell types, DC-3F/TG2 cells contain only an oxalate-impermeable Ca pool within which Ca can be accumulated. Thus, the oxalate-permeable pool present in the parent line is clearly absent in the resistant DC-3F/TG2 cells. These data may provide some information to distinguish between the two possibilities described above, that is, whether in DC-3F/TG2 cells it is InsP receptors as opposed to Ca pumps that are being expressed in organelles that are different from those in parent DC-3F cells. Thus, from the oxalate data, it appears perhaps more likely that in these cells InsP receptors are being expressed within an enlarged version of the TG-insensitive, InsP-nonreleasable, oxalate-impermeable pool that exists as a relatively minor component of pools within parent DC-3F cells. Conversely, it appears less likely that TG-insensitive Ca pumps are being diverted to pump Ca into pre-existing InsP-sensitive Ca pools.


Figure 6: Measurement of GTP-mediated translocation of Ca between pools in DC-3F cells (A) and DC-3F/TG2 cells (B). Ca uptake was measured as described under ``Experimental Procedures'' under control conditions () or in the presence of either 5 mM oxalate () or 5 mM oxalate together with 20 µM GTP (). Data are from a single experiment representative of four separate experiments with each cell type.



Although it is clear that the resistant DC-3F/TG2 cells contain functional InsP-sensitive Ca pools, the oxalate permeability data indicate the pool is not identical with that in normal DC-3F cells. A further functional parameter of Ca signaling pools is their coupling to activation of Ca entry across the plasma membrane. Thus, in many cells, depletion of intracellular Ca pools results in activation of capacitative entry of Ca via an as yet undetermined coupling process (4, 5) ; this Ca entry is essential in the generation of cytosolic Ca signals and the refilling of depleted stores. We undertook experiments to assess coupling between Ca pool emptying and Ca entry in both normal DC-3F and resistant DC-3F/TG2 cells. The results() revealed that capacitative Ca entry is activated in normal DC-3F cells after emptying pools with thapsigargin. Although influx becomes deactivated with time, a small residual component appears to be maintained for at least 60 min. During this time, a much larger component of Ca influx can be transiently reactivated by a short-term (1 min) depletion of extracellular Ca, as has been shown in other cells (38) . Interestingly, in resistant DC-3F/TG2 cells, a similar small component of capacitative Ca entry appears to be constitutively activated, presumably as a result of an empty Ca pool. Moreover, in these cells, a similar large transient activation of Ca influx occurs upon short-term external Ca depletion. This suggests that the resistant DC-3F/TG2 cells do contain an emptied pool that is coupled to activation of capacitative Ca entry. Presumably, therefore, this is not the same pool that accumulates Ca via the thapsigargin-resistant pump. Curiously, it would also appear to be distinct from the InsP-sensitive Ca pool which in these cells accumulates Ca through the thapsigargin-insensitive Ca pump. Alternatively, the pool activating Ca entry may be nonexistent (as opposed to merely empty) in these cells, and, instead, the downstream pathway triggering Ca entry constitutively activated.

The ability of the channel to be deactivated and reduce its conductance state may be an important factor in determining whether cells can survive in the continuous presence of emptied pools which activate capacitative Ca entry. Thus, as mentioned earlier, the level of cytosolic Ca following pool depletion may be critical to whether cells have a cytostatic as opposed to a cytotoxic response to thapsigargin. In DDTMF-2 smooth muscle cells which have little Ca entry in response to pool emptying, cells respond to Ca pump blockers by entering a G-like state from which they can be rescued (12, 13) ; in contrast, thapsigargin induces an irreversible blockade of proliferation of prostatic cancer cells which then undergo apoptosis (14).

Concluding Remarks

The major conclusion of the studies presented here is that a novel thapsigargin-insensitive Ca pump can be expressed within cells which have been selected to grow in the presence of thapsigargin. This pump appears to be endogenously expressed within these cells as well as other cells (18); however, its function in normal cells does not appear to be associated with Ca signaling events (18, 29, 30, 31) . After cells are induced to become resistant to thapsigargin, the thapsigargin-insensitive Ca pump accumulates Ca within a Ca pool that functions to release Ca in response to InsP. This pool appears distinct from that which normally functions to generate InsP-induced Ca signals with respect to anion permeability and with respect to coupling to capacitative Ca entry. It may be that during the selection for thapsigargin resistance, cells are selected that redirect functional InsP receptors from their normal thapsigargin-sensitive Ca pumping target pool to one that can still operate in the presence of thapsigargin. Certainly, the presence of functional Ca pumping pools within the thapsigargin-resistant DC-3F/TG2 cells is consistent with our previous assertion that cell proliferation requires functional Ca pools (2, 11, 12, 13) . The question of the molecular identity of the thapsigargin-insensitive Ca pump remains open. Although it has similar Ca affinity, ATP dependence, and vanadate sensitivity to known SERCA pumps, the thapsigargin insensitivity of this pump distinguishes it from most SERCA pumps that have so far been identified (15, 16). Interestingly, evidence for a SERCA-like 97-kDa protein that may function with low thapsigargin sensitivity has been presented in platelets (39, 40) ; its relationship to the activity in resistant DC-3F/TG2 cells is unknown. From Western analysis, the resistant DC-3F/TG2 cells have only slightly increased expression of SERCA-2 type pump protein (20) , all of which is presumed nonfunctional as a consequence of irreversible thapsigargin binding. The plasma membrane Ca pump is also a possible candidate for the pumping observed within DC-3F/TG2 cells, having a greatly reduced sensitivity to thapsigargin that appears to be in the low micromolar range (41) . Obviously, this pump protein is assembled in the ER; whether it can be retained within a subcompartment inside cells and whether this process might be accentuated during the selection of thapsigargin-resistant cells are intriguing questions. Whatever the nature of the pump protein, the present study reveals that thapsigargin-insensitive Ca pumps can accumulate Ca within functional intracellular Ca pools.

  
Table: Comparison of Ca uptake in parent DC-3F and thapsigargin-resistant DC-3F/TG2 cells

Maximal Ca accumulation was measured in both cell types in the absence and presence of 100 nM thapsigargin in the Ca uptake assay. Total ATP-dependent intracellular Ca uptake was measured after 9 min of incubation using either permeabilized DC-3F cells (grown without thapsigargin) or DC-3F/TG2 cells (grown in the presence of 2 µM thapsigargin) as described under ``Experimental Procedures.'' Results shown are in nanomoles of Ca/10 cells and are means ± S.D. of 10 separate experiments.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant NS19304 and National Science Foundation Grant DCB 9307746, and by the award of a postdoctoral fellowship from the Maryland Heart Association, Maryland Affiliate (to A. D. S.). 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.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biological Chemistry, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Office Tel.: 410-706-2593; Laboratory Tel.: 410-706-7247; Fax: 410-706-8297.

The abbreviations used are: ER, endoplasmic reticulum; TG, thapsigargin; InsP, inositol 1,4,5-trisphosphate; fura-2/AM, fura-2 acetoxymethyl ester; Pgp, P-glycoprotein; SERCA, sarcoplasmic/endoplasmic reticulum.

R. T. Waldron, A. D. Short, and D. L. Gill, unpublished observations.


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