Luminal Ca2+ protects against thapsigargin inhibition in neuronal endoplasmic reticulum.

Thapsigargin is a specific and potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases. However, in whole rat brain microsomes, 1 microM thapsigargin had no significant effect on the 10-min time course of ATP-dependent Ca2+ uptake in the absence of the luminal Ca2+ chelator oxalate. In contrast, 50 mM oxalate resolved a thapsigargin-sensitive Ca2+ uptake rate (IC50 approximately 1 nM thapsigargin) five times that of a thapsigargin-insensitive rate. This remaining approximately 20% of the total ATP-dependent accumulation was insensitive to thapsigargin (up to 10 microM), slightly less sensitive to vanadate inhibition, and unresponsive to 5 microM inositol 1,4,5-trisphosphate or 10 mM caffeine. Measuring both 12-min Ca2+ uptake and initial Ca2+ uptake rates, the apparent thapsigargin sensitivity increased as oxalate concentrations increased from 10 to 50 mM, corresponding to a range of luminal free Ca2+ concentrations of approximately 300 down to 60 nM. Addition of oxalate during steady-state 45Ca accumulation rapidly resolved the aforementioned thapsigargin sensitivity. These results strongly suggest that luminal Ca2+ may protect a large portion of neuronal endoplasmic reticulum Ca2+ pumps against thapsigargin inhibition. Although high [Ca2+] has been previously shown to protect against thapsigargin inhibition in several reticular membrane preparations, our results suggest that luminal Ca2+ alone is responsible for mediating this effect in neurons.

The endoplasmic and sarcoplasmic reticula (ER 1 and SR, respectively) actively accumulate Ca 2ϩ via the sarco/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA) family of Ca 2ϩ pumps. These pumps are encoded by at least three different genes, and alternative splicing creates a total of at least five isoforms (SERCA1a, SERCA1b, SERCA2a, SERCA2b, and SERCA3). The isoforms found in brain ER are SERCA2b and SERCA3, whereas the skeletal muscle isoforms are exclusively SERCA1 and SERCA2a (1)(2)(3). The isoforms appear to share overall structure-function similarities: all are thought to be asymmetrical transmembrane proteins with similar structure (4, 5) that can translocate two Ca 2ϩ ions into the lumen by hydrolyzing one ATP molecule and forming an enzyme-phosphorylated (EϳP) intermediate (6). Thapsigargin, a naturally occurring, tumor-promoting sesquiterpene lactone, has been shown to release Ca 2ϩ from the ER by specifically inhibiting these Ca 2ϩ pumps (7). Lytton et al. (8), using a COS expression system and cDNA clones for SERCA1, -2a, -2b, and -3, demonstrated a stoichiometric, potent, and essentially irreversible inhibition of each of the SERCA isoforms by thapsigargin. Similarly, Campbell et al. (9) detected no difference in the Ca 2ϩ affinities or inhibitor effects for avian subtypes 1, 2a, and 2b when expressed in COS cells. A protective effect of high [Ca 2ϩ ] against thapsigargin inhibition has been described for the skeletal muscle SERCA1 pump (10,11) and a SERCA-type ATPase in ER microsomes of bovine adrenal chromaffin cells (12). Although most of the Ca 2ϩ uptake into the ER and SR is likely due to the action of SERCAs, some recent evidence suggests the existence of thapsigargin-resistant ATP-dependent mechanisms capable of sequestering Ca 2ϩ in some cell and microsome preparations (11,(13)(14)(15)(16)(17)(18)(19).
In this study, we were interested in characterizing neuronal ER Ca 2ϩ accumulation both in terms of thapsigargin sensitivity and the way by which high [Ca 2ϩ ] might protect against thapsigargin inhibition. We employed 45 Ca flux studies to characterize both thapsigargin-sensitive and -resistant ATPdependent Ca 2ϩ uptake processes by their kinetics, vanadate sensitivities, and responsiveness to inositol 1,4,5-trisphosphate (IP 3 ) and caffeine. Most important, we found that lowering the luminal free Ca 2ϩ concentration ([Ca 2ϩ ] i ) relieved a protection against thapsigargin inhibition. This study provides new insights into the nature of neuronal ER Ca 2ϩ pumps as well as new information about the protective effect of high [Ca 2ϩ ], which until now has largely been attributed to the binding of extramicrosomal, or cytosolic, free Ca 2ϩ to the E1 conformation of the pump (10,11).

EXPERIMENTAL PROCEDURES
Microsomal Isolation-Whole rat brain microsomes were isolated as described previously (20 In experiments where oxalate and/or digitonin are indicated, microsomes were preincubated in Buffer A along with 50 mM oxalate and/or 10 M digitonin. For the experiment shown in Fig. 5, however, oxalate was added 5 min after the addition of isotope (time 0). Where thapsigargin is indicated, it was added at the beginning of preincubation at a concentration of 1 M; however, for preloading experiments, 1 M thapsigargin was added to the preincubation mixture 2 min prior to time 0. Any other drugs were added at time 0 (in Buffer B) unless stated otherwise.
Ca 2ϩ Preloading Technique-Some microsomes were loaded with Ca 2ϩ prior to time 0 by preincubating in Buffer A, 50 M Mg⅐ATP, and 50 M Ca 2ϩ without EGTA. To measure uptake in the presence of ATP, Buffer B was added at time 0, adjusting final concentrations to 3 mM Mg⅐ATP, 90 M CaCl 2 , and 100 M EGTA. To measure uptake in the absence of ATP, 1 ml of Buffer B alone was added at time 0 so as to dilute the [Mg⅐ATP] down to ϳ10 M, a concentration that in our preparation did not significantly increase 45 Ca accumulation as compared with that measured in the absence of ATP (data not shown). Therefore, for preloaded microsomes, the ATP-dependent uptake (see Fig. 3) represents uptake in the presence of 3 mM ATP minus uptake in the presence of 10 M ATP.

RESULTS
Oxalate is a Ca 2ϩ -precipitating anion that accumulates in mitochondrial and ER-type vesicles, maintaining very low [Ca 2ϩ ] i by acting as a high-capacity Ca 2ϩ buffer (22,23). In the absence of oxalate, rat brain microsomes accumulate 45 Ca in an extramicrosomal free Ca 2ϩ -and ATP-dependent manner, taking up Ca 2ϩ rapidly during the first 1-2 min and reaching a steady-state after ϳ3 min (20,24). In our preparation, most of this ATP-dependent uptake would presumably be due to the pumping action of SERCAs, which should be inhibited by thapsigargin. However, as illustrated in Fig. 1A, preincubation with and continued exposure to 1 M thapsigargin (a maximal concentration) had no significant effect on the 10-min time course of ATP-dependent 45 Ca uptake. In contrast, at a similar [Ca 2ϩ ] o of ϳ0.3 M, the addition of 50 mM oxalate significantly increased the magnitude of total ATP-dependent uptake and resolved a large thapsigargin sensitivity (Fig. 1B). In the presence of oxalate, these two apparently distinct ATP-dependent uptake processes had significantly different kinetics: the thapsigargin-sensitive component (slope ϭ 41 Ϯ 12 nmol⅐g Ϫ1 ⅐s Ϫ1 ) took up Ca 2ϩ approximately five times faster than the thapsigargin-insensitive component (slope ϭ 8 Ϯ 2 nmol⅐g Ϫ1 ⅐s Ϫ1 ). In the presence of 50 mM oxalate, ϳ80% of the total ATP-dependent 45 Ca uptake was potently and specifically inhibited by thapsigargin, having an IC 50 of ϳ1 nM and being maximally inhibited in the range of 10 -100 nM (Fig. 1C). The thapsigargin-insensitive portion of the uptake was resistant to inhibition up to 10 M (data not shown).
To ensure that the apparent lack of thapsigargin sensitivity in the absence of oxalate was not due to an increased lipophilic association of the drug with membranes, we compared the effects of thapsigargin in microsomal suspensions that were either more dilute or more concentrated than typically used. The differential thapsigargin sensitivity, observed in the presence versus absence of oxalate, was unaffected by either halving (ϳ0.05 mg/ml) or doubling (ϳ0.2 mg/ml) the final microsomal protein concentration (data not shown). Regardless of the presence of oxalate, a 10 M concentration of the Ca 2ϩ ionophore A23187 released Ն90% of the actively accumulated Ca 2ϩ , indicating that membrane vesicles were responsible for our measured 45 Ca uptake. Addition of a 100 nM concentration of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had no effect on measured uptake, indicating that none of our ATP-dependent Ca 2ϩ accumulation could be attributed to mitochondrial contamination. Both the thapsigargin-sensitive and -insensitive processes were detected in the presence of 10 M digitonin, a detergent known to selectively permeabilize plasma membrane vesicles at the concentration used (23). Although digitonin caused a small consistent decrease of 10 -20% in the total ATP-dependent 45 Ca uptake, the proportional effect of thapsigargin, with or without oxalate, remained unaltered by digitonin. Because our preparation may have been slightly contaminated with plasma membrane vesicles, all further experiments were carried out in the presence of 10 M digitonin, unless otherwise stated.
To further investigate whether these thapsigargin-sensitive Both curves plateaued to the same steady-state Ca 2ϩ level (A ϭ 3.8 mol/g of protein) and had the same value ( ϭ 55 s); r i (ϪTG) ϭ 70 Ϯ 26 nmol⅐g Ϫ1 ⅐s Ϫ1 and r i (ϩTG) ϭ 70 Ϯ 22 nmol⅐g Ϫ1 ⅐s Ϫ1 , where the initial rate (r i ) was calculated as A/. B, data were fit by linear regression (n ϭ 7 for each point). The slope in the absence of thapsigargin was 49 Ϯ 6 nmol⅐g Ϫ1 ⅐s Ϫ1 , and the slope in the presence of thapsigargin was 8 Ϯ 2 nmol⅐g Ϫ1 ⅐s Ϫ1 . C, data were fit to the sigmoidal function f ϭ (a Ϫ d)/(1 ϩ(x/c) b ) ϩ d (n ϭ 3 for each point, and error bars represent S.D.). The approximate values for a, b, c, and d were 23 mol/g of protein, 1, 1 nM, and 3 mol/g of protein, respectively. and -insensitive uptake processes were in fact distinct, we compared their vanadate, IP 3 , and caffeine sensitivities. Both processes were susceptible to vanadate inhibition, but differed slightly in their sensitivities ( Fig. 2A): vanadate inhibited the thapsigargin-sensitive accumulation with an IC 50 of 16 Ϯ 1 M, whereas the IC 50 for the thapsigargin-insensitive accumulation was 30 Ϯ 6 M. Additionally, as illustrated in Fig. 2B, only the thapsigargin-sensitive accumulation was affected by either 5 M IP 3 or 10 mM caffeine. After 10 min, IP 3 decreased the total thapsigargin-sensitive ATP-dependent uptake from 20 Ϯ 1 to 10 Ϯ 1 mol/g, and caffeine decreased it to 13 Ϯ 1 mol/g.
We were primarily interested in understanding why thapsigargin could have such a profound inhibitory effect in the presence of oxalate, but not in its absence (Fig. 1). To account for this behavior, we assumed that oxalate, by maintaining an extremely low free [Ca 2ϩ ] inside the microsomes, was removing any inhibitory effect of rising [Ca 2ϩ ] i on pump activity, a phenomenon we (20) and others (22) had previously documented. The constant uptake rate in Fig. 1B could be explained by two effects of oxalate: relief of any [Ca 2ϩ ] i -dependent inhibition of the pump and at the same time elimination of the normal leak of Ca 2ϩ out of the store (due to "trapping" of Ca 2ϩ in the lumen). Our initial hypothesis, then, was that the thapsigargin-sensitive portion of the ATP-dependent uptake was more responsive to feedback inhibition by increasing [Ca 2ϩ ] i than was the thapsigargin-insensitive component. If true, the thapsigargin-sensitive pump activity in the absence of oxalate would be depressed early in the time course by increasing [Ca 2ϩ ] i ; after several minutes, the total ATP-dependent uptake would be largely due to the activity of the thapsigargin-insensitive mechanism, and this could account for the observation that 1 M thapsigargin had little or no effect on the prolonged time course of uptake (Fig. 1A). To directly test this hypothesis, we preloaded microsomes with Ca 2ϩ and measured ATP-dependent initial uptake rates in the absence or presence of thapsigargin. Because preloading required 50 M unchelated Ca 2ϩ (see "Experimental Procedures"), digitonin was eliminated from these experiments due to adverse interactions with high [Ca 2ϩ ]. As shown in Fig. 3, preloading microsomes resulted in a 40 -50% decrease in the initial rate of ATP-depend-ent uptake, regardless of the presence of thapsigargin.
Because the thapsigargin-sensitive and -insensitive uptake processes were equally affected by Ca 2ϩ preloading, we then focused our attention on an alternative hypothesis: that oxalate was capable of "transforming" apparently thapsigargin-insensitive pumps into thapsigargin-sensitive ones, presumably by removing a protective effect of Ca 2ϩ . If, in fact, luminal calcium ions were protecting thapsigargin-susceptible pumps against thapsigargin inhibition, there should be a range of [Ca 2ϩ ] i in which the profile of thapsigargin sensitivity changed. Therefore, as illustrated in Fig. 4, we measured ATP-dependent 45 Ca accumulation as well as ATP-dependent initial uptake rates in the presence and absence of thapsigargin at various oxalate concentrations. The relative contribution of the thapsigarginsensitive and -insensitive uptakes changed most over the range of 10 -50 mM oxalate. This sensitivity was unaffected by oxalate concentrations of 0.01-1.0 mM, and concentrations greater than 50 mM had no additional effect. Both the magnitude of total ATP-dependent uptake (Fig. 4A) and the apparent thapsigargin sensitivity, in terms of 12-min accumulation (Fig. 4B) as well as initial Ca 2ϩ uptake rates (Fig. 4C), increased with increasing oxalate concentrations. These data, in combination with the results of Fig. 3, were not consistent with our initial hypothesis, but did support the second hypothesis, that increasing [Ca 2ϩ ] i could protect against thapsigargin inhibition. To illustrate this point (Fig. 4B), we calculated an estimated maximal [Ca 2ϩ ] i by dividing the solubility product for Ca 2ϩ and oxalate (ϳ3 ϫ 10 Ϫ9 M 2 ) by each oxalate concentration. To ensure that it was in fact only luminal Ca 2ϩ , and not extramicrosomal Ca 2ϩ , that was mediating this protective effect, we adjusted the total Ca 2ϩ concentration in the presence of 100 M EGTA and 50 mM oxalate to give a measured [Ca 2ϩ ] o identical to that measured in the absence of oxalate, 0.3 M. The results shown in Fig. 4D confirmed that luminal Ca 2ϩ was responsible for the protective effect: the apparent thapsigargin-insensitive uptake as a percent of the total ATP-dependent uptake was 121 Ϯ 25% in the absence of oxalate, but was 31 Ϯ 5% and 37 Ϯ 7% in the presence of 50 mM oxalate for 0.1 and 0.3 M free Ca 2ϩ , respectively. To determine if this protective effect of Ca 2ϩ i was reversible, microsomes were allowed to accumulate Thapsigargin-insensitive uptake (plotted as a percent of the total thapsigargininsensitive uptake) was the measured ATP-dependent uptake in the presence of 1 M thapsigargin. Thapsigargin-sensitive uptake (plotted as a percent of the total thapsigargin-sensitive uptake) was calculated by subtracting the ATP-dependent uptake in the presence of thapsigargin from the ATP-dependent uptake in the absence of thapsigargin. B, thapsigargin-sensitive accumulation was 20.57 Ϯ 0.91 mol/g for the control (CON), 15.68 Ϯ 0.58 mol/g for 5 M IP 3 , and 13.06 Ϯ 0.75 mol/g for 10 mM caffeine (CAFF); thapsigargin-insensitive accumulation was 5.47 Ϯ 0.26 mol/g for the control, 5.59 Ϯ 0.32 mol/g for IP 3 , and 5.34 Ϯ 0.27 mol/g for caffeine. The thapsigargin-sensitive and -insensitive data points were measured and calculated as described for A. 45 Ca for 5 min, reaching steady state, in the absence of oxalate and either with or without thapsigargin; at 5 min, the microsomal suspensions were injected with either 50 mM oxalate or an equal volume of Buffer A (see "Experimental Procedures"). As illustrated in Fig. 5, the addition of oxalate almost immediately transformed the 45 Ca accumulation into a linear function and resolved thapsigargin-sensitive (ϳ30 nmol⅐g Ϫ1 ⅐s Ϫ1 ) and thapsigargin-insensitive (ϳ8 nmol⅐g Ϫ1 ⅐s Ϫ1 ) uptake rates similar to those measured when microsomes had been preincubated with 50 mM oxalate (Fig. 1B). Therefore, the protection against thapsigargin due to luminal Ca 2ϩ was reversible.

DISCUSSION
Several studies have documented a protective effect of Ca 2ϩ against thapsigargin inhibition. For example, Sagara et al. (10) showed a protection of skeletal muscle SERCA1 phosphorylation against thapsigargin inhibition when the enzyme was preincubated with Ca 2ϩ . In studies on neurally derived chromaffin cells of the adrenal medulla, Caspersen and Treiman (12) found that preincubation of microsomes with thapsigargin in the absence of Ca 2ϩ resulted in a complete inhibition of Ca 2ϩ pump EϳP formation. But when the microsomes were prein-cubated with thapsigargin and 0.6 mM free Ca 2ϩ , a differential sensitivity to thapsigargin emerged, and this sensitivity varied among the fractions of these ER membranes on an isopycnic sucrose gradient. This work suggests that Ca 2ϩ may have a protective effect against thapsigargin inhibition in neurally derived cells.
In neuronal ER vesicles, we observed an oxalate concentration dependence of thapsigargin sensitivity, consistent with a gradual removal of Ca 2ϩ protection. Our data contribute new information about the nature of this protective effect, namely, that [Ca 2ϩ ] i mediates this protection from thapsigargin inhibition. We eliminated any contributing effect of [ We propose that the bulk of the ATP-dependent 45 Ca uptake observed over 10 min in the absence of oxalate (Fig. 1A) is largely attributable to the activity of typical thapsigargin-sus- FIG. 3. Effects of Ca 2؉ preloading on initial ATP-dependent uptake rates in the presence or absence of thapsigargin. Microsomes were preincubated without digitonin for 12-14 min in Buffer A alone (Unloaded) or in Buffer A with 50 M Ca 2ϩ and 50 M Mg⅐ATP (Preloaded) and then allowed to accumulate 45 Ca in the presence or absence of 3 mM Mg⅐ATP (see "Experimental Procedures"). Data were fit to exponentials, and initial uptake rates (r i ) were calculated as described in the legend to Fig. 1A, constraining b ϭ 0. A, in the absence of thapsigargin, r i values for unloaded and preloaded microsomes were 52 Ϯ 10 and 33 Ϯ 5 nmol⅐g Ϫ1 ⅐s Ϫ1 , respectively (n ϭ 21 for each point). B, in the presence of thapsigargin, r i values for unloaded and preloaded microsomes were 41 Ϯ 6 and 19 Ϯ 2 nmol⅐g Ϫ1 ⅐s Ϫ1 , respectively (n ϭ 15 for each point). ceptible SERCA pumps. The fact that thapsigargin had no apparent effect on this time course would be understandable if the isolated microsomes had a sufficiently high [Ca 2ϩ ] i (prior to exposure to radioactive buffer) to elicit the protective behavior against thapsigargin inhibition. If this were in fact true, one would expect, in the absence of thapsigargin, the rate of ATPdependent uptake in the presence of oxalate to be similar to the initial rate of uptake in the absence of oxalate. Comparing Fig.  1A (without oxalate) with Fig. 1B (with oxalate), the uptake rates are indeed similar: the initial uptake rate in the absence of oxalate was 70 Ϯ 26 nmol⅐g Ϫ1 ⅐s Ϫ1 , and the uptake rate in 50 mM oxalate was 49 Ϯ 6 nmol⅐g Ϫ1 ⅐s Ϫ1 . Measuring only early time points, to arrive at better estimates of initial uptake rates, revealed only small differences (Fig. 4C): 33 Ϯ 3 nmol⅐g Ϫ1 ⅐s Ϫ1 in the absence of oxalate and 50 Ϯ 16 nmol⅐g Ϫ1 ⅐s Ϫ1 in 50 mM oxalate. This slight increase in the initial rate in the presence of oxalate would be expected since lowering luminal free [Ca 2ϩ ] eliminates the normal negative feedback on pump activity (20,22). In the absence of oxalate, 1 M thapsigargin slightly decreased the initial rate of ATP-dependent 45 Ca uptake from 33 Ϯ 3 to 22 Ϯ 3 nmol⅐g Ϫ1 ⅐s Ϫ1 (Fig. 4C). This slight effect of thapsigargin is probably due to sufficiently low luminal free Ca 2ϩ in a small portion of the microsomes, prior to time 0, such that thapsigargin is capable of binding to and inhibiting a nominal fraction of the thapsigargin-susceptible pumps. Because thapsigargin seems to affect a relatively small percentage of these pumps in the absence of oxalate, a decrease in the measured ATP-dependent uptake was detectable only at early time points, providing a plausible explanation for the apparent lack of thapsigargin sensitivity at later times (Fig. 1A). The noticeable difference in the apparent thapsigargin-insensitive uptake rate in the absence versus presence of 50 mM oxalate (Figs. 1, 3, and 4) further supports our second hypothesis and bolsters the notion that most of the uptake observed in the absence of oxalate is likely due to the action of thapsigarginsusceptible pumps.
The various SERCA-type pumps are thought to share a common mechanism of pump cycling (6), involving a conformational change from E1 (with two cytosolic facing, high-affinity Ca 2ϩ -binding sites) to E2-P (with two luminal facing, lowaffinity Ca 2ϩ -binding sites) following cytosolic Ca 2ϩ binding and ATP-dependent phosphorylation; E2-P then loses two Ca 2ϩ ions to the lumen. In skeletal muscle SR, thapsigargin is thought to preferentially bind the E2 conformation of the pump, thereby shifting the E1-E2 equilibrium toward E2 (10, 25-28). Sagara et al. (10,26) suggested that thapsigargin preferentially binds the enzyme conformation that exists in the absence of Ca 2ϩ , thereby forming a very stable "dead-end complex." The protective effect of high [Ca 2ϩ ] against thapsigargin inhibition might be explained by a Ca 2ϩ -induced shift in the E1-E2 equilibrium to the E1 (Ca 2ϩ -bound) conformation of the pump (10,11). In skeletal muscle, the protective effect of Ca 2ϩ was lost after very short periods (1 s) of pump activity (10). In contrast, in ER from chromaffin cells, there was no change in the degree of thapsigargin-sensitive EϳP formation for each membrane fraction, even when measuring pump phosphorylation up to 120 s (12). These diverging observations may be due to differences in the SERCA pump subtypes that exist in neurally derived ER and in skeletal muscle SR. Our results suggest that the majority of Ca 2ϩ -pumping activity observed in neuronal ER is attributable to thapsigargin-sensitive SERCA subtypes that are susceptible to luminal Ca 2ϩ protection against thapsigargin inhibition. In our system, it seems unlikely that this protection would be due to a cytosolic Ca 2ϩ -induced shift in E1-E2 equilibrium toward the E1 conformation, as suggested for the SR. Assuming thapsigargin favors the E2 conformation of the pump, an alternative explanation is that increasing [Ca 2ϩ ] i , which slows pump activity, leads to pumps that exist in the E2 conformation longer; more Ca 2ϩ ions in the lumen may result in E2-P pump conformations that have Ca 2ϩ bound for longer periods of time, and thapsigargin may have a reduced affinity for or be incapable of inactivating this Ca 2ϩ -bound E2-P conformation of the pump.
In most whole cell preparations, as well as in many isolated microsome systems, thapsigargin treatment results in a significant release of accumulated Ca 2ϩ , even in the absence of Ca 2ϩ -precipitating anions like oxalate. In our preparation of whole rat brain microsomes, however, we saw little or no effect of thapsigargin (at maximal doses) in the absence of oxalate. We believe that this apparent discrepancy is most likely due to our particular isolation procedure and experimental system, which probably result in isolated microsomes having sufficient luminal free [Ca 2ϩ ] to mediate protection against thapsigargin inhibition, even prior to initiation of ATP-dependent 45 Ca uptake. It is conceivable, during an isolation procedure that involves homogenization and thus disruption of ER membranes, that endogenous luminal Ca 2ϩ buffers (which in living cells may subserve the function of oxalate) are lost or that microsomes accumulate and store small amounts of Ca 2ϩ during the isolation protocol (the results of Fig. 4 suggest that very low levels of luminal free Ca 2ϩ could mediate protection against thapsigargin). In fact, it is possible that oxalate is relieving a protection against thapsigargin inhibition in a number of microsomal studies reported in the literature. Particularly in FIG. 5. Effect of the addition of oxalate following steady-state 45 Ca accumulation in the absence of oxalate. Microsomes were preincubated with 10 M digitonin and either no thapsigargin (A) or 1 M thapsigargin (B). For each point, n ϭ 3 and error bars represent S.D., except for t ϭ 300 s, for which n ϭ 1, and the data point is included only as an approximation of the steady-state microsomal Ca 2ϩ load just prior to the addition of either oxalate or buffer. Isotope was added at time 0 with or without 3 mM Mg⅐ATP and without or without 1 M thapsigargin. Microsomes accumulated 45 Ca in an ATP-dependent manner for 5 min (achieving steady-state accumulation), at which time either 50 mM oxalate or an equal volume of Buffer A was added. isolated subcellular membrane preparations, oxalate is often automatically included when measuring thapsigargin sensitivity of 45 Ca uptake (8,9,14), and whether the same effect of thapsigargin is observed in the absence of oxalate is unclear.
Several groups have documented thapsigargin-resistant ATPdependent uptake in a variety of cell and membrane preparations, including PC12 cells (13)(14)(15), dog brain microsomes (11), saponin-permeabilized DDT 1 MF-2 smooth muscle cells (16), permeabilized cell and microsome preparations of rat pituitary GH 4 C 1 cells (17), cultured arterial myocytes (18), and DC-3F Chinese hamster lung cells (19). Kijima et al. (11) found that in dog brain microsomes, ϳ70% of their Ca 2ϩ -loading activity was inhibited by thapsigargin, and only the thapsigargin-sensitive portion of this activity was responsive to 10 M IP 3 (which released ϳ27% of the total preloaded Ca 2ϩ ). Our observations using rat brain microsomes were similar: ϳ80% of the oxalateand ATP-dependent Ca 2ϩ uptake was thapsigargin-sensitive; 5 M IP 3 released ϳ40% of the total ATP-dependent 45 Ca accumulation; and only the thapsigargin-sensitive portion of this uptake was responsive to IP 3 . In contrast to other studies that found a caffeine or ryanodine sensitivity associated with the thapsigargin-resistant Ca 2ϩ pools (17,18), we observed no effect of 10 mM caffeine on the thapsigargin-insensitive pool. Caffeine did, however, cause a 37% reduction in the thapsigargin-sensitive 45 Ca accumulation. Our results suggest that the thapsigargin-sensitive and -insensitive uptake mechanisms are functionally segregated. Poulsen et al. (29) have also demonstrated a thapsigargin-sensitive Ca 2ϩ pool in adrenal chromaffin cells responsive to both IP 3 and caffeine. In two of the aforementioned studies (16,19), no differential sensitivity to vanadate was observed for these two uptake processes. In contrast, we observed a small but significant difference in vanadate concentration-response curves: the thapsigargin-insensitive portion of the uptake was shifted slightly to the right of the thapsigargin-sensitive portion of the uptake. While suggesting that these two processes may have different vanadate sensitivities, these data also provided compelling evidence that our thapsigargin-resistant uptake was not due to the presence of contaminating plasma membrane-type Ca 2ϩ pumps, which have been shown to have a higher affinity for vanadate than ER-or SR-type Ca 2ϩ pumps (29,30). Also, the fact that 10 M digitonin had no effect on the relative amounts of thapsigarginsensitive (ϳ80%) and -insensitive (ϳ20%) uptakes ruled out contaminating plasma membrane vesicles as the source of the thapsigargin-resistant vesicular accumulation.
In summary, our data contribute further evidence of a thapsigargin-resistant mechanism for ATP-dependent Ca 2ϩ accumulation in the ER of neuronal cells. When [Ca 2ϩ ] i is kept very low (in the presence of oxalate), the uptake kinetics of this thapsigargin-insensitive process are much slower than those of the thapsigargin-sensitive pump. This study also suggests that in neural cells, the thapsigargin-insensitive pool may be func-tionally segregated from both the IP 3 and ryanodine receptor Ca 2ϩ release channels. Finally, our results demonstrate for the first time that the ability of high [Ca 2ϩ ] to protect some neuronal ER Ca 2ϩ pumps against thapsigargin inhibition is conferred by luminal free Ca 2ϩ and cannot be explained by an effect of cytosolic Ca 2ϩ . These new data suggest that either the mechanism by which Ca 2ϩ can protect against thapsigargin inhibition is different for the SERCA subtypes that exist in neuronal ER as compared with skeletal muscle SR or that a generalized model for Ca 2ϩ protection should be re-evaluated in terms of possible luminal Ca 2ϩ effects.