Luminal Ca2+ Protects against Thapsigargin Inhibition in Neuronal Endoplasmic Reticulum*

Karen M. Wells and Ronald F. AbercrombieDagger

From the Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Thapsigargin is a specific and potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases. However, in whole rat brain microsomes, 1 µM 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 sime  1 nM thapsigargin) five times that of a thapsigargin-insensitive rate. This remaining ~20% of the total ATP-dependent accumulation was insensitive to thapsigargin (up to 10 µM), slightly less sensitive to vanadate inhibition, and unresponsive to 5 µM 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 ~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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The endoplasmic and sarcoplasmic reticula (ER1 and SR, respectively) actively accumulate Ca2+ via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family of Ca2+ 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 (1-3). 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 Ca2+ ions into the lumen by hydrolyzing one ATP molecule and forming an enzyme-phosphorylated (E~P) intermediate (6). Thapsigargin, a naturally occurring, tumor-promoting sesquiterpene lactone, has been shown to release Ca2+ from the ER by specifically inhibiting these Ca2+ 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 Ca2+ affinities or inhibitor effects for avian subtypes 1, 2a, and 2b when expressed in COS cells. A protective effect of high [Ca2+] 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 Ca2+ 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 Ca2+ in some cell and microsome preparations (11, 13-19).

In this study, we were interested in characterizing neuronal ER Ca2+ accumulation both in terms of thapsigargin sensitivity and the way by which high [Ca2+] might protect against thapsigargin inhibition. We employed 45Ca flux studies to characterize both thapsigargin-sensitive and -resistant ATP-dependent Ca2+ uptake processes by their kinetics, vanadate sensitivities, and responsiveness to inositol 1,4,5-trisphosphate (IP3) and caffeine. Most important, we found that lowering the luminal free Ca2+ concentration ([Ca2+]i) relieved a protection against thapsigargin inhibition. This study provides new insights into the nature of neuronal ER Ca2+ pumps as well as new information about the protective effect of high [Ca2+], which until now has largely been attributed to the binding of extramicrosomal, or cytosolic, free Ca2+ to the E1 conformation of the pump (10, 11).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Microsomal Isolation-- Whole rat brain microsomes were isolated as described previously (20). The final microsomal pellet was resuspended in Buffer A, a Ca2+-free buffered solution (pH 7.4) containing 150 mM KCl, 1.4 mM MgCl2, 20 mM HEPES, and 2 mM KH2PO4. The total protein content of microsomal suspensions was determined by the Bradford method (21). A typical test volume (300 µl) of microsomes contained between 100 and 200 µg of protein; therefore, the addition of 1 ml of radioactive Buffer B (see below) resulted in a final microsomal protein concentration of ~0.1 mg/ml.

45Ca Technique-- Microsomal suspensions were preincubated at 37 °C for 12-14 min. An experiment was initiated by the addition of 1 ml of warmed Buffer B to a 300-µl volume of microsomes. Buffer B (pH 7.4) contained tracer 45Ca, 150 mM KCl, 1.4 mM MgCl2, 20 mM HEPES, 2 mM KH2PO4, 0.13 mM EGTA, and 0.117 mM CaCl2 (to give a final free [Ca2+] of 0.3-0.4 µM, as measured with mini-electrodes), ±Mg·ATP (to a final concentration of 3 mM), ±0.01 mM digitonin, and ±50 mM oxalate (addition of oxalate gave a free [Ca2+] of 0.1-0.2 µM, as measured with mini-electrodes). Microsomes accumulated 45Ca for various time intervals. An experiment was terminated by the addition of 3 ml of ice-cold wash solution (pH 7.4) containing 150 mM KCl, 1.4 mM MgCl2, 20 mM HEPES, and 2 mM KH2PO4. The sample was then passed over a Whatman GF/B glass-fiber filter over a vacuum and washed three times with 5 ml of the same ice-cold buffer solution. ATP-dependent Ca2+ uptake was determined by subtracting 45Ca accumulation in the absence of Mg·ATP from 45Ca accumulation in the presence of Mg·ATP. Unless otherwise noted, data are means ± S.E. and are expressed as µmol or nmol of Ca2+/g of protein.

In experiments where oxalate and/or digitonin are indicated, microsomes were preincubated in Buffer A along with 50 mM oxalate and/or 10 µM 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 µM; however, for preloading experiments, 1 µM 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.

Ca2+ Preloading Technique-- Some microsomes were loaded with Ca2+ prior to time 0 by preincubating in Buffer A, 50 µM Mg·ATP, and 50 µM Ca2+ without EGTA. To measure uptake in the presence of ATP, Buffer B was added at time 0, adjusting final concentrations to 3 mM Mg·ATP, 90 µM CaCl2, and 100 µM 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 [Mg·ATP] down to ~10 µM, a concentration that in our preparation did not significantly increase 45Ca 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 µM ATP.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Oxalate is a Ca2+-precipitating anion that accumulates in mitochondrial and ER-type vesicles, maintaining very low [Ca2+]i by acting as a high-capacity Ca2+ buffer (22, 23). In the absence of oxalate, rat brain microsomes accumulate 45Ca in an extramicrosomal free Ca2+- and ATP-dependent manner, taking up Ca2+ rapidly during the first 1-2 min and reaching a steady-state after ~3 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 µM thapsigargin (a maximal concentration) had no significant effect on the 10-min time course of ATP-dependent 45Ca uptake. In contrast, at a similar [Ca2+]o of ~0.3 µM, 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 = 41 ± 12 nmol·g-1·s-1) took up Ca2+ approximately five times faster than the thapsigargin-insensitive component (slope = 8 ± 2 nmol·g-1·s-1). In the presence of 50 mM oxalate, ~80% of the total ATP-dependent 45Ca uptake was potently and specifically inhibited by thapsigargin, having an IC50 of ~1 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 µM (data not shown).


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Fig. 1.   Effects of oxalate and thapsigargin on microsomal ATP-dependent 45Ca uptake. Microsomes were preincubated for 12-14 min in Buffer A with 10 µM digitonin, ±1 µM thapsigargin (TG), and either without (A) or with (B) 50 mM oxalate and then, at time 0, incubated in Buffer B (45Ca ± 3 mM Mg·ATP) for various times (see "Experimental Procedures"). A, data points represent average uptakes from four separate experiments and were fit to the exponential function f = A(1 - exp-t/tau ) + b (n = 10 for each point). Both curves plateaued to the same steady-state Ca2+ level (A = 3.8 µmol/g of protein) and had the same tau  value (tau = 55 s); ri(-TG) = 70 ± 26 nmol·g-1·s-1 and ri(+TG) = 70 ± 22 nmol·g-1·s-1, where the initial rate (ri) was calculated as A/tau . B, data were fit by linear regression (n = 7 for each point). The slope in the absence of thapsigargin was 49 ± 6 nmol·g-1·s-1, and the slope in the presence of thapsigargin was 8 ± 2 nmol·g-1·s-1. C, data were fit to the sigmoidal function f = (a - d)/(1 +(x/c)b) + d (n = 3 for each point, and error bars represent S.D.). The approximate values for a, b, c, and d were 23 µmol/g of protein, 1, 1 nM, and 3 µmol/g of protein, respectively.

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 (~0.05 mg/ml) or doubling (~0.2 mg/ml) the final microsomal protein concentration (data not shown). Regardless of the presence of oxalate, a 10 µM concentration of the Ca2+ ionophore A23187 released >= 90% of the actively accumulated Ca2+, indicating that membrane vesicles were responsible for our measured 45Ca 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 Ca2+ accumulation could be attributed to mitochondrial contamination. Both the thapsigargin-sensitive and -insensitive processes were detected in the presence of 10 µM 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 45Ca 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 µM digitonin, unless otherwise stated.

To further investigate whether these thapsigargin-sensitive and -insensitive uptake processes were in fact distinct, we compared their vanadate, IP3, 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 IC50 of 16 ± 1 µM, whereas the IC50 for the thapsigargin-insensitive accumulation was 30 ± 6 µM. Additionally, as illustrated in Fig. 2B, only the thapsigargin-sensitive accumulation was affected by either 5 µM IP3 or 10 mM caffeine. After 10 min, IP3 decreased the total thapsigargin-sensitive ATP-dependent uptake from 20 ± 1 to 10 ± 1 µmol/g, and caffeine decreased it to 13 ± 1 µmol/g.


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Fig. 2.   Effects of vanadate, IP3, and caffeine on oxalate-sensitive ATP-dependent 45Ca uptake. Microsomes were preincubated with 10 µM digitonin, 50 mM oxalate, and ±1 µM thapsigargin and then allowed to accumulate 45Ca for 10 min (n = 8 for each condition). A, thapsigargin (TG)-sensitive uptake (open circles) and thapsigargin-insensitive uptake (filled circles) were fit to sigmoidal functions (see legend to Fig. 1C), giving inflection points of 16 ± 1 and 30 ± 6 µM vanadate, respectively. Thapsigargin-insensitive uptake (plotted as a percent of the total thapsigargin-insensitive uptake) was the measured ATP-dependent uptake in the presence of 1 µM 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 ± 0.91 µmol/g for the control (CON), 15.68 ± 0.58 µmol/g for 5 µM IP3, and 13.06 ± 0.75 µmol/g for 10 mM caffeine (CAFF); thapsigargin-insensitive accumulation was 5.47 ± 0.26 µmol/g for the control, 5.59 ± 0.32 µmol/g for IP3, and 5.34 ± 0.27 µmol/g for caffeine. The thapsigargin-sensitive and -insensitive data points were measured and calculated as described for A.

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 [Ca2+] inside the microsomes, was removing any inhibitory effect of rising [Ca2+]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 [Ca2+]i-dependent inhibition of the pump and at the same time elimination of the normal leak of Ca2+ out of the store (due to "trapping" of Ca2+ 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 [Ca2+]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 [Ca2+]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 µM thapsigargin had little or no effect on the prolonged time course of uptake (Fig. 1A). To directly test this hypothesis, we preloaded microsomes with Ca2+ and measured ATP-dependent initial uptake rates in the absence or presence of thapsigargin. Because preloading required 50 µM unchelated Ca2+ (see "Experimental Procedures"), digitonin was eliminated from these experiments due to adverse interactions with high [Ca2+]. As shown in Fig. 3, preloading microsomes resulted in a 40-50% decrease in the initial rate of ATP-dependent uptake, regardless of the presence of thapsigargin.


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Fig. 3.   Effects of Ca2+ 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 µM Ca2+ and 50 µM Mg·ATP (Preloaded) and then allowed to accumulate 45Ca in the presence or absence of 3 mM Mg·ATP (see "Experimental Procedures"). Data were fit to exponentials, and initial uptake rates (ri) were calculated as described in the legend to Fig. 1A, constraining b = 0. A, in the absence of thapsigargin, ri values for unloaded and preloaded microsomes were 52 ± 10 and 33 ± 5 nmol·g-1·s-1, respectively (n = 21 for each point). B, in the presence of thapsigargin, ri values for unloaded and preloaded microsomes were 41 ± 6 and 19 ± 2 nmol·g-1·s-1, respectively (n = 15 for each point).

Because the thapsigargin-sensitive and -insensitive uptake processes were equally affected by Ca2+ 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 Ca2+. If, in fact, luminal calcium ions were protecting thapsigargin-susceptible pumps against thapsigargin inhibition, there should be a range of [Ca2+]i in which the profile of thapsigargin sensitivity changed. Therefore, as illustrated in Fig. 4, we measured ATP-dependent 45Ca 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 thapsigargin-sensitive 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 Ca2+ 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 [Ca2+]i could protect against thapsigargin inhibition. To illustrate this point (Fig. 4B), we calculated an estimated maximal [Ca2+]i by dividing the solubility product for Ca2+ and oxalate (~3 × 10-9 M2) by each oxalate concentration. To ensure that it was in fact only luminal Ca2+, and not extramicrosomal Ca2+, that was mediating this protective effect, we adjusted the total Ca2+ concentration in the presence of 100 µM EGTA and 50 mM oxalate to give a measured [Ca2+]o identical to that measured in the absence of oxalate, 0.3 µM. The results shown in Fig. 4D confirmed that luminal Ca2+ was responsible for the protective effect: the apparent thapsigargin-insensitive uptake as a percent of the total ATP-dependent uptake was 121 ± 25% in the absence of oxalate, but was 31 ± 5% and 37 ± 7% in the presence of 50 mM oxalate for 0.1 and 0.3 µM free Ca2+, respectively. To determine if this protective effect of Ca2+i was reversible, microsomes were allowed to accumulate 45Ca 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 45Ca accumulation into a linear function and resolved thapsigargin-sensitive (~30 nmol·g-1·s-1) and thapsigargin-insensitive (~8 nmol·g-1·s-1) 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 Ca2+ was reversible.


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Fig. 4.   Thapsigargin sensitivity as a function of oxalate concentration and luminal [Ca2+]. Microsomes were preincubated with 10 µM digitonin, ±1 µM thapsigargin (TG), and 0, 10, 25, or 50 mM oxalate. A, ATP-dependent uptake as a function of oxalate concentration. Microsomes accumulated 45Ca for 12 min (n = 5 for each condition). B, the apparent thapsigargin-insensitive ATP-dependent uptake as a percent of the total ATP-dependent uptake at each given oxalate concentration, based on the data shown in A. [Ca2+]i is the approximated luminal free Ca2+ concentration at each oxalate concentration. C, initial 45Ca uptake rates as a function of oxalate concentration. Rates were determined by fitting a line through measured 30- and 60-s ATP-dependent uptake values (n >=  12 for each point). D, microsomal accumulation of 45Ca for 12 min in Buffer B, adjusting the final total concentrations to 100 µM EGTA and the following: I, no oxalate and 90 µM Ca2+; II, 50 mM oxalate and 90 µM Ca2+; or III, 50 mM oxalate and 95 µM Ca2+, giving the measured free Ca2+ concentrations indicated (n = 4 for each condition).


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Fig. 5.   Effect of the addition of oxalate following steady-state 45Ca accumulation in the absence of oxalate. Microsomes were preincubated with 10 µM digitonin and either no thapsigargin (A) or 1 µM thapsigargin (B). For each point, n = 3 and error bars represent S.D., except for t = 300 s, for which n = 1, and the data point is included only as an approximation of the steady-state microsomal Ca2+ load just prior to the addition of either oxalate or buffer. Isotope was added at time 0 with or without 3 mM Mg·ATP and without or without 1 µM thapsigargin. Microsomes accumulated 45Ca 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.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Several studies have documented a protective effect of Ca2+ 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 Ca2+. 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 Ca2+ resulted in a complete inhibition of Ca2+ pump E~P formation. But when the microsomes were preincubated with thapsigargin and 0.6 mM free Ca2+, 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 Ca2+ 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 Ca2+ protection. Our data contribute new information about the nature of this protective effect, namely, that [Ca2+]i mediates this protection from thapsigargin inhibition. We eliminated any contributing effect of [Ca2+]o by adjusting the total [Ca2+] in the presence of 50 mM oxalate to give a [Ca2+]o identical to that in the absence of oxalate, 0.3 µM, as measured with Ca2+ mini-electrodes (Fig. 4D). Indeed, we found that the proportional effect of thapsigargin in the presence of oxalate and 100 µM EGTA with either 90 µM Ca2+ (0.1 µM [Ca2+]o) or 95 µM total Ca2+ (0.3 µM [Ca2+]o) was the same.

We propose that the bulk of the ATP-dependent 45Ca uptake observed over 10 min in the absence of oxalate (Fig. 1A) is largely attributable to the activity of typical thapsigargin-susceptible 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 [Ca2+]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 ATP-dependent 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 ± 26 nmol·g-1·s-1, and the uptake rate in 50 mM oxalate was 49 ± 6 nmol·g-1·s-1. Measuring only early time points, to arrive at better estimates of initial uptake rates, revealed only small differences (Fig. 4C): 33 ± 3 nmol·g-1·s-1 in the absence of oxalate and 50 ± 16 nmol·g-1·s-1 in 50 mM oxalate. This slight increase in the initial rate in the presence of oxalate would be expected since lowering luminal free [Ca2+] eliminates the normal negative feedback on pump activity (20, 22). In the absence of oxalate, 1 µM thapsigargin slightly decreased the initial rate of ATP-dependent 45Ca uptake from 33 ± 3 to 22 ± 3 nmol·g-1·s-1 (Fig. 4C). This slight effect of thapsigargin is probably due to sufficiently low luminal free Ca2+ 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 thapsigargin-susceptible 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 Ca2+-binding sites) to E2-P (with two luminal facing, low-affinity Ca2+-binding sites) following cytosolic Ca2+ binding and ATP-dependent phosphorylation; E2-P then loses two Ca2+ 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 Ca2+, thereby forming a very stable "dead-end complex." The protective effect of high [Ca2+] against thapsigargin inhibition might be explained by a Ca2+-induced shift in the E1-E2 equilibrium to the E1 (Ca2+-bound) conformation of the pump (10, 11). In skeletal muscle, the protective effect of Ca2+ 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 E~P 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 Ca2+-pumping activity observed in neuronal ER is attributable to thapsigargin-sensitive SERCA subtypes that are susceptible to luminal Ca2+ protection against thapsigargin inhibition. In our system, it seems unlikely that this protection would be due to a cytosolic Ca2+-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 [Ca2+]i, which slows pump activity, leads to pumps that exist in the E2 conformation longer; more Ca2+ ions in the lumen may result in E2-P pump conformations that have Ca2+ bound for longer periods of time, and thapsigargin may have a reduced affinity for or be incapable of inactivating this Ca2+-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 Ca2+, even in the absence of Ca2+-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 [Ca2+] to mediate protection against thapsigargin inhibition, even prior to initiation of ATP-dependent 45Ca uptake. It is conceivable, during an isolation procedure that involves homogenization and thus disruption of ER membranes, that endogenous luminal Ca2+ buffers (which in living cells may subserve the function of oxalate) are lost or that microsomes accumulate and store small amounts of Ca2+ during the isolation protocol (the results of Fig. 4 suggest that very low levels of luminal free Ca2+ 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 isolated subcellular membrane preparations, oxalate is often automatically included when measuring thapsigargin sensitivity of 45Ca 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 ATP-dependent uptake in a variety of cell and membrane preparations, including PC12 cells (13-15), dog brain microsomes (11), saponin-permeabilized DDT1MF-2 smooth muscle cells (16), permeabilized cell and microsome preparations of rat pituitary GH4C1 cells (17), cultured arterial myocytes (18), and DC-3F Chinese hamster lung cells (19). Kijima et al. (11) found that in dog brain microsomes, ~70% of their Ca2+-loading activity was inhibited by thapsigargin, and only the thapsigargin-sensitive portion of this activity was responsive to 10 µM IP3 (which released ~27% of the total preloaded Ca2+). Our observations using rat brain microsomes were similar: ~80% of the oxalate- and ATP-dependent Ca2+ uptake was thapsigargin-sensitive; 5 µM IP3 released ~40% of the total ATP-dependent 45Ca accumulation; and only the thapsigargin-sensitive portion of this uptake was responsive to IP3. In contrast to other studies that found a caffeine or ryanodine sensitivity associated with the thapsigargin-resistant Ca2+ 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 45Ca 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 Ca2+ pool in adrenal chromaffin cells responsive to both IP3 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 Ca2+ pumps, which have been shown to have a higher affinity for vanadate than ER- or SR-type Ca2+ pumps (29, 30). Also, the fact that 10 µM digitonin had no effect on the relative amounts of thapsigargin-sensitive (~80%) and -insensitive (~20%) 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 Ca2+ accumulation in the ER of neuronal cells. When [Ca2+]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 functionally segregated from both the IP3 and ryanodine receptor Ca2+ release channels. Finally, our results demonstrate for the first time that the ability of high [Ca2+] to protect some neuronal ER Ca2+ pumps against thapsigargin inhibition is conferred by luminal free Ca2+ and cannot be explained by an effect of cytosolic Ca2+. These new data suggest that either the mechanism by which Ca2+ 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 Ca2+ protection should be re-evaluated in terms of possible luminal Ca2+ effects.

    ACKNOWLEDGEMENTS

We thank Drs. Peter Becker, Douglas Eaton, and Fernan Jaramillo for thoughtful insights and suggestions during the course of this project.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant NS19194.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 404-727-7425; Fax: 404-727-2648.

1 The abbreviations used are: ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; IP3, inositol 1,4,5-trisphosphate; [Ca2+]i, luminal free Ca2+ concentration(s); [Ca2+]o, extramicrosomal (or cytosolic) free Ca2+ concentration(s).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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