Abstract
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.
Highlights
Release Ca2ϩ from the ER by inhibiting these Ca2ϩ pumps [7]
In the absence of oxalate, rat brain microsomes accumulate 45Ca in an extramicrosomal free Ca2ϩ- and ATP-dependent manner, taking up Ca2ϩ rapidly during the first 1–2 min and reaching a steady-state after ϳ3 min [20, 24]. Most of this ATP-dependent uptake would presumably be due to the pumping action of sarco/endoplasmic reticulum Ca2ϩ-ATPase (SERCA), which should be inhibited by thapsigargin
Several studies have documented a protective effect of Ca2ϩ against thapsigargin inhibition
Summary
Release Ca2ϩ from the ER by inhibiting these Ca2ϩ 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. The thapsigargin-sensitive pump activity in the absence of oxalate would be depressed early in the time course by increasing [Ca2ϩ]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).
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