Mobilization of Ca²⁺ by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone in permeabilized insulin-secreting RlNm5F cells: Evidence for separate uptake and release compartments in inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool

We characterized and directly compared the Ca²⁺-releasing actions of two inhibitors of endoplasrnic-reticulum (ER) Ca²⁺-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydro-quinone (tBuBHQ), in electropermeabilized insulin-secreting RINm5F cells. Ambient free calcium concentration ([Ca²⁺]) was monitored by Ca²⁺-selective mini-electrodes. After ATP-dependent Ca²⁺ uptake, thapsigargin and tBuBHQ released Ca²⁺ with an EC₅₀ of ~ 37 nM and ~ 2 μM respectively. Both agents mobilized Ca²⁺ predominantly from the Ins(1,4,5)P₃-sensitive Ca²⁺ pool, and in this respect thapsigargin was more specific than tBuBEIQ. The total increase in [Ca²⁺] obtained with thapsigargin and Ins(1,4,5)P₃ was, on the average, only 7% greater than that with Ins(1,4,5)P₃ alone. In contrast, the total increase in [Ca²⁺] obtained with tBuBHQ and Ins(1,4,5)P₃ was 33 % greater than that obtained with only InsP₃ (P < 0.05). Although Ca²⁺ was rapidly mobilized by thapsigargin and tBuBHQ,complete depletion of the Ins(1,4,5)P₃-sensitive Ca²⁺ pool was difficult to achieve. After the release by thapsigargin or tBuBHQ, Ins(1,4,5)P₃ induced additional Ca²⁺ release. The additional Ins(1,4,5)P₃-induced Ca²⁺ release was not altered by supramaximal concentrations of thapsigargin and tBuBHQ, or by Bafilomycin A₁, an inhibitor of V-type ATPases, but was decreased by prolonged treatment with the ER Ca²⁺-ATPase inhibitors. These results suggest the existence of distinct uptake and release compartments within the Ins(1,4,5)P₃-sensitive Ca²⁺ pool. When treated with the inhibitors, the two compartments became distinguishable on the basis of their Ca²⁺ permeability. Apparently, thapsigargin and tBuBHQ readily mobilized Ca²⁺ from the uptake compartment, whereas Ca²⁺ from the release compartment could be mobilized only very slowly, in the absence of Ins(1,4,5)P₃.