FIGURE 1.

Store-operated Ca²⁺ entry in MIN6 cells. A, MIN6 cells were loaded with Fura 2 and perifused with test solutions containing 2 mm glucose at 37 °C. Cells were initially bathed in normal, 2.5 mm Ca²⁺ extracellular solution to establish a baseline. The bath solution was then exchanged for Ca²⁺-free solution (10 μm EGTA), Ca²⁺-free with 250 μm carbachol (CCh) followed by Ca²⁺-free with 10 μm cyclopiazonic acid (CPA) to discharge and prevent refilling of intracellular Ca²⁺ stores. Reintroduction of 2.5 mm Ca²⁺ caused a rise of cytosolic [Ca²⁺] ([Ca²⁺]_c) representing SOCE. B, the addition of 5 μm nimodipine (Nimod) before the reintroduction of extracellular Ca²⁺ caused a small decrease in the peak amplitude. The efficacy of nimodipine to block voltage-gated Ca²⁺ channels was confirmed by applying a pulse of 56 mm KCl solution (indicated by arrow). Whereas under control conditions KCl elicited a large rise in [Ca²⁺]_c, this rise was inhibited by nimodipine. C, the AUC of the SOCE response shown in B was measured for the 160-s period after Ca²⁺ addition and was significantly reduced by nimodipine (52 control cells, 24 nimodipine-treated; *, p = 0.002, ANOVA). D, MIN6 cells in a 96-well plate were loaded with Fura 2 in Ca²⁺-free solution containing 2 mm glucose and 1 μm Tg. The cells were then washed with fresh bath solution containing Tg. Ca²⁺-free bath solution (Cont) or Ca²⁺-containing solution with Tg and SKF96365 (final, 2 mm [Ca²⁺], SKF96365 concentration indicated in μm) was injected at 100 s. SOCE was dose-dependently blocked by SKF96365.Data are plotted as the mean ± S.E., error bars shown are larger than symbols, averaged from 12 wells for each solution and are representative of three independent experiments.