Caffeine stimulated calcium mobilization from internal stores in intact INS-1 cells. The representative traces show the effects of repetitive 30 mM caffeine stimulation on [Ca²⁺]_i increases in the presence (A) and absence (B) of extracellular Ca²⁺. The data were obtained from 5 and 7 separate experiments, respectively. INS-1 cells were responsive to repetitive caffeine stimulation in normal extracellular Ca²⁺ buffer, but only responded to the first caffeine stimulation in Ca²⁺ free solution. (C) A 50 μM of ruthenium red markedly reduced the [Ca²⁺]_i peak in the absence of extracellular Ca²⁺. Data were normalized to control values and expressed as mean %±S.E. Asterisk indicates the value is significantly different from the corresponding value of caffeine alone (p<0.05).

KCl triggered Ca²⁺ release from internal stores in intact INS-1 cells. (A) The representative trace shows the effect of 45 mM KCl on [Ca²⁺]_i increases in the presence and absence of extracellular Ca²⁺. The data were obtained from 6 separate experiments. [Ca²⁺]_i elevation was not observed in Ca²⁺ free medium. (B) Effects of CPA plus caffeine, CPA alone or caffeine alone on KCl-induced [Ca²⁺]_i peaks in the presence of extracellular Ca²⁺. The data were obtained from at least 5 separate experiments. Data were normalized to the initial [Ca²⁺]_i peak and expressed as mean % ± S.E. Asterisks indicate that the values are significantly different from the corresponding value for control (p<0.05). Intracellular Ca²⁺ store depletion reduced depolarization-induced Ca²⁺ mobilization. (C) Representative trace shows the effect of ruthenium red on KCl-induced [Ca²⁺]_i elevations. The data were obtained from 6 separate experiments. A 50 μM of ruthenium red significantly reduced depolarization-induced Ca²⁺ mobilization; the effect was restored after washout of the ruthenium red.

Effects of internal calcium store depletion on caffeine-induced calcium release. (A) The representative trace shows the 30 mM caffeine-induced [Ca²⁺]_i rise after internal store depletion by 10 μM carbamylcholine (CCh) in Ca²⁺ free solution. (B) The representative trace shows the 10 μM CCh-induced [Ca²⁺]_i rise under store depleted conditions induced by pretreatment with 30 mM caffeine in the absence of extracellular Ca²⁺. (C) A representative trace of the caffeine effect under store depleted condition induced by pretreatment of cells with 10 μM cyclopiazonic acid (CPA) in Ca²⁺ free solution. All data were obtained from at least 5 separate experiments. Caffeine failed to increase [Ca²⁺]_i after internal Ca²⁺ store depletion induced by pretreatment with CCh or CPA.

Caffeine, Ca²⁺ or ryanodine-induced calcium release in permeabilized INS-1 cells. (A) 10 mM caffeine (☐), 10 μM Ca²⁺ (⋄) or 1 μM ryanodine (Δ) significantly stimulated Ca²⁺ release from internal stores in permeabilized INS-1 cells. Arrows indicate the starting point of each drug perfusion. (B) Summarized Ca²⁺ release rates (S^–1) induced by caffeine, Ca²⁺ or ryanodine. Data were summarized from at least 5 experiments. Asterisks indicate that the values are significantly different from the corresponding value for control (p<0.05). (C) The blocking effect of 50 μM ruthenium red on 10 μM Ca²⁺-induced Ca²⁺ release in permeabilized INS-1 cells. (D) Summarized data showing the effects of ruthenium red on Ca²⁺-induced Ca²⁺ release rates in permeabilized cells. Asterisk indicates that the value is significantly different from the corresponding value of Ca²⁺ (p<0.05). Ca²⁺ release induced by elevated Ca²⁺ was completely blocked by ruthenium red, a RyR blocker, in permeabilized INS-1 cells.

The expression of ryanodine receptors in INS-1 rat insulinoma cells. The fluorescence image (A) and the bright image (B) show the expression and distribution of RyRs in the intracellular compartments. The images were obtained from 4 separate experiments. Immunocytochemistry was done using primary RyR antibody as described under experimental procedures. The scale bar is 10 μm.