-induced Release from Internal Stores in INS-1 Rat Insulinoma Cells

The secretion of insulin from pancreatic ${\beta}$-cells is triggered by the influx of $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channels. The resulting elevation of intracellular calcium ($[Ca^{2+}]_i$) triggers additional $Ca^{2+}$ release from internal stores. Less well understood are the mechanisms involved in $Ca^{2+}$ mobilization from internal stores after activation of $Ca^{2+}$ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic ${\beta}$-cell line, INS-1 cells. To measure cytosolic and stored $Ca^{2+}$, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. $[Ca^{2+}]_i$ was repetitively increased by caffeine stimulation in normal $Ca^{2+}$ buffer. However, peak $[Ca^{2+}]_i$ was only observed after the first caffeine stimulation in $Ca^{2+}$ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in $[Ca^{2+}]_i$ were reduced by pretreatment with ruthenium red, as well as by depletion of internal $Ca^{2+}$ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced $Ca^{2+}$ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells,$Ca^{2+}$ release from internal stores was activated by caffeine, $Ca^{2+}$, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in perrneabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic ${\beta}$-cells.