Ca<SUP>2＋</SUP>-induced Ca<SUP>2＋</SUP> Release from Internal Stores in INS-1 Rat Insulinoma Cells

Ca<SUP>2＋</SUP>-induced Ca<SUP>2＋</SUP> Release from Internal Stores in INS-1 Rat Insulinoma Cells

The secretion of insulin from pancreatic &#1346;-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 &#1346;-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＋]_iwere 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 permeabilized 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 &#1346;-cells.