The role of calcium-induced calcium release in pancreatic beta cell function

The role of the Ca2+-induced Ca2+ release channel (ryanodine receptor) in MIN6 pancreatic beta cells was investigated. The presence of ryanodine receptors was demonstrated by immunocytochemistry and by competitive ryanodine binding studies. Treatment with caffeine, a ryanodine receptor agonist, resulted in a transient increase in cytoplasmic Ca 2+. This Ca2+ transient persisted when extracellular Ca2+ was removed, and was diminished by pre-treatment with agents that decrease the endoplasmic reticulum Ca2+ concentration. An endoplasmic reticulum-targeted “chameleon” was used to report lumenal free Ca2+. Depolarization of MIN6 cells with KCl led to release of Ca2+ from the endoplasmic reticulum. This endoplasmic reticulum Ca2+ release was mimicked by treatment with caffeine, prevented by the voltage-gated Ca2+ channel antagonist diltiazem, and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca2+ was also blunted by ryanodine treatment, and both endoplasmic reticulum and cytoplasmic Ca 2+ changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca2+ but no detectable change in endoplasmic reticulum Ca2+. Carbamyl choline caused release of endoplasmic reticulum Ca2+. The effect of carbamyl choline was not altered by ryanodine or by glucose, but ryanodine did diminish the plateau phase of the carbamyl choline-induced increase in cytoplasmic Ca2+. The ryanodine-induced change in the cytoplasmic Ca2+ response was mimicked by treatment with diazoxide or diltiazem, or by chelation of extracellular Ca2+, but ryanodine did not have inhibitory effects on the voltage-dependent calcium channel, suggesting that ryanodine receptor activation is involved in the plateau phase of the carbamyl choline response. MIN6 cells also exhibited cytoplasmic Ca 2+ transients, which were not dependent on extracellular Ca 2+, in response to thyrotropin-releasing hormone. Thyrotropin-releasing hormone responses were dose-dependent. Treatment of MIN6 cells with the incretin hormone, glucagon-like peptide-1, or with forskolin, both of which raise intracellular cAMP concentrations, resulted in oscillations in cytoplasmic Ca2+ concentrations, but no effect on endoplasmic reticulum Ca2+ was observed. Ca2+-induced Ca2+ release was also observed in another pancreatic beta cell line, INS-1. Ryanodine significantly inhibited both KCl- and glucose-induced insulin secretion, but had no effect on carbamyl-choline-induced insulin secretion, and there was no effect of ryanodine on basal rates of insulin secretion. Taken together, these results provide strong evidence that Ca2+-induced Ca2+ release augments depolarization-induced cytoplasmic Ca2+ signals in pancreatic beta cells, and contributes to the signal for insulin secretion.