Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic β-cells (MIN6)

We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca2+ concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 muM (n = 4, p < 0.01), while having no effect on ER Ca2+ concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca2+ concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca2+ from a thapsigargin-insensitive Ca2+ store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca2+-induced Ca2+ release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.