title: Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6).
abstract: 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 Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) 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 Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.
authors.affiliation: Henry Wellcome Laboratories of Integrated Cell Signaling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Kathryn J
authors.initials: KJ
authors.lastname: Mitchell
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D017931:DNA Primers; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D007328:Insulin; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D019837:Ryanodine Receptor Calcium Release Channel; D009249:NADP; C024376:NAADP; D002118:Calcium