Regulation of exocytosis by Ca2+ and cAMP - A study on pancreatic beta- and alpha-cells

Type-2 diabetes is characterized by impaired insulin secretion associated with excess glucagon release. Exocytosis of insulin- or glucagon-containing granules is initiated by Ca2+-influx through voltage-dependent Ca2+-channels and is modulated by the second messengers such as cAMP. Membrane capacitance measurements were used in this study to investigate the cellular mechanisms behind cAMP-regulated Ca2+-dependent exocytosis in pancreatic beta-and alpha-cells. In pancreatic beta-cells, the secretory granules have been demonstrated to co-localize with L-type Ca2+-channels. Experiments using photorelease of caged Ca2+ revealed that exocytosis of insulin-containing granules requires an increase in [Ca2+]i with a Kd of 17 µM, which is most likely achieved in the vicinity of the Ca2+-channels. It was further proved that a close association between the alpha1c L-type Ca2+-channels and the secretory granules is a prerequisite for rapid exocytosis and ensures maximum usage of Ca2+ influx in a cell with few Ca2+-channels (~500) as is the case in the beta-cell. Pharmacologically, L-type Ca2+-channels are suppressed by organic Ca2+-channel blockers such as dihydropyridines. Glacontryphan- M was isolated from the venom of Conus Marmoreus and was detected to reduce L-type Ca2+ currents and associated exocytosis. It was discovered that this novel antagonist needs the binding of Ca2+ to the Gla-residues for its function. Ca2+-dependent exocytosis in the beta-cell is enhanced by cAMP through both PKA-independent granular priming and PKA-dependent recruitment of granules from a reserve pool. This study revealed a concentration-dependent activation of the two cAMP-signalling pathways and that 5-fold higher [cAMP]i was needed for the PKA-independent mechanism. Interestingly, the sulfonylurea receptor SUR1 was required in the regulation of cAMP-stimulated PKA-independent exocytosis. This is an additional function of SUR1 apart from being a subunit of the KATP channel. Enhancement of exocytosis by cAMP in alpha-cells was discovered to involve similar mechanisms as in the beta-cell. In the present study glucagon was detected to elevate cAMP resulting in an enhanced Ca2+-dependent exocytotic response by binding to the glucagon-receptors in the plasma membrane of the alpha-cell. The stimulatory effect of glucagon was mainly mediated by PKA-dependent mechanisms, possibly due to the inability of glucagon to elevate [cAMP]i to the sufficient level needed for activation of the PKA-independent pathway