Multiple sites of purinergic control of insulin secretion in mouse pancreatic beta-cells.

In mouse pancreatic beta-cells, extracellular ATP (0.1 mmol/l) effectively reduced glucose-induced insulin secretion. This inhibitory action resulted from a direct interference with the secretory machinery, and ATP suppressed depolarization-induced exocytosis by 60% as revealed by high-resolution capacitance measurements. Suppression of Ca2+-dependent exocytosis was mediated via binding to P2Y1 purinoceptors but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. Inhibition of exocytosis by ATP resulted from G-protein-dependent activation of the serine/threonine protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. In contrast to the direct inhibitory action on exocytosis, ATP reduced the whole-cell ATP-sensitive K+ (K(ATP)) current by 30% (via activation of cytosolic phospholipase A2), leading to membrane depolarization and stimulation of electrical activity. The stimulatory effect of ATP also involved mobilization of Ca2+ from thapsigargin-sensitive intracellular stores. We propose that the inhibitory action of ATP, by interacting with the secretory machinery at a level downstream to an elevation in [Ca2+]i, is important for autocrine regulation of insulin secretion in mouse beta-cells

Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells

Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells.When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 μm and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis.The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mm) and quinine (10 μm). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 μm), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels.Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim.Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide.We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization