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Insulin secretion is controlled by the β cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulincontaining secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P[sub 2]] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P[sub 2]-binding protein Ca[sup 2+]-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca[sup 2+]- and PI(4,5)P[sub 2]-induced exocytosis. We conclude that ADPinduced changes in PI 4-kinase activity, via generation of PI(4,5)P[sub 2], represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic β-cells by stimulation of Ca2+-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A2 (cPLA2) inhibitors ACA and AACOCF3 and in cells treated with antisense oligonucleotide against cPLA2α. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF3, and cPLA2α antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA2 activity. We propose that cPLA2α plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting β-cells. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]