Regulation of Exocytosis

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We have obtained the following results from this research project on the regulation of exocytosis.1) We have previously demonstrated that myosin lightchain kinase is essential for ATP-dependent priming of exocytosis in adrenal chromaffin cells. By use of mychalolide B,a toxin that selectively inhibit actin-myosin interaction, we demonstrated that actin-myosin interaction is important in ATP-dependent priming mechanism. It was also suggested that ATP-dependent priming is regulated by a trimeric G-protein, Go, and GAP43.2) In order to understand the dynamics aspects of exocytosis, we analyzed the releasing process by combined use of the carbon fiber DC-amperometry and the video-enhanced contrast DIC microscopy. When a cell was stimulated by acetylcholine or by electrode attachment, current spikes as large as 350 pA appeared and rapid flickers of granule image also appeared simultaneously. About 80% of current spikes were correlated with video microscopic responses, and 20% of spikes were assigned to invisible responses presumably taking place off focus. We conclude that (i) in bovine chromaffin cells, current transients recorded with DC amperometry can be securely considered to represent single vesicular fusion events ; and (ii) that the amplitute distribution is determined primarily by the diffusional profile of the catecholamine.3) Uridine 5'-triphosphate (UTP)-evoked increase in intracellular Ca^<2+> concentration ([Ca]i) and release of dopamine were investigated using rat pheochromocytoma PC12 cells. The data demonstrated that UTP stimulates P2U-purinoceptors and induces a rise in [Ca]i both by Ca^<2+>-mobilization and Ca^<2+>-influx in PC12 cells. The UTP-evokes dopamine release requires external Ca^<2+> which may enter the cells through pathway sensitive to Zn^<2+>, but insensitive to Cd^<2+> or nicardipine. These results suggested that ATP modulates transmitter release through different types of purinoceptors.