Control of granular fusion by small molecular GTP-binding protein

1)表面负电荷激活Ca^<2+>-动员受体膜表面的负电荷可以修饰各种类型的功能蛋白，包括膜受体或g蛋白/效应复合物。电荷被溶液中的正离子中和或屏蔽，所以我引入了二价或三价阳离子来中和表面电荷，并研究了它们的作用。过量的二价（Ca、Mg、Ba、Sr、Mn）、三价（La）或多价有机阳离子（鱼精蛋白）均诱导肝素敏感性升高[Ca^<2+>]i，表明电荷中和产生肌醇三磷酸从而增加[Ca^<2+>]i，可能是通过激活表面受体和/或受体复合物。2)通过使用依赖Ca^<2+>的离子电流监测[Ca^<2+>]i的升高，研究了将肌醇多磷酸（InsP3或InsP4）引入单个胰腺腺泡细胞的作用。在磷脂酶抑制剂4-BPB的存在下，Ca^<2+>反应明显增强。相反，这种反应被外源性花生四烯酸酯的存在所消除。结果表明，增加[Ca^<2+>]i后，花生四烯酸酯的形成可以抑制[Ca^<2+>]i的进一步升高。3) [Ca^<2+>]i的局部性升高直接触发胞外颗粒融合胰腺腺泡细胞在ACh或InsP3刺激后显示[Ca^<2+>]i的局部性升高。在乙酰胆碱刺激下研究了膜电容的增加，它们与[Ca^<2+>]i的局部升高有时间相关性。因此，我们认为[Ca^<2+>]i的升高足以触发极化外分泌细胞分泌胞外酶。

1)Negative surface charges activating Ca^<2+>-mobilizing receptorsThe negative charges on the membrane surface may modify various types of functional protein including membrane receptors or G-protein/effector complex. The charges are neutralize or screened by positively charged ions in the solution, so I have introduced divalent or trivalent cations to neutralize the surface charges and studied effect of them. Excess divalent (Ca, Mg, Ba, Sr, Mn), trivalent(La), or polyvalent organic cation(protamine) all induced heparin-sensitive rise in [Ca^<2+>]i, suggesting that the charge neutralization produces inositol trisphosphate thereby increasing [Ca^<2+>]i, presumably through activation of surface receptors and/or receptor complex.2)Control of inositol polyphosphate receptors by arachidonateEffect of inositol polyphosphates (InsP3 or InsP4) introduced into single pancreatic acinar cells were studied by monitoring a rise in [Ca^<2+>]i with Ca^<2+>-dependent ionic currents. In the presence of phospholipase inhibitor, 4-BPB, the Ca^<2+> response was markedly enhanced. In contrast, the response was abolished by the presence of exogenous arachidonate. The results suggest that formation of arachidonate after increases in [Ca^<2+>]i can inhibit further rises in [Ca^<2+>]i.3)Localized rise in [Ca^<2+>]i directly triggering exocytotic granular fusionPancreatic acinar cells shows a localized luminal increase in [Ca^<2+>]i after stimulation of either ACh or InsP3. Increases in membrane capacitance were studied in ACh stimulation and they were temporally correlated with the localized rise in [Ca^<2+>]i. Therefore we conclude that such rise in [Ca^<2+>]i is enough to trigger the exocytotic enzyme secretion in polarized exocrine cells.