ION CHANNELS IN PANCREATIC ISLET CELLS

一种可检验的餐后诱导“代谢假说” 胰岛素分泌的关键是B细胞营养燃料的新陈代谢， 尤其是葡萄糖，-(1)-&GT；细胞内的变化 中间体--(2)--代谢调节K+的关闭 通道_(3)-细胞去极化-(4)-&gt；动作电位 电压依赖性钙通道的启动和开放- (5)-&GT；钙离子进入-(6)-&GT；胰岛素颗粒胞吐。超过了 在过去的五年里，在识别 这个假说中的“分子玩家”包括ATP1 敏感的K+通道，其闭合使细胞去极化；电压 依赖的钙离子和钾离子通道，这可能参与了 动作电位，细胞内钙离子浓度升高。 B细胞去极化的条件。我们建议进一步 通过调查几个动态链接来验证这一假设(4- 6)将细胞去极化与颗粒胞吐联系起来 同时应用几种新的“综合”技术 成人身体胰岛B细胞的分离纯化 捐赠者。这些技术是当前的“穿孔补丁”或 电压钳记录.使用钙离子的微量荧光分光光度测定法 敏感染料；以及用“相”测量膜电容 检测技术。首先，我们将检查离子通道 代谢物诱发电流模式的基础电流 活性，尤其是钙通道。第二，我们将相互关联 细胞内钙离子与电活动模式的变化 由营养燃料分泌物引起。第三，我们将尝试 将电活动和钙离子内流的模式与 胰岛素颗粒胞吐的变化反映了胰岛素颗粒的类型 膜电容。最后一种方法将需要我们 建立胰岛素分泌的最佳条件 “反向溶血空斑试验”和验证电容 观察到的变化反映了胰岛素颗粒的实时掺入 膜进入细胞表面。这些实验应该进一步 我们对正常人刺激-分泌耦合的理解 人B细胞，并作为研究人类B细胞的基础 非胰岛素依赖者胰岛素分泌减少的病理生理学研究 依赖型糖尿病。

A testable "metabolic hypothesis" for the postprandial induction of insulin secretion is that B cell metabolism of nutrient fuels, especially glucose, -(1)-> alteration of intracellular intermediates -(2)-> closure of metabolically regulated K+ channels _(3)-> cell depolarization -(4)-> action potential initiation and the opening of voltage dependent Ca2+ channels - (5)-> Ca2+ entry -(6)-> insulin granule exocytosis. Over the past five years important advances have been made in identifying "molecular players" in this hypothesis including an ATP1 sensitive K+ channel, whose closure depolarizes the cell; voltage dependent Ca2+, and K+ channels, which probably contribute to the action potential; and increases in cytosolic Ca2+ under conditions where B cells depolarize. We propose to further verify this hypothesis by investigating several dynamic links (4- 6) which connect cell depolarization to granule exocytosis by simultaneously applying several new "integrative" techniques to single normal adult human islet B cells harvested from cadaver donors. These techniques are "perforated patch" current or voltage clamp recording; microspectrofluometry, using Ca2+- sensitive dyes; and membrane capacitance measurements by "phase detection" techniques. First we shall examine the ionic channel currents underlying patterns of metabolite induced electrical activity, especially Ca2+ channels. Second, we shall correlate changes in cytosolic Ca2+ with patterns of electrical activity induced by nutrient fuel secretogogues. Third, we shall attempt to correlate patterns of electrical activity and Ca2+ entry with patterns of insulin granule exocytosis as reflected by changes in membrane capacitance. The last approach will require us to establish optimal conditions for insulin secretion using the "reverse hemolytic plaque assay" and to verify that capacitance changes seen reflect real-time incorporation of insulin granule membrane into the cell surface. The experiments should further our understanding of stimulus-secretion coupling in the normal human B cell and serve as a basis for studies on the pathophysiology of diminished insulin secretion in non-insulin dependent diabetes mellitus.