ION CHANNELS IN PANCREATIC ISLET CELLS

胰岛细胞中的离子通道

• 批准号: 3236259
• 负责人: STANLEY MISLER
• 金额: $ 13.33万
• 依托单位: JEWISH HOSPITAL OF SAINT LOUIS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3236259
• 关键词: B lymphocyte; acidity /alkalinity; action potentials; adenosine triphosphate; calcium channel; endoplasmic reticulum; exocytosis; glucagon; glucose transport; hormone regulation /control mechanism; insulin; laboratory rat; membrane channels; membrane model; membrane permeability; membrane potentials; noninsulin dependent diabetes mellitus; pancreatic islet neoplasm; pancreatic islets; potassium channel; secretion; voltage /patch clamp

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.

一个可检验的餐后诱导“代谢假说” 是B细胞代谢营养燃料， 特别是葡萄糖，-（1）->细胞内 中间体-（2）->代谢调节K+的关闭 通道_（3）->细胞去极化-（4）->动作电位 启动和开放电压依赖性Ca 2+通道- (5)-> Ca 2+内流-（6）->胰岛素颗粒胞吐。 来 在过去五年中， 在这个假设中的“分子参与者”包括ATP 1 敏感的K+通道，其关闭使细胞去极化;电压 依赖的Ca 2+和K+通道，这可能有助于 动作电位;和增加胞浆Ca 2+下 B细胞去增殖的条件。 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)其通过以下方式将细胞去极化与颗粒胞吐作用联系起来： 同时应用几种新的“一体化”技术， 从尸体中收获的单个正常成人胰岛B细胞 捐助者。 这些技术是“穿孔贴片”电流或 电压钳记录;显微荧光分光光度法，使用Ca 2 +- 敏感染料和膜电容测量的“相 检测”技术。 首先我们将检查离子通道 代谢物诱导的电流模式的基础 活性，尤其是Ca 2+通道。 第二，我们将 细胞质Ca 2+的变化与电活动模式 由营养燃料促分泌素引起。 第三，我们将尝试 将电活动和Ca 2+进入的模式与 胰岛素颗粒胞吐作用的模式，如通过 膜电容。 最后一种方法要求我们 建立胰岛素分泌的最佳条件， “反向溶血斑块测定”并验证电容 观察到的变化反映了胰岛素颗粒的实时掺入 进入细胞表面。 实验应进一步 我们对正常人中刺激-分泌偶联的理解 人B细胞，并作为研究 非胰岛素依赖型糖尿病患者胰岛素分泌减少的病理生理学 依赖性糖尿病