HORMONE REGULATION OF (CA+2)I IN PANCREATIC ACINAR CELLS

这项建议的主要目的是扩大我们对钙离子的研究 静息和刺激的胰腺腺泡细胞的动态平衡。这 构成了理解以下机制的基本阶段 [Ca~(2+)]i过程中细胞内钙离子储存的释放和重新加载 振荡，并在细胞刺激终止时。在过去的几年里 多年来，我们已经开发出了一些技术：专门标记激动剂-- 在完整细胞中含有(45)Ca的可流动细胞内池；测量 (45)未通透性和通透性条件下传导通路介导的钙通量 细胞和钙泵介导的通透性细胞内的~(45)Ca通量； 用Fura-2测定细胞悬液中游离钙浓度 单发胰腺腺泡。我们还发现了一种 胞内钙结合蛋白，抑制IP3介导的钙释放。 使用这些技术，提案的直接目标是1) 确定钙离子跨质膜进入的途径 刺激和重新装填。这应该可以揭示胞浆内钙离子的作用。 2控制细胞内钙库重新加载的池，2) 胰腺腺泡细胞和单细胞的[Ca~(2+)]_i振荡特性 在腺泡内，试图确定池中钙含量的作用 和[Ca~(2+)]i调节Ca~(2+)振荡，3)定义 激动剂和蛋白激酶的细胞内钙泵，4)研究 对IP3结合和IP3介导的钙释放的全面调节 胞内抑制性钙结合蛋白，5)纯化抑制性 蛋白质及其与IP3可动员钙离子相互作用的研究 水池及其在不同细胞中的分布。相互作用的调节 这种蛋白与IP3激活的钙通道具有潜在的 为调节IP3介导的钙释放提供一个时间方面的方面 从而导致钙振荡。 我们希望我们的基础研究提供了检验假说的技术 细胞内游离钙调控的激素调节途径 并将使我们更接近理解钙动态平衡和 受刺激的胰腺腺泡细胞产生的钙振荡。

The principal objective of this proposal is to extend our studies of Ca2+ homeostasis in resting and stimulated pancreatic acinar cells. This constitutes an essential stage towards understanding the mechanisms of release and reloading of intracellular Ca2+ stores during [Ca2+]i oscillation, and upon termination of cell stimulation. Over the last few years we have developed techniques: to specifically label the agonist-- mobilizable intracellular pool with (45)Ca in intact cells; to measure (45)Ca fluxes mediated by conductive pathways in intact and permeabilized cells and Ca2+ pump-mediated-(45)Ca fluxes in permeabilized cells; to measure free cytosolic Ca 2+ concentration with Fura 2 in cell suspension and in single pancreatic acinus. We have also identified the presence of a cytosolic Ca2+ binding protein which inhibits IP3-mediated Ca 2+ release. Using these techniques the immediate aims of the proposal are to 1) determine the route of Ca 2+ entry across the plasma membrane during stimulation and reloading. This should reveal the role of the cytosolic Ca 2+ pool in controlling reloading of intracellular Ca 2+ stores, 2) characterize [Ca2+]i oscillation of pancreatic acinus and single cells within the acinus in an attempt to determine the role of pool Ca 2+ content and [Ca2+]i in regulating Ca2+ oscillation, 3) define the activation of the intracellular Ca2+ pump by agonists and protein kinases, 4) study the overall regulation of IP3 binding and IP3-mediated Ca 2+ release by the cytosolic inhibitory Ca 2+ binding protein, 5) purify the inhibitory protein and characterize its interaction with the IP3-mobilizable Ca 2+ pool and its distribution in various cells. Regulation of the interaction of this protein with the IP3-activated Ca 2+ channel has the potential of providing a temporal aspect to regulation of IP3-mediated Ca 2+ release and thus resulting in Ca2+ oscillation. We hope that our basic studies are providing techniques to test hypotheses on hormonal regulation of cellular Ca2+ pathways controlling free cytosolic Ca 2+ and will bring us closer toward understanding Ca 2+ homeostasis and Ca 2+ oscillation by the stimulated pancreatic acinar cell.