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

这一建议的主要目的是扩展我们对Ca 2+的研究 在静息和刺激的胰腺腺泡细胞中的稳态。这 这是了解其机制的一个重要阶段， [Ca ~（2+）]i过程中细胞内Ca ~（2+）库的释放和重新加载 振荡，并在细胞刺激终止时。过去几 多年来，我们已经开发了技术：专门标记激动剂-- 完整细胞中含有（45）Ca的可动员细胞内池;以测量 （45）在完整和透化的细胞中由传导途径介导的Ca通量 细胞和透化细胞中Ca 2+泵介导的-（45）Ca通量; 用Fura 2测定细胞悬液中游离Ca ~（2+）浓度 单个胰腺腺泡。我们还发现了一种 胞浆Ca 2+结合蛋白，抑制IP 3介导的Ca 2+释放。 使用这些技术，该提案的直接目标是：1） 确定钙离子进入细胞膜的途径， 刺激和重新加载。这将揭示细胞质Ca ~（2+） 2+库在控制细胞内Ca 2+库再负荷中的作用; 胰腺腺泡和单个细胞[Ca 2 +]i振荡的特征 在腺泡内，试图确定钙池含量的作用 和[Ca ~（2+）]i在调节Ca ~（2+）振荡中的作用; 细胞内钙泵的激动剂和蛋白激酶，4）研究 IP 3结合和IP 3介导的Ca 2+释放的整体调节 胞浆抑制性钙结合蛋白; 5）纯化抑制性钙结合蛋白 蛋白质，并表征其与IP 3-可动员的Ca 2+的相互作用 池及其在各种细胞中的分布。相互作用的调节 这种具有IP 3激活的Ca 2+通道的蛋白质具有以下潜力 为IP 3介导的Ca 2+释放的调节提供时间方面， 从而导致Ca 2+振荡。 我们希望我们的基础研究能提供检验假设的技术 对细胞内游离钙通道的激素调节 这将使我们更接近于理解Ca 2+的稳态， 胰腺腺泡细胞受刺激后的Ca ~（2+）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.