PANCREATIC SECRETION--G PROTEIN MEDIATED CA++ SIGNALING

这一提议旨在详细阐明信号转导。 由胰腺促分泌剂激活的通路，因此直接 与外分泌的生理学和病理生理学相关 胰腺。胰腺促分泌剂，如缩胆囊素、神经递质 C和乙酰胆碱启动信号级联反应，导致 [Ca~(2+)]i的增加。这种[Ca~(2+)]i的增加主要是由于 细胞内储存部位的钙离子释放。中的高程 [Ca2+]i携带重要的时间和空间信息， 每一种激动剂，作为主要信号在控制 胰腺腺泡细胞的消化酶分泌。这项建议 将专注于了解配体如何利用相同的通用 信号转导通路实现信号特异性。这是假设的 这种信号的特异性是重要的分子 存在于转导系统的所有元素中的多样性，如 用一种特殊的促分泌剂刺激会导致激活 这些信号蛋白的特定子集。个人 负责从受体传递这一信号的蛋白质 质膜占位对细胞内钙离子释放的影响 将定义细胞内存储。个体G蛋白 Alpha/Betagamma亚基和磷脂酶Cβ同工酶表达 胰腺腺泡细胞和胰腺的功能意义 细胞系AR4-2J将通过免疫印迹和聚合酶链式反应进行鉴定。特定的 Alphaq家族亚基和Betagamma亚基之间的相互作用将 通过免疫共沉淀和组成亚基进行研究 与As/Betagamma杂三聚体相比。实验将是 用来阐明磷脂酶-Cβ激活的机制 在腺泡细胞中，假设G蛋白α和G蛋白 贝塔格玛亚基也参与其中。在单个大鼠胰腺腺泡中， 单个G蛋白α亚基、β亚基和PLC-β亚基 同工酶将被选择性地拮抗利用特定的 抗体和反义寡核苷酸。测量的方法 测定磷脂酰肌醇的水解度和[钙]i的波动 将利用荧光数字成像作为读数 促分泌剂与其各自的转导机制的偶联。 G-亚基与PLC-β效应物酶的关系 刺激将通过免疫共沉淀法进行研究。 这些技术将确定特定的刺激是否 促分泌剂导致信号的一个独特子集的激活 腺泡细胞中导致钙离子特殊模式的蛋白质 在单个促分泌剂刺激下观察到的信号。

This proposal is designed to elucidate in detail signal transduction pathways activated by pancreatic secretagogues and thus has direct relevance to both the physiology and pathophysiology of the exocrine pancreas. Pancreatic secretagogues such as cholecystokinin, neuromedin C and acetylcholine initiate a signaling cascade resulting in an increase in [Ca 2+]i. This increase in [Ca2+]i is primarily the result of Ca2+ release from an intracellular storage site. The elevation in [Ca2+]i carries important temporal and spatial information, unique to each agonist, which serves as a primary signal in the control of digestive enzyme secretion from pancreatic acinar cells. This proposal will focus on understanding how ligands utilizing the same general transduction pathway achieve signal specificity. It is hypothesized that signal specificity is a result of the significant molecular diversity which exists in all elements of the transduction system, such that stimulation with a particular secretagogue results in activation of particular subset of these signaling proteins. The individual proteins responsible for transducing this signal from receptor occupation on the plasma membrane to the release of Ca2+ from the intracellular store will be defined. The individual G-protein alpha/betagamma subunits and phospholipase C beta isozymes expressed and functionally significant in pancreatic acinar cells and the pancreatic cell-line AR4-2J will be elucidated by immunoblotting and PCR. Specific interactions between alphaq family subunits and betagamma subunits will be investigated by co-immunoprecipitation and the constituent subunits compared to the as/betagamma heterotrimer. Experiments will be performed to elucidate the mechanism of phospholipase-C beta activation in acinar cells; it is hypothesized that both G protein alpha and betagamma subunits are involved. In single rat pancreatic acini, individual G-protein alpha subunits, betagamma subunits and PLC-beta isozymes will be selectively antagonized by utilizing specific antibodies and antisense oligonucleotides. The measurement of phosphoinositide hydrolysis and fluctuations in [Ca2+]i measured by fluorescence digital imaging will be utilized as a readout of the coupling of secretagogues to their respective transduction mechanisms. The association of G-subunits with the PLC-beta effector enzyme on stimulation will be investigated by a co-immunoprecipitation protocol. These techniques will determine if stimulation by a specific secretagogue results in activation of a unique subset of the signaling proteins in acinar cells leading to the specific pattern of Ca2+ signaling observed on stimulation by individual secretagogues.