PANCREATIC SECRETION--G PROTEIN MEDIATED CA++ SIGNALING

胰腺分泌--G蛋白介导的CA信号传导

• 批准号: 6381256
• 负责人: David I Yule
• 金额: $ 16.04万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381256
• 关键词: G protein; acetylcholine; antisense nucleic acid; biological signal transduction; calcium ion; cell line; cholecystokinin; enzyme activity; immunoprecipitation; isozymes; laboratory rat; pancreas hormone; phospholipase C; polymerase chain reaction; receptor coupling; secretory protein; western blottings

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.

这个建议旨在详细阐明信号转导 胰腺促分泌素激活的途径，因此具有直接作用 与外分泌的生理学和病理生理学相关 胰腺 胰腺促分泌素，如胆囊收缩素、神经介肽 C和乙酰胆碱启动信号级联反应， 增加[Ca 2+]i. [Ca 2 +]i的增加主要是由于 从细胞内储存位点释放的Ca 2+。 的提升 [Ca2+]i携带重要的时间和空间信息，是 每种激动剂，其作为控制 胰腺腺泡细胞分泌消化酶。 这项建议 将侧重于了解如何利用相同的一般配体 信号转导途径具有信号特异性。 据推测 这种信号特异性是由于 存在于转导系统的所有元件中的多样性，例如 特定促分泌素的刺激会激活 这些信号蛋白的特定子集。 个人 负责将这种信号从受体 细胞膜上Ca ~（2+）的释放 将定义细胞内储存。单个G蛋白 α/β γ亚基和磷脂酶C β同工酶表达， 在胰腺腺泡细胞和胰腺 细胞系AR 4 - 2 J将通过免疫印迹和PCR来阐明。具体 α q家族亚单位和β γ亚单位之间的相互作用将 通过免疫共沉淀和组成亚基来研究 与As/β γ异源三聚体相比。 实验将 阐明磷脂酶-C β激活的机制 在腺泡细胞中，假设G蛋白α和 β-γ亚单位参与其中。 在单个大鼠胰腺腺泡中， 单个G蛋白α亚基、β γ亚基和PLC-β 同工酶将通过利用特异性的 抗体和反义寡核苷酸。 的测量 磷酸肌醇水解和[Ca 2 +]i波动， 荧光数字成像将被用作 促分泌素与它们各自的转导机制的偶联。 G-亚基与PLC-β效应酶的相关性 通过免疫共沉淀方案研究刺激。 这些技术将确定是否通过特定的刺激 促分泌素导致信号传导的独特子集的激活 腺泡细胞中的蛋白质导致特定的Ca 2+模式 通过个体促分泌素刺激观察到的信号传导。