SECOND MESSENGERS AND PROTEIN PHOSPHORYLATION IN CAROTID CHEMOTRANSDUCTION

颈动脉化学转导中的第二信使和蛋白质磷酸化

• 批准号: 6273606
• 负责人: SALVATORE J FIDONE
• 金额: $ 13.1万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6273606
• 关键词: SDS polyacrylamide gel electrophoresis; atrial natriuretic peptide; biological signal transduction; calcium channel; carotid body; catecholamines; chemoreceptors; enzyme inhibitors; fluorescence microscopy; immunocytochemistry; laboratory rabbit; laboratory rat; membrane potentials; neurotransmitter biosynthesis; nitric oxide; phosphoprotein phosphatase; phosphoproteins; phosphorylation; potassium channel; protein kinase; second messengers; voltage /patch clamp

The carotid body, an arterial chemorecptor organ, possesses specialized preneural type I cells which contain an array of excitatory and inhibitory neurotransmitter agents. Current views suggest that chemostimuli initiate a complex cascade of primary transductive events in type I cells, which culminates in transmitter release and excitation of nearby chemosensory nerve endings. Type I cell activity is also modulated by neuroactive substances released from chemosensory nerve fibers. We showed previously that excitation and inhibition of type I cells is mediated by classical second messengers, including Ca 2+ and cyclic nuicleotides. The current application extends our studies of signal transduction to consider the role of protein kinases, phosphatases, and important target phosphoproteins. A multidisciplinary approach will investigate these cellular mechanisms, as formulated in the following three specfic aims: 1) The cellular machinery which mediates protein phosphorylation in the carotid body will be identified and localized using immunocytochemical techniques to study protein kinases, phosphoprotein phosphatases and important target phosphoproteins. In addition, gel electrophoretic techniques (SDS-PAGE) combined with 32 P-gamma-ATP labeling will be used charaterize changes in phosphorylation of diverse proteins in response to of chemoreceptor and second messenger stimuli, respectively. 2) Studies of the role of protein phosphorylation in synthesis and release of neurotransmitters from the carotid body will examine the effects of specific kinases and phosphatase inhibitors on catecholamine and nitric oxiden (N0) metabolism. The net physiological effects of altered transmitter release will be monitored as the chemoreceptor activity of the carotid sinus nerve. 3) The role of protein phosphorylation in modulation of type I cell membrane currents and intracellular ionic activities will be examined with patch- clamp and fluorescent dye cell-imaging techniques. These studies will focus on the regulation of potassium and calcium channels, as well as the cellular mechanisms underlying chemoreceptor inhibition mediated both by endogenous N0, and atrial natriuretic peptide (ANP).

颈动脉体是动脉化学感受器， 特化的前神经I型细胞，其包含一系列兴奋性的 和抑制性神经递质剂。目前的观点认为， 化学刺激物启动初级转导事件的复杂级联 在I型细胞中，它在递质释放和兴奋中达到高潮 化学感受神经末梢的位置 I型细胞活性也是 由化学感受器释放的神经活性物质调节 神经纤维我们以前表明，兴奋和抑制 I型细胞由经典的第二信使介导，包括Ca 2+和环状核苷酸。 当前的应用程序扩展了我们的 信号转导的研究考虑蛋白激酶的作用， 磷酸酶和重要的靶磷蛋白。 一 多学科方法将研究这些细胞机制， 制定了以下三个具体目标：1）细胞 介导颈动脉体中蛋白磷酸化的机制 将使用免疫细胞化学技术进行鉴定和定位， 研究蛋白激酶，磷蛋白磷酸酶和重要的 靶向磷蛋白。 此外，凝胶电泳技术 将使用结合32 P-γ-ATP标记的SDS-PAGE 不同蛋白质磷酸化的特征变化， 对化学感受器和第二信使刺激的反应， 分别 2)蛋白质磷酸化作用的研究 神经递质从颈动脉体的合成和释放将 检查特定激酶和磷酸酶抑制剂对 儿茶酚胺和一氧化氮（N 0）代谢。 网络生理 将监测发射器释放改变的影响，因为 化学感受器活动的颈动脉窦神经。 3)的作用 I型细胞膜调节中的蛋白质磷酸化 电流和细胞内离子活性将用贴片- 钳位和荧光染料细胞成像技术。这些研究将 专注于钾和钙通道的调节，以及 化学感受器抑制介导的细胞机制 内源性NO和心房钠尿肽（ANP）。