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器官，具有 特化的 I 型前神经细胞，含有一系列兴奋性细胞 和抑制性神经递质剂。目前的观点认为 化学刺激启动初级转导事件的复杂级联 在 I 型细胞中，最终导致递质释放和兴奋 附近的化学感应神经末梢。 I 型细胞活性也 由化学感应释放的神经活性物质调节 神经纤维。我们之前表明，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 研究信号转导以考虑蛋白激酶的作用， 磷酸酶和重要的靶磷蛋白。 一个 多学科方法将研究这些细胞机制，如 制定了以下三个具体目标： 1) 细胞 介导颈动脉体中蛋白质磷酸化的机制 将使用免疫细胞化学技术进行识别和定位 研究蛋白激酶、磷蛋白磷酸酶和重要的 目标磷蛋白。 此外，凝胶电泳技术 (SDS-PAGE) 结合 32 P-gamma-ATP 标记将被使用 表征不同蛋白质磷酸化的变化 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 颈动脉体合成和释放神经递质 检查特定激酶和磷酸酶抑制剂的影响 儿茶酚胺和一氧化氮 (N0) 代谢。 净生理学 改变发射机释放的影响将被监测 颈动脉窦神经的化学感受器活性。 3）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动将通过补丁进行检查 钳和荧光染料细胞成像技术。这些研究将 重点关注钾通道和钙通道的调节，以及 化学感受器抑制介导的细胞机制 均由内源性 N0 和心房钠尿肽 (ANP) 产生。