SECOND MESSENGERS AND PROTEIN PHOSPHORYLATION IN CAROTID CHEMOTRANSDUCTION

颈动脉小体是一个动脉化学感受器，具有 特化的I型创业细胞，含有一系列兴奋性细胞 和抑制性神经递质制剂。目前的观点表明， 化学刺激启动一系列复杂的初级转导事件 在I型细胞中，最终导致递质释放和兴奋 附近的化学感觉神经末梢。I型细胞活性也是 受化学感觉释放的神经活性物质的调节 神经纤维。我们之前已经证明，刺激和抑制 I型细胞由经典的第二信使介导，包括 2+和环状核苷酸。当前应用程序扩展了我们的 考虑蛋白激酶作用的信号转导研究， 磷酸酶，和重要的靶向磷蛋白。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 颈动脉小体中调节蛋白质磷酸化的机制 将使用免疫细胞化学技术进行鉴定和定位 研究蛋白激酶、磷酸蛋白磷酸酶及其重要作用 靶向磷蛋白。此外，凝胶电泳技术 (十二烷基硫酸钠-PAGE)结合32P-伽马-三磷酸腺苷标记 不同蛋白质磷酸化的变化特征 对化学感受器和第二信使刺激的反应， 分别进行了分析。2)蛋白质磷酸化作用的研究 颈动脉小体神经递质的合成和释放 检测特定的激酶和磷酸酶抑制剂对 儿茶酚胺和一氧化氮(N0)代谢。网络生理学 改变发射器释放的影响将被监测为 颈动脉窦神经的化学感受器活性。3)的角色 蛋白磷酸化在I型细胞膜调控中的作用 电流和细胞内离子活度将用贴片- 钳位和荧光染料细胞成像技术。这些研究将 关注钾和钙通道的调节，以及 化学感受器抑制介导的细胞机制 均由内源性NO和心钠素(ANP)引起。

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).