SECOND MESSENGERS AND PROTEIN PHOSPHORYLATION IN CAROTID CHEMOTRANSDUCTION

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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).