Adenylyl cyclase isoform expression in hair cells and their neural contacts, both efferent axons and afferent dendrites, will determine the pharmacology of cAMP-mediated signal transduction at the specified cellular sites, hypothesized to modulate receptoneural transmission and adaptation of mechanosensory transduction. (1) A goal is to ascertain this expression, utilizing RT-PCR applied to the organ of Corti and spiral ganglion tissue fractions and PCR with nested primers applied to lambdaZAP cDNA libraries of inner hair cells and outer hair cells and in a teleost hair cell preparation. (2) G-protein coupling to adenylyl cyclase enzymatic activity will be determined at an ultrastructural level with pertussis/cholera toxins, and if evidence of Galphas is absent, the effect of calcium ionophores will be examined as a putative mechanism for activating adenylyl cyclase isoforms, specifically, in hair cells. Inhibition of adenylyl cyclase enyzmatic activity by glutamate metabotropic agonists will be analyzed. (3) Protein targets of adenylyl cyclase action in hair cells will be identified including those proteins that are substrates for protein kinase A phosphorylation and cyclic nucleotide gated ion channels. (4) Dopaminergic efferent regulation via D2L receptor coupling of Galphai2 to adenylyl cyclase will be ascertained with agonist-elicited modulation of intracellular cAMP in the intact rat organ of Corti compared to the intact sensory epithelia and separately, hair cells, of the teleost saccular macula. Proteins specifically targeted by D2L receptor occupation for PKA-driven phosphorylation (or inhibition of PKA-driven phosphorylation) will be identified. A consequential presynaptic modulation of dopamine synthesis and content in the lateral efferents will be determined with radioactive precursors and electrochemical detection of dopamine chromatographically resolved by HPLC. The elucidation of adenylyl cyclase-mediated signal transduction in inner-ear sensory epithelia through dopaminergic innervation may allow eventual pharmacological amelioration of sensorineural deafness and tinnitus.
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