Recent studies from our laboratory identify previously unappreciated complexities in cardiac beta-adrenergic receptor subtype signaling, with profound developmental changes beta/2-receptor activation of effector mechanisms. At least two aspects of beta-receptor subtype signaling can not be explained in the context of traditional concepts of receptor- activated signaling pathways: [1] The observation that beta/2-receptors increase cAMP accumulation only in neonatal (not in adult) cardiomyocytes. [2] The observation that individual beta-receptor subtypes in neonatal cardiomyocytes display differential susceptibility to the inhibitory effects of muscarinic cholinergic agonists. In an attempt to understand the mechanisms that underlie these intriguing complexities in beta- receptor subtype signaling, studies in the renewal of this Project will apply newer concepts related to molecular heterogeneity of components of beta-receptor subtype signaling cascades (G protein subunits, adenylyl cyclase isoforms, and components of the PKA enzyme) as well as the concept that compartmentalization of second messenger molecules to caveolae (specialized membrane subdomains that serve as 'signaling processing center') serves to facilitate and/or restrict receptor signaling events. The first two Specific Aim will elucidate the molecular and/or cellular basis for differences in beta-adrenergic receptor subtype signaling in neonatal and adult ventricular myocytes. The third Specific Aim will build upon our exciting recent observation that beta/2-receptors influence contraction in adult ventricular myocytes via a cAMP-independent signaling pathway leading to HCO/3-dependent intracellular alkalinization to investigate pH/i-regulation by beta-receptor subtypes. The fourth Specific Aim will test the hypothesis that sympathetic innervation influence beta/2-receptor action. These studies will combine a broad range of biochemical/molecular techniques, fluorescence microscopy with ion- sensitive probes, and measurements of unloaded cell shortening; this permits an analysis of the functional role of intracellular signaling intermediates in beta-receptor subtype responsiveness. By considering novel signal transduction mechanisms that regulate cardiomyocyte autonomic responses in the context of normal growth, development, and/or sympathetic innervation of the ventricle, studies in this Project will contribute to the Program's long-range goal, which is to understand the structural, molecular, biochemical, and/or ionic determinants that lead to distinct catecholamine-dependent electrophysiologic and contractile responses in newborn and adult hearts.
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