The overall aim of the proposed research is to determine the mechanisms of adenosine A1 receptor modulation of signal transduction in normal and ischemic ventricular myocardium. Although the adenosine A1 receptor is a typical G protein coupled receptor (GPCR), it appears to differ from other GPCR in that it exerts no direct effects in ventricular myocardium. There is significant evidence that GPCR, heterotrimeric GTP binding (G) proteins, and associated second messengers, such as adenylyl cyclase and protein kinase C (PKC) isoforms, are enriched in discrete intracellular compartments, including cholesterol-enriched, flask-shaped invaginations of the plasma membrane, referred to as caveolae. Based on the applicant's preliminary data, that ventricular myocyte adenosine A1 receptors are enriched in caveolae, it is hypothesized that A1 receptor-mediated signaling in normal and ischemic myocardium is regulated by subcellular compartmentation.
Specific Aim 1 will determine the effects of receptor agonists and endogenous adenosine on the subcellular localization of adenosine A1 receptors and associated G proteins in normal and ischemic myocytes.
Specific Aim 2 will test the hypothesis that the adenosine A1 receptor anti-adrenergic effect is co-localized with the components of the B-adrenergic receptor second messenger system.
Specific Aim 3 will determine whether adenosme A1 receptor activation modulates the activation of localized PKC isoforms or serine-threonine protein phosphatases in normal and ischemic myocytes. Studies will be conducted in adult rat isolated ventricular myocytes under normoxic and simulated ischemia conditions. The role of subcellular compartmentalization of adenosine A1 receptor signaling will be determined by subcellular fractionation of cell lysates. Subcellular compartmentation of adenosine A1 and B-adrenergic receptors, G protein subunits, PKC isoforms, and protein phosphatases, assessed with immunoblotting, immunoprecipitation, and immunofluorescence will be correlated with adenosine A1 receptor modulation of excitation-contraction coupling studied by measuring myocyte twitch amplitude, intracellular calcium, total and particulate cAMP levels, and cytosolic and particulate protein phosphatase activities. Adenosine A1 receptor modulation of PKC isoform translocation and protein phosphatases will be correlated with protection against cell death during simulated ischemia-reperfusion. There is substantial evidence that adenosine has numerous beneficial effects in ischemic-reperfused ventricular myocardium, but there is little information available regarding mechanism of action. The proposed studies will provide a better understanding of GPCR regulation of signal transduction in normal and ischemic cardiac myocytes which may aid in the development of new treatment regimens for the ischemic heart.
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