The overall hypothesis for this project is that cardiac myocytes constitutively release and metabolize adenine nucleotides in the extracellular cardiac interstitial space under basal conditions, and that this nucleotide release and metabolism is significantly increased in response to ischemic and hypoxic stress. It is proposed that released adenine nucleotides play two important roles in cardiac regulation: 1) as autocrine/paracrine agonists of myocyte P2 nucleotide receptors; and 2) as substrates for the localized generation of adenosine used in autocrine/paracrine activation of A1 and A3 receptors. By inducing signaling pathways that counteract the effects of hypoxia and ischemia, activation of these myocyte receptors will provide a rapid feedback response to metabolic stress. A corollary of this hypothesis is that cardiac myocytes per se are major sites of th ecto-ATPase and other ectonucleotidase activities that both scavenge released ATP and generate adenosine at the myocyte cell surface. The proposed studies are physiologically significant because they will provide new insights regarding now highly localized changes in nucleotide and nucleoside levels at the myocyte cell surface contribute to the autocrine regulation of signaling pathways that modulate basal cardiac contractility and bioenergetic adaptation to hypoxic and ischemic stress. The studies are novel because the analysis of nucleotide and nucleoside levels at the myocyte cell surface contribute to the autocrine regulation of signaling pathways that modulate basal cardiac contractility and bioenergetic adaptation to hypoxic and ischemic stress. The studies are novel because the analysis of nucleotide release and extracellular metabolism will utilize newly developed methods that provide a quantitative sensitivity, temporal resolution, and spatial localization not possible in previous studies with cardiac myocytes or other cells. The studies are timely because recent research has identified many new genes encoding distinct ecto-nucleotidases, but the expression and function of these genes in cardiac myocytes has not been explored. Finally, the proposed project is highly relevant to the overall theme of the Program Project because it seeks to define how adenine nucleotide flux, a central aspect of intracellular cardiac bioenergetics, may be coupled to extracellular signaling cascades that regulate adaptation to bioenergetic stress.
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