application): The long-term goal of this project is to study the mechanisms by which cGMP-elevating agents (cGMP agonists) inhibit DNA synthesis in primary cultures of aortic smooth muscle cells isolated from newborn rats. Preliminary experiments indicate that cGMP agonists inhibit both basal and growth factor simulated DNA synthesis. Moreover, these agonists cause reduction in phosphotyrosine phosphorylation in selected proteins, suggesting activation of one or more protein tyrosine phosphatases. Peroxovanadate, a potent and selective inhibitor of PTPases, reverses the antimitogenic effect as well as the protein phosphotyrosine dephosphorylation induced by cGMP agonists, thus linking the biochemical event of phosphotyrosine dephosphorylation to the physiological endpoint of decreased DNA synthesis. Additional recent work performed since the last submission indicates that cGMP agonists, and other agents which decrease calcium, induce phosphotyrosine dephosphorylation and increase PTPase activity in cell homogenates or cytosolic fractions. One such agent, nifedipine, also inhibits DNA synthesis, providing further evidence linking the biochemical and physiological endpoints. Chelerythrine, a selective inhibitor of protein kinase C, also induces protein phosphotyrosine dephosphorylation, suggesting the involvement of PKC in these events. Collectively, these results suggest the antimitogenic effect of cGMP agonists and calcium entry blockers may be mediated by a decreased cytoplasmic calcium followed by decreased PKC activity and increased activation of one or more PTPases. The purpose of the current proposal is to investigate the mechanism underlying these effects. To this end, the PI proposes to test the following hypothesis: Can cyclic GMP agonists and other agents that decrease cytoplasmic calcium levels activate a specific PTPase that is inhibited by calcium via PKC? Activation of this PTPase then causes the phosphotyrosine dephosphorylation of specific proteins. To test this hypothesis the aims are: 1) to determine whether inhibition of PKC mimics and downregulation or activation of PKC blocks or attenuates the effect of cGMP agonists and other agents that decrease calcium on protein phosphotyrosine dephosphorylation; 2) to identify the protein substrates that are phosphotyrosine dephosphorylated in response to cGMP agonists and other agents that reduce calcium; and 3) to identify a PTPase that is activated by cGMP agonists and other agents that decrease calcium.
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