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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Experimental Cardiovascular Sciences Study Section (ECS)
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University of Tennessee Health Science Center
Schools of Medicine
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Brown, C; Lin, Y; Hassid, A (2001) Requirement of protein tyrosine phosphatase SHP2 for NO-stimulated vascular smooth muscle cell motility. Am J Physiol Heart Circ Physiol 281:H1598-605
Brown, C; Pan, X; Hassid, A (1999) Nitric oxide and C-type atrial natriuretic peptide stimulate primary aortic smooth muscle cell migration via a cGMP-dependent mechanism: relationship to microfilament dissociation and altered cell morphology. Circ Res 84:655-67
Hassid, A; Yao, J; Huang, S (1999) NO alters cell shape and motility in aortic smooth muscle cells via protein tyrosine phosphatase 1B activation. Am J Physiol 277:H1014-26
Hassid, A; Huang, S; Yao, J (1999) Role of PTP-1B in aortic smooth muscle cell motility and tyrosine phosphorylation of focal adhesion proteins. Am J Physiol 277:H192-8
Kaur, K; Yao, J; Pan, X et al. (1998) NO decreases phosphorylation of focal adhesion proteins via reduction of Ca in rat aortic smooth muscle cells. Am J Physiol 274:H1613-9
Dhaunsi, G S; Matthews, C; Kaur, K et al. (1997) NO increases protein tyrosine phosphatase activity in smooth muscle cells: relationship to antimitogenesis. Am J Physiol 272:H1342-9
Dhaunsi, G S; Hassid, A (1996) Atrial and C-type natriuretic peptides amplify growth factor activity in primary aortic smooth muscle cells. Cardiovasc Res 31:37-47
Cahill, P A; Hassid, A (1994) ANF-C-receptor-mediated inhibition of aortic smooth muscle cell proliferation and thymidine kinase activity. Am J Physiol 266:R194-203
Hassid, A; Arabshahi, H; Bourcier, T et al. (1994) Nitric oxide selectively amplifies FGF-2-induced mitogenesis in primary rat aortic smooth muscle cells. Am J Physiol 267:H1040-8
Cahill, P A; Hassid, A (1993) Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: relationship to expression of ANF-C receptors. J Cell Physiol 154:28-38

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