SIGNAL TRANSDUCTION IN VASCULAR SMOOTH MUSCLE

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Adenosine receptors (AR) play a major role in maintaining and regulating cardiovascular function. The physiological effects of adenosine range from control of chronotropy and ionotropy to vasoregulation and the modulation of growth and migration of smooth muscle cells. Congestive heart failure is associated with decreased vasoregulatory response to adenosine and small vessel vasospasm. In this project, the investigators will characterize AR mediated transmembrane signalling in vascular smooth muscle cells (VSMC) including a delineation of which AR are present, which effector systems are utilized in signalling, how AR regulate growth and differentiation, how elevated levels of adenosine regulate these receptor systems and how these functions can be modified through creation of mutant ARs with particular emphasis on AZARs. Evidence will also be sought to determine if the newly found A2b and A3AR are present in VSMC since physiological data are consistent with their presence. Studies will be carried out in primary cultures of VSMCs, a transformed VSMC line and in CHO and COS7 cells transfected with wild type or mutant receptors. The process of desensitization will be probed in detail concerning the role of phosphorylation of the receptor, mechanisms by which the cell cycles AR and how mutation of potential regulatory sites on the AR changes the patterns of desensitization. Strong evidence exists that the A1AR can regulate multiple effector systems in VSMCs including adenylylcyclase, phospholipase C and guanylate cyclase. A2AR receptors appear to only stimulate adenylylcyclase. Evidence suggests that activation of A2ARs inhibit and A1ARs stimulate growth and DNA synthesis in VSMC. These pathways will be studied in detail and approaches to selectively differentiate pathways that lead to regulation of muscle tone such as inhibition of adenylylcyclase versus regulation of growth and DNA synthesis which may well be mediated by phospholipase C will be undertaken. Attempts to modulate growth of SMCs through transfection (infection) of mutant receptors such as constitutively active AR or dominant negative AR will be undertaken using high efficiency adenovirus vectors. The overall goal will be to understand AR mediated transmembrane signalling in VSMCs, how it is regulated and how it is possible to manipulate these pathways via recombinant DNA techniques for potential therapeutic benefit.