The goal is to study the molecular mechanisms of hormonal signalling in vascular smooth muscle cells (VSMC). The focus will be on the cellular events involved in maintenance of the tonic phase of vessel contraction. Previous work has shown that mitogens such as PDGF and EGF cause contraction of rat aortic strips. The current application is based on the proposal that the intracellular pathways activated by mitogens in VSMC involve mechanisms also shared by classic vasoconstrictors which may be particularly relevant to mediation of tonic contraction. In many cells, mitogens have been shown to be potent activators of phospholipase C, causing a rapid, transient increase in intracellular free calcium concentration ([Ca2+]i) thought due to inositol trisphosphate, and a slower, prolonged rise in intracellular pH thought secondary to diacylglycerol activation of C kinase with increased Na+/H+ exchange. In VSMC, it is proposed that activation of the diacylglycerol/C kinase limb of this system by mitogens and vasoconstrictors leads to increased transmembrane Ca2+ permeability (perhaps due to increased Na+ influx causing increased Na+/Ca2+ exchange) which mediates the tonic phase of contraction. To test this hypothesis, the following investigations are planned. The effects of vasoconstrictors and mitogens on Na+/H+ exchange in VSMC will be analyzed using fluorescent pH indicators. New ionic indicators for fluorescent measurements in single cells and NMR measurements in tissue will be studied to look at changes in Na+. 2) A series of protein phosphorylations mediated by C kinase in VSMC will be generated by 2 dimensional gel electrophoresis. C kinase will be purified and then in vitro reconstitution experiments will be performed to identify those phosphoproteins essential to cation movement. 3) The time course and magnitude of changes in phospholipid metabolism in response to mitogens and vasoconstrictors will be studied and compared with simultaneous changes in [Ca2+]i. 4) The roles of Na+/H+ exchange and C kinase activity in mediating Ca2+ flux in tonic contraction will be characterized. In summary, the proposed research program is designed to gain insight into the mechanisms of action of mitogens and vasoconstrictors in VSMC which mediate the tonic contractile response.
The specific aims are consistent with the long-range goals of the applicant which are focused on relating the cell biology of VSMC response to mitogens with the clinical features of atherosclerotic coronary artery disease.
