The purpose of the proposal is to understand the role of ET and its receptors in modulating retinal hemodynamics in the normal and diabetic state in vivo and in cultured retinal vascular cells. Since the first report showing the existence of ETA receptors in the retinal pericytes and the regulation of ET-1 expression by insulin, ET-1 and ET-3 have been shown to have many actions in retinal vascular cells. The investigator's results have established the fact that ET-1 via ETA receptor is a major regulator of vascular tone in the retina during normal homeostasis and hyperoxia and in the early stages of diabetic retinopathy. The increased vasoconstriction observed in these states are due to either overexpression of ET-1 or increase in its processing by endothelin converting enzyme (ECE) in the retina. Recently, the applicant has shown that insulin can decrease ET-1 gene expression in pericytes and smooth muscle cells which could partially explain the in vivo vasodilatory effect of insulin. At the signal transduction level, ET-1 and angiotensin can affect phospholipase C (PLC)-protein kinase C (PKC) activation and the tyrosine kinase cascade involving MAP kinase in pericytes and smooth muscle cells. The investigator has recently found that angiotensin and ET-1 can also activate a G-protein associated phosphatidylinositol 3-kinase (PI 3-kinase), thereby identifying a whole new signal transduction pathway for ET-1. Lastly, the finding that hyperglycemia can blunt ET-1's effect on PLC activation has been confirmed by smooth muscle cells and mesangial cells as well. The investigator proposes: (1) to characterize the expression and processing of ET-1 in retinal vascular cells as regulated by insulin, oxygen tension, angiotensin and hyperglycemia; (2) to identify the various signal transduction pathways used by ET-1 in retinal pericytes and in smooth muscle cells specifically on the activation of G-protein-associated PI 3-kinase and the association of these pathways to biological actions such as calcium flux, NO production and DNA synthesis; and (3) to determine the expression and the effect of ET-1 and ET-3 and their receptors in vivo on normal retinal hemodynamics and in diabetes.
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