Endothelial-derived nitric oxide (EDNO) modulates vascular tone, platelet aggregation, and smooth muscle cell proliferation. Alteration of its release has been implicated in several models of atherogenesis, but he mechanism(s) by which this occurs remain unknown. Recent evidence suggest that lipoproteins an inhibit EDNO release and activate neuclear binding of this research proposal is to explore possible mechanism(s0 of how lipoproteins alter G-protein expression and/or function in bovine aortic endothelial cells. We will characterize the acute and chronic effects of lipoproteins on EDNO release in bovine aortic endothelial cells and determine whether lipoprotein treatment affects the expression of alphas, alphai2,3,alphaq/11,beta1-4,gamma1-3 subunit and nitric oxide synthase. Several classes of lipoproteins (LDL, VLDL, HDL) as well as serum from hyperlipidemic patients will be examined. The effects of lipoproteins on alpha2-adrenergic, beta2-adrenergic and bradykinin receptor-G-protein coupling will be assessed by the formation of high-affinity agonist binding sites and agonist-stimulated GTP hydrolysis. The effects of lipoproteins on G-protein-phospholipase C will be assessed by phosphatidylinositol 4,5- bisphosphate hydrolysis and increases in intracellular calcium by Fura-2 AM fluorescence. The effects of lipoproteins on G-protein adenylyl cyclase coupling will be assessed by forskolin and cAMP assays. Finally, for the G-protein components which are affected y lipoproteins, we will overexpress these components (alpha, beta, and gamma subunits) and nitric oxide synthase in cultured bovine aortic endothelial cells and NIH3T3 fibroblasts to determine if G-protein function and EDNO release can be restored in the presence of lipoprotein treatment. Both transient and stable transfections will be attempted using the endothelin and a variety of available viral promoters. This project will help create a better understanding of how lipoproteins can affect G-protein-mediated processes such as EDNO release and NF-kappaB activation . Inhibition of EDNO release and activation of NF-kappaB may mediate some of the atherogenic effects of lipoproteins. Thus, by knowing the mechanism(s0 behind how lipoproteins alter G-protein function, we may gain greater insight into how hypercholesterolemia can produce changes in vascular function and predispose to atherosclerosis. This may ultimately lead to therapeutic modalities which can restore vascular function through enhancing G-protein interaction with its receptors and effectors.
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