This research proposal addresses possible mechanism(s) underlying the control of blood flow and the effects of cholesterol on vascular tone. The specific questions concern how signals are transduced by mediators (G proteins) across cellular membranes for the release of a putative substance (EDRF) which causes vasodilation. Inhibition of EDRF release can produce vasoconstriction and ischemia. Certain pathological conditions such as hypercholesterolemia has been shown to inhibit EDRF release, but cellular mechanism(s) responsible for this is not presently known. The purpose of this study is to determine whether LDL inhibits EDRF release through changes in membrane fluidity and receptor-G protein-effector interaction. Bovine aortic endothelial cells grown on microcarrier beads in the presence and absence of LDL will be stimulated to release EDRF in a perfused bioassay system. Specific receptors will be characterized by radioligand binding studies. The G proteins will be identified by toxin labelling, Western blotting, and activated an inactivated by treating permeabilized cells with nonhydrolyzable GTP analogues, GTPgammaS and GDPbetaS, respectively. Endothelial cells will be transfected by various methods with available cDNA clones coding for the receptors and G proteins(s) which may be responsible for EDRF release. The resulting expressions of these cDNAs, the release of EDRF, and the receptor-G protein coupling in transfected cell lines will be determined. The effects of LDL on receptor- G protein coupling will also be examined by performing competitive agonist binding studies. The effects of LDL on G protein-phospholipase C coupling will be inferred by introducing GTPgammaS which directly stimulates G proteins and by the use of a calcium ionophore which directly stimulates EDRF release without G protein involvement. Phospholipase C activity is determined by measuring the increase in intracellular calcium and phosphoinositide turnover. Membrane fluidity will be assessed using spin- labelled fatty acids ESR spectroscopy before and after LDL treatment, and any changes will be correlated with alterations in G protein function and EDRF release. This project will help define the role of G proteins in mediating EDRF release in endothelial cells. It will provide a better understanding of the cellular mechanism(s) responsible for EDRF release. Information derived from this research project has potential benefits in several areas of cardiovascular medicine such as coronary artery disease, strokes, and hypertension.
