This is a new application which represents a continuation of a K08 award to the principal investigator. The central hypothesis of the proposal is that nitric oxide causes relaxation of vascular smooth muscle cells and inhibition of platelet aggregation through phosphorylation of receptors for thromboxane and thrombin by stimulating a cyclic guanosine monophosphate sensitive G-kinase. This mechanism is novel in that it does not require prior binding of ligand to the receptors; instead, desensitization occurs through uncoupling of the receptors from their respective G-proteins. This represents a novel mechanism for heterologous receptor desensitization. Vascular smooth muscle cells derived from explanted human saphenous veins, internal mammary arteries, and platelets will be used to investigate the three specific aims. The first specific aim will determine the phosphorylation of G-protein-coupled receptors in cultured smooth muscle cells or platelets. Immunoblotting techniques will identify phosphorylation of the receptors as they relate to time and dose of nitric oxide and the ability of various inhibitors of G-kinase to prevent the phosphorylation initiated by nitric oxide. The second specific aim will identify specific amino acid sequence residues which are phosphorylated by the G-kinase in thromboxane and thrombin receptors. These studies will involve analysis of phosphoamino acid of receptor immunoprecipitates. Two-dimensional phosphopeptide mapping to identify the phosphorylated domains in each receptor. Microsequencing techniques will be used to identify precisely which residues are phosphorylated.
Specific aim three will determine the functional and physiological consequences of G-kinase-dependent phosphorylation. Measurements of the relative ability of the phosphorylated proteins to increase intracellular calcium would be measured as an endpoint of functional response. Tranfected mammalian (CHO) cells expressing either the native receptor or a receptor with a site-specific mutant in the G-kinase target region will be studied. The ability of G-kinase or cyclic GMP to phosphorylate the receptors or to activate the calcium signal will be compared between the two types of cells. Results of these experiments are important in determining and defining a specific mechanism by which receptors are inactivated or desensitized; it will provide new information regarding the mechanism by which nitric oxide inhibits vascular tone and there are important implications for development of therapeutic interventions for diseases associated with desensitization of receptors such as hypertension or atherosclerosis.
