Recent pharmacological studies have suggested that cholinergic stimulation of blood vessel strips with low doses of acetylcholine results in relaxation through release of endothelial vasodilator substance(s), while higher doses directly lead to contraction. Because of the structural complexity of the vessel wall, cholinergic biochemical and cellular mechanisms underlying these responses can be best approached using homogeneous cultures of its major functional elements - endothelial cell (EC) and smooth muscle cell (SMC). In the first phase of the project I shall characterize the cholinergic mechanisms in cultured EC and SMC by utilizing ligand binding techniques, adenylate cyclase and cyclic nucleotide assays, as well as assay for the phosphorylation of the SMC myosin light chain - the major regulatory locus of SMC contraction. During the second phase of the project I shall explore the production of vasoactive mediators (prostacyclin, angiotensin II, bradykinin, adenosine) by EC in response to cholinergic stimulation. Utilizing the technique of growing EC on solid plastic beads and arranging these into a chromatography column, high concentrations of vasoactive mediators should be obtainable. During the latter stages of the project, I shall investigate the effects of cholinergic stimulation on aspects of endothelial function not directly related to the control of vascular tone, such as pinocytosis and the maintenance of nonthrombogenic surface. The proposed project should elucidate the mechanisms by which the parasympathetic system modulates vascular tone and at the clinical level contribute to better understanding of vasospatic diseases. The proposed research is an integral part of my long-range interests in mechanisms of vascular reactivity.
