Endothelin (ET) is a peptide secreted by endothelial cells which has potent constrictor and mitogenic effects on vascular smooth muscle (VSM) cells. The signal transduction mechanisms which mediate the effects of ET in VSM are poorly understood. The goal of this proposal is to elucidate the signal transduction mechanisms which couple ET receptors on VSM cells to the generation of two important classes of second messengers, diacylglycerols (DAG) and arachidonic acid (AA), both of which regulate the contractile and proliferative state of VSM cells. All of the proposed studies will be performed with cultured VSM cells derived from rabbit renal artery (RRA-VSM). In RRA-VSM cells prelabelled with [3H]-AA, ET-stimulated [3H]-AA release occurs in two phases. One phase is rapid and transient, the other is delayed in onset and sustained for long periods. This proposal will test the hypothesis that these two phases of AA release are mediated by different signalling mechanisms. These mechanisms will be investigated with respect to the role of 1) phospholipase A2 activation, 2) DAG lipase activation, 3) protein kinase C activation, 4) elevation of cytosolic calcium, and 5) RNA and/or protein synthesis. Another major hypothesis is that the sustained phase of ET-stimulated AA release is entirely dependent on sustained formation of DAG. Furthermore, it is hypothesized that this DAG is the product of several phospholipase effector systems, including 1) phosphoinositide hydrolysis by phospholipase C, 2) phosphatidylcholine hydrolysis by a different phospholipase C, and 3) phosphatidylcholine hydrolysis by a phospholipase D. Each of these putative pathways of ET-stimulated DAG formation will be characterized with respect to the role of calcium influx, the role of protein kinase C activation, and the involvement of each pathway in mediating sustained ET-stimulated AA release. The activation of these phospholipases will be measured in intact radiolabelled RRA-VSM cells by quantitating the formation of radiolabelled metabolites of each pathway after TLC separation. ET is synthesized and secreted by endothelial cells in human blood vessels, and may play is crucial role in the regulation of the contractile and proliferative state of VSM cells. Pathological disturbances in the signal transduction mechanisms which mediate the action of ET may lead to the development of hypertension, vasospasm, and atherosclerosis. Understanding these signalling mechanisms may contribute to the development of novel therapeutic agents to treat these disorders.
