Numerous cardiovascular diseases involve alterations in vascular tone and resistance to blood flow. These include systemic and pulmonary hypertension and acute coronary and cerebral artery vasospasm. It is now known that the vascular endothelium produces important vaso-regulatory substances which mediate vascular smooth muscle tone. One such substance is endothelin, the most potent vasoconstrictor polypeptide known to date. The long term goal of this proposal is to understand the role of endothelin in vascular disease and to develop methods of blocking its pathological effects. Endothelin acts by binding to high affinity receptors on the surface of vascular smooth muscle cells. The aim of this project is to purify the endothelin receptor and use it to study the mechanism of endothelin action. In order to do this, our specific aims are to determine the important structural features of the endothelin molecule, to determine the endothelin binding characteristics of various tissues, to purify, clone, and sequence the endothelin receptor, and to use mutated forms of the receptor to study endothelin's signaling pathways. This work will utilize newer biochemical techniques including the use of nuclear magnetic resonance spectroscopy to determine the three dimensional structure of endothelin and expression cloning as a means to isolate the endothelin receptor. We have the ability to synthesize functional endothelin derivatives, which may allow the production of specific endothelin antagonists. Preliminary studies have shown that endothelin receptor stimulation causes a marked increase in intracellular calcium and may be coupled to opening of the voltage activated calcium channel. Site directed mutagenesis will allow us to study the mechanism of this and other signaling pathways important to cell biology. The results of these investigations should provide a better understanding of the basic aspects of endothelin action and should aid in the development of new approaches to the study and treatment of vascular disease.