Vasoactive agents play a pivotal role in the autoregulation of regional blood flow and in the pathogenesis of hypertension and vasospasm. These agents, some of which also have mitogenic properties, act on smooth muscle cells by inducing a variety of enzymatic processes and ion transport events. Endothelin 1 (ET-1), is a 21-amino acid peptide synthesized by endothelial cells, with highly potent vasoconstrictive and mitogenic properties. ET-1 has been implicated in the genesis of renal vasoconstriction that leads to post-ischemic renal failure and in graft rejection after renal transplantation. On a cellular level, ET-1, binds to its specific receptor on the plasma membrane which is a G-protein coupled receptor. After binding to its receptor, activation of phospholipase C leads to the generation of inositol 1,4,5-triphosphate (lP3), and release of intracellular Ca2+. Subsequent to this process there is an influx of Ca2+ from the outside the cell. The ionic mechanisms and the regulation of this process has not been clearly delineated. The primary objective of this grant is to determine the mechanism and mode of entry of extracellular Ca2+ in cells stimulated by ET-1. The signaling system will also be studied. Studies will be performed using vascular smooth muscle cells and Chinese Hamster Ovary (CHO) cell line. The latter cell line has been manipulated to overexpress endothelin receptor and causes a robust entry of extracellular calcium on stimulation with ET-1. Techniques such as microinjection of antibody to PIP2 which blocks phosphoinositide metabolism, as well as microinjection of heparin which blocks intracellular release of calcium will be used. In addition, calcium entry in response to metabolites of phosphatidylinositol will be examined. Results of these experiments will provide valuable insights into how ET-1 functions at a molecular level. Understanding of the normal response is critical to understanding the response in the deranged state.
