This grant is requested to support a program in basic research aimed at elucidating at a molecular level the nature, mode of functioning, and molecular mechanisms of regulation of receptors for kinins. The major mammalian kinins, bradykinin and kallidin, are generated in plasma and tissues from large molecular weight hormogens, kininogens, by specific serine proteases, kallikreins, in response to e.g. tissue damage and inflammatory reactions. In vivo, the predominant result of kinin action is a dramatic fall in blood pressure due to arteriolar vasodilatation, edema due to increased vascular permeability, and pain. Indeed, kinins mimic all the cardinal signs of inflammation. In addition, kinins have been implicated in the genesis of cardiovascular shock, hyper- tension, and rheumatoid arthritis. At the cellular level, kinins stimulate inositol phospholipid metabolism followed by increased intracellular Ca2+ levels, and the release of arachidonic acid and prostaglandins by stimulating the activity of phospholipase C and A2, respectively. This research proposal has four major goals all aimed at an increased understanding of the function of kinin receptors in various physiological and pathophysiological conditions. The goals are: 1) to develop high affinity radioligands for kinin receptors; 2) to elucidate the molecular mechanisms involved in translation of agonist occupancy of receptors on the exterior of the cell into generation of intracellular signals. Initially, these studies will be pursued using the cultured smooth muscle cell line, DDT1 MF-2. The characteristics of kinin receptor binding and receptor activation of phospholipase A2 and C will be studied both in membrane and intact cell preparations; 3) to elucidate the molecular properties of the kinin receptor protein. This goal involves development of tools such as solid receptor affinity resins and affinity and photoaffinity probes. These tools will then be used for purification of the receptor and identification and characterization of the receptor ligand binding peptide; 4) to elucidate the molecular mechanisms involved in the regulation of kinin receptor function. This goal involves identification of various factors such as homologous and heterologous agonists, tumor-promoting phorbol esters, noxious stimuli, and metabolic state that may regulate the kinin receptor responsiveness and the mechanisms underlying such regulation. The goals presented in this proposal constitute an integral part of the long term objective of this laboratory which is to understand the molecular mechanisms of action of vasoactive hormones.
