Interaction of vasoactive, inflammatory agents, such as bradykinin, vasopressin, angiotensin, endothelin and the cytokines, with their cellular receptors and the subsequent mechanism(s) leading to their function is at present little understood. Understanding the mechanism of action of these G-protein associated seven transmembrane receptors, which share a number of transmembrane signaling mechanisms responses, yet differ in their cellular responses, will eventually lead to predictable manipulation of such disorders as the adult respiratory distress syndrome and pulmonary hypertension. In this proposal we set out to determine the signaling mechanisms of a typical such receptor, the B2 receptor for bradykinin, a multipotent receptor which is known to regulate a number of target signaling systems. Binding of bradykinin to it's receptor induces vasodilation in vivo, in part through release of prostaglandins. We have achieved the functional expression of the bradykinin B2 receptor in cells which lacked this receptor i.e. the Chinese hamster lung fibroblasts (CCL39) and Rat-1 cells. We are using approaches such as site directed mutagenesis, chimera formation and antisense against G-protein to map the motifs that determine the phenotypic response of the bradykinin B2 receptor. In the process, as we generate chimeras between bradykinin and lL-8 cDNA we hope to also obtain better understanding of the function of this important chemotactic, inflammatory agent. Pharmaco-physiologic studies with pulmonary artery endothelial cells suggest that at least 2 G-proteins are related to the action of the bradykinin B2 receptor. We find that bradykinin stimulates Ca2+ mobilization and polyphosphoinositide (PI) metabolism, which is regulated by a pertussis toxin insensitive G-protein. Bradykinin also stimulates arachidonate release through a pertussis toxin sensitive G-protein. Our overall aim is to map the motifs of the bradykinin receptor responsible for G-protein activation by bradykinin and to determine which G-protein alpha subunits are involved in bradykinin stimulation of phospholipase A2 (PLA2) and phospholipase C (PLC). We expect that understanding of the mechanism(s) involved in the action(s) of the bradykinin receptor will provide insights into the action of vasoactive and inflammatory peptides in general and into the regulation of the pulmonary vascular pressures.
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