Recently much evidence has been published that vasopressin and norepinephrine increase cytosolic calcium levels in vascular smooth muscle by means of receptor-mediated breakdown of phosphatidyl inositol 4,5 bisphosphate, followed by inositol 1,4,5 tris phosphate stimulated Ca+2 release from sarcoplasmic reticulum and the resulting contraction of the smooth muscle. Many questions remain concerning the precise molecular mechanism by which this system operates. There is some doubt concerning the rate at which IP3 level increase in smooth muscle in response to agonist. It is also currently unclear whether G proteins interact with the system, whether IP4, PC or PIP play important role in this system. Our hypothesis is that the breakdown of inositol phospholipid is required for agonist-mediated smooth muscle contraction. As a result of this hypothesis we have formulated the following specific aims: 1) To determine the rapid kinetics of IP3 formation in rat tail artery using laser activated caged phenylephrine, in order to determine if the agonist- stimulated production of IP3 in the tissue is rapid enough to produce the contractile response. 2) To measure increases in IP3 levels in these cells with the aid of a purified IP3 binding protein, so as to prove that IP3 levels actually increase without the need to use isotopically prelabeled tissue. 3) To determine the role of G proteins in this system by determining if GTP-gammaS, pertussis toxin, or AIF4-stimulate IP3 production in permeabilized rat tail artery cells. 4) To determine the rate at which PIP is broken down in these cells, by determining the effect of added IP3 on the production of IP2 in permeabilized cells. 5) To measure the kinetics of IP4 production in these cells, using high pressure liquid chromatography. 6) To determine if the stimulated production of inositol phosphates that we observe in these cells is sensitive to the removal of endothelial cells. 7) To determine if vasopressin stimulates Pc breakdown, in order to determine if the main source of DAG in these cells is inositol lipid. These proposed studies should greatly increase our understanding of the role that inositol lipids play in controlling the contraction of vascular smooth muscle.
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