Alpha-1 adrenergic receptors cause increased vascular tone via a G-protein mediated hydrolysis of phosphatidylinositoldiphosphate (PIP2), IP3-induced Ca2+ release and diacylglycerol (DAG) activation of protein kinase C (PKC). The coupling pathway for alpha-2 receptors is not as well established although work form other tissues has focused interest on a receptor associated increase of Na+/H+ exchange. Recent evidence indicated that PKC may act as a modulator of alpha-1 receptor coupling via phosphorylation of the receptor itself or at subsequent steps. Receptor-activated hydrolysis of phospholipids other than PIP2 (e.g. phosphatidylinositol or phosphatidylcholine) may be a function of the level of kinase activity. This proposal is directed toward elucidating the importance of PKC and cGMP-dependent protein kinase regulation at arterial alpha-1 receptors. In addition, a study of alpha-2 receptor coupling pathways and its regulation by these kinases will be undertaken to permit a more comprehensive view of the function of these closely related receptor systems in vascular tissues. The approach taken will be to measure the comparative hydrolysis of different phospholipids in isolated tissues upon receptor activation under conditions where protein kinase C or protein kinase G have been elevated (by phorbol ester or nitroprusside respectively) or reduced (by staurosporine or LY 83583). The specific influence of these kinase on receptor binding will be assayed in radioligand binding studies using plasma membrane fractions isolated from bovine aorta after prior treatment to provide kinase activation or inhibition conditions. In these membrane preparations we will also measure agonist-induced phospholipid hydrolysis in order to correlate binding of agonists with functional responses. These studies will include examination of the GTP and Ca2+ requirements, as well as the influence of pH on phospholipase activity. Studies on alpha-2 receptors will focus on the possible role of changes in pHi mediated by increased Na+/H+ exchange and protein kinase C activation in contractile responses of the isolated saphenous vein. Binding studies will be utilized to demonstrate or exclude involvement of a G-protein in receptor coupling and the influence of kinase activation on agonist binding. Results from these experiments will provide further insight into the role of protein kinase and Na+/H+ exchange activities in regulating agonist-induced arterial contraction.
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