Products of the LO pathway of AA metabolism, the hydroxyeicosatetraenoic acids (HETEs), may play an important role in the mechanism of action of pressor-induced vascular activity. The applicants have shown that LO inhibitors markedly reduce responses to angiotensin II (Ang II) in vivo, vascular contractility in vitro, and cytosolic calcium ([Ca2+]i) increments in cultured vascular smooth muscle cells (VSMC). Of particular importance is their observation that LO inhibitor attenuate Ang II-induced [Ca2+]i responses and phospholipase C activity in VSMC, and that 12(S)HETE pre- treatment potentiates the Ang II [Ca2+]i response. New data suggest that HETEs substitution on membrane phospholipids may be a control point for their actions on [Ca2+]i. These studies strongly suggest a role for HETEs as modulators of pressor hormone-induced second messenger signal transduction. In whole animal studies, LO inhibitors also prevent hypertension in 2 Kidney-1 Clip renovascular and in SHR. They have also demonstrated elevated HETEs in blood and vascular tissues in SHR, and a pronounced acute hypotensive effect of LO inhibitors. In this proposal, they will investigate two potential pathways of LO product mediation of agonist action in vascular tissue. They originally proposed that LO products act directly as obligatory intermediates for signal transduction. They have now advanced this hypothesis to include the concept that LO products may also act indirectly to modulate the activity of other signal transducers such as inositol triphosphate (IP3), diacylglycerol (DAG), and protein kinase C (PKC). The applicants' approach will test the effects of LO inhibition and HETE substitution on signal transduction. The sources and control mechanisms of HETE modulated [Ca2+]i will be defined. LO products (12- and 15-HETE) will be quantified in vascular tissue using improved extraction, HPLC, and RIA techniques to measure HETE products, and expression of LO enzyme, and mPNA expression and transcription rate in VSMC. The physiologic significance for blood pressure regulation by LO products will be evaluated in isolated blood vessels and whole animals. The effects of LO pathway inhibition on vascular responsiveness will examine the role of LO inhibition on the endothelium, and on the interactions of LO products and inhibitors with other eicosanoids, as they relate to vascular reactivity. Finally, they will compare Ang II-induced (Ca2+)i, phosphoinositide metabolism, contractility, HETE products, and LO enzyme expression in vessels and VSMC from SHR, WKY and Sprague-Dawley rats. These experiments are designed to comprehensively study the role of LO products, HETEs, in vascular tone control. In addition, novel mechanisms of cell signaling for pressor agents in the vasculature may be uncovered.
