application) Systemic hypertension is the major risk factor for coronary, cerebral and renal vascular disease. Recent studies have emphasized the role of arachidonic acid in modulating vascular contractility both through its actions on myosin phosphatase activity as well as its ability to modulate K+ channel function. The PI has demonstrated that the majority of agonist-induced release of arachidonic acid in vascular smooth cells is due to a calcium-independent phospholipase A2 which is regulated by ATP and calmodulin. Moreover, specific mechanism-based inhibition of calcium-independent phospholipase A2 results in the ablation of acetylcholine-induced relaxation of phenylephrine-constricted mesenteric arteries and ablation of agonist-induced contractile oscillatory activity. Accordingly, the overall goal of the proposed research is the identification of the molecular mechanisms through which calcium-independent phospholipase A2 participates in the regulation of vascular contractility and responsiveness.
In Specific Aim I, the molecular mechanisms through which calcium-independent phospholipase A2 facilitates nitric oxide-mediated smooth muscle vascular relaxation and oscillatory contractile activity will be identified through inhibition of calcium-independent phospholipase A2 in isolated mesenteric resistance arteries.
In Specific Aim II, the biochemical mechanisms underlying the activation of calcium-independent phospholipase A2 isoforms during agonist stimulation will be examined utilizing microbore chromatography, immunoprecipitation and phosphopeptide mapping. The molecular details of the domains of calcium-independent phospholipase A2 which mediate catalysis, activation by ATP, and interaction with calmodulin will be determined through deletional and site-directed mutagenesis.
In Specific Aim III, the role of calcium-independent phospholipase A2 in mediating the nitric oxide-induced activation of Ca2+-activated K+ channels will be examined in Sf9 cells coexpressing both K+ channel protein and calcium-independent phospholipase A2. The chemical loci through which arachidonic acid mediates its effects on the major smooth muscle K+ channels will be determined utilizing the parallel approaches of photoaffinity labeling, deletional mutagenesis and site-directed mutagenesis. Collectively, these studies constitute a multidisciplinary approach aimed at elucidating the molecular mechanisms through which calcium-independent phospholipase A2 modulates vascular contractility and responsiveness.
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