The long term objective of this proposal is to investigate the signaling mechanisms of norepinephrine (NE), and angiotensin II (AII) in releasing arachidonic acid (AA) for prostaglandin (PG) synthesis in blood vessels. Recent findings in rabbit aortic vascular smooth muscle cells (VSMC) indicate that NE and AII promote AA release by activating calcium- calmodulin dependent protein kinase (CaMKII), which, both directly and through mitogen activated protein kinase (MAPK), stimulates Cytosolic phospholipase A2 (cPLA2). The present proposal is to elucidate the mechanism by which NE and AII stimulated CaMKII activates MAPK and cPLA2. The overall hypothesis, based on preliminary studies with inhibitors of AA metabolism and exogenous eicosanoids, is that NE- and AII- stimulated CaMKII directly activates cPLA2 and releases AA; products of AA generated via cytochrome P-450 (CYP-450) and lipoxygenase (LO) activate MAPK, which further stimulates cPLA2 and also phospholipase D (PLD). The following are the specific aims. I. Signaling Mechanisms Involved in NE-Induced AA Release.
Aim 1. To Investigate Whether CaMKII-Induced Activation of MAPK, which further Activates cPLA2, is Mediated by the Products of AA Generated via CYP-450 and LO in Response to NE in VSMC.
Aim 2. To Determine the Contribution of GTPase Activating Proteins (GAP), Phosphatidylinositol 3- Kinase (PI3-Kinase) and Syk Kinase in Mediating the Action of AA Metabolites Derived via CYP-450 and LO in NE Induced Ras and Rho/MAPK and cPLA2 Activation in VSMC.
Aim 3. To Investigate Whether CaMKII directly Activates cPLA2 and the Underlying Mechanism of its Activation.
Aim 4. To Investigate the Contribution of PLD to NE-Stimulated AA Release and the Mechanism of PLD Activation in VSMC. II. Signaling Mechanisms Involved in Angiotensins Induced AA Release. Previous work and preliminary data suggest that the mechanisms involved in AII stimulated AA release in VSMC is similar to that of NE. Therefore, an approach identical to that described for NE will be used. Studies will be conducted using techniques currently operative in the P.I'S laboratory. The activity of the lipases, kinases and GTP binding proteins will be determined by established procedures. HPLC and GCMS, procedures will be used to identify AA metabolites. The proposed studies should further our understanding of how NE and AII activate cPLA2 and PLD and AA release for PG synthesis in VSMC. Furthermore, information generated from these studies should allow formulation of rational approaches to the development of novel agents with greater selectivity for the treatment of vascular diseases.
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