: Our long-term goal is to investigate the mechanism of interaction of the adrenergic nervous system, angiotensin II (Ang II) and eicosanoids in the regulation of cardiovascular function and in the development of hypertension. Over the past grant period, we have shown that norepinephrine (NE)- and Ang II stimulate calcium/calmodulin dependent protein kinase II (CaMKII) phosphorylate cytosolic phospholipase A2 (cPLA2) and release AA in vascular smooth muscle cells (VSMC); AA metabolltes formed via cytochrome P450 (CYP45O) and lipoxygenase (LO) activate phosphoilpase D (PLD), amplify cPLA2 activity and contribute to VSMC hyperplasia and/or hypertropby and hypertension. This proposal is an extension of these studies and will test the hypotheses that: 1) Phosphorylation of cPLA2 at Ser-515 by CaMKII is responsible for its activation, translocation to the nuclear envelope and AA release in response to NE- and Ang II. 2) Translocation of cPLA2 to the nuclear envelope is dependent upon vimentin phosphorylation and actin filament organization, which is maintained by phosphorylation of heat shock protein 27 (HSP27) by ERK1/2. 3) AA metabolites derived via CYP45O and LO by transactivating epidermal growth factor receptor (EGFR) and/or NAD(P)H oxidase, activate ERK1/2, which causes further phosphorylation of cPLA2 at Ser505/727, and of PLD2 at a Tyr-residue(s), and promotes VSMC proliferation and/or hypertrophy. The specific alms of the proposal are: I. Signaling Mechanisms Involved in NE-induced cPLA2 and PLD Activation and their Functional Significance.
Aim 1. To determine if NE-stimulated CaMKII, by phosphorylating cPLA2 on Ser-515, promotes AA release, Ras/ERK1/2 and PLD activation and VSMC proliferation.
Aim 2. To investigate the contribution of CaMKII, Ras-ERK1/2, actin, vimentin and heat shock protein (HSP27) to NE-induced cPLA2 translocation to the nuclear envelope and the underlying mechanism involved Aim 3. To examine the contribution of epidermal growth factor receptor (EGFR) transactivation and NAD(P)H oxidase to AA metabolite-induced activation of Ras-ERK1/2 and PLD2, amplification of cPLA2 activity and VSMC proliferation in response to NE.
Aim 4. To elucidate the mechanism of PLD activation by tyrosine phosphorylation caused by cPLA2 derived AA metabolites via ERK1/2 and p38 MAPK in response to NE. II. Signaling Mechanisms Involved in Ang II-Induced cPLA2 and PLD Activation and their Functional Significance. We will also investigate the effects of Ang II on the above signaling mechanisms in VSMC using an approach similar to that described for NE. We will include Ang II and NE in the same protocols. These studies should further our understanding of the cellular mechanisms involved in NE-and Ang II- induced cPLA2 activation and AA release and the mechanism by which AA metabolites cause PLD activation, amplification of cPLA2 activity and VSMC proliferation and hypertrophy.
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