Previously, the applicant has shown that treatment of endothelial cells with vascular endothelial growth factor (VEGF) stimulates NO production through a phosphotidylinositol 3' kinase (PI-3 kinase), pathway suggesting that kinases downstream of PI-3 kinase may regulate the activation state of eNOS. In preliminary experiments presented here, we show that VEGF activates the protein kinase, Akt (or protein kinase B) in endothelial cells and that Akt can phosphorylate eNOS, both in vitro and in vivo, resulting in enhanced NO release. Akt phosphorylates serine 1179 on bovine eNOS and mutation of serine to alanine results in a loss of function while mutation of serine to aspartate results in a form of eNOS that is constitutively active. Moreover, infection of endothelial cells with adenovirus expressing dominant negative Akt attenuates VEGF driven NO production and infection with activated Akt further enhances stimulated NO synthesis. With this background in mind, we hypothesize that agonist stimulated Akt activation and subsequent eNOS serine phosphorylation links endothelial cell signal transduction events with NO release and that phosphorylation dramatically alters the calcium sensitivity of the enzyme. To examine this pathway in more detail, the following Specific Aims are proposed.
Aim I. Examine the signaling pathways leading to eNOS phosphorylation by Akt and determine the localization of eNOS and Akt in resting and stimulated endothelial cells;
Aim II. Elucidate the cell biology and function of constitutively activated eNOS and mechanisms of how phosphorylation of eNOS influences the calcium sensitivity of the enzyme;
and Aim III. Examine the importance of Akt regulation of eNOS in transgenic mice. These proposed studies will use a multi-disciplinary approach (cellular, molecular, protein chemistry and functional physiology) to understand the importance of eNOS serine phosphorylation in the regulation of NO production by the endothelium. Results from these studies will assist in our understanding of potential mechanisms leading to endothelial dysfunction, a common manifestation of a variety of cardiovascular diseases.
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