Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biochemistry and Molecular Biology

Major Professor

Duane C. Eichler, Ph.D.

Keywords

Nitric oxide, Enos, Endothelial, Apoptosis, tnf-alpha, ppar-gamma

Abstract

While cellular levels of arginine greatly exceed the apparent Km for endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production is limited by availability of arginine. Results from this work have provided a unique understanding of endothelial NO production, showing that arginine regeneration, that is the recycling of citrulline back to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL), defines the essential source of arginine for NO production. Using RNA interference analysis, selective reduction of AS expression was shown to directly correspond with a diminished capacity of endothelial cells to produce NO, despite saturating levels of arginine in the medium. In addition, the viability of AS siRNA-treated endothelial cells was compromised due to apoptotic cell death.AS expression was also investigated in response to two major vascular effectors. Tumor necrosis factor (TNF)-alpha; which is known to impair endothelial NO production, was shown to provoke a dose-dependent reduction of AS expression that corresponded to a decrease in NO production. Furthermore, TNF-alpha was shown to suppress AS expression through a NFkappaB mediated pathway, which involves three essential Sp1 elements in the proximal AS gene promoter. On the other hand, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, troglitazone and ciglitazone, which are known to elicit a vascular protective response against TNF-alpha effects, were shown to coordinately induce NO production and AS expression via a PPARgamma response element in the distal AS gene promoter. Importantly, these PPARgamma agonists were shown to restore AS expression and NO production following down-regulation by TNF-alpha, consistent with their vascular protective properties.

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