Graduation Year


Document Type




Degree Granting Department

Molecular Medicine

Major Professor

Larry P. Solomonson, Ph.D.


Arginine, Citrulline, Argininosuccinate synthase, Vascular biology, Caveolae


The discovery of nitric oxide (NO) as the endothelial-derived relaxing factor has led to significant research on NO and the proteins involved in its function, generation, location and regulation. Synthesis of NO by blood vessel endothelial cells results from the enzymatic oxidation of arginine by endothelial nitric oxide synthase (eNOS) resulting in the formation of equimolar amounts of NO and citrulline. Citrulline is sequentially recycled to arginine by successive reactions involving the enzymes argininosuccinate synthase (AS) and argininosuccinate lyase (AL), respectively.

eNOS activity has been shown to be regulated by post-translational modifications including dynamic phosphorylation on multiple serine/threonine and tyrosine residues and dynamic O-linked beta-N-acetylglucosamine (O-GlcNAc) modifications on serine/threonine residues.Previous studies showed that even though intracellular endothelial arginine levels range from 0.1 to 0.8 mM and the Km of eNOS for arginine is 3 uM, the addition of exogenous arginine caused an increase in NO production. To explain this "arginine paradox" we hypothesize that there is a separate and distinct cellular source of arginine substrate directed to NO production and that this source is maintained through the regeneration of arginine via a citrulline-NO cycle. The presented research has provided the following evidence in support of this hypothesis: Citrulline stimulates NO production in an arginine-rich medium, without an increase in intracellular arginine.

The enzymes of the citrulline-NO cycle, eNOS, AS and AL, co-fractionate with caveolin-1 in an endothelial cell caveolar membrane fraction. In vitro interaction assays demonstrate protein-protein interactions between fusion tagged AS or AL with eNOS or caveolin-1. Simultaneous monitoring of apparent citrulline and NO production demonstrates an efficient and essential coupling of the reactions of the citrulline-NO cycle. Glucosamine treatment of endothelial cells results in increased NO production in the basal state and decreased NO production in the stimulated state.Our findings demonstrate the enzymes of the citrulline-NO cycle, eNOS, AS and AL, are functionally associated, the reactions are efficiently coupled and enzyme activities are changed by post-translational modifications based on nutrient levels. These alterations ensure a constant and distinct source of arginine which is available for NO production to ensure vascular health.