There is abundant evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. One of the major endothelium-derived vasoactive mediators is nitric oxide (NO), formed in healthy vascular endothelium from the amino acid precursor L-arginine. Endothelial dysfunction is increased by various cardiovascular risk factors, metabolic diseases, and systemic or local inflammation. One mechanism that has been implicated in the development of endothelial dysfunction is the presence of elevated levels of asymmetric dimethylarginine (ADMA). Free ADMA, which is formed during proteolysis, is actively degraded by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH) which catalyzes the conversion of ADMA to citrulline and dimethylamine. It has been estimated that more than 70% of ADMA is metabolized by DDAH 1 . Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA mediated eNOS impairment. However, recent studies suggest that DDAH may regulate eNOS activity and endothelial function through both ADMAdependent and independent mechanisms. In this regard, elevated plasma ADMA may serve as a marker of impaired methylarginine metabolism and the pathology previously attributed to elevated ADMA may be manifested, at least in part, through altered activity of the enzymes involved in ADMA regulation, specifically DDAH and PRMT.

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and N (G)-monomethyl- l-arginine (L-NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH 4) depletion eNOS also generates (*)O 2 (-); however, the effects of methylarginines on eNOS-derived (*)O 2 (-) generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose-dependent effects of ADMA and L-NMMA on (*)O 2 (-) production from eNOS under conditions of BH 4 depletion. In the absence of BH 4, ADMA dose-dependently increased NOS-derived (*)O 2 (-) generation, with a maximal increase of 151% at 100 microM ADMA. L-NMMA also dose-dependently increased NOS-derived (*)O 2 (-), but to a lesser extent, demonstrating a 102% increase at 100 microM L-NMMA. Moreover, the native substrate l-arginine also increased eNOS-derived (*)O 2 (-), exhibiting a similar degree of enhancement as that observed with ADMA. Measurements of NADPH consumption from eNOS demonstrated that binding of either l-arginine or methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies suggest, just as for l-arginine and L-NMMA, the binding of ADMA shifts the eNOS heme to the high-spin state, indicative of a more positive heme redox potential, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and (*)O 2 (-) generation from eNOS. These observations have important implications with regard to the therapeutic use of l-arginine and the methylarginine-NOS inhibitors in the treatment of disease.