In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for L-arginine is 3.14 μM with a Vmax of 0.14 μmol mg-1 min-1, whereas Ki values of 0.9 μM and 1.1 μM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 μM level of L-arginine, MA levels of >10μM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 μM. Similar effects were observed in isolated vessels where 5 μM ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and L-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. L-Arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, ∼4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.
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