Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function.
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 microM with a Vmax of 0.14 micromol mg-1 min-1, whereas Ki values of 0.9 microM and 1.1 microM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 microM level of L-arginine, MA levels of >10 microM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 microm. Similar effects were observed in isolated vessels where 5 microm 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, approximately 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.
Cardounel
Arturo J
AJ
Department of Pharmacology, Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA. cardounel-1@medctr.osu.edu
Cui
Hongmei
H
Samouilov
Alexandre
A
Johnson
Wesley
W
Kearns
Patrick
P
Tsai
Ah-Lim
AL
Berka
Vladomir
V
Zweier
Jay L
JL
D000818:Animals; D001011:Aorta / Q000166:cytology; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D020212:Carotid Artery Injuries / Q000378:metabolism* / Q000503:physiopathology; D002404:Catheterization / Q000009:adverse effects; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D008297:Male; D008745:Methylation; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*; D019323:omega-N-Methylarginine / Q000378:metabolism / Q000493:pharmacokinetics
D004791:Enzyme Inhibitors; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III