title: Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis.
abstract: 1. The metabolism of methylarginines by human cultured endothelial cells and human saphenous vein was studied in vitro. The human endothelial cell line (SGHEC-7), primary cultures of human umbilical vein endothelial cells (HUVEC) and human saphenous vein were incubated with [14C]-monomethyl-L-arginine ([14C]-L-NMMA) and the cytosolic extract analysed by high performance liquid chromatography (h.p.l.c.) with on-line radioisotope detection. 2. SGHEC-7, HUVEC and human saphenous vein metabolized [14C]-L-NMMA to a compound which co-eluted with [14C]-citrulline. A second metabolite which co-eluted with [14C]-arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts. 3. The intracellular levels of [14C]-L-NMMA and [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA (0.5 microCi ml-1: 8.9 microM) for 1 h were 113 +/- 22 and 67.6 +/- 6.2 pmol mg-1 cell protein respectively (n = 7). Co-incubation with NGNGdimethyl-L-arginine (ADMA; 100 microM) but not NGNGdimethyl-L-arginine (SDMA; 100 microM) reduced the intracellular level of [14C]-citrulline to 26.3 +/- 3.7 pmol mg-1 cell protein (P < 0.01; n = 3) without reducing the intracellular level of [14C]-L-NMMA. 4. The intracellular levels of [14C]-citrulline in SGHEC-7 cells incubated with [14C]-L-NMMA for 1 h were reduced following co-incubation with NGnitro-L-arginine methylester (L-NAME; 1 mM), NGnitro-L-arginine (L-NOARG; 1 mM) and L-canavanine (1 mM) to 47.1 +/- 6.2, 24.7 +/- 3.6 and 12.5 +/- 2.8% of control levels (P < 0.001; n = 9). ADMA (1 mM; n = 3) reduced intracellular [14C]-citrulline levels to4 +/- 4% of control (P<0.01) but SDMA (1 mM; n = 3) had no effect.5. The accumulation of endogenously synthesized ADMA in the culture supernatant of SGHEC-7 cells was increased by co-incubation with L-NMMA (1 mM) from 1.98 +/- 0.08 to 2.74 +/- 0.36 nmol mg- cell protein, an increase of 40%.6. These results demonstrate that human vasculature possesses an enzyme which has similar properties to dimethylarginase; human endothelial cells and human saphenous vein metabolize L-NMMA to citrulline via a process inhibited by ADMA but not SDMA. The increase in endothelium-derivedADMA following co-incubation with L-NMMA is consistent with competition between ADMA and L-NMMA for dimethylarginase. Inhibition of this enzyme might increase the intracellular concentration of ADMA, an endogenously produced compound that inhibits nitric oxide synthesis.
authors.affiliation: Department of Pharmacology, St. George's Hospital Medical School, London.
authors.forename: R J
authors.initials: RJ
authors.lastname: MacAllister
authors.forename: S A
authors.initials: SA
authors.lastname: Fickling
authors.forename: G S
authors.initials: GS
authors.lastname: Whitley
authors.forename: P
authors.initials: P
authors.lastname: Vallance
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001119:Arginase / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D012501:Saphenous Vein / Q000187:drug effects / Q000378:metabolism; D014471:Umbilical Veins / Q000378:metabolism; D019323:omega-N-Methylarginine
chemical_list: D019323:omega-N-Methylarginine; D002956:Citrulline; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D001119:Arginase; D019331:NG-Nitroarginine Methyl Ester