METABOLISM OF METHYLARGININES BY HUMAN VASCULATURE - IMPLICATIONS FOR THE REGULATION OF NITRIC-OXIDE SYNTHESIS

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 [C-14]-monomethyl-L-arginine ([C-14]-L-NMMA) and the cytosolic extract analysed by high performance liquid chromatogaphy (h.p.l.c.) with on-line radioisotope detection. 2 SGHEC-7, HWEC and human saphenous vein metabolized [C-14]-L-NMMA to a compound which co-eluted with [C-14]-citrulline. A second metabolite which co-eluted with [C-14]-arginine was evident on the radiochromatograms of HUVEC cytosol and saphenous vein extracts. 3 The intracellular levels of [C-14]-L-NMMA and [C-14]-citrulline in SGHEC-7 cells incubated with [C-14]-L-NMMA (0.5 mu C ml-(1): 8.9 mu M) for 1 h were 113 +/- 22 and 67.6 +/- 6.2 pmol mg(-1) cell protein respectively (n=7). Co-incubation with N(G)N(G)dimethyl-L-arginine (ADMA; 100 mu M) but not N(G)N(G)dimethyl-L-arginine (SDMA; 100 mu M) reduced the intracellular level of [C-14]-citrulline to 26.3 +/- 3.7 pmol mg(-1) cell protein (P<0.01; n = 3) without reducing the intracellular level of [C-14]-L-NMMA. 4 The intracellular levels of [C-14]-citrulline in SGHEC-7 cells incubated with [C-14]-L-NMMA for Ih were reduced following co-incubation with N(G)nitro-L-arginine methylester (L-NAME; 1 mM), N(G)nitro-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 [C-14]-citrulline levels to 4 +/- 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(-1) 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-derived ADMA 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.