A possible role of endogenous inhibitor for nitric oxide synthesis in the bovine ciliary muscle

The present experiments were designed to investigate the possible role of endogenous methylarginine derivatives such as N-G-monomethyl-L-arginine, asymmetrical N-G,N-G-dimethyl-L-arginine and symmetrical N-G,N-G'-dimethyl-L-arginine for the nitric oxide synthesis in the bovine ciliary muscle. The contents of asymmetrical N-G,N-G-dimethyl-L-arginine and symmetrical N-G,N-G'-dimethyl-L-arginine in the bovine ciliary muscle were determined to be 370.2 +/- 27.6 (n = 5) and 182.4 +/- 22.9 (n = 5) pmoles g(-1) wet weight, respectively by means of the automated high-performance liquid chromatography. N-G-Monomethyl-L-arginine was below the assay limits. On the basis of the total tissue wafer content (0.792 +/- 0.006 ml g(-1) wet weight, n = 14), the concentrations of asymmetrical N-G,N-G-dimethyl-L-arginine and symmetrical N-G,N-G'-dimethyl-L-arginine were tentatively estimated to be (4.7 +/- 0.3) x 10(-7) ha (n = 5) and (2.3 +/- 0.3) x 10(-7) M (n = 5), respectively. A23187 (10(-7)-3 x 10(-6) M) produced a concentration-dependent relaxation of the ciliary muscle strips which had been contracted with 10(-5) M carbachol. Authentic asymmetrical N-G,N-G-dimethyl-L-arginine (3 x 10(-6)-3 x 10(-4) M), but not symmetrical N-G,N-G-dimethyl-L-arginine (3 x 10(-4) M), inhibited the 10(-6) M A23187-induced relaxation in a concentration-dependent manner. The inhibition with asymmetrical N-G,N-G-dimethyl-L-arginine (10(-4) M) was reversed by an addition of 3 x 10(-3) M L-arginine, but not by 3 x 10(-3) M D-arginine. The A23187 (10(-6) M)-induced relaxation was enhanced by 3 x 10(-3) M L-arginine or superoxide dismutase (50 U ml(-1)), whereas it was inhibited by carboxy-PTIO (3 x 10(-4) M), a scavenger of nitric oxide, or methylene blue (10(-5) M), an inhibitor of guanylate cyclase. The carbachol-induced contraction was enhanced by asymmetrical, N-G,N-G-dimethyl-L-arginine (10(-5) M) and inhibited by 3 x 10(-3) M L-arginine. Any effect of prostanoid formation during the A23187-induced relaxation was ruled out by using indomethacin (10(-5) M). Sodium nitroprusside (10(-5) M), a donor of nitric oxide, also produced a relaxation, which was inhibited by methylene blue (10(-5) M) or carboxy-PTIO (3 x 10(-4) M) and was augmented by superoxide dismutase (50 U ml(-1)), but unaffected by asymmetrical N-G,N-G-dimethyl-L-arginine (3 x 10(-4) M) or L-arginine (3 x 10(-3) M). These results lead us to speculate that the nitric oxide synthesized endogenously from L-arginine may play a role for mediating relaxation of the bovine ciliary muscle and that the endogenous asymmetrical N-G,N-G-dimethyl-L-arginine may be involved in inhibiting the biosynthesis of nitric oxide when there are increased intracellular concentrations of the methylarginine under certain circumstances. (C) 1997 Academic Press Limited.