A Possible Role of Endogenous Inhibitor for Nitric Oxide Synthesis in the Bovine Ciliary Muscle

Abstract

The present experiments were designed to investigate the possible role of endogenous methylarginine derivatives such asNG-monomethyl-L-arginine, asymmetricalNG,NG-dimethyl-L-arginine and symmetricalNG,NG-dimethyl-L-arginine for the nitric oxide synthesis in the bovine ciliary muscle. The contents of asymmetricalNG,N″G-dimethyl-L-arginine and symmetricalNG,NG-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−1wet weight, respectively by means of the automated high-performance liquid chromatography.NG-Monomethyl-L-arginine was below the assay limits. On the basis of the total tissue water content (0.792±0.006 ml g−1wet weight,n=14), the concentrations of asymmetricalNG,NG-dimethyl-L-arginine and symmetricalNG,NG-dimethyl-L-arginine were tentatively estimated to be (4.7±0.3)×10−7M(n=5) and (2.3±0.3)×10−7M(n=5), respectively. A23187 (10−7–3×10−6M) produced a concentration-dependent relaxation of the ciliary muscle strips which had been contracted with 10−5Mcarbachol. Authentic asymmetricalNG,NG-dimethyl-L-arginine (3×10−6–3×10−4M), but not symmetricalNG,N″G-dimethyl-L-arginine (3×10−4M), inhibited the 10−6MA23187-induced relaxation in a concentration-dependent manner. The inhibition with asymmetricalNG,NG-dimethyl-L-arginine (10−4M) was reversed by an addition of 3×10−3ML-arginine, but not by 3×10−3MD-arginine.>The A23187 (10−6M)-induced relaxation was enhanced by 3×10−3ML-arginine or superoxide dismutase (50 U ml−1), whereas it was inhibited by carboxy-PTIO (3×10−4M), a scavenger of nitric oxide, or methylene blue (10−5M), an inhibitor of guanylate cyclase. The carbachol-induced contraction was enhanced by asymmetrical,NG,N″G-dimethyl-L-arginine (10−5M) and inhibited by 3×10−3ML-arginine. Any effect of prostanoid formation during the A23187-induced relaxation was ruled out by using indomethacin (10−5M). Sodium nitroprusside (10−5M), a donor of nitric oxide, also produced a relaxation, which was inhibited by methylene blue (10−5M) or carboxy-PTIO (3×10−4M) and was augmented by superoxide dismutase (50 U ml−1), but unaffected by asymmetricalNG,NG-dimethyl-L-arginine (3×10−4M) orL-arginine (3×10−3M). These results lead us to speculate that the nitric oxide synthesized endogenously fromL-arginine may play a role for mediating relaxation of the bovine ciliary muscle and that the endogenous asymmetricalNG,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.