Diabetes does not alter the activity and localisation of nitric oxide synthase in the rat anococcygeus muscle

Functional studies have revealed diabetes specifically impairs smooth muscl e reactivity to nitric oxide in the rat anococcygeus muscle. The present st udy was conducted to examine whether concurrent prejunctional defects in ni trergic neurotransmission exist in anococcygeus muscles from diabetic rats. Nitric oxide synthase (NOS) activity was assessed by the conversion of H-3 -L-arginine to H-3-L-citrulline in homogenates of anococcygeus muscles obta ined from 8-week diabetic rats and control rats. NOS activity measured in a ll tissue samples was dependent on the presence of calcium (2 mM), NADPH (1 mM), tetrahydrobiopterin (100 mu M) and flavin adenine dinucleotide (10 mu M); however, removal of calmodulin (50 U/ml) did not reduce L-citrulline p roduction. Both N-G-nitro-L-arginine (100 mu M) and N-G-nitro-L-arginine me thyl ester (100 mu M) produced significant inhibition of enzyme activity. N OS activity measured in tissue samples from diabetic rats (369.6 +/- 75.9 f mol L-citrulline/mg protein) did not significantly differ from that measure d in samples from control rats (423.9 +/- 110.6 fmol L-citrulline/mg protei n). However, NOS activity measured after removal of the cofactor tetrahydro biopterin, was significantly greater in samples from control rats than that from the diabetic group. NOS-immunoreactive and NADPH-diaphorase reactive nerve terminals were found to be sparsely distributed throughout longitudin al sections or whole mounts of anococcygeus muscles from both control and d iabetic rats. Quantification of NADPH-diaphiorase positive fibres intersect ing transects of whole tissue mounts, revealed no significant difference in fibre number between the treatment groups. All NOS-immunoreactive fibres a lso showed vasoactive-intestinal-polypeptide immunoreactivity. In conclusio n, the findings together provide no evidence to indicate that diabetes can induce prejunctional changes in NOS activity or localisation, concurrent wi th the reported postjunctional impairment in smooth muscle reactivity to ni tric oxide, in the rat anococcygeus muscle. (C) 1999 Elsevier Science B.V. All rights reserved.