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

Abstract

Functional studies have revealed diabetes specifically impairs smooth muscle reactivity to nitric oxide in the rat anococcygeus muscle. The present study was conducted to examine whether concurrent prejunctional defects in nitrergic neurotransmission exist in anococcygeus muscles from diabetic rats. Nitric oxide synthase (NOS) activity was assessed by the conversion of 3 H - l-arginine to 3 H - l-citrulline in homogenates of anococcygeus muscles obtained from 8-week diabetic rats and control rats. NOS activity measured in all tissue samples was dependent on the presence of calcium (2 mM), NADPH (1 mM), tetrahydrobiopterin (100 μM) and flavin adenine dinucleotide (10 μM); however, removal of calmodulin (50 U/ml) did not reduce l-citrulline production. Both N G-nitro- l-arginine (100 μM) and N G-nitro- l-arginine methyl ester (100 μM) produced significant inhibition of enzyme activity. NOS activity measured in tissue samples from diabetic rats (369.6±75.9 fmol l-citrulline/mg protein) did not significantly differ from that measured in samples from control rats (423.9±110.6 fmol l-citrulline/mg protein). However, NOS activity measured after removal of the cofactor tetrahydrobiopterin, 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 longitudinal sections or whole mounts of anococcygeus muscles from both control and diabetic rats. Quantification of NADPH-diaphorase positive fibres intersecting transects of whole tissue mounts, revealed no significant difference in fibre number between the treatment groups. All NOS-immunoreactive fibres also showed vasoactive-intestinal-polypeptide immunoreactivity. In conclusion, the findings together provide no evidence to indicate that diabetes can induce prejunctional changes in NOS activity or localisation, concurrent with the reported postjunctional impairment in smooth muscle reactivity to nitric oxide, in the rat anococcygeus muscle.