Location and characteristics of nitric oxide synthase in sheep spinal cord and its interaction with alpha(2)-adrenergic and cholinergic antinociception

Background: Nitric oxide synthase is located in the spinal cord dorsal horn and intermediolateral cell column, where it may modulate sensory and sympathetic neuronal activity. However, the biochemical characteristics of this enzyme have not been examined in these different areas in the spinal cord. Although alpha(2)-adrenergic agonists, muscarinic agonists, and nitric oxide may interact in the spinal cord to produce antinociception, these interactions have not been characterized. Methods: Sheep spinal cord tissue was homogenized and centrifuged at high speed to separate soluble and membrane-bound fractions. Nitric oxide synthase activity was determined by conversion of [C-14]-L-arginine to [C-14]-L-citrulline and its kinetic characteristics, dependency on cofactors, and sensitivity to inhibitors determined. Sheep spinal cord was stained for nicotinamide adenine dinucleotide phosphate diaphorase as a marker for nitric oxide synthase. Antinociception to a mechanical stimulus from intrathecal clonidine alone and with neostigmine was determined and the effects of L-arginine and n-methyl-L-arginine were determined. Results: More than 85% of nitric oxide synthase activity was present in the soluble form and its kinetic, cofactor, and antagonist properties were similar to those of the neuronal isoform of nitric oxide synthase, Biochemical and histochemical studies localized nitric oxide synthase to the superficial dorsal horn and the intermediolateral cell column. Clonidine antinociception was enhanced by L-arginine and neostigmine, but not by D-arginine. Neostigmine's enhancement of clonidine antinocieption was blocked by n-methyl-L-arginine. Conclusions: These results confirm those of previous studies demonstrating localization of nitric oxide synthase to superficial dorsal horn and intermediolateral cell column of mammalian spinal cord, and suggesting its identity as the neuronal isoform. Spinal alpha(2)-adrenergic agonist antinociception may be partly dependent on cholinergic and nitric oxide mechanisms.