Segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal nitric oxide synthase-immunoreactive neurons versus radioassay detection of catalytic nitric oxide synthase activity in the rabbit spinal cord

Abstract

The distributions of neuronal nitric oxide synthase-immunoreactive neurons and of nicotinamide adenine dinucleotide phosphate-diaphorase activity were studied in the C6, Th2, L1, L5, S2 and S3 segments and laminae in the rabbit spinal cord and compared with the catalytic nitric oxide synthase activity, determined by monitoring the conversion of [ 3H]arginine to [ 3H]citrulline in the same segments and laminae. Morphologically, a heterogeneous population of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal nitric oxide synthase-immunoreactive neurons was detected in the superficial and deep dorsal horn and the pericentral region in all segments studied, and in the intermediolateral cell column of the thoracic and lumbosacral segments. A disproportionate distribution of both neuronal categories which had a significantly higher number of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing rather than neuronal nitric oxide synthase-immunoreactive cell bodies was found in all segments. The catalytic nitric oxide synthase activity was distributed unequally in the C6, Th2, L1, L5, S2 and S3 segments, with a comparatively low value in the Th2 segment (70±5.1 d.p.m./μg protein) in comparison with the S3 segment, where the highest level (140±5.5 d.p.m./μg protein) was found. A close correlation between the number of neuronal nitric oxide synthase-immunoreactive somata and catalytic nitric oxide synthase activity was revealed in the dorsal horn (laminae I–VI). Whereas a low number of neuronal nitric oxide synthase-immunoreactive somata in laminae VII–X was found in the L5, S2 and S3 segments, the values of catalytic nitric oxide synthase activity in the same laminae and segments were found to be exceedingly high. These findings indicate that the occurrence of many neuronal nitric oxide synthase-immunoreactive fibers (mainly axons), and dense, punctate, non-somatic neuronal nitric oxide synthase immunopositivity in the neuropil staining of the same laminae and segments, can substantially enhance catalytic nitric oxide synthase activity.