NADPH DIAPHORASE STAINING SUGGESTS LOCALIZATION OF NITRIC-OXIDE SYNTHASE WITHIN MATURE VERTEBRATE OLFACTORY NEURONS

Nitric oxide, a simple gas which serves as a neurotransmitter in the C NS, has been proposed to serve as an interneuronal second messenger in olfactory transduction. However, the role of nitric oxide in olfactio n has been questioned by experiments in which nitric oxide synthase, t he enzyme that generates nitric oxide, could not be localized to the o lfactory epithelium. We have localized nitric oxide synthase to the ol factory neurons in adult rat and catfish olfactory epithelia using a m odified nicotinamide adenine dinucleotide phosphate diaphorase techniq ue. In the rat, staining was also found in cells with morphology remin iscent of microvillar olfactory cells. In contrast, the respiratory ep ithelium and the sustentacular cells in the olfactory epithelium displ ayed no staining. The nicotinamide adenine dinucleotide phosphate diap horase reaction, which has been shown to co-localize with immunohistoc hemical staining for nitric oxide synthase in the brain, was stimulate d by addition of the nitric oxide synthase substrate L-arginine, and w as inhibited by the nitric oxide synthase inhibitor L-N-G-nitro argini ne, indicating that staining was specific for nitric oxide synthase. U nilateral bulbectomy, which causes degeneration of mature olfactory ne urons on the bulbectomized side, markedly reduced nicotinamide adenine dinucleotide phosphate diaphorase staining. These observations were s ubstantiated by biochemical assays for nitric oxide synthase by monito ring the production of [H-3]-L-citrulline from [H-3]-L-arginine. This is the first demonstration of specific NADPH diaphorase staining of ma ture olfactory neurons in rat and catfish olfactory epithelia suggesti ng the presence of nitric oxide synthase in these cells. Our histologi cal and biochemical Endings, in conjunction with data from other resea rch, are supportive of a role for nitric oxide synthase in olfactory f unction.