The neuronal form of the enzyme nitric oxide synthase (nNOS) synthesizes the messenger molecule nitric oxide (NO). In addition to NO formation, nNOS exhibits a so-called NADPH-diaphorase (NADPH-d) activity. This study focused on the characterization of NADPH-d activity with regard to NO formation in the rat olfactory bulb. In this area of the brain pronounced staining is localized in discrete populations of neuronal somata and in olfactory glomeruli. Diaphorase staining combined with demonstration of nNOS by polyclonal antibodies revealed that NADPH-d activity of neuron somata is associated with nNOS immunoreactivity. It is concluded that neuron somata exhibit NADPH-d activity of nNOS. NADPH-d activity of nNOS did not utilize beta-NADH or alpha-NADPH. Moreover, NADPH-d activity was inhibited in the presence of alpha-NADPH. Dichlorophenolindophenol (DPIP), an artificial electron acceptor and an inhibitor of NO formation, totally suppressed NADPH-d staining of neurons, supporting the concept that the NADPH-d of neuron somata is due to nNOS. Cytochrome C, miconazole, EGTA, and trifluoperazine, which have been reported to inhibit cytochrome P450 reductase activity of NOS, did not affect NADPH-d staining. Hence, NADPH-d activity of NOS does not involve cytochrome P450 reductase activity as required for NO formation. Contrary to NADPH-d activity of neuron somata, staining of olfactory glomeruli was not co-localized with nNOS immunoreactivity. Glomerular staining was also observed in the presence of beta-NADH and alpha-NADPH. Further, it was unchanged in the presence of the NO formation inhibitor DPIP. Hence, the glomerular staining in the presence of NADPH is not due to the NADPH-d activity of NOS. We conclude that staining of neuronal structures in the presence of NADPH does not necessarily represent NADPH-d activity of NOS.
Fixation conditions affect the intensity but not the pattern of NADPH-diaphorase staining as a marker for neuronal nitric oxide synthase in rat olfactory bulb.
