NADPH-DIAPHORASE ACTIVITY OF NITRIC-OXIDE SYNTHASE IN THE OLFACTORY-BULB - COFACTOR SPECIFICITY AND CHARACTERIZATION REGARDING THE INTERRELATION TO NO FORMATION

The neuronal form of the enzyme nitric oxide synthase (nNOS) synthesiz es the messenger molecule nitric oxide (NO). In addition to NO formati on, nNOS exhibits a so-called NADPH-diaphorase (NADPH-d) activity. Thi s study focused on the characterization of NADPH-d activity with regar d 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 d emonstration of nNOS by polyclonal antibodies revealed that NADPH-d ac tivity of neuron somata is associated with nNOS immunoreactivity. It i s 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. Dichl orophenolindophenol (DPIP), an artificial electron acceptor and an inh ibitor of NO formation, totally suppressed NADPH-d staining of neurons , supporting the concept that the NADPH-d of neuron somata is due to n NOS. Cytochrome C, miconazole, EGTA, and trifluoperazine, which have b een 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 formatio n. Contrary to NADPH-d activity of neuron somata, staining of olfactor y glomeruli was not co-localized with nNOS immunoreactivity. Glomerula r staining was also observed in the presence of beta-NADH and alpha-NA DPH. further, it was unchanged in the presence of the NO formation inh ibitor DPIP. Hence, the glomerular staining in the presence of NADPH i s not due to the NADPH-d activity of NOS. We conclude that staining of neuronal structures in the presence of NADPH does not necessarily rep resent NADPH-d activity of NOS.