L-NNA INHIBITS THE HISTOCHEMICAL NADPH-D REACTION IN RAT SPINAL-CORD NEURONS

In nerve tissue the histochemical nicotinamide adenine dinucleotide ph osphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown tha t the NOS-specific inhibitor L-nitroarginine (L-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; s uch a reaction might serve as a control for the presence of a NOS-rela ted catalytic activity, i.e., L-NNA-dependent NO synthesis in these ne urons. However, L-NNA inhibition of neuronal NADPH-d is inconsistent a nd is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibit ion experiments. In IML neurons of formaldehyde-fixed spinal cord tiss ue, inhibition of NADPH-d reaction was tested by preincubation of froz en sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 mu M-l mM) which blocked the NADPH-d reaction in a concentration-d ependent way, suggesting an inverse relationship of inhibitor concentr ation and final reaction product generated. Preincubation with the NOS -specific inhibitor L-NNA in glycine-NaOH buffer (pH 8.5-9.5) but not L-nitroarginine methyl ester (L-NAME) revealed a concentration-depende nt blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of a L-NNA sensitive NADPH-d activi ty. Blocking with L-NNA (100 mu M-10 mM) was prevented by excess L-arg inine (10-100 mM), suggesting competitive binding sites. NADPH-d stain ing was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus , in formaldehyde-fixed nervous tissue both DPI and L-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-de pendent conversion of nitroblue tetrazolium to formazan.