L-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons.
In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitor L-nitroarginine (L-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord: such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e., L-NNA-dependent NO synthesis in these neurons. However, L-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 microM-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration 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-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of a L-NNA sensitive NADPH-d activity. Blocking with L-NNA (100 microM-10 mM) was prevented by excess L-arginine (10-100 mM), suggesting competitive binding sites. NADPH-d staining 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-dependent conversion of nitroblue tetrazolium to formazan.
Blottner
D
D
Institut für Anatomie, Freie Universität Berlin, Germany.
Baumgarten
H G
HG
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D005629:Frozen Sections; D006651:Histocytochemistry; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors*; D009474:Neurons / Q000201:enzymology*; D019335:Nitroarginine; D051381:Rats; D017208:Rats, Wistar; D013116:Spinal Cord / Q000166:cytology* / Q000201:enzymology
D019335:Nitroarginine; D001120:Arginine; D009252:NADPH Dehydrogenase