In situ measurement of neuronal nitric oxide synthase activity in the spinal cord by NADPH-diaphorase histochemistry.
NADPH-diaphorase (NADPH-d) histochemistry has provided a simple method to stain neuronal nitric oxide synthase (nNOS)-containing neurons in the central nervous system. In the spinal cord, NO formation following activation of N-methyl-D-asparate (NMDA) receptors plays a crucial role in nociceptive processing. To investigate the molecular mechanisms, we attempted to evaluate nNOS activity in situ using isolated intact spinal cord preparation and NADPH-d histochemistry. NADPH-d activity in the superficial layer of the spinal cord increased gradually with ages from P10 to P30 and NMDA enhanced the NADPH-d staining in a time- and concentration-dependent manner. The NMDA-stimulated NADPH-d staining was inhibited by NMDA receptor antagonists, but not by non-NMDA and metabotropic glutamate receptor antagonists. The NADPH-d staining showed a pronounced stereospecificity for beta-NADPH and completely suppressed by dichlorophenolindophenol, an artificial electron acceptor. NMDA-evoked NO formation in the spinal cord was confirmed by the fluorescent NO indicator diaminofluorescein-FM (DAF-FM). These results demonstrate that NADPH-d activity in the superficial spinal cord is ascribed to nNOS activity and is dependent on NMDA. A combination of isolated intact spinal cord preparations and NADPH-d histochemistry may provide a unique system to elucidate biochemical and molecular mechanisms for nNOS activation in the spinal cord.
Xu
Li
L
Department of Medical Chemistry, Kansai Medical University, Moriguchi, 570-8506 Osaka, Japan.
Matsumura
Shinji
S
Mabuchi
Tamaki
T
Takagi
Kunio
K
Abe
Testuya
T
Ito
Seiji
S
D000818:Animals; D004789:Enzyme Activation / Q000502:physiology; D006651:Histocytochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D009252:NADPH Dehydrogenase / Q000032:analysis*; D009474:Neurons / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D009924:Organ Culture Techniques; D013116:Spinal Cord / Q000378:metabolism*
D052248:Nitric Oxide Synthase Type I; D009252:NADPH Dehydrogenase