In situ measurement of neuronal nitric oxide synthase activity in the spinal cord by NADPH-diaphorase histochemistry |
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Authors: | Xu Li Matsumura Shinji Mabuchi Tamaki Takagi Kunio Abe Testuya Ito Seiji |
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Affiliation: | Department of Medical Chemistry, Kansai Medical University, Moriguchi, 570-8506 Osaka, Japan. |
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Abstract: | 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. |
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Keywords: | N-Methyl- smCaps" >d-asparate (NMDA) Nitric oxide synthase (NOS) Nitric oxide NADPH-diaphorase Spinal cord |
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