HISTOCHEMICAL METHODS FOR DETECTING NITRIC-OXIDE SYNTHASE

The three isoforms of nitric oxide synthase (NOS), neuronal (nNOS), en dothelial (iNOS), and inducible (iNOS), can be visualized in cells and tissues by NADPH-diaphorase (NADPH-d) histochemistry, immunocytochemi stry and in situ hybridization. Histochemical demonstration of NADPH-d shows the formazan final reaction product as a solid blue deposit. Th e ultrastructural localization of NADPH-d in the rat hippocampus showe d an electron-dense deposit on membranes predominantly of the endoplas mic reticulum. The immunohistochemical demonstration of nNOS, using th e nickel enhancement technique, shows positive reaction product over t he dendrites and the soma of the nerve cell in the rat brain. Ultrastr uctural localization of nNOS in whole mount preparations of myenteric plexus and circular smooth muscle from guinea-pig ileum shows that NOS immunoreactivity was patchily distributed in myenteric neurones and w as not specifically associated with any intracellular organelles or wi th plasma membranes. In situ hybridization, using radio-labelled probe s, was used to study nNOS mRNA in lumbar dorsal root ganglia after per ipheral transection of the sciatic nerve in rats. Labelling of the NOS mRNA-positive neurones is observed as a series of dense granules over the entire cell. NADPH-d histochemistry, immunocytochemistry and in s itu hybridization each have a significant role to play in the localiza tion of NOS. NADPH-d detects an enzyme associated with the NOS molecul e, immunocytochemistry detects the NOS molecule, and in situ hybridiza tion detects mRNA for NOS. Therefore, if each of these techniques is a pplied in carefully controlled experiments, consideration of the accum ulated data should be valuable in revealing insights into the biology of NOS.