Nitric oxide synthase (NOS), argininosuccinate synthetase (ASS), and argininosuccinate lyase (ASL) compose a cyclic pathway to form nitric oxide (NO). These enzymes, however, are localized differentially in most regions of the brain. To find out whether NOS, ASS, and ASL are colocalized in neurons of the spinal cord, we examined the distribution of these enzymes by using a double-labeling procedure combining fluorescent immunohistochemistry with an assay for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d).
Results indicate that neurons in the dorsal horn, the intermediolateral nucleus, and the central canal region were NADPH-d active (1) and NOS-, ASS-, and ASL-like immunoreactive (-LI). In laminae II and III of the dorsal horn, some NADPH-d (1) neurons were ASL-LI (8-30%) but only a few were ASS-LI (0.5-7%). In the nucleus intermediolateralis, a large portion of NADPH-d (1) neurons were ASL-LI (30-60%), whereas only a small portion of NADPH-d (1) neurons were ASS-LI (10-20%). In the central canal region, some NADPH-d (1) neurons were ASL-LI (15-40%), and a few NADPH-d (1) neurons were ASS-LI (3-16%). Thus, the results suggest that, in the nucleus intermediolateralis and the central canal region, NOS, ASS, and ASL are colocalized and form a cyclic pathway to produce NO, whereas, in the dorsal horn, these enzymes are more characteristically localized in different neurons, which may transport the substrates intercellularly.