NADPH diaphorase and nitric oxide synthase colocalization in enteric neurons of canine proximal colon

Abstract

Considerable evidence has recently been presented that suggests that nitric oxide (NO) is a nonadrenergic noncholinergic (NANC) neurotransmitter in gastrointestinal tissues. One of the criteria that must be satisfied before this hypothesis can be accepted is that enteric neurons must be shown to contain the enzymatic apparatus necessary to synthesize NO. Specific antibodies have been developed for NO synthase (NOS) isolated from rat cerebellum, and studies have shown that NOS copurifies and colocalizes with NADPH diaphorase activity, a commonly used neural marker. We used antibodies raised against the cerebellar NOS to determine the distribution of NOS-like immunoreactivity (NOS-LI) in enteric neurons of the canine proximal colon. We also tested whether NADPH diaphorase staining would label the population of neurons containing NOS-LI in this species. A subpopulation of neurons in myenteric and submucosal ganglia displayed NOS-LI and were colabeled with NADPH diaphorase. Labeled neurons had morphological characteristics similar to the Dogiel type I morphology. Cryostat sections showed NOS-positive nerve trunks throughout the circular and longitudinal muscle layers, but a high density of NOS-LI was observed within the submucosal pacemaker region, as predicted from physiological studies. These studies provide the first morphological support for the hypothesis that NO serves as a NANC neurotransmitter in the canine colon. The study also shows that the NADPH diaphorase reaction provides a useful method to label cells with NOS-LI.

Nitric oxide synthase in the enteric nervous system of the rainbow trout, Salmo gairdneri .

“…Thus, NOS immunoreactivity and NADPH diaphorase enzyme activity correspond 100% in the myenteric neurons of the trout as they do in mammals, amphibians and birds (BELAI et al, 1992;LI et al, 1992LI et al, , 1993WARD et al, 1992;YOUNG et al, 1992). There are several possible explanations for the lack of NOS immunoreactivity in some nerve cell bodies of the vagus.…”

“…In the central and enteric nervous system of mammals, NOS immunoreactivity is co-localized with NADPH diaphorase staining, and it has been suggested that NADPH diaphorase reactivity reveals the NADPH binding site of NOS (DAWSON et al, 1991;HOPE et al, 1991;BELAI et al, 1992;WARD et al, 1992;YOUNG et al, 1992). A similar one-to-one correlation between the NOS immunoreactivity and NADPH diaphorase staining has been noted to occur in neurons of the myenteric and submucous plexuses of the gastrointestinal tract of both an amphibian and a bird (LI et al, , 1993.…”

“…Thus, the presence of nitric oxide synthase (NOS) in neurons is strong evidence that the neurons utilise nitric oxide as a transmitter. NOS has been demonstrated to occur in enteric neurons of mammals COSTA et al, 1992;FURNESS et al, 1992;WARD et al, 1992;YOUNG et al, 1992) and has recently been localized in enteric neurons of an amphibian ) and a bird (LI et al, 1993).…”

Nitric oxide, and not vasoactive intestinal peptide, as the main neurotransmitter of vagally induced relaxation of the guinea pig stomach

“…This has been confirmed in a number of studies by immunocytochemical localization of nitric oxide synthase (NOS), the enzyme catalysing the formation of NO from endogenous L-arginine, to neurones of the myenteric plexus in the guinea pig and rat ileum, rat stomach, duodenum, canine proximal colon and the human gut (Bredt et al, 1990;Schmidt et al, 1992;Springall et al, 1992;Ward et al, 1992;Forster & Southam, 1993). In the guinea pig ileum, NOS-immunoreactive neurones in the myenteric plexus synapse with other neurones and innervate the circular muscle (Llewellyn-Smith et al, 1992).…”

“…In the guinea pig ileum, NOS-immunoreactive neurones in the myenteric plexus synapse with other neurones and innervate the circular muscle (Llewellyn-Smith et al, 1992). In the rat duodenum and canine proximal colon the NOS-positive neurones have a Dogiel type I morphology and axons going towards the muscle, which is characteristic of motor neurones (Ward et al, 1992;Aimi et al, 1993). In addition, NOS has been localized to the vagal efferent fibres innervating the rat stomach (Forster & Southam, 1993).…”