COLOCALIZATION OF NADPH-DIAPHORASE STAINING AND VIP IMMUNOREACTIVITY IN NEURONS IN OPOSSUM INTERNAL ANAL-SPHINCTER

Nitric oxide and vasoactive intestinal polypeptide (VIP) are important inhibitory neurotransmitters mediating relaxation of the internal ana l sphincter. The location and coexistence of these two neurotransmitte rs in the internal anal sphincter has not been examined. We performed a double-labeling study to examine the coexistence of nitric oxide syn thase and VIP in the opossum internal anal sphincter using the NADPH-d iaphorase technique which is a histochemical stain for nitric oxide sy nthase. In perfusion-fixed, frozen-sectioned tissue, VIP-immunoreactiv e neurons were labeled using immunofluorescence histochemistry. After photographing the VIP-immunoreactive neurons, nitric oxide synthase wa s labeled using the NADPH-diaphorase technique. Ganglia containing neu ronal cell bodies were present in the myenteric plexus for the entire extent of the internal anal sphincter. VIP-immunoreactive and NADPH-di aphorase-positive neurons were present in ganglia in the myenteric as well as the submucosal plexuses. Most of the VIP-immunoreactive neuron s were also NADPH-diaphorase positive. VIP and nitric oxide synthase a re present and frequently coexist in neurons in the internal anal sphi ncter of the opossum. These neurons may be an important source of inhi bitory innervation mediating the rectoanal reflex-induced relaxation o f the sphincter. The demonstration of the coexistence of these two neu rotransmitters will be of fundamental importance in unraveling their r elationship and interaction in the internal anal sphincter as well as other systems.