ULTRASTRUCTURAL-LOCALIZATION OF NITRIC-OXIDE SYNTHASE IN CANINE SMALL-INTESTINE AND COLON

The ultrastructural distribution and subcellular localization of nitri c oxide synthase (NOS) immunoreactivity and its possible colocalizatio n with vasoactive intestinal polypeptide (VIP) and substance P in the muscularis externa in canine ileum and colon were studied by using pol yclonal antisera raised against VIP, substance P, and cerebellar NOS. Immunogold staining, with or without silver enhancement, was carried o ut directly on ultrathin sections using single and two-faced double im munogold methods. NOS immunoreactivity was observed in nerve profiles in myenteric plexus and circular muscle layer. Immunoreactivity was oc casionally detected in smooth muscle cells and interstitial cells of C ajal. The double immunostaining revealed NOS and VIP in the same nerve varicosities but never in the same organelles. NOS was localized in e lectron-dense material of undetermined nature, whereas VIP was associa ted with large granular vesicles. Substance P and NOS were never found in the same nerves. These results indicate that NOS is present in the enteric nerves containing VIP but in different organelles and that ni tric oxide release probably does not occur by an exocytotic mechanism.