IMMUNOHISTOCHEMICAL LOCALIZATION OF PERIPHERAL NITRIC-OXIDE SYNTHASE-CONTAINING NERVES USING ANTIBODIES RAISED AGAINST SYNTHESIZED C-TERMINAL AND N-TERMINAL FRAGMENTS OF A CLONED ENZYME FROM RAT-BRAIN

Antibodies were raised in rabbits against C- or N-terminal fragments o f a cloned nitric oxide synthase (NOS) enzyme from rat cerebellum, and used for demonstration of NOS-immunoreactive (NOS-IR) nerves in diffe rent tissues from the rat (colon, duodenum, adrenal gland, aorta, cava l vein, penis and urethra). Both antisera demonstrated the same neuron al elements, although with differences in intensity in the immunoreact ion in some tissues. Sections incubated with antisera preabsorbed with excess of the antigens showed no NOS immunoreactivity. In duodenum an d colon, NOS-immunoreactivity was found in the cytoplasm of numerous c ell bodies in myenteric ganglia and in some nerve cell bodies in the s ubmucosa. NOS-IR nerve fibres were numerous in the circular muscle lay er, while few were found in the longitudinal layer or the mucosa and s ubmucosa. In the penis, strong NOS immunoreactivity was found in nerve s surrounding the deep penile and dorsal arteries, and in nerves in th e stroma of the cavernous tissue. In the urethra, NOS immunoreactivity was found in nerves in the mucosa. No NOS immunoreactivity was found in the urothelium. The adrenal medulla, and occasionally the cortex, c ontained nerve cell bodies with strong cytoplasmic NOS immunoreactivit y as well as scattered nerve fibres. No NOS immunoreactivity was found in the abdominal aorta or inferior caval vein. Combined NOS immunosta ining and NADPH diaphorase staining showed that virtually all NOS-IR n erve structures were also NADPH diaphorase-positive. However, thin ner ve fibres and cell linings were sometimes better visualized by NOS-imm unohistochemistry. Furthermore, the adrenal cortex, which only occasio nally showed NOS immunoreactivity, was strongly NADPH diaphorase-posit ive. A positive NADPH diaphorase reaction, but a negative NOS immunore activity, was also found in other structures, such as urothelium, epit helial cells in duodenum and colon, and endothelium of some vessels. I t is concluded that the antibodies raised against the synthesized sequ ences of neuronal NOS are highly specific and may be used in immunohis tochemistry in order to detect neuronal NOS.