IMMUNOHISTOCHEMICAL LOCALIZATION OF 3',5'-CYCLIC GUANOSINE-MONOPHOSPHATE IN THE CANINE PROXIMAL COLON - RESPONSES TO NITRIC-OXIDE AND ELECTRICAL-STIMULATION OF ENTERIC INHIBITORY NEURONS

There is growing evidence that nitric oxide serves as a neurotransmitt er released from enteric inhibitory nerves in the gastrointestinal tra ct. The distribution of nitric oxide synthase suggests that nitric oxi de may also be a neurotransmitter within enteric ganglia. Since many a ctions of nitric oxide are mediated by stimulation of soluble guanylat e cyclase and a subsequent increase in 3',5'-cyclic guanosine monophos phate (cGMP) concentration, targets for nitric oxide in the canine pro ximal colon were investigated by immunohistochemical localization of c GMP. In the presence of phosphodiesterase inhibitors (M&B 22948, 100 m uM and 3-isobutyl-1-methyl-xanthine, 1 mM), exogenous nitric oxide and electrical field stimulation caused an accumulation of cGMP-like immu noreactivity in several cell-types including colonic smooth muscle cel ls. cGMP-like immunoreactivity was also observed in a subpopulation of neurons in both myenteric and submucosal ganglia. Sequential labeling with the NADPH diaphorase technique showed that 94% of neurons that r esponded to exogenous nitric oxide with an increase in cGMP-like immun oreactivity were NADPH diaphorase negative. None of the myenteric neur ons that responded to electrical field stimulation with an increase in cGMP-like immunoreactivity were NADPH diaphorase positive, and only o ne submucosal neuron with cGMP-like immunoreactivity was also NADPH di aphorase positive. The electrical field-stimulated increase in cGMP-li ke immunoreactivity was blocked by nitroarginine (100 muM). An increas e in cGMP-like immunoreactivity also occurred in interstitial cells lo cated at the submucosal surface of the circular muscle layer. These ce lls are interposed between nerve varicosities and smooth muscle cells and may partially mediate neuromuscular transmission. Sodium nitroprus side and nitric oxide also caused an accumulation of cGMP-like immunor eactivity in smooth muscle cells of intramural arterioles and venules. The results of this study further support the role of nitric oxide as a neurotransmitter in colonic muscles, and provide support for the hy pothesis that interstitial cells are functionally innervated by enteri c inhibitory neurons. The data also suggest that nitric oxide may serv e as a neurotransmitter in enteric ganglia.