DETECTION OF NITRIC-OXIDE RELEASE FROM CANINE ENTERIC NEURONS

Previous pharmacological and immunohistochemical studies have suggeste d that nitric oxide (NO) is a mediator of enteric neuromuscular transm ission in the canine proximal colon. The present study demonstrates th e release of nitric oxide (NO) and/or its oxidation products (collecti vely termed NOx) following stimulation of intrinsic neurons with the n icotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP). Str ips of muscularis externa, including the ganglionated myenteric and su bmucosal plexuses were suspended under tension in modified Krebs solut ion. DMPP (1-100 mu M) produced concentration-dependent inhibition of circular muscle contractile activity and this effect was antagonized b y L-nitroarginine methyl ester or L-nitroarginine(100 mu M). Tetrodoto xin (TTX, 1 mu M) significantly reduced mechanical responses to DMPP ( 10 mu M) but had no effect on responses to high concentrations of DMPP (100 mu M) Aliquots of the bathing medium were assayed for NOx after regeneration of NO from NO; and/or NO3-;. NO was measured as chemilumi nescence produced by reaction with ozone. Detection was linear over th e range 6-25000 pmol of added NO2- or NO3-. In the absence of drugs, a basal release of 1113.5+/-100.4 pmol NOx/g tissue (n = 27) was detect ed after a 30-min incubation period. DMPP (1-100 mu M) stimulated a co ncentration-dependent increase in NOx to 5099.9+/-430 pmol/g per 30 mi n (n = 17) at 100 mu M. NOx release was inhibited by an inhibitor of n itric oxide synthase activity (N-G-monomethyl-L-arginine) or by reduct ion in extracellular Ca2+ concentration. DMPP-stimulated NOx accumulat ion was significantly reduced, but not abolished by TTX (1 mu M). Thes e results provide further evidence that NO is released following stimu lation of intrinsic neurons in canine proximal colon and support previ ous suggestions that nicotinic agonists may directly stimulate termina ls of NO-releasing neurons.