This study examined the role of nitric oxide (NO) in tonic inhibition of motor activity in isolated, perfused canine ileal segments. Brief addition of N omega-nitro-L-arginine methyl ester (L-NAME) to the perfusate caused, after a delay, a concentration-dependent persistent increase in tonic and phasic activity of circular muscle. This increased motor activity was prevented or reversed by addition of L- but not D-arginine to the perfusate. Removal of Ca2+ or addition of 10(-7) M omega-conotoxin (GVIA) to the perfusate markedly reduced this response. The motor activity induced by L-NAME was accompanied by loss of distal inhibition and enhanced excitation to low-frequency field stimulation. L-NAME infusion significantly reduced tonic vasoactive intestinal polypeptide (VIP) output, sodium nitroprusside increased VIP output, but L-arginine infusion did not restore VIP output. Atropine (10(-7) M) and/or hexamethonium (10(-4) M) reduced the motor response to L-NAME by 75%. Atropine reduced and hexamethonium nearly abolished VIP output. We conclude that there is tonic Ca(2+)-dependent NO output from perfused intestinal segments dependent on nerves with N-Ca channels, that NO acts to inhibit muscle directly and by inhibiting release of excitatory mediators, and that this output is the primary inhibitory determinant of contractile activity.

1 The degradation of tritiated and unlabelled neurotensin (NT) following close intra-arterial infusion of the peptides in ileal segments of anaesthetized dogs was examined. 2 Intact NT and its catabolites recovered in the venous effluents were purified by chromatography on Sep-Pak columns followed by reverse-phase h.p.l.c. and identified by their retention times or by radioimmunoassay. 3 The half-life of neurotensin was estimated to be between 2 and 6 min. Four labelled catabolites, corresponding to free tyrosine, neurotensin (1-8), neurotensin (1-10) and neurotensin (1-11), were detected. 4 Neurotensin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) was mainly generated by a phosphoramidon-sensitive cleavage, probably elicited by endopeptidase 24-1. 5 Two endopeptidase 3.4.24.16 inhibitors, phosphodiepryl 03 and the dipeptide Pro-Ile, dose-dependently potentiated the recovery of intact neurotensin. Furthermore, both agents inhibited the formation of neurotensin (1-10), the product that results from the hydrolysis of neurotensin by purified endopeptidase 3.4.24.16. In contrast, the endopeptidase 3.4.24.15 inhibitor Cpp-AAY-pAB neither protected neurotensin from degradation nor modifed the production of neurotensin (1-10). 6 Our study is the first evidence to indicate that endopeptidase 3. Experimental procedureAn equilibration period preceded the start of each experiment until the venous effluent became free of haematies by inspection. Dog ileum segments were pretreated for 10 min by perfusion of Krebs-Ringer bicarbonate buffer either without (control) or with freshly prepared solutions of peptidase inhibitors before neurotensin injection. At zero time, a mixture of tritiated (106 c.p.m., 107 Ci mmol ') and unlabelled (10 nmol) neurotensin was injected in 1 ml. After 20 s, the peristaltic pump was stopped for 2 min in order to facilitate neurotensin diffusion inside ileal tissues, then the perfusion was continued for a period of 8 min (from injection). Perfusates corresponding to 2 min time intervals were collected. Venous effluents were passed through ODS-Silica cartridges (C18 Sep-pak, Millipore) that had been previously activated by several washes with acetonitrile (2 x 5 ml) then with 4 x 5 ml of 0.1% (v/v) trifluoroacetic acid (TFA), 0.05% (v/v) triethylamine (TEA) in water. After loading of samples, resin was washed with 0.1% (v/v) TFA, 0.05% (v/v) TEA in water, then peptides were eluted with 4 ml of 80% acetonitrile containing 0.1% (v/v) TFA, 0.05% (v/v) TEA. More than 90% of the radioactivity applied was recovered after the various step-elutions. Eluates were Iyophilized and reconstituted in 2 ml of water containing 0.02% bovine serum albumen. Aliquots containing about 6000 c.p.m. were submitted to reverse-phase h.p.l.c.Reverse-phase h.p.l.c. analysisThe h.p.l.c. procedure has been described previously . Briefly, aliquots of reconstituted samples were loaded onto a RP18 lichrosorb column (Merck, France) and eluted at room temperature. Elutions were performed at a flow rate of 1 ml min-' and absorbing material was...

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Glucagon-like peptide-1 (GLP-1) modulates glucose levels following a meal, including by inhibition of gastric emptying and intestinal transport. Intra-arterial injection of GLP-1 into the gastric corpus, antrum, or pylorus of anesthetized dogs had no effect on the contractile activity of the resting or neurally activated stomach. GLP-1 injected intra-arterially inhibited intestinal segments when activated by enteric nerve stimulation but not by acetylcholine. Isolated ileum segments were perfused intra-arterially, instrumented with strain gauges to record circular muscle activity and with subserosal electrodes to stimulate enteric nerves. GLP-1 caused concentration-dependent inhibition of nerve-stimulated phasic but not tonic activity. This was absent during TTX-induced activity and partly prevented by N G-nitro-l-arginine. Exendin-(9—39), the GLP-1 antagonist, had no intrinsic activity and did not affect the actions of GLP-1. Capsaicin mimicked the effects of GLP-1 and may have reduced the effect of subsequent GLP-1. GLP-1 may mediate paracrine action on afferent nerves in the canine ileal mucosa using an unusual receptor.