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.

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PYY receptors were characterized and their loci determined in canine small intestine. The density of 125I-labeled peptide tyrosine tyrosine (PYY) binding was highest in myenteric (MY) and submucosal (SUB) plexus fractions enriched in synaptosomes. Two binding sites [high affinity (H) and low affinity (L)] were found in the submucosal synaptosome-enriched membrane: dissociation constant (Kd)H = 7.6 pM, maximal binding capacity (Bmax)H = 28 fmol/mg; KdL = 0.18 nM, BmaxL = 120 fmol/mg protein. The binding of 125I-PYY reached a maximum within 30 min; dissociation was incomplete in the presence of unlabeled PYY. The rate of dissociation was enhanced after exposure of synaptosomes to guanosine 5'-O-(3-thiotriphosphate). Binding of 125I-PYY was completely inhibited by neuropeptide Y (NPY)-(13-36) (in SUB and MY) and by [Leu31,Pro34]NPY (in MY) but only partially by [Leu31,Pro34]NPY in SUB, suggesting the presence of Y2 receptor in SUB and the presence of Y1 and Y2 receptors in MY. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PYY receptor complex revealed a radioactive band at 70 kDa. The PYY receptors in the canine small intestinal myenteric and submucosal plexus correspond in location to that of PYY in synaptosomes and are coupled with G proteins. Different subtypes are present in different loci.

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