1 The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied.2 An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP638, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 MM.3 PACAP638 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIPIO 28 at 3 Mm, which inhibited the relaxation by 44%.4 An antagonist of large conductance Ca2+-activated K+ channels, charybdotoxin, concentrationdependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM.5 An antagonist of small conductance Ca2 +-activated K+ channels, apamin, concentration-dependently inhibited the relaxation (58% at 1 gM).6 Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS-induced relaxation, which is comparable to the extent of inhibition induced by PACAP638 plus VIPI0O28. 7 The inhibitory effect of VIP1028 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6-38 and charybdotoxin, but not apamin. 8 Exogenously added VIP (100 nM-1 Mm) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP-induced relaxation. VIPI0O28, but not PACAP638 selectively inhibited the VIP-induced relaxation.9 Exogenously added PACAP (10-100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP-induced relaxation. PACAP638, but not VIPI0O28 selectively inhibited the PACAP-induced relaxation.10 Apamin at 100 nM inhibited inhibitory junction potentials (ij.ps) induced by a single pulse of EFS. Apamin also inhibited a rapid phase, but not a delayed phase of ij.ps induced by two pulses at 10 Hz. VIPIO28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIPIO28 abolished the ij.ps induced by two pulses.11 Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP-, but not VIP-induced hyperpolarization. 12 From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin-and apaminsensitive K+ channels, respectively.

The mediators of non‐adrenergic non‐cholinergic (NANC) relaxation of the longitudinal muscle of rat proximal, middle and distal colon were examined in vitro. Electrical transmural stimulation (TMS) of proximal, middle and distal segments of rat colon induced NANC relaxations which were inhibited by tetrodotoxin (1 μm), but not by atropine (1 μm) or guanethidine (4 μm). In the proximal colon, l‐nitro‐arginine (N5‐nitroamidino‐l‐2,5‐diaminopentanoic acid) inhibited the TMS‐induced NANC relaxation and l‐arginine (1 mm) reversed this inhibition. Nitric oxide (0.3–10 μm) induced relaxation of the proximal segment. NANC relaxation of the proximal segments was still evident after desensitization to vasoactive intestinal peptide (VIP). A VIP antagonist (VIP 10–28, 10 μm) had no effect on the TMS‐induced NANC relaxation, which was also resistant to α‐chymotrypsin (2 units ml−1) and a substance P antagonist ([d‐Pro2, d‐Trp7,9]substance P, 1 μm). In the middle colon, l‐nitro‐arginine did not inhibit the TMS‐induced NANC relaxation in 6 of 9 preparations tested and partially inhibited the relaxation in the other 3 preparations. l‐Arginine did not reverse the partial inhibition. Complete desensitization to VIP was not achieved in the middle colon. The VIP antagonist had no effect on the TMS‐induced NANC relaxation. After α‐chymotrypsin treatment of the segment, desensitization of the segments to substance P, or in the presence of the substance P antagonist, the TMS‐induced NANC relaxation was augmented. In the distal colon, l‐nitro‐arginine did not have any significant effect on the TMS‐induced relaxation and nitric oxide did not induce relaxation. The VIP antagonist significantly inhibited TMS‐induced NANC relaxation. α‐Chymotrypsin‐treatment of the distal segments resulted in significant inhibition of NANC relaxation. No desensitization to substance P was achieved. Treatment with the substance P antagonist had no effect. These results suggest that nitric oxide is the mediator of the NANC inhibitory response in the proximal region of rat colon; in the middle colon, substance P acts as an excitatory neurotransmitter, antagonizing the NANC relaxation caused by the mediator of the response, which is still uncertain. Our results also suggest that VIP is the most likely candidate as a NANC transmitter in the distal colon.

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1 We studied the relation of nitric oxide-mediated relaxation of smooth muscle to changes in membrane potential of cells in the proximal colon of rats. 2 The resting membrane potential and electrical field stimulation (EFS)-induced junction potentials were recorded from the circular and longitudinal muscle cells. 3 Localized distension with a small balloon caused relaxation of the circular muscle on the anal side of the distended region (descending relaxation). Relaxation of the longitudinal muscle was also induced by EFS. 4 Inhibitory junction potentials (ij.ps) were recorded from all circular muscle cells tested, but rarely from the longitudinal muscle cells. 5 The ij.ps were recorded only in the presence of atropine but relaxations of both muscles were induced even in the absence of atropine. 6 Apamin (100 nM) completely abolished the ij.ps recorded in both circular and longitudinal muscle cells, but had no significant effect on the relaxations of either.7 In contrast to apamin, N' nitro-L-arginine (10 JLM) inhibited the relaxations of both muscles, but did not affect the ij.ps. 8 Exogenously added nitric oxide (0.1-101AM) induced relaxations of both muscles concentrationdependently, but did not affect the membrane potentials at these concentrations. 9 These data strongly suggest that nitric oxide-mediated relaxation of rat proximal colon is not associated with the ij.ps of the cell membrane.

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