We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M1-M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M1-M5 receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M1-M5 receptor single KO mice. In M2 and M4 receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M2 receptor immunoreactivity was located in both smooth muscle cells and enteric neurons. M4 receptor immunoreactivity was located in the enteric neurons, being in co-localization with M2 receptor immunoreactivity. These results indicate that both M2 and M4 receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. M1, M3, and M5 receptors do not seem to be involved in this mechanism.

1 The mediators of nonadrenergic, noncholinergic (NANC) relaxation in longitudinal muscle of the jejunum and ileum of Wistar rats were examined in vitro. 2 Treatment of the jejunal and ileal segments with a-chymotrypsin resulted in decreases in the NANC relaxations induced by electrical ®eld stimulation (EFS) by about one half. 3 The NANC relaxations were also decreased by about one half after the segments had been desensitized to neurotensin. A neurotensin receptor antagonist, SR48692 (10 mM) inhibited the NANC relaxation by 56 and 34% in the jejunal and ileal segments, respectively. 4 An inhibitor of small conductance Ca 2+ -activated K + channel (SK channel), apamin (100 nM) also inhibited the NANC relaxation by 83 and 63%, respectively. Exogenous neurotensin-induced relaxations of the two segments were abolished by apamin. 5 In the ileal segments, N G -nitro-L-arginine (L-NOARG, 100 mM), inhibited the NANC relaxation by 43%. L-NOARG, but not apamin, further inhibited the relaxation which persisted after the desensitization to neurotensin. Apamin with SR48692 inhibited the relaxation only to the same extent as apamin alone. 6 EFS induced inhibitory junction potentials (i.j.ps) in the longitudinal muscle cells of the ileum. I.j.ps consisted of a rapid and a delayed phase. L-NOARG signi®cantly inhibited only the delayed phase. 7 EFS induced only a rapid i.j.ps in the jejunum. SR48692 and apamin inhibited the i.j.ps. 8 These ®ndings suggest that neurotensin and unknown substance(s) mediate NANC relaxation via SK channels in the jejunum of Wistar rats, and that neurotensin via SK channels and nitric oxide not via SK channels separately mediate the relaxation in the ileum.

Abstract. The role of interstitial cells of Cajal (ICC) in electrical field stimulation (EFS)-induced neurogenic responses in ileum was studied by using the ICC-deficient mutant (SLC-W / W V ) mouse and its wild type. In the immunohistochemical study with anti-c-Kit antibody, ICC was observed in the myenteric plexus (MY) and deep muscular plexus (DMP) region in the wild type. In the mutant, ICC-MY were lost, only ICC-DMP were present. EFS induced a rapid contraction of the ileal segments from the wild type mouse in the direction of longitudinal muscle. In the mutant mouse, onset of contraction was delayed and its rate was slowed. EFS induced nonadrenergic, noncholinergic (NANC) relaxation in the presence of atropine and guanethidine in the wild type. A nitric oxide synthase inhibitor inhibited the relaxation and L-arginine reversed it. In the mutant, EFS did not induce NANC relaxation. There was no difference between the responsiveness of the segments from wild type and mutant mice to exogenously added acetylcholine or Nor-1. Taking into account the selective loss of ICC-MY in the mutant mice, it seems likely that ICC-MY have an essential role in inducing nitric oxidemediated relaxation of longitudinal muscle of the mouse ileum and that ICC-MY partly participate in EFS-induced contraction.

Recently an essential role of interstitial cells of Cajal (ICC) within myenteric plexus (ICC-MY) was suggested in ascending contraction and descending relaxation in the mouse ileum. The role of ICC in these neural reflexes was examined in the distal colonic segments prepared from the wild type and c-kit mutant, W/W V mice, in the present study. Localized distension of the segments from the wild type mice by using a small balloon resulted in ascending contraction and descending relaxation. In the segments from the mutant mice, localized distension also induced these neural reflexes similar to those observed in the wild type mice. Immunohistochemical examination demonstrated that ICC-MY and ICC present in muscle layers (ICC-IM) were severely disrupted in the mutant mouse, but only ICC, present within submucosal plexus (ICC-SMP), remained unchanged. In the small strips with ICC-SMP absent prepared from the mutant mouse, electrical field stimulation induced contraction or relaxation in the absence or presence of atropine, respectively. It was suggested that ICC have no important role in the ascending and descending neural reflexes in the mouse distal colon, this is in direct contrast to the role of ICC-MY in the ileum.

Abstract. We examined the role of interstitial cells of Cajal (ICC) in the ascending and descending neural reflexes in the ileal segments prepared from wild type mice and c-kit mutant W/W V mice. Localized distension of the ileal segments from wild type mice with a small balloon caused contraction or relaxation of the circular muscle on the oral or anal side of the distended region, respectively. However, these intestinal reflexes were not induced in the ileal segments from the mutant mice. In the small strips that include the step of the pathways from efferent motor neurons to smooth muscle cells, nerve stimulation induced contraction of circular muscle in the absence of atropine and relaxation in the presence of atropine. The extent of nerve stimulation-induced contractions and relaxations of the ileal circular muscle were similar in wild type and W/W V mice. The responsiveness of ileal circular muscle to exogenously added acetylcholine and Nor-1, a nitric oxide donor, was also unaffected in the mutant ileum. Since previous immunohistochemical study had revealed selective loss of ICC within the myenteric plexus (ICC-MY) in the mutant ileum, it was concluded that ICC-MY have an essential role in ascending and descending neural pathways in the mouse ileum.