Examination of the role of cholinergic myenteric neurons with the impairment of neural reflexes in the ileum of c-kit mutant mice.

Our previous study showed that impairment of ascending and descending neural reflexes in the ileum of the c-kit mutant, W/W(V), mice is due to a loss of interstitial cells of Cajal present at the myenteric plexus region (ICC-MY) in the mutant. In the present study, cholinergic interneurons were thought to be involved in these pathways, since hexamethonium, an antagonist of the nicotinic ACh receptor, significantly inhibited both neural reflexes in wild type mice. Therefore, we examined whether the loss of ICC-MY affects cholinergic interneurons involved in these pathways. Immunohistochemistry with anti-choline acetyltransferase revealed that there was no difference in the numbers of immunopositive cells in the myenteric plexus region between the wild type and mutant mice. In addition, there was no difference in the extent of spontaneous and EFS-evoked ACh release from longitudinal muscle with myenteric plexus preparations between the wild type and mutant mice. Exogenously added nicotine induced contraction or relaxation of ileal circular muscle in the absence or presence of atropine, respectively, to a similar extent in both the wild type and mutant mice. These results suggest that loss of ICC-MY resulted in an impairment of the ascending and descending reflex pathways at the step before activation of cholinergic interneurons.     

Absence of peristalsis in the ileum of W/W(V) mutant mice that are selectively deficient in myenteric interstitial cells of Cajal.

It is well known that the enteric nervous system plays a key role in the generation of gastrointestinal peristaltic movements. Recently, the networks of interstitial cells of Cajal (ICC) have been found to be essential in the generation of spontaneous gastrointestinal movements. However, the role of ICC in the mechanisms involved in the generation of peristaltic movements is still controversial. The aim of the present study was to reveal how pacemaker myenteric ICC (ICC-MY) and the enteric nervous system contribute to the mechanisms involved in the generation of intestinal peristalsis. We compared spontaneous peristaltic movements of the ileum in wild type (WT) mice with those in W/W(V) mutant mice which are selectively deficient in ICC-MY. Simultaneous recordings were made from both the circular and longitudinal muscle of a 4-cm long segment of ileum under hydrostatic pressure of 0--0.5 cm H(2)O. Mechanical activity and continuous video-images of the ileum were compared between WT and W/W(V) mutant mice under control conditions, in the presence of N-nitro-L-arginine methyl ester (L-NAME) and after tetrodotoxin (TTX). In the WT mouse ileum, peristaltic waves to propagate from the oral to the anal end were frequently observed. The frequency of these peristaltic waves and their associated synchronous longitudinal and circular muscle contractions was increased by L-NAME. The peristaltic waves were abolished by TTX. In the W/W(V) mutant mouse ileum, no peristaltic waves to propagate from the oral to the anal end were observed in control and even after L-NAME, although the local spontaneously generated longitudinal and circular muscle contractions were enhanced by L-NAME. These local contractions were not abolished by TTX. The results presented here suggested that ICC-MY are essential for the generation of spontaneous intestinal peristaltic movements. It is conceivable that ICC-MY may determine the polarity of the excitation of the intestine such that longitudinal and circular muscle contractions propagate from the oral to the anal end of the intestinal segments, although the question of why ICC-MY are necessary for the neural pathways remains unresolved.     

Interstitial cells of Cajal in the gastrointestinal musculature of W mutant mice

Interstitial cells of Cajal (ICC) are important regulatory cells generating electrical rhythmicity and transducing neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and can be examined morphologically using the c-Kit antibody. The c-kit gene is allelic with the murine white-spotting locus W, and the c-kit mutation (W mutation) affects various aspects of hematopoietic cells, germ cells, melanocytes, mast cells, and ICC. Heterozygous W/W( v) mutant mice lack a specific type of ICC and have been used to reveal its function. To search for a new model that lacks a specific type of ICC, we examined homozygous W( v)/W( v) black-eyed-white mice that are viable with anemia. Results showed the principal patterns of ICC deficiency were the same between the W/W( v) and W( v)/W( v) mutants. In the stomach of both mice, intramuscular ICC (ICC-IM) were missing and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced in spite of a normal distribution of deep muscular plexus ICC (ICC-DMP). The cecum also exhibited fewer reduced. ICC-IM in the colon were almost entirely missing, whereas ICC-MY were reduced only in the distal colon. In the small intestine and colon, the number of remaining ICC-MY in W( v)/W( v) mice was greater than that in W/W( v) mice. The enteric nervous system of the two mutant mice showed normal characteristics. From these findings, we conclude that W( v)/W( v) mice represent a new genotype that lacks a part of the ICC in its gastrointestinal musculature.     

Long Vasodilator Reflexes Projecting Through the Myenteric Plexus in Guinea-Pig Ileum

This study examined enteric neural reflexes activating submucosal cholinergic vasodilator motoneurons, which innervate the final resistance vessels regulating mucosal blood flow. Videomicroscopy was employed to monitor dilatation of submucosal arterioles in in vitro preparations from guinea-pig ileum. Balloon distension of intact lumen evoked reflex vasodilatation and flat sheet preparations were employed to separate mucosal mechanical stimulation from intestinal distension. Mucosal stroking and balloon distension of the orad segment evoked vasodilatations > 1.5 cm from the stimulating site. Mucosal stimulation was blocked by combined 5-HT₃/5HT₄ antagonists but distension-evoked responses were unaffected. Distension-evoked responses were also unaffected by nifedipine (5 μ m ) or nifedipine (1 μ m ) and wortmannin (300 n m ), suggesting stretch activation rather than stretch-activated contraction was involved. Mucosal and distension-evoked responses were completely blocked when the myenteric plexus was surgically lesioned and were significantly inhibited by hexamethonium. The muscarinic antagonist 4-DAMP, which inhibits vasodilatations evoked by submucosal cholinergic vasodilator neurons, blocked dilatations elicited by mucosal stimulation and balloon distension. Maximal dilatations evoked with either sensory modality could be further enhanced when stimulated with the second modality. Dilatations evoked by stimulation of the aborad segment were similar to those elicited in the orad segment. In conclusion, sensory mechanisms in the mucosa and muscularis propria activate vasodilator pathways in the myenteric plexus which project for significant distances in both ascending and descending directions before innervating submucosal arterioles. These reflexes could co-ordinate mucosal blood flow during multiple motor events such as peristalsis and intestinal mixing between propulsive events.     

Polar innervation of enteric non-cholinergic and non-adrenergic neurons in the isolated guinea-pig ileum

To study the polarity of the efferent pathway of the myenteric plexus, recordings were made of the mechanical activity of the longitudinal muscle of isolated guinea-pig ileal segments upon stimulation with an electrical field around the myenteric plexus contained within strips of longitudinal muscle (LM-MP) continuous with each end of ileal segment. The amplitude of the contractile response to stimulation of the anal LM-MP was always larger than that to the oral LM-MP. After cholinergic and adrenergic transmission was suppressed by atropine (10 microM) and guanethidine (1 microM), and the tone of the segment was enhanced by histamine (1 microM), the LM-MP stimulation produced non-cholinergic, non-adrenergic (NCNA) ascending contraction and NCNA descending relaxation. The NCNA contraction, but not the NCNA relaxation, was abolished or reduced by desensitization to substance P. The present results suggest that the NCNA innervation of the myenteric plexus participates in the polar effects observed in the guinea-pig ileum, that the NCNA excitatory response may be mediated at least in part by myenteric substance P neurons, and that the NCNA inhibitory response is mediated by non-adrenergic neurons.     

Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine

Interstitial cells of Cajal (ICC) provide important regulatory functions in the motor activity of the gastrointestinal tract. In the small intestine, ICC in the myenteric region (ICC-MY), between the circular and longitudinal muscle layers, generate and propagate electrical slow waves. Another population of ICC lies in the plane of the deep muscular plexus (ICC-DMP), and these cells are closely associated with varicose nerve terminals of enteric motor neurons. Here we tested the hypothesis that ICC-DMP mediate excitatory and inhibitory neural inputs in the small bowel. ICC-DMP develop largely after birth. ICC-DMP, with receptor tyrosine kinase Kit-like immunoreactivity, appear first in the jejunum and then in the ileum. We performed electrophysiological experiments on mice immediately after birth (P0) or at 10 days post partum (P10) to determine whether neural responses follow development of ICC-DMP. At P0, slow-wave activity was present in the jejunum, but neural responses were poorly developed. By P10, after ICC-DMP developed, both cholinergic excitatory and nitrergic inhibitory neural responses were intact. Muscles of P0 mice were also put into organotypic cultures and treated with a neutralizing Kit antibody. Neural responses developed in culture within 3–6 days in control muscles, but blocking Kit caused loss of ICC and loss of cholinergic and nitrergic neural responses. Non-cholinergic excitatory responses remained after loss of ICC-DMP. Our observations are consistent with the idea that cholinergic and nitrergic motor neural inputs are mediated, to a large extent, via ICC-DMP. Thus, ICC-DMP appear to serve a function in the small intestine that is similar to the role of the intramuscular ICC in the stomach.     

Kit signaling is essential for development and maintenance of interstitial cells of Cajal and electrical rhythmicity in the embryonic gastrointestinal tract.

Interstitial cells of Cajal (ICC) are specialized cells in smooth muscle organs that generate and propagate pacemaker activity, receive inputs from motor neurons, and serve as mechanosensors. In the gastrointestinal tract, development and maintenance of the ICC phenotype have been linked to intracellular signaling via Kit, but its role in development of ICC during embryogenesis is controversial. Here we have studied the development of functional ICC-MY during the late gestational period in mice. Blocking Kit with a neutralizing antibody before and after development of spontaneous electrical activity (E17 to P0) caused loss of ICC-MY networks and pacemaker activity. ICC-MY and pacemaker activity developed normally in W/+ and W(V)/+ heterozygotes, but failed to develop between E17 to P0 in W/W(V) embryos with compromised Kit function. Muscles treated with Kit neutralizing antibody or the tyrosine kinase inhibitor, imatinib mesylate (STI571), from E17-P0 for 3 days caused loss of functionally developed ICC-MY networks, but ICC-MY and pacemaker activity recovered within 9 days after discontinuing treatment with neutralizing antibody or imatinib mesylate. These data suggest that Kit signaling is an important factor in lineage decision and in the development of functional ICC in late gestation. ICC-MY demonstrate significant plasticity in gastrointestinal tissues. Manipulation of the ICC phenotype might provide useful therapies in gastrointestinal disease where the Kit-positive cell population is either lost or amplified.     

Interstitial cells of cajal are involved in neurotransmission in the gastrointestinal tract

Interstitial cells of Cajal (ICC) are important cells which coordinate gastrointestinal motility. ICC express Kit receptor tyrosine kinase, and Kit immunohistochemistry reveals ICC morphology and distribution in the gastrointestinal musculature. ICC show a highly branched morphology and form unique networks. Myenteric ICC (ICC-MY) are located at the layer of the myenteric plexus and serve as electrical pacemakers. Intramuscular ICC (ICC-IM) and ICC in the deep muscular plexus (ICC-DMP) are distributed within the muscular layers, and are densely innervated by excitatory and inhibitory enteric motor neurons and in close contact with nerve terminals. Recent studies combined with morphological and functional techniques directly revealed that ICC-IM and ICC-DMP are mediators of enteric motor neuro-transmission. These types of ICC express several receptors for neurotransmitters such as acetylcholine and substance P and show responses to excitatory nerve stimulations. ICC also express receptive mechanisms for nitric oxide, which is an inhibitory neurotransmitter in the gastrointestinal tract. They can respond to nitrergic nerve stimulation by cyclic GMP production. Kit mutant mice lack ICC-IM and show attenuated postsynaptic responses after intrinsic nerve stimulation. These findings indicate the importance for ICC in neurotransmission in the gastrointestinal tract.     

Release of vasoactive intestinal polypeptide anally of a local distension of the feline small intestine

The peristaltic reflex is elicited by a local distension of the intestine and consists of an ascending contraction and a descending inhibition of the smooth muscle layer. The transmitter mediating the inhibition of the smooth muscle cells is not known but vasoactive intestinal polypeptide (VIP) has, among others, been proposed to mediate the descending inhibition of a distension. An in vivo preparation of a segment of the feline intestine was designed to allow collection of the venous effluent and determination of the VIP release orally and anally of a distension. The release of VIP rose significantly more on the anal side of the intestinal distension, and so did the blood flow. Division of the myenteric plexus prevented the asymmetry of the VIP release to a large extent. These effects of distension on the release of VIP are consistent with the hypothesis that VIP mediates the descending inhibition of the peristaltic reflex.     

Migrating Motor Complexes do not Require Electrical Slow Waves in the Mouse Small Intestine

We have investigated whether migrating motor complexes (MMCs) are impaired or absent in the small intestine of W/W^v mutant mice, which lack pacemaker interstitial cells of Cajal (ICC) and electrical slow waves. The intracellular electrical and mechanical activities of the small intestines of wild-type (+/+) and W/W^v mutant mice were recorded. Electrical recordings from circular muscle cells confirmed the absence of slow waves in W/W^v mice, whereas slow waves were always recorded from +/+ muscle cells. Spontaneous phasic contractions were recorded from W/W^v muscles in the absence of slow waves, but these events occurred at a lower frequency than in +/+ tissues. MMC activity was recorded consistently from the ileum of +/+ mice, and normal MMCs were also recorded from W/W^v mice. MMCs in both +/+ and W/W^v mice were abolished by tetrodotoxin (1 μ m ), hexamethonium (300 μ m ) or atropine (1 μ m ), suggesting that the neural control mechanisms responsible for MMCs in +/+ mice are intact and are responsible for MMCs in W/W^v mice. Transmural nerve stimulation demonstrated intact inhibitory and excitatory neural regulation of W/W^v intestinal muscles. Prolonged trains of cholinergic motor nerve stimulation failed to activate slow waves in the intestinal muscles of W/W^v mice. Our findings show that the generation and directional propagation of MMC activity in mouse small intestine does not require slow-wave activity or an intact network of myenteric ICC. The generation and propagation of MMCs appear to be an intrinsic capability of the enteric nervous system and are not related to slow waves or the gradient in slow-wave frequency.     

Gut pacemaker cells: the interstitial cells of Cajal (ICC).

This review will focus on the pacemaker mechanisms underlying gastrointestinal autonomic rhythmicity in an attempt to elucidate the differences and similarities between the pacemaker mechanisms in the heart and gut. Interstitial cells of Cajal (ICC) form networks that are widely distributed within the submucosal (ICC-SM), intra-muscular (ICC-IM, ICC-DMP) and inter-muscular layers (ICC-MY) of the gastrointestinal tract from the esophagus to the internal anal sphincter. The ICC generate spontaneously active pacemaker currents that may be recorded as plateau and slow potentials. These pacemaker currents drive the spontaneous electrical and mechanical activities of smooth muscle cells. The enteric nervous system, composed of both the myenteric (inter-muscular) plexus and the submucosal plexus, is also distributed in the gastrointestinal tract from the esophagus to the internal anal sphincter. The role of the ICC and the enteric nervous system in the integrative control of gastrointestinal function and especially of spontaneous rhythmic activity, is still unknown. Nevertheless, at least from the results presented in this review of studies of the jejunum, ileum and proximal colon of the mouse, it is convincing that the ICC drive spontaneous rhythmic motility, although a role for the enteric nervous system in the regulation of spontaneous rhythmic motility cannot be overlooked. Furthermore, intracellular Ca2+ handling has a critical role in the generation of pacemaker activity in the gut and heart, although respective players such as the Ca2+-ATPase of the sarcoplasmic reticulum (endoplasmic reticulum), IP3 receptors, ryanodine receptors and plasma membrane ion channels may have divergent roles in the Ca2+-release refilling cycles. In conclusion, intracellular Ca2+ handling plays a key role in the gut pacemaker responsible for spontaneous rhythmicity, as well as in the cardiac pacemaker responsible for spontaneous beating. Pharmacotherapeutic targeting of intracellular Ca2+ handling mechanisms may be a promising approach to the treatment and cure of gut motility dysfunction.     

长爪沙鼠胃肠道Cajal间质细胞的形态学

目的 探讨长爪沙鼠胃肠道Cajal间质细胞（ICCs）的形态和分布规律。 方法 采用10只成年长爪沙鼠,体重50~70g,取胃、小肠、结肠制作冷冻切片,结合全层铺片的c-Kit免疫荧光染色。结果 ICCs呈网络状分布于整个胃肠道,不同部位ICCs的分布及形态有所不同。在胃底部,仅见肌内ICCs(ICC-IM）,而在胃体和胃窦部除ICC-IM外,可见肌间ICCs(ICC-MY)分布在肌间神经丛周围;其细胞密度胃底ICC-IM最多,由胃底至胃窦逐渐减少,而ICC-MY由胃体至胃窦逐渐增多。在小肠可见ICC-IM, ICC-MY和深肌层ICCs(ICC-DMP)3个亚群,结肠管壁内也分布有ICC-IM、ICC-MY和黏膜下ICCs(ICC-SM)3个亚群。结论 沙鼠可用于有关ICCs正常形态、结构及功能的研究。     

A ‘new’ mechanism for oedema generation: strongly negative interstitial fluid pressure causes rapid fluid flow into thermally injured skin

Release of vasoactive intestinal polypeptide anally of a local distension of the feline small intestine. Acta Physiol Scand 130 , 433–438. Received 12 February 1987, accepted 27 February 1987. ISSN 0001–6772. Department of Physiology, University of Göteborg, Sweden, and Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark. The peristaltic reflex is elicited by a local distension of the intestine and consists of an ascending contraction and a descending inhibition of the smooth muscle layer. The transmitter mediating the inhibition of the smooth muscle cells is not known but vasoactive intestinal polypeptide (VIP) has, among others, been proposed to mediate the descending inhibition of a distension. An in vivo preparation of a segment of the feline intestine was designed to allow collection of the venous effluent and determination of the VIP release orally and anally of a distension. The release of VIP rose significantly more on the anal side of the intestinal distension, and so did the blood flow. Division of the myenteric plexus prevented the asymmetry of the VIP release to a large extent. These effects of distension on the release of VIP are consistent with the hypothesis that VIP mediates the descending inhibition of the peristaltic reflex.     

Anterograde labeling of the corticospinal tract in jimpy mutant mice by Di I injection into the motor cortex

Carbocyanine fluorescent dye, DiI, is an excellent anterograde/retrograde neural tracer, but its efficacy for the anterograde labeling of neural circuits in the adult brain tends to decrease with ages. The present study shows that an injection of DiI into the motor cortex of the young adult jimpy mutant mice (Plp1(jp)/+) resulted in successful anterograde labeling of corticospinal tract fibers. Furthermore, an injection of Fast Blue into the lumbar spinal cord of the mutant mice resulted in retrograde labeling of layer 5 corticospinal tract neurons within the motor cortex. Since no abnormality except for myelin deficiency is known in the long descending and ascending tracts of jimpy mutant mouse, this mutant is suitable for neural tracing studies of long axonal trajectories with the use of carbocyanine dye, DiI, although these males die between 20 and 40 days of age.     

Interstitial cells of Cajal: primary targets of enteric motor innervation

For many years morphologists have noted the close relationship between interstitial cells of Cajal (ICC) and nerve fibers within the tunica muscularis of gastrointestinal (GI) organs. These observations led to speculations about a role for ICC in mediating neural inputs to the GI tract. Immunohistochemical and functional studies demonstrated the presence of receptors for the neurotransmitters utilized by enteric motor neurons, and changes in second messengers in ICC after field stimulation of intrinsic enteric neurons showed that ICC were functionally innervated in GI muscles. Recent double labeling experiments have shown that both excitatory and inhibitory enteric motor neurons are closely associated with ICC in the deep muscular plexus (IC-DMP) of the small intestine and intramuscular ICC (IC-IM) of the proximal and distal GI tract. Enteric motor neurons form synaptic-like structures with IC-IM and IC-DMP. Far fewer close contacts are found between enteric motor neurons and smooth muscle cells. Experiments on W/W(V) mutants that lack IC-IM in the stomach, lower esophageal sphincter, and pylorus have shown that these ICC are critical components of the neuromuscular junction. Cholinergic excitatory and nitrergic inhibitory neurotransmission are severely decreased in tissues lacking IC-IM, yet there is no loss of cholinergic or nitrergic neurons in W/W(V) mutants. These data suggest that either the post-junctional mechanisms responsible for receiving and transducing neurotransmitter signals are specifically expressed by ICC, or that the large extracellular spaces typically between nerve terminals and smooth muscle cells may not allow effective concentrations of neurotransmitters to reach receptors expressed by smooth muscle cells. These findings indicate an important role for certain classes of ICC in enteric neurotransmission and predict that loss of ICC in human motor disturbances may significantly compromise neural regulation of GI motility.     

Effects of Dai-kenchu-to on spontaneous activity in the mouse small intestine.

The effects of Dai-kenchu-to (DKT), a Chinese medicine, on spontaneous activity of mouse small intestine were investigated. Experiments were carried out with tension recording and intracellular recording. DKT contracted mouse longitudinal smooth muscles in a dose dependent manner (0.1-10 mg/ml). Low concentration of DKT (0.1 mg/ml) did not contract the longitudinal muscles of mouse small intestine. DKT (0.1 mg/ml) inhibited contraction elicited by transmural nerve stimulation (TNS). DKT (1 mg/ml) evoked relaxation before contraction. The initial relaxation was abolished by Nomega-nitro-L-arginine (L-NNA). DKT (10 mg/ml)-induced contraction had two components: a transient rapid contraction and a following slow contraction. Atropine inhibited DKT (1 mg/ml)-induced contraction to about 50% of control. In the presence of atropine, tetrodotoxin (TTX) inhibited the contraction elicited by DKT (1 mg/ml) to about 80%. DKT depolarized the membrane and decreased the amplitude of pacemaker potentials recorded from in situ myenteric interstitial cells of Cajal (ICC-MY) with no alteration to the frequency, duration and maximum rates of rise in the presence of nifedipine and TTX. The same results were obtained in slow waves recorded from circular smooth muscle cells. These results indicate that DKT evoked both contraction and relaxation by releasing acetylcholine, nitric oxide and other excitatory neurotransmitters in mouse small intestine. DKT had no effects on pacemaker mechanisms and electrical coupling between ICC-MY and smooth muscle cells in mouse small intestine. The results also suggest that DKT may contract smooth muscles by depolarizing the membrane directly.     

Postnatal development of interstitial cells of Cajal in mouse colon in response to Kit signal blockade with Imatinib (Glivec)

This study investigated the response of interstitial cells of Cajal (ICC) in postnatal mouse colon to treatment with Imatinib (Glivec®, a potent inhibitor of Kit receptor). ICC were revealed by immunofluorescent staining on frozen cross-sections and whole-mount preparations by anti-Kit and DOG1 antibodies. Kit and p-Kit protein were also evaluated by Western blot. After administration of Imatinib for 4 days beginning at 8 days post-partum (P8), the mean density of Kit⁺ ICC, which were localized around the myenteric nerve plexus (ICC-MY), within smooth muscle layers (ICC-IM) and in the connective tissue beneath the serosa (ICC-SS), was dramatically decreased to about 50% when compared with controls, but those Kit⁺ cells located at the submucosal border of circular smooth muscle layer (ICC-SM) seemed to be unchanged in both cell number and morphology. A small number of DOG1⁺/Kit⁻ cells appeared during Imatinib administration. However, these Kit⁺ ICC were not changed in mice even after 12 days of Imatinib treatment from P24. When Imatinib was discontinued, the number of ICC recovered to normal within 4 days. Our results indicate that the postnatal development of ICC in the mouse colon is Kit dependent, but ICC-SM are unlikely, and the Kit dependence of ICC development is also age-dependent.     

Association of Salmonella typhimurium with, and its invasion of, the ileal mucosa in mice.

A wild-type strain of Salmonella typhimurium and three mutant rough colonial variants of the wild type were compared for their ability to become associated with and invade the ileal mucosa of germfree and specific-pathogen-free mice. The rough-mutant strains differed from the wild type in having incomplete lipopolysaccharides lacking one or more sugars in the polysaccharide moiety. The wild-type and mutant strains also differed one from the other in the types of appendages (flagella, pili) on their surfaces. Depending upon the dosage of bacteria given, all mutant strains as well as the wild type could associate with and invade the intestinal mucosa of infected gnotobiotic mice. If the infecting dosage was high enough, at least two of the mutant strains and the wild type invade the intestinal mucosa of the specific-pathogen-free animals. O antigen, flagella, or pili do not appear to be essential for the association of S. typhimurium with the mucosal surface of the mouse ileum. O antigen on the bacterial cell surface may be important, but not essential, for invasion of the ileal mucosa.     

A mouse model of a human multiple GIST family with KIT-Asp820Tyr mutation generated by a knock-in strategy

Several families exhibiting multiple gastrointestinal stromal tumours (GISTs) and germline c-kit gene mutations at exons 8, 11, 13, or 17 have been reported. These patients also exhibit diffuse hyperplasia of the interstitial cells of Cajal (ICCs) as a pre-existing lesion of multiple GISTs. We generated a mouse model of a family with germline c-kit gene mutation at exon 17, and compared the phenotypes between the mice and humans. The mouse counterpart (KIT-Asp818Tyr) of the human KIT-Asp820Tyr mutation was transmitted into germline by a knock-in strategy. Mating of male and female heterozygotes (KIT-Asp818Tyr/+) resulted in the generation of homozygotes (KIT-Asp818Tyr/KIT-Asp818Tyr). Histological examination revealed that all heterozygotes had both a small KIT-positive mesenchymal tumour at the caecum, consistent with GIST, and KIT-positive diffuse spindle-shaped cell proliferation in the distal oesophagus, stomach, proximal duodenum, and colon consistent with ICC hyperplasia. All homozygotes exhibited a larger caecal tumour and more prominent spindle-shaped cell proliferation compared with the heterozygous mice, and they usually died within 10 weeks after birth, likely due to ileus. The small intestine of both genotypes showed no apparent morphological abnormality, and autonomous contraction of the ileal segments appeared normal. Western blotting demonstrated that the caecal tumours expressed phosphorylated KIT, MAPK, Stat1, and Stat5. These mutant mice are considered to be useful for further investigation of the mechanism of GIST development as a result of ICC hyperplasia and for assessment of the in vivo effects of drugs against molecular targets.     

Mediators of nonadrenergic, noncholinergic relaxation in longitudinal muscle of the intestine of ICR mice.

Mediators of nonadrenergic, noncholinergic (NANC) relaxation in longitudinal muscle of several regions of ICR mouse intestine were studied. An inhibitor of synthesis of nitric oxide, N(G)-nitro-L-arginine (L-NOARG) at 10 microM significantly inhibited NANC relaxations induced by electrical field stimulation (EFS) in the jejunum, ileum, and the proximal and distal colon. Especially in the ileum extent of the inhibition was more than 80%. An antagonist of vasoactive intestinal peptide (VIP) receptors, VIP(10-28) at 3 microM partially inhibited the EFS induced relaxations in the jejunum and proximal colon, but very slightly in the distal colon and had no effect in the ileum. An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptor, PACAP(6-38) at 3 microM partially inhibited the EFS-induced relaxations in the proximal and distal colon, but not in the jejunum and ileum. Totals of the percentages of relaxant components mediated by nitric oxide, VIP and PACAP in every region are roughly equal to a hundred percent. In another series of experiments, EFS-induced relaxations were almost completely inhibited by the treatment of the segments with L-NOARG and VIP(10-28) in the jejunum, with L-NOARG, VIP(10-28) and PACAP(6-38) in the proximal colon, and with L NOARG and PACAP(6-38) in the distal colon. The present results suggest that nitric oxide solely mediates the relaxation of longitudinal muscle of the ileum of ICR mice, whereas nitric oxide and VIP co-mediate it in the jejunum, nitric oxide, VIP and PACAP in the proximal colon, and nitric oxide and PACAP in the distal colon.