Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.     

Constitutive expression and function of cyclooxygenase-2 in murine gastric muscles

BACKGROUND & AIMS: Cyclooxygenase enzymes (COX) generate intermediates in the prostaglandin (PG) cascade. COX-1 is constitutively expressed in many cells, and COX-2 is typically thought to be an inducible isoform. METHODS: We evaluated constitutive expression and function of COX-2 in murine gastric muscles. RESULTS: Immunohistochemistry showed COX-2-like immunoreactivity (COX-2-LI) in myenteric neurons. Half the neurons with COX-2-LI expressed nitric oxide synthase (NOS). COX-2-LI was not observed in smooth muscle cells. Interstitial cells of Cajal within muscle layers (IC-IM) expressed COX-2-LI, suggesting a novel role for IC-IM. Molecular studies verified expression of COX-2 in gastric muscles. Quantitative polymerase chain reaction (PCR) showed equal expression of COX-1 and COX-2 in the antrum. COX-2 was more abundant in fundus. Indomethacin and GR253035X, a COX-2 inhibitor, increased antral phasic contractions and potentiated responses to ACh. Indomethacin, but not GR253035X, increased contractions and potentiated responses in tissues of COX-2 knockout mice. Indomethacin and GR253035X reduced tone in the fundus. CONCLUSIONS: COX-2 is constitutively expressed by IC-IM and neurons in the stomach and at levels similar to COX-1. Prostanoids produced by COX-2 regulate mechanical activities of fundus and antral muscles.     

Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/W mutant mice

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) generate and propagate slow waves in the stomach. Gastric peristalsis depends on a proximal-to-distal gradient in slow wave frequency. We tested whether the gastric frequency gradient was an intrinsic property of ICC and whether dysrhythmias result from disruptions of ICC networks. METHODS: We studied wild-type (WT) and W/W(V) mice, which have only myenteric (pacemaker) ICC in the stomach. ICC distributions were analyzed by Kit immunofluorescence. Pacemaking in tissues was studied by intracellular electrophysiologic recording and in cultured ICC by monitoring mitochondrial [Ca(2+)] oscillations with rhod-2 fluorescence or membrane potential with DiBAC(4)(3) fluorescence. RESULTS: Slow wave frequencies were constant throughout WT gastric muscle sheets containing corpus and antrum. Separating the antrum from the corpus caused a significant drop in antral slow wave frequency. ICC from WT antrums also displayed significantly slower pacemaker frequencies than corpus ICC, but the corpus pacemaker frequency dominated in cocultures of corpus and antrum ICC. Myenteric ICC networks were reduced in W/W(V) mice, particularly in the corpus. In W/W(V) mice, separating the antrum from the corpus failed to reduce antral slow wave frequency. Antral pacemaker frequency in ICC from W/W(V) stomachs was the same as in corpus ICC. CONCLUSIONS: The proximal-to-distal slow wave frequency gradient and entrainment of distal electrical activity by proximal pacemakers are fundamental properties of gastric ICC. Chronic depletion of ICC networks disrupts the proximal-to-distal frequency gradient, and emergence of ectopic pacemakers in the antrum may be caused by "reprogramming" of the ICC pacemaker apparatus.     

Novel genes and functional relationships in the adult mouse gastrointestinal tract identified by microarray analysis

BACKGROUND & AIMS: A genome-level understanding of the molecular basis of segmental gene expression along the anterior-posterior (A-P) axis of the mammalian gastrointestinal (GI) tract is lacking. We hypothesized that functional patterning along the A-P axis of the GI tract could be defined at the molecular level by analyzing expression profiles of large numbers of genes. METHODS: Incyte GEM1 microarrays containing 8638 complementary DNAs (cDNAs) were used to define expression profiles in adult mouse stomach, duodenum, jejunum, ileum, cecum, proximal colon, and distal colon. Highly expressed cDNAs were classified based on segmental expression patterns and protein function. RESULTS: 571 cDNAs were expressed 2-fold higher than reference in at least 1 GI tissue. Most of these genes displayed sharp segmental expression boundaries, the majority of which were at anatomically defined locations. Boundaries were particularly striking for genes encoding proteins that function in intermediary metabolism, transport, and cell-cell communication. Genes with distinctive expression profiles were compared with mouse and human genomic sequence for promoter analysis and gene discovery. CONCLUSIONS: The anatomically defined organs of the GI tract (stomach, small intestine, colon) can be distinguished based on a genome-level analysis of gene expression profiles. However, distinctions between various regions of the small intestine and colon are much less striking. We have identified novel genes not previously known to be expressed in the adult GI tract. Identification of genes coordinately regulated along the A-P axis provides a basis for new insights and gene discovery relevant to GI development, differentiation, function, and disease.     

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.

BACKGROUND & AIMS: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS: Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS: ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.     

Screening and identification of proteins mediating senna induced gastrointestinal motility enhancement in mouse colon

AIM: To isolate the proteins involved in pharmacologic action of senna extract (SE) from mouse gastrointestinal tract and to explore the molecular mechanism of gastrointestinal motility change induced by SE. METHODS: SE was administrated to mice by different routes. Gastrointestinal motility of mice was observed using cathartic, gastrointestinal propellant movement experiments and X-ray analysis. Mouse model for gastrointestinal motility enhancement was established through continuous gastric administration of SE at progressively increased dose. At 3 h and week 3, 4, 6 and 10, morphological changes of gastrointestinal tissues were found under light microscope. Ultrastructural changes of intestinal and colonic tissues at week 6 were observed under transmission electron microscope. The colonic proteomic changes in model mice were examined by two-dimension polyacrylamide gel electrophoresis with immobilized pH gradient isoelectric focusing to screen the differentially expressed proteins, and their molecular masses and isoelectric points were determined. Two N-terminal sequences of the samples were also determined by mass spectrometry. RESULTS: SE (0.3g) caused diarrhea after gastric administration in 1-6h and enhanced gastrointestinal propellant (65.1+/-7.5%; 45.8+/-14.6%, P<0.01) in mice, but intramuscular and hypodermic injection had no cathartic effect. X-ray analysis of gastrointestinal motility demonstrated that gastric administration of SE enhanced gastric evacuation and gastrointestinal transferring function. At 3 h and week 3 and 4 after gastric administration of SE, light microscopic examination revealed no apparent change in gastrointestinal mucosal tissues, but transmission electron microscopic examination revealed inflammatory changes in whole layer of intestinal and colonic wall. Twenty differential proteins were detected in the colonic tissues of the model mice by two-dimensional electrophoresis, and the N-terminal amino acid sequences of two proteins were determined. CONCLUSION: SE causes diarrhea and enhances gastrointestinal motility through digestive tract administration. Long-term gastric administration of SE induces inflammatory changes and cell damage in the whole gastrointestinal tract. The differential proteins screened from the colonic tissues of the model mice might mediate the enhancing effect of SE on gastrointestinal motility.     

Cajal间质细胞在胃癌胃肠动力障碍中作用的实验研究

目的 探讨Cajal间质细胞在胃癌胃肠动力障碍发病机制中的作用.方法 将20只裸鼠随机分为正常对照组、胃癌组,每组各10只.将人胃癌SGC-7901细胞悬液皮下接种于胃癌组裸鼠背部,当接种部位肿瘤长至最大径4～5 mm时作为胃癌动物模型.采用文献报道的方法,取幽门环形肌和下食管括约肌肌条,记录正常对照组、胃癌组幽门环形肌和下食管括约肌的机械收缩运动的变化.采用免疫组化的方法,比较正常对照组、胃癌组胃壁Cajal间质细胞含量及形态学改变.结果 与正常对照组相比,胃癌组大鼠幽门环形肌和下食管括约肌肌条肌肉张力和收缩振幅明显降低（P＜0.01）.免疫组化染色显示,胃癌组Cajal间质细胞在胃体及胃窦的分布稀疏,数量减少,且细胞突起变少、变钝.结论 胃肠Cajal间质细胞异常变化可能是胃癌出现胃肠动力障碍性疾病的重要发病机制.     

Roles of Interstitial Cells of Cajal in Intestinal Transit and Exogenous Electrical Pacing

The aims of this study were to investigate the role of interstitial cells of Cajal (ICCs) on small intestinal transit and its responses to exogenous pacing in W/W^v mice. Eleven W/W^v mice and their controls implanted with four pairs of gastrointestinal electrodes were used for testing the entrainment of slow waves. Another 20 W/W^v mice and their controls equipped with a duodenal catheter and one pair of intestinal electrodes were used to test small intestinal transit represented by the geometric center (GC). Results were as follows. (1) The effect of pacing on slow wave frequency was sustained only in controls, and not in W/W^v mice. (2) Both gastric and intestinal slow waves were completely entrained in controls and W/W^v mice. Higher energy was required for pacing the stomach than the small intestine. (3) There was no significant difference in small intestinal transit between the controls and the W/W^v mice (GC: 5.4 vs. 5.5). (4) Pacing showed no effects on small intestinal transit in either wild-type (GC: 5.4 vs. 5.6) or W/W^v mice (GC: 5.5 vs. 5.7). We conclude that myenteric ICCs may not play an important role in the regulation of small intestinal transit in conscious mice. Gastric and intestinal pacing can be achieved without ICCs.     

In Vivo Gastric and Intestinal Slow Waves in W/W<Superscript>v</Superscript> Mice

The aim was to investigate whether there are regular gastric and intestinal slow waves in conscious W/W^v mice. Eleven W/W^v mice and 11 wild-type mice were implanted with two pairs of electrodes in the stomach and small intestine. Gastrointestinal slow waves were recorded both under anesthesia and in the conscious state. Atropine and verapamil were given separately to an additional 10 W/W^v mice. Results were as follows. (1) The conscious W/W^v mice showed lower rhythmic slow waves in the small intestine (77.1 vs 93.5%; P < 0.001). However, the frequency (10.7 vs 18.8 cpm; P < 0.0001) and the antregrade propagation of intestinal slow waves in W/W^v mice were significantly lower than in the controls. In the stomach, regular slow waves were recorded in both groups, with no difference between the two groups. (2) Anesthesia significantly impaired both gastric and intestinal slow waves in both groups. (3) Atropine and verapamil had no effects on the rhythmicity of intestinal slow waves. We conclude that ICC-MY may not be the sole pacemaker cells for slow waves in the small intestine.There may be some abnormality of smooth muscle cells in W/W^v mice that causes a reduction in the frequency, rhythmicity, and antegrade propagation of slow waves.Xiaohua Hou is a Visiting Scientist from the Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.     

Changes of the gastric endocrine cells in the C57BL／6 mouse after implantation of murine lung carcinoma： An immunohistochemical quantitative study

AIM: The regional distributions and relative frequencies of some gastric endocrine cells of C57BL/6 mice were studied by immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon and human pancreatic polypeptide (HPP) after subcutaneous implantation of murine lung carcinoma (3LL) cells. METHODS: The experimental animals were divided into two groups, one is non-implanted sham and the other is 3LL-implanted group. Samples were collected from the two regions of stomach (fundus and pylorus) at 28 d after implantation of 3LL cells (1x105 cell/mouse). RESULTS: In this study, all the seven types of immunoreactive (IR) cells were identified except for HPP. Most of these IR cells in the gastric portion were generally spherical or spindle in shape (open-type cell) while cells showing round in shape (closed-type cell) were found occasionally. The regional distributions of gastric endocrine cells in the 3LL-implanted group were similar to those of non-implanted sham. However, significant decreases of some types of IR cells were detected in 3LL-implanted group compared to those of non-implanted sham. In addition, the IR cells showing degranulation were numerously detected in 3LL-implanted group. CGA-, serotonin- and somatostatin-IR cells in the fundus and pylorus regions, and gastrin-IR cells in the pylorus regions of 3LL-implanted groups significantly decreased compared to those of non-implanted sham. However, no changes on frequencies of CCK-8- and glucagon-IR cells were demonstrated between 3LL-implanted and non-implanted groups. CONCLUSION: Endocrine cells are the anatomical units responsible for the production of gut hormones, and the change in their density would reflect a change in the capacity of producing these hormones. Implantation of tumor cell mass (3LL) induced severe quantitative changes of gastric endocrine cell density, and the abnormality in density of gastric endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.     

Effects of magnolol and honokiol derived from traditional Chinese herbal remedies on gastrointestinal movement

AIM: To study the effects of magnolol and honokiol on isolated smooth muscle of gastrointestinal tract and their relationship with Ca2+, and on the gastric emptying and the intestinal propulsive activity in mice. METHODS: Routine experimental methods using isolated gastric fundus strips of rats and isolated ileum segments of guinea pigs were adopted to measure the smooth muscle tension. The effects of magnolol 10-3,10-4,10-5 mol/L, and honokiol 10-4, 10-5,10-6 mol/L on the contractility of gastric fundus strips of rats and ileum of guinea pigs induced by acetylcholine (Ach) and 5-hydroxvtryptamine (5-HT) was assessed respectively. The method using nuclein and pigment methylene blue was adopted to measure the gastric retention rate of nuclein and the intestinal propulsive ratio of a nutritional semi-solid meal for assessing the effect of magnolol and honokiol (0.5, 2, 20 mg/kg) on gastric emptying and intestinal propulsion. RESULTS: Magnolol and honokiol significantly inhibited the contractility of isolated gastric fundus strips of rats treated with Ach or 5-HT and isolated ileum guinea pigs treated with Ach or CaCI2, and both of them behaved as non-competitive muscarinic antagonists. Magnolol and honokiol inhibited the contraction induced by Ach in Ca2+-free medium and extracellular Ca2+-dependent contraction induced by Ach. Each group of magnolol and honokiol experiments significantly decreased the residual rate of nuclein in the stomach and increased the intestinal propulsive ratio in mice. CONCLUSION: The inhibitory effect of magnolol and honokiol on contractility of the smooth muscles of isolated gastric fundus strips of rats and isolated ileum of guinea pigs is associated with a calcium-antagonistic effect. Magnolol and honokiol can improve the gastric emptying of a semi-solid meal and intestinal propulsive activity in mice.     

Restoration of gut motility in Kit-deficient mice by bone marrow transplantation

Purpose  Interstitial cells of Cajal (ICC) play important roles in autonomic gut motility as electrical pacemakers and mediators of neural regulation of smooth muscle functions. Insufficiency of ICC has been reported in a wide range of gut dysmotilities. Thus, restoration of ICC may be a therapeutic modality in these diseases. Here we provide evidence that transplanted bone marrow (BM) cells can restore gut dysmotility in part via transdifferentiation to ICC. Methods  Bone marrow cells obtained from Kit insufficient W/W ^v mice or syngeneic GFP-transgenic mice with wild-type Kit were transferred to W/W ^v recipients. Whole gut transit time and gastric emptying were examined 5 and 6 weeks after BM transplantation, respectively, and ICCs were identified in whole mounts, frozen sections and transmission electron immunomicroscopy of the gut smooth muscle layers using specific antibodies. Results  Transplantation of wild-type BM into W/W ^v mice significantly improved whole gut transit time and gastric emptying. Fluorescent immunohistochemistry revealed GFP⁺Kit⁺ cells in the myenteric plexus, deep muscular plexus, and submucosal plexus smooth muscle layers of the stomach, small intestine, and colon, respectively. In the whole mounts, GFP⁺Kit⁺ cells were bipolar and spindle shaped, and transmission electron immunomicroscopy showed GFP⁺ cells rich in mitochondria and endoplasmic reticulum between gut smooth muscle layers, suggesting the presence of GFP⁺ cells with morphological characteristics of ICC. Conclusions  These results suggest that BM contains cells that may incorporate into ICC networks and improve dysmotility in W/W ^v mice. Thus, BM transplantation may become to a new therapeutic modality for gut dysmotilities due to ICC insufficiency.     

旋复代赭汤促胃肠动力作用的实验研究

目的：研究旋复代赭汤对小鼠胃肠动力的作用及其作用机制。方法：通过整体动物实验与离体器官实验相结合,整方实验和拆方实验相结合,多层次综合研究与分析旋复代赭汤的促胃肠动力作用及其作用机制。结果：旋复代赭汤能促进正常状态的小鼠胃排空,对其小肠推进无明显影响;能够拮抗芬氟拉明、左旋麻黄碱、多巴胺引起的小鼠胃排空抑制和小肠推进减慢;对阿托品引起的胃排空抑制有拮抗作用,对其造成的小肠推进减慢无明显影响;不能拮抗吗啡引起的小鼠胃排空抑制和小肠推进减慢。采用正交设计法拆方研究发现,党参、代赭石、大枣和旋复花对大鼠胃底条肌收缩具有显著促进作用,旋复花与甘草、大枣分别合用,有明显协同促进作用。党参、旋复花和半夏能显著拮抗阿托品引起的胃底条肌舒张作用,代赭石和生姜合用,具有协同拮抗作用。结论：旋复代赭汤具有确切的促胃动力作用。     

Protease-activated receptors mediate apamin-sensitive relaxation of mouse and guinea pig gastrointestinal smooth muscle

BACKGROUND & AIMS: Protease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms. METHODS: Responses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2 and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli. RESULTS: In mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2 and SLIGRL-NH2 caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2 and SLIGRL-NH2 caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine. CONCLUSIONS: The mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+ channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.