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The c-Kit receptor tyrosine kinase regulates the development and differentiation of several progenitor cells. In the gastrointestinal (GI) tract, the c-Kit regulates the development of the interstitial cells of Cajal (ICC) that are responsible for motility regulation of the GI musculature. W-sash (W sh ) is an inversion mutation upstream of the c-kit promoter region that affects a key regulatory element, resulting in cell-type-specific altered gene expression, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the GI tract of W sh /W sh mice using immunohistochemistry and electron microscopy. Although the musculature of the W sh /W sh mice did not show any c-Kit immunoreactivity, we detected intensive immunoreactivity for transmembrane member 16A (TMEM16A, anoctamin-1), another ICC marker. TMEM16A immunopositive cells were observed as ICC-MY in the gastric corpus-antrum and the large intestine, ICC-DMP in the small intestine, and ICC-SM in the colon. Electron microscopic analysis revealed these cells as ICC from their ultrastructural features, such as numerous mitochondria and caveolae, and their close contact with nerve terminals. In the developmental period, we examined 14.5 and 18.5 day embryos but did not observe c-Kit immunoreactivity in the W sh /W sh small intestine. From this study, ICC subtypes developed and maturated structurally without c-Kit expression. W sh /W sh mice are a new model to investigate the effects of c-Kit and unknown signaling on ICC development and function.

c-Kit receptor tyrosine kinase and its ligand stem cell factor (SCF) play critical roles in regulating the development and proliferation of various cells, including the interstitial cells of Cajal (ICC) in the gastrointestinal tract. Many subtypes of ICC are known to be lacking in c-Kit-SCF-insufficient mice, such as W/W v and Sl/Sl d , whereas ICC-deep muscular plexus (DMP) in small intestine are not lacking. In this study, we examine ICC-DMP development in normal and c-Kit-SCF signal-insufficient mice. In normal mice, numerous ICC-DMP labeled with c-Kit and neurokinin 1 receptor (NK1R) antibodies were observed only in the duodenum on the day of birth, in the duodenum and the jejunum on postnatal day 4 and throughout the small intestine after postnatal day 6. In W mutant mice (W/W v , W v /W v , W/W), ICC-DMP investigated using c-Kit and NK1R immunoreactivities were similar to that in normal mice. c-Kit ligand SCF-deficient mice (Sl/Sl) also showed almost identical ICC-DMP development and proliferation as normal mice. These results show that the development and proliferation of ICC-DMP occur in the postnatal period independent of c-Kit-SCF signaling.

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Interstitial cells of Cajal (ICC) generate electrical rhythmicity and transduce neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and are identified morphologically by c-Kit immunoreactivity. The c-kit gene is allelic with the murine white-spotting locus W, and mutations of c-kit are known as W mutations. W mutations affect various developmental aspects of hematopoietic cells, germ cells, melanocytes, mast cells and ICC. We examined W(jic)/W(jic) mutant mice that have a mutation in the tyrosine kinase domain resulting in severe loss of protein function. W(jic)/W(jic) homozygotes exhibited white coats and black eyes. The gross morphology of the gastrointestinal tract showed no abnormality in mutant mice other than a forestomach papilloma. In the stomach, 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; however there was a normal distribution of deep muscular plexus ICC (ICC-DMP). In the cecum, the numbers of ICC-IM and ICC-MY were severely depleted. ICC-IM were almost entirely absent in the colon, whereas ICC-MY loss was restricted to the distal colon. Patterns of ICC deficiency were generally similar between W(jic)/W(jic) mice and W/W(v) mutants, which lack a specific type of ICC. The enteric nervous system of the mutant mice appeared normal. From these findings, we conclude that W(jic)/W(jic) mice represent a distinct, novel genotype resulting in a lack of a specific type of ICC in the gastrointestinal musculature.

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are the regulatory cells of gut movement. W/W mutant mice that have receptor tyrosine kinase KIT mutation lack ICC along the myenteric plexus layer of small intestine. The development and maintenance of the ICC phenotype have been related to KIT, but the other genes involved in ICC development during embryogenesis are not clear. Our aim was to identify ICC-specific genes in the embryonic stage. We examined genes that are expressed less in ICC-deficient W/W mice than in wild type (WT) at embryonic day 14 (E14) in order to clarify the genes associated with the ICC development using subtractive hybridization and microarray. Among them, we identified msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2). Using real-time PCR, msx2 and ntrk2 were found to be expressed at significantly lower levels in W/W than in WT during embryogenesis. Msx2 immunoreactivity was high in the WT small intestine. These data suggest that the gene expressions of ntrk2 and msx2 were significantly suppressed in KIT mutant mouse embryo and neonate and that these genes are likely to regulate ICC development., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Background: Gastrointestinal tract is one of the most susceptible organ systems to ischaemia. Not only mucosal injury but also alterations of the intestinal motility and loss of interstitial cells of Cajal (ICC) have been reported in response to ischaemia and reperfusion (I/R). However, there are few reports on the changes in the gastric motility after gastric I/R. The present study was designed to investigate the alterations in gastric emptying, the ICC and enteric nerves that regulate smooth muscle function in response to gastric I/R., Methods: Seven-week-old male Wistar rats were exposed to gastric I/R, and the gastric emptying rates at 12 and 48 h after I/R were evaluated by the phenol red method. Expressions of gene product of c-kit receptor tyrosine kinase (c-Kit), a marker of ICC, and of neuronal proteins were also examined., Key Results: Gastric emptying was transiently delayed at 12 h after I/R, but returned to normal by 48 h. Expression of c-Kit protein as assessed by Western blotting and immunofluorescent staining of the smooth muscle layer, as well as expression of the mRNA of stem cell factor, the ligand for c-Kit, were reduced at both 12 and 48 h after I/R. The expression of neuronal nitric oxide synthase (nNOS) protein as assessed by Western blotting and immunofluorescent staining was also decreased at 12 h after I/R, but was restored to normal by 48 h., Conclusions & Inferences: Gastric I/R evokes transient gastroparesis with delayed gastric emptying, associated with disruption of the ICC network and nNOS-positive neurons.

Abstract: Background: Interstitial cells of Cajal (ICC) serve as intestinal pacemakers. Postoperative ileus (POI) is a gastrointestinal motility disorder that occurs following abdominal surgery, which is caused by inflammation‐induced dysfunction of smooth muscles and enteric neurons. However, the participation of ICC in POI is not well understood. In this study, we investigated the functional changes of ICC in a mouse model of POI. Methods: Intestinal manipulation (IM) was performed to induce POI. At 24 h or 48 h after IM, the field potential of the intestinal tunica muscularis was investigated. Tissues were also examined by immunohistochemistry and electron microscopic analysis. Key Results: Gastrointestinal transit was significantly decreased with intestinal tunica muscularis inflammation at 24 h after IM, which was ameliorated at 48 h after IM. The generation and propagation of pacemaker potentials were disrupted at 24 h after IM and recovered to the control level at 48 h after IM. ICC networks, detected by c‐Kit immunoreactivity, were remarkably disrupted at 24 h after IM. Electron microscopic analysis revealed abnormal vacuoles in the ICC cytoplasm. Interestingly, the ICC networks recovered at 48 h after IM. Administration of aminoguanidine, an inducible nitric oxide synthase inhibitor, suppressed the disruption of ICC networks. Ileal smooth muscle tissue cultured in the presence of nitric oxide donor, showed disrupted ICC networks. Conclusions and Inferences: The generation and propagation of pacemaker potentials by ICC are disrupted via nitric oxide after IM, and this disruption may contribute to POI. When inflammation is ameliorated, ICC can recover their pacemaker function. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells. [ABSTRACT FROM AUTHOR]