c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine

 c-kit immunohistochemistry was performed on unfixed frozen sections of human small (duodenum, jejunum, and ileum) and large intestine (ascending, transverse, descending, and sigmoid colon). The c-kit immunoreactive cells in the muscularis externa of the intestinal wall were identified as interstitial cells of Cajal (ICC) and mast cells. ICC were identified by their morphology, localization, and organization based on previous light and electron microscopic studies. In the small intestine, ICC were located primarily in relation to the myenteric plexus of Auerbach, but also in septa between circular muscle lamellae. In the large intestine, ICC were seen in relation to Auerbach’s plexus, but also and in great numbers in the circular muscle layer and in teniae of the longitudinal muscle layer. The morphology of the ICC was similar in the small and large intestine, but the pattern of distribution was obviously different. c-kit immunoreactive mast cells were found predominantly in the inner part of the circular muscle layer. The anti-c-kit method is found to be an easy and reliable method to study at least most of the interstitial cells of Cajal and thereby contribute to further normal and pathological studies. Accepted: 31 July 1997     

Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.     

Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon

The role of the interstitial cells of Cajal (ICC) associated with the myenteric plexus (ICC-MP) as regulators of the motility of the colonic external muscle remains unclear. Ultrastructural studies of myenteric interstitial cells are lacking in human colon. We therefore characterized the distinctive ultrastructure of these cells in the myenteric region of the colon by transmission electron microscopy of the region between the main muscle layers in all parts of the colon in unaffected areas of resected specimens from nine adult human patients. ICC-MP were similar in various colonic regions and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells, and close contacts (<100 nm) between ICC and nerves. No gap junctions were observed. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, but lacked prominent intermediate filaments and caveolae. FLC had a patchy basal lamina, and peg and socket junctions were observed between them. Macrophage-like cells frequently occurred in close apposition with FLC and, more seldomly, with ICC-MP. The ultrastructure of ICC and FLC in the myenteric region of the human colon thus differs characteristically, but significant overlaps in the ultrastructure between ICC and FLC might complicate any interpretation in pathological ultrastructural studies of the human colonic muscle layer. An erratum to this article can be found at     

Inflammatory response to Dentitruncus truttae (Acanthocephala) in the intestine of brown trout

Brown trout, Salmo trutta L., were infected with the acanthocephalan Dentitruncus truttae with the most affected areas being the anterior (near the pyloric caeca) and middle intestine. The parasite attached with a proboscis which usually penetrated the mucosa, lamina propria, stratum compactum, stratum granulosum and, sometimes, the muscularis layer. Around the parasite's body was an area of inflammatory tissue. At the point of attachment the lamina propria was thickened and the stratum compactum, stratum granulosum and muscularis layer were disrupted by proboscis penetration. Rodlet cells were more numerous in infected fish (P<0.01), and were found in the epithelial layer away from the worm. Infected intestines had larger numbers of mast cells (P<0.01), often in close proximity to, and inside, the blood capillaries and associated with fibroblasts of the muscularis layer and the stratum granulosum. Their migration toward the site of infection was suggested. Intense degranulation of mast cells was encountered in all intestinal layers especially near the parasite's body. Immunohistochemical tests were conducted on sections of intestinal tissue of uninfected and infected fish revealing the presence of met-enkephalin and serotonin (5-HT) in immuno-related cells of the intestine wall. Infected trout had larger numbers of elements positive to met-enkephalin and serotonin antisera. These data provided evidence for the role of the immune system of brown trout in the modulation of the inflammatory response to D. truttae. Results are discussed with respect to host immune response to an intestinal helminth.     

Histological and histochemical observations on the forestomach of goat during pre-natal life.

Non-keratinized, stratified squamous epithelium lined the mucosa of the fore-stomach. The mucosal appendages in the form of ruminal papillae did not develop in the rumen during this study, however, omasal laminae and reticular ribs were evident in groups I and II, respectively. Cytoplasmic accumulations were evident in the middle layer of the epithelium, which is characteristic of this region. The thickness of the epithelium increased constantly in the rumen and reticulum, but in the omasum it remained constant with the increase in the size of the fetus. The corial papillae in the rumen, reticulum and omasum appeared at the 12.7-, 11.5-and 14.6-cm CVR stage, respectively. The muscularis mucosae appeared in the omasum at the 14.6-cm CVR stage in the first-order laminae. The tunica muscularis consisted of spirally arranged muscle fibre bundles. Cross-cut muscle fibre bundles below the tip of the omasal laminae descended downward with the increase in the size of the fetus and continued with the inner circular layer. Acid and alkaline phosphatases and fat could not be observed. The concentration of glycogen in the epithelium was maximum in group III and minimum in group II.     

Development of c‐kit immunopositive interstitial cells of Cajal in the human stomach

Interstitial cells of Cajal (ICC) include several types of specialized cells within the musculature of the gastrointestinal tract (GIT). Some types of ICC act as pacemakers in the GIT musculature, whereas others are implicated in the modulation of enteric neurotransmission. Kit immunohistochemistry reliably identifies the location of these cells and provides information on changes in ICC distribution and density. Human stomach specimens were obtained from 7 embryos and 28 foetuses without gastrointestinal disorders. The specimens were 7–27 weeks of gestational age, and both sexes are represented in the sample. The specimens were exposed to anti‐c‐kit antibodies to investigate ICC differentiation. Enteric plexuses were immunohistochemically examined by using anti‐neuron specific enolase and the differentiation of smooth muscle cells (SMC) was studied with anti‐α smooth muscle actin and anti‐desmin antibodies. By week 7, c‐kit‐immunopositive precursors formed a layer in the outer stomach wall around myenteric plexus elements. Between 9 and 11 weeks some of these precursors differentiated into ICC. ICC at the myenteric plexus level differentiated first, followed by those within the muscle layer: between SMC, at the circular and longitudinal layers, and within connective tissue septa enveloping muscle bundles. In the fourth month, all subtypes of c‐kit‐immunoreactivity ICC which are necessary for the generation of slow waves and their transfer to SMC have been developed. These results may help elucidate the origin of ICC and the aetiology and pathogenesis of stomach motility disorders in neonates and young children that are associated with absence or decreased number of these cells.     

Ultra-structural identification of interstitial cells of Cajal in the zebrafish Danio rerio

The interstitial cells of Cajal (ICCs) are important mediators of gastrointestinal (GI) motility because of their role as pacemakers in the GI tract. In addition to their function, ICCs are also structurally distinct cells most easily identified by their ultra-structural features and expression of the tyrosine kinase receptor c-KIT. ICCs have been described in mammals, rodents, birds, reptiles, and amphibians, but there are no reports at the ultra-structural level of ICCs within the GI tract of an organism from the teleost lineage. We describe the presence of cells in the muscularis of the zebrafish intestine; these cells have similar features to ICCs in other vertebrates. The ICC-like cells are associated with the muscularis, are more electron-dense than surrounding smooth muscle cells, possess long cytoplasmic processes and mitochondria, and are situated opposing enteric nervous structures. In addition, immunofluorescent and immunoelectron-microscopic studies with antibodies targeting the zebrafish ortholog of a putative ICC marker, c-KIT (kita), showed c-kit immunoreactivity in zebrafish ICCs. Taken together, these data represent the first ultra-structural characterization of cells in the muscularis of the zebrafish Danio rerio and suggest that ICC differentiation in vertebrate evolution dates back to the teleost lineage.     

Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks

Interstitial cells of Cajal (ICC) are the pacemaker cells in gastrointestinal (GI) muscles. They also mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium. What is known about functional roles of ICC comes from developmental studies based on the discovery that ICC express c-kit. Functional development of ICC networks depends on signaling via the Kit receptor pathway. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells within the tunica muscularis, and the loss of ICC in some motility disorders. Manipulating Kit signaling with reagents to block the receptor or downstream signaling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies of the development of these cells within the tunica muscularis and also allowed the study of specific functions of different classes of ICC in several regions of the GI tract. This article examines the role of ICC in GI motility, focusing on the functional development and maintenance of ICC networks in the GI tract and the phenotypic changes that can occur when the Kit signaling pathway is disrupted.     

Subtractive hybridization unravels a role for the ion cotransporter NKCC1 in the murine intestinal pacemaker

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.     

Down-Regulation of Hydrogen Sulfide Biosynthesis Accompanies Murine Interstitial Cells of Cajal Dysfunction in Partial Ileal Obstruction

Purpose

To investigate the role of endogenous hydrogen sulfide (H₂S) in partial obstruction-induced dysfunction of the interstitial cells of Cajal (ICC) in mice ileum.

Materials and Methods

Partial intestinal obstruction was induced surgically in male imprinting control region (ICR) mice. ICC networks were studied by Immunohistochemistry. Electrical activity was recorded by intracellular recording techniques. The expression of ICC phenotype marker c-kit receptor tyrosine kinase (c-kit), membrane binding stem cell factor (mSCF), the endogenous H₂S biosynthesis enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) was studied by Western blotting. The expression of tumor necrosis factor-α (TNF-α) mRNA was observed by using real-time polymerase chain reaction.

Results

Partial intestinal obstruction resulted in ICC networks were disrupted above obstruction 14 days after the operation. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed and their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized. The expression of c-kit and mSCF was significantly decreased, also suggesting the disruption of the ICC network. The expression of TNF-α was significantly increased in the tunica muscularis of the obstructed intestine. Treatment of cultured intestinal smooth muscle cells with TNF-α caused dramatic down regulation of mSCF. The expression of CBS and CSE was significantly decreased in the tunica muscularis of the obstructed intestine. Intraperitoneal injection (i.p) of DL-propargylglycine, an irreversible inhibitor of CSE, and aminooxyacetic acid, an inhibitor of CBS, elevated the expression of TNF-α mRNA in the tunica muscularis of the ileum. Obstruction-induced over expression of TNF-α was significantly improved by supplementation of NaHS, but not the expressions of mSCF and c-kit.

Conclusions

The down regulation of endogenous H₂S biosynthesis is related to over expression of TNF-α in obstructed small intestine. TNF-α-mediated mSCF down-regulation is not the only reason of partial intestinal obstruction-induced loss of ICC.     

Interstitial cells of Cajal,macrophages and mast cells in the gut musculature: morphology,distribution, spatial and possible functional interactions

• ? Introduction
• ? Identification of the cells
  • ‐ ICC
  • ‐ Macrophages
    • ‐ Activation
    • ‐ Identification
  • ‐ Mast cells
    • ‐ Activation
    • ‐ Identification
• ? Cell distribution
  • ‐ ICC in rodent gastrointestinal tract
  • ‐ ICC in human gastrointestinal tract
  • ‐ Macrophages in rodent gastrointestinal tract
  • ‐ Macrophages in human gastrointestinal tract
  • ‐ Mast cells in rodent gastrointestinal tract
  • ‐ Mast cells in human gastrointestinal tract
• ? Inflammation
  • ‐ Models of inflammation
    • ‐ LPS administration
    • ‐ Surgical anastomosis
    • ‐ Ileal obstruction
    • ‐ Post‐operative ileus
    • ‐ Helminth infections
  • ‐ Inflammatory bowel disease
  • ‐ Achalasia
• ? Diabetes mellitus
  • ‐ NOD/LtJ mice
  • ‐ STZ‐DM rats
• ? Conclusions

Interstitial cells of Cajal (ICC) are recognized as pacemaker cells for gastrointestinal movement and are suggested to be mediators of neuromuscular transmission. Intestinal motility disturbances are often associated with a reduced number of ICC and/or ultrastructural damage, sometimes associated with immune cells. Macrophages and mast cells in the intestinal muscularis externa of rodents can be found in close spatial contact with ICC. Macrophages are a constant and regularly distributed cell population in the serosa and at the level of Auerbach’s plexus (AP). In human colon, ICC are in close contact with macrophages at the level of AP, suggesting functional interaction. It has therefore been proposed that ICC and macrophages interact. Macrophages and mast cells are considered to play important roles in the innate immune defence by producing pro‐inflammatory mediators during classical activation, which may in itself result in damage to the tissue. They also take part in alternative activation which is associated with anti‐inflammatory mediators, tissue remodelling and homeostasis, cancer, helminth infections and immunophenotype switch. ICC become damaged under various circumstances – surgical resection, possibly post‐operative ileus in rodents – where innate activation takes place, and in helminth infections – where alternative activation takes place. During alternative activation the muscularis macrophage can switch phenotype resulting in up‐regulation of F4/80 and the mannose receptor. In more chronic conditions such as Crohn’s disease and achalasia, ICC and mast cells develop close spatial contacts and piecemeal degranulation is possibly triggered.     

Studies on 5'-nucleotidase histochemistry. III. 5'-Nucleotidase activity in smooth muscle cells of the rat's gastrointestinal tube.

The distribution pattern of histochemically detectable 5'-nucleotidase (5'-Nase) activity is described in smooth muscle cells of the rat's gastrointestinal tube (esophagus, stomach, small intestine, large intestine). Both, light and electron microscopic methods are used. Faint positive 5'-Nase activity is observed on smooth muscle cells of the lamina muscularis mucosae in the thoracal esophagus whereas it is completely absent from smooth muscle cells of the abdominal esophagus and the stomach. In the small and large intestine strong positive 5'-Nase reaction is found on smooth muscle cells of the lamina muscularis mucosae and the innermost part of the lamina muscularis externa. In the circular and longitudinal layer of the lamina muscularis externa a slight increase in 5'-Nase activity is observed from the proximal to the distal segments. The reaction product is restricted to the outer cell surface of smooth muscle cells. In the small intestine the strong enzymatic activity in the innermost part of the muscularis externa is found to be localized at small and dense muscle cells (sd-cells). Common morphological and histochemical characteristics of sd-cells and smooth muscle cells of the lamina muscularis mucosae are emphasized. Hypothetical functions e.g. uptake of precursors of nucleosidephosphates, possible functional connection to a high glycogen content, correlation between 5'-Nase activity and proliferation capacity and local vasodilatory effect are discussed.     

Ultrastructural characterization of interstitial cells of Cajal associated with the submucosal plexus in the proximal colon of the guinea pig

Interstitial cells of Cajal (ICC) associated with the submucosal (submucous) plexus (ICC-SP) in the proximal colon of the guinea pig were studied by immunohistochemistry and electron microscopy. Whole-mount stretch preparations with c-Kit immunohistochemistry revealed that a number of ICC-SP constituted a dense cellular network around the submucosal plexus. Some of these ICC-SP were observed in the vicinity of the muscularis mucosae in sections immunostained for c-Kit and α-smooth muscle actin. Ultrastructural observation demonstrated, for the first time, that ICC-SP of the proximal colon of the guinea pig retained typical ultrastructural characteristics of ICC repeatedly reported in association with the tunica muscularis of the gastrointestinal tract: a basal lamina, caveolae, many mitochondria, abundant intermediate filaments and the formation of gap junctions with the same type of cells. The most remarkable ultrastructural finding was the presence of thick bundles composed of the processes of ICC-SP connected to each other via large gap junctions. These ICC-SP might be involved in the main mucosal functions of the proximal colon of the guinea pig, namely the transportation of water and electrolytes, possibly via their involvement in the spontaneous contractions of the muscularis mucosae.     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.     

Macrophages in the Small Intestinal Muscularis Externa of Embryos, Newborn and Adult Germ-Free Mice

Previously, we demonstrated the presence of a constant and regularly distributed macrophage population of ramified cells in the intestinal muscle layers of smaller rodents. The function of these resident macrophages under normal conditions remains unknown. Histochemistry, immunohistochemistry and electron microscopy were applied to the muscularis externa of 15- and 17-day-old embryos, 2-day-old mice, adult germ-free and conventional mice. Since lipopolysaccharides (LPS) activates macrophages and inflammation affects gut motility, LPS-treated mice were also included in the study. Two macrophage antibodies, F4/80 and 2F8 were used to demonstrate the presence of macrophages in the muscle layers. The localization was confirmed by electron microscopy. In contrast to conventional adult mice, the muscle layers in embryos, newborn and germ-free adult mice were devoid of class II MHC antigen reactive cells. The acid phosphatase reaction and antibodies directed towards a lysosomal protein (Lamp-2) were used in order to verify other activation markers. None of these showed specific staining of the muscularis macrophages. Only LPS-treated adult mice showed iNOS-positive cells in whole mounts. We conclude that the characteristic organization and distribution of muscularis macrophages in adult mice are also present in embryos, newborn and germ-free mice and thus develop independently of foreign antigens. Further, these macrophages are truly resident and appear to have differential responses to exogene stimuli.     

Muscularis inflammation and the loss of interstitial cells of Cajal in the endothelin ETB receptor null rat

Endothelin receptor null rats [ETB(-/-)] are a model for long-segment Hirschsprung's disease. These animals have significant intestinal distension (megaileum) proximal to a constricted region of the gastrointestinal tract lacking enteric ganglia. Experiments were performed to determine the pathophysiological changes that occur in these animals and to examine the tunica muscularis as a unique, immunologically active compartment. We observed abnormal intestinal flora in ETB(-/-) rats, which included a marked increase in gram-negative aerobes (Enterobacteriaceae) and anaerobes (Bacteroidaceae) in the distended region of the small intestine. Histochemical observations showed that neutrophilic infiltration was rarely or not observed, but the number of ED2 positive macrophages was increased in the tunica muscularis. Expression of IL-1beta and IL-6 mRNA was also significantly increased, and the level of CD14 (LPS receptors) were increased significantly in the tunica muscularis. Spontaneous phasic contractions were irregular in the distended intestinal regions of ETB(-/-) rats, and this was associated with an increased number of macrophages and damage to interstitial cells of Cajal (ICC) as revealed by using Kit-like immunoreactivity and electron microscopy. These results suggest that ED2-positive resident macrophages may play an important role in the inflammation of tunica muscularis in ETB(-/-) rats. Increased numbers and activation of macrophages may result in damage to ICC networks leading to disordered intestinal rhythmicity in regions of the gut in which myenteric ganglia are intact.     

Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation finally leading to extensive tissue fibrosis and resulting in a stiff colon unable to carry out peristalsis or to resorb fluids. Telocytes, a peculiar type of stromal cells, have been recently identified in the human gastrointestinal tract. Several roles have been proposed for telocytes, including mechanical support, intercellular signalling and modulation of intestinal motility. The aim of the present work was to investigate the presence and distribution of telocytes in colonic specimens from UC patients compared with controls. Archival paraffin‐embedded samples of the left colon from UC patients who underwent elective bowel resection and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were identified by CD34 immunohistochemistry. In early fibrotic UC cases, fibrosis affected the muscularis mucosae and submucosa, while the muscularis propria was spared. In advanced fibrotic UC cases, fibrosis extended to affect the muscle layers and the myenteric plexus. Few telocytes were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. In the muscle layers and myenteric plexus of early fibrotic UC, telocytes were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of telocytes was reduced or even completely absent around smooth muscle bundles and myenteric plexus ganglia, paralleling the loss of the network of interstitial cells of Cajal. In UC, a loss of telocytes accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility.     

Immunohistochemical analysis of connexin26 and 43 expression in the mouse alimentary tract

The spatial pattern of connexins26 (Cx26) and 43 (Cx43) expressions were investigated in the mouse digestive tract by immunocytochemistry. High levels of connexin43 in the epithelium of the oesophagus, non-glandular part of the stomach, and the circular layer of duodenal and ilea muscularis externa were detected. Cx26 was expressed in stratum granulosum of oesophagal folds and in the non-glandular part of the stomach. A low level of immunoreactivity of Cx43 was observed in the circular, and very low in the longitudinal layer of the muscularis externa in the stomach and colon. No immunoreactivity for Cx26 and Cx43 was found in the entire muscularis externa of the oesophagus or in the longitudinal muscle layers of the duodenum and ileum.