Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study

Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases) were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.     

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.     

东方铃蟾消化道组织学的初步研究

采用组织学方法对东方铃蟾的消化道进行了研究。结果表明:肠分为十二指肠、空肠和大肠。消化道管壁由粘膜层、粘膜下层、肌层和浆膜层构成。食道、胃和肠均为单层柱状上皮。胃和十二指肠的粘膜皱褶最丰富。食道腺为复泡状腺,胃腺属于单管状腺,肠的各段无多细胞腺体,但空肠和大肠有丰富的杯状细胞。肌层均为平滑肌,内层环肌较厚,外侧纵肌较薄,其中大肠的外侧纵肌最发达。     

Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.     

Immunohistochemical localization of a gap junction protein (connexin43) in the muscularis externa of murine,canine, and human intestine

Electron-microscopic studies have revealed a heterogeneous distribution of gap junctions in the muscularis externa of mammalian intestines. This heterogeneity is observed at four different levels: among species; between small and large intestines; between longitudinal and circular muscle layers; and between subdivisions of the circular muscle layer. We correlated results obtained with two immunomethods, using an antibody to the known gap-junctional protein (connexin43) with ultrastructural findings, and further evaluated the respective sensitivity of these two approaches. For comparative reasons we also included the vascular smooth muscle of coronary arteries into our study. Two versions of the immunotechnique (peroxidase-antiperoxidase and fluorescence methods) were applied to frozen sections of murine, canine, and human small and large intestines, as well as to pig coronary artery. In the small intestine of all three species a very strong reactivity marked the outer main division of the circular muscle layer, while the longitudinal muscle layer as well as the inner thin division of the circular muscle layer were negative. In murine and human colon both muscle layers were negative, while in canine colon the border layer between the circular muscle and the submucosa reacted strongly, and scattered activity was found in the portion of the circular muscle layer (one tenth of its thickness) closest to the submucosa. The remainder of the circular muscle layer and the entire longitudinal muscle layer were negative in the canine colon. In the coronary artery we could not confirm the positive, specific labeling reported by other investigators (l.c.). In conclusion, we found close correlations at all four above-mentioned levels in the distribution of gap junctions in the gut musculature, as determined by binding of anticonnexin43 in comparison to conventional ultrastructural studies. Since no significant immunostaining was found in (i) the outer border of the circular muscle layer of the canine colon and (ii) the border layer between the submucosa and the circular muscle layer of human colon, where rare gap junctions have been identified at the ultrastructural level, we conclude that the electron-microscopic analysis is the more sensitive of the two methods.     

日本沼虾消化道形态和组织学特点

应用石蜡切片和扫描电镜技术对日本沼虾消化道进行了研究。结果表明,食道壁向腔内形成四个纵突,食道上皮由单层柱状细胞构成,上皮下的结缔组织中具有放射肌和皮肤腺,环肌层近于连续。食道和胃连结处的管腔背方具食道瓣,胃内具胃磨、滤器和滤沟等结构,胃的组织学结构中除无皮肤腺分布外与食道相似。中肠较长,约占整个消化道的７１７％,具一对中肠前盲囊。中肠上皮细胞大致有两种类型,基膜着色深,环肌层连续,纵肌成束分散排列。后肠为一短管,管腔呈迷路状,其中部为一球形膨大的直肠。后肠的组织学结构与前肠相似。     

Ablation of connexin43 in smooth muscle cells of the mouse intestine: functional insights into physiology and morphology

Connexin43 (Cx43) gap-junction channels are highly abundant in intestinal smooth muscle but their functional impact has not been studied so far. Here, we have aimed to elucidate the functional role of Cx43 in the tunica muscularis of the mouse intestine in vivo. Transgenic mice with conditional deletion of Cx43 in smooth muscle cells (SMC) were generated. Histological investigations by immunofluorescence analyses and organ-bath recordings to assess the contractility of intestinal tissue strips were carried out. Measurements of gastrointestinal transit and of the visceromotor response by utilizing a standardized colorectal distension model to quantify alterations of visceral sensory function were also performed in SMC-specific Cx43 null mice and control littermates. Histologically, we found thickening of the tunica muscularis and a 13-fold increase of neutrophil infiltration of the gastrointestinal wall of SMC-specific Cx43 null mice. These animals also exhibited a decrease of 29% in gastrointestinal transit time. In contrast, the visceromotor response to a standardized colorectal distension was elevated, as was the contractility in SMC-specific Cx43 null mice, compared with controls. Thus, SMC-specific ablation of Cx43 in mice leads to morphological and functional alterations of the intestinal tunica muscularis, to gastrointestinal motor dysfunction and to altered visceral sensory function. This study was supported by a grant from the German Research Association (Wi 270/25-1,2) to K.W. and in part by the IFORES program of the University Hospital, Essen, Germany.     

Development of the external muscle coats in the digestive tract of the opossum, Didelphis virginiana

Development of the external musculature of the gastrointestinal tract has been studied in the stomach, small intestine and colon of the postnatal opossum. The muscle support is thin and poorly developed at birth, especially in the stomach and small intestine where only the inner layer is completely formed. The outer layer is discontinuous and formed by scattered myoblasts. The muscularis externa of the colon at birth is considerably thicker and both layers are present. Subsequent development of the muscularis externa consists of an early period of proliferative activity followed by hypertrophy. A low rate of mitotic activity continues throughout development and into the adult. Elements of the myenteric plexus are present at birth.     

Structure and Function of the Gastrointestinal Tract of the Green Turtle(Chelonia mydas) Hatchling

The gastrointestinal tracts of four Chelonia mydas hatchlings were examined at the anatomical, histological and ultrastructural level. Our results show that the gastrointestinal tract(GI) is composed by esophagus, stomach, small intestine(SI) and large intestine(LI), and histologically of mucosa, submucosa, muscularis externa(ME) and serosa. The esophagus is marked by conical papillae lined by keratinized stratified squamous epithelium, whereas the remaining GI by simple columnar epithelium; esophageal diverticulum is absent. The stomach covered with mucous granule cells, contains cardia, fundic regions and pylorus, which are separately characterized by cardiac glands, fundic glands and pyloric glands, and have the thickest submucosa and ME of the GI. The ME of the esophagus mainly consist of one layer of circular smooth muscle whereas the rest of GI of two layers, inner circular muscle and outer longitudinal muscle. The SI is slightly longer than the LI and the GI is approximately 5.11 times of the carapace length. The SI is lined with longitudinal zigzag folds and characterized by absorptive cells with longer and denser microvilli, whereas the LI by transversal folds, goblet cells and lymphoid nodules. Only intestinal glands appear in duodenum. Endocrine cells are observed in all sections of the GI and accounted for the largest proportion in duodenum. The results demonstrate a perfect combination of the structure and function of the GI and reveal that the digestion and absorption primarily occurs in the foregut. C. mydas hatchling may prefer carnivorous diet.     

The microscopic anatomy of the oesophagus, stomach and intestine of the channel catfish, Ictalurus punctatus

An anatomical study of the digestive tract of the channel catfish revealed that the oesophageal mucosa was longitudinally folded and that secondary folds were occasionally located on the primary longitudinal folds. The infoldings were more numerous near the stomach. The stratified squamous epithelium covering the folds was made up of a basal layer, large mucous cells and simple squamous cells on the surface. The epithelium on the side of the folds consisted primarily of mucous secreting cells. Taste buds were observed between mucous cells on the apical portion of the oesophageal folds and were more prevalent in the cranial part of the oesophagus. The remaining layers of the oesophagus were: a lamina propria-submucosa, tunica muscularis and adventitia or serosa.
The J-shaped stomach had two regions: a large sac-shaped region containing gastric glands and a smaller, nonglandular pyloric region. The large rugae of the stomach became gradually smaller near the pylorus. There was a well developed pyloric sphincter. The mucosa included a simple columnar epithelium, a lamina propria and adventitia or serosa.
The intestine could be differentiated into a thick ascending segment, a descending segment, a thin convoluted segment and a thicker terminal segment, the rectum. Many mucosal folds containing branched villi characterized the ascending segment of the intestine. The descending and convoluted segments contained fewer folds with shorter and less-branching villi and were smaller in diameter and thinner walled. Descending and convoluted segments were also mildly convoluted and accounted for 80% of the total length of the intestine. An intestinal valve with a sphincter marked the beginning of the rectum. There was an approximately four-fold increase in the thickness of the tunica muscularis of the terminal segment of the intestine.     

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     

Intramural distribution of immunoreactive vasoactive intestinal polypeptide (VIP), substance P,somatostatin and mammalian bombesin in the oesophago-gastro-pyloric region of the human gut

Summary The intramural distribution of vasoactive intestinal polypeptide (VIP), substance P, somatostatin and mammalian bombesin was studied in the oesophago-gastro-pyloric region of the human gut. At each of 21 sampling sites encompassing this entire area, the gut wall was separated into mucosa, submucosa and muscularis externa, and extracted for radioimmunoassay. VIP levels in the mucosa were very high in the proximal oesophagus (1231±174 pmol/g, mean±SEM) and showed varied, but generally decreasing concentrations towards the stomach, followed by a clear-cut increase across the pyloric canal (distal antrum: 73±16 pmol/g, proximal duodenum: 366±62 pmol/ g); consistent levels were found in submucosa and muscle (200–400 pmol/g) at most sites, the stomach again showing lower concentrations. By contrast, substance P was present in small amounts as far as the proximal stomach, but sharply increased across the pyloric canal, especially in mucosa and submucosa (distal antrum: 20±6.5 and 5.5±1.3 pmol/g; proximal duodenum: 62±8.5 and 34±11 pmol/g, respectively). Somatostatin concentrations were very low in the mucosa of the oesophagus and stepwise increased in the cardiac, mid-gastric and pyloric mucosa (cardia: 224±72 pmol/g; distal antrum: 513±152 pmol/g; proximal duodenum: 1013±113 pmol/g); concentrations in the submucosa and muscularis were generally low, with the exception of antrum and duodenum. Mammalian bombesin was comparatively well represented throughout the oesophageal muscularis (5–8 pmol/g), but most abundant in the stomach in all layers (oxyntic mucosa: 24±2.7 pmol/g; submucosa: 20±5.7 pmol/g; muscle: 28±5.0 pmol/g). In conclusion, a distinct differential distribution of the four peptides studied was revealed, indicating a diffuse, but highly differentiated peptide-containing innervation of the proximal human gut.     

Cholinesterase histochemistry of the rabbit distal colon

Summary The distribution of cholinesterase in the rabbit distal colon has been studied using histochemical methods. Non-specific cholinesterase predominates in muscle tissue, the longitudinal layer of the muscularis externa staining slightly more strongly than the circular layer. The muscularis mucosae stains the most intensely but its non-specific cholinesterase is markedly inactivated by 20 hr fixation in formaldehyde. Specific cholinesterase is present in ganglion cells in the myenteric and submucous plexuses, and these cells vary in staining intensity. Nerve fibres which stain for specific cholinesterase are present in the nerve plexuses and in the muscle layers. It is concluded that there are cholinergic nervous elements in the myenteric and submucous plexuses and in the muscle layers of the rabbit distal colon.M. R. C. Junior Research Fellow.     

金丝猴食管和胃连接部的组织学研究

本文研究了金丝猴食管和胃连接处的组织结构。金丝猴的食管粘膜为典型的复民鳞状上皮,食管末端含有粘膜腺,粘膜表面有轻微的角质化。管壁外纵肌层有少量的横纹肌。与胃粘膜的连接均位于胃的贲门部以内,两种上皮的连接是突然的,不存在过渡。 贲门腺为少量的分枝管状腺,短而直,由粘膜细胞构成,对PAS染色呈阳性反应。     

Comparative immunohistolocalization of carbonic anhydrase isozymes I, II and III in the equine and bovine digestive tract

Summary Immunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in equine and bovine digestive tracts were studied. In the horse, epithelial cells in both the oesophagus and non-glandular part of the stomach lacked all three isozymes. In contrast, surface epithelial and parietal cells in the glandular region of the stomach showed reactivity for CA-II. In the small intestine, absorptive columnar cells covering the villi in the duodenum were positive for CA-II. The epithelium of the jejunum and ileum lacked all three isozymes. In the large intestine, CA-II was detected in the columnar cells in the upper part of the crypt. In cattle, epithelial cells of the oesophagus showed reactions for CA-I and CA-III but not for CA-II. Although the absorptive epithelial cells of the small intestine lacked CA-I, CA-II and CA-III, those of the upper part of large intestine crypts were heavily stained for all three isozymes.     

Intercellular junctions between circular and longitudinal intestinal muscle layers

Summary Different intercellular relationships between smooth muscle cells are observed in the tunica muscularis externa of the ileum of guinea-pig. a) simple adjoining of the two muscle cells; b) nexus; c) desmosome-like attachment with dense aggregations on both membranes; d) projection from one muscle cell protruding into another muscle cell. All four types are found in the circular muscle layer. On the contrary, in the longitudinal layer clear-cut nexuses are not observed. Simple adjoining of muscle cells, nexuses, and desmosome-like attachments are observed between muscle cells of the circular layer and the longitudinallayer.Dedicated to Professor Angelo Bairati, on the occasion of his sixtieth birthday.I thank Professor E. G. Gray for discussion, Professor J. Z. Young for use of facilities, and the Wellcome Trust for a fellowship.     

Identification of interstitial cells in canine proximal colon using NADH diaphorase histochemistry

In gastrointestinal muscles special cells, referred to as interstitial cells, may be involved in pacemaking and transduction of inputs from the enteric nervous system. We have used a modification of the NADH diaphorase method to characterize the distribution of interstitial cells in the muscularis externa of the canine colon. The staining product of the NADH diaphorase reaction is useful because it allows light and electron microscopic studies to be performed with the same marker. Therefore rigorous identification of the cells observed at the light microscopic level could be made by electron microscopy. We were able to lable at least three classes of interstitial cells: (1) at the submucosal surface of the circular muscle layer; (2) within the thickness of the circular and longitudinal muscle layers; and (3) in the region of the myenteric plexus. This technique also labeled cell bodies and initial segments of processes of Dogiel type II neurones in enteric ganglia. Nerve fibres within the muscle layers did not exhibit NADH diaphorase activity. This study has identified the interstitial cells within the circular and longitudinal muscle layers and shows the arrangement of these cells in a three-dimensional network.     

白胸苦恶鸟消化管的组织学观察

采用石蜡切片技术对白胸苦恶鸟(Amaurornis phoenicurus)的消化道进行了组织学观察.结果表明,食管皱襞发达,黏膜上皮为复层扁平上皮,食管腺发达,颈段多于胸段,黏膜肌层为一层纵肌,厚约0.06 ～0.26 mm.肌层为一层厚约0.19～0.27mm的环肌.腺胃被覆单层柱状上皮,固有层内有单管腺和复管腺两种腺体,单管腺仅深约0.11 ～0.20 mm;复管腺厚约1.19～1.26 mm,占管壁的77.8％ ～80.4％.肌胃的类角质层发达,厚约0.16～0.24 mm.肌胃腺呈管状,与类角质层突起形成皱襞.肌层发达,由内环外纵两层平滑肌构成.肠绒毛无分支和中央乳糜管,十二指肠绒毛长而密集,空肠绒毛呈细长指状,直肠绒毛长且呈叶状.十二指肠与直肠肠绒毛内有大量致密淋巴小结,盲肠绒毛短,肠腺少.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.