Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine

In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R ^epi). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R ^epi, followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R ^trans or TER) overestimated true R ^epi by ~60%. In colon, strongest expression of “tightening” claudins 1, 3, 4, 5, and 8 was detected. In accordance with R ^epi the most proximal of the small intestinal segments, duodenum exhibited highest expression of “tightening” claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.     

Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine

Multidrug resistance-associated protein 3 (MRP3; symbol ABCC3), has been shown to mediate ATP-dependent transport of organic anions including 17beta-glucuronosyl estradiol, glucuronosyl bilirubin, monovalent, and sulfated bile salts. MRP3 mRNA expression was reported in rat intestine suggesting a role of MRP3 in the intestinal transport. We examined the expression and localization of MRP3 in rat small and large intestine by RT-PCR, immunofluorescence, and immunoblot analysis. MRP3 was identified in all intestinal segments by RT-PCR. MRP3 expression was low in duodenum and jejunum but markedly increased in ileum and colon. With the use of a rat MRP3 specific antibody, MRP3 was localized to the basolateral domains of enterocytes. Immunofluorescence analysis and immunoblot analysis confirmed a strong expression of rat MRP3 in ileum and colon. In contrast, MRP2 was predominantly expressed in the proximal segments of rat small intestine. Our findings demonstrate a high expression of rat MRP3 in ileum and colon and provide evidence for an involvement of MRP3 in the ATP-dependent transport of organic anions, including bile salts from the enterocyte into blood.     

Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment

Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.     

EpCAM contributes to formation of functional tight junction in the intestinal epithelium by recruiting claudin proteins

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell-cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell-cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell-cell junctions.     

Effect of age and dietary calcium on intestinal calbindin D-9k expression in the rat

The capacity of rats and humans to adapt to low dietary Ca by increasing intestinal Ca absorption declines with age. The intestinal calbindin-D-9k protein (calbindin) is thought to play a role in the transcellular transport of Ca across the mammalian intestine. The purpose of these studies was to determine the effect of age and diet on the expression of calbindin at the protein and mRNA levels. Young (2 month) and adult (12 month) male F344 rats were placed on either a high Ca diet (1.2%) or a low Ca diet (0.02%) for four weeks. In the duodenum, the level of intestinal calbindin protein induced by a low Ca diet was 8-fold higher in young rats compared to adult rats. In the ileum, expression of calbindin protein was only about 10% that of the duodenum. In addition, the adult ileum showed the same decreased adaptation to a low Ca diet that was seen in the adult duodenum. In both the duodenum and the ileum, the changes in calbindin protein expression were highly correlated with calbindin mRNA expression and the correlations in each segment were quantitatively similar. In the duodenum, the changes in calbindin protein levels were strongly correlated with both Ca transport and Ca uptake. This quantitative correlation suggests a role for calbindin protein in the age-related decline in Ca absorption. In the ileum, the decreased adaptation to a low Ca diet may also be important given the long transit time through the distal intestine. The changes in both intestinal segments may contribute to the negative Ca balance seen in adult rats fed a low Ca diet.     

Protein Kinase C Activation Has Distinct Effects on the Localization, Phosphorylation and Detergent Solubility of the Claudin Protein Family in Tight and Leaky Epithelial Cells

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell–cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.     

Mesenteric Lymphoblast Localization Throughout the Murine Small Intestine: Temporal Analysis Relating Intestinal Length and Lymphoblast Division

Abstract. Mesenteric lymphoblasts (MLN) have a predilection to selectively localize in the lamina propria and epithelium of the small intestine. Using an adoptive transfer method, we examined the localization kinetics of these blasts in the intestinal wall with respect to their distribution from duodenum to terminal ileum and also assessed their mitotic activity by autoradiographic techniques. ³H-thymidine-labelled MLN cells were found throughout the small intestine by 6 hr post-transfer and reached a maximum frequency in this organ by 24 hr post-transfer.
Donor blasts were most frequent in the duodenum and terminal ileum regions of the gut. Subsequently, the frequency of labelled cells throughout the intestinal wall declined to near zero. the apparent accumulation of MLN blasts in the gut was not related to either a temporary retention and departure from the pulmonary vasculature or to mitotic division of labelled cells in the gut wall. A model describing the relationship between MLN blast localization kinetics in various segments of the intestine was formulated.     

Mesenteric lymphoblast localization throughout the murine small intestine: temporal analysis relating intestinal length and lymphoblast division

Mesenteric lymphoblasts (MLN) have a predilection to selectively localize in the lamina propria and epithelium of the small intestine. Using an adoptive transfer method, we examined the localization kinetics of these blasts in the intestinal wall with respect to their distribution from duodenum to terminal ileum and also assessed their mitotic activity by autoradiographic techniques. 3H-thymidine-labelled MLN cells were found throughout the small intestine by 6 hr post-transfer and reached a maximum frequency in this organ by 24 hr post-transfer. Donor blasts were most frequent in the duodenum and terminal ileum regions of the gut. Subsequently, the frequency of labelled cells throughout the intestinal wall declined to near zero. The apparent accumulation of MLN blasts in the gut was not related to either a temporary retention and departure from the pulmonary vasculature or to mitotic division of labelled cells in the gut wall. A model describing the relationship between MLN blast localization kinetics in various segments of the intestine was formulated.     

Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.     

Morphology and stress-strain properties along the small intestine in the rat

The stress-strain relationship is determined by the inherent mechanical properties of the intestinal wall, the geometric configurations, the loading conditions and the zero-stress state of the segment. The purpose of this project was to provide morphometric and biomechanical data for rat duodenum, jejunum and ileum. The circumferential strains were referenced to the zero-stress state. Large morphometric variations were found along the small intestine with an increase in the outer circumferential length and luminal area and a decrease in wall thickness in distal direction. The serosal residual strain was tensile and decreased in distal direction (P < 0.05). The mucosal residual strain was compressive and the absolute value decreased in distal direction (P < 0.001). The stress-strain experiments showed that the duodenum was stiffest. All segments were stiffest in longitudinal direction (P < 0.05). In conclusion, axial variation in morphometric and biomechanical properties was found in the small intestine. The zero-stress state must be considered in future biomechanical studies in the gastrointestinal tract.     

Proteomic characterization of the site-dependent functional difference in the rat small intestine

To investigate the site-dependent functional difference in the small intestine, proteomic analysis was carried out on the three distinct parts of the rat small intestine. Male Wistar rats (7 weeks old) were fed a semi-purified diet ad libitum for 1 week. Intestinal tissues from the proximal, middle and distal regions of the small intestine were subjected to two-dimensional polyacrylamide gel electrophoresis, and the abundance of each spot was determined fluorometrically. MALDI-TOF/MS and LC-MS/MS analysis of the tryptic peptides were performed to identify the proteins. Many of the 180 identified proteins showed a distinctive distribution pattern along the small intestine. Glutathione S-transferase, Catechol O-methyltransferase and Villin 2 decreased gradually from the jejunum to the ileum, in contrast, non-specific dipeptidase and Keratin 19 increased gradually toward the ileum. The voltage-dependent anion channel 2 was most abundant in the duodenum while the L- and I-Fatty acid binding protein (FABP) and Cellular retinol binding protein (CRBP-II) were in the jejunum, and the Bile acid binding protein (BABP) was only observed in the ileum. The findings of these and of another proteins identified in this study may contribute to further understanding of the small intestinal function, and to clinical applications of small intestinal diseases.     

Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract

Summary Glucose is actively absorbed in the intestine by the action of the Na⁺-dependent glucose transporter. Using an antibody against the rabbit intestinal Na⁺-dependent glucose transporter (SGLT1), we examined the localization of SGLT1 immunohistochemically along the rat digestive tract (oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum). SGLT1 was detected in the small intestine (duodenum, jejunum and ileum), but not in the oesophagus, stomach, colon or rectum. SGLT1 was localized at the brush border of the absorptive epithelium cells in the small intestine. Electron microscopical examination showed that SGLT1 was localized at the apical plasma membrane of the absorptive epithelial cells. SGLT1 was not detected at the basolateral plasma membrane. Along the crypt-villus axis, all the absorptive epithelial cells in the villus were positive for SGLT1, whose amount increased from the bottom of the villus to its tip. On the other hand, cells in the crypts exhibited little or no staining for SGLT1. Goblet cells scattered throughout the intestinal epithelium were negative for SGLT1. These observations show that SGLT1 is specific to the apical plasma membrane of differentiated absorptive epithelial cells in the small intestine, and suggest that active uptake of glucose occurs mainly in the absorptive epithelial cells in the small intestine.     

不同途径下MIMP对 炎症性肠病小鼠肠道菌群结构的影响

目的通过葡聚糖硫酸钠诱导小鼠炎症性肠病(IBD)模型并观察不同途经下乳杆菌微小膜蛋白(MIMP)对炎症性肠病小鼠的紧密连接蛋白及菌群结构的影响。方法 C57BL/6小鼠24只根据DSS和MIMP不同干预组合将其分为4组:MIMP腹腔注射组(n=6)、MIMP灌胃组(n=6)、诱导肠炎组(n=6)和健康对照组(n=6),利用Western blot对各组小鼠肠道中紧密连接蛋白(Occludin、JAM-1和ZO-1)的表达进行检测,采用16SrRNA测序技术检测V4区鉴定细菌,并进行菌群差异分析。结果 MIMP腹腔注射及灌胃均可显著提高IBD小鼠肠道中紧密连接蛋白的表达水平,灌胃组效果更为显著;MIMP干预后小鼠肠道中拟杆菌门(Bacteroidetes)丰度增高、厚壁菌门(Firmicutes)及变形菌门(Proteobacteria)丰度降低,LEfSe分析、PCoA分析和PCA分析提示4组小鼠肠道菌群结构差异显著。结论不同途径下MIMP均可显著提高IBD小鼠肠道中紧密连接蛋白的表达水平,改善肠黏膜屏障功能,纠正小鼠肠道菌群结构紊乱。     

Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins

Tight junctions (TJs) are dynamic, multiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiological development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a biosensor of TJ remodeling, we observed increased claudin 4 fluorescence recovery after photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon γ and tumor necrosis factor α increased the proportion of mobile claudin 4 in the TJ. Up-regulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin-binding incompatibility.