The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

Absorption of alpha-ketoglutarate by the gastrointestinal tract of pigs

Only a small percentage of alpha-ketoglutarate (AKG) administered lumenally to pigs appears in the portal circulation. This has been attributed to mucosal metabolism, and possibly by limited absorption. Although transporters for di- and tricarboxylic acids, which includes the sodium-dependent transporter NaDC-1, have been detected in the small intestine, correlations with functional assays are lacking. Therefore, intact tissues from three regions of the small intestine, stomach, and colon of weaned pigs were used to measure rates of AKG absorption. Western analysis was used to detect NaDC-1 in the three regions of small intestine. Rates of AKG absorption were highest in the small intestine, lowest in the colon, and intermediate in the stomach. Immunoreactive NaDC-1 was detected in the small intestine and this coincided with a component of AKG absorption that was inhibited by AKG and succinate. In contrast, absorption of AKG was inhibitable by unlabeled AKG, but not succinate, in the stomach, and by neither in the colon. Feeding studies indicated that the amounts of AKG that might be included in practical diets for pigs would not (1) upregulate rates of AKG absorption or (2) exceed estimated capacities of the small intestine to absorb AKG. The present findings indicate that the efficacy of AKG as an alternative metabolic fuel for enterocytes to spare dietary amino acids is not limited by absorption.     

Histochemical and electron microscopical studies on the epithelium of the fetal colon with special reference to the occurrence of lipids

Summary Absorbing cells of the colon of 28 human fetuses were studied histochemically while 4 were studied ultrastructurally. Cellular ultrastructure included: microvilli with a fuzz, typically lateral specializations, small mitochondria placed at the peripheral part of the cells, apically placed lysosomes, basally placed lipid droplets and disperse accumulations of glycogen. Only a slight pinocytosis was observed and the endoplasmic reticulum and Golgi apparatus were only poorly developed. Comparing the enzymatic findings with the ultrastructural findings, it is suggested that some absorption may take place in the fetal large intestine. It is further considered whether an intracellular synthesis of lipids may occur.This study has been supported by a grant from the Association of the Aid of the Crippled Children, New York.     

Differences in the reducing power along the rat GI tract: Lower antioxidant capacity of the colon

The ability of the gastrointestinal (GI) tract, as well as other tissues, to cope with reactive oxygen species (ROS) efflux in pathological events is determined partly by epithelial antioxidant levels. These levels are comprized of tissue antioxidant enzymes and low molecular weight antioxidants (LMWA). While glutathione levels and the activity of enzymatic antioxidants along the GI tract have been studied, the contribution of the overall LMWA to the total antioxidant capacity has not yet been determined. In this study the overall antioxidant activity in the mucosa/submucosa and muscularis/serosa of various sections along the small intestine and colon of the rat was evaluated by determining the reducing power, which reflects the total antioxidant activity derived from LMWA, using cyclic voltammetry. The activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase was also measured. The reducing power (total antioxidant activity) was higher in the mucosa/submucosa of the small intestine as compared to the mucosa/submucosa of the colon. Similarly, catalase and SOD activity in the mucosa/submucosa of the small intestine was significantly higher than in the mucosa/submucosa of the colon. Differences were also observed in the reducing power and SOD activity in the muscularis/serosa of the rat small intestine as compared to the colon. The low antioxidant capacity in the colon may facilitate reactive oxygen species (ROS)-mediated injury and lead to inflammatory diseases such as ulcerative colitis, specifically in the colon.     

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.     

1-Naphthol beta-D-glucuronide formed intraluminally in rat small intestine mucosa and absorbed into the colon

UDP-glucuronosyltransferase expressed in the rat intestinal epithelial cells is important as the first barrier against chemicals. The distribution of 1-naphthol and its glucuronide formed in rat intestine was estimated by using everted intestine. Roughly 60% of the 1-naphthol added to the mucosal fluid was absorbed into the mucosa of the small intestine and colon within 30 min. Approximately 66% of the 1-naphthol absorbed in the proximal intestine was secreted intraluminally as a glucuronide, and a minimal 9% was transported into the serosal fluid as a glucuronide. In the distal intestine, approximately 34% was secreted intraluminally and 30% was transported into the serosal fluid as a glucuronide. The greatest amount of the glucuronide (37% of the absorbed 1-naphthol) was transported into the serosal fluid, whereas a minimal 7% was secreted intraluminally in the colon. In marked contrast, the colon was found to transport 1-naphthol-glucuronide from the mucosal fluid into the serosal fluid at an approximately 8-fold higher rate than that of the small intestine. These results suggest that, in the small intestine, phenolic xenobiotics are mostly glucuronidated and secreted intraluminally and that the resulting glucuronide is absorbed and transported into the serosal side of the colon.     

Tissue-specific induction of the carcinogen inducible cytochrome P450 isoform, P450IAI, in colonic epithelium

The epithelial cells of the gastrointestinal tract have the capacity to engage in biotransformation of ingested chemicals. A principal component of phase I metabolism of xenobiotics is the family of hemeproteins referred to as cytochrome(s) P450. The presence of cytochrome P450 isoforms was examined by Western blot analysis in the epithelial cells of the colon and proximal small intestine of male rats following oral administration with either beta-naphthoflavone or phenobarbital. The appearance of beta-naphthoflavone-inducible cytochrome P450IAI was observed in the colon and small intestine. The appearance of this cytochrome P450 isoform was concurrent with increases (up to 150-fold) in cytochrome P450-related O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin in both tissues. Following administration of phenobarbital, cytochrome P450IIBI was identified immunochemically in the small intestine. However, this isozyme could not be detected in colon. These data suggest that the epithelial cells of the proximal small intestine respond to beta-naphthoflavone and phenobarbital in a manner similar to the liver, whereas colonic epithelial cells may have a greater capacity to respond to P450IAI-type inducers such as beta-naphthoflavone. Evidence exists that differences in cytochrome P450 isozyme composition can affect the ultimate metabolic fate of ingested chemicals, including carcinogens, and thus a role for colonic P450-dependent monooxygenase activity in the biogenesis of cancer in this tumor-susceptible tissue is suggested.     

The distribution of endocrine cells along the mouse intestine: A quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30–60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the botom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3–4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

Isoforms of the human PDZ-73 protein exhibit differential tissue expression

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.     

长爪沙鼠肠道生长抑素和P 物质细胞密度的年龄变化

胃肠激素对调节小哺乳动物的消化功能具有重要作用。本文应用卵白素- 生物素- 过氧化物酶复合物(Avidin-biotin-peroxidase complex,ABC)免疫组织化学法,对冬季幼年、成年和老年长爪沙鼠肠道生长抑素(Somtostatin,SS)和P 物质(Substance P,SP)细胞进行了定位和比较。结果显示:不同年龄组两种内分泌细胞的形态学特征相似,呈圆形、椭圆形、梭形、锥形或不规则形。SS 细胞随年龄增加而分布范围缩小,主要分布于小肠和十二指肠,老年鼠的结肠和直肠无分布;盲肠段老年鼠高于幼年鼠和成年鼠,其余各段均无年龄差异。幼年鼠、成年鼠和老年鼠SP 细胞分别以结肠、盲肠和直肠密度为最高,成年鼠结肠和直肠无分布;肠道各段的密度均有年龄差异。这些结果表明,长爪沙鼠肠道SS 和SP 细胞的分布模式和发育特点有年龄差异,这可能与其生存环境的食物质量和两种内分泌细胞相互拮抗的消化生理功能有关。     

Partial characterization of the gastrointestinal weight changes produced in the female rat by 16,16-dimethylprostaglandin E2

Oral and subcutaneous administration of 16,16-dimethylprostaglandin E₂ (16,16-dimethyl PGE₂) resulted in an increase in the dry weight of the stomach and small intestine of the female rat. This weight response was rapid, controlled rather than continuously progressing, dose dependent and reversible. The dry weight of the colon also increased but this was not studied in detail.Two-day treatment with 16,16-dimethyl PGE₂ caused an increase in the incorporation of ³H-thymidine into the duodenum, jejunum and colon suggesting an increase in cell number. Incorporation into the stomach and ileum was not changed.The number of goblet cells per crypt was increased by prostaglandin treatment in all parts of the small intestine. Since these are mucus producing cells, the small intestine may have increased in cell number and mucus production.Both anti-secretory and cytoprotective doses of 16,16-dimethyl PGE₂ caused weight increases in the stomach and small intestine. However, the weight gain by itself was not sufficient to protect the stomach or small intestine from necrotic agents after the prostaglandin was discontinued.     

c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a netword similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.     

Impacts of Dietary Pleurotus eryngii Polysaccharide on Nutrient Digestion,Metabolism, and Immune Response of the Small Intestine and Colon—An iTRAQ‐Based Proteomic Analysis

Pleurotus eryngii polysaccharides have been shown to exert significant biological activities to the host. However, few studies have been conducted on its effects on gastrointestinal tract (GIT) health alteration. In the present study, small intestinal and colonic proteome alterations generated by dietary supplementation with a novel homogeneous P. eryngii polysaccharide (PEP) in C57BL/6 mice, based on the isobaric tag for relative and absolute quantification (iTRAQ) proteomics, are investigated. Compared to the control group, PEP supplementation result in a total of 113 and 194 significant differential proteins (DPs) in the small intestine and colon, respectively. Interestingly, DPs in small intestine are mainly related to the transport and biosynthetic process, along with the digestion and absorption pathway of nutrients, whereas the colonic DPs are significantly found participating in numerous metabolic processes. Moreover, the alterations of some DPs in small intestine and colon are speculated to correlate with the colonic microbiota structure and are involved in the regulation of host immune response. Subsequently, some critical DPs of small intestine and colon are selected and validated by Western blotting. The current research facilitated the generation of potential insights into the health benefit activities and functional mechanisms of polysaccharides from P. eryngii.     

Expression and localization of Muc4/sialomucin complex (SMC) in the adult and developing rat intestine: implications for Muc4/SMC function

Muc4/sialomucin complex (SMC) is a high molecular mass heterodimeric membrane mucin, encoded by a single gene, and originally discovered in a highly metastatic ascites rat mammary adenocarcinoma. Subsequent studies have shown that it is a prominent component of many accessible and vulnerable epithelia, including the gastrointestinal tract. Immunoblot and immunofluorescence analyses demonstrated that Muc4/SMC expression in the rat small intestine increases from proximal to distal regions and is located predominantly in cells at the base of the crypts. These cells were postulated to be Paneth cells, based on their location, morphology, and secretory granule content. Immunohistochemistry indicated the presence of Muc4/SMC in these granules. Muc4/SMC expression was higher in the rat colon than small intestine and was abundantly present in colonic goblet cells, but not in goblet cells in the small intestine. Immunohistochemistry also suggested the presence of MUC4 in human colonic goblet cells. Biochemical analyses indicated that rat colonic Muc4/SMC is primarily the soluble form of the membrane mucin. Analyses of Muc4/SMC during development of the rat gastrointestinal tract showed its appearance at embryonic day 14 of the esophagus and at day 15 at the surface of the undifferentiated stratified epithelium at the gastroduodenal junction, then later at cell surfaces in the more distal regions of the differentiated epithelium of the small intestine, culminating in expression as an intracellular form in the crypts of the small intestine at about day 21. Limited expression in the colon was observed during development before birth at cell surfaces, with expression as an intracellular form in the goblet cells arising during the second week after birth. These results suggest that membrane mucin Muc4/SMC serves different functions during development of the intestine in the rat, but is primarily a secreted product in the adult animal.     

Excretion of bisphenol A-glucuronide into the small intestine and deconjugation in the cecum of the rat

The environmental estrogen bisphenol A (BPA) is regarded as a modulator of endocrine systems and has been reported to have adverse effects on the reproductive organs of animals. In rats, BPA is metabolized to glucuronide by UDP-glucuronosyltransferase UGT2B1 in the liver and excreted into the bile. In the present study, we found that most of the bisphenol A-glucuronide (BPA-GA) excreted into the small intestine was deconjugated in the contents of the cecum. After BPA administration, BPA-GA was (immediately should be 15 min) found in the contents of the upper part of the small intestine, and then it moved to the lower part of the small intestine. However, only free BPA was found in the content of the cecum, and there was smaller amount of free BPA in the colon contents, indicating that BPA had been reabsorbed in the colon. BPA-GA was deconjugated by extract prepared from the cecum content which included highest beta-glucuronidase (beta-Gase) observed in Western blot analysis using antibodies against bacterial beta-Gase.These results indicate enterohepatic circulation of BPA and suggest that the adverse effects of BPA are enhanced by repeated exposure.     

Giardia lamblia: stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold (P less than 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.