Somatostatin binding sites in cytosolic fraction of rabbit intestinal mucosa: distribution throughout the intestinal tract

Specific binding sites for somatostatin have been identified in cytosolic fraction of both small and large intestinal mucosa. The stoichiometric data suggested the presence of two classes of binding sites in each part of the intestine. The binding capacity varied depending on the segment considered (rectum greater than duodenum = jejunum greater than ileum, caecum and colon). However, the affinities of the binding sites were similar throughout the whole intestinal mucosa, with the exception of rectum which showed higher Kd values. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as vasoactive intestinal peptide, neurotensin, substance P and Leu-enkephalin did not show any effect upon somatostatin binding.     

Characterization of specific binding sites for vasoactive intestinal peptide in rat intestinal epithelial cell membranes

Specific binding sites for vasoactive intestinal peptide were characterized in plasma membranes from rat intestinal epithelial cells. At 30°C, the interaction of ¹²⁵I-labelled peptide with intestinal membranes was rapid, reversible, specific and saturable. At equilibrium, the binding of ¹²⁵I-labelled peptide was competitively inhibited by native peptide in the 3 · 10^?11?3 · 10^?7 M range concentration. Scatchard analysis of binding data suggested the presence of two distinct classes of vasoactive intestinal peptide binding sites: a class with a high affinity K_d = 0.28 nM) and a low capacity (0.8 pmol peptide/mg membrane protein) and a class with a low affinity (K_d = 152 nM) and a high capacity (161 pmol peptide/mg membrane protein). Secretin competitively inhibited binding of ¹²⁵I-labelled peptide but its potency was 1/1000 that of native peptide. Glucagon and the gastric inhibitory peptide were ineffective. The guanine nucleotides, GTP and Gpp(NH)p inhibited markedly the interaction of ¹²⁵I-labelled peptide with its binding sites, by increasing the rate of dissociation of peptide bound to membranes. The other nucleotides triphosphate tested (ATP, ITP, UTP, CTP) were also effective in inhibiting binding of ¹²⁵I-labelled peptide to membranes but their potencies were 1/100-1/1000 that of guanine nucleotides.The specificity and affinity of the vasoactive intestinal peptide-binding sites in plasma membranes prepared from rat intestinal epithelial cells, which is in agreement with an adenylate cyclase highly sensitive to the peptide recently characterized in these membranes (Amiranoff, B., Laburthe, M., Dupont, C. and Rosselin, G. (1978) Biochim. Biophys. Acta 544, 474–481) further argue for a physiological role of the peptide in the regulation of intestinal epithelial function.     

The effects of starvation and refeeding on intestinal cell proliferation in the mouse

The effects of starvation and refeeding on intestinal cell proliferation were studied in four sites of the mouse intestine. Control mice were studied at different times of day in order to compensate for any circadian variations in proliferation. A circadian rhythm in crypt cell production rate was observed in all the sites of the small intestine and colon, and this rhythm appeared to be entrained to the food intake. The fractional crypt cell production rate decreased in all sites of the intestine after 24 h starvation, and remained low until 9 h after refeeding, when there was a marked increase in the crypt cell production rate of all the small intestinal sites, especially the proximal sites. There was little change in colonic crypt cell production rate until 12 h after refeeding, when there was a large increase in cell production. The crypt cell production rate of all sites then returned to control values for the remainder of the investigation. Crypt cell number decreased after refeeding and villus cell number increased, however a similar effect was observed in the control animals, nevertheless the changes in villus cell population of the refed mice occurred before any increase in crypt cell production, suggesting that cell migration from crypt to villi is not immediately dependent on cell proliferation.     

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.     

Lipid-soluble and water-soluble antioxidant activities of the avian intestinal mucosa at different sites along the intestinal tract

The antioxidant capacity of the avian intestinal mucosa is potentially important in protecting the gut wall from the harmful actions of reactive oxygen species originating from the diet, mucosal metabolism and the inflammatory response to enteric microbes. To assess this capacity, we determined the total lipid-soluble and water-soluble antioxidant activities of mucosal extracts, using tissue from different parts of the intestinal tract of the chicken. The lipid-soluble antioxidants, vitamin E and carotenoids, were also measured in the same samples. Total lipid-soluble antioxidant activity was highest in mucosa from the duodenum followed by the jejunum, with much lower activities in the ileum, ceca and colon. Total water-soluble antioxidant activity of the mucosa was at least an order of magnitude greater than the lipid-soluble activity under the assay conditions and did not differ significantly among the different parts of the intestinal tract. High concentrations of vitamin E were present in the mucosa of the duodenum and jejunum, with a trend to lower levels in the ileum and ceca, and significantly less in the colon. Similarly, the mucosa of the duodenum and jejunum contained the highest concentrations of carotenoids, with much lower levels in the ileum and colon. The different isoforms of vitamin E were absorbed from the digesta by the mucosa without any major selectivity. However, the liver was greatly enriched with alpha-tocopherol over the other isoforms, indicating a high degree of discrimination by this tissue. The results indicate major differences in the relative contributions of lipid- and water-soluble antioxidants in the mucosa along the different parts of the intestinal tract, most likely reflecting the sites of vitamin E and carotenoid absorption.     

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.     

Vasoactive intestinal peptide (VIP) receptors in the canine gastrointestinal tract

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of ¹²⁵I-VIP receptor binding sites in the canine gastrointestinal tract. While the distribution of VIP binding sites was different for each segment examined, specific VIP binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, lymph nodules, and the circular and longitudinal smooth muscle of the muscularis externa. These results identify putative target tissues of VIP action in the canine gastrointestinal tract. In correlation with physiological data, VIP sites appear to be involved in the regulation of a variety of gastrointestinal functions including epithelial ion transport, gastric secretion, hemodynamic regulation, immune response, esophageal, gastric and intestinal motility.     

Alterations in surface ultrastructure and anionic sites of rat dimethylhydrazine-induced intestinal tumors

The relative roles of cell surface shedding and electronegative charge as determinants of metastatic capacity were studied in experimentally produced intestinal tumors. The ultrastructural organization and distribution of anionic sites on the luminal plasma membrane surface components were examined in small intestinal and colonic tumors induced in male Sprague-Dawley rats with 1,2-dimethylhydrazine. The overall distribution of negatively charged groups was demonstrated with ruthenium red staining. Compared to normal epithelial cells, neoplastic cells revealed evidence of decreased cell surface shedding as manifested by decreased numbers of membrane-bound bodies, and an increased quantity of glycocalyx. Malignant cell surfaces were directly exposed to the intestinal lumen as a result of losing the enteric surface coat covering. The exposed microvilli appeared damaged with shortening and blunting. The glycocalyx and surface coat both reacted strongly with ruthenium red indicating the presence of anionic sites. As a result of surface coat loss, the malignant cell surface components revealed an overall decrease in net negative charge. These alterations in cell surface component ultrastructure and electronegative charge appear to be consistent with the low capacity for chemically induced rat intestinal tumors to metastasize.     

Binding of Shigella to rat and human intestinal mucin

Invasion of epithelial cells by Shigella is an early step in their pathogenesis. Adherence is generally presumed to be a prerequisite for invasion. This study examined the possibility of intestinal mucins serving as initial binding sites for clinical isolates of S. boydii and S. sonnei. The interactions of Shigella with rat and human small intestinal and colonic mucin were investigated. In solid phase binding assays, [³⁵S] labelled Shigella did not show any preferential binding to rat/human small intestinal mucin or to rat colonic mucin. On the other hand, Shigella bound specifically to human colonic mucin in a concentration-dependent manner. This specific binding to human colonic mucin was not by weak hydrophobic interactions and could not be attributed to the presence of contaminating glycolipids in the mucin preparation. The human colonic mucin receptor was sensitive to periodate treatment suggesting the involvement of the carbohydrate portion of the mucin. Reduction and alkylation of mucin enhanced adherence probably by exposing buried binding sites. The monosaccharides present in mucins were ineffective as hapten inhibitors as was the lectin wheat germ agglutinin suggesting that the mucin receptor is a more complex one. This study identifies, for the first time, the presence of a specific Shigella-binding site on the carbohydrate portion of human colonic mucin, which is not present in rat colonic mucin or in rat/human small intestinal mucin.     

The origin of IgA-containing cells in intestinal lymph of sheep

The origin of IgA-containing cells in sheep intestinal lymph was determined by antigenically stimulating a mesenteric lymph node and by studying afferent intestinal lymph. Since antigenic stimulation of the node resulted almost exclusively in the appearance of IgG1 antibody-containing cells, it was proposed that IgA-containing cells are normally produced either in the Peyer's patches or lamina propria of the intestine. These conclusions were supported by studying lymph obtained by cannulation of a lymphatic duct afferent to the mesenteric lymph nodes. Study of the cells in afferent lymph revealed the presence of a significant population of IgA-containing blast cells. This was convincing evidence that the IgA-containing cells normally found in intestinal lymph originate from sites in the intestine.     

The SP1 sites of the human apoCIII enhancer are essential for the expression of the apoCIII gene and contribute to the hepatic and intestinal expression of the apoA-I gene in transgenic mice

We have generated transgenic mice carrying wild-type and mutant forms of the apolipoprotein (apo)A-I/apoCIII gene cluster. Mutations were introduced either in one or in three SP1 binding sites of the apoCIII enhancer. In mice carrying the wild-type transgene, major sites of apoA-I mRNA synthesis were liver and intestine and minor sites were kidney and, to a lesser extent, other tissues. The major site of chloramphenicol acetyl transferase (CAT) activity (used as a reporter for the apoCIII gene) was liver and minor sites intestine and kidney. A mutation in one SP1 binding site reduced the expression of the apoA-I gene to ~23 and 19% in the liver and intestine, respectively, as compared to the control wild-type. The hepatic expression of the CAT gene was not affected whereas the intestinal expression was nearly abolished. Mutations in three SP1 binding sites reduced the hepatic and intestinal expression of the apoA-I and CAT genes to 14 and 4%, respectively, as compared to the wild-type control, and abolished CAT expression in all tissues. The findings suggest that the SP1 sites of the apoCIII enhancer are required for the expression of the apoCIII gene and also contribute significantly to the hepatic and intestinal expression of the apoA-I gene in vivo.     

Observations on the binding of lanthanides and calcium to vitamin D-dependent chick intestinal calcium-binding protein. Implications regarding calcium-binding protein function

The binding of calcium and terbium to purified chick vitamin D-dependent intestinal calcium-binding protein was studied by terbium fluorescence, circular dichroism, and intrinsic protein fluorescence techniques. Calcium-binding protein bound, with high affinity, at least 3 mol of terbium/mol of protein; numerous low affinity terbium-binding sites were also noted. The three highest affinity sites were resolved into one very high affinity site (site A) and two other sites (sites B and C) with slightly lower affinity. Resonance energy transfer from tryptophan residues to terbium occurred only with site A. This site was filled before sites B and C. Competition experiments in which calcium was used to displace terbium bound to the protein showed that larger amounts of calcium were needed to displace terbium from site A than from sites B and C. Energy transfer from terbium to holmium indicated that the terbium-binding sites (B and C) were located close to each other (about 7-12 A) but were distant (greater than 12 A) from site A. The addition of EDTA to calcium-binding protein resulted in a 25% decrease in intrinsic protein fluorescence, suggesting a conformational change in the protein. The titration of EDTA-treated calcium-binding protein with calcium resulted in recovery of intrinsic protein fluorescence. A reversible calcium-dependent change in the ellipticity of calcium-binding protein in circular dichroism experiments was also seen. These observed properties suggest that vitamin D-dependent chick intestinal calcium-binding protein behaves in a manner similar to other well-known calcium-binding regulatory proteins.     

Autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors

Autophagy has been demonstrated to be associated with the pathogenesis of cancer, but no consensus has been reached about its precise role. Therefore, we investigated whether autophagy in the intestinal epithelium is involved in the pathogenesis of intestinal tumors. To evaluate the relationship between autophagy and intestinal tumors, GFP-LC3-APC(min/+) mice were generated by mating GFP-LC3 transgenic mice with APC(min/+) mice. Autophagy was weakly induced in the intestinal polyp regions of the mice in comparison to their non-polyp regions. Under starved conditions, autophagy was not induced in the polyp regions, whereas it was observed in the non-polyp regions. Then, to examine whether a lack of autophagy in the intestinal epithelium enhances the induction of intestinal tumor, Atg7flox/flox:vil-cre-APC(min/+) mice, in which Atg7 had been conditionally deleted in the intestinal epithelium, were generated by mating Atg7flox/flox:vil-cre mice with APC(min/+) mice. However, there was no significant difference in the number of intestinal polyps between the Atg7flox/flox:vil-cre-APC(min/+) and the corresponding control Atg7flox/flox-APC(min/+) mice. These results indicate that autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors, and future research should focus on regulating autophagy as a form of cancer therapy.     

Association of Treponema hyodysenteriae with porcine intestinal mucosa

The association of Treponema hyodysenteriae with porcine caecal and colonic mucosal surfaces was studied by electron microscopy after orogastric inoculation of pigs with pure cultures. Examination of caecal and colonic mucosa from infected and control animals revealed that large numbers of the spirochaete were associated only with intestinal mucosal surfaces of infected animals. Further examination of the intestinal mucosa from infected pigs showed that T. hyodysenteriae colonized two sites preferentially: the mucus-filled crypts of Lieberkühn and the mucus gel covering the epithelium. Furthermore, no evidence of either specific or nonspecific adhesion to the epithelium proper was found, suggesting that penetration of, or trapping in the mucus gel may be the predominant mechanism of mucosal association by T. hyodysenteriae. Moreover, T. hyodysenteriae was also observed to be highly motile in intestinal mucus, moving faster than any other organism present, and this 'high speed' motility appeared to facilitate penetration into the mucosa. The pattern of motility observed was also highly suggestive of chemotaxis, and this was subsequently confirmed using an in vitro assay to porcine mucus material. It is suggested, therefore, that motility and chemotaxis are important factors/mechanisms in the association and colonization of porcine intestinal mucosa by T. hyodysenteriae.     

Oxalate binding to rat intestinal brush-border membrane in pyridoxine deficiency: a kinetic study

Oxalate bound specifically to the intestinal brush-border membrane (BBM) of pyridoxine-deficient rats, but not to BBM of control rats. The binding of oxalate to intestinal BBM of pyridoxine-deficient rats was rapid, reversible, dependent on concentration of oxalate, temperature sensitive and competitively inhibited by oxalate analogues. Kinetic analysis of the oxalate binding data revealed induction of two distinct classes of receptor site for oxalate. The high-affinity oxalate binding sites, reached saturation at 60-70 nM oxalate, had a Kd of 24.29 nM and the number of binding sites were 30 pmoles (i.e., 1.8.10(13) molecules). The low-affinity oxalate binding sites, could not be saturated under experimental conditions upto 1 microM oxalate. It had a Kd of 487.5 nM and the number of binding sites were 156 pmoles (i.e., 9.4.10(13) molecules). The apparent energy of activation was 19 kcal/mol. The half-saturation concentration of inhibitor (IC50) of oxalate was 0.4.10(-5) M, while all other structural analogues of oxalate had higher IC50 values. Among the competitive inhibitors tested IC50 was in the following order, pyruvate greater than maleate greater than oxaloacetate greater than glyoxylate greater than parabonate greater than oxalate. These kinetic characteristics indicate involvement of a membrane protein in oxalate binding and transport in rat intestinal brush-border membrane in pyridoxine deficiency.     

The rat liver vasoactive intestinal peptide binding site. Molecular characterization by covalent cross-linking and evidence for differences from the intestinal receptor.

To identify the molecular components of the vasoactive intestinal peptide (VIP) binding sites in the liver, 125I-labelled VIP was covalently linked to liver membranes by using the cleavable cross-linker dithiobis(succinimidylpropionate). Purified rat liver plasma membranes were incubated with 125I-VIP, washed and treated with 1 mM-cross-linker. Polyacrylamide-gel electrophoresis of membrane proteins followed by autoradiography revealed a major 125I-VIP-protein complex of Mr 51 000. A minor Mr 89 000 complex was also observed. An identical pattern of protein labelling was obtained using crude membranes from rat liver. Labelling of the Mr 51 000 and 89 000 species was specific in that it could be abolished by native VIP, but was unaffected by 1 microM-glucagon and cholecystokinin octapeptide. Densitometric scanning of autoradiographs indicated that the labelling of the two species was abolished by similar low VIP concentrations (0.1-100 nM). It was also reduced by two VIP agonists, peptide histidine isoleucine amide and secretin, with a potency that is 1/7 and 1/200 that of native VIP, respectively. The guanine nucleotide GTP in the concentration range between 10(-7) and 10(-3) M reduces the labelling of the major Mr 51 000 protein and that of the minor Mr 89 000 protein, but with a slightly higher potency. Assuming one molecule of 125I-VIP was bound per molecule of protein, a major Mr 48 000 protein and a minor Mr 86 000 protein were identified as components of the high-affinity VIP binding sites in liver. This contrasts markedly with the pattern of labelling of rat intestinal epithelial membranes, where a Mr 73 000 protein was identified as a high-affinity VIP receptor and a Mr 33 000 protein as a low-affinity VIP binding site [Laburthe, Bréant & Rouyer-Fessard (1984) Eur. J. Biochem. 139, 181-187], suggesting structural differences between VIP binding sites in rat liver and intestinal epithelium.     

Angiotensin I converting enzyme of rat intestinal and vascular surface membrane

High levels of angiotensin I converting enzyme are present in rat intestinal mucosa and in intestinal arteries. Homogenates of both tissues were subfractionated and fractions enriched in vascular plasma membrane or intestinal brush border were prepared. The preparations were identified and their purities established by marker enzyme enrichment and/or electron microscopy. Converting enzyme activity was highly enriched on both the vascular plasma membrane and the intestinal brush border. Subsequently the properties of these membrane-bound enzymes were compared. Both surface membrane-bound enzymes were highly sensitive to inhibition by captopril (SQ 14225) and teprotide (SQ 20881). Similar to converting enzyme isolated from other sources, they were also inhibited by bradykinin, angiotensin I, EDTA and o-phenanthroline. Finally, both membrane-bound enzymes were relatively resistant to activation by sonication, freezing and thawing or detergent. These results demonstrate significant similarities between surface membrane-bound converting enzyme from vascular and non-vascular sites. In addition, in view of the possible relationship of kinins and angiotensins to gastrointestinal function and blood flow, inhibition of gastrointestinal converting enzyme by captopril may affect some aspects of intestinal physiology.     

肠道菌群变化对实验小鼠肠黏膜免疫的影响

目的探讨肠道菌群变化对肠黏膜相关淋巴组织的影响。方法通过变性梯度凝胶电泳（Denatu-ring gradient gel electrophoresis,DGGE）法研究了三种不同级别实验小鼠即清洁级小鼠、SPF小鼠和普通小鼠肠道菌群的组成,并用免疫组织化学（immunohistochemistry,IHC）方法研究了此三种不同级别的实验小鼠肠黏膜相关淋巴组织sIgA阳性细胞分布情况。结果普通小鼠肠道细菌种类最多,其sIgA阳性细胞分布最多,肠道不同部位之间sIgA分布情况差异有显著性（P〈0.05）,小肠和大肠之间的阳性细胞分布差异极显著（P〈0.01）;其次是清洁级小鼠,其肠道不同部位之间菌种组成差异无显著性,小肠和大肠之间的阳性细胞分布差异有显著性（P〈0.05）;SPF小鼠肠道细菌种类最少,故其sIgA阳性细胞分布最少,且其肠道不同部位之间菌种组成差异无显著性,小肠和大肠之间的阳性细胞分布差异无显著性（P〉0.05）。结论随着动物微生物控制级别的增高,肠道微生物多样性递减;sIgA阳性细胞与肠道细菌种类正相关。     

Identification of cholera toxin-binding sites in the nucleus of intestinal epithelial cells

Post-embedding immunogold electron microscopy shows several binding sites for cholera toxin in mouse intestinal epithelial cells, particularly in the heterochromatin of the nucleus as well as in the plasma membrane. Anti-ganglioside GM1 antibodies also bound to the nucleus, but did not interfere with the binding of toxin. 125I-labelled toxin bound specifically to a nuclear preparation from rabbit intestinal cells.