Intestinal Absorptive Cells Research Articles

Adherence of Helicobacter pylori to areas of type II intestinal metaplasia in Korean gastric mucosa.

The aim of this study was to examine whether Helicobacter pylori (H. pylori) attaches to areas of intestinal metaplasia in Korean patients. Gastric biopsy specimens with intestinal metaplasia from 8 gastric cancers, 24 gastric ulcers, 11 duodenal ulcers, and 57 chronic gastritis were examined. The specimens were stained with periodic acid-Schiff/alcian blue pH 2.5 and high-iron diamine/alcian blue pH 2.5 to identify the subtype of intestinal metaplasia, and then immunohistochemical stain was done with rabbit anti-H. pylori polyclonal antibody. In 17 patients, H. pylori attached to areas of type II intestinal metaplasia. All areas of intestinal metaplasia showing adherence contained sialomucin, and H. pylori was not detected in the areas of intestinal absorptive cells and sulfomucin-containing metaplastic cells.

Hereditary haemochromatosis as an immunological disease.

Hereditary haemochromatosis as an immunological disease.

Anteroposterior Gradient of Epithelial Transformation during Amphibian Intestinal Remodeling: Immunohistochemical Detection of Intestinal Fatty Acid-Binding Protein

To determine whether the remodeling of the well-organized intestinal epithelium during amphibian metamorphosis is regionally regulated along the anteroposterior axis of the intestine, we raised a polyclonal antibody against theXenopus laevisintestinal fatty acid-binding protein (IFABP), which is known to be specifically expressed in intestinal absorptive cells, and examined immunohistochemically the differentiation, proliferation, and apoptosis of the epithelial cells throughoutX. laevissmall intestine. During pre- and prometamorphosis, IFABP-immunoreactive (ir) epithelial cells were localized only in the anterior half of the larval intestine. At the beginning of metamorphic climax, apoptotic cells detected by nick end-labeling (TUNEL) suddenly increased in number in the entire larval epithelium, concurrently with the appearance of adult epithelial primordia. Subsequently, the adult primordia in the anterior part of the intestine developed more rapidly by active cell proliferation than those in the posterior part, and replaced the larval epithelial cells earlier than those in the posterior part. IFABP-ir cells in the adult epithelium were first detectable at the tips of newly formed folds in the proximal part of the intestine. Thereafter, IFABP expression gradually progressed both in the anteroposterior direction and in the crest–trough direction of the folds. These results suggest that developmental processes of the adult epithelium in theX. laevisintestine are regionally regulated along the anteroposterior axis of the intestine, which is maintained throughout metamorphosis, and along the trough–crest axis of the epithelial folds, which is newly established during metamorphosis. Furthermore, the regional differences in IFABP expression along the anteroposterior axis of the intestine were reproduced in organ culturesin vitro.In addition, IFABP expression was first down-regulated and then reactivatedin vitrowhen the anterior part, but not the posterior part, of the larval intestine was treated with thyroid hormone (TH) for extended periods. Therefore, it seems that, in addition to TH, an endogenous factor(s) localized in the intestine itself with an anteroposterior gradient participates in the development of the adult epithelium during amphibian metamorphosis.

The alternate iron transport pathway: mobilferrin and integrin in reticulocytes.

Iron transport in reticulocytes is known to occur via the well-described transferrin-receptor-endosome pathway. An alternative pathway for iron transport independent of transferrin has been postulated in reticulocytes and other cells. Transport of iron into reticulocytes from ferric citrate solutions was shown to be saturable and independent of transferrin. During transport of iron from ferric citrate, both cell surface integrins, and a soluble protein, mobilferrin, were labelled. This demonstrated that the reticulocyte transferrin independent pathway for iron transport involved integrins and mobilferrin similar to intestinal absorptive cells. This pathway would be expected to transport iron into cells under conditions of iron overload and was capable of providing iron for haemoglobin synthesis. Mobilferrin was also radiolabelled when radioiron labelled transferrin was incubated with reticulocytes and this occurred with a different time course than was observed following reticulocyte exposure to radiolabelled ferric citrate. This suggested that mobilferrin may serve as an intermediary in both pathways.

Structure and expression of aminopeptidase N.

Aminopeptidase N (APN) is a very abundant membrane protein in the microvillar membrane of the small intestinal absorptive epithelial cell the enterocyte 1, 2. APN is an ectopeptidase and from its position in the brush border membrane it faces the small intestinal lumen. The enzyme is therefore readily supplied with substrate molecules in the form of oligopeptides derived from nutritional proteins following the actions of gastric and pancreatic proteases. It is generally accepted that the physiological function of APN (and other brush border peptidases) is to convert oligopeptides in the small intestinal lumen into amino acids which can subsequently be absorbed by amino acid carriers. Besides the small intestine APN is also found in a wide variety of other tissues such as the endometrium3, the kidney, the spleen and the brain 2. The physiological role of APN in these alternative locations is unknown but it has been suggested that APN might be involved in the degradation of regulatory peptides2. In the recent years the realisation that APN is expressed in cells of the immune system has lent support to the idea that APN might be involved in the processing of antigens4.

Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway ( K m, 0.91 ± 0.11 mM; ∼90% at 1 mM) together with a minor passive component ( k J, 32.3 ± 6.6 ng (10 6 cells) −1 (20 min) −1. Uptake was inhibited by competition with dipeptides such as l-AIa- l-Pro (K i, 2.96 mM) and l-Phe-Gly ( K i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert −1 min −1 ( k 1) and a terminal value of 1.65 ± 0.08 ng insert −1 min −1 ( (k 2 ) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t max in vivo following oral administration.

Peptide transporters in the intestine and the kidney.

Even though the existence of a transport process for intact peptides in the brush border membrane of intestinal and renal absorptive epithelial cells has been known for almost three decades, it is only recently that the molecular nature of the proteins responsible for the transport process has been elucidated. Two peptide transporters, PEPT 1 and PEPT 2, have been cloned. The cloned transporters catalyze active transport of intact di- and tripeptides and utilize a transmembrane electrochemical H+ gradient as the driving force. The characteristic of H+ coupling makes PEPT 1 and PEPT 2 unique among the transporters thus far identified in mammalian cells. In addition, the peptide transporters have immediate pharmacologic relevance because a number of peptide-like drugs are recognized as substrates by these transporters. Recently, cultured cell lines of intestinal and renal origin that express PEPT 1 and PEPT 2 have been identified. These cell lines are likely to facilitate studies on the regulatory aspects of the peptide transporters.

Fatty Acid Transfer from Liver and Intestinal Fatty Acid-binding Proteins to Membranes Occurs by Different Mechanisms

Intestinal absorptive cells contain high levels of expression of two homologous fatty acid-binding proteins (FABP), liver FABP (L-FABP), and intestinal FABP (I-FABP). Both bind long chain fatty acids with relatively high affinity. The functional distinction, if any, between these two proteins remains unknown. It is often hypothesized that FABP are important in intracellular transport of fatty acids. To assess whether fatty acid transport properties might differ between the two enterocyte FABPs, we examined the rate and mechanism of transfer of fluorescent anthroyloxy fatty acids (AOFA) from these proteins to model membranes using a resonance energy transfer assay. The results show that the absolute rate of AOFA transfer from I-FABP is faster than from L-FABP. Moreover, the apparent mechanism of fatty acid transfer is different between the two proteins. The rate of AOFA transfer from I-FABP is independent of ionic strength, directly dependent on the concentration of acceptor membrane vesicles, and dramatically regulated by the lipid composition of the membranes. These data strongly suggest that fatty acid transfer from I-FABP to membranes occurs by direct collisional interaction of the protein with the phospholipid bilayer. In contrast, the characteristics of fatty acid transfer from L-FABP are consistent with an aqueous diffusion-mediated process. Thus the two enterocyte FABPs may perform different functions within the intestinal absorptive cell in the regulation of fatty acid transport and utilization. It is hypothesized that L-FABP may act as a cytosolic buffer for fatty acids, maintaining the unbound fatty acid concentration, whereas I-FABP may be involved in the uptake and/or specific targeting of fatty acid to subcellular membrane sites.

Ultrastructural Changes in the Intestinal Absorptive Cells of the Mouse Induced by Capsaicin in Relation to Thiamine Absorption

The correlation between an inhibition of thiamine absorption and ultrastructural changes in the intestinal absorptive cells of the mouse jejunum induced by capsaicin was examined. In mice fed with capsaicin 1 and 2 mg/kg BW/day for 12 weeks, thiamine absorption was inhibited by 2.7% and 12.5% (p<0.001) respectively. Ultrastructural alterations were observed in villus absorptive cells of jejunum in mice fed with capsaicin 1 and 2 mg/kg BW/day for 12 weeks. The most striking ultrastructural changes occurred in mice fed with capsaicin 2 mg/kg BW/day for 12 weeks. The organelle most affected by capsaicin was the mitochondria. The result shows that the ultrastructural changes of intestinal absorptive cells may be associated with the inhibition of thiamine absorption.

Ultrastructural Changes in the Intestinal Absorptive Cells of the Mouse Induced by Capsaicin in Relation to Thiamine Absorption

The correlation between an inhibition of thiamine absorption and ultrastructural changes in the intestinal absorptive cells of the mouse jejunum induced by capsaicin was examined. In mice fed with capsaicin 1 and 2 mg/kg BW/day for 12 weeks, thiamine absorption was inhibited by 2.7% and 12.5% (p<0.001) respectively. Ultrastructural alterations were observed in villus absorptive cells of jejunum in mice fed with capsaicin 1 and 2 mg/kg BW/day for 12 weeks. The most striking ultrastructural changes occurred in mice fed with capsaicin 2 mg/kg BW/day for 12 weeks. The organelle most affected by capsaicin was the mitochondria. The result shows that the ultrastructural changes of intestinal absorptive cells may be associated with the inhibition of thiamine absorption.

Heavy metal toxicity following apical and basolateral exposure in the human intestinal cell line Caco-2

Caco-2 is a cell line, derived from a human colon carcinoma, that retains the ability to differentiate in culture into absorptive intestinal cells. Caco-2 cells were used to evaluate the toxicity of three heavy metals—the essential trace elements zinc and copper, and the xenobiotic cadmium. The cells were cultivated on permeable filters until differentiated and were then exposed to the metals either from the apical (luminal) or from the basolateral (serosal) side. Toxicity was measured in dose-effect experiments with reference to cell survival and integrity of the cell monolayer. The metals were more toxic when presented to the basolateral than to the apical cell side. The toxicity ranking was cadmium > > copper > zinc. The cell's ability to transport each metal across the monolayer and the resulting intracellular accumulation could account for the cytotoxic effects. A specific toxic effect observed on a specialized function of these cells was the interference of cadmium in tight-junction integrity as shown by changes in the transepithelial electrical resistance, in the rate of transport of a specific marker across the cell monolayer, and by morphological alterations of the tight junctions.

A monoclonal antibody to mammalian angiotensin II AT1 receptor recognizes one of the angiotensin II receptor isoforms expressed by the eel (Anguilla anguilla)

Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (Ang II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6.5 and 6.7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6.5 form. To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT1 subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-3H]Tyr4-Ile5-Ang II binding. Moreover, Ang II-receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6.5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6.5 in eel intestine and liver preparations, but not the liver pI 6.7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa in intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membranes in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT1 subtype.

Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Rat intestinal mucosa contains high diacylglycerol-synthesizing activity (monoacylglycerol acyltransferase (MGAT) activity) due to monoacylglycerol and fatty acid, independently of coenzyme A and ATP. MGAT activity was purified from rat intestinal mucosa by successive chromatography separations on DEAE-cellulose, CM- Sephadex, and anti-IgG-Sepharose against rat pancreatic lipase. The enzyme was electrophoretically homogeneous, and its molecular weight was 49,000, which is identical with that of rat pancreatic lipase. Immunoblotting analysis with antibody against rat pancreatic lipase showed one immunoreactive protein with an estimated molecular weight of 49,000. The activity of the purified enzyme was completely inhibited by addition of the antibody. Using immunocytochemical techniques, it was found that immunoreactive protein against rat pancreatic lipase was uniformly distributed within the absorptive cells of the intestine but was absent from the microvillar membrane. The MGAT activity of intestinal mucosal homogenate was inhibited by about 65% by addition of antibody against rat pancreatic lipase. Trioleoylglycerol- and dioleoylglycerol-hydrolyzing activities of the purified enzyme and pancreatic lipase were inhibited by addition of intestinal mucosa extract. These results suggest that pancreatic lipase is present in intestinal absorptive cells and that it may contribute to resynthesis of diacylglycerol from monoacylglycerol and fatty acids in these cells.

Paraferritin: a protein complex with ferrireductase activity is associated with iron absorption in rats.

Recent studies reported that iron salts were absorbed in the duodenum utilizing a pathway involving membrane-associated integrin and a cytosolic protein named mobilferrin. In addition, a large molecular weight cytoplasmic complex was labeled with radioiron during mucosal uptake of iron in the duodenum. The molecular mass of this protein was 520 000 daltons, slightly larger than ferritin. On denaturing SDS-PAGE, the purified protein complex appeared to consist of at least four polypeptides, closely associated with each other. This complex was called paraferritin because its hydrodynamic volume resembled ferritin. In the present work, antibody studies demonstrate the presence of integrin, mobilferrin, and flavin monooxygenase in the water-soluble complex. Biochemical studies demonstrate the presence of a NADPH-dependent flavin monooxygenase ferrireductase activity that reduces Fe(III) to Fe(II). Antibodies against either integrin or mobilferrin inhibit monooxygenase activity. Inhibition of monooxygenase activity decreases radioiron uptake by tissue culture intestinal cells. Thus, we postulated that paraferritin plays a role in the mucosal uptake and transport of inorganic iron in small intestinal absorptive cells and is a mechanism for both the internalization of integrin from membranes to cellular cytosol and the delivery of iron to cellular constituents in an appropriate redox state.

Inhibition of purified human sucrase and isomaltase by ethanolamine derivatives.

Sucrase-isomaltase complex was purified from human intestinal mucosa. Immunostaining shows that sucrase-isomaltase is confined to the area of the striated cell borders of human small intestinal absorptive cells of the villus. Inhibition of sucrase and isomaltase activity by ethanolamine derivatives was investigated. Tris inhibits both types of enzyme activity and is the strongest inhibitor of the ethanolamine derivatives investigated. Bis-Tris inhibited sucrase more than isomaltase. On the other hand, mono-, di- and tri-ethanolamine were weak inhibitors of sucrase but not isomaltase.

The transport of acidic amino acids and their analogues across monolayers of human intestinal absorptive (Caco-2) cells in vitro

The X − AG system, a sodium-dependent, acidic amino-acid transport system has been implicated in the transport of l-aspartate and l-glutamate across monolayers of human Caco-2 cells, an in vitro model of intestinal absorption. This system, which shares many properties with the l-glutamate carrier present in the human jejunum, is highly saturable (> 95% at 50 μM), vectorial (apical-to-basolateral ⪢ basolateral-to-apical) and sodium-, pH- and temperature-dependent. l-Aspartate was also transported against a 10-fold reverse concentration gradient. These data are consistent with a major (saturable) carrier-mediated pathway superimposed onto a minor non-saturable (diffusional) pathway. The carrier has an absolute sodium-dependence and the Michaelis constants for the sodium-dependent transport component ( K m) for l-aspartate and l-glutamate were 56 ± 3 μM and 65 ± μM, respectively. Cross-inhibition studies showed that strong interaction with the carrier was limited to close analogues of the natural substrates. Potent inhibitors included l-aspartate, d-aspartate ( K i, 70 μM), l-glutamate ( K i, 180 μM) and threo-ß-hydroxy- dl-aspartate ( K i, 55 μM), while partial inhibitors included α-methyl- dl-aspartate, d-glutamate, l-asparagine, l-proline and l-alanine. Replacement of the side-chain -COO − group (aspartate) with -SO − 3 ( l-cysteate, K i, 65 μM) or -(H)P(O)O − ( dl-3-(hydroxyphosphoryl)alanine, K i, 0 60 μM) maintained strong interaction with the carrier while -As(O)(OH)O − ( dl-3-arsonoalanie, K i, 1100 μM) and -P(O)(OH)O − ( dl-3-phosphonoalanine, K i, 3270 μM) were much more weakly bound, with the larger, but probably less ionised, arsono analogue being more tightly bound than the phosphoro compound. The corresponding analogues of glutamate (homologous extension of the methylene chain) showed negligible interaction. We conclude that Caco-2 monolayers are a relevant experimental model for the study of the transport of acidic amino acids and their analogues in man.

Inhibitory effect of steviol, a metabolite of stevioside, on glucose absorption in everted hamster intestine in vitro

The effects of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) on intestinal glucose absorption were examined in hamster jejunum. By using the everted sac technique, we found that stevioside (1 and 5 mM) had no inhibitory effect on glucose absorption. In contrast, glucose absorption was inhibited 29% by 1 mM steviol. The inhibition of glucose absorption by steviol was related to steviol concentration and incubation time. The possible mechanism of steviol inhibitory action of glucose absorption was also investigated. Reductions in the intestinal mucosal ATP content and absorptive surface area were responsible for the inhibition of glucose absorption by steviol. The decrease in the intestinal mucosal ATP content was accompanied by a decrease in the activities of mitochondrial NADH cytochrome c reductase and cytochrome oxidase. Morever, no inhibitory effects of steviol on the activity of intestinal Na +,K +-ATPase and glucose uptake in the intestinal brush-border membrane vesicles were seen. These results suggest that inhibition of intestinal glucose absorption by steviol in hamsters is due to the reduction in mucosal ATP content and an alteration of the morphology of the intestinal absorptive cells.

Co-cultures of human intestinal goblet (HT29-H) and absorptive (Caco-2) cells for studies of drug and peptide absorption

A co-culture model of the two most abundant intestinal epithelial cell types (absorptive and goblet cells) was developed on permeable supports using the human Caco-2 and HT29-H cell lines. Seeding conditions were selected to give a proportion of goblet cells comparable to the 25–55% found in human colon. The co-cultures comprised small clusters of HT29-H goblet cells embedded in Caco-2 absorptive cells. Electron microscopy showed that tight junctions formed between the two cell types in co-culture. Mono-cultures of HT29-H goblet cells were more permeable to ions and hydrophilic marker molecules and peptides and had a lower Isc than mono-cultures of Caco-2 absorptive cells. The permeability of the co-cultures was intermediate between those of the two mono-cultures. The co-cultures provide a model where absorptive and goblet cells can be studied simultaneously in studies on e.g. drug binding and transport, as well as in studies on absorption enhancement.

Intestinal absorption of radioactive long-chain saturated fatty acids by the larval dragon-fly Aeshna cyanea and co-absorptive effects of other stable fatty acids

In contrast with unsaturated fatty acids, ingestions of long-chain saturated fatty acids by Aeshna cyanea larvae failed to induce an accumulation of lipid droplets in the intestinal absorptive cells, so that it remained questionable whether they were absorbed or not (Bauerfeind and Komnick, 1989). Therefore, we reinvestigated the question with the use of radioactive tracers, which were orally infused under two conditions: at low doses and at high doses prepared by the addition of other stable fatty acids. We found that carboxyl- 14C labelled long-chain stearic and palmitic acids, as well as the medium-chain lauric acid studied for comparison, were indeed absorbed from low doses of 7 nmol. They were incorporated predominantly into the phospholipids of the midgut wall and released into the haemolymph mainly as diacylglycerols. A substantial part was also transported as cholesterylester. The increase in total fatty acids of the infusates by the addition of 1.8 μmol stable oleic, arachidonic, myristoleic acids and lauric acid ethylester, which at high doses induce the formation of triacylglycerol droplets within the midgut enterocytes, markedly reduced the absorption rates of the radioactive saturated fatty acids and greatly favoured their incorporation into di- and triacylglycerols on the expense of the phospholipids and, at a minor part, on the expense of cholesterylester. By contrast, the addition of 1.8 μmol stable caproic acid yielded little, if any, effect on the absorption pattern of the radioactive palmitic acid. The results suggest that the co-absorption of medium- and long-chain saturated and unsaturated fatty acids is interdependent utilizing the same or similar uptake mechanisms and dose-dependent esterification pathways, while it is largely independent of and probably different from the absorption of short-chain fatty acids.

Morphological Alterations of the Intestinal Villi and Absorptive Epithelial Cells in each Intestinal part in Fasted Chickens.

To demonstrate morphologically the functional difference in each intestinal part, 27 (starter stage)-and 113 (developer stage)-day-old male Single Comb White Leghorn chickens were fasted for 3 or 7 days. After the end of each fasting period, the villous height and the fine structure of absorptive epithelial calls in the duodenum, jejunum and ileum were compared using light and electron microscopies. On the post-hatching developmental changes of the villous height in the normal control, the duodenum had the highest villi at starter stage followed by undeveloped lower jejunal villi, while the latter showed a marked growth rate up to the developer stage. This explains that the vigorous absorptive part would be mainly the duodenum in early life stage and then extend to the jejunum with increasing age. However, after fasting treatment these proximal intestines had thought to have a high absorptive ability revealed a dramatic reduction of the villous height. This suggests that among the intestinal parts, the higher the intestinal absorptive ability during the normal feeding is, the more rapidly the intestinal villous height is reduced by fasting. In fine structural alterations, the epithelial cells in the duodenum had large lysosomal supranuclear vacuoles even after 3 days fasting. On the other hand, by the first 3 days fasting the jejunal cells showed the lamellar bodies thought as a precursor of vacuoles and had typical vacuoles after next 4 days fasting. These suggest that among the intestinal epithelial cells, the higher the cellular absorptive ability during the normal feeding is, the more rapidly the lysosomal vacuoles grow larger in size by fasting. Compared with the alterations of villous height and ultrastructure of epithelial cells observed in the proximal intestines, the ileum showed an almost constant vilious height and no typical vacuoles but well-developed Golgi apparatuses in cells after fasting. These findings lead to the possibility that the ileum appears to be inactive in absorptive function and it might have another specific function in addition to the conventional absorptive function.