Villus Epithelial Cells Research Articles

Effect of Shiga toxin 2 on water and ion transport in human colon in vitro.

Shiga toxin-producing Escherichia coli (STEC) colonize the lower segments of the human gastrointestinal tract, causing gastrointestinal and systemic diseases. In this study, the effects of Shiga toxin 2 (Stx2) on fluid absorption and ion transport in the human colon were examined. Net water movement (Jw) and short-circuit current (Isc) were simultaneously measured across the colonic mucosa incubated with crude or purified Stx2. Stx2 significantly inhibited the absorptive J(w) with no effect on the basal I(sc) after 60 min of exposure. These effects may be due to the inhibition of a nonelectrogenic transport system present in the surface colonic villus cells. Morphological studies of the colonic mucosa treated with crude or purified Stx2 demonstrated a selective damage in the absorptive villus epithelial cells. These findings suggest that Stx2 inhibits water absorption across the human colon by acting on a specific cell population: the mature, differentiated absorptive villus epithelium.

Cytokine Regulation of CD44 Expression on rat Intestinal Epithelial Cells

CD44 comprises a family of type I transmembrane glycoproteins that is expressed on a wide range of cells including those of epithelial, lymphoid and myeloid lineage. Although expression of CD44 in the small intestine is typically localised in the crypts of Lieberkühn, we have reported the expression of CD44 on mature, intestinal villus epithelial cells during the development of small bowel allograft rejection. The mechanisms underlying CD44 up-regulation are unknown, although it may be influenced by localised cytokine production. This study used flow cytometry to assess the effects of recombinant IFN-γ and TNF-α on CD44 expression and hyaluronan binding by the rat small intestinal epithelial cell lines, RIE and IEC 6. IFN-γ upregulated CD44 expression on RIE (155% of unstimulated control) and IEC 6 (209% of unstimulated control) cells, whereas TNF-α had no effect. IFN-γ had no qualitative effect on CD44, as binding of the ubiquitously expressed extracellular matrix polysaccharide hyaluronan was unchanged. RIE and IEC 6 cells expressed the 82 kDa and 130 kDa major isoforms of CD44, however cytokine stimulation did not affect the expression of these, nor did stimulation induce the expression of other variants. In summary, these findings demonstrate that CD44 expression by intestinal epithelial cells can be regulated by cytokines, yet their ability to bind hyaluronan and the isoform of the expressed CD44 remains unaltered. It appears that localised inflammatory conditions and cytokine production may modify epithelial cell expression of CD44, however the physiological role for such a response has yet to be elucidated.

Ca(2+)/Calmodulin kinase II and decreases in intracellular pH are required to activate K(+) channels after substantial swelling in villus epithelial cells.

To assess the activation of the charybdotoxin-insensitive K(+) channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH(i)), intracellular calcium ([Ca(2+)](i)) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH(i) and [Ca(2+)](i) of cells in suspension, loaded with 2, 7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca(2+)](i) to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of approximately 0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 microm), an inhibitor of Ca(2+)/calmodulin kinase II, blocked RVD. Gramicidin (0.5 microm bypassed this inhibition suggesting that the K(+) channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K(+) gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 microm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K(+) channels utilized for RVD after substantial (>7%) 'nonphysiological' volume increases requires phosphorylation mediated by Ca(2+)/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca(2+)](i) are a critical determinant of this activation.

Increases in intracellular pH and Ca(2+) are essential for K(+) channel activation after modest 'physiological' swelling in villus epithelial cells.

We studied the relationship between changes in intracellular pH (pH(i)), intracellular Ca(2+)([Ca(2+)](i)) and charybdotoxin sensitive (CTX) maxi-K(+) channels occurring after modest 'physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH(i) and [Ca(2+)](i) by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 x isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 x isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 x isotonic dilution, the pH(i) alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this DeltapH(i) and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH(4)Cl permitted complete RVD which could be inhibited by CTX. The rate of (86)Rb efflux which also increased after this 0.93 x isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na(+)-free medium. Modest swelling transiently increased [Ca(2+)](i) and Ca(2+)-free medium or blocking alkalinization by MIA or Na(+)-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca(2+)-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca(2+)](i) and volume changes became sensitive to CTX. We conclude that both alkalinization of pH(i) and increased [Ca(2+)](i) observed with 'physiological' volume increase are essential for the activation of CTX-sensitive maxi-K(+) channels required for RVD.

Functional characterization and localization of AQP3 in the human colon.

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo.

cAMP activated insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) channels from endosomes to the apical plasma membrane has been hypothesized to regulate surface expression and CFTR function although the physiologic relevance of this remains unclear. We previously identified a subpopulation of small intestinal villus epithelial cells or CFTR high expressor (CHE) cells possessing very high levels of apical membrane CFTR in association with a prominent subapical vesicular pool of CFTR. We have examined the subcellular redistribution of CFTR in duodenal CHE cells in vivo in response to the cAMP activated secretagogue vasoactive intestinal peptide (VIP). Using anti-CFTR antibodies against the C terminus of rodent CFTR and indirect immunofluorescence, we show by quantitative confocal microscopy that CFTR rapidly redistributes from the cytoplasm to the apical surface upon cAMP stimulation by VIP and returns to the cytoplasm upon removal of VIP stimulation of intracellular cAMP levels. Using ultrastructural and confocal immunofluorescence examination in the presence or absence of cycloheximide, we also show that redistribution was not dependent on new protein synthesis, changes in endocytosis, or rearrangement of the apical cytoskeleton. These observations suggest that physiologic cAMP activated apical membrane insertion and recycling of CFTR channels in normal CFTR expressing epithelia contributes to the in vivo regulation of CFTR mediated anion transport.

Guinea pig 5-HT transporter: cloning, expression, distribution, and function in intestinal sensory reception.

Studies of the guinea pig small intestine have suggested that serotonin (5-HT) may be a mucosal transmitter that stimulates sensory nerves and initiates peristaltic and secretory reflexes. We tested the hypothesis that guinea pig villus epithelial cells are able to inactivate 5-HT because they express the same 5-HT transporter as serotonergic neurons. A full-length cDNA, encoding a 630-amino acid protein (89.2% and 90% identical, respectively, to the rat and human 5-HT transporters) was cloned from the guinea pig intestinal mucosa. Evidence demonstrating that this cDNA encodes the guinea pig 5-HT transporter included 1) hybridization with a single species of mRNA ( approximately 3.7 kb) in Northern blots of the guinea pig brain stem and mucosa and 2) uptake of [3H]5-HT by transfected HeLa cells via a saturable, high-affinity (Michaelis constant 618 nM, maximum velocity 2.4 x 10(-17) mol . cell-1 . min-1), Na+-dependent mechanism that was inhibited by chlorimipramine > imipramine > fluoxetine > desipramine > zimelidine. Expression of the 5-HT transporter in guinea pig raphe and enteric neurons and the epithelium of the entire crypt-villus axis was demonstrated by in situ hybridization and immunocytochemistry. Inhibition of mucosal 5-HT uptake potentiates responses of submucosal neurons to mucosal stimulation. The epithelial reuptake of 5-HT thus appears to be responsible for terminating mucosal actions of 5-HT.

Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease.

Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied. COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry. COX-1 protein was expressed at equal levels in normal, Crohn's disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohn's disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohn's colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohn's colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohn's ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase. COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.

Potential role of epithelial cell-derived histone H1 proteins in innate antimicrobial defense in the human gastrointestinal tract.

In the human gastrointestinal tract, microorganisms are present in large numbers in the colon but are sparse in the proximal small intestine. In this study, we have shown that acid extracts of fresh human terminal ileal mucosal samples mediate antimicrobial activity. Following cation-exchange chromatography, one of the eluted fractions demonstrated antibacterial activity against bacteria normally resident in the human colonic lumen. This activity was further fractionated by reverse-phase high-performance liquid chromatography and identified as histone H1 and its fragments. We have also shown that in tissue sections, immunoreactive histone H1 is present in the cytoplasm of villus epithelial cells. In vitro culturing of detached (from the basement membrane) villus epithelial cells led to the release of antimicrobial histone H1 proteins, while the cells demonstrated ultrastructural features of programmed cell death. Our studies suggest that cytoplasmic histone H1 may provide protection against penetration by microorganisms into villus epithelial cells. Moreover, intestinal epithelial cells released into the lumen may mediate antimicrobial activity by releasing histone H1 proteins and their fragments.

Cellular expression and regulation of iron transport and storage proteins in genetic haemochromatosis.

Genetic haemochromatosis is a common iron overload disorder of unknown aetiology. To characterize the defect of iron metabolism responsible for this disease, this study localized and semi-quantified the mRNA and protein expression of transferrin, transferrin receptor and ferritin in the liver and duodenum of patients with genetic haemochromatosis. Biopsies were obtained from iron-loaded non-cirrhotic patients with genetic haemochromatotic and control patients with normal iron stores. Additional duodenal biopsies were obtained from patients with iron deficiency. Immunohistochemical and in situ hybridization analysis for transferrin, transferrin receptor and ferritin was performed. Hepatic transferrin, transferrin receptor and ferritin protein expression was localized predominantly to hepatocytes and was increased in patients with genetic haemochromatosis when compared with normal controls. Interestingly, hepatic ferritin mRNA expression was not increased in these same patients. In the genetic haemochromatotic duodenum, ferritin mRNA and protein was localized mainly to crypt and villus epithelial cells and the level of expression was decreased compared with normal controls, but similar to iron deficiency. Duodenal transferrin receptor mRNA and protein levels colocalized to epithelial cells of the crypt and villus were similar to normal controls. Early in the course of genetic haemochromatosis and before the onset of hepatic fibrosis, transferrin receptor-mediated iron uptake by hepatocytes contributes to hepatic iron overload. Increased hepatic ferritin expression suggests this is the major iron storage protein. While persisting duodenal transferrin receptor expression may be a normal response to increased body iron stores in patients with genetic haemochromatosis, decreased duodenal ferritin levels suggest that duodenal mucosa is regulated as if the patient were iron deficient.

Villus Epithelial Cell Migration is Delayed in Hypothyroid C.RF/hyt Mice • 579

The intestinal epithelium is established and maintained by continuous production of new cells, their migration along the villi and extrusion at the villus tip. In newborn mice replicating and differentiating cells are located throughout the intervillus epithelium, and mature cells slowly traverse the length of the villi. During the suckling period the proliferative epithelium becomes organized and confined to developing crypts. As crypts mature near weaning the replicating cell populations expand, and villus cell migration accelerates eventually reaching rates in adults that are 4-5X greater than in newborns. Glucocorticoid and thyroid hormones mediate changes in digestive enzyme expression during the suckling-weaning transition period, but mechanisms regulating epithelial renewal kinetics during development are not known. Our prior studies show glucocorticoids exert little effect on cell migration and epithelial renewal rates. This study examines thyroid hormone effects on villus epithelial cell migration in C.RF/hyt mice, a strain with primary hypothyroidism, undetectable serum T4 and normal corticosterone production. Proliferating cells were labeled with the thymidine analog, BrdUrd, on day of life 2, 5, 9, 14 or 18, or adults, and migration examined at intervals after labeling using immunohistochemical staining. Migration rates in C.RF/hyt and control euthyroid, Balb/cJ, mice are comparable in the first week of life when endogenous T4 levels are normally low. As T4 rises in the second week, migration rates accelerate rapidly in Balb/cJ mice. In contrast, migration rates in C.RF/hyt mice increase only gradually through weaning and remain delayed in adult animals. In all regions of the small bowel, villus epithelial cell migration rates in adult C.RF/hyt mice reach only 50% of the rates attained in adult euthyroid mice. Correction of hypothyroidism with dietary supplementation adequate to establish fertility and normal serum T4 results in more rapid epithelial cell migration and renewal rates approaching those observed in euthyroid mice. These studies show normal acceleration of villus epithelial cell migration rates during intestinal development are regulated by the physiologic rise in thyroid hormone levels during the suckling period.

Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-γ, TNF) in the induction of epithelial cell death and renewal

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN-γ and TNF; these activities are increased by in vitro exposure to IL-12. Mice injected with IL-12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus-dependent as well as natural killer-T cell IEL) and the release of IFN-γ. The critical role of IFN-γ may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN-γ into mutant mice lacking IEL showed that IFN-γ can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL-12-induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL-12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN-γ and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.

Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal.

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN-gamma and TNF; these activities are increased by in vitro exposure to IL-12. Mice injected with IL-12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus-dependent as well as natural killer-T cell IEL) and the release of IFN-gamma. The critical role of IFN-gamma may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN-gamma into mutant mice lacking IEL showed that IFN-gamma can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL-12-induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL-12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN-gamma and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.

Ferritin gene expression and transferrin receptor activity in intestine of rats with varying iron stores.

Expression of transferrin receptor and ferritin genes has been shown previously to be under transcriptional and posttranscriptional regulation, the latter being reciprocally regulated according to cellular iron levels. This study examined transferrin receptor function and ferritin gene expression along the crypt-villus axis of the intestinal tract in rats with varying iron stores. Altered iron stores were produced by feeding a control diet and diets low or high in iron (2% carbonyl iron) for 8-10 wk. Expression and activity of the ferritin genes were assessed by in situ hybridization and immunohistochemical localization, respectively. Transferrin receptor activity was determined by the uptake of intravenously injected transferrin-bound iron and was shown to increase with the level of iron loading. In all iron status groups, ferritin mRNA was seen at highest levels in the epithelial cells of the crypt and macrophages within the lamina propria and at lower levels in villus epithelial cells. In all groups, ferritin protein was not seen in the crypt region but was seen with increasing staining in the apical two-thirds of the villus cells of control and iron-loaded, but not iron-deficient, rats. Ferritin staining increased with iron loading. We conclude that in undifferentiated crypt cells ferritin genes are transcribed, but the message is not translated. After differentiation, these genes appear to be controlled posttranscriptionally by cellular iron stores.

Mesenchyme-mediated effects of retinoic acid during rat intestinal development.

In previous experiments we showed that intestinal development was dependent upon epithelial-mesenchymal cell interactions. The aim of this study was to investigate the possible role of retinoic acid (RA), a morphogenetic and differentiating agent, on the gut epithelial-mesenchymal unit. For this purpose we first analyzed the effects of a physiological dose of RA on 14-day fetal rat intestine using short-term organ culture experiments, or long-term grafts under the skin of nude mice. In these conditions, RA accelerated villus outgrowth and epithelial cell differentiation as assessed by the onset of lactase expression, and it also stimulated muscle and crypt formation. In order to analyze potential effects of RA mediated by mesenchymal cells, we isolated and characterized gut mucosa mesenchyme-derived cell cultures (mesenchyme-derived intestinal cell lines, MIC). These cells were shown to express mRNAs for retinoid binding proteins similar to those expressed in situ in the intestinal mesenchyme. MIC cells co-cultured with 14-day intestinal endoderms promoted endodermal cell adhesion and growth, and the addition of exogeneous RA enhanced epithelial cell polarization and differentiation assessed by cytokeratin and lactase immunostaining. Such a differentiating effect of RA was not observed on endodermal cells when cultured without a mesenchymal feeder layer or maintained in conditioned medium from RA-treated MIC cells. In the co-cultures, immunostaining of laminin and collagen IV with polyclonal antibodies, as well as alpha1 and beta1 laminin chains mRNAs (analyzed by RT-PCR) increased concurrently with the RA-enhanced differentiation of epithelial cells. It is worth noting that this stimulation by RA was also obvious on the mesenchymal cells cultured alone. These results show that RA plays a role in intestinal morphogenesis and differentiation. In addition, they indicate that RA acts on the mesenchymal cell phenotype and suggest that RA may modify the mesenchymal-epithelial cell interactions during intestinal development.

Enhanced growth of small bowel in transgenic mice expressing human insulin-like growth factor 1.

Insulin‐like growth factor 1 (IGF‐1) plays an important role in the regulation of intestinal growth and maturation, whereas the direct effects of growth hormone (GH) on intestinal growth are less clear. In this study the authors utilize a transgenic mouse model to assess the extent to which longterm excess of IGF‐1 in a background of GH deficiency stimulates small bowel growth.IGF‐1 transgenic mice that overexpress an IGF‐1 transgene driven by the mouse metallothionein I promotor were compared with wild‐type littermates and GH transgenic mice. The IGF‐1 transgenics showed elevation of circulating IGF‐1, undetectable levels of plasma GH, and a decrease in pituitary GH messenger RNA (mRNA) caused by negative feedback effects of IGF‐1 excess. Growth of small bowel, abundance and localization of mRNAs for the IGF‐1 transgene, and IGF binding protein 3 (IGFBP‐3) were assayed to determine whether enterotrophic effects of excess IGF‐1 correlate with local overexpression of IGF‐1 in small bowel.Small bowel length and mass were greater in IGF‐1 transgenic than in wild‐type mice. Villus height, crypt depth, and crypt cell mitoses were greater in IGF‐1 transgenics than wild‐type mice. Jejunal dissacharidase activities were not different among the IGF‐1 transgenics and wild‐type mice. The IGF‐1 transgene was expressed strongly in villus epithelial cells and at much lower, but significant levels in crypt epithelial cells, lamina propria, and smooth muscle layers. IGFBP‐3 was strongly expressed and localized in the lamina propria.Regional expression of IGF‐1 and IGFBP‐3 correlated with the increase in mucosal mass in IGF‐I transgenics.Effects of IGF‐I overexpression on intestinal length and mucosal mass were similar to effects of GH overexpression, although IGF‐I transgenics showed greater small bowel growth relative to somatic growth compared with GH transgenics. Excess of IGF‐I increased crypt cell proliferation, whereas excess of GH did not increase crypt cell proliferation but did stimulate jejunal sucrase activity. These observations suggest that GH has greater differentiative actions, whereas IGF‐I exerts primarily mitogenic actions in small bowel.

Endothelin‐1 potently stimulates chloride secretion and inhibits Na(+)‐glucose absorption in human intestine in vitro.

1. Serosally added synthetic endothelin-1 (ET-1) increased short-circuit current (Isc) across isolated muscle-stripped human colonic mucosa in vitro. Bumetanide inhibited Isc responses, indicating that ET-1 stimulates electrogenic Cl- secretion. 2. In isolated human jejunal mucosa, ET-1 exhibited a concentration-dependent dual action. At low concentrations it induced rapid increases in Isc and these were inhibited by bumetanide. At a higher concentration (0.1 microM), ET-1 provoked a drastic and progressive decrease in Isc below the baseline value. 3. Pretreatment with phlorizin or omission of glucose from the Krebs-Ringer solution at the apical (luminal) side of the jejunal mucosa prevented the decreases in Isc evoked by ET-1, suggesting that the peptide inhibits the glucose-coupled electrogenic Na+ absorption. Indeed, flux experiments with D-[14C]glucose demonstrated that ET-1 decreases jejunal glucose absorption by approximately 80% within 30 min. 4. Electron microprobe analyses of cryosections of human jejunum showed that ET-1 (0.1 microM) evokes a significant decrease in intracellular Na+ concentrations of villus (not crypt) epithelial cells, suggesting that the peptide attenuates apical Na(+)-glucose entry by reducing the activity of the Na(+)-glucose cotransporter, SGLT1. 5. In the presence of tetrodotoxin (TTX), ET-1-induced Cl- secretion was significantly reduced, in both human jejunal and colonic mucosa. However, the inhibitory effect on jejunal Na(+)-glucose absorption was not affected by TTX. 6. ET-1 increases electrogenic Cl- secretion across human intestinal mucosa in vitro. This effect is mediated in part via the activation of enteric nerves. Responses of the human jejunal mucosa to high ET-1 concentrations exhibit a second component, namely the rapid inhibition of electrogenic Na(+)-glucose absorption, which might be mediated by an inhibition of the transport activity of SGLT1. This effect is independent from neuronal mediators. Our results suggest different cellular action sites for ET-1 in human small and large intestine.

Enhanced growth of small bowel in transgenic mice expressing human insulin-like growth factor I

BACKGROUND & AIMS: Growth hormone and insulin-like growth factor I (IGF- I) stimulate small bowel growth. The aim of this study was to analyze whether IGF-I mediates enterotrophic actions of growth hormone.METHODS: IGF-I transgenic mice that overexpress an IGF-I transgene driven by the mouse metallothionein I promoter and are growth hormone deficient were compared with wild-type littermates. Growth of small bowel, abundance and localization of messenger RNAs for the IGF-I transgene, and insulin-like growth factor-binding protein 3 were assayed.RESULTS: Small bowel length and mass were greater in IGF-I transgenic mice than in wild-type mice. Villus height, crypt depth, and crypt cell mitoses were greater in jejunum of transgenics than wild- type mice, but jejunal disacharidase activities were not increased. The transgene was expressed strongly in villus epithelial cells. Insulin- like growth factor-binding protein 3 messenger RNA was localized in the lamina propria. Regional expression of both correlated with the increase in mucosal mass.CONCLUSIONS: Effects of IGF-I overexpression on intestinal length and mucosal mass were similar to effects of growth hormone overexpression observed previously. Excess of IGF-I increased crypt cell proliferation, whereas excess of growth hormone did not increase crypt cell proliferation. IGF-I excess stimulated differentiation of intestinal epithelial cells less effectively than growth hormone excess.(Gastroenterology 1997 Feb;112(2):444-54)

Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa.

It has been demonstrated previously that the inducible isoform of nitric oxide synthase (iNOS) is present throughout the intestinal tract in various inflammatory disease processes. Here we demonstrate that iNOS mRNA is present in the ileum but not in the jejunum or colon of normal mice. By Western blot analysis, iNOS protein is also detected in normal ileum, but not in the normal jejunum. However, by 3 h postinjection of 0.5 mg/kg lipopolysaccharide (LPS), iNOS mRNA is also detectable in the jejunum and colon. The enzyme message and protein, localized immunohistochemically by in situ hybridization and iNOS expression, is normally restricted to the villus epithelial cells. The iNOS mRNA was also present in the ilea of mice with defined intestinal flora (anaerobes only), germ-free mice, nude mice, and to a lesser extent in mice with severe combined immunodeficiency. These results suggest that the constitutive presence of iNOS in ileal epithelium indicates a role for this enzyme in maintaining intestinal homeostasis.

The Na+/H+ exchange isoform NHE3 regulates basal canine ileal Na+ absorption in vivo

BACKGROUND & AIMS: Three different Na+/H+ exchange isoforms have been localized in mammalian ileum: Na+/H+ exchange isoform 1 (NHE1) on the basolateral membrane and NHE2 and NHE3 on the brush border membrane. The aim of this study was to determine whether NHE3 is the predominant isoform serving basal ileal Na+ absorption. METHODS: Immunocytochemistry was performed on canine ileum with specific polyclonal antibodies. Absorption studies (n = 58) were performed in dogs with 25-cm ileal Thiry-Vella fistulas. In groups 1-4, luminal 5- (N,N-dimethyl)-amiloride (DMA) was administered at doses of 100 nmol/L, 5 mumol/L, and 0.7 mmol/L to inhibit NHE1, NHE2, and NHE3, respectively. In groups 5-8, 1 mmol/L phlorizin (PHLZ), 0.7 mmol/L DMA, and 1 mmol/L PHLZ plus 0.7 mmol/L DMA were administered intraluminally. RESULTS: NHE2 and NHE3 were present in the brush border of canine ileal villus epithelial cells. The highest concentration of DMA (0.7 mmol/L) caused a significant reduction (P > 0.05) in basal ileal water and sodium absorption. PHLZ and 0.7 mmol/L DMA each alone significantly reduced basal ileal absorption (P > 0.05), whereas PHLZ plus 0.7 mmol/L DMA had an additive effect. CONCLUSIONS: NHE3 seems to be the Na+/H+ exchange isoform involved in the modulation of basal ileal water and sodium absorption. Both Na+/H+ exchange mediated through NHE3 and Na(+)- glucose cotransport contribute to basal ileal water and sodium absorption. (Gastroenterology 1997 Jan;112(1):174-83)