89 Evaluating the effect of Ca-gluconate and Ca-butyrate on SCFA absorption and permeability of the gastrointestinal tract

The objective was to evaluate short-chain fatty acid (SCFA) absorption and permeability of the gastrointestinal tract (GIT) of heifers infused either with Ca-gluconate or Ca-butyrate. Thirty-two ruminally cannulated beef heifers were fed a common diet (forage-to-concentrate ratio of 50:50) for 28 d and once daily infused with water (ruminal infusion; control), Ca-gluconate embedded in a fat matrix (ruminal infusion; 0.192% BW), unprotected Ca-gluconate (abomasal infusion; 0.077% of BW), and unprotected Ca-butyrate (abomasal infusion; 0.029% BW). Treatments were designed to provide the same amount of butyrate to the small intestine. DMI was restricted to 95% of voluntary DMI on d 8 and was recorded until heifers were slaughtered on d 28. Rumen, jejunum, and colon tissues were collected to determine the rate and pathway of SCFA transport, and for measurement of permeability.14C-acetate and 3H-butyrate absorption across the ruminal and colonic epithelium was measured with no inhibition and under maximal inhibition. Permeability was assessed for the rumen, jejunum and colon using mucosal-to-serosal flux of 14C-mannitol. Initial and final BW were not different (P > 0.60) averaging 388 ± 5.5 kg and 409 ± 6.6 kg, respectively. DMI was not affected by treatment averaging 7.7 kg/d (P = 0.77). Treatment did not affect the flux of acetate or butyrate across the ruminal or colonic epithelium (P > 0.33), but flux rates of SCFA were numerically greater across the ruminal epithelium. Moreover, approximately 50% of the ruminal acetate was transported via bicarbonate-dependent mechanisms. Mannitol flux was not affected by treatment (P > 0.29) and was numerically lower in the rumen and colon than in the jejunum, supporting previous research evaluating permeability across the GIT. According to this experiment, provision of Ca-butyrate or Ca-gluconate in an attempt to increase intestinal butyrate supply does not impact SCFA absorption or permeability of the rumen, jejunum, or colon.

Effects of the ProbioticEnterococcus faeciumand PathogenicEscherichia coliStrains in a Pig and Human Epithelial Intestinal Cell Model

The aim of this study has been to elucidate the effect of the probioticEnterococcus faeciumNCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC) and enteropathogenicEscherichia coli(EPEC). Porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER) and mannitol flux rates.E. faeciumalone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas theE. colistrains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation withE. faeciumhad no effect on the TEER decrease induced byE. coliin preliminary experiments. However, in a second set of experiments using a slightly different protocol,E. faeciumameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude thatE. faeciumpositively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods.

Characterising barrier function among regions of the gastrointestinal tract in Holstein steers

The objective of this study was to characterise the regional variation in the barrier function of the gastrointestinal tract in Holstein calves using the flux rates of mannitol and inulin as permeability markers and tissue conductance (Gt) as an electrophysiological indicator of barrier function. Six Holstein steer calves (6 months of age) fed a common diet were used. Calves were killed by captive bolt stunning and pithing, and tissues were collected from the rumen, omasum, duodenum, jejunum, ileum, caecum, proximal colon, and distal colon. Tissues were carefully washed using a pre-heated (38.5°C) buffer solution (pH 7.4) saturated with oxygen and then transported to the laboratory. The mucosa was prepared by hand stripping and mounted between two halves of an Ussing chamber (n = 3/region with an exposed surface area of 3.14 cm2 for rumen and omasum and 1 cm2 for all other tissues). All tissues were incubated under short-circuit conditions and exposed to a similar buffer solution except for the energy source; rumen, omasum, caecum, and colon tissues were incubated with buffer containing short-chain fatty acids while tissues from the small intestine were bathed in buffer containing glucose. The Gt and the serosal-to-mucosal flux rates of 14C-inulin and 3H-mannitol were measured as indicators of barrier function. The serosal-to-mucosal flux rate of mannitol was greatest (P < 0.001) in the jejunum [104.8 nmol/(cm2 × h)] and least in the rumen and omasum [20.3 and 18.6 nmol/(cm2 × h), respectively]. In contrast, the serosal-to-mucosal flux rate of inulin was greatest (P < 0.001) in the omasum [158.6 nmol/(cm2 × h)] followed by the rumen [87.3 nmol/(cm2 × h)] with no differences among the other regions [18.7 – 62.0 nmol/(cm2 × h)]. The Gt was greatest (P < 0.001) in the jejunum (34.6 mS/cm2) and least for the rumen (3.67 mS/cm2) and omasum (3.23 mS/cm2). The Gt was correlated with both inulin and mannitol flux rates in duodenum, caecum and proximal colon (P < 0.05); whereas, no such correlations existed in jejunum, ileum and distal colon. The Gt was correlated with the mannitol flux rate but not the inulin flux rate in rumen and omasum. For all regions but the rumen and omasum there was a positive correlation between mannitol and inulin flux rates. These data indicate that the translocation of a large molecule (inulin) across the omasum and rumen is greatest despite having an apparently tight epithelium based on Gt and mannitol flux rate, while the jejunum appears to have greatest potential for paracellular permeability.

Physiological concentrations of bile salts inhibit recovery of ischemic-injured porcine ileum

We have previously shown rapid in vitro recovery of barrier function in porcine ischemic-injured ileal mucosa, attributable principally to reductions in paracellular permeability. However, these experiments did not take into account the effects of luminal contents, such as bile salts. Therefore, the objective of this study was to evaluate the role of physiological concentrations of deoxycholic acid in recovery of mucosal barrier function. Porcine ileum was subjected to 45 min of ischemia, after which mucosa was mounted in Ussing chambers and exposed to varying concentrations of deoxycholic acid. The ischemic episode resulted in significant reductions in transepithelial electrical resistance (TER), which recovered to control levels of TER within 120 min, associated with significant reductions in mucosal-to-serosal 3H-labeled mannitol flux. However, treatment of ischemic-injured tissues with 10−5 M deoxycholic acid significantly inhibited recovery of TER with significant increases in mucosal-to-serosal 3H-labeled mannitol flux, whereas 10−6 M deoxycholic acid had no effect. Histological evaluation at 120 min revealed complete restitution regardless of treatment, indicating that the breakdown in barrier function was due to changes in paracellular permeability. Similar effects were noted with the application of 10−5 M taurodeoxycholic acid, and the effects of deoxycholic acid were reversed with application of the Ca2+-mobilizing agent thapsigargin. Deoxycholic acid at physiological concentrations significantly impairs recovery of epithelial barrier function by an effect on paracellular pathways, and these effects appear to be Ca2+ dependent.

Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} ion transport

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na+/H+ exchange, [Formula: see text]/[Formula: see text] exchange and [Formula: see text]/[Formula: see text] co-transport, and Na+-K+-2Cl- cotransport, respectively. Ion substitution experiments with Na+-free, Cl--free, and [Formula: see text]-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl--free, or [Formula: see text]-free solutions did not affect restitution. In Na+-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na+-dependent [Formula: see text] transport. Because [Formula: see text]-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in [Formula: see text] ion transport but essential for cell migration after injury and restitution in the gastric mucosa.

Regulation of acid secretion and paracellular permeability by F-actin in the bullfrog,Rana catesbeiana

G526, 2002. First published December 5, 2001; 10.1152/ajpgi.00393. 2001.—Many studies have implicated F-actin in the regulation of gastric acid secretion using cytochalasin D (CD) to disrupt apical actin filaments in oxyntic cells. However, it is known that CD also affects mucosal permeability by disrupting tight junction structure. Here we investigated the contribution of F-actin to mucosal permeability and acid secretion in the stomach using CD. Stomachs were mounted in Ussing chambers and acid secretion (stimulated or inhibited), transepithelial resistance (TER), mannitol flux, bicarbonate transport, and dual mannitol/sodium fluxes were determined with or without CD. H+back diffusion was predicted from its diffusion coefficient. Incubation with CD resulted in a significant reduction in stimulated acid secretion. TER was unchanged in stimulated tissues but significantly reduced in inhibited tissues. Mannitol flux, bicarbonate transport, and H+-back diffusion increased significantly with CD. However, the rates of bicarbonate and H+flux were not large enough to account for the inhibition of acid secretion. These findings demonstrate that actin filaments regulate paracellular permeability and play an essential role in the regulation of acid secretion in the stomach.

Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Occludin is a functional component of the tight junction

Occludin's role in mammalian tight junction activity was examined by ‘labeling’ the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30–40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30–40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.