[Show abstract][Hide abstract]ABSTRACT: Aim:
To study whether the inflammatory bowel disease (IBD) colon which exhibits varying severity and cytokine levels across its mucosa create varying types of transepithelial leak.
We examined the effects of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1-β (IL1β) and hydrogen peroxide (H2O2) - singly and in combinations - on barrier function of CACO-2 cell layers. Our focus was on the type (not simply the magnitude) of transepithelial leak generated by these agents as measured by transepithelial electrical resistance (TER) and transepithelial flux of (14)C-D-mannitol, (3)H-Lactulose and (14)C-Polyethylene glycol as radiolabeled probe molecules. The isoquinoline alkaloid, berberine, was then examined for its ability to reduce specific types of transepithelial leak.
Exposure to TNF-α alone (200 ng/mL; 48 h) induced a 50% decrease in TER, i.e., increased leak of Na(+) and Cl(-) - with only a marginal but statistically significant increase in transepithelial leak of (14)C-mannitol (Jm). Exposure to TNF-α + IFN-γ (200 ng/mL; 48 h) + IL1β (50 ng/mL; 48 h) did not increase the TER change (from TNF-α alone), but there was now a 100% increase in Jm. There however was no increase in transepithelial leak of two larger probe molecules, (3)H-lactulose and (14)C-polyethylene glycol (PEG). However, exposure to TNF-α + IFN-γ + IL1β followed by a 5 h exposure to 2 mmol/L H2O2 resulted in a 500% increase in (14)C-PEG leak as well as leak to the luminal mitogen, epidermal growth factor.
This model of graded transepithelial leak is useful in evaluating therapeutic agents reducing IBD morbidity by reducing barrier leak to various luminal substances.
[Show abstract][Hide abstract]ABSTRACT: Dietary methionine restriction (MR) has been found to affect one of the most primary tissue-level functions of an organism: the efficiency with which the epithelial linings of major organs separate the fluid compartments that they border. This process, epithelial barrier function, is basic for proper function of all organs, including the lung, liver, gastrointestinal tract, reproductive tract, blood-brain barrier, and kidney. Specifically, MR has been found to modify the protein composition of tight junctional complexes surrounding individual epithelial cells in a manner that renders the complexes less leaky. This has been observed in both a renal epithelial cell culture model and in gastrointestinal tissue. In both cases, MR increased the transepithelial electrical resistance across the epithelium, while decreasing passive leak of small nonelectrolytes. However, the specific target protein modifications involved were unique to each case. Overall, this provides an example of the primary level on which MR functions to modify, and improve, an organism.
Full-text · Article · Dec 2015 · Annals of the New York Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.
[Show abstract][Hide abstract]ABSTRACT: Background:
Delivery of a pharmacologically effective drug dosage to a target tissue is critical. Barrett's epithelia are a unique challenge for drug delivery of orally administered zinc due to rapid transit down the esophageal lumen, incomplete absorptive differentiation of these epithelia, and the use of proton-pump inhibitor drugs abrogating intestinal uptake of supplemental zinc.
Barrett's esophagus patients were administered oral zinc gluconate (26 mg zinc twice daily) for 14 days prior to biopsy procurement. Barrett's biopsies were analyzed for total zinc content by atomic absorption spectroscopy and by western immunoblot for cellular proteins known to be regulated by zinc.
Cellular levels of both the Znt-1 transport protein and the alpha isoform of PKC were over 50% lower in the zinc treatment group.
Oral zinc administration can result in effective delivery of zinc to Barrett's epithelia with resulting effects on intracellular signal transduction.
Full-text · Article · Mar 2014 · Therapeutic delivery
[Show abstract][Hide abstract]ABSTRACT: Amid an increasing number of reports in the literature concerning epithelial barrier enhancement by various nutrient compounds, there has never been a study performing side-by-side comparisons of these agents in a single epithelial model. We compare five nutrient compounds (previously reported in various epithelial models to enhance barrier function) regarding their ability to increase transepithelial electrical resistance (Rt) and decrease transepithelial mannitol permeability (Jm) across LLC-PK1 renal epithelial cell layers. The effects of these nutrients on the abundance of various tight junctional proteins are also compared. In the overall group of nutrients tested - zinc, indole, quercetin, butyrate and nicotine - only nicotine failed to improve barrier function by either parameter. Nicotine also was without effect on tight junctional proteins. Quercetin simultaneously increased Rt and decreased Jm. Zinc, butyrate and indole only exhibited statistically significant enhancement of Rt. Each of these four effective nutrient compounds had unique patterns of effects on the panel of tight junctional proteins studied. No two compounds produced the same pattern of effects. This unique pattern of effects on tight junctional complex composition by each compound establishes the chance for additive or even synergistic improvement of barrier function by combinations of compounds. A synergistic effect of the combination of quercetin and zinc on Rt is shown.
[Show abstract][Hide abstract]ABSTRACT: Background and aims:
Earlier work by our group and others has documented improvement of epithelial barrier function in human gastrointestinal models. Here we tested zinc's ability to improve a renal epithelial model. Our aim was to compare the functional and structural effects of zinc on the tight junctional (TJ) complexes of these two very distinct epithelial cell types. Zinc's ability to achieve barrier enhancement in very different epithelial cell types by action upon distinct molecular targets in each epithelial model may suggest a fundamental general role for supplemental zinc in epithelial barrier improvement throughout the body.
Cell layers were exposed to 50 or 100 μM zinc on both cell surfaces for 48 h followed by measurement of transepithelial electrical resistance (Rt) and transepithelial (14)C-mannitol flux (Jm). TJ proteins in cell layers were analyzed by Western immunoblot.
Results and conclusions:
Zinc supplementation improved the basal TJ barrier function of LLC-PK1 renal cell layers, exemplified by increased Rt and decreased Jm. These zinc-induced changes were also accompanied by decreased NaCl dilution potentials. Of the tight junctional proteins that were tested (occludin, claudins 1, 2, 3, 4, and 5, and tricellulin), we did not observe a zinc-induced change in abundance of any of them, in detergent-soluble fractions of lysates of confluent differentiated cell layers. However, examination of cytosolic fractions showed concentration-dependent increases in the levels of claudins -2 and -4 in this compartment as a result of supplemental zinc. The effects of supplemental zinc on the tight junctional complexes and barrier properties of this renal epithelial model are contrasted with zinc effects on the CACO-2 gastrointestinal model.
[Show abstract][Hide abstract]ABSTRACT: Background
Zinc deficiency is known to result in epithelial barrier leak in the GI tract. Precise effects of zinc on epithelial tight junctions (TJs) are only beginning to be described and understood. Along with nutritional regimens like methionine-restriction and compounds such as berberine, quercetin, indole, glutamine and rapamycin, zinc has the potential to function as a TJ modifier and selective enhancer of epithelial barrier function.
The purpose of this study was to determine the effects of zinc-supplementation on the TJs of a well-studied in vitro GI model, CACO-2 cells.
Barrier function was assessed electrophysiologically by measuring transepithelial electrical resistance (Rt), and radiochemically, by measuring transepithelial (paracellular) diffusion of 14C-D-mannitol and 14C-polyethyleneglycol. TJ composition was studied by Western immunoblot analyses of occludin, tricellulin and claudins-1 to -5 and -7.
Fifty- and 100-μM zinc concentrations (control medium is 2 μM) significantly increase Rt but simultaneously increase paracellular leak to D-mannitol. Claudins 2 and 7 are downregulated in total cell lysates, while occludin, tricellulin and claudins-1, -3, -4 and -5 are unchanged. Claudins-2 and -7 as well as tricellulin exhibit decreased cytosolic content as a result of zinc supplementation.
Zinc alters CACO-2 TJ composition and modifies TJ barrier function selectively. Zinc is one of a growing number of “nutraceutical” substances capable of enhancing epithelial barrier function, and may find use in countering TJ leakiness induced in various disease states.
Full-text · Article · Aug 2012 · Digestive Diseases and Sciences
[Show abstract][Hide abstract]ABSTRACT: Supplemental Figure 1. Effect of Bin1 loss on immune cytokine expression. Serum was collected from mice administered 3 % DSS in drinking water for 7 days and the level of the immune cytokines indicated was determined by a cytometric bead array. The data represent the determination of at least three data points per sample
[Show abstract][Hide abstract]ABSTRACT: To determine if the observed paracellular sucrose leak in Barrett's esophagus patients is due to their proton pump inhibitor (PPI) use.
The in vivo sucrose permeability test was administered to healthy controls, to Barrett's patients and to non-Barrett's patients on continuous PPI therapy. Degree of leak was tested for correlation with presence of Barrett's, use of PPIs, and length of Barrett's segment and duration of PPI use.
Barrett's patients manifested a near 3-fold greater, upper gastrointestinal sucrose leak than healthy controls. A decrease of sucrose leak was observed in Barrett's patients who ceased PPI use for 7 d. Although initial introduction of PPI use (in a PPI-naïve population) results in dramatic increase in sucrose leak, long-term, continuous PPI use manifested a slow spontaneous decline in leak. The sucrose leak observed in Barrett's patients showed no correlation to the amount of Barrett's tissue present in the esophagus.
Although future research is needed to determine the degree of paracellular leak in actual Barrett's mucosa, the relatively high degree of leak observed with in vivo sucrose permeability measurement of Barrett's patients reflects their PPI use and not their Barrett's tissue per se.
Full-text · Article · Jun 2012 · World Journal of Gastroenterology
[Show abstract][Hide abstract]ABSTRACT: Inflammatory bowel disease (IBD) is associated with defects in intestinal barriers that rely upon cellular tight junctions. Thus, identifying genes that could be targeted to enforce tight junctions and improve barrier function may lead to new treatment strategies for IBD.
This preclinical study aimed to evaluate an hypothesized role for the tumor suppressor gene Bin1 as a modifier of the severity of experimental colitis.
We ablated the Bin1 gene in a mosaic mouse model to evaluate its effects on experimental colitis and intestinal barrier function. Gross pathology, histology and inflammatory cytokine expression patterns were characterized and ex vivo physiology determinations were conducted to evaluate barrier function in intact colon tissue.
Bin1 attenuation limited experimental colitis in a sexually dimorphic manner with stronger protection in female subjects. Colitis suppression was associated with an increase in basal transepithelial electrical resistance (TER) and a decrease in paracellular transepithelial flux, compared to control wild-type animals. In contrast, Bin1 attenuation did not affect short circuit current, nor did it alter the epithelial barrier response to non-inflammatory permeability enhancers in the absence of inflammatory stimuli.
Bin1 is a genetic modifier of experimental colitis that controls the paracellular pathway of transcellular ion transport regulated by cellular tight junctions. Our findings offer a preclinical validation of Bin1 as a novel therapeutic target for IBD treatment.
[Show abstract][Hide abstract]ABSTRACT: The beneficial effects of caloric restriction in increasing longevity and forestalling age-related diseases are well known. Dietary restriction of methionine also renders similar benefits. We recently showed in a renal epithelial cell culture system that reduction of culture medium methionine by 80% resulted in altered tight junctional (TJ) claudin composition and also improved epithelial barrier function (51). In the current study, we examined the effect of dietary restriction of methionine on TJ barrier function in rat gastrointestinal tissue to see whether this phenomenon also holds true in a tissue model and for a different epithelial cell type. After 28 days on methionine-restricted (MR) diet, rats showed small but significant reductions in the plasma and (intracellular) colonocyte levels of methionine. Colon mucosal sheets from rats on the MR diet showed increased transepithelial electrical resistance with concomitant decrease in paracellular diffusion of (14)C-D-mannitol, suggesting improved barrier function relative to rats on control diet. This improved barrier function could not be explained by changes in colon crypt length or frequency. Neither was the colonocyte mitotic index nor the apoptotic frequency altered significantly. However, TJ composition/structure was being altered by the MR diet. RT-PCR and Western blot analysis showed an increase in the abundance of claudin-3 and an apparent change in the posttranslational modification of occludin, data reinforcing a paracellular barrier alteration. Overall, our data suggest that reduction in dietary intake of methionine results in improved epithelial barrier function by inducing altered TJ protein composition.
Full-text · Article · Nov 2010 · AJP Cell Physiology
[Show abstract][Hide abstract]ABSTRACT: To investigate omeprazole-induced transepithelial gastric leak and its effects on the permeability of the peptides bradykinin and oxytocin.
Rat gastric corpus tissue was isolated and mounted in an Ussing chamber apparatus to evaluate the permeability of (3)H-bradykinin, (3)H-oxytocin, and (14)C-EDTA in the presence or absence of omeprazole. Thin-layer chromatography was performed to identify any metabolic breakdown products of the peptides resulting from permeation through the gastric tissue, and thereby calculate the true flux of the peptide.
The flux rate of intact (3)H-bradykinin increased substantially after omeprazole addition (109.5%) compared to the DMSO vehicle control (14%). No corresponding change in flux of intact (3)H-oxytocin was observed under the same conditions (11.9% and 6.4% in the DMSO- and omeprazole-treated conditions, respectively). After exposure to omeprazole, the flux rate of (14)C-EDTA also increased dramatically (122.3%) compared to the DMSO condition (36.3%).
The omeprazole-induced gastric leak allows for transmucosal permeability to charged molecules as well as non-electrolytes. This induced leak will allow certain peptides to permeate.
Full-text · Article · Mar 2010 · World Journal of Gastroenterology
[Show abstract][Hide abstract]ABSTRACT: Despite their remarkable safety profile and lack of clinical side effects, proton pump inhibitors (PPIs) induce a transmucosal
gastric leak to non-electrolyte probes of various sizes. The exvivo addition of PPIs to isolated rat gastric corpus increases
transmucosal permeability in a dose-dependent manner, which corresponds with PPIs’ dose-dependent inhibition of acid secretion.
Upon the addition of omeprazole, lansoprazole, or esomeprazole, a small decrease in transepithelial resistance and the concomitant
stimulation of short circuit current was observed. Additionally, transepithelial flux of 14C-[d]-mannitol (MW 182.17) across the gastric mucosa increased by a mean of 68% immediately following the addition of 200μM omeprazole.
This flux increase was bidirectional. Omeprazole also increased the paracellular permeability to larger radiolabeled probes,
including 14C-sucrose (MW 342.3) and 14C-polyethylene glycol (MW 4,000) by 118% and 350%, respectively. However, the flux of still larger probes, 10,000 and 70,000MW
dextrans, was not increased. Because PPIs are so widely used and are assumed to be innocuous, this transmucosal gastric leak
must be further investigated, as it may carry considerable biomedical implications.
[Show abstract][Hide abstract]ABSTRACT: Previous animal and patient-based studies have shown that omeprazole induces a transepithelial paracellular gastric leak. This study reports on the potential for an omeprazole-induced leak of drugs with narrow therapeutic windows. Ussing chamber experiments investigated the effects of omeprazole on rat gastric corpus permeability to the drugs, digoxin and phenytoin. Digoxin (780 MW) permeated the gastric mucosa at an accelerated rate in the presence of omeprazole. This leak could contribute to dangerous elevations of blood digoxin levels in certain situations. Omeprazole was found to have no effect on the flux rate of phenytoin (252 MW). The tight-junctional leak generated by omeprazole thus exhibits specificity to the types of molecules it allows to permeate through the gastric mucosa. This leak may pose a clinical danger by increasing drug uptake into the bloodstream, a phenomenon which would act synergistically with the effect of omeprazole on inhibiting liver cytochrome P450s that remove drugs from the bloodstream, thereby elevating drug blood levels.
[Show abstract][Hide abstract]ABSTRACT: The relative abundance of various claudin proteins of LLC-PK(1) renal epithelial tight junctions (TJs) is modulated by culturing the cells in a medium that is sharply reduced in the sulfur-containing amino acids, cysteine, cystine, and methionine. The functional result is an epithelial barrier that has a higher transepithelial electrical resistance and a decreased paracellular leak to D-mannitol (i.e., improved barrier function). This is accomplished without affecting the culture's confluent cell density, its short circuit current, or its hallmark differentiated property, Na(+)-dependent sugar transport. The implications of being able to enhance epithelial TJ barrier function by nutritional means are discussed, particularly in light of the ability of methionine-restrictive diets to enhance life span and forestall age-related morbidity.
Full-text · Article · Jun 2009 · Annals of the New York Academy of Sciences