Comparison of the cytotoxic effects of cadmium chloride and cadmium-metallothionein in LLC-PK1 cells

Recent studies have shown that ionic cadmium (Cd²⁺) can selectively damage the tight junctions between LLC-PK₁ cells. The objective of the present studies was to determine if cadmium that is bound to metallothionein (Cd-Mt) can also damage the junctions between these cells. Cells on Falcon Cell Culture Inserts were exposed to Cd²⁺ or Cd-Mt from the apical and basolateral compartments. The integrity of cell junctions was assessed by monitoring the transepithelial electrical resistance, and cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. Exposure to Cd²⁺ for 1–4 hours caused a pronounced decrease in the transepithelial resistance without affecting cell viability. By contrast, exposure to Cd-Mt had little effect on the electrical resistance until the cells began to die, which did not occur until 24–48 hours of exposure. Additional results showed that the cells accumulated Cd²⁺ more rapidly than Cd-Mt. These results indicate that Cd-Mt does not damage the junctions between LLC-PK₁ cells, but that it can kill the cells after prolonged exposure.     

Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture

Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFbeta) into the basolateral chamber ( approximately 70% > apical) and tumor necrosis factor-alpha (TNFalpha) into the apical compartment ( approximately 30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24-48 h increased epithelial cell TER ( approximately 70% higher than control) and decreased TNFalpha release into both the apical and basolateral chambers ( approximately 30%-50%). In contrast, TGFbeta release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFalpha release persisted for 5-7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNFalpha without effecting TGFbeta release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.     

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells

Summary Previous studies from our laboratory have shown that Cd²⁺ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK₁. The objective of the present studies was to determine whether or not Cd²⁺ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd²⁺ by adding CdCl₂ to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10–40 μM Cd²⁺ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd²⁺ was added basolaterally than when it was added apically. Moreover, the effects of Cd²⁺ were qualitatively similar to those observed when the cells were incubated in Ca²⁺-free medium. These results show that Cd²⁺ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.     

A C-NMR study on the influxes into the tricarboxylic acid cycle of a renal epithelial cell line, LLC-PK1/Cl4: the metabolism of [2-C]glycine, l-[3-C]alanine and l-[3-C]aspartic acid in renal epithelial cells

Perchloric acid extracts of LLC-PK₁/Cl₄ cells, a renal epithelial cell line, incubated with either [2-¹³C]glycine l-[3-¹³C]alanine, or d,l-[3-¹³C]aspartic acid were investigated by ¹³C-NMR spectroscopy. All amino acids, except labelled glycine, gave rise to glycolytic products and tricarboxylic acid cycle (TCA) intermediates. For the first time we also observed activity of γ-glutamyltransferase activity and glutathione synthetase activity in LLC-PK₁ cells, as is evident from enrichment of reduced glutathione. Time courseS showed that only 6% of the labelled glycine was utilized in 30 min, whereas 31% of l-alanine and 60% of l-aspartic acid was utilized during the same period. ¹³C-NMR was also shown to be a useful tool for the determination of amino acid uptake in LLC-PK₁ cells. These uptake experiments indicated that glycine alanine and aspartic acid are transported into Cl₄ cells via a sodium-dependent process. From the relative enrichment of the glutamate carbons, we calculated the activity of pyruvate dehydrogenase to be about 61% of when labelled l-alanine was the only carbon source for LLC-PK₁/Cl₄ cells. Experiments with labelled d,l-aspartic, however, showed that about 40% of C-3-enriched oxaloacetate (arising from a de-amination of aspartic acid) reached the pyruvate pool.     

Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils

In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S. typhimurium. We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport. However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued. In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration. Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN. Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells. Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8). However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration. These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium. Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.     

Effect of TaiCatoxin (TCX) on the electrophysiological,mechanical and biochemical characteristics of spontaneously beating ventricular cardiomyocytes

TaiCatoxin (TCX), a complex toxin isolated from Taipan snake venom, is believed to have a specific blocking activity on voltage-dependent cardiac calcium channels. The aim of this study was to investigate the effects of TCX on a broad range of heart muscle cell functions, i.e. electrophysiology, contractility, automaticity and the related biochemical modifications. Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded with glass microelectrodes. The contractions were monitored photometrically. TCX decreased the action potential amplitudes, mainly by lowering the plateau. The action potential duration and the contraction parameters were decreased. Although TCX has a minor overall negative chronotropic effect, it evoked transient but severe arrhythmias and prolonged changes in the intercellular electrical coupling. Moreover, the action of TCX appeared to be dose-dependent. These effects are consistent with a specific blockade of the L-type, voltage-dependent calcium channels, but effects of other components of the toxin complex cannot be excluded. TCX also exhibits phospholipase A₂ activity leading to the release of lysophospholipids and FFA (acyl CoA and acyl carnitine), which have detrimental effects on cellular integrity and function.     

Transepithelial transport in cell culture:d-Glucose transport by a pig kidney cell line (LLC-PK1)

Summary The pig kidney cell line LLC-PK₁ cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5mm d-glucose, a PD of 2.8 mV, apical surface negative, a SCC of 13 A cm^–2 and transepithelial resistance of 211 ohm·cm² were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13mm -methyl-d-glucoside, 0.28mm d-glucose, 0.65mm -methyl-d-glucoside, 0.77mm 6-deoxy-d-glucose, 4.8mm d-galactose, and 29mm 3-O-methyl-glucose. When [Na] was reduced, the concentration ofd-glucose required for half-maximal SCC increased. Isotopically labeled³H and¹⁴Cd-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeledd-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK₁, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

Enhancement of Tight Junctional Barrier Function by Micronutrients: Compound-Specific Effects on Permeability and Claudin Composition

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 (R_t) and decrease transepithelial mannitol permeability (J_m) 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 R_t and decreased J_m. Zinc, butyrate and indole only exhibited statistically significant enhancement of R_t. 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 R_t is shown.     

Effects of diacylglycerols on LLC-PK1 renal epithelia: Similarity to phorbol ester tumor promoters

As previously shown using phorbol ester tumor promoters (see Mullin and O'Brien: Am. J. Physiol., 251:C597–C602, 1986), diacylglycerols induce leakiness in LLC-PK₁ renal epithelial tight junctions. The similarity between phorbol ester and diacylglycerol action includes effects on (1) cell morphology, (2) dome formation, (3) transepithelial resistance and potential difference, (4) transepithelial flux of D-mannitol, and (5) mitogenesis. Four diacylglycerols have been tested: 1,2-dioctanoylglycerol; 1,2-dicaprylglycerol; 1,2-dioleoylglycerol; and 1-oleoyl-2-acetoyl-sn-3-glycerol. Their relative effectiveness depended upon the phenomenon being observed. Unlike phorbol esters, diacylglycerol effects were reversible within hours at 37°C in the continued presence of diacylglycerol, and effects were more pronounced when cell sheets were exposed to diacylglycerols from the basolateral cell surface. Overall, these findings indicate that previous results with phorbol esters may be attributed to the protein kinase C signal transduction system, and this system may therefore exert a role in transepithelial permeability.     

Toxigenic C. difficile induced inflammatory marker expression by human intestinal epithelial cells is asymmetrical

Clostridium difficile infection of the intestinal epithelium and consequent pseudomembranous colitis is an important cause of morbidity and mortality. Pathogenesis has been ascribed exclusively to toxin production. Using in vitro models of human intestinal epithelial layers, we show that exposure to toxigenic C. difficile upregulates epithelial expression of IL-8 and ICAM-1, two molecules important in neutrophil chemoattraction and adhesion and subsequent inflammation. IL-8 production was also stimulated by toxin-containing supernatants. C difficile induced IL-8 release was inhibited by specific antiserum. Increased ICAM-1 expression only occurred after basolateral exposure to C. difficile while apical exposure had no effect on ICAM-1 expression. However, transepithelial electrical resistance was impaired by apical exposure to bacterial suspensions. We suggest that apical exposure to C. difficile induces changes in epithelial layer integrity which allows the bacteria and/or the toxin access to the basolateral compartment where pathogenic inflammatory mechanisms are activated.     

Toxicity and cellular responses of intestinal cells exposed to titanium dioxide

The increasing use of nanomaterials in healthcare and industrial products heightens the possibility of their ingestion by humans, other mammals, and fish. While toxicity of many nanomaterials has recently been studied, reports of non-lethal effects of nanomaterials remain ill-defined. This study investigates possible pathways by which nanoparticles, titanium dioxide (TiO₂), could cross the epithelium layer by employing both toxicity and mechanistic studies. This study provides evidence that at 10 μg/mL and above, TiO₂ nanoparticles cross the epithelial lining of the intestinal model by transcytosis, albeit at low levels. TiO₂ was able to penetrate into and through the cells without disrupting junctional complexes, as measured by γ-catenin. To monitor the epithelial integrity, transepithelial electrical resistance (TEER) was employed and determined low concentrations (10 or 100 μg/mL) of TiO₂ do not disrupt epithelial integrity. Live/dead analysis results did not show cell death after exposure to TiO₂. In addition, at 10 μg/mL (and above) TiO₂ nanoparticles begin alteration of both microvillar organization on the apical surface of the epithelium as well as induce a rise in intracellular-free calcium. The latter is a mechanism cells use to respond to extracellular stimuli and may be linked to the alteration of the apical microvilli. Although TiO₂ does not show cell death, the implication of other, non-lethal, effects could lead to undesired outcomes (i.e., disease, malnutrition, shortened life span, etc.).     

A Note on the Anti-Pseudomonas Activity of Cephalosporins with Chlorhexidine

Thirty clinical isolates of Pseudomonas aeruginosa were tested for susceptibility to cephaloridine, cefuroxime, cefotaxime and cefoxitin with and without chlorhexidine in agar dilution 'chequerboard' titrations and antibiotic disc tests. A synergistic response (σFIC = < 0.7) in combined titration with chlorhexidine was seen in 50% of the strains with cefuroxime and cefoxitin and 13% with cefotaxime. Cephaloridine MICs mostly exceeded the tested range but a definite reduction in combination was shown with 33% of strains. Susceptibility to cephaloridine, cefuroxime and cefoxitin was not enhanced to clinically useful levels and the effect was seen only in chlorhexidine concentrations close to the MIC. Disc tests failed to show the antibacterial activity of the combinations. It is suggested that the level of the principal lesion induced by chlorhexidine in the inner membrane might be too deep to affect the penetration of β-lactam antibiotics to their site of action.     

Different effects of cGMP and cAMP in the intestine of the European eel,Anguilla anguilla

The regulation of salt absorption in the sea water cell intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E ₁ on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increase the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying Cl^– transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine vasoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.Abbreviations AP III rat artriopeptin III - 8 Br cAMP 8 Br cyclic adenosine monophosphate - 8 Br cGMP 8 Br cyclic guanosine monophosphate - g _t transepithelial conductance - I _sc short circuit current - IC ₅₀ half-maximal inhibitory concentration - NaK ATPase Na-K-adenosine monophosphate - NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid - PGE prostaglandin E ₁ - R _t tissue resistance - SITS 4-acetamide-4-isothiocyano-stilbene-2,2-disulfonic acid - V _t transepithelial voltage difference - VIP porcine vasoactive intestinal peptide     

Vaccinia virus entry, exit, and interaction with differentiated human airway epithelia

Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors.     

Human intestinal epithelial cells exhibit a cellular response indicating a potential toxicity upon exposure to hematite nanoparticles

This study examined the effects of different-sized nanoparticles on potential cytotoxicity in intestinal epithelia. Three sizes of hematite nanoparticles were used for the study at a 10?ppm concentration: 17, 53, and, 100?nm. Results indicate that, of the hematite nanoparticles tested, 17?nm was more toxic to the epithelial integrity than 53 or 100?nm. In addition, the epithelial integrity was affected by disruption of epithelial structures such as apical microvilli, and by disruption of the cell-cell junctions leading to reduction in transepithelial electrical resistance measurements (TEER). The drop in TEER was caused by disruption of the adhering junctions not by cell death, as determined by immunocytochemistry, and by using a cell viability assay. Epithelial integrity was also affected at the molecular level as shown by differential expression of genes related to cell junction maintenance, which was assessed by microarray analysis. In conclusion, the 17- and 100-nm hematite nanoparticles caused significant structural changes in the epithelium but not the 53?nm nanoparticles. Also, different-sized hematite nanoparticles each had different effects both at the cellular level and genetic level.     

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells

Summary The growth and differentiation of an established renal epithelial cell line, LLC-PK₁, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK₁ monolayers grown on ECM-coated membranes displayed the highest transepitheliald-glucose transport (333 ± 22 ng·cm⁻²·min⁻¹) whereas cells plated on collagen-coated membranes displayed the lowest (94 ± 23 ng·cm⁻²·min⁻¹). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.     

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets

By observing increases in the transepithelial paracellular permeability of a range of radiolabeled solutes and electron dense dyes, changes in molecular sieving caused by the cytokine, TNF (tumor necrosis factor), and the phorbol ester, TPA (12-0-tetra-decanoylphorbol-13-acetate), were characterized. Using ¹⁴C-labeled mannitol (mw 182), raffinose (mw 504), PEG (polyethylene glycol; mw 4000), and dextran (mw 10,000, 70,000 and 2,000,000), the transepithelial flux rates of these compounds were determined at the peak of the transepithelial electrical resistance (TER) changes caused by these two agents. TNF treatment resulted in increased permeability across LLC-PK₁ epithelial cell sheets only to relatively small solutes, with an upper limit of approximately 4,000 mw. The low molecular weight “ceiling” for the TNF-treated epithelium is further evidence against TNF increasing transepithelial permeability by means of inducing nonspecific, microscopic “holes” in the epithelium, for which a “ceiling” would not exist. TPA treatment increases transepithelial paracellular permeability to a much broader range of solutes, extending well beyond 2 million mw. Transmission electron micrographs provide evidence that even the electron-dense dye complex, ruthenium red, can cross tight junctions of TPA-treated cell sheets. However, cationic ferritin cannot cross tight junctions of TPA-treated cell sheets. This shows that there is an upper limit to solutes able to cross TPA-treated cell sheets, but that this upper limit will include most proteins, which would then be able to cross tumor promoter-exposed (protein kinase C-activated) epithelial layers at accelerated rates. The biomedical implications for a high molecular weight cutoff in tumor promoter action in epithelial carcinogenesis, and for a low molecular weight cutoff in cytokine-induced epithelial apoptosis in inflammation, are discussed. J. Cell. Physiol. 171:226–233, 1997. © 1997 Wiley-Liss, Inc.     

Enzyme leakage due to change of membrane permeability of primary cultured rat hepatocytes treated with various hepatotoxins and its prevention by glycyrrhizin

Various hepatotoxins were added to the medium of primary cultures of adult rat hepatocytes and the release of the cytosolic enzymes lactic dehydrogenase, glutamic-oxaloacetic and glutamic-pyruvic aminotransferases were measured 24 h later. CCl₄ at low concentrations caused dose-dependent release of soluble enzymes into medium without appreciable cytolysis of the hepatocytes. Mitochondrial enzymes were not released under these conditions. At 5 mM CCl₄, both soluble and mitochondrial glutamic-oxaloacetic aminotransferase were found in the culture medium. Glycyrrhizin, a triterpenoid glycoside of licorice roots, prevented the enzyme release caused by CCl₄.Abbreviations CCl₄ carbon tetrachloride - GDH glutamic dehydrogenase - GOT glutamic-oxaloacetic aminotransferase - GPT glutamic-pyruvic aminotransferase - LDH lactic dehydrogenase     

Epithelial Barrier Resistance is Increased by the Divalent Cation Zinc in Cultured MDCKII Epithelial Monolayers

Topical zinc applications promote wound healing and epithelialization. "Leaky" MDCKII epithelia exposed to apical ZnCl? (10 mM) showed a time-dependent increase (t (0.5) 22.2 ± 2.7 min) of transepithelial resistance (R (t)) from 82.3 ± 2.4 Ω cm2 to 1,551 ± 225.6 Ω cm2; the increase was dose-dependent, being observed at 3 mM but not at 1 mM. Basal Zn2+ applications also increased epithelial resistance (at 10 mM to 323 ± 225.6 Ω cm2). The linear current-voltage relationship in control epithelia changed after apical 10 mM ZnCl? to show rectification. Voltage deflections resulting from inward currents showed time-dependent relaxation (basal potential difference (p.d.)-positive), with outward currents being time-independent. Cation selectivity was tested after apical ZnCl? elevated resistance; both the NaCl:mannitol (basal replacement) dilution p.d. and the choline:Na bi-ionic p.d. decreased (P(Na)/P(Cl) from 4.9 to 2.3 and P(Na)/P(choline) from 3.8 to 2.1, respectively). Transepithelial paracellular basal to apical ??Ca fluxes increased approximately twofold when driven by a basal positive Na:NMDG bi-ionic p.d., but with basal 10 mM ZnCl?, ??Ca fluxes decreased approximately twofold. Neither ZO-1 nor occludin distribution was altered after ~2-h exposure to apical 10 mM ZnCl?. However, claudin-2, though present at the tight junction, increased within the cell. Increased epithelial barrier resistance by Zn2+ is due to modification of the paracellular pathway, most probably by multiple mechanisms.