Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells.

When rotavirus infects the mature villus tip cells of the small intestine, it encounters a highly polarized epithelium. In order to understand this virus-cell interaction more completely, we utilized a cell culture-adapted rhesus rotavirus (RRV) to infect human intestinal (Caco-2) and Madin-Darby canine kidney (MDCK-1) polarized epithelial cells grown on a permeable support. Filter-grown Caco-2 cells and MDCK-1 cells, producing a transepithelial resistance of 300 to 500 and greater than 1,000 omega . cm2, respectively, were infected from either the apical or basolateral domain with RRV or Semliki Forest virus. Whereas Semliki Forest virus infection only occurred when input virions had access to the basolateral domain of MDCK-1 or Caco-2 cells, RRV infected MDCK-1 and Caco-2 monolayers in a symmetric manner. The effect of rotavirus infection on monolayer permeability was analyzed by measuring the transepithelial electrical resistance. Rotavirus infection on filter-grown Caco-2 cells caused a transmembrane leak at 18 h postinfection, before the development of the cytopathic effect (CPE) and extensive virus release. Electrical resistance was completely abolished between 24 and 36 h postinfection. Although no CPE could be detected on RRV-infected MDCK cells, the infection caused a transmembrane leak that totally abolished the electrical resistance at 18 to 24 h postinfection. Cell viability and the CPE analysis together with immunohistochemistry and immunofluorescence data indicated that the abolishment of resistance across the monolayer was due not to an effect on the plasma membrane of the cells but to an effect on the paracellular pathway limited by tight junctions. Attachment and penetration of rotavirus onto Caco-2 cells caused no measurable transmembrane leak during the first hour of infection.     

Basolateral entry and release of Crimean-Congo hemorrhagic fever virus in polarized MDCK-1 cells

Crimean-Congo hemorrhagic fever virus (CCHFV) is an etiological agent of a disease with mortality rates in patients averaging 30%. The disease is characterized by fever, myalgia, and hemorrhage. Mechanisms underlying the hemorrhage have to our knowledge not been elucidated for CCHFV. Possibly, a direct or indirect viral effect on tight junctions (TJ) could cause the hemorrhage observed in patients, as TJ play a crucial role in vascular homeostasis and can cause leakage upon deregulation. Moreover, there is no knowledge regarding the site of entry and release of CCHFV in polarized epithelial cells. Such cells represent a barrier to virus dissemination within the host, and as a site of viral entry and release, they could play a key role in further spread. For the first time, we have shown preferential basolateral entry of CCHFV in Madin-Darby canine kidney 1 (MDCK-1) epithelial cells. Furthermore, we demonstrated basolateral release of CCHFV in polarized epithelial cells. Interestingly, by measuring transepithelial electrical resistance, we found no effect of CCHFV replication on the function of TJ in this study. Neither did we observe any difference in the localization of the TJ proteins ZO-1 and occludin in CCHFV-infected cells compared to mock-infected cells.     

Induction of the IFN-gamma gene and protein is linked to the establishment of cell polarity in a porcine epithelial cell line

We report here original properties of a porcine trophectoderm cell line, TBA B4-3, that developed a polarized phenotype with high transepithelial electrical resistance (TER) values and functional tight junctions (TJs) when grown on a microporous membrane. We found that treatment of polarized TBA B4-3 cells with a strong protein kinase C (PKC) agonist, phorbol 12-myristate-13-acetate (PMA), induced 3-4 days later a transient interferon-gamma (IFN-gamma) mRNA expression and vectorial IFN-gamma protein secretion toward the apical side of the monolayer. Exposure of TBA B4-3 cells to PMA first resulted in a rapid and profound disorganization of the monolayer structure mainly characterized by the appearance of multilayered polyp-like foci structures, a strong decrease of the TER, and a increase of permeability correlated with changes in the organization and localization of the TJ-associated proteins (ZO-1 and occludin) and filamentous actin (f-actin). After PMA removal, spontaneous return to the initial polarized monolayer state occurred, characterized by TER rising to prestimulation values, TJ protein relocalization, and multilayered cell structures fading. This return was strictly correlated with transient IFN-gamma gene induction. Our report represents the first example of an inducible expression of IFN-gamma by a polarized epithelial cell. After PMA treatment, the close correlation between establishment of cell polarity and IFN-gamma gene expression suggests a link between these phenomena. This also suggests a novel biological mechanism by which transient and reversible disorganization of a polarized monolayer of epithelial cells could trigger regulated expression of a cytokine gene by these cells.     

Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells

The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellular distribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.     

Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4

Birds are uricotelic and, like humans, maintain high plasma urate concentrations (approximately 300 microM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.     

The virotoxin model of HIV-1 enteropathy: Involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line

BACKGROUND: Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS: We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION: We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.     

Mammalian lin-7 stabilizes polarity protein complexes

Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have a role in polarity determination in epithelial cells. In this study we have found that loss of Lin-7 protein from the polarized epithelial cell line Madin-Darby canine kidney II by small hairpin RNA results in defects in tight junction formation as indicated by lowered transepithelial electrical resistance and mislocalization of the tight junction protein ZO-1 after calcium switch. The knock down of Lin-7 also resulted in the loss of expression of several Lin-7 binding partners, including PALS1 and the polarity protein PATJ. The effects of Lin-7 knock down were rescued by the exogenous expression of murine Lin-7 constructs that contained the L27 domain, but not the PDZ domain alone. Furthermore, exogenously expressed PALS1, but not other Lin-7 binding partners, also rescued the effects of Lin-7 knock down, including the restoration of PATJ protein in rescued cell lines. Finally, the effects of Lin-7 knock down appeared to be due to instability of PALS1 protein in the absence of Lin-7, as indicated by an increased rate of PALS1 protein degradation. Taken together, these results indicate that Lin-7 functions in tight junction formation by stabilizing its membrane-associated guanylate kinase binding partner PALS1.     

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.     

Effects of iron on rainbow trout gill cells in primary culture

This study investigated the effects of iron in the form of iron sulphate (FeSO₄·7H₂O), over the range 0.01–1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO₄ did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO₄ reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron.     

Cadherin-related protein 24 induces morphological changes and partial cell polarization by facilitating direct cell-cell interactions

Cadherin-related protein 24 (CDHR24) is a potential tumor suppressor located apically as well as laterally in polarized cells. Here, the role of CDHR24 in contributing to cell morphology and polarity is examined. CDHR24 was predominantly localized at the nonattached part of nonpolarizing cells while another apically sorted protein, aminopeptidase N, was equally distributed over the plasma membrane. Furthermore, CDHR24 expression induced cell aggregation capacity, indicating direct cell-cell interaction. The transepithelial resistance, however, was elevated in polarized MDCK cells, but not in nonpolarizing CHO cells. Our data propose a model in which CDHR24 is directly involved in cell and tissue morphogenesis.     

The avian influenza virus NS1 ESEV PDZ binding motif associates with Dlg1 and Scribble to disrupt cellular tight junctions

The influenza A virus NS1 protein contains a conserved 4-amino-acid-residue PDZ-ligand binding motif (PBM) at the carboxyl terminus that can function as a virulence determinant by targeting cellular PDZ proteins. The NS1 proteins from avian and human viral isolates have consensus PBM sequences ESEV and RSKV, respectively. Currently circulating highly pathogenic H5N1 viruses contain the ESEV PBM which specifically associates with the PDZ proteins Scribble, Dlg1, MAGI-1, MAGI-2, and MAGI-3. In this study, we found NS1 proteins from viral isolates that contain the PBM sequence RSKV, KSEV, or EPEV are unable to associate with these PDZ proteins. Other results showed that the ESEV PBM mediates an indirect association with PDZ protein, Lin7C, via an interaction with Dlg1. Infection with a virus that expresses a NS1 protein with the ESEV PBM results in colocalization of NS1, Scribble, and Dlg1 within perinuclear puncta and mislocalization of plasma membrane-associated Lin7C to the cytoplasm. Infection of polarized MDCK cells with the ESEV virus additionally results in functional disruption of the tight junction (TJ) as measured by altered localization of TJ markers ZO-1 and Occludin, decreased transepithelial electrical resistance, and increased fluorescein isothiocyanate (FITC)-inulin diffusion across the polarized cell monolayer. A similar effect on the TJ was observed in MDCK cells depleted for either Scribble or Dlg1 by small interfering RNA (siRNA). These findings indicate that ESEV PBM-mediated binding of NS1 to Scribble and Dlg1 functions to disrupt the cellular TJ and that this effect likely contributes to the severe disease associated with highly pathogenic H5N1 influenza A viruses.     

Up-regulation of ZO-1 expression and barrier function in cultured human corneal epithelial cells by substance P

The effects of the sensory neurotransmitter substance P on the expression of tight junction proteins and on barrier function in human corneal epithelial cells were investigated. The expression of ZO-1, but not that of occludin or claudin-1, was increased by substance P in a concentration- and time-dependent manner. This effect was inhibited by the NK-1 receptor antagonist GR82334 and by KN62, an inhibitor of Ca²⁺- and calmodulin-dependent protein kinase II. Substance P also increased the transepithelial electrical resistance of a cell monolayer in a manner sensitive to GR82334. Substance P may therefore play a role in maintenance of tight junctions in the corneal epithelium.     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.     

Glycoprotein E of varicella-zoster virus enhances cell-cell contact in polarized epithelial cells

Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca(2+) binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts.     

Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line

Sj?gren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.     

Tauroursodeoxycholic acid reduces damaging effects of taurodeoxycholic acid on fundus gastric mucosa

We investigated the effects of tauroursodeoxycholic acid (TUDCA) to assess whether this acid may also have "protective" effects similar to those found with ursodeoxycholic acid (UDCA). We used a well-known amphibian model of gastric mucosa, and studied the effects of taurodeoxycholic acid (TDCA) on electrical transepithelial parameters, acid secretion and histology in absence or in presence of TUDCA. Mucosal exposure to TDCA, after stimulation with histamine, caused a reduction in transepithelial potential difference (V(t)) and transepithelial resistance (R(t)) and a decrease in acid secretion while mucosal exposure to TUDCA did not cause a significant change in the electrical parameters. Moreover, TDCA primarily affected the neck cells, while TUDCA affected only oxyntic cells, causing a similar degree of injury to that observed in controls. Mucosal exposure to TUDCA plus TDCA caused a reduction in short circuit current (I(sc)) and R(t), whereas acid secretion did not change. These results suggest that: (1) TUDCA reduces the damaging effects of TDCA on fundus gastric mucosa; (2) TUDCA may play an important role in the treatment of gastritis associated with bile reflux.     

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1 -expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resitance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition. © 1995 Wiley-Liss, Inc.     

Nuclear localization of cytoplasmic poly(A)-binding protein upon rotavirus infection involves the interaction of NSP3 with eIF4G and RoXaN

Rotavirus nonstructural protein NSP3 interacts specifically with the 3′ end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.     

H+-peptide cotransport in the human bile duct epithelium cell line SK-ChA-1

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant (Kt) value of 1.1 mM. Several dipeptides, cefadroxil, and delta-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 +/- 29 omega x cm(2). The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.