Transepithelial Resistance Values Research Articles

Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system.

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5-7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

Assessment of Intestinal Transcytosis of Neonatal <em>Escherichia coli</em> Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Assessment of Intestinal Transcytosis of Neonatal <em>Escherichia coli</em> Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Cells of the Caco-2 line as a model for studying the absorption of medicinal substances

Cells of the Caco-2 line have the basic properties of enterocytes of the small intestine, and therefore can be used to study the absorption of medicinal substances. Aim. To characterize the properties of the Caco-2 cell line from the Institute of Cytology of the Russian Academy of Sciences and to evaluate with its help the mechanism of absorption of the original domestic drug - ethylmethylhydroxypyridine succinate (EMGPS). Materials and methods. The study was performed on Caco2 cells that were cultured for 21 days, since at this time their spontaneous differentiation into polarized cells similar to enterocytes of the small intestine occurs. The density of the cell monolayer was estimated by the value of transepithelial resistance. The number of major efflux proteins of glycoprotein-P transporters (Pgp) and breast cancer resistance protein (BCRP) in Caco-2 cells was analyzed using enzyme immunoassay. In specialized transwell systems, the transport of the Pgp substrate fexofenadine (40, 150 and 300 microns), the BCRP substrate methotrexate (5, 10, 50 microns) and EMGPS (10, 100 and 250 microns) through the cell monolayer was studied. The results of the study. By day 21 of cultivation, cells of the Caco2 line formed a merging monolayer with pronounced dense contacts. The amount of Pgp and BCRP was 110.8±14.1 ng/mg and 4.39±0.12 ng/mg, respectively, which correlates with the amount of these proteins in the human small intestine. Transport of fexofenadine (40, 150 and 300 microns) and methotrexate (5 microns) from the basolateral chamber to the apical chamber (corresponding to transport from enterocytes to the intestinal lumen) prevailed over transport in the opposite direction, which is associated with the work of Pgp and BCRP. The transport of EMGPS significantly exceeded the transport of fexofenadine and methotrexate and was symmetrical with respect to the cellular monolayer. Conclusion. Thus, the cells of the Caco-2 line, commercially available in the Russian Federation, have the basic properties of enterocytes of the small intestine, and can be used to study the absorption of medicinal substances in vitro. EMGPS quickly passes through the cellular monolayer, and the mechanism of its absorption is passive diffusion, without the participation of specific transporters.

Caco-2 cells monolayer as an in-vitro model for probiotic strain translocation

Background: Caco-2 cells monolayer is one of invitro models to evaluate the translocation capacity of Lactobacillus spp probioticstrains. The translocation is influenced by mucosa permeability of enterocytes as shown by increasing transepithelial resistance (TER) and formation of tight junction proteins. The pore size of the supported permeable membrane used in in vitro assay was one of the crucial factors in performing bacterial translocation assay. Almost no study has been conducted using Caco-2 cells monolayer grown on 8-μm pore size polycarbonate membrane for evaluating probiotics translocation. Therefore this study aimed to determine whether the Caco-2 cells monolayer model was suitable as an in vitro translocation model.Methods: Caco-2 cells monolayer was seeded onto 8-μm collagen-coated polycarbonate membrane insert Transwell®. Differentiation of Caco-2 cells was detected by measuring the TER, while the ZO-1 protein (the tight junction proteins) was detected by immunofluorescence. H2O2 was used as a tight junction disruptive agent. Data were analyzed using SPSS version 23 software to compare the mean of TER measurement between untreated and H2O2-treated Caco-2 cells monolayer.Results: The result showed that the TER of Caco-2 cells monolayer was gradually increasing until day 14, reaching more than 800 ohm.cm2. Furthermore, the ZO-1 protein was successfully detected, indicated the tight junction formation.  TER value of H2O2-treated cells showed significantly lower than that of untreated cells (P<0.05), indicating a disturbance of cells monolayer integrity. Lactobacillus rhamnosus FBB81 was used for validating the translocation. There was no translocation observed; however, translocation was observed in H2O2-treated cells.Conclusion: Altogether suggests that Caco-2 cells grown on 8 μm-pore size permeable filters could be considered as a suitable in vitro model for probiotics strains translocation.

Stabilization of Hypoxia Inducible Factor by Cobalt Chloride Can Alter Renal Epithelial Transport

Around 600,000 people in the United States alone have polycystic kidney disease (PKD) which is characterized by the formation of fluid‐filled pouch‐like structures, called cysts. These cysts tend to expand over the years, finally leading to kidney failure. Previously, kidney cysts were reported to be hypoxic [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply can result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of Hypoxia Inducible Factor 1α (HIF1α), a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular transport in normal renal epithelia, transforming the normal absorptive epithelia to a secretory and leaky phenotype, leading to cyst expansion.Using cobalt chloride (CoCl2), HIF1α was stabilized in cellular nucleus of a mouse cortical collecting duct cell line (mCCD 1296 (d)), which resulted in an increased level of erythropoietin, an effector molecule of HIF1α. The mCCD monolayers have high transepithelial resistance (TER) value around 3000 Ω‐cm2 and around 95% amiloride sensitive voltage value. Equivalent current (Ieq) was calculated to compare active ion transport and FITC‐conjugated dextran molecules (3 and 70 kDa) were used to quantify transepithelial transport in terms of permeability values. MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same levels of metabolically active cells. Our results showed that TER values decreased significantly after 48 and 72 hours of HIF stabilization by CoCl2. The decrease of TER value was consistent with the increase in the permeability of large‐size 70 kDa FITC‐dextran molecules, supporting HIF stabilization actually altered paracellular transport. Western blot analysis showed HIF stabilization caused a significant decrease in the protein level of one of the major tight junction proteins, zonula occludin 1 (ZO1). Decrease in the ZO1 protein level was consistent with the decreased TER value and the increased paracellular permeability. HIF stabilization by CoCl2 for 48 hours caused almost complete loss of active sodium ion transport, and very interestingly, 72 hours of HIF stabilization caused a switch in the direction of active ion transport. Western blot analysis showed that HIF stabilization caused a decrease in the protein level of sodium‐potassium‐ATPase (Na+‐K+‐ATPase) α1 subunit, the catalytic subunit of the enzyme responsible for active sodium ion transport. Decreased level of Na+‐K+‐ATPase α1 subunit due to HIF stabilization was consistent with the loss of active sodium ion transport. Thus, HIF stabilization by CoCl2 can affect Na+‐K+‐ATPase α1 subunit and ZO1 protein levels, leading to alterations of transcellular and paracellular transport through normal kidney epithelial monolayers. Thus, our results indicate that localized ischemia‐hypoxia can transform a normal absorptive epithelia to a secretory and leaky epithelia and may contribute to cyst expansion.Support or Funding InformationThis research was supported by the Dr. John Vitullos Pilot and Bridge Funding Program at the Center for Gene Regulation in Health and Disease (GRHD), NIH DK092716 award (both to AR) and Cleveland State University Dissertation Research Award to SN.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Epithelial Function Can Be Altered by Hypoxia Inducible Mechanisms

Polycystic Kidney Disease (PKD) is characterized by fluid filled cysts which interfere with renal function and can lead to kidney failure. In cystic epithelia, hypoxia inducible factor 1α (HIF1α) and HIF2α were reported to be elevated [1]. Cyst expansion, stagnant fluid accumulation, and insufficient vascular supply may result in localized chronic ischemia‐hypoxia in cysts and as well as in normal epithelia adjacent to a cyst. We hypothesize that in normal epithelia near a cyst, the stabilization of HIF1α, a major regulator of cellular response to hypoxia, can cause altered paracellular and transcellular epithelial transport. Thus, increased level of HIF1α can be a triggering factor in the transformation of normal renal epithelia from an absorptive to a secretory phenotype, leading to cyst expansion.Cobalt chloride (CoCl2) was used at a concentration of 100 μM to stabilize HIF1α in the cellular nucleus. Using western blots, we confirmed that increased HIF1α levels in the nuclear extracts by CoCl2 also increased cellular erythropoietin (EPO) levels. Application of a HIF1α inhibitor, methyl 3‐[[2‐[4‐(2‐adamantyl)phenoxy]acetyl]amino]‐4‐hydroxybenzoate was found to suppress the increased level of EPO expression by CoCl2. Transcellular and paracellular transport studies were performed in a mouse cortical collecting duct cell line (mCCD 1296 (d)) which has a high monolayer transepithelial resistance (TER) value around 3000 Ω‐cm2 and a detectable 95% amiloride sensitive voltage value. A few paracellular transport studies were also carried out in Mardin‐Darby Canine Kidney (MDCK) epithelial cell line which has relatively low monolayer TER value around 400 Ω‐cm2 with no detectable voltage value. Short circuit current (Isc) was calculated using Ohm's law (I = V/R) to compare active ion transport. TER and voltage values were recorded using an Endohm chamber. Higher TER values in mCCD cells can reflect reduced paracellullar permeability which was verified by permeability assay using FITC‐conjugated dextran molecules (3 and 70 kDa). MTT assay was used in order to make sure CoCl2 treated and untreated groups maintained same level of cellular viability. Cells exposed to CoCl2 showed altered active ion transport in mCCD cells. Treatment with CoCl2 caused increased 70 kDa FITC‐dextran paracellular permeability both in mCCD and MDCK monolayers. In MDCK cell line, we performed a paracellular permeability experiment in which we used the HIF1α inhibitor along with CoCl2. The HIF1α inhibitor was found to block the effect of CoCl2 treatment on FITC‐dextran permeability to a certain extent. Thus, CoCl2 can alter epithelial transport most likely due to hypoxia inducible mechanisms. Currently, we are trying to strengthen the connection between HIF1α levels and renal epithelial function using HIF1α inhibitors in active and passive transport experiments in the presence of CoCl2 or another HIFα stabilizer, the iron chelator desferrioxamine.Support or Funding InformationThe project is supported by National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716 to AR and Cleveland State University Dissertation Research Award to SN.

Characterization of primary cultures of adult human epididymis epithelial cells

To establish cultures of epithelial cells from all regions of the human epididymis to provide reagents for molecular approaches to functional studies of this epithelium. Experimental laboratory study. University research institute. Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement. Human epididymis epithelial cells harvested from adult epididymis tissue. Establishment of a robust culture protocol for adult human epididymal epithelial cells. Cultures of caput, corpus, and cauda epithelial cells were established from epididymis tissue of seven donors. Cells were passaged up to eight times and maintained differentiation markers. They were also cryopreserved and recovered successfully. Androgen receptor, clusterin, and cysteine-rich secretory protein 1 were expressed in cultured cells, as shown by means of immunofluorescence, Western blot, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The distribution of other epididymis markers was also shown by means of qRT-PCR. Cultures developed transepithelial resistance (TER), which was androgen responsive in the caput but androgen insensitive in the corpus and cauda, where unstimulated TER values were much higher. The results demonstrate a robust in vitro culture system for differentiated epithelial cell types in the caput, corpus, and cauda of the human epididymis. These cells will be a valuable resource for molecular analysis of epididymis epithelial function, which has a pivotal role in male fertility.

Sheep rumen and omasum primary cultures and source epithelia: barrier function aligns with expression of tight junction proteins

The forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Cultured cells were characterized by transepithelial resistance (TER) measurements and patch clamping. Cells developed TER values of 729±134 Ω cm(2) (rumen) and 1522±126 Ω cm(2) (omasum). Both primary cells and source epithelia of rumen and omasum expressed cytokeratin, occludin and claudins 1, 4 and 7 (but not claudins 2, 3, 5, 8 and 10), consistent with the observed paracellular sealing properties. Staining for claudin-1 reached the stratum basale. The full mRNA coding sequence of claudins 1, 4 and 7 (sheep) was obtained. Patch-clamp analyses of isolated cells proved expression of an anion conductance with a permeability sequence of gluconate<acetate<chloride. This is in accordance with a model that ruminal and omasal transport of anions such as chloride and acetate has to occur via a transcellular route and involves channel-mediated basolateral efflux, driven by Na(+)/K(+)-ATPase.

IN VITRO THROMBIN DOSE RESPONSE ON MADIN DARBY CANINE KIDNEY CELL MONOLAYER

Epithelial cells are known to play an important role in sustaining the airway barrier that may be impaired in certain inflammatory conditions. Recently, the use of thrombin has been reported to open the airway of patients with asthma as well as enhance the permeability of endothelial cell monolayers. We designed an in vitro model of Madin Darby Canine Kidney (MDCK) cells and the physiological functions of this model were evaluated by measuring the transepithelial resistance (TER). The epithelial cytoskeletal organization was observed by staining with Bisbenzimide and Rodamine-Phalloidin (BBZ-Phalloidin) and confirmed by fluorescence microscopy. Measurements of the TER generated values up to 2020 Ω/cm2. A dose response of thrombin was observed, showing the permeability changes in the MDCK monolayer and subsequent recovery. A relationship between TER values and cytoskeletal organization was also observed and discussed.

Evaluation of oesophageal mucosa integrity by the intraluminal impedance technique

BackgroundOesophageal intraluminal impedance is currently used for assessment of reflux in gastro-oesophageal reflux disease (GORD). Oesophageal mucosa integrity may have a key role in heartburn perception in non-erosive reflux disease...

Maintenance of paracellular barrier function by insulin-like growth factor-I in submandibular gland cells

Insulin-like growth factor-I (IGF-I) is expressed in salivary glands. We examined the effects of IGF-I on cell number, the expression and distribution of tight junction proteins and the paracellular barrier function in cells derived from rat submandibular glands. When those cells were cultured in medium containing 10% foetal bovine serum (FBS) or IGF-I, the number of cells was comparable at 10 days. However, in the presence of inhibitor of IGF-I receptors, the number of cells cultured with FBS only was clearly reduced. The tight junction proteins occludin and claudin-3 were similarly detected by Western blotting in cells cultured with IGF-I or FBS. Immunostaining revealed that occludin and another tight junction protein (ZO-1) were similarly localized at intracellular junctions of cells cultured with IGF-I or FBS. The barrier functions were evaluated by transepithelial resistance (TER) and by FITC-dextran permeability. The TER values and FITC-dextran permeability of cells cultured with IGF-I or FBS were comparable. These observations suggest that IGF-I contributes to the maintenance not only of the cell number of salivary gland cells but also of their paracellular barrier function via the expression and distribution of tight junction proteins.

Human α-Defensins Inhibit Clostridium difficile Toxin B

Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Inactivation of C difficile toxins by alpha-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine K(m) and median inhibitory concentration (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K(m) values for uridine 5'-diphosphate-glucose up to 10-fold. The IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.

Study of Claudin Function by RNA Interference

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na+ permeation. Removal of claudin-2 depressed the permeation of Na+ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na+ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl- permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl- and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na+ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na+ or as paracellular channels to Cl-, depending upon the cellular background, to decrease the cation selectivity of the tight junction.

Hyperoxia-induced changes in human airway epithelial cells: The protective effect of perflubron

To determine the protective effect of perflubron (PFB), a type of perfluorochemical liquid, in hyperoxia-induced cellular injury in the human airway epithelial cells. A controlled, in vitro laboratory study. Tertiary-care children's hospital. Human airway epithelial cells. Human airway epithelial cells, Calu-3 cells, grown on polycarbonate porous filters at an air-liquid interface culture were exposed to normoxic (Fico(2) = 5%, balance air) or hyperoxic (Fio(2) = 95%, balance CO(2)) conditions. Hyperoxia-induced cellular changes were monitored by measuring transepithelial resistance (TER) of monolayers, histology of cells, total protein, and interleukin-8 (IL-8) secretion in apical surface fluid (ASF) washings. Under hyperoxic conditions, the protective effect of PFB was assessed by directly adding PFB liquid to the apical surface of monolayers. During hyperoxic gas-liquid interface culture, Calu-3 monolayers exhibited a loss of cellular integrity morphologically, decreased protein concentration, and IL-8 level in ASF washings. During hyperoxic PFB-liquid interface culture, there was an overall increase in TER value of monolayers, improved histology, decreased total protein secretion in ASF washings, and unaltered IL-8 secretion. Cytomorphologic observations of PFB-treated Calu-3 cells indicated the presence of varying numbers of differently sized intracellular vacuoles during both normoxic and hyperoxic conditions. We conclude that the air-liquid interface culture of Calu-3 may be helpful in understanding mechanisms of lung injuries caused in clinical practice, and PFB protects against hyperoxia-induced airway epithelial cell injury by promoting cellular integrity as well as cytologic modifications. PFB-liquid interface culture of Calu-3 may be a useful in vitro model for studying the cytoprotective role of liquid ventilation.

Response of developing cultured freshwater gill epithelia to gradual apical media dilution and hormone supplementation

We investigated gradual dilution of the apical medium (Leibovitz's L15 to fresh water [FW], analogous to gradual reduction in environmental salinity) and basolateral hormone support on the electrophysiological and ion-transporting properties of "developing" FW trout gill epithelia cultured on filter inserts. Epithelia were of the double-seeded type, containing both pavement cells and mitochondria-rich cells. In these experiments we were able to circumvent "symmetrical development" (typically L15 apical/L15 basolateral for 6-9 days) by commencing dilution of apical media (unchanged L15 basolateral, i.e., asymmetrical conditions) at culture-day 3, the time when transepithelial resistance (TER) and potential (TEP) would normally be increasing rapidly under symmetrical conditions. In Series 1 (without basolateral hormone support), epithelia were exposed to progressively diluted apical media (100%, 75%, 50% L15) at 24-hr intervals, thereafter cultured in 50% L15 apical media for 4 days, and then in apical FW. In Series 2, epithelia were exposed to progressively diluted apical media (100%, 75%, 50%, 25%, 12.5% L15, and FW) at 24-hr intervals with physiologically relevant doses of cortisol (500 ng ml(-1)), prolactin (50 ng ml(-1)), or cortisol + prolactin (500 ng ml(-1) + 50 ng ml(-1), respectively) added to basolateral media (100% L15). In Series 1, TER reached a plateau phase over 25 kohms cm2 under 50% L15/L15 culture conditions (after 4 days of culture) but fell to approximately 6 kohms cm2 after 24 hr in FW/L15 conditions. In Series 2, TER stabilized at 4-11 kohms cm2 depending on treatment. In general, apical media dilution during epithelial development was well tolerated. Preparations exhibited continued integrity right down to apical FW, indicated by only modest increases in net ion losses (i.e., basolateral to apical movement of ions), relatively stable TER values, and the expected changeover from positive to negative TEP in FW. Cortisol was clearly beneficial to FW adaptation, promoting greater TER, reduced unidirectional and net Na+ and Cl- flux rates, and elevated Na+, K+ -ATPase activity. Prolactin also offered some support, where its actions on TER were less than but additive to those of cortisol. There was no direct evidence that prolactin limited ion movements during gradual dilution. These in vitro studies demonstrate that "developing epithelia" were able to tolerate gradual dilution of apical media, the remarkable barrier properties of gill epithelia, and the importance of cortisol and prolactin in promoting integrity of this barrier during FW adaptation.

Hormone-regulated transepithelial Na+ transport in mammalian CCD cells requires SGK1 expression.

To study the role of serum and glucocorticoid-inducible kinase-1 (SGK1) in mammalian cells, we compared Na(+) transport rates in wild-type (WT) M1 cortical collecting duct cells with M1 populations stably expressing human full-length SGK1, NH(2)-terminal truncated (DeltaN-60) SGK1, "kinase-dead" (K127M) SGK1, and cells that have downregulated levels of SGK1 mRNA (antisense SGK1). Basal rates of transepithelial Na(+) transport were highest in full-length SGK1 populations, compared among the above populations. Dexamethasone treatment increased Na(+) transport in WT and full-length SGK1 cells 2.7- and 2-fold, respectively. Modest stimulation of Na(+) absorption was detected after dexamethasone treatment in DeltaN-60 SGK1 populations. However, DeltaN-60 SGK1 transport rates remained substantially lower than WT values. Importantly, a combination of high insulin, dexamethasone, and serum failed to significantly stimulate Na(+) transport in antisense or K127M SGK1 cells. Additionally, expression of antisense SGK1 significantly decreased transepithelial resistance values. Overall, we concluded that SGK1 is a critical component in corticosteroid-regulated Na(+) transport in mammalian cortical collecting duct cells. Furthermore, our data suggest that the NH(2) terminus of SGK1 may contain a Phox homology-like domain that may be necessary for effective Na(+) transport.

Cell–Substrate Contact: Another Factor May Influence Transepithelial Electrical Resistance of Cell Layers Cultured on Permeable Filters

Transepithelial resistance (TER) measurement has often been used to study the paracellular transport properties of epithelia grown on permeable filters, especially the barrier function of tight junctions. However, the TER value includes another source, the resistance caused by cell–substrate contact, that may give rise to a high TER value if cell–substrate separation is small. In this study we use electric cell–substrate impedance sensing (ECIS) to measure both paracellular resistance and the average cell–substrate distance of MDCK (II), HEp-2, and WI-38 VA13 cells. Comparing ECIS data with those from TER measurements of cell layers cultured on polycarbonate filters, we can obtain the approximate extra resistance resulting from cell–substrate contact for each cell type. The value of cell–substrate resistance was also estimated by two theoretical calculations that bracket the true values. Our results demonstrate that cell–substrate contact substantially influences the TER data measured using polycarbonate filters and that the extra resistance due to cell–substrate spaces depends on both cell type and filter property.

Involvement of a Heterotrimeric G Protein α Subunit in Tight Junction Biogenesis

The tight junction (TJ) of polarized epithelial cells is critical for maintaining an impermeant barrier and epithelial cell polarity. The signaling events important for TJ assembly require regulated calcium stores and protein kinase C (PKC), but the earliest signaling events in the cascade have not been well defined. We now show that Galphai2 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Galpha subunits in epithelial cells, rat Galphao, Q205Lalphao (Galphao "activated" by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy. Similar to endogenous Galphai2, Galphao-MDCK cells localize Galphao, (84% similar to Galphai2) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Galphao. In Galphao-MDCK cells, a physical association of Galphao with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Galphao-MDCK cells consistently coprecipitated Galphao. Constitutively active Q205LGalphao localized to the subapical lateral membrane similar to wild-type Galphao. To determine if constitutively activated Galpha subunits can affect TJ biogenesis, the formation of tight junctions in PC, Galphao, and Q205Lalphao-MDCK cells was followed by measurement of transepithelial resistance (TER) during the Ca2+ switch, a model widely used to study mechanisms of junctional assembly. Baseline and post Ca2+ switch TER values did not differ among the cell lines. However, constitutively activated Q205Lalphao-MDCK cells developed TER significantly faster than PC and Galphao cells in the early phase (0-4 h) (54 +/- 4 versus 23 +/- 3 (PC); 12 +/- 1 (Galphao) Omega.m2/h) and late phase (4-h peak) (117 +/- 10 versus 45 +/- 5 (PC); 66 +/- 7 (Galphao) Omega.m2/h) after Ca2+ switch. Peak TER values were significantly higher in Q205Lalphao-MDCK cells (1168 +/- 107 versus 437 +/- 37 (PC); 548 +/- 54 (Galphao) Omega.cm2). These results indicate that Galphao and Q205Lalphao expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Galphao accelerates TJ biogenesis without significantly affecting the maintenance of the TJ. Together, these results suggest an important role for heterotrimeric G proteins in TJ assembly.

A method to quantify and correct for edge leaks in Ussing chambers.

A technique is presented which allows edge leaks in Ussing chambers to be detected and quantified. It is based on the fact that in leaky chambers direct current or low frequency alternating current passes preferentially around the edge of the tissue, while high frequency alternating current shunts the cell membranes and distributes homogeneously through the entire chamber. By measuring the current density in the center of the chamber with low and high frequency alternating current and using correction factors which account for the shape of the electrical field as a function of chamber geometry and of estimated tissue resistance, edge leaks can be quantified. This technique allows correct transepithelial resistance values to be obtained from leaky measurements, and enables the question of whether a given discrepancy between cellular and transepithelial resistances reflects leaky tight junctions or a leaky chamber or both to be answered.