Transepithelial Flux Research Articles

P.3.c.039 Effects of neuroleptic treatment on oxidative stress parameters in schizophrenia patients

To clarify whether the epithelium of the human urinary tract possesses an active transepithelial sodium transport, the unidirectional sodium fluxes and the electrical parameters of the epithelium were determined on isolated pieces of renal pelvis. The epithelium was incubated in isotonic Ringer’s solution at 37.3C in Ussing-type chambers. The transepithelial potential difference was found to be 15.8 ± 3.0 mV and the transepithelial electrical resistance 760 ± 48Ω.-cm.−2. The transepithelial, unidirectional sodium fluxes were measured under short-circuit conditions. From both the unidirectional sodium influx from the luminal to the serosal side (7.01 ± 2.17) and from the efflux in the opposite direction (0.45 ± 0.07) a transepithelial sodium net flux of 6.56 ± 2.20 nmol. · minutes−1 · cm.−2 was calculated. Since there are no passive forces across the epithelium under short circuited conditions to explain the net flux, an active sodium transport has to be postulated. In all experiments, the simultaneously measured short circuit current (6.43 ± 2.18 nmol. · minutes−1·cm.−2) was not significantly different from the net sodium flux, and can therefore be taken as a measure of the net flux.

Claudin-2, a component of the tight junction, forms a paracellular water channel

Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na(+) gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na(+)-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH

TASK-2 (KCNK5 or K(2P)5.1) is a background K(+) channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH(o)) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH(i)) is independent from that under the command of pH(o). It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH(o) had normal pH(i)-dependent gating. Increasing extracellular K(+) concentration acid shifts pH(o) activity curve of TASK-2 yet did not affect pH(i) gating of TASK-2. pH(o) modulation of TASK-2 is voltage-dependent, whereas pH(i) gating was not altered by membrane potential. These results suggest that pH(o), which controls a selectivity filter external gate, and pH(i) act at different gating processes to open and close TASK-2 channels. We speculate that pH(i) regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys(245)) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH(i). We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH(i)-dependence curve of TASK-2 as expected from its lower pK(a). We conclude that intracellular pH, together with pH(o), is a critical determinant of TASK-2 activity and therefore of its physiological function.

VEGF Antagonists Decrease Barrier Function of Retinal Pigment Epithelium In Vitro: Possible Participation of Intracellular Glutathione

To investigate the influence of VEGF antagonists on the barrier function of the retinal pigment epithelium and underlying mechanisms. Porcine RPE cells were cultured on six-well membrane inserts. The cells were exposed to bevacizumab (62.5 microg/mL) or ranibizumab (25 microg/mL) for 24 hours (short term) or 9 days (long term). Transepithelial flux of FITC-dextran and intracellular levels of reduced glutathione (GSH) at normal and low-glucose conditions were investigated at different points in time. The influence of the addition of triamcinolone acetonide (TA) was investigated. The effect of GSH depletion on RPE permeability was examined using L-buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthethase inhibitor. After short-term exposure, VEGF antagonists increased the transepithelial flux of FITC-dextran significantly on day 2. Bevacizumab, but not ranibizumab, increased permeability up to 9 days. Under long-term exposure, both drugs enhanced permeability for 7 days; bevacizumab had the stronger effect. The addition of TA inhibited this increase. At the ninth day of short- and long-term exposure, bevacizumab-exposed cells, but not ranibizumab-exposed cells, exhibited a significantly lower GSH level. In the low-glucose condition, both drugs accelerated the decrease of intracellular GSH for the first 48 hours. GSH depletion increased the permeability of retinal pigment epithelium. TA had no effect on BSO-induced GSH depletion. The results suggest that bevacizumab and ranibizumab may decrease RPE barrier function, with bevacizumab exhibiting a prolonged and more profound effect. Combination with TA is thought to be beneficial because of its protective effect on stabilizing RPE junctional integrity.

The apical (hPepT1) and basolateral peptide transport systems of Caco-2 cells are regulated by AMP-activated protein kinase

The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) activation of the AMP-activated protein kinase (AMPK) on the transport of the model radiolabeled dipeptide [3H]-D-Phe-L-Gln was investigated in the human epithelial colon cancer cell line Caco-2. Uptake and transepithelial fluxes of [3H]-D-Phe-L-Gln were carried out in differentiated Caco-2 cell monolayers, and hPepT1 and glucose transporter 2 (GLUT2) protein levels were quantified by immunogold electron microscopy. AICAR treatment of Caco-2 cells significantly inhibited apical [3H]-D-Phe-L-Gln uptake, matched by a decrease in brush-border membrane hPepT1 protein but with a concomitant increase in the facilitated glucose transporter GLUT2. A restructuring of the apical brush-border membrane was seen by electron microscopy. The hPepT1-mediated transepithelial (A-to-B) peptide flux across the Caco-2 monolayers showed no significant alteration in AICAR-treated cells. The electrical resistance in the AICAR-treated monolayers was significantly higher compared with control cells. Inhibition of the sodium/hydrogen exchanger 3 (NHE3) had an additive effect to AICAR, suggesting that the AMPK effect is not via NHE3. Fluorescence measurement of intracellular pH showed no reduction in the proton gradient driving PepT1-mediated apical uptake. The reduction in apical hPepT1 protein and dipeptide uptake after AICAR treatment in Caco-2 cells demonstrates a regulatory effect of AMPK on hPepT1, along with an influence on both the microvilli and tight junction structures. The absence of an associated reduction in transepithelial peptide movement implies an additional stimulatory effect of AICAR on the basolateral peptide transport system in these cells. These results provide a link between the hPepT1 transporter and the metabolic state of this model enterocyte.

199 TNF-α Induced Expression of Enteric Neuronal Neuropeptide Y (NPY) Influences Intestinal Epithelial Barrier Functions

Introduction: The integrity of the intestinal epithelium is crucial to maintain boundaries between luminal contents and mucosal immune system. Increased epithelial permeability has been noted in 10-20% of pre-symptomatic Crohn's disease patients. We investigated if enteric neurons producing neuropeptide Y (NPY) could influence epithelial permeability and modulate TNF-α signaling. Methods: Caco2BBE epithelial cells were grown on transwell plates and NPY was added to the basolateral medium (0.1 μM). The epithelial permeability was measured by transepithelial resistance (TER) and flux of FITC-Dextran (4 Kd) at 2 and 24 h. The changes in the expression of tight junction proteins were compared in control and NPY-treated cells by real-time polymerase chain reaction (qRT-PCR) and Western blotting. The enteric neurons transfected with NPY promoter constructs (-1078 (full length), -952, -836, -769, -728, -597, -448, -278) were treated with TNF-α, and NPY promoter activity was assessed by luciferase assay. NPY expression in enteric neurons treated with TNF-α was also determined by qRT-PCR. Results: The addition of NPY increased the TER and FITC-Dextran flux across the CaCo2-BBE monolayer (p<0.05), thus increasing colonic epithelial permeability. NPY also induced a two-fold increase in the claudin-2 expression at mRNA (p< 0.01) and protein (p< 0.05) levels. NPY mRNA was up regulated by two-fold in TNF-α treated enteric neurons (p< 0.05) as seen by qRT-PCR. Luciferase assay demonstrated a significant increase in the NPY promoter activity, specifically in the regions between -728 and -836 of the NPY promoter (p< 0.05. Transcription factor (TRANSFAC) analysis showed Activator Protein-1 (AP-1) binding sites in this region of the NPY promoter, supporting the responsiveness to TNF-α. Conclusions: TNF-α upregulates NPY expression in enteric neurons; NPY in turn alters barrier functions of the colonic epithelium via modulation of leaky tight junction proteins like Claudin-2. Taken together, enteric neurons producing NPY aggravate pro-inflammatory signaling and increases epithelial permeability. Our studies demonstrate a role of NPY regulation of epithelial permeability and participation of enteric neuronal signaling in propagating inflammation during inflammatory bowel disease.

M1850 Geranylgeranylacetone Prevents Cytokine-Induced Down-Regulation of Heat Shock Protein 70 and Ameliorates Oxidative Injury of Colonic Epithelial Cells In Vitro

Background and Aims: Heat shock protein (HSP) 70 plays an important role in protection of gastrointestinal cells from a variety of stresses. Recent studies have shown that HSP70 is upregulated in inflamed intestinal mucosa in patients with inflammatory bowel diseases(IBD)(Luding, Dig Dis Sci, 1999), while other studies have shown that proinflammatory cytokines, which are highly expressed in intestinal mucosa in IBD patients, down-regulate expression of HSP 70(Sien H, Gastroenterology 2007). In the present study, we examined if proinflammatory cytokines inhibit HSP 70 expression in colonic mucosa, thereby exaggerating oxidative injury in colonic mucosa. We also examined if induction of heat shock protein 70 (HSP 70) by geranylgeranyacetone (GGA), a non-toxic HSP inducer, could protect colonic mucosa from oxidative injury In Vitro.Methods: Confluent monolayers of Caco-2 cells were incubated on membrane permeable supports. HSP 70 expression was evaluated by Western blot analysis and real time PCR. Cell viability was assessed by MTT assay. Apoptosis was evalutated by Caspase-3 assay. Integrity of the Caco-2 cell monolayers was assessed by measuring electrical transepithelial electrical resistance (TER) and transmucosal flux of FITClabeled inulin across the monolayers. Cells were incubated with or without cytokines (IFNγ and TNF-α) and/or Hydrogen peroxide (H2O2). In some experiments, the cells were preincubated with GGA. Results: 1. Treatment with either IFN-γ alone or TNF-α alone did not affect either HSP 70 expression or cell viability, and did not affect TER or FITC-labeled inulin flux in the Caco-2 cell monolayers. 2. Combination of IFN-γ (100 nM) and TNF-α (100 nM) downregulated HSP 70 expression, but did not affect cell viability or mucosal integrity. 3. In the absence of the cytokines, lower doses of H2O2 (< 10 mM) upregulated HSP 70 expression, and did not cause cell death or disruption of the mucosal integrity. 4. In the presence of the cytokine cocktail, lower doses of H2O2 failed to increase in HSP 70 expressions, enhanced cell death, and impaired mucosal integrity. 4. Pretreatment of the cells with GGA up-regulated HSP 70 expression, and prevented oxidative injury induced by H2O2, in both the absence and the presence of the cytokine cocktails. Conclusions: The present study suggest that (1)IFN-γ and TNF-α synergistically downregulate HSP 70 expression in colonic mucosa, and exaggerate colonic mucosal injury induced by oxidative stress, (2)induction of HSP70 by GGA appears to be useful in treatment and/or prevention of colonic mucosal injury in IBD patients.

M1851 Roles of Cyclooxygenase/Prostaglandin E2, Nitric Oxide Synthase/Nitric Oxide, and Enterobacteria in Pathogenesis of Ischemic Enteritis in Rats

Background and Aims: Heat shock protein (HSP) 70 plays an important role in protection of gastrointestinal cells from a variety of stresses. Recent studies have shown that HSP70 is upregulated in inflamed intestinal mucosa in patients with inflammatory bowel diseases(IBD)(Luding, Dig Dis Sci, 1999), while other studies have shown that proinflammatory cytokines, which are highly expressed in intestinal mucosa in IBD patients, down-regulate expression of HSP 70(Sien H, Gastroenterology 2007). In the present study, we examined if proinflammatory cytokines inhibit HSP 70 expression in colonic mucosa, thereby exaggerating oxidative injury in colonic mucosa. We also examined if induction of heat shock protein 70 (HSP 70) by geranylgeranyacetone (GGA), a non-toxic HSP inducer, could protect colonic mucosa from oxidative injury In Vitro.Methods: Confluent monolayers of Caco-2 cells were incubated on membrane permeable supports. HSP 70 expression was evaluated by Western blot analysis and real time PCR. Cell viability was assessed by MTT assay. Apoptosis was evalutated by Caspase-3 assay. Integrity of the Caco-2 cell monolayers was assessed by measuring electrical transepithelial electrical resistance (TER) and transmucosal flux of FITClabeled inulin across the monolayers. Cells were incubated with or without cytokines (IFNγ and TNF-α) and/or Hydrogen peroxide (H2O2). In some experiments, the cells were preincubated with GGA. Results: 1. Treatment with either IFN-γ alone or TNF-α alone did not affect either HSP 70 expression or cell viability, and did not affect TER or FITC-labeled inulin flux in the Caco-2 cell monolayers. 2. Combination of IFN-γ (100 nM) and TNF-α (100 nM) downregulated HSP 70 expression, but did not affect cell viability or mucosal integrity. 3. In the absence of the cytokines, lower doses of H2O2 (< 10 mM) upregulated HSP 70 expression, and did not cause cell death or disruption of the mucosal integrity. 4. In the presence of the cytokine cocktail, lower doses of H2O2 failed to increase in HSP 70 expressions, enhanced cell death, and impaired mucosal integrity. 4. Pretreatment of the cells with GGA up-regulated HSP 70 expression, and prevented oxidative injury induced by H2O2, in both the absence and the presence of the cytokine cocktails. Conclusions: The present study suggest that (1)IFN-γ and TNF-α synergistically downregulate HSP 70 expression in colonic mucosa, and exaggerate colonic mucosal injury induced by oxidative stress, (2)induction of HSP70 by GGA appears to be useful in treatment and/or prevention of colonic mucosal injury in IBD patients.

Transport of a Hydrophilic Paclitaxel Derivative, 7-Xylosyl-10-Deacetylpaclitaxel, by Human Intestinal Epithelial Caco-2 Cells

7-Xylosyl-10-deacetylpaclitaxel is an active compound used in traditional Chinese medicine to treat cancer. However, pharmacokinetic studies yielded low plasma concentrations of 7-xylosyl-10-deacetylpaclitaxel after its oral administration in preclinical trials. Therefore, we investigated whether the observed low oral bioavailability of this compound is due to poor absorption. We studied the transepithelial flux of 7-xylosyl-10-deacetylpaclitaxel using the human colonic cell line Caco-2 as a model and found out that its flux (at a concentration range of 0.5-20 µM) across the Caco-2 cell layer was linear with time for up to 3 hr. The apparent maximal concentration (K (M)) of the active efflux component was 93.4 µM. Verapamil (50 µM) and tetrandrine (25 µM) significantly decreased the active transport component. These data support the conclusion that rapid passive diffusion of 7-xylosyl-10-deacetylpaclitaxel through the intestinal epithelium is partially counteracted by the action of an outwardly directed efflux pump, presumably P-glycoprotein. The relatively high apparent permeability coefficient ( P(app)) for the apical to basolateral 7-xylosyl-10-deacetylpaclitaxel transport (16.3 ± 6.3 × 10 (-6) cm/s; n = 3) suggests that the drug may still be effectively absorbed in the intestinal tract.

Impact of amino acid replacements on in vitro permeation enhancement and cytotoxicity of the intestinal absorption promoter, melittin

Melittin is an amphipathic α-helical peptide known to cause the non-cell selective perturbation of cell membranes, especially erythrocytes. The well characterised interaction of the peptide with phospholipid bilayers has led to its use as a model to study lipid-peptide interactions. In recent years, melittin has emerged as a potential intestinal absorption promoter that increases paracellular marker permeability across both in vitro and in situ intestinal drug delivery models. Like many other promoters, inherent toxicity limits the drug delivery potential of melittin. The purpose of this study was to examine the effect of amino acid modifications of melittin on viability and drug permeation in human intestinal epithelial cell monolayers (Caco-2), where each structural change made to the peptide is known to reduce the cytolytic action of the peptide on cell membranes composed of zwitterionic phospholipids. Each of the 4 peptide analogues (PA) demonstrated reduced cytotoxicity in the methylthiazolyldiphenyl-tetrazolium bromide (MTT) conversion assay and lactate dehydrogenase (LDH) membrane integrity assay, which was correlated with a reduction in amphipathicity and hydrophobicity, as measured by RP-HPLC. The selected amino acid changes however, also attenuated the epithelial permeation enhancement activity of melittin, as measured by transepithelial electrical resistance (TEER) and flux of FITC-dextran-4 kDa across Caco-2 monolayers. This data suggests that the cytolytic action of melittin is responsible in part for permeation enhancement and that these effects are related to transcellular perturbation in addition to effects on tight junctions.

Effect of Different Metal Ions on the Biological Properties of Cefadroxil.

The effect of different metal ions on the intestinal transport and the antibacterial activity of cefadroxil [(6R,7R)-7-{[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino}-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid] was investigated. The [14C]Gly-Sar uptake via PEPT1 was inhibited by Zn2+ and Cu2+ treatment in a concentration-dependent manner (Ki values 107 ± 23 and 19 ± 5 µM, respectively). Kinetic analysis showed that the Kt of Gly-Sar uptake was increased 2-fold in the presence of zinc sulphate (150 µM) whereas the Vmax value were not affected suggesting that zinc ions inhibited Gly-Sar uptake by PEPT1 in a competitively manner. Ni2+ exhibited moderate inhibitory effect, whereas Co2+, Mg2+, Al3+ ions showed no inhibitory effect on Gly-Sar uptake via PEPT1. Subsequently, we examined the effect of Zn2+ and Al3+ ions on the transepithelial transport of cefadroxil across Caco-2 cells cultured on permeable supports. The results showed that zinc ions inhibited the transepithelial flux of cefadroxil at Caco-2 cell monolayers while Al3+ ions had no effect. The interaction of cephalosporins with the metal ions could suggest negative effects of some metal ions on the clinical aspects of small intestinal peptide and drug transport. Finally, the effect of Zn2+, Cu2+ and Al3+ ions on the antibacterial activity of cefadroxil was tested. It was found that there is no significant difference between the activity of cefadroxil and the cefadroxil metal ion complexes studied against the investigated sensitive bacterial species.

Transport of phenylethylamine at intestinal epithelial (Caco-2) cells: Mechanism and substrate specificity

This study was performed to characterize the intestinal transport of β-phenylethylamine (PEA). Uptake of [ 14C]PEA into Caco-2 cells was Na +-independent but strongly stimulated by an outside directed H + gradient. At extracellular pH 7.5, the concentration-dependent uptake of PEA was saturable with kinetic parameters of 2.6 mM ( K t) and 96.2 nmol/min per mg of protein ( V max). Several biogenic amines such as harmaline and N-methylphenylethylamine as well as cationic drugs such as phenelzine, tranylcypromine, d, l -amphetamine, methadone, chlorphenamine, diphenhydramine and promethazine strongly inhibited the [ 14C]PEA uptake with K i values around 1 mM. Tetraethylammonium, N-methyl-4-phenylpyridinium and choline had no effect. We also studied the bidirectional transepithelial transport of [ 14C]PEA at cell monolayers cultured on permeable filters. Net transepithelial flux of [ 14C]PEA from apical-to-basolateral side exceeded basolateral-to-apical flux 5-fold. We conclude that PEA is transported into Caco-2 cells by a highly active, saturable, H +-dependent (antiport) process. The transport characteristics do not correspond to those of the known carriers for organic cations of the SLC22, SLC44, SLC47 and other families.

Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6-8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox(2-), Cl(-), and SO(4)(2-) whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO(3)(-) exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox(2-), Cl(-), or SO(4)(2-) was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl(-)-dependent HCO(3)(-) efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC(50) approximately 5 microM) and that mucosal Cl(-) inhibits oxalate uptake with an EC(50) < 20 mM. Transepithelial Cl(-) gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl(-) gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.

Transport of Free and Peptide-Bound Pyrraline at Intestinal and Renal Epithelial Cells

Pyrraline is a quantitatively dominating glycation compound of the advanced Maillard reaction in foods and can be found in urine after consumption of pyrraline-containing food items. The purpose of this study was to investigate the transport of pyrraline and its dipeptide derivatives alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala) at intestinal and renal cell lines. Pyrraline inhibited the l-[(3)H]lysine uptake with IC(50) values of 0.3 mM (Caco-2 cells) and 3.5 mM (OK cells), respectively, but not the uptake of [(14)C]Gly-Sar (Caco-2 and SKPT cells). In contrast, Ala-Pyrr strongly inhibited the uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells with IC(50) values of 0.19 and 0.017 mM, respectively. Pyrr-Ala inhibited the carrier-mediated uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells by 50% at concentrations of 0.03 and 0.008 mM, respectively. The transepithelial flux of peptide-bound pyrraline across Caco-2 cell monolayers was up to 15-fold higher compared to the flux of free pyrraline. We conclude that free pyrraline is not a substrate for the intestinal lysine transporter and that the absorption of dietary pyrraline occurs most likely in the form of dipeptides rather than as the free amino acid.

Novel bUT-B2 urea transporter isoform is constitutively activated

Our previous studies have detailed a novel facilitative UT-B urea transporter isoform, bUT-B2. Despite the existence of mouse and human orthologs, the functional characteristics of UT-B2 remain undefined. In this report, we produced a stable MDCK cell line that expressed bUT-B2 protein and investigated the transepithelial urea flux across cultured cell monolayers. We observed a large basal urea flux that was significantly reduced by known inhibitors of facilitative urea transporters; 1,3 dimethylurea (P < 0.001, n = 17), thionicotinamide (P < 0.05, n = 11), and phloretin (P < 0.05, n = 9). Pre-exposure for 1 h to the antidiuretic hormone vasopressin had no effect on bUT-B2-mediated urea transport (NS, n = 3). Acute vasopressin exposure for up to 30 min also failed to elicit any transient response (NS, n = 9). Further investigation confirmed that bUT-B2 function was not affected by alteration of intracellular cAMP (NS, n = 4), intracellular calcium (NS, n = 3), or protein kinase activity (NS, n = 4). Finally, immunoblot data suggested a possible role for glycosylation in regulating bUT-B2 function. In conclusion, this study showed that bUT-B2-mediated transepithelial urea transport was constitutively activated and unaffected by known regulators of renal UT-A urea transporters.

Inhibition of anion fluxes in Calu‐3 cells by polystyrene nanoparticles: role of lipid rafts

IntroductionThere is growing concern about the potential toxicity of non‐engineered and engineered nanoparticles. Lungs are particularly susceptible to the injury brought about by inhalation of gases and aerosols containing nanoparticles. Aims: We investigated the effects of apical exposure (up to 60 min) of cells to polystyrene nanoparticles (PNPs) (positive, negative, uncharged; 20, 50, 100nm) on transepithelial anion fluxes.MethodsCalu‐3 cells grown on inserts were mounted into modified Ussing chambers. Anion fluxes (measured as short‐circuit current, Isc) were stimulated with forskolin (10µM) in the presence or absence of PNPs (88‐104 µg/ml). In some experiments lipid rafts were disrupted by removing cell surface cholesterol with cyclodextrin (CD).ResultsForskolin resulted in mean peak increase in Isc from 29.4 to 108.4µA/cm2. All charged PNPs caused a significant (p&lt;0.05, n.≥3) reduction of forskolin‐stimulated anion fluxes (from 24 to 40‰), an effect undetectable with uncharged particles. The CD treatment led to a significant potentiation of the inhibitory effects of 20 and 50nm PNPs. In contrast, the treatment abolished the inhibitory effects of 100nm PNPs.ConclusionOur results suggest that charge, size and interactions with lipid rafts are essential factors in determining the extent of PNPs‐mediated inhibition of forskolin‐stimulated anion fluxes in Calu‐3 cells.

Slc26a3(Down‐regulated in adenoma) Cl/HCO3 exchanger is responsible for Cl absorption and HCO3 secretion in the murine large intestine

In the large intestine a Cl/HCO3 exchanger is suggested to be involved in Cl absorption which is closely associated with Na absorption mediated by Na/H exchanger. A Cl/HCO3 exchanger is also responsible for HCO3 secretion. Molecular identity of these Cl/HCO3 exchanger(s) was investigated using mice with gene‐targeted knockout (KO) of Cl/HCO3 exchangers: Slc26a3, down‐regulated in adenoma (Dra). Isolated cecum and distal colon were mounted in Ussing chambers. We measured transepithelial fluxes of 22Na and 36Cl, and by using a pH‐stat device a luminal alkalinization rate. Essentially similar findings were obtained for both the cecum and distal colon. In control (ddy strain) and wild type mice, mucosa‐to‐serosa flux (Jms) was larger than serosa‐to‐mucosa flux (Jsm) for both 22Na and 36Cl. In contrast, in DraKO mice Jms and Jsm for 36Cl were both markedly reduced and net 36Cl absorption was almost, if not entirely, abolished, while Jms and Jsm for 22Na were not noticeably affected. Cl‐dependent HCO3 secretion was completely abolished in DraKO mice. These results suggest that, under Ussing chamber condition, Dra is a major transporter responsible for both the absorptive and secretory transport of Cl, and for most of the net Cl absorption. Dra is also involved in Cl‐dependent HCO3 secretion.

Transepithelial fluxes of adenosine and 2′-deoxyadenosine across human renal proximal tubule cells: roles of nucleoside transporters hENT1, hENT2, and hCNT3

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2'-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities (11). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine (9-beta-d-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2'-deoxyadenosine), and clofarabine (2-chloro-2'-fluoro-deoxy-9-beta-d-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral hENT2, whereas transepithelial fluxes of 2'-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2'-deoxyadenosine.

Acute regulation of tight junction ion selectivity in human airway epithelia

Electrolyte transport through and between airway epithelial cells controls the quantity and composition of the overlying liquid. Many studies have shown acute regulation of transcellular ion transport in airway epithelia. However, whether ion transport through tight junctions can also be acutely regulated is poorly understood both in airway and other epithelia. To investigate the paracellular pathway, we used primary cultures of differentiated human airway epithelia and assessed expression of claudins, the primary determinants of paracellular permeability, and measured transepithelial electrical properties, ion fluxes, and La(3+) movement. Like many other tissues, airway epithelia expressed multiple claudins. Moreover, different cell types in the epithelium expressed the same pattern of claudins. To evaluate tight junction regulation, we examined the response to histamine, an acute regulator of airway function. Histamine stimulated a rapid and transient increase in the paracellular Na(+) conductance, with a smaller increase in Cl(-) conductance. The increase was mediated by histamine H(1) receptors and depended on an increase in intracellular Ca(2+) concentration. These results suggest that ion flow through the paracellular pathway can be acutely regulated. Such regulation could facilitate coupling of the passive flow of counter ions to active transcellular transport, thereby controlling net transepithelial salt and water transport.

NH 4+ secretion in the avian colon. An actively regulated barrier to ammonium permeation of the colon mucosa

Experiments were designed to characterize an active, electrogenic transport of NH 4 + ions across the colonic epithelium of the domestic fowl ( Gallus gallus). Colonic segments were isolated and stripped of underlying muscle. The mucosal epithelia were mounted in Ussing chambers and voltage-clamped to measure the short-circuit currents ( I SC) associated with transport. Bilateral addition of NH 4 + caused a dose-dependent outward current (negative I SC), with a Km of 34 ± 8 mM and a maximal current response of 311 ± 47 µA cm − 2 (12 ± 2 µEq cm − 2 h − 1 ). A similar effect was seen with unilateral addition of NH 4 + to the serosal (s) side, but not with mucosal (m) addition. Pre-treatment with 10 − 4 M amiloride exposed a net outward (negative) I SC, and serosal NH 4 + addition further increased this outward current with a Km of 53 ± 24 mM. Decreasing the bath pH from 7.3 to 6.0 did not affect the I SC response to NH 4 +. Unidirectional NH 4 + flux measurements revealed a net secretory flux (8.8 ± 3.1 µmol cm − 2 h − 1 s–m, versus 2.6 ± 1.4 µmol cm − 2 h − 1 m–s). Furthermore, the secretory flux closely matched the resulting change in I SC with serosal NH 4 +, showing that the transepithelial flux of NH 4 + could account for the outward current response. Addition of 50 nM bafilomycin A to the mucosal solution completely eliminated serosal to mucosal NH 4 + transport, implicating an apical V-type H +-ATPase in this transport process. The I SC response to NH 4 + was partially inhibited by ouabain, a blocker of the Na +/K +-ATPase, but only minimally affected by bumetanide, an inhibitor of the serosal Na +–K +–2Cl − cotransporter. Active NH 4 + extrusion across the mucosal membrane, combined with low permeability to NH 3 in this tissue, allow for maintenance of steep ammonia gradients across the colonic epithelium and protection from ammonia toxicity. Furthermore, these studies indicate that the hen colon may be a useful new model system for the study of NH 4 + transport.