Transepithelial Flux Research Articles

In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph

The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4 1-6, due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4 2-8, due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4 1-6 and cydiastatin 4 2-8. Similarly, 1–10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.

MULTISPECIES PROBIOTICS ABOLISH ACUTE PANCREATITIS-ASSOCIATED MUCOSAL BARRIER FAILURE IN MURINE ILEUM

Intestinal mucosal barrier failure during acute pancreatitis is associated with translocation of luminal bacteria to extra-intestinal sites. Resulting infectious complications are a major cause of morbidity and mortality. Aim of this study was to determine if multispecies probiotics can diminish mucosal barrier failure in the course of experimental acute pancreatitis (AP). Mice were treated daily with a mixture of six probiotic strains (Ecologic 641®, Winclove BioIndustries, Amsterdam, The Netherlands, 2.5×109 bacteria/oral gavage) or placebo, from 2 days prior to cerulein induced AP (hourly intraperitoneal injections, 50μg/kg, for 10 hours) until termination 72 hours after the first injection. Sham procedure consisted of 10 hourly sterile saline injections, without probiotic or placebo treatment. Mucosal barrier function of the distal ileum was assessed by measurement of mucosal integrity (electrical resistance, R: Ω.cm2) and permeability (steady state transepithelial flux of 0.01 gr/l Na-fluorescin, flux: ng/cm2/h) at 36°C, in Ussing chambers. AP reduced mucosal integrity and increased permeability (AP (n = 13) vs. sham (n = 15): R = 28.1 ± 1.4 Ω.cm2 (mean ± SEM) vs. 37,5 ± 1.7, P = 0.002; flux = 400 ± 48 ng/cm2/h vs. 189 ± 18, P = 0.001, ANOVA). Probiotic treatment completely abolished the AP-induced mucosal barrier failure (placebo vs. probiotics (n = 15): R = 26.4 ± 2.6 vs. 34.9 ± 1.8, P = 0.003; flux = 427 ± 62 vs. 246 ± 32, P = 0.014). Mucosal resistance and permeability of probiotic treated mice were not different from those of healthy controls or sham (P = 0.551 and P = 0.194, respectively). Conclusions: Experimental acute pancreatitis results in decreased integrity and increased permeability of ileal mucosa. This mucosal barrier failure can be abolished by the used regime of selected multispecies probiotics. Support of mucosal barrier function by multispecies probiotics may provide a novel strategy to reduce infectious complications during acute pancreatitis.

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P‐glycoprotein substrates in Malpighian tubules of Drosophila melanogaster

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.

In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

BackgroundCeliac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity.Methods and FindingsIn our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [3H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines.ConclusionsOur findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4.

Culture of Calu-3 Cells at the Air Interface Provides a Representative Model of the Airway Epithelial Barrier

The aim of this study was to compare the effect of liquid-covered culture (LCC) and air-interfaced culture (AIC) on Calu-3 cell layer morphology and permeability, thus assessing the fitness of these culture systems as models of airway epithelium barrier function. Cell layers were grown on 0.33 cm2 Transwell polyester cell culture supports. Cell layers grown using LCC and AIC were evaluated by using light and electron microscopy, transepithelial electrical resistance (TER), and permeability to the transepithelial flux of fluorescein sodium (flu-Na), and by varying molecular weight dextrans labeled with fluorescein isothiocyanate (FITC-dex). The tight junction protein, zona occludens protein-1 (ZO-1), was visualized by confocal microscopy and apical glycoprotein secretions were identified by using alcian blue. Cells grown via AIC produced a more columnar epithelium with a more rugged apical topography and greater glycoprotein secretion compared to cells grown via LCC. Apical protrusions appearing to be cilia-like structures were observed on occasional cells using AIC, but typical airway ciliated cell phenotypes were not produced under either condition. Secretory granules were observed in cells cultured under both conditions. Cells cultured using LCC exhibited higher levels of ZO-1 protein than the AIC counterpart. The maximal TER of cells using LCC, 1,086 +/- 113 ohms cm2 at 11-16 days, was significantly greater than the TER of cells cultured using AIC, 306 +/- 53 ohms cm2 at 11-13 days. Apparent permeability (P(app)) values for the transport of flu-Na using LCC and AIC were 1.48 +/- 0.19x10(-7) and 3.36 +/- 0.47x10(-7) cm s(-1), respectively. Transport rates of flu-Na and FITC-dex were inversely proportional to molecular weight, and were significantly lower (p < 0.05) in cell layers grown using LCC than AIC. Renkin analysis fitted the data to single pore populations of radii 7.7 and 11.0 nm for LCC and AIC, respectively. Distinct differences in morphology and permeability result when Calu-3 cells are grown using AIC or LCC. Cells cultured using AIC generate a model more morphologically representative of the airway epithelium than cells cultured using LCC.

Transepithelial flux of early and advanced glycation compounds across Caco-2 cell monolayers and their interaction with intestinal amino acid and peptide transport systems

Maillard products arise from condensation reactions between amino acids or proteins with reducing sugars during food processing. As ubiquitous components of human food, these early or advanced glycation products may be subject to intestinal absorption. The present study was performed to investigate the intestinal uptake of Maillard products and to determine whether they are substrates for peptide and amino acid transporters expressed at the apical membrane of Caco-2 cells. At a concentration of 10 mM, N(epsilon)-(carboxymethyl)-L-lysine, N(alpha)-hippuryl-N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine, N(alpha)-hippuryl-N(epsilon)-(carboxymethyl)-L-lysine and N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine inhibited the [(14)C]glycylsarcosine uptake mediated by the H(+)-peptide co-transporter PEPT1 by 13 to 45%. For N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine, an inhibitory constant of 8.7 mM was determined, reflecting a low affinity to PEPT1 in comparison with natural dipeptides. Uptake of L-[(3)H]lysine was weakly affected by N(epsilon)-(carboxymethyl)-L-lysine, N(alpha)-hippuryl-L-lysine and N(alpha)-hippuryl-N(epsilon)-(carboxymethyl)-L-lysine but strongly inhibited by N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine (81%). None of the Maillard products was able to inhibit the uptake of L-[(3)H]leucine by more than 15%. We also studied the transepithelial flux of Maillard products across Caco-2 cell monolayers cultured on permeable filters. The flux rates of Maillard products ranged from 0.01 to 0.3%/cm(2) per h and were shown to be much lower than those of carrier substrates such as glycylsarcosine, L-proline and the space marker [(14)C]mannitol. We conclude that the Maillard products investigated in the present study are neither transported by PEPT1 nor by carriers for neutral amino acids. The low transepithelial flux measured for these compounds most probably occurs by simple diffusion.

Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells

Transepithelial [(14)C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol.cm(-2).min(-1) in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within approximately 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30-40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V(2) receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to approximately 5 nmol.cm(-2).min(-1). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 microM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 microM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A.

Ubiquitination regulates urea transporter UT‐A1 cell membrane expression

The ubiquitin (Ub)-proteasome pathway, which selectively degrades intracellular proteins, plays an important role in diverse cellular functions. To identify candidate UT-A1 binding proteins, we used a 48-aa fragment of the intracellular C-terminus of UT-A1 as bait in a yeast two-hybrid screen. One positive clone encoded ubiquitin specific protease 24 (USP24), suggesting that ubiquitination may serve as a regulatory marker for degradation. To test this hypothesis, we performed a Ub pull-down assay and demonstrated ubiquitinated UT-A1 in UT-A1-MDCK cells. Inhibition of the Ub-proteasome pathway by MG132 dose and time-dependently increased total UT-A1 protein abundance. Next, we treated UT-A1-MDCK cells with 100 μg/ml cycloheximide (CHX) to block protein synthesis and measured UT-A1 degradation. The UT-A1 half-life is about 10–12 hours. When UT-A1-MDCK cells were treated with CHX and 10 μM MG132, the half-life of UT-A1 increased. Both cell surface biotinylation and imunostaining showed that cells treated with MG132 increased UT-A1 cell surface expression. Transepithelial urea flux experiments demonstrated that the cells with increased cell membrane UT-A1 by MG132 treatment have significantly increased urea transport activity. Transfection of UT-A1-MDCK cells with exogenous Ub (pCW7) greatly reduced UT-A1 abundance in the cell surface. In contrast, lysosomal pathway inhibitors (10 mM methylamine or 50 μg/ml chloroquine) did not affect UT-A1 abundance. Our studies demonstrate, for the first time, that UT-A1 is a ubiquitinated protein and is degraded by the proteasome pathway. These findings suggest that ubiquitination plays an important role in the regulation of UT-A1 bioactivity.

Characterisation of creatinine secretion across monolayers of mouse proximal tubule cells

The Renal clearance of creatinine is dependent upon the complex interplay between transport proteins at the basolateral and apical membranes of proximal tubule cells which act in concert to form an effective secretory mechanism. The role of OCT2 in the tubular uptake of creatinine is well established, but there is almost no information on the identity of the transporters involved in the apical exit step for creatinine secretion into the urine. The aim of this investigation was use primary cultures of mouse proximal tubule cell monolayers to identify the importance of individual transporters to the renal secretion of creatinine. To do this, steady state transepithelial fluxes of [14C]creatinine and [3H] mannitol were measured in cell monolayers grown on permeable filter supports. Results are expressed as mean ± SEM. For creatinine, the basolateral to apical flux (Jba) was ~2 fold greater the apical to baolateral flux (Jab) resulting in a net secretion (Jnet) of creatinine across the monolayer (1.2±0.35 nmole/cm2/hr, P<0.001). Creatinine fluxes were inhibited by a range of organic cations and decynium-22 at the basolateral membrane. Importantly the net secretion of creatinine was abolished in the presence of either verapamil or vinblastine (10μM) at the apical membrane face suggesting that the apical efflux of creatinine was mediated by MDR1 and/or MRP2. These data suggest that primary cultures of mouse tubule cells will prove an important model with which to gain understanding of creatinine secretion in an intact system.

Plasma membrane depolarization: a possible coupling factor between epithelial transcellular and paracellular transport

The coordinated transport through transcellular and paracellular pathways ensures efficient transepithelial flux of water and solutes. Despite the fact that the coupling between these processes is of vital importance, their link is not well understood. To gain insight into the mechanism we asked whether 1) depolarization, induced by apical electrogenic transport processes, can serve as a signal coupling transcellular and paracellular transport; 2) increased cell contractility might play a role in this process. Paracellular permeability across confluent monolayer of proximal tubular (LLC-PK1) cells was followed as apical-to-basolateral transport of 4 kDa FITC-dextran. We show that depolarization, elicited either by high extracellular [K+], or by glucose or alanine, substrates of apical electrogenic transporters, substantially increased paracellular permeability. The effect of glucose required Na+-dependent cotransport, since 2-deoxy-glucose, which is not a substrate of the Na+/glucose cotransporter, failed to increase paracellular permeability. Importantly, depolarization was accompanied with Rho activation and Rho kinase (ROK)-dependent MLC phosphorylation. Inhibitors of ROK and myosin ATPase (Y27632, and blebbistatin) partially prevented the depolarization-induced rise in paracellular permeability, suggesting that the Rho-ROK-MLC pathway and the consequent increase in contractility contribute to the effect. Thus, plasma membrane depolarization is a good candidate as a coupling signal between apical and paracellular transport. Supported by Canadian Institute of Health Research.

C-type natriuretic peptide and its receptor are downregulated in pulmonary epithelium following birth

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family and acts through the membrane bound guanylyl cyclase linked natriuretic peptide receptor B (NPR-B) to increase intracellular cGMP. Activation of the CNP/NPR-B pathway in pulmonary epithelium has been linked to the inhibition of amiloride-sensitive sodium absorption and to the stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR). Given the importance of ion movement across the pulmonary epithelium of the fetal and newborn lung, we sought to examine the expression of CNP and NPR-B in pulmonary epithelium of the developing fetal lamb and following the transition to air breathing. Lambs were sacrificed at 100 and 136 days of gestation and at 3 days, and 4 weeks after full term delivery. Lung sections were immunostained for CNP and NPR-B. At 100 days of gestation, staining for CNP and NPR-B was absent within all pulmonary epithelium. At 136 days of gestation, prominent staining for both CNP and NPR-B was seen within alveolar type II cells, non-ciliated cells of the distal airways (Clara cells), and ciliated epithelium of the upper airways. At both 3 days and 4 weeks following birth, staining for CNP and NPR-B was absent in alveolar type II cells, ciliated bronchial epithelium and was markedly reduced in Clara cells. The presence of CNP and NPR-B within the pulmonary epithelium in the nearterm fetal period and its rapid downregulation following birth suggests that CNP may contribute to the maintenance of the fluid-filled lung through the regulation of trans-epithelial ion flux.

Pigment epithelium-derived factor protects retinal pigment epithelium from oxidant-mediated barrier dysfunction

Retinal pigment epithelium (RPE) cells form a monolayer at the blood–retina barrier between the retina and choriocapillaries. The barrier function may be damaged by multiple stresses to the cell, including the repeated exposure to oxidants that are generated by photoreceptor cell turnover. The purpose of our study was to document the protective effect of pigment epithelium-derived factor (PEDF), a tropic factor produced by the RPE, on H 2O 2-induced RPE barrier dysfunction. When assayed by a FITC-labeled dextran transepithelial flux, the increased permeability of the RPE barrier (induced by H 2O 2) was prevented by PEDF pretreatment. To further explore the mechanism leading to this permeability change, we investigated the distribution of cytoskeleton and junctional proteins. The redistribution of the two junctional proteins occludin, and N-cadherin and actin reorganization in RPE, induced by H 2O 2, can be prevented by PEDF pretreatment. PEDF can also prevent H 2O 2-induced stress kinase p38/27-kDa heat shock protein signaling which is known to mediate actin rearrangement. These findings indicated that PEDF can stabilize actin, maintain normal membrane occludin and N-cadherin structure, and preserve the barrier function of RPE cells against oxidative stress.

Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets

Transport of fluorescent substrates of p-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) by insect Malpighian tubules was examined using confocal laser scanning microscopy (CLSM). Isolated tubules of the cricket Teleogryllus commodus accumulated the MRP2 substrate Texas Red in the cells and lumen at concentrations up to 20 and 40 times, respectively, those in the bathing medium. Quantitative CLSM analysis of fluorochrome transport in some cricket tubules and all Drosophila tubules was not practical because of interfering effects of concretions in the cells and lumen. Samples of fluid secreted by tubules set up in Ramsay assays were therefore collected in hollow rectangle glass capillaries. Transepithelial dye flux was calculated as the product of fluid secretion rate (measured in the Ramsay assay) and dye concentration (measured by CLSM of the fluid samples). Dose-response curves for transport and the ratio of dye concentration in the secreted fluid to that in the bathing medium (S/M) were determined for Texas Red as well as for P-gp substrates (rhodamine 123, daunorubicin), the organic anion fluorescein and the organic cation quinacrine. Transepithelial transport of Texas Red was reduced by the MRP2 inhibitors MK571 and probenecid. Transport of daunorubicin was reduced by the P-gp inhibitors verapamil and quinacrine and also by the organic cation tetraethylammonium. The results indicate the presence of P-gp-like and MRP2-like transporters in the Malpighian tubules of both species.

Eosinophils alter colonic epithelial barrier function: role for major basic protein

Mucosal eosinophils increase in a number of gastrointestinal diseases that are often associated with altered epithelial barrier function, including food allergic enteropathies and inflammatory bowel diseases. Although eosinophils are known to secrete biologically active mediators including granule proteins, their role in gastrointestinal diseases is uncertain. The aim of this study was to determine the impact of eosinophils on intestinal barrier function. Epithelial barrier function was determined in a coculture of eosinophils and T84 epithelial cells and in a murine model of T helper (Th) type 2-mediated colitis. Coculture conditions resulted in decreased transepithelial resistance (TER) and increased transepithelial flux. Cell-free coculture supernatants contained a > or =5-kDa soluble factor that also diminished TER; these supernatants contained the eosinophil-granule proteins major basic protein (MBP) and eosinophil-derived neurotoxin (EDN). T84 barrier function decreased significantly when basolateral surfaces were exposed to native human MBP but not EDN. Additional studies identified downregulation of the tight junctional molecule occludin as at least one mechanism for MBP action. MBP-null mice were protected from inflammation associated with oxazolone colitis compared with wild-type mice. In conclusion, MBP decreases epithelial barrier function and in this manner contributes to the pathogenesis of inflammatory bowel diseases.

Interferon-γ selectively increases epithelial permeability to large molecules by activating different populations of paracellular pores

Impairment of the gut epithelial barrier by agents such as IFNgamma may play a key role in the pathogenesis of inflammatory disorders by increasing the paracellular penetration of luminal macromolecules, potentially including bacterial antigens. Owing to limitations of current paracellular probes, little is known about the precise functional changes induced by IFNgamma and how these relate to the development of increased macromolecular permeability. Here we investigate how IFNgamma modulates this pathway in T84 monolayers using a novel profiling technique that resolves different populations of paracellular pores by simultaneous analysis of 24 permeability probes of defined molecular size. Two types of functional pore present in control monolayers, an abundant restrictive pore with a radius of approximately 4.5 Angstrom and a much larger but infrequent, non-restrictive pore, were differentially regulated by IFNgamma. Incubation with IFNgamma dose-dependently and reversibly increased the frequency of the non-restrictive pores while having no significant effect on the restrictive component. Cytokine-induced increases in beta, the descriptor of the non-restrictive pore, correlated closely with increased permeability to large molecules (10 kDa) including E. coli-derived lipopolysaccharide, but not small (0.182 kDa) molecules. This effect was associated with changes in expression of the tight junction proteins occludin and claudin-1. These data suggest that IFNgamma selectively increases the transepithelial flux of large molecules by activating specific pathways within the junctional pore. One hypothesis is that this process may be activated in the early stages of the inflammatory response, facilitating the passage of large and potentially antigenic molecules across the gut without gross disruption of the barrier to small molecules.

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

Transition from antidiuresis to diuresis exposes cortical collecting duct cells (CCD) to asymmetrical changes in environment osmolality, inducing an osmotic stress, which activates numerous membrane-associated events. The aim of the present work was to investigate, either in the presence or not of AQP2, the transepithelial osmotic water permeability (P(osm)) following cell exposure to asymmetrical hyper- or hypotonic gradients. For this purpose, transepithelial net volume fluxes were recorded every minute in two CCD cell lines: one not expressing AQPs (WT-RCCD(1)) and another stably transfected with AQP2 (AQP2-RCCD(1)). Our results demonstrated that the rate of osmosis produced by a given hypotonic shock depends on the gradient direction (osmotic rectification) only in the presence of apical AQP2. In contrast, hypertonic shocks elicit P(osm) rectification independently of AQP2 expression, and this phenomenon may be linked to modulation of basolateral membrane permeability. No asymmetry in transepithelial resistance was observed under hypo- or hypertonicity, indicating that rectification cannot be attributed to a shunt through the tight junction path. We conclude that osmotic rectification may be explained in terms of dynamical changes in membrane permeability probably due to activation/incorporation of AQPs or transporters to the plasma membrane via some mechanism triggered by osmolality.

Jejunal Creatine Absorption: What is the Role of the Basolateral Membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

Oxidant stress suppresses CFTR expression

Epithelial mucous membranes are repeatedly exposed to oxidants and xenobiotics. CFTR plays a role in glutathione transepithelial flux and in defining the hydration and viscoelasticity of protective mucus. We therefore hypothesized that CFTR expression and function may be modulated by oxidant stress. A sublethal oxidant stress (tert-butylhydroquinone, BHQ) in CFTR-expressing epithelial cells (T84) induced a significant increase in cellular glutathione that was associated with an increase in expression of the gene encoding the heavy subunit of the rate-limiting enzyme for glutathione synthesis, gamma-glutamylcysteine synthetase (gamma-GCShs). CFTR gene expression was markedly decreased according to a time course that mirrored the changes in gamma-GCShs. Western blot analysis confirmed that the decrease in CFTR gene expression was associated with a decrease in CFTR protein. cAMP-dependent iodide efflux was also decreased by the oxidant stress. Nuclear run-on assays indicated that the oxidant stress had no effect on CFTR gene transcription, but the mRNA stability in the oxidant-stressed cells was markedly reduced. Furthermore, BHQ increased gamma-GCShs mRNA while decreasing CFTR mRNA in Calu-3 cells, and taurine chloramine induced similar effects in T84 cells. We conclude that suppression of CFTR expression may represent an adaptive response of mucosal epithelium to an exogenous oxidant stress.

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

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

Mast Cell Tryptase Controls Paracellular Permeability of the Intestine: ROLE OF PROTEASE-ACTIVATED RECEPTOR 2 AND β-ARRESTINS

Tight junctions between intestinal epithelial cells prevent ingress of luminal macromolecules and bacteria and protect against inflammation and infection. During stress and inflammation, mast cells mediate increased mucosal permeability by unknown mechanisms. We hypothesized that mast cell tryptase cleaves protease-activated receptor 2 (PAR2) on colonocytes to increase paracellular permeability. Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i. Supernatant from degranulated mast cells increased [Ca2+]i in colonocytes, which was prevented by a tryptase inhibitor, and desensitized responses to PAR2 agonist, suggesting PAR2 cleavage. When applied to the basolateral surface of colonocytes, PAR2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of tight junction ZO-1 and occludin and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increased paracellular permeability of colonocytes. This was prevented by a tryptase inhibitor. We determined the role of ERK1/2 and of beta-arrestins, which recruit ERK1/2 to PAR2 in endosomes and retain ERK1/2 in the cytosol, on PAR2-mediated alterations in permeability. An ERK1/2 inhibitor abolished the effects of PAR2 agonist on permeability and redistribution of F-actin. Down-regulation of beta-arrestins with small interfering RNA inhibited PAR2-induced activation of ERK1/2 and suppressed PAR2-induced changes in permeability. Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2. PAR2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation.