Ussing Chamber Studies Research Articles

Augmentation of Cystic Fibrosis Transmembrane Conductance Regulator Function in Human Bronchial Epithelial Cells via SLC6A14-Dependent Amino Acid Uptake. Implications for Treatment of Cystic Fibrosis.

SLC6A14-mediated l-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia. We sought to determine the role of l-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from patients with cystic fibrosis (CF). Human bronchial epithelial (HBE) cells from lung explants of patients without CF (HBE) and those with CF (CF-HBE) were used for H3-flux, airway surface liquid, and Ussing chamber studies. We used α-methyltryptophan as a specific inhibitor for SLC6A14. CFBE41o-, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR. SLC6A14 is functionally expressed in CF-HBE cells. l-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14-mediated l-arginine uptake also increased the airway surface liquid in CF-HBE cells. Using CFBE41o cells, we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide (NO) synthase activity, nitrogen oxides, including NO, and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE, as inducible NO synthase inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR. In summary, SLC6A14-mediated l-arginine transport augments residual F508del-CFTR channel function via a noncanonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be used for the development of companion therapy in CF.

The Fast Lane of Hypoxic Adaptation: Glucose Transport Is Modulated via A HIF-Hydroxylase-AMPK-Axis in Jejunum Epithelium.

The intestinal epithelium is able to adapt to varying blood flow and, thus, oxygen availability. Still, the adaptation fails under pathologic situations. A better understanding of the mechanisms underlying the epithelial adaptation to hypoxia could help to improve the therapeutic approach. We hypothesized that the short-term adaptation to hypoxia is mediated via AMP-activated protein kinase (AMPK) and that it is coupled to the long-term adaptation by a common regulation mechanism, the HIF-hydroxylase enzymes. Further, we hypothesized the transepithelial transport of glucose to be part of this short-term adaptation. We conducted Ussing chamber studies using isolated lagomorph jejunum epithelium and cell culture experiments with CaCo-2 cells. The epithelia and cells were incubated under 100% and 21% O2, respectively, with the panhydroxylase inhibitor dimethyloxalylglycine (DMOG) or under 1% O2. We showed an activation of AMPK under hypoxia and after incubation with DMOG by Western blot. This could be related to functional effects like an impairment of Na+-coupled glucose transport. Inhibitor studies revealed a recruitment of glucose transporter 1 under hypoxia, but not after incubation with DMOG. Summing up, we showed an influence of hydroxylase enzymes on AMPK activity and similarities between hypoxia and the effects of hydroxylase inhibition on functional changes.

LC478, a Novel Di-Substituted Adamantyl Derivative, Enhances the Oral Bioavailability of Docetaxel in Rats.

P-glycoprotein (P-gp)-mediated efflux of docetaxel in the gastrointestinal tract mainly impedes its oral chemotherapy. Recently, LC478, a novel di-substituted adamantyl derivative, was identified as a non-cytotoxic P-gp inhibitor in vitro. Here, we assessed whether LC478 enhances the oral bioavailability of docetaxel in vitro and in vivo. LC478 inhibited P-gp mediated efflux of docetaxel in Caco-2 cells. In addition, 100 mg/kg of LC478 increased intestinal absorption of docetaxel, which led to an increase in area under plasma concentration-time curve (AUC) and absolute bioavailability of docetaxel in rats. According to U.S. FDA criteria (I, an inhibitor concentration in vivo tissue)/(IC50, inhibitory constant in vitro) >10 determines P-gp inhibition between in vitro and in vivo. The values 15.6–20.5, from (LC478 concentration in intestine, 9.37–12.3 μM)/(IC50 of LC478 on P-gp inhibition in Caco-2 cell, 0.601 μM) suggested that 100 mg/kg of LC478 sufficiently inhibited P-gp to enhance oral absorption of docetaxel. Moreover, LC478 inhibited P-gp mediated efflux of docetaxel in the ussing chamber studies using rat small intestines. Our study demonstrated that the feasibility of LC478 as an ideal enhancer of docetaxel bioavailability by P-gp inhibition in dose (concentration)-dependent manners.

IN VITRO AND IN SILICO CHARACTERIZATION OF HUMAN NASAL EPITHELIAL ION AND LIQUID TRANSPORT MECHANISMS

IntroductionCystic Fibrosis (CF) is a genetic disease caused by mutations in the CFTR gene, which codes for a widely expressed anion channel located in the apical membrane of epithelial cells. Loss of CFTR function results in an osmotic imbalance due to defective ion and water transport. Human bronchial epithelial cell cultures (HBE) are a well‐established experimental model of CF and show the same transport defects of the CF airway. More recently, human nasal epithelial cell cultures (HNE) have emerged as a novel alternative due to their accessibility and potential to allow for matched in vitro studies analogous to common clinical measurements. Here we implement a combination of experimental and computational approaches to facilitate the description of the ion and liquid transport functions of HNEs, and the role they may play in CF.MethodsWe have performed extensive characterization studies on well‐differentiated non‐CF HNE (n=9) and non‐CF HBE (n=12) to determine the extent of any possible physiological differences. This includes Ussing chamber studies to assess epithelial short‐circuit currents and resistance, apical volume regulation and paracellular flux assays, as measured by Tc99m‐DTPA (DTPA), to study response to volume challenges, and standard protein expression. We have also used this data to generate parametric fits of individual ion channel permeabilities, as predicted by a mathematical model of cell‐scale electrophysiology. Best parameter sets from each donor were estimated and compared against those from the opposite group to establish population differences according to a 2‐sample Kolmogorov‐Smirnov test.ResultsOur study shows HNE have a significant increase in the ENaC current (p<0.05), and a decrease in stimulated CFTR current (p<0.01) versus non‐CF HBEs. Similarly, thin film studies revealed significantly decreased DTPA retention, (p < 0.05), as well as apical volume at 12 and 24 hrs following an apical volume challenge (p<0.01). Donor‐specific parameter fits predicted statistically different parametric distributions for CFTR, ENaC, BKCa, and a basolateral Cl‐channel between the HBE and HNE population (p<0.01). Of note are the increased basolateral Cl− and BKCA permeabilities relative to CFTR permeability.ConclusionOur studies showed non‐CF HNEs have higher liquid absorption rates than non‐CF HBEs. This behavior may be correlated with the lower CFTR dependent Cl− current also seen in HNE. It could suggest that HNEs operate at a different homeostatic level than HBEs due to environmental differences between the bronchi and the paranasal sinus, or perhaps it is the result of distinct functional levels of members of the epithelial electrophysiology network. Similar studies exploring the extent of these physiological differences in CF HNEs, and carriers of a single CF mutation, are currently underway.Support or Funding InformationSupported by NIH R01‐HL108929‐01, U01‐HL131046‐01, P30‐DK‐072506This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P115 THE IBD CANDIDATE GENE, PTPN2, RESTRICTS INTESTINAL BARRIER DEFECTS AND CLAUDIN-2 EXPRESSION VIA STAT INHIBITION

Loss-of-function mutations in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene locus increase IBD susceptibility. We reported that PTPN2 knockdown (PTPN2-KD) in intestinal epithelial cells (IECs): (i) increased IFN-g activation of STAT signaling; (ii) reduced IEC barrier function; (iii) increased expression of the tight junction (TJ) protein, claudin-2, by IFN-g; and that the claudin-2 promoter contains a STAT binding sequence. Increased claudin-2 expression occurs in IBD and increases permeability to cations thus reducing transepithelial electrical resistance (TER). To confirm PTPN2 regulation of barrier function in vivo and identify the mechanisms of STAT regulation of claudin-2 expression. In Ussing chamber studies, TER was significantly reduced in cecum from Ptpn2 knockout (KO) and heterozygous (Het) mice vs. wild-type (WT) littermates indicating increased electrolyte permeability. Immunostaining and western blotting showed progressive increases in claudin-2 expression and STAT-1 phosphorylation (Y701) in Het and KO mice vs. wild-type intestine. Claudin-2 was also elevated in intestinal enteroids from Ptpn2-deficient mice. STAT-1 siRNA significantly decreased the elevated claudin-2 expression in PTPN2-KD IECs. Chromatin immunoprecipitation (ChIP) analysis showed increased STAT-1 binding to the claudin-2 promoter in PTPN2-KD vs. control cells. Claudin-2 promoter-luciferase reporter construct activity was significantly elevated in PTPN2-KD vs. Con-shRNA IEC and was further increased by IFN-g (1000 U/ml). This effect was lost by two different mutations of the STAT binding site thus confirming the requirement of STAT binding for increased claudin-2 transcription in PTPN2-KD IECs. Loss of functional PTPN2 increases susceptibility to inflammation-induced tight junction reorganization, providing an explanation for the association of PTPN2 mutants with UC and CD.

Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells.

Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research, particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y), termed conditionally reprogrammed cell (CRC) technology, enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings, we then created six CF cell CRC populations, three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted, and cells were subcultured, without irradiated feeders plus Y, into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y, dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage, but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus, CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers.

Soybean Milk Inhibits Absorption and Intestinal Transmembrane Transport of Gegen in Rats.

Puerariae Lobatae Radix, known as Gegen in Chinese, is widely used to treat cardiovascular diseases, diabetes, and many other chronic illnesses. Flavonoids are the main active components in Gegen and are found in high concentrations in soybeans. Few studies, however, have focused on the effects of flavonoid-rich food on the absorption of Gegen. Here, we report an in vivo pharmacokinetic study on rats to explore the effects of soybean milk on the absorption of Gegen and an in vitro Ussing chamber study of puerarin intestinal transmembrane absorption. Area under the plasma concentration-time curve (AUC0–t) and maximum plasma concentration (Cmax) values of puerarin in rats were significantly decreased after drinking soybean milk, when taking Gegen decoction or a Gegen patent medicine (P < 0.01). In the Ussing chamber experiment, cumulative transmission (Qtn) after 2 h and apparent permeability coefficient (Papp) were lower in the puerarin-daidzin and puerarin-soybean milk solution groups than in the puerarin group. Daidzin in soybean milk inhibited the transmembrane transport of puerarin, resulting in decreased bioavailability of puerarin in Gegen. The results of this study strongly suggest that Gegen should not be taken with flavonoid-rich food, particularly soybean products.

Epigenetic regulation of CFTR in salivary gland

Cystic fibrosis transmembrane conductance regulator (CFTR) plays a key role in exocrine secretion, including salivary glands. However, its functional expression in salivary glands has not been rigorously studied. In this study, we investigated the expression pattern and regulatory mechanism of CFTR in salivary glands using immunohistochemistry, western blot analysis, Ussing chamber study, methylation-specific PCR, and bisulfite sequencing. Using an organ culture technique, we found that CFTR expression was first detected on the 15th day at the embryonic stage (E15) and was observed in ducts but not in acini. CFTR expression was confirmed in HSG and SIMS cell lines, which both originated from ducts, but not in the SMG C-6 cell line, which originated from acinar cells. Treatment of SMG C-6 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored the expression level of CFTR mRNA in a time-dependent manner. Restoration of CFTR was further confirmed by a functional study. In the Ussing chamber study, 10 μM Cact-A1, a CFTR activator, did not evoke any currents in SMG C-6 cells. In contrast, in SMG C-6 cells pretreated with 5-Aza-CdR, Cact-A1 evoked a robust increase of currents, which were inhibited by the CFTR inhibitor CFTRinh-172. Furthermore, forskolin mimicked the currents activated by Cact-A1. In our epigenetic study, SMG C-6 cells showed highly methylated CG pairs in the CFTR CpG island and most of the methylated CG pairs were demethylated by 5-Aza-CdR. Our results suggest that epigenetic regulation is involved in the development of salivary glands by silencing the CFTR gene in a tissue-specific manner.

Excised segments of rat small intestine in Ussing chamber studies: A comparison of native and stripped tissue viability and permeability to drugs

Excised rat intestinal tissue mounted in an Ussing chamber can be used for intestinal permeability assessments in drug development. The outer layer of the intestine, the serosa and part of the muscle layer, is traditionally removed since it is considered a barrier to the diffusion of nutrients and oxygen as well as to that of pharmaceutical substances. However, the procedure for removing the serosal-muscle layer, i.e. stripping, is a technically challenging process in the pre-experimental preparation of the tissue which may result in tissue damage and reduced viability of the segment. In this study, the viability of stripped and native (non-stripped) rat small intestine tissue segments mounted in Ussing chambers was monitored and the apparent permeability of the tissue to a set of test compounds across both tissue preparations was determined. Electrical measurements, in particular the potential difference (PD) across the intestinal membrane, were used to evaluate the viability. In this study, there were no differences in initial PD (health status of the tissue) or PD over time (viability throughout the experiment) between native and stripped rat jejunum segments. Overall, there were also no significant differences in permeability between stripped and native rat intestinal tissue for the compounds in this study. Based on these results, we propose that stripping can be excluded from the preparation procedures for rat jejunal tissue for permeability studies when using the Ussing chamber technique.

CHLORIDE‐INDEPENDENT, UNIDIRECTIONAL FLOW OF FLUID INTO THE SMALL INTESTINE: OPENING THE FLOODGATES

The volume of fluid in the gastrointestinal (GI) tract is the result of the carefully regulated dynamic balance between the counter processes of absorption and secretion. The ability to reversibly regulate fluid secretion is imperative for maintaining the normal gut fluid state. Diarrhea results from dysregulation of these processes and remains a leading cause of death worldwide. Although chloride secretion is generally considered as the mechanism for fluid secretion, it does not account for the large volumes of fluid and electrolytes that move into the intestinal lumen during diarrhea. We discovered using cultured freshly harvested mouse enterocytes in Ussing chamber studies a heretofore unreported unidirectional pathway for the movement of fluid into the small intestine. The fluid flow (FF) is directly related with mechanical force provided byaeration (none, minimal, intense, P&lt;0.001), rapidly (&lt;5 min) establishes apical to basolateral hydrostatic gradients that can exceed 2 cm that are sustained, is independent of ion gradients and channel inhibitors for Cl (P=0.4) and Na and K (P&gt;0.50), does not require osmotic gradients, and is unidirectional based on movement of FITC‐dextran toward the apical chamber, but not from the apical to the basolateral chamber. The FF is related to extent of tight junction development based on the lack of FF by cultured fibroblasts (without tight junctions) and diminished FF by epithelia prepared using cells of colonic origin with extensive tight junction development (&lt;20% for Caco‐2 and T84 cells compared with enterocytes). Exposure to zinc (100 microM) caused a rapid (&lt;10 sec) inhibition of FF (&gt;90%; P&lt;0.001) that could be reversed by Zn chelation with citrate (~85% recovery; P&lt;0.001). Sacs prepared from intact mouse small intestine were used to confirm cell data for FF based on loss of weight and leakage of FITC‐dextran. FF was increased by exposure to aeration and stirring (P=0.02), was higher for everted than uneverted sacs verifying FF is toward the lumen (P=0.02), and was inhibited ~40% by zinc (P=0.003).ConclusionsThe tight junction complex of enterocytes includes a “one‐way valve” for paracellular, unidirectional flow of fluid into the gut. This newly‐described paracellular pathway requires mechanical force that in vivo would be provided by contractions of smooth muscle, is sufficient to explain the massive fluid shifts that occur during diarrhea, and explains why the most effective anti‐diarrheal agents reduce smooth muscle contraction (and thus, mechanical force) and why agents that only inhibit chloride secretion have not proven effective. The reversible inhibition of FF by zinc, a safe and effective antidiarrheal agent, provides a promising target for therapeutics to treat diarrhea and constipation. Hypotheses tested, experimental approaches, and results for fluid flow (FF) by cultured mouse enterocytes and sacs of intact small intestine. Hypothesis Experiment Results Experiments with cultured mouse enterocytes FF requires aeration. Record FF at no, low, and vigorous aeration P&lt;0.001 FF is related to aeration intensity FF is unidirectional Record movement of FF and FITC‐dextran when added to the apical vs the basolateral chamber P&lt;0.001 FF is paracellular and directed toward the lumen FF is coupled with Cl flux Record FF with Cl gradients and channel inhibitors P&gt;0.50 FF is independent of Cl flux FF is coupled with Na or K flux Record FF with Na and K gradients and inhibitors of channel and transporters P&gt;0.50 FF is independent of Na and K fluxes FF responds to an osmotic gradient Determine if an apical to basolateral osmotic gradient reduces FF P&gt;0.50 FF is independent of an osmotic gradient Zinc inhibits FF Measure FF before and after adding zinc sulfate P&lt;0.0001 zinc rapidly inhibits FF Zinc inhibition of FF is reversible Measure FF after adding citrate to chelate zinc P&lt;0.0001 citrate reverses Zn inhibition Experiments with sacs prepared from mouse small intestine Agitation increases FF Record weight and FITC‐dextran leak with and without agitation by aeration and stirring P&lt;0.01 agitation increases weight loss and FITC‐dextran leakage FF by intact tissue is unidirectional Compare FF of evened and non‐everted sacs P&lt;0.01 FF greater for everted sacs Zinc inhibits FF by intact tissue Compare FF with and without zinc P&lt;0.001 zinc inhibits FF

FXYD1 negatively regulates Na+/K+-ATPase activity in lung alveolar epithelial cells

Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na+/K+-ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na+/K+-ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-β, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-β signaling altered Na+/K+-ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na+/K+-ATPase activity, and contribute to edema persistence.

Linaclotide-induced electrolyte secretion in human and rat colon: Ussing chamber studies

Background Linaclotide is a guanylyl cyclase C (GC-C) agonist approved for treatment of adult patients with irritable bowel syndrome with constipation (IBS-C). Linaclotide binding to the extracellular receptor domain of GC-C activates the intracellular catalytic domain of GC-C, resulting in the generation of cyclic GMP (cGMP). cGMP, via a pathway leading to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel, stimulates Cland HCO3-secretion and concomitant inhibition of Na+ absorption by sodium/hydrogen exchanger 3 (NHE3), driving the efflux of water into the lumen and accelerating bowel transit. However, the effect of linaclotide on electrolyte transport in the lower gastrointestinal tract has not yet been characterized. This study specifically examined the effect of linaclotide on electrolyte transport in the colon. We used an in vitro Ussing chamber assay to measure the secretion of electrolytes in human ascending colon segments and in rat proximal colon in response to linaclotide treatment.

WS06.2 R117H-CFTR has a defect in channel gating activity that can be potentiated by ivacaftor

Background The R117H mutation is associated with residual chloride transport and a delayed onset of CF symptoms. Although generally called a conductance mutation, R117H-CFTR exhibits defective channel gating. This study aimed to more fully explore the mechanistic defect of R117H-CFTR and to evaluate the effect of ivacaftor, a CFTR potentiator, on R117H-CFTR. Methods and Results In single-channel, patch-clamp studies, the channel open probability (gating activity) of R117H-CFTR was 22% of normal, whereas the channel conductance was 88% of normal. The channel gating activity of R117H-CFTR was characterized by frequent but brief openings of the channel compared with normal CFTR. The brief openings are consistent with a low channel open probability due in part to destabilization of the channel open state by R117H. The addition of ivacaftor increased the channel open probability of R117H-CFTR from 22% to 42% of normal. The increase in open probability was due to an increase in the frequency of channel opening and a decreased closed duration. Channel open duration and conductance were not increased. In Ussing chamber studies using R117H-5T/F508del-human bronchial epithelial cells from two donor bronchi, chloride transport was 23% of normal, which is consistent with residual chloride transport. Ivacaftor treatment for 18–24 hours increased chloride transport to 36% of normal. Conclusion These data confirm that R117H is a residual function CFTR mutation that causes a predominant defect in channel gating activity that is potentiated by ivacaftor. Based on these in vitro data, ivacaftor would be expected to enhance CFTR activity in people with the R117H mutation.

Mechanisms of Intestinal Serotonin Transporter (SERT) Upregulation by TGF-β1 Induced Non-Smad Pathways.

TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer’s or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM) attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders.

Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-fiber subtypes in guinea pig esophagus.

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE.

Examining urea flux across the intestine of the spiny dogfish, Squalus acanthias

Recent examination of urea flux in the intestine of the spiny dogfish shark, Squalus acanthias, has shown that feeding significantly enhances urea uptake across the intestine, and this was significantly inhibited following mucosal addition of phloretin. The present study examined potential mechanisms of urea uptake across the dogfish intestine in starved and fed dogfish. Unidirectional flux chambers were used to examine the kinetics of urea uptake, and to determine the influence of sodium, ouabain, competitive urea analogues, and phloretin on urea uptake across the gut of fed dogfish. Intestinal epithelial preparations from starved and fed dogfish were mounted in Ussing chambers to examine the effect of phloretin on bidirectional solute transport across the intestine. In the unidirectional studies, the maximum uptake rate of urea was found to be 35.3±6.9μmol.cm-2.h-1 and Km was found to be 291.8±9.6mM in fed fish, and there was a mild inhibition of urea uptake following mucosal addition of competitive agonists. Addition of phloretin, Na-free Ringers and ouabain to the mucosal side of intestinal epithelia also led to a significant reduction in urea uptake in fed fish. In the Ussing chamber studies there was a net influx of urea in fed fish and a small insignificant efflux in starved fish. Addition of phloretin blocked urea uptake in fed fish when added to the mucosal side. Furthermore, phloretin had no effect on ion transport across the intestinal epithelia with the exception of the divalent cations, magnesium and calcium.

Abstract 2963: Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development

Abstract Background: Inactivation of the TGF-β signaling occurs in 50-75% of all colorectal cancer cases. Down-regulation of Smad4, the central mediator of TGF-β signaling, occurs in &amp;gt;50% of stage III patients. How Smad4 contributes to normal intestinal homeostasis and functions as a tumor suppressor are incompletely understood. We reported that β-catenin mRNA expression is inhibited by BMP-mediated Smad4 signaling, and loss of Smad4 signaling results in biologically significant amplification of the Wnt/β-catenin signal. Here, we report on the consequences of Smad4 loss for intestinal homeostasis. Methods: We generated mice genetically engineered to undergo inducible, tissue-specific deletion of Smad4 within intestinal epithelial cells. K19-CreERT x Smad4 flx/flx and Lrig1-CreERT x Smad4 flx/flx mice were treated with vehicle or tamoxifen to induce recombination at the Smad4 locus in the intestinal stem cell compartment. Intestines from sacrificed mice were cleansed for enteroid isolation, Ussing chamber studies, RNA-seq analysis and preservation by formalin fixation and paraffin embedding (FFPE). We validated Smad4 status via immunohistochemistry and stained FFPE sections for different cell populations within the crypt. To evaluate inflammatory cytokines, we performed Luminex cytokine array analysis on colonic mucosa from control and tamoxifen treated animals. To determine the role for Smad4 in intestinal response to injury, we also treated control and tamoxifen treated animals with dextran sodium sulfate (DSS). We utilized RNA-Seq analysis to determine an intestinal Smad4 loss gene signature. Results: Smad4 deficient crypts exhibited an expansion of the Ki67 positive cells in the proliferative zone and fewer Carbonic anhydrase II staining cells suggesting fewer mature enterocytes. We found that loss of Smad4 in the intestinal crypt is accompanied by an accumulation of pericryptal CD3+ lymphocytes and F4/80+ macrophages and is associated with increased intestinal permeability as measured by low molecular weight FITC Dextran using the Ussing chamber. Cytokine analysis revealed Macrophage Inflammatory Protein 3a (MIP3a) to be up-regulated in Smad4 knockout mice. Guided by our RNA-Seq analysis, we observe increased levels of pErk in Smad4 deplete tissue. We also observed that mice with intestinal epithelial Smad4 depletion developed advanced mucinous adenocarcinoma after recovery from an inflammatory stimulus induced by DSS. Conclusions: These results suggest that Smad4 loss disrupts intestinal homeostasis by enabling persistently high levels of Erk signaling to interfere with cell maturation, thereby contributing to increased inflammation. We postulate that the increased inflammation observed in Smad4 knockout mice leads to a tumorigenic environment. Citation Format: Tanner J. Freeman, Jillian Pope, Josh Smith, Daniel Sharbel, Kay Washington, Xi Chen, Rupesh Chaturvedi, Keith Wilson, Noah Shroyer, Punita Dhawan, Anna Means, Natasha G. Deane, R. Daniel Beauchamp. Loss of Smad4 within the intestinal tract results in altered intestinal homeostasis and tumor development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2963. doi:10.1158/1538-7445.AM2014-2963

An amino acid mixture mitigates radiation-induced gastrointestinal toxicity.

Electrolyte and nutrient absorption occur in villous epithelial cells. Radiation often results in reduced electrolyte and nutrient absorption, which leads to gastrointestinal toxicity. Therefore, the authors studied: (1) radiation-induced changes in glucose and amino acid absorption across ileal tissues and (2) the effect of amino acid mixtures on absorptive capacity. NIH Swiss mice were irradiated (0, 1, 3, 5, or 7 Gy) using a ¹³⁷Cs source at 0.9 Gy min⁻¹. Transepithelial short circuit current (I(sc)), dilution potential, and isotope flux determinations were made in Ussing chamber studies and correlated to plasma endotoxin and IL-1β levels. Amino acids that increased electrolyte absorption and improved mucosal barrier functions were used to create a mitigating amino acid mixture (MAAM). The MAAM was given to mice via gastric gavage; thereafter, body weight and survival were recorded. A significant decrease in basal and glucose-stimulated sodium absorption occurred after 0, 1, 3, 5, and 7 Gy irradiation. Ussing chamber studies showed that paracellular permeability increased following irradiation and that the addition of glucose resulted in a further increase in permeability. Following irradiation, certain amino acids manifested decreased absorption, whereas others were associated with increased absorption. Lysine, aspartic acid, glycine, isoleucine, threonine, tyrosine, valine, tryptophan, and serine decreased plasma endotoxins were selected for the MAAM. Mice treated with the MAAM showed increased electrolyte absorption and decreased paracellular permeability, IL-1β levels, and plasma endotoxin levels. Mice treated with MAAM also had increased weight gain and better survival following irradiation. The MAAM has immediate potential for use in mitigating radiation-induced acute gastrointestinal syndrome.

Effects of age and controlled oral dosing of Enterococcus faecium on epithelial properties in the piglet small intestine.

Enterococcus faecium NCIMB 10415 is a licensed probiotic for piglets that has been shown to positively affect diarrhoea incidence and to act on transport properties and immunological parameters in the porcine intestine. The aim of the present study was to examine its effects on jejunal absorptive and secretory capacities around weaning. Furthermore, the possible involvement of heat shock proteins in the effects of probiotics on epithelial functions was investigated. A significant part of the probiotic was dosed orally to reduce the variability of intake of the probiotic. The piglets were randomly assigned to a control and a probiotic feeding group, the latter receiving 4.5×109 cfu/day of E. faecium directly into the mouth for 34 days starting after birth. Additionally, their feed was supplemented with the probiotic strain. Piglets were weaned at day 29 after birth. Ussing chamber studies were conducted with the mid-jejunum of piglets aged 14, 28, 31, 35 and 56 days. Changes in short-circuit current (ΔIsc) were measured after stimulation of Na+-coupled absorption with L-glutamine or glucose or with the secretagogue prostaglandin E2 (PGE2). The mRNA expression for SGLT1, CFTR and various heat shock proteins was determined. The transport properties changed significantly with age. The glucose-, L-glutamine- and PGE2-induced changes in Isc were highest at day 31 after birth. No significant differences between the feeding groups were observed. The mRNA of HSP60, HSC70, HSP70 and HSP90 was expressed in the jejunal tissues. The mRNA expression of HSC70 was higher and that of HSP60 was lower in the probiotic group. HSC70 expression increased with age. In conclusion, whereas age effects were observed on absorptive and secretory functions, controlled E. faecium dosing had no measurable effects on these functional parameters in this experimental setup. The possible role of heat shock proteins should be further evaluated.

ASK1 and p38 MAP kinase regulate proteolytic processing of ENaC

These Ussing chamber studies were undertaken to investigate the mechanism by which hydrogen peroxide (0.5 mM) inhibits mouse epithelial Na+ channels (ENaC) transfected into FRT cell monolayers.We found the inhibition was blocked by expression of a dominant negative construct of the kinase ASK1, and was replicated by expression of a constitutively active mutant of ASK1. The effects of both hydrogen peroxide and of the constitutively active mutant of ASK1 were blocked by the p38 MAP kinase inhibitor, SB203580, and by expression of a dominant negative mutant of p38 MAP kinase.Western blotting with an antibody for phosphorylated p38 MAP kinase confirmed that ASK1 modulates the amount of phosphorylated p38 MAP kinase. Epithelial Na+ channels are regulated by the Nedd4–2 system that controls their surface expression. We found, using surface biotinylation methods, however, that neither ASK1 nor p38 MAP kinase affected surface expression of the channels.Epithelial Na+ channels are normally activated by proteolysis of the α‐ and γ‐subunits during their intracellular processing. When we used ENaC subunit constructs in which the N‐ and C‐terminals are labelled with V5 or FLAG tags to examine the effect of the kinases on proteolytic processing, we found that they inhibit proteolytic cleavage of both α‐ and γ‐ENaC.In summary, we have found that hydrogen peroxide inhibits epithelial Na+ channels expressed in FRT cell monolayers via the kinases ASK1 and p38 MAP kinase, which reduce the activity of the channels by inhibiting the normal activation of the channels by proteolysis.