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Author(s):  
Ciaran A Shaughnessy ◽  
Sangya Yadav ◽  
Preston E Bratcher ◽  
Pamela L Zeitlin

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current (ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to levels comparable to forskolin. Pre-treatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pre-treated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) co-treatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ~60% compared to treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that co-treatment with lubiprostone can further restore modulator-rescued CFTR.


2022 ◽  
Vol 52 (6) ◽  
Author(s):  
Vinicius Duarte ◽  
Adriano Olnei Mallmann ◽  
Camila Tonini ◽  
Diogo Liberalesso ◽  
Cristiane Rosa da Silva ◽  
...  

ABSTRACT: In vitro tests are performed to evaluate the efficacy of antimycotoxins additives (AMAs); nevertheless, such assays show a low correlation with in vivo trials, which are also required to determine AMAs’ efficacy. In search of an alternative method, the current study investigated the use of an ex vivo technique. Six AMAs (AMA1 to AMA6) had their ability to reduce intestinal absorption of aflatoxin B1 (AFB1) evaluated. Jejunal explants were obtained from broilers and subjected to two treatments per AMA in Ussing chambers: T1 (control) - 2.8 mg/L AFB1, and T2 - 2.8 mg/L AFB1 + 0.5% AMA. AMAs were also tested in vitro to assess adsorption of AFB1 in artificial intestinal fluid. In the ex vivo studies, AMA1 to AMA6 decreased intestinal absorption of AFB1 by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively. As for the in vitro results, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. The evaluated ex vivo model proved useful in the assessment of AMAs. No correlation was reported between ex vivo and in vitro findings. Further studies are needed to elucidate the correlation between ex vivo and in vivo results seeking to reduce animal testing.


2021 ◽  
Vol 55 (6) ◽  
pp. 784-804

BACKGROUND/AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity. METHODS: We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]i) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers. RESULTS: PDE8 inhibition elevated [cAMP]i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators. CONCLUSION: These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.


2021 ◽  
Vol 12 ◽  
Author(s):  
Andre Barany ◽  
Milagrosa Oliva ◽  
Silvia Filipa Gregório ◽  
Gonzalo Martínez-Rodríguez ◽  
Juan Miguel Mancera ◽  
...  

Aflatoxin B1 (AFB1) is a mycotoxin often present in food. This study aimed to understand the physiological effects of AFB1 on the seabream (Sparus aurata) gastrointestinal system. In a first in vitro approach, we investigated ion transport using the short-circuit current (Isc) technique in Ussing chambers in the anterior intestine (AI). Application of apical/luminal AFB1 concentrations of 8 and 16 μM to healthy tissues was without effect on tissue transepithelial electrical resistance (TER), and apparent tissue permeability (Papp) was measured using fluorescein FITC (4 kD). However, it resulted in dose-related effects on Isc. In a second approach, seabream juveniles fed with different AFB1 concentrations (1 and 2 mg AFB1 kg−1 fish feed) for 85 days showed significantly reduced gill Na+/K+-ATPase (NKA) and H+-ATPase (HA) activities in the posterior intestine (PI). Moreover, dietary AFB1 modified Isc in the AI and PI, significantly affecting TER in the AI. To understand this effect on TER, we analyzed the expression of nine claudins and three occludins as markers of intestinal architecture and permeability using qPCR. Around 80% of the genes presented significantly different relative mRNA expression between AI and PI and had concomitant sensitivity to dietary AFB1. Based on the results of our in vitro, in vivo, and molecular approaches, we conclude that the effects of dietary AFB1 in the gastrointestinal system are at the base of the previously reported growth impairment caused by AFB1 in fish.


2021 ◽  
Vol 22 (22) ◽  
pp. 12535
Author(s):  
Francesca Piccapane ◽  
Andrea Gerbino ◽  
Monica Carmosino ◽  
Serena Milano ◽  
Arduino Arduini ◽  
...  

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.


Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1419
Author(s):  
Sarina Koehler ◽  
Andrea Springer ◽  
Nicole Issel ◽  
Stefanie Klinger ◽  
Michael Wendt ◽  
...  

Ascaris suum, the most important pig parasite, also infects humans as a zoonotic pathogen. Malabsorption upon infection probably results from impaired nutrient transport, presumably mediated by the parasite´s excretory-secretory (ES) or cuticle somatic (CSO) antigens. The present study investigated the electrogenic transport (∆Isc) of glucose, alanine and the dipeptide glycyl-l-glutamine (glygln), as well as glucose net flux rates in pig jejunal tissue after in vitro exposure to adult A. suum total ES or CSO antigens in Ussing chambers. ∆Isc of glucose, alanine and glucose net flux rate were significantly decreased after one hour of exposure to total ES antigen. In contrast, CSO antigens increased the transport of glygln. Additionally, nutrient uptake and ES antigen pattern were compared in culture medium from untreated adult worms and those with sealed mouth and anal openings. Untreated worms completely absorbed glucose, while cuticular absorption in sealed worms led to 90% reduction. Amino acid absorption was 30% less effective in sealed worms, and ammonia excretion decreased by 20%. Overall, the results show that A. suum total ES antigen rapidly impairs nutrient transport in vitro. Future studies confirming the results in vivo, narrowing down the ES components responsible and investigating underlying molecular mechanisms are needed.


2021 ◽  
Vol 8 ◽  
Author(s):  
Tim Vanuytsel ◽  
Jan Tack ◽  
Ricard Farre

An increased intestinal permeability has been described in various gastrointestinal and non-gastrointestinal disorders. Nevertheless, the concept and definition of intestinal permeability is relatively broad and includes not only an altered paracellular route, regulated by tight junction proteins, but also the transcellular route involving membrane transporters and channels, and endocytic mechanisms. Paracellular intestinal permeability can be assessed in vivo by using different molecules (e.g., sugars, polyethylene glycols, 51Cr-EDTA) and ex vivo in Ussing chambers combining electrophysiology and probes of different molecular sizes. The latter is still the gold standard technique for assessing the epithelial barrier function, whereas in vivo techniques, including putative blood biomarkers such as intestinal fatty acid-binding protein and zonulin, are broadly used despite limitations. In the second part of the review, the current evidence of the role of impaired barrier function in the pathophysiology of selected gastrointestinal and liver diseases is discussed. Celiac disease is one of the conditions with the best evidence for impaired barrier function playing a crucial role with zonulin as its proposed regulator. Increased permeability is clearly present in inflammatory bowel disease, but the question of whether this is a primary event or a consequence of inflammation remains unsolved. The gut-liver axis with a crucial role in impaired intestinal barrier function is increasingly recognized in chronic alcoholic and metabolic liver disease. Finally, the current evidence does not support an important role for increased permeability in bile acid diarrhea.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mandy Laube ◽  
Diana Dornis ◽  
Fine Wenzel ◽  
Ulrich H. Thome

AbstractMale sex remains an independent risk factor for respiratory distress syndrome (RDS) in preterm infants. Insufficient Na+ transport-mediated alveolar fluid clearance contributes to RDS development and we previously demonstrated sex-specific differences in Na+ transport. The epidermal growth factor (EGF) is important during fetal lung development with possible influence on Na+ transport. Sex-specific effects of EGF during surfactant synthesis were shown. We thus determined whether EGF exerts sex-specific effects on Na+ transport in fetal alveolar cells. We analyzed sex-specific fetal distal lung epithelial (FDLE) cells exposed to EGF and related ligands with Ussing chambers, RT-qPCR and Western blots. EGF strongly reduced the epithelial Na+ channel (ENaC) mRNA levels in both male and female FDLE cells. This was corroborated by a markedly reduced ENaC activity, while amiloride-insensitive pathways as well as barrier function were raised by EGF. In contrast to chronic effects, acute effects of EGF were sex-specific, because Na+ transport was reduced only in males. AKT phosphorylation was elevated only in female cells, while pERK1/2 was increased in both male and female cells. EGF showed certain sex- and time-dependent effects in FDLE cells. Nevertheless, the results suggest that EGF is an unlikely cause for the sex-specific differences in Na+ transport.


2021 ◽  
Vol 12 ◽  
Author(s):  
Yinghui Cui ◽  
Fenglan Chu ◽  
Kai Yin ◽  
Xiongying Chen ◽  
Hanxing Wan ◽  
...  

As little is known about the role of calcium (Ca2+) signaling mediating the small intestinal epithelial anion secretion, we aimed to study its regulatory role in secretagogue-stimulated duodenal anion secretion and the underlying molecular mechanisms. Therefore, intestinal anion secretion from native mouse duodenal epithelia was examined with Ussing chambers to monitor PGE2-, 5-HT-, and CCh-induced short-circuit currents (Isc). PGE2 (10 μM) and 5-HT (10 μM) induced mouse duodenal Isc, markedly attenuated by serosal Ca2+-free solution and selective blockers of store-operated Ca2+ channels on the serosal side of the duodenum. Furthermore, PGE2- and 5-HT-induced duodenal Isc was also inhibited by ER Ca2+ chelator TPEN. However, dantrolene, a selective blocker of ryanodine receptors, inhibited PGE2-induced duodenal Isc, while LiCl, an inhibitor of IP3 production, inhibited 5-HT-induced Isc. Moreover, duodenal Isc response to the serosal applications of both PGE2 and 5-HT was significantly attenuated in transient receptor potential vanilloid 4 (TRPV4) knockout mice. Finally, mucosal application of carbachol (100 μM) also induced duodenal Isc via selective activation of muscarinic receptors, which was significantly inhibited in serosal Ca2+-free solution but neither in mucosal Ca2+-free solution nor by nifedipine. Therefore, the serosal TRPV4-constituted SOCE mechanism is likely universal for the most common and important secretagogues-induced and Ca2+-dependent intestinal anion secretion. These findings will enhance our knowledge about gastrointestinal (G.I.) epithelial physiology and the associated G.I. diseases, such as diarrhea and constipation.


2021 ◽  
Vol 11 (14) ◽  
pp. 6384
Author(s):  
Carlotta Giromini ◽  
Marco Tretola ◽  
Cinzia Cristiani ◽  
Elisabetta Finocchio ◽  
Paolo Silacci ◽  
...  

Supplemental dietary amino acids (AAs) need to be provided in a form that prevents their degradation along the gastrointestinal tract to guarantee their high bioavailability and bioactivity. In this study, methionine (Met) protected via organo-clay intercalation (natural carriers) has been developed as a sustainable alternative to polymeric coating. Specifically, two different bentonite-zeolite-based mineral clays were tested, Adsorbene (ADS) and BioKi (BIO). Briefly, 1 g of the carrier (ADS or BIO) was contacted with 50 mL of an aqueous solution at a pH of 3.0, 5.8, and 8.9. Solid-liquid separation was conducted. The released Met in the liquid phase was analysed by Chemical Oxygen Demand, while residual Met in the solid phase was analysed by Fourier Transform Infra-Red (FT-IR) spectroscopy. The effect of Met-ADS complex on cell viability was tested on IPEC-J2 cells incubated 3 h with Met-ADS 2.5 mM. Jejunum segments obtained by entire male pigs (Swiss Large White, body weight 100 ± 5 kg) were used as ex vivo models to compare the absorption of 2.5 mM Met released by ADS with 2.5 mM free Met and its influence on epithelial integrity in perfusion Ussing chambers. The carriers released a very low amount of Met and Met-BIO interaction was stronger than Met-ADS. The maximum release of Met was at pH 3, with 3% and 6% of Met release from Met-BIO and Met-ADS, respectively. Cell viability experiments revealed that Met-ADS did not alter cell metabolic activity. No differences in Met absorption and intestinal epithelial integrity were observed ex vivo between free Met and Met-ADS. This study provided new insights into the release of Met from natural clays such as ADS and BIO, the safety of its use in the porcine intestine and the ability of ADS-released Met to absorb to the same extent as the free Met in porcine jejunum.


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