Apical Membrane Of Epithelial Cells Research Articles

Purinergic control of apical ion conductance by luminal ATP in rat colonic epithelium

ATP, released e.g. after cell damage or during inflammation, can alter ion transport across the intestinal mucosa via stimulation of purinergic receptors in the basolateral as well as in the apical membrane of epithelial cells. When ATP acts from the serosal side, it induces an increase in short-circuit current (Isc) via Cl− secretion across the colonic epithelium. In contrast, mucosal ATP or its derivative, BzATP, predominantly stimulating ionotropic P2X4 and P2X7 receptors, evoke an increase in Isc, which could not be explained by Cl− secretion. The underlying ion currents after stimulation of apical purinergic receptors in rat distal colon are still unclear and were investigated in the present study.Ussing chamber experiments revealed that the Isc induced by mucosal ATP was dependent on the presence of mucosal Ca2+ and inhibited by the K+ channel blocker, Ba2+, indicating the involvement of Ca2+-dependent K+ channels. Blockade of the transepithelial Isc by lanthanides (La3+, Gd3+) suggests that Ca2+ enters the epithelium via nonselective cation channels. Experiments with basolaterally depolarized epithelia confirmed the activation of apical lanthanide-sensitive Na+- and Ca2+-permeable cation channels by ATP. Putative candidates might be TRP channels, from which several subtypes were detected in colonic tissue in RT-PCR experiments. In addition, the activation of an apical Cl− conductance was observed when suitable Cl− concentration gradients were applied. Consequently, mucosal ATP, acting as ‘danger signal’, stimulates cation and anion channels in the apical membrane to induce a secretory response as part of the local defence mechanism in the intestinal epithelium.

Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder.

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by the amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of cystic fibrosis transmembrane conductance regulatory protein (CFTR) in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR were compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR were quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish a significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared with control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR were accompanied by decreases in mRNA for the Cl-/[Formula: see text] exchanger SLC26A3, K+ channels (KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor (VIPR1), which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low-risk, high-risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by the underlying failure of CFTR function.NEW & NOTEWORTHY Cystic fibrosis transmembrane conductance regulatory protein (CFTR) genomic variants and expression of mRNA, protein, and electrogenic anion secretory activity of CFTR were characterized in dog gallbladder. Acquired inhibition of CFTR expression by gallbladder epithelium was identified as underpinning a naturally occurring muco-obstructive disease of the dog gallbladder that bears striking pathological similarity to animal models of cystic fibrosis.

Targeted Therapy for Cystic Fibrosis in Children

A mutation in the CFTR gene causes the malfunctioning of the CFTR protein that is located on the apical membrane of epithelial cells and functions as a chloride channel. The discovery of CFTR modulators is an advance in cystic fibrosis treatment.Objective: To analyze modern Russian and foreign literature on targeted therapy for cystic fibrosis in children. We also describe the history of cystic fibrosis, evolution in its diagnosis and treatment, and mechanisms behind targeted therapy for cystic fibrosis.

Glucose absorption activity and gene expression of sugar transporters in the trophotaenia of the viviparous teleost Xenotoca eiseni

In viviparous reproductive systems, nutrient transfer from mother to embryo plays a critical role in the generation of offspring. Herein, we investigated the mother-to-embryo nutrient transfer machinery in the viviparous teleost Xenotoca eiseni, which belongs to the family Goodeidae. The intraovarian embryo absorbs maternal supplements via the hindgut-derived placental structure termed the trophotaenia. Tracer analysis indicated that the trophotaenia can take up glucose analogs in ex vivo cultured embryos. The candidate genes for absorption, sglt1, glut2, atp1a, and atp1b, were determined from published transcriptomes. These genes were expressed in the trophotaenia of X. eiseni embryos. Fluorescent immunohistochemistry of Na+/K+ ATPase indicated the polarity of epithelial cells in the trophotaenia. The presented evidence suggests that the epithelial cell layer transports monosaccharides from the apical membrane of epithelial cells in a basolateral direction. Taken together, this study provides insight into how maternal fish maintain their offspring during gestation and will aid in the development of strategies to improve offspring generation in these fish.

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Epithelial sodium channel (ENaC) in GtoPdb v.2023.1

Profile of the intestinal microbiota of patients with cystic fibrosis: A systematic review

Cystic fibrosis (CF) is a multisystem disease that can compromise several human body organs. The autosomal recessive genetic disorder is caused by different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for chloride ion transport across apical membranes of epithelial cells in tissues and bicarbonate secretion. In this study, we provide a systematic review of the profile of the intestinal microbiota of cystic fibrosis individuals. The review was conducted according to Preferred Items of Reports for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. PubMed/MEDLINE and Scopus databases were searched for relevant articles until Jully 2022. Eighteen studies (1304 participants) met the inclusion criteria. The quality and bias was assessed using the Methodological index for non-randomized studies (MINORS) tool, with the majority of the studies indicating medium to high quality. Results showed significant changes in the composition of the intestinal microbiota of the individuals with CF compared with healthy controls, with increased of Enterococcus, Veillonella, and Streptococcus, and decreased of Bifidobacterium, Roseburia, and Alistipes genus. The intestinal bacterial community of CF patients was marked by a reduction in its richness and diversity. The systematic review suggests a change in the intestinal microbiota of CF individuals, characterized by a reduction in microbial diversity and abundance of some bacterial markers.

Intracellular Helix-Loop-Helix Domain Modulates Inactivation Kinetics of Mammalian TRPV5 and TRPV6 Channels.

TRPV5 and TRPV6 are calcium-selective ion channels expressed at the apical membrane of epithelial cells. Important for systemic calcium (Ca2+) homeostasis, these channels are considered gatekeepers of this cation transcellular transport. Intracellular Ca2+ exerts a negative control over the activity of these channels by promoting inactivation. TRPV5 and TRPV6 inactivation has been divided into fast and slow phases based on their kinetics. While slow inactivation is common to both channels, fast inactivation is characteristic of TRPV6. It has been proposed that the fast phase depends on Ca2+ binding and that the slow phase depends on the binding of the Ca2+/Calmodulin complex to the internal gate of the channels. Here, by means of structural analyses, site-directed mutagenesis, electrophysiology, and molecular dynamic simulations, we identified a specific set of amino acids and interactions that determine the inactivation kinetics of mammalian TRPV5 and TRPV6 channels. We propose that the association between the intracellular helix-loop-helix (HLH) domain and the TRP domain helix (TDh) favors the faster inactivation kinetics observed in mammalian TRPV6 channels.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

CD10 and Das1: a biomarker study using immunohistochemistry to subtype gastric intestinal metaplasia

BackgroundIntestinal metaplasia (IM) is considered a key pivot point in the Correa model of gastric cancer (GC). It is histologically subtyped into the complete and incomplete subtypes, the latter being associated with a greater risk of progression. However, the clinical utility of IM subtyping remains unclear, partially due to the absence of reliable defining biomarkers.MethodsBased on gene expression data and existing literature, we selected CD10 and Das1 as candidate biomarkers to distinguish complete and incomplete IM glands in tissues from patients without GC (IM-GC) and patients with GC (IM + GC). Immunohistochemical staining of individually subtyped IM glands was scored after blinding by two researchers using tissue belonging to both IM-GC and IM + GC patients. Whole tissue Das1 staining was further assessed using digital image quantification (cellSens Dimension, Olympus).ResultsAcross both cohorts CD10 stained the IM brush border and was shown to have a high sensitivity (87.5% and 94.9% in IM-GC and IM + GC patients respectively) and specificity (100.0% and 96.7% respectively) with an overall AUROC of 0.944 for complete IM glands. By contrast Das1 stained mainly goblet cells and the apical membrane of epithelial cells, mostly of incomplete IM glands with a low sensitivity (28.6% and 29.3% in IM-GC and IM + GC patients respectively) but high specificity (98.3% and 85.1% respectively) and an overall AUROC of 0.603 for incomplete IM glands. A combined logistic regression model showed a significant increase in AUROC for detecting complete IM glands (0.955 vs 0.970). Whole tissue digital quantification of Das1 staining showed a significant association with incomplete IM compared to complete IM, both in IM-GC and in IM + GC patients (p = 0.016 and p = 0.009 respectively, Mann–Whitney test and unpaired t test used). Additionally, complete IM in IM + GC patients exhibited significantly more Das1 staining than in IM-GC patients (p = 0.019, Mann–Whitney test).ConclusionsThese findings suggest that CD10 is an outstanding biomarker for complete IM and Das1 may be useful as a secondary biomarker for IM glands at greater risk of progression irrespective of IM subtype. Overall, the clinical use of these biomarkers could lead to improved patient stratification and targeted surveillance.

The use of targeted therapy lumacaftor/ivacaftor in patients with cystic fibrosis

Accepted, basic therapy of cystic fibrosis (CF), until recently, was symptomatic and aimed at slowing down pathological processes, mainly from the respiratory system and gastrointestinal tract, caused by a defect in the CFTR gene. New strategic opportunities have emerged since 2012 and are aimed at correcting a defect in a gene or its product. A mutation in the cystic fibrosis gene disrupts the function of the cystic fibrosis transmembrane conductance regulator protein (CFTR or CFTR in English transcription), which is located on the surface of the apical membrane of epithelial cells and functions as a chloride channel. The basic achievement for the new CF therapy was the discovery of small molecules that restore the processes of synthesis, transport to the membrane, or the functioning of the defective CFTR protein. The effectiveness of drugs that restore CFTR function is related to the ability of the molecules to deliver an adequate amount of the CFTR protein to the surface of the epithelial cell and/or improve its functional activity. Among them, correctors and potentiators for pharmacological modulation of ion transport are distinguished in clinical practice. Correctors are medicinal substances that allow the mutant CFTR protein to pass through the system of intracellular quality control and take the correct location on the apical membrane (with class II mutations). The action of potentiators is aimed at restoring (activating) the function of the ion channel formed by the mutant CFTR protein (class III–IV mutations). The purpose of this article is to analyze the literature sources, the results of clinical trials on the use of CFTR modulators, including combinations of a potentiator and a corrector. Their effectiveness and safety were evaluated. Literature sources (20) are used and own clinical observation is given. A positive assessment of the action of the modulator, the combination of a potentiator and a corrector, its safety and good tolerability is given. In conclusion, the place and indications for prescribing drugs of this class, the number of patients in the Russian Federation in need of such treatment are determined.

A Shaving Proteomic Approach to Unveil Surface Proteins Modulation of Multi-Drug Resistant Pseudomonas aeruginosa Strains Isolated From Cystic Fibrosis Patients.

Cystic fibrosis (CF) is the most common rare disease caused by a mutation of the CF transmembrane conductance regulator gene encoding a channel protein of the apical membrane of epithelial cells leading to alteration of Na+ and K+ transport, hence inducing accumulation of dense and sticky mucus and promoting recurrent airway infections. The most detected bacterium in CF patients is Pseudomonas aeruginosa (PA) which causes chronic colonization, requiring stringent antibiotic therapies that, in turn induces multi-drug resistance. Despite eradication attempts at the first infection, the bacterium is able to utilize several adaptation mechanisms to survive in hostile environments such as the CF lung. Its adaptive machinery includes modulation of surface molecules such as efflux pumps, flagellum, pili and other virulence factors. In the present study we compared surface protein expression of PA multi- and pan-drug resistant strains to wild-type antibiotic-sensitive strains, isolated from the airways of CF patients with chronic colonization and recent infection, respectively. After shaving with trypsin, microbial peptides were analyzed by tandem-mass spectrometry on a high-resolution platform that allowed the identification of 174 differentially modulated proteins localized in the region from extracellular space to cytoplasmic membrane. Biofilm assay was performed to characterize all 26 PA strains in term of biofilm production. Among the differentially expressed proteins, 17 were associated to the virulome (e.g., Tse2, Tse5, Tsi1, PilF, FliY, B-type flagellin, FliM, PyoS5), six to the resistome (e.g., OprJ, LptD) and five to the biofilm reservoir (e.g., AlgF, PlsD). The biofilm assay characterized chronic antibiotic-resistant isolates as weaker biofilm producers than wild-type strains. Our results suggest the loss of PA early virulence factors (e.g., pili and flagella) and later expression of virulence traits (e.g., secretion systems proteins) as an indicator of PA adaptation and persistence in the CF lung environment. To our knowledge, this is the first study that, applying a shaving proteomic approach, describes adaptation processes of a large collection of PA clinical strains isolated from CF patients in early and chronic infection phases.

Functional interplay between CFTR and pendrin: physiological and pathophysiological relevance.

The transport of chloride and bicarbonate across epithelia controls the pH and volume of the intracellular and luminal fluids, as well as the systemic pH and vascular volume. The anion exchanger pendrin (SLC26A4) and the cystic fibrosis transmembrane conductance regulator (CFTR) channel are expressed in the apical membrane of epithelial cells of various organs and tissues, including the airways, kidney, thyroid, and inner ear. While pendrin drives chloride reabsorption and bicarbonate, thiocyanate or iodide secretion within the apical compartment, CFTR represents a pathway for the apical efflux of chloride, bicarbonate, and possibly iodide. In the airways, pendrin and CFTR seems to be involved in alkalinization of the apical fluid via bicarbonate secretion, especially during inflammation, while CFTR also controls the volume of the apical fluid via a cAMP-dependent chloride secretion, which is stimulated by pendrin. In the kidney, pendrin is expressed in the cortical collecting duct and connecting tubule and co-localizes with CFTR in the apical membrane of β intercalated cells. Bicarbonate secretion occurs via pendrin, which also drives chloride reabsorption. A functional CFTR is required for pendrin activity. Whether CFTR stimulates pendrin via a direct molecular interaction or other mechanisms, or simply provides a pathway for chloride recycling across the apical membrane remains to be established. In the thyroid, CFTR and pendrin might have overlapping functions in driving the apical flux of iodide within the follicular lumen. In other organs, including the inner ear, the possible functional interplay between pendrin and CFTR needs to be explored.

Concentration of fecal β-defensin-2 in children with cystic fibrosis: how the inneral intestinal immune response?

Cystic fibrosis is a disease caused by mutations in a gene encoding CFTR-protein (Cystic Fibrosis Transmembrane conductance Regulator), located in the apical membrane of epithelial cells of the respiratory tract, intestines and pancreas. Defensins serve as important components of the innate human immune system, they play a key role in providing the first line of defense of a macroorganism against infection; they have high antimicrobial, antiviral, cytotoxic activity.Objective. To determine the values of fecal β-defensin-2 in children with cystic fibrosis and to reveal the dependence of its level on the exocrine function of the pancreas and the severity of the patient’s condition.Characteristics of children and research methods. The study included 57 children with cystic fibrosis, the average age was 20.93 ± 2.9 months. Cystic fibrosis was diagnosed on the basis of an increase in immunoreactive trypsin, sweat chlorides by Cook’s method (>60 meq / l). To assess the exocrine function of the pancreas the scientists determined the activity of fecal elastase. They evaluated the levels of fecal β-defensin-2 and calprotectin using a quantitative enzyme immunoassay.Results. The levels of fecal β-defensin-2 were increased (108.2 ± 11.3 ng / ml) in all children under examination. The researchers found no correlation between the levels of fecal β-defensin-2 and fecal elastase. The level of fecal calprotectin was significantly higher in the group of children with cystic fibrosis as compared to the control group. There was a significant correlation between the levels of fecal calprotectin and fecal β-defensin-2 (r=0.57; p <0.05), however, no correlations were found between the levels of fecal β-defensin-2 and fecal elastase. The group of children with a severe course of the disease demonstrated an increase in the level of fecal β-defensin-2, fecal calprotectin significantly more frequent.Conclusion. Children with cystic fibrosis demonstrated a significant increase in the concentration of β-defensin-2 as compared to the control group, which confirms the activation of the innate immune system of the intestinal mucosa. The researchers traced the relationship between high levels of fecal β-defensin-2 and the severity of the disease. The levels of fecal β-defensin-2 directly correlated with the concentration of fecal calprotectin and there was no correlation between the severity of pancreatic insufficiency and the concentration of fecal β-defensin-2.

Macrophages and Epithelial Cells Mutually Interact through NLRP3 to Clear Infection and Enhance the Gastrointestinal Barrier

Activation of the nod-like receptor protein 3 (NLRP3) leads to the release of the proinflammatory cytokine IL-1β, which then facilitates pathogen control by macrophages. The role of NLRPs in controlling infection of epithelial cells is not well understood. Our hypothesis was that activation of the NLRP3 inflammasome in colonic epithelial cells would promote macrophage-mediated epithelial recovery after infection with the pathogen Citrobacter rodentium. We devised a co-culture model using mouse colonic epithelial cells (CMT-93) and macrophages (J774A.1) during infection with C. rodentium. Inflammasome was activated using LPS and ATP and inhibited by YVAD. We assessed cytokine secretion (ELISA), macrophage recruitment and pathogen penetration (immunofluorescence), and epithelial barrier integrity (transepithelial electrical resistance). Macrophages were recruited to the apical membrane of epithelial cells, associated with tight junctions, promoted epithelial barrier recovery, and displaced C. rodentium. While NLRP3 was expressed in infected epithelial cells, IL-18 or IL-1β secretion remained unchanged. Supernatants from infected epithelial cells promoted infection clearance by macrophage; while this was inflammasome-independent, ATP significantly improved epithelial barrier recovery. The inflammasome appears to promote epithelial barrier function, independent of IL-18 and IL-1β secretion. Inflammasome activation in macrophages plays a dual role of promoting pathogen clearance and improving epithelial barrier integrity.

Epithelial sodium channel (ENaC) in GtoPdb v.2021.2

The epithelial sodium channels (ENaC) are located on the apical membrane of epithelial cells in the kidney tubules, lung, respiratory tract, male and female reproductive tracts, sweat and salivary glands, placenta, colon, and some other organs [9, 13, 22, 21, 42]. In these epithelia, Na+ ions flow from the extracellular fluid into the cytoplasm of epithelial cells via ENaC. The Na+ ions are then pumped out of the cytoplasm into the interstitial fluid by the Na+/K+ ATPase located on the basolateral membrane [36]. As Na+ is one of the major electrolytes in the extracellular fluid (ECF), osmolarity change initiated by the Na+ flow is accompanied by a flow of water accompanying Na+ ions [6]. Thus, ENaC has a central role in regulating ECF volume and blood pressure, primarily via its function in the kidney [37]. The expression of ENaC subunits, hence its activity, is regulated by the renin-angiotensin-aldosterone system, and other factors involved in electrolyte homeostasis [37, 30]. In the respiratory tract and female reproductive tract, large segments of the epithelia are composed of multi-ciliated cells. In these cells, ENaC is located along the entire length of the cilia that cover the cell surface [15]. Cilial location greatly increases ENaC density per cell surface and allows ENaC to serve as a sensitive regulator of osmolarity of the periciliary fluid throughout the whole depth of the fluid bathing the cilia [15]. In contrast to ENaC, CFTR (ion transporter defective in cystic fibrosis) is located on non-cilial cell-surface [15]. In the vas deferens segment of the male reproductive tract, the luminal surface is covered by microvilli and stereocilia projections with backbones composed of actin filament bundles [42]. In these cells, both ENaC and the water channel aquaporin AQP9 are localized on these projections and also in the basal and smooth muscle layers [42]. Thus, ENaC function is also essential for the clearance of respiratory airways, transport of germ cells, fertilization, implantation, and cell migration [15, 22].

Villin expression in human tumours: a tissue microarray study on 12,429 tumours

Villin protein is part of the brush border of the apical membrane of epithelial cells. Villin is used as an immunohistochemical marker for colorectal and gastrointestinal neoplasms although other tumour entities can also express villin. To comprehensively determine villin expression in normal and neoplastic tissues, a tissue microarray containing >14,000 samples from 118 different tumour types and subtypes as well as 608 samples of 76 different normal tissue types was analysed. Villin positivity was found in 54 of tumour categories and was most commonly seen in adenocarcinomas and neuroendocrine tumours of the colorectum (60–100%), upper gastrointestinal tract (61–100%), bilio-pancreatic system (25–86%), yolk sack tumours (76%), and in mucinous carcinomas of the ovary (67%). Villin was also common in various categories of extra-gastrointestinal neuroendocrine neoplasms (22–41%), kidney tumours (up to 18%), hepatocellular carcinoma (25%), endometroid carcinoma of the ovary (20%) and of the endometrium (12%) as well as in adenocarcinomas of the lung (12%). These data demonstrate that villin is frequently expressed in gastrointestinal, biliopancreatic, and neuroendocrine neoplasms, but at least weak villin positivity occurred in 54 different tumour categories. Villin immunohistochemistry should, thus, be applied as a part of a panel for the distinction of tumours.

Mechanisms of Epidermal Growth Factor Effect on Animal Intestinal Phosphate Absorption: A Review.

Phosphorus is one of the essential mineral elements of animals that plays an important role in animal growth and development, bone formation, energy metabolism, nucleic acid synthesis, cell signal transduction, and blood acid–base balance. It has been established that the Type IIb sodium-dependent phosphate cotransporters (NaPi-IIb) protein is the major sodium-dependent phosphate (Pi) transporter, which plays an important role in Pi uptake across the apical membrane of epithelial cells in the small intestine. Previous studies have demonstrated that epidermal growth factor (EGF) is involved in regulating intestinal Pi absorption. Here we summarize the effects of EGF on active Pi transport of NaPi-IIb under different conditions. Under normal conditions, EGF inhibits the active transport of Pi by inhibiting the expression of NaPi-IIb, while, under intestinal injury condition, EGF promotes the active absorption of Pi through upregulating the expression of NaPi-IIb. This review provides a reference for information about EGF-regulatory functions in Pi absorption in the animal intestine.

Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed on the apical membrane of epithelial cells, where it plays a pivotal role in chloride transport and overall tissue homeostasis. CFTR constitutes a unique member of the ATP-binding cassette transporter superfamily, due to its distinctive cytosolic regulatory (R) domain carrying multiple phosphorylation sites that allow the tight regulation of channel activity and gating. Mutations in the CFTR gene cause cystic fibrosis, the most common lethal autosomal genetic disease in the Caucasian population. In recent years, major efforts have led to the development of CFTR modulators, small molecules targeting the underlying genetic defect of CF and ultimately rescuing the function of the mutant channel. Recent evidence has highlighted that this class of drugs could also impact on the phosphorylation of the R domain of the channel by protein kinase A (PKA), a key regulatory mechanism that is altered in various CFTR mutants. Therefore, the aim of this review is to summarize the current knowledge on the regulation of the CFTR by PKA-mediated phosphorylation and to provide insights into the different factors that modulate this essential CFTR modification. Finally, the discussion will focus on the impact of CF mutations on PKA-mediated CFTR regulation, as well as on how small molecule CFTR regulators and PKA interact to rescue dysfunctional channels.

Loss of Myosin Vb Results in Decreased Apical P‐Glycoprotein in vivo and in vitro

Background & Aims P-glycoprotein (p-gp), also known as multidrug resistance protein 1, is a xenobiotic efflux pump. In the small intestine, p-gp is localized to the apical membrane of enterocytes. Changes in p-gp expression is linked to inflammatory bowel diseases (IBD) and impairment of drug response in cancer. Regulating the trafficking of p-gp to the plasma membrane represents a plausible approach to improve therapy for cancer treatment. At present, the trafficking mechanism responsible for delivery of p-gp to the apical membrane of epithelial cells is unclear. Myosin Vb (Myo5b) is a molecular motor that is crucial for the proper transport of vesicles and proteins to the apical membrane of epithelial cells. Loss of functional Myo5b results in a congenital diarrheal disorder termed Microvillus Inclusion Disease (MVID). Individuals with MVID present with severe, unremitting diarrhea requiring parenteral nutrition and eventual small bowel transplantation. Studies using animal models of MVID and MVID patient tissue have revealed that loss of Myo5b results in a dramatic decrease in key transporters on the apical membrane of enterocytes. Therefore, we investigated whether proper localization of p-glycoprotein was impeded by loss of Myo5b. Methods Intestinal tissue was analyzed from neonatal control and germline Myo5b knockout (Myo5b KO) mice. Additionally, a tamoxifen inducible, intestinal specific mouse model of loss of Myo5b was also used (VillinCreERT2;Myo5bflox/flox). Adult Myo5bflox/flox mice treated with tamoxifen were used as controls for VillinCreERT2;Myo5bflox/flox mice. Intestinal organoids were generated from VillinCreERT2;Myo5bflox/flox mice and treated with either ethanol as a control or 4 hydroxy tamoxifen to induce loss of Myo5b. Results Immunostaining of gamma actin staining was performed to delineate the actin rich microvilli and identify the apical membrane. In control mice, p-gp was restricted to the apical membrane and co-localized with gamma actin as expected. In germline Myo5b KO mice, p-gp had increased immunofluorescence intensity and was observed below the apical membrane of small intestinal enterocytes. Lamp1 immunostaining also revealed that subapical p-gp was surrounded by lamp1 positive lysosomes in Myo5b KO mice. The association of lysosomes with p-gp suggests that improper localization of p-gp resulting from loss of Myo5b may promote lysosomal degradation of p-gp. Adult Myo5bflox/flox control mice treated with tamoxifen showed normal distribution of p-gp on the apical membrane of small intestinal enterocytes demonstrating co-localization with gamma actin. In contrast, tamoxifen induced VillinCreERT2;Myo5bflox/flox mice showed no p-gp at the apical brush border and no overlap with gamma actin immunostaining. Tamoxifen induced VillinCreERT2;Myo5bflox/flox mice displayed a large accumulation of p-gp below the apical membrane. Complementing our in vivo data, in vitro induction of Myo5b loss using 4 hydroxy tamoxifen resulted in a similar subapical localization of p-gp in induced VillinCreERT2;Myo5bflox/flox intestinal organoids compared to ethanol treated organoids. Conclusions Loss of Myo5b results in improper localization of p-gp. Collectively, these data suggest that Myo5b is necessary for the proper trafficking of p-gp to the apical membrane of intestinal cells.

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl− and HCO3−. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E2 (PGE2), a major prostaglandin in the semen. Luminal administration of PGE2 triggered a remarkable and sustained increase of short-circuit current (ISC) in rat vaginal epithelium, which was mainly due to Cl− and HCO3− secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the ISC response evoked by PGE2, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl− transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl− signals. Conclusively, T. vaginalis restrained exogenous PGE2-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.