Chloride transport modulators as drug candidates.

Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.

Antiviral Drug Ivermectin at Nanomolar Concentrations Inhibits Glycine-Induced Chloride Current in Rat Hippocampal Neurons.

Ivermectin (IVM) belongs to the class of macrocyclic lactones, which is used as an antiparasitic agent. At present, the researchers focus on possibility to use IVM in treatment of certain forms of cancer and viral diseases such as COVID-19. The mechanisms of IVM action are not clear. It is assumed that IVM affects chloride channels and increases cytoplasmic concentration of chloride. This study examines the effect of IVM on chloride currents induced by glycine (IGly). Experiments were carried out on isolated pyramidal neurons of the rat hippocampus with whole-cell patch clamp. A short-term (600 msec) application of IVM in a concentration of 10 µM induced a slow inward current, which persisted after washing the neurons. The low concentrations (0.1-1000 nM) of IVM did not induce any novel current, but it rapidly and reversibly reduced the peak amplitude and accelerated desensitization of IGly in a dose-dependent manner. The threshold concentrations of IVM sufficient to reduce peak amplitude of IGly and to accelerate desensitization of IGly were 100 nM and 0.1 nM, respectively. The study revealed a high sensitivity of neuronal glycine receptors to IVM.

Ivermectin: An Anthelmintic, an Insecticide, and Much More.

Here we tell the story of ivermectin, describing its anthelmintic and insecticidal actions and recent studies that have sought to reposition ivermectin for the treatment of other diseases that are not caused by helminth and insect parasites. The standard theory of its anthelmintic and insecticidal mode of action is that it is a selective positive allosteric modulator of glutamate-gated chloride channels found in nematodes and insects. At higher concentrations, ivermectin also acts as an allosteric modulator of ion channels found in host central nervous systems. In addition, in tissue culture, at concentrations higher than anthelmintic concentrations, ivermectin shows antiviral, antimalarial, antimetabolic, and anticancer effects. Caution is required before extrapolating from these preliminary repositioning experiments to clinical use, particularly for Covid-19 treatment, because of the high concentrations of ivermectin used in tissue-culture experiments.

G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations.

G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.

Regulation of Cl- Electrolyte Permeability in Epithelia by Active Traditional Chinese Medicine Monomers for Diarrhea.

The epithelial layer, lining the inner surface of the mammalian alveolar, kidney, brain and colon, is a typical electrolyte transporting tissue. Large quantities of salt and fluid are actively moved from the mucosal side toward the blood vessel. Transepithelial salt re-absorption in epithelial tissues plays an important role in maintaining fluid homeostasis. In absorptive epithelium, fluid and salt flux is controlled by the machinery mainly composed of epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl- cotransporter, Na+/H+ exchanger, and Na+/K+-ATPase. Dysregulation of salt permeability across epithelium contributes to the pathogenesis of organ edema. In numerous ion transporters, epithelial Cl- transportation plays an important role in water secretion across epithelial tissues and regulation of body fluid content. Many traditional Chinese medicines treat diarrhea by regulating the Cl- electrolyte transport. We systematically summarized the recent progress regarding the traditional Chinese medicine on Cl- electrolyte transport in the intestinal epithelial tissues. The pharmaceutical relevance of developing advanced strategies to mitigate edematous disorders is also implicated. In conclusion, the crosstalk between Cl- electrolyte transport and active traditional Chinese medicine monomers may lead to the development of new strategies for diarrhea by manipulating the function and expression of ion channels.

PAC proton-activated chloride channel contributes to acid-induced cell death in primary rat cortical neurons.

Severe local acidosis causes tissue damage and pain, and is associated with many diseases, including cerebral and cardiac ischemia, cancer, infection, and inflammation. However, the molecular mechanisms of the cellular response to extracellular acidic environment are not fully understood. We recently identified a novel and evolutionarily conserved membrane protein, PAC (also known as PACC1 or TMEM206), encoding the proton-activated chloride (Cl-) channel, whose activity is widely observed in human cell lines. We demonstrated that genetic deletion of Pac abolished the proton-activated Cl- currents in mouse neurons and also attenuated the acid-induced neuronal cell death and brain damage after ischemic stroke. Here, we show that the proton-activated Cl- currents are also conserved in primary rat cortical neurons, with characteristics similar to those observed in human and mouse cells. Pac gene knockdown nearly abolished the proton-activated Cl- currents in rat neurons and reduced the neuronal cell death triggered by acid treatment. These data further support the notion that activation of the PAC channel and subsequent Cl- entry into neurons during acidosis play a pathogenic role in acidotoxicity and brain injury.

Nanomolar Potency Aminophenyltriazine CFTR Activator Reverses Corneal Epithelial Injury in a Mouse Model of Dry Eye.

Purpose: Dry eye disorders are a major health care burden. We previously reported the identification of N-methyl-N-phenyl-6-(2,2,3,3-tetrafluoropropoxy)-1,3,5-triazine-2,4-diamine [cystic fibrosis transmembrane conductance regulator (CFTR)act-K267], which activated human wild-type CFTR chloride conductance with EC50 ∼ 30 nM. Here, we report in vivo evidence for CFTRact-K267 efficacy in an experimental mouse model of dry eye using a human compatible ophthalmic vehicle. Methods: CFTR activation in mice in vivo was demonstrated by ocular surface potential difference (OSPD) measurements. Ocular surface pharmacodynamics was measured in tear fluid samples obtained at different times after topical administration of CFTRact-K267. Dry eye was produced by lacrimal duct cautery (LDC) and corneal epithelial injury and was assessed by Lissamine green (LG) staining. Results: OSPD measurements demonstrated a hyperpolarization of -8.6 ± 3 mV (standard error of the mean, 5 mice) in response to CFTRact-K267 exposure in low chloride solution that was reversed by a CFTR inhibitor. Following single-dose topical administration of 2 nmol CFTRact-K267, tear fluid CFTRact-K267 concentration was >500 nM for more than 6 h. Following LDC, corneal surface epithelial injury, as assessed by LG staining, was substantially reversed in 10 of 12 eyes receiving 2 nmol CFTRact-K267 3 times daily starting on day 2, when marked epithelial injury had already occurred. Improvement was seen in 3 of 12 vehicle-treated eyes. Conclusion: These studies provide in vivo evidence in mice for the efficacy of a topical, human use compatible CFTRact-K267 formulation in stimulating chloride secretion and reversing corneal epithelial injury in dry eye.

Insights into the impact of Ivermectin on some protein aspects linked to Culex pipiens digestion and immunity.

In developing countries, low-cost control and treatment programs that offer combined approaches against diseases and their vectors are certainly needed. Ivermectin (IVM) has been well known for its role in the treatment of parasitic diseases, due to its effect on glutamate-gated chloride channels. These same channels are also present in the mosquito vector, and thus, research has focused on the insecticidal effects of this drug. Possible alternative mechanisms of IVM on the physiology of mosquitoes, however, have not been sufficiently elaborated. We assessed the protease activity, lipid peroxidation, and local expression of STAT, p53, caspase-3, and Bax markers to study the effect of this antibiotic on digestion and immunity in Culex pipiens. Sugar- and blood-feeding assays were employed to investigate the potential influence of blood feeding on the dynamics of these parameters. IVM was found to have an effect on protease activity, lipid peroxidation as well as the expression of different markers investigated in this work. The focus on the detailed effect of this drug certainly opens the gate to broadening the spectrum of IVM and expanding its health and economic benefit, especially that it is relatively more affordable than other antibiotics on the market.

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties.

There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.

Molecular, biophysical, and pharmacological properties of calcium-activated chloride channels.

Calcium-activated chloride channels (CaCCs) are a family of anionic transmembrane ion channels. They are mainly responsible for the movement of Cl- and other anions across the biological membranes, and they are widely expressed in different tissues. Since the Cl- flow into or out of the cell plays a crucial role in hyperpolarizing or depolarizing the cells, respectively, the impact of intracellular Ca2+ concentration on these channels is attracting a lot of attentions. After summarizing the molecular, biophysical, and pharmacological properties of CaCCs, the role of CaCCs in normal cellular functions will be discussed, and I will emphasize how dysregulation of CaCCs in pathological conditions can account for different diseases. A better understanding of CaCCs and a pivotal regulatory role of Ca2+ can shed more light on the therapeutic strategies for different neurological disorders that arise from chloride dysregulation, such as asthma, cystic fibrosis, and neuropathic pain.

Activation of ClC-3 chloride channel by 17ß-estradiol relies on the estrogen receptor α expression in breast cancer.

Although extensively studied, the mechanisms by which estrogen promotes breast cancer growth remain to be fully elucidated. Tamoxifen, an antiestrogen agent to treat ERα+ breast cancer, is also a high-affinity blocker of the chloride channels. In this study, we explored the involvement of the chloride channels in the action of estrogen in breast cancer. We found that 17ß-estradiol (17ß-E2) concentration-dependently activated the chloride currents in ERα+ breast cancer MCF-7 cells. Extracellular hypertonic challenge and chloride channel blockers, NPPB and DIDS inhibited the 17ß-E2-activated chloride currents. Decreased the ClC-3 protein expression caused the depletion of the 17ß-E2-activated chloride currents. 17ß-E2-activated chloride currents which relied on the ERα expression were demonstrated by the following evidences. Firstly, 17ß-E2-activated chloride currents could not be observed in ERα- breast cancer MDA-MB-231 cells. Secondly, ER antagonists, tamoxifen and ICI 182,780, and downregulation of ERα expression inhibited or abolished the 17ß-E2-activated chloride currents. Thirdly, ERα expression was induced in MDA-MB-231 cells by ESR1 gene transfection, and then 17ß-E2-activated chloride currents could be observed. In MCF-7 cells, ERα and ClC-3 mainly located in nucleus and translocated to cell plasma and membrane with respect to co-localization following treatment of 17ß-E2. Downregulation of ERα expression could decrease the expression of ClC-3 protein. Conversely, downregulation of ClC-3 expression did not influence the ERα expression. Taken together, our findings demonstrated that ClC-3 is a potential target of 17ß-E2 and is modulated by the ERα in breast cancer cell. Pharmacological modulation of ClC-3 may provide a deep understanding in antiestrogen treatment of breast cancer patients.

Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Altered ion transport in normal human bronchial epithelial cells following exposure to chemically distinct metal welding fume particles.

Welding fume inhalation causes pulmonary toxicity, including susceptibility to infection. We hypothesized that airway epithelial ion transport is a target of fume toxicity, and investigated the effects of fume particulates from manual metal arc-stainless steel (MMA-SS) and gas metal arc-mild steel (GMA-MS) on ion transport in normal human bronchial epithelium (NHBE) cultured in air-interface. MMA-SS particles, more soluble than GMA-MS particles, contain Cr, Ni, Fe and Mn; GMA-MS particles contain Fe and Mn. MMA-SS or GMA-MS particles (0.0167-166.7µg/cm2) were applied apically to NHBEs. After 18h transepithelial potential difference (Vt), resistance (Rt), and short circuit current (Isc) were measured. Particle effects on Na+ and Cl¯ channels and the Na+,K+,2Cl¯-cotransporter were evaluated using amiloride (apical), 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, apical), and bumetanide (basolateral), respectively. MMA-SS (0.0167-16.7µg/cm2) increased basal Vt. Only 16.7µg/cm2 GMA-MS increased basal Vt significantly. MMA-SS or GMA-MS exposure potentiated Isc responses (decreases) to amiloride and bumetanide, while not affecting those to NPPB, GMA-MS to a lesser degree than MMA-SS. Variable effects on Rt were observed in response to amiloride, and bumetanide. Generally, MMA-SS was more potent in altering responses to amiloride and bumetanide than GMA-MS. Hyperpolarization occurred in the absence of LDH release, but decreases in Vt, Rt, and Isc at higher fume particulate doses accompanied LDH release, to a greater extent for MMA-SS. Thus, Na+ transport and Na+,K+,2Cl¯-cotransport are affected by fume exposure; MMA-MS is more potent than GMA-MS. Enhanced Na+ absorption and decreased airway surface liquid could compromise defenses against infection.

Regulating effect of TongXie-YaoFang on colonic epithelial secretion via Cl- and HCO3- channel.

AIM: To investigate the pharmacological effect of TongXie-YaoFang (TXYF) formula, a Chinese herbal formula, on Diarrhea-predominant irritable bowel syndrome (D-IBS) rats. METHODS: In a neonatal maternal separation plus restraint stress (NMS + RS) model of D-IBS, male Sprague Dawley rats were randomly divided into two groups (NMS + RS group and TXYF-formula group) with no handlings were used as controls (NH group). Starting from postnatal day 60, rats in TXYF-formula group were administered TXYF-formula (4.92 g/100 g bodyweight) orally twice a day for 14 consecutive days while NH group and NMS + RS group were given distilled water. Using short-circuit current technology, we observed 5-HT-induced changes of current across ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, epithelial Na+ channel (ENaC), Ca2+-dependent Cl- channel (CACC), Na+-K+-2Cl- co-transporter (NKCC), and Na+-HCO3- co-transporter (NBC), in the colonic epithelium of three groups after exposure to drugs and specific blockers with a Power Lab System (AD Instruments International). RESULTS: Under basal conditions, the changes of short-circuit current (∆Isc, µA/cm2) induced by 5-HT were similar in NH group and TXYF-formula group, and both higher than NMS + RS group (70.86 µA/cm2 ± 12.32 µA/cm2, 67.67 µA/cm2 ± 11.68 µA/cm2vs 38.8 µA/cm2 ± 7.25 µA/cm2, P < 0.01, respectively). When CACC was blocked by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-HT-induced ∆Isc was smaller in NMS + RS group than in NH group and TXYF-formula group, respectively (48.41 µA/cm2 ± 13.15 µA/cm2vs 74.62 µA/cm2 ± 10.73 µA/cm2, 69.22 µA/cm2 ± 11.7 µA/cm2, P < 0.05, respectively). The similar result could be obtained when ENaC was blocked by Amiloride (44.69 µA/cm2 ± 12.58 µA/cm2vs 62.05 µA/cm2 ± 11.26 µA/cm2, 62.11 µA/cm2 ± 12.01 µA/cm2, P < 0.05, respectively). However, when CFTR Cl- channel was blocked by 1,1-dimethyl piperidinium chloride (DPC), 5-HT-induced ∆Isc did not significantly differ in three groups (42.28 µA/cm2 ± 10.61 µA/cm2vs 51.48 µA/cm2 ± 6.56 µA/cm2vs 47.75 µA/cm2 ± 7.99 µA/cm2, P > 0.05, respectively). The similar results could also be obtained in three groups when NBC and NKCC were respectively blocked by their blockers. CONCLUSION: TXYF-formula can regulate the Cl- and HCO3- secretion of colonic mucosa via CFTR Cl- channel, Cl-/HCO3- exchanger, NBC and NKCC co-transporters.

Walker 256 tumor-bearing rats demonstrate altered interstitial cells of Cajal. Effects on ICC in the Walker 256 tumor model.

BACKGROUND: Cachexia is a significant problem in patients with cancer. The effect of cancer on interstitial cells of Cajal (ICC) and neurons of the gastrointestinal tract have not been studied previously. Although supplementation with L-glutamine 2% may have beneficial effects in cancer-related cachexia, and be protective of ICC in models of oxidative stress such as diabetes, its effects on ICC in cancer have also not been studied. METHODS: Twenty-eight male Wistar rats were divided into four groups: control (C), control supplemented with L-glutamine (CG), Walker 256 tumor (WT), and Walker 256 tumor supplemented with L-glutamine (WTG). Rats were implanted with tumor cells or injected with saline in the right flank. After 14 days, the jejunal tissues were collected and processed for immunohistochemical techniques including whole mounts and cryosections and Western blot analysis. KEY RESULTS: Tumor-bearing rats demonstrate reduced numbers of Myenteric ICC and deep muscular plexus ICC and yet increased Ano1 protein expression and enhanced ICC networks. In addition, there is more nNOS protein expressed in tumor-bearing rats compared to controls. L-glutamine treatment had a variety of effects on ICC that may be related to the disease state and the interaction of ICC and nNOS neurons. Regardless, L-glutamine reduced the size of tumors and also tumor-induced cachexia that was not due to altered food intake. CONCLUSIONS & INFERENCES: There are significant effects on ICC in the Walker 256 tumor model. Although supplementation with L-glutamine has differential and complex effects of ICC, it reduces tumor size and tumor-associated cachexia, which supports its beneficial therapeutic role in cancer.

Targeting ion channels in cystic fibrosis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause a characteristic defect in epithelial ion transport that plays a central role in the pathogenesis of cystic fibrosis (CF). Hence, pharmacological correction of this ion transport defect by targeting of mutant CFTR, or alternative ion channels that may compensate for CFTR dysfunction, has long been considered as an attractive approach to a causal therapy of this life-limiting disease. The recent introduction of the CFTR potentiator ivacaftor into the therapy of a subgroup of patients with specific CFTR mutations was a major milestone and enormous stimulus for seeking effective ion transport modulators for all patients with CF. In this review, we discuss recent breakthroughs and setbacks with CFTR modulators designed to rescue mutant CFTR including the common mutation F508del. Further, we examine the alternative chloride channels TMEM16A and SLC26A9, as well as the epithelial sodium channel ENaC as alternative targets in CF lung disease, which remains the major cause of morbidity and mortality in patients with CF. Finally, we will focus on the hurdles that still need to be overcome to make effective ion transport modulation therapies available for all patients with CF irrespective of their CFTR genotype.

Efficacy and safety of ivacaftor in patients with cystic fibrosis who have an Arg117His-CFTR mutation: a double-blind, randomised controlled trial.

BACKGROUND: Ivacaftor has been previously assessed in patients with cystic fibrosis with Gly551Asp-CFTR or other gating mutations. We assessed ivacaftor in patients with Arg117His-CFTR, a residual function mutation. METHODS: We did a 24-week, placebo-controlled, double-blind, randomised clinical trial, which enrolled 69 patients with cystic fibrosis aged 6 years and older with Arg117His-CFTR and percentage of predicted forced expiratory volume in 1 s (% predicted FEV1) of at least 40. We randomly assigned eligible patients (1:1) to receive placebo or ivacaftor 150 mg every 12 h for 24 weeks. Randomisation was stratified by age (6-11, 12-17, and ≥18 years) and % predicted FEV1 (<70, ≥70 to ≤90, and >90). The primary outcome was the absolute change from baseline in % predicted FEV1 through week 24. Secondary outcomes included safety and changes in sweat chloride concentrations and Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain scores. An open-label extension enrolled 65 of the patients after washout; after 12 weeks, we did an interim analysis. FINDINGS: After 24 weeks, the treatment difference in mean absolute change in % predicted FEV1 between ivacaftor (n=34) and placebo (n=35) was 2·1 percentage points (95% CI -1·13 to 5·35; p=0·20). Ivacaftor treatment resulted in significant treatment differences in sweat chloride (-24·0 mmol/L, 95% CI -28·01 to -19·93; p<0·0001) and CFQ-R respiratory domain (8·4, 2·17 to 14·61; p=0·009). In prespecified subgroup analyses, % predicted FEV1 significantly improved with ivacaftor in patients aged 18 years or older (treatment difference vs placebo: 5·0 percentage points, 95% CI 1·15 to 8·78; p=0·01), but not in patients aged 6-11 years (-6·3 percentage points, -11·96 to -0·71; p=0·03). In the extension study, both placebo-to-ivacaftor and ivacaftor-to-ivacaftor groups showed % predicted FEV1 improvement (absolute change from post-washout baseline at week 12: placebo-to-ivacaftor, 5·0 percentage points [p=0·0005]; ivacaftor-to-ivacaftor, 6·0 percentage points [p=0·006]). We did not identify any new safety concerns. The studies are registered with ClinicalTrials.gov (the randomised, placebo-controlled study, number NCT01614457; the open-label extension study, number NCT01707290). INTERPRETATION: Although this study did not show a significant improvement in % predicted FEV1, ivacaftor did significantly improve sweat chloride and CFQ-R respiratory domain scores and lung function in adult patients with Arg117His-CFTR, indicating that ivacaftor might benefit patients with Arg117His-CFTR who have established disease. FUNDING: Vertex Pharmaceuticals Incorporated.

Resveratrol ameliorates abnormalities of fluid and electrolyte secretion in a hypoxia-Induced model of acquired CFTR deficiency.

OBJECTIVE/HYPOTHESIS: Ineffective mucociliary clearance (MCC) is a common pathophysiologic process that underlies airway inflammation and infection. A dominant fluid and electrolyte secretory pathway in the nasal airways is governed by the cystic fibrosis transmembrane conductance regulator (CFTR). Decreased transepithelial Cl(-) transport secondary to an acquired CFTR deficiency may exacerbate respiratory epithelial dysfunction by diminishing MCC and increasing mucus viscosity. The objectives of the present study are to 1) develop a model of acquired CFTR deficiency in sinonasal epithelium using hypoxia, 2) investigate whether the polyphenol resveratrol promotes CFTR-mediated anion transport, 3) explore resveratrol mechanism of action and determine therapeutic suitability for overcoming acquired CFTR defects, and 4) test the drug in the hypoxic model of acquired CFTR deficiency in preparation for a clinical trial in human sinus disease. We hypothesize that hypoxia will induce depletion of airway surface liquid (ASL) secondary to acquired CFTR deficiency and that resveratrol will restore transepithelial Cl(-) secretion and recover ASL hydration. STUDY DESIGN: Basic science. METHODS: Murine nasal septal (MNSE) and human sinonasal epithelial (HSNE) cultures were incubated under hypoxic conditions (1% O2 , 5% CO2 ) and transepithelial ion transport (change in short-circuit current = ΔISC ) evaluated in Ussing chambers. Resveratrol was tested using primary cells and HEK293 cells expressing human CFTR by Ussing chamber and patch clamp techniques under both phosphorylating and nonphosphorylating conditions. CFTR activation was evaluated in human explants and by murine in vivo (nasal potential difference) assessment. Cellular cyclic adenosine monophosphate (cAMP) (ELISA) and subsequent CFTR regulatory domain (R-D) phosphorylation (gel-shift assay) were also evaluated. Effects of hypoxia and resveratrol on ASL were tested using confocal laser scanning microscopy (CLSM) and micro-optical coherence tomography (µOCT). RESULTS: Hypoxia significantly decreased ΔISC (in µA/cm(2) ) attributable to CFTR at 12 and 24 hours of exposure in both MNSE (13.55 ± 0.46 [12 hours]; 12.75 ± 0.07 [24 hours] vs. 19.23 ± 0.18 [control]; P < 0.05) and HSNE (19.55 ± 0.56 [12 hours]; 17.67 ± 1.13 [24 hours] vs. 25.49 ± 1.48 [control]; P < 0.05). We have shown that resveratrol (100 µM) enhanced CFTR-dependent Cl(-) secretion in HSNE to an extent comparable to the recently Food and Drug Administration-approved CFTR potentiator, ivacaftor. Cl(-) transport across human sinonasal explants (78.42 ± 1.75 vs. 1.75 ± 1.5 [control]; P < 0.05) and in vivo murine nasal epithelium (-4 ± 1.8 vs. -0.8 ± 1.7 mV [control]; P < 0.05) were also significantly increased by the drug. No increase in cAMP or CFTR R-D phosphorylation was detected. Inside-out patches showed increased CFTR open probability (NPo/N (N = channel number]) compared to controls in both MNSE (0.329 ± 0.116 vs. 0.119 ± 0.059 [control]; P < 0.05) and HEK293 cells (0.22 ± 0.048 vs. 0.125 ± 0.07 [control]; P < 0.05). ASL thickness was decreased under hypoxic conditions when measured by CLSM (4.19 ± 0.44 vs. 6.88 ± 0.67 [control]; P < 0.05). A 30-minute apical application of resveratrol increased ASL depth in normal epithelium (8.08 ± 1.68 vs. 6.11 ± 0.47 [control]; P < 0.05). Furthermore, hypoxia-induced abnormalities of fluid and electrolyte secretion in sinonasal epithelium were restored with resveratrol treatment (5.55 ± 0.74 vs. 3.13 ± 0.17 [control]; P < 0.05). CONCLUSIONS: CFTR activation with a leading edge Cl(-) secretagogue such as resveratrol represents an innovative approach to overcoming acquired CFTR defects in sinus and nasal airway disease. This exciting new strategy bears further testing in non-CF individuals with chronic rhinosinusitis. LEVEL OF EVIDENCE: N/A. Laryngoscope, 125:S1-S13, 2015.

Effect of entacapone on colon motility and ion transport in a rat model of Parkinson's disease.

AIM: To study the effects of entacapone, a catechol-O-methyltransferase inhibitor, on colon motility and electrolyte transport in Parkinson's disease (PD) rats. METHODS: Distribution and expression of catechol-O-methyltransferase (COMT) were measured by immunohistochemistry and Western blotting methods. The colonic smooth muscle motility was examined in vitro by means of a muscle motility recording device. The mucosal electrolyte transport of PD rats was examined by using a short-circuit current (ISC ) technique and scanning ion-selective electrode technique (SIET). Intracellular detection of cAMP and cGMP was accomplished by radioimmunoassay testing. RESULTS: COMT was expressed in the colons of both normal and PD rats, mainly on the apical membranes of villi and crypts in the colon. Compared to normal controls, PD rats expressed less COMT. The COMT inhibitor entacapone inhibited contraction of the PD rat longitudinal muscle in a dose-dependent manner. The ß2 adrenoceptor antagonist ICI-118,551 blocked this inhibitory effect by approximately 67% (P < 0.01). Entacapone increased mucosal ISC in the colon of rats with PD. This induction was significantly inhibited by apical application of Cl(-) channel blocker diphenylamine-2, 2'-dicarboxylic acid, basolateral application of Na(+)-K(+)-2Cl(-)co-transporter antagonist bumetanide, elimination of Cl(-) from the extracellular fluid, as well as pretreatment using adenylate cyclase inhibitor MDL12330A. As an inhibitor of prostaglandin synthetase, indomethacin can inhibit entacapone-induced ISC by 45% (P < 0.01). When SIET was applied to measure Cl(-) flux changes, this provided similar results. Entacapone significantly increased intracellular cAMP content in the colonic mucosa, which was greatly inhibited by indomethacin. CONCLUSION: COMT expression exists in rat colons. The ß2 adrenoceptor is involved in the entacapone-induced inhibition of colon motility. Entacapone induces cAMP-dependent Cl(-) secretion in the PD rat.

Cyclic AMP prevents decrease of phosphorylated ezrin/radixin/moesin and chloride intracellular channel 5 expressions in injured podocytes.

BACKGROUND: Our previous in vitro studies suggested that cyclin AMP (cAMP) signaling protects against podocyte injury. However, the molecular mechanisms remain unknown. The aim of the present study was to explore the role of forskolin, an agonist for adenylate cyclase, on ezrin/radixin/moesin (ERM) phosphorylation and chloride intracellular channel 5 (CLIC5) expressions in injured podocytes. METHODS: ADR nephrosis model were induced by adriamycin (ADR) injection in BalB/C mice. Parts of ADR nephrosis mice were pretreated with forskolin. Albuminuria was estimated by urine Coomassie blue stain. Nephrin, synaptopodin, CLIC5, phosphorylated ERM and podocalyxin were measured by confocal microscopy. CLIC5 and phosphorylated ERM also were studied using western blotting. RhoA and Rac1 were estimated by G-Lisa kit. RESULTS: We found that forskolin partially alleviated albuminuria and width of foot processes. Nephrin, synaptopodin, phosphorylated-ERM (p-ERM) and CLIC5 expression were decreased in ADR mice, which were improved by forskolin pretreatment. In vitro studies, pretreatment of podocytes with pCPT-cAMP(PKA-selective cAMP analogue)prevented puromycin aminonucleoside (PAN)-induced CLIC5 downregulation. 8-pCPT-2'-O-Me-cAMP (2Me-cAMP, an Epac-selective cAMP analogue) blocked PAN-induced p-ERM downregulation. PAN inhibited RhoA activation in podocytes, which could be prevented by pCPT-cAMP pretreatment. Y-27632, a Rho inhibitor, decreased CLIC5 expression in podocytes. CONCLUSION: Activation cAMP signaling might attenuate albuminuria in ADR-induced nephrosis mice. Different downstream signaling pathway might mediate cAMP protection on CLIC5 and p-ERM expression, respectively.