Jay R. Thiagarajah

Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin–induced intestinal fluid secretion

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

Chloride Concentration in Endosomes Measured Using a Ratioable Fluorescent Cl− Indicator EVIDENCE FOR CHLORIDE ACCUMULATION DURING ACIDIFICATION

A novel long wavelength fluorescent Cl− indicator was used to test whether endosomal Cl− conductance provides the principal electrical shunt to permit endosomal acidification. The green fluorescent Cl−-sensitive chromophore 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate (BAC) was conjugated to aminodextran together with the red fluorescent Cl−-insensitive chromophore tetramethylrhodamine (TMR). BAC fluorescence is pH-insensitive and quenched by Cl−with a Stern-Volmer constant of 36 m −1. Endosomes in J774 and Chinese hamster ovary (CHO) cells were pulse-labeled with BAC-TMR-dextran by fluid-phase endocytosis. Endosomal [Cl−] increased over 45 min from 17 to 53 mm in J774 cells and from 28 to 73 mm in CHO cells, during which time endosomal pH decreased from 6.95 to 5.30 (J774) and 6.92 to 5.60 (CHO). The acidification and increased [Cl−] were blocked by bafilomycin. Together with ion substitution and buffer capacity measurements, we conclude that Cl− transport accounts quantitatively for the electrical shunt during vacuolar acidification. Measurements of relative endosomal volume by a novel ratio imaging method involving fluorescence self-quenching indicated a 2.5-fold increase in volume during early acidification and Cl− accumulation, which was blocked by bafilomycin. These experiments provide the first direct measurement of endosomal [Cl−] and indicate that endosomal acidification is accompanied by significant Cl− entry and volume increase.

Inhibition of Ca2+-activated Cl− channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea

TMEM16A was found recently to be a calcium‐activated CU channel (CaCC). CaCCs perform important functions in cell physiology, including regulation of epithelial secretion, cardiac and neuronal excitability, and smooth muscle contraction. CaCC modulators are of potential utility for treatment of hypertension, diarrhea, and cystic fibrosis. Screening of drug and natural product collections identified tannic acid as an inhibitor of TMEM16A, with IC50 ∼ 6 µM and ∼100% inhibition at higher concentrations. Tannic acid inhibited CaCCs in multiple cell types but did not affect CFTR Cl− channels. Structure‐activity analysis indicated the requirement of gallic or digallic acid substituents on a macromolecular scaffold (gallotannins), as are present in green tea and red wine. Other polyphenolic components of teas and wines, including epicatechin, catechin, and malvidin‐3‐glucoside, poorly inhibited CaCCs. Remarkably, a 1000‐fold dilution of red wine and 100‐fold dilution of green tea inhibited CaCCs by >50%. Tannic acid, red wine, and green tea inhibited arterial smooth muscle contraction and intestinal Cl− secretion. Gallotannins are thus potent CaCC inhibitors whose biological activity provides a potential molecular basis for the cardioprotective and antisecretory benefits of red wine and green tea.—Namkung, W., Thiagarajah, J. R., Phuan, P.‐W., Verkman, A. S. Inhibition of Ca2+‐activated Cl− channels by gallotannins as a possible molecular basis for health benefits of red wine and green tea. FASEB J. 24, 4178–4186 (2010). www.fasebj.org

A small molecule CFTR inhibitor produces cystic fibrosis-like submucosal gland fluid secretions in normal airways

Airway submucosal glands have been proposed as a primary site for initiating and sustaining airway disease in cystic fibrosis (CF). However, it has been difficult to define the role of CFTR in gland fluid secretion because of concerns in interpreting experiments on diseased CF human airways subjected to chronic infection and inflammation. Here, we test the role of CFTR in gland fluid secretion by using a selective CFTR inhibitor (CFTRinh‐172) in pig and human airways. Measurements of single‐gland fluid secretion rates showed inhibition of both cholinergic and cAMP‐stimulated fluid secretion by CFTRinh‐172. Secreted fluid [Na+] and [Cl–] measured by fluorescence ratio imaging were 101 and 116 mM, respectively, and not significantly altered by secretory agonists or CFTR inhibition. Gland fluid pH was 7.1 and reduced by 0.4 units after CFTR inhibition. Gland fluid viscosity, determined by photobleaching of FITC‐dextran, was threefold increased in pilocarpine‐stimulated gland fluid after CFTR inhibition, and protein concentration was increased from 12 to 20 mg/ml. Our data provide strong evidence that gland fluid secretion is CFTR‐dependent. The relatively hyper‐viscous and acidic fluid secretions produced by acute CFTR inhibition support a role for defective gland function in CF lung disease and provide a rational basis for pharmacological creation of a large animal model of CF.

Submucosal gland dysfunction as a primary defect in cystic fibrosis

It has been proposed that defective submucosal gland function in CF airways is a major determinant of CF airway disease. We tested the hypothesis that submucosal gland function is defective early in CF subjects with minimal clinical disease. Functional assays of gland fluid secretion rate and viscosity were performed on freshly obtained nasal biopsies from 6 CF subjects and 5 non‐CF controls (age range 2–22 years). Secretions from individual submucosal glands were visualized by light/fluorescence microscopy after orienting and immobilizing biopsy specimens in a custom chamber. The viscosity of freshly secreted gland fluid after pilocarpine, measured by fluorescence recovery after photobleaching of microinjected FITC‐dextran, was 4.9 ± 0.2‐ vs. 2.2 ± 0.2‐fold greater than water viscosity in CF vs. non‐CF specimens, respectively (se, P<10−4). Gland fluid secretion rate in CF specimens, measured by video imaging (4.5±0.5 nL/min/gland, n=6), was 2.7–fold reduced compared to non‐CF specimens (n=3, P<0.05). Quantitative histology revealed similar size and morphology of submucosal glands in CF and non‐CF specimens. Our results suggest that defective airway submucosal gland function is an early, primary defect in CF. Therapies directed at normalizing gland fluid secretion early in CF may thus reduce lung disease.

Impaired enterocyte proliferation in aquaporin-3 deficiency in mouse models of colitis

Background/Aims: Recent evidence has implicated the involvement of aquaporins (AQPs) in cellular functions that are unrelated to transepithelial water transport. Although AQPs are expressed in the gastrointestinal tract, their importance has so far been unclear. AQP3 is a water/glycerol transporter expressed at the basolateral membrane of colonic epithelial cells. The aim of this study was to investigate the involvement of AQP3 in enterocyte proliferation using mouse models of inflammatory bowel disease. Methods: Expression and function of AQP3 in mouse colonic epithelium were established. Colitis was induced in wild-type and AQP3 null mice by oral dextran sulphate administration or intracolonic acetic acid administration. Outcome measures included clinical disease severity, survival, pathology and cellular responses. Some mice were administered glycerol to test whether disease progression could be altered. Results: AQP3 null mice given dextran sulphate developed severe colitis after 3 days, with colonic haemorrhage, marked epithelial cell loss and death. Wild-type mice, which had comparable initial colonic damage as assessed by cell apoptosis, developed remarkably less severe colitis, surviving to >8 days. Cell proliferation was greatly reduced in AQP3 null mice. Oral glycerol administration significantly improved survival and reduced the severity of colitis in AQP3 null mice. Survival was also reduced in AQP3 null mice in the acetic acid model. Conclusions: The results implicate a novel role for AQP3 in enterocyte proliferation that is probably related to its glycerol-transporting function. AQP3 is thus a potential target for therapy of intestinal diseases associated with enterocyte destruction.

Noninvasive early detection of brain edema in mice by near-infrared light scattering

Brain edema accounts for significant morbidity and mortality in many neurologic conditions such as head trauma, stroke, meningitis, and brain tumor. The water channel aquaporin‐4 (AQP4) has been found to be an important determinant of brain water accumulation and clearance of excess brain water. We report the development of a noninvasive near‐infrared (NIR) light‐scattering method to compare the early kinetics of brain swelling in normal and AQP4‐deficient mice. Brain tissue was illuminated through the intact skull with NIR light at 850 nm, and steady‐state scattered light intensity was monitored at an angle of 90 degrees at a position on the skull ∼10 mm from the illuminated site. NIR light scattering reversibly increased with brain swelling (ΔI/Io ∼25% per 1% increase in brain water content), but was insensitive to changes in cerebral blood flow, blood oxygenation, or blood flow‐related changes in intracranial pressure (ICP). ΔI/Io increased approximately linearly with brain water content as measured by wet‐to‐dry weight ratios. Acute water intoxication (intraperitoneal water, 20% body weight) produced a gradual increase in ΔI/Io of 12 ± 4% in wild‐type mice at 5 min, much greater than that of 2 ± 1% in AQP4‐null mice. Correlation of the NIR signal with ICP showed that increased ΔI/Io preceded measurable increases in ICP, indicating the ability of the NIR method to detect early brain edema before ICP elevation. NIR light scattering provides a simple noninvasive method to monitor brain edema in mice, with potential clinical applications.

Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways

The composition and depth of the airway surface liquid (ASL) are key parameters in airway physiology that are thought to be important in the pathophysiology of cystic fibrosis and other diseases of the airways. We reported novel fluorescent indicator and microscopy methods to measure [Na+], [Cl−], pH, and depth of the ASL in large airways (Jayaraman, S., Y. Song, L. Vetrivel, L. Shankar, and A.S. Verkman. 2001. J. Clin. Invest. 107:317–324.). Here we report a stripped-lung preparation to measure ASL composition and depth in small distal airways. Distal ASL was stained with ion- or pH-sensitive fluorescent indicators by infusion into mouse trachea of a perfluorocarbon suspension of the indicator. After stripping the pleura and limited microdissection of the lung parenchyma, airways were exposed for measurement of ASL [Na+], [Cl−], and pH by ratio imaging microscopy, and depth by confocal microscopy. The stripped-lung preparation was validated in stability and tissue viability studies. ASL [Na+] was 122 ± 2 mM, [Cl−] was 123 ± 4 mM and pH was 7.28 ± 0.07, and not dependent on airway size (<100- to >250-μm diameter), ENaC inhibition by amiloride, or CFTR inhibition by the thiazolidinone CFTRinh-172. ASL depth was 8–35 μm depending on airway size, substantially less than that in mouse trachea of ∼55 μm, and not altered significantly by amiloride. These results establish a novel lung preparation and fluorescence approach to study distal airway physiology and provide the first data on the composition and depth of distal ASL.

Radiation induced cytochrome c release causes loss of rat colonic fluid absorption by damage to crypts and pericryptal myofibroblasts

BACKGROUND Therapeutic or accidental exposure to radiation commonly causes gastrointestinal disturbances, including diarrhoea. Rats subjected to whole body ionising radiation at a dose of 8 Gy lose their capacity to absorb fluid via the descending colon after four days. After seven days, fluid absorption recovers to control levels. AIMS To investigate the effect of ionising radiation on colonic permeability together with its effect on mitochondria dependent apoptotic signals and intercellular adhesion molecules. METHODS Rats were irradiated with doses of 0–12 Gy. Colonic permeability was measured by accumulation of fluorescein isothiocyanate (FITC) dextran in crypt lumens. Changes in levels of cytochrome c, caspase 3, E and OB cadherin, β-catenin smooth muscle actin, and collagen IV were assessed using immunocytochemistry with confocal microscopy. RESULTS Cytosolic cytochrome c increased after 8 Gy (t1/2 1.4 (0.6) hours) and peaked at approximately six hours. Caspase 3 increased more slowly, particularly in crypt epithelial cells (t1/2 57 (14.5) hours). Pericryptal myofibroblasts disintegrated within 24 hours as was evident from loss of OB cadherin and smooth muscle actin. This coincided with increased crypt permeability to dextran. Intercellular adhesion between crypt luminal cells was not lost until day 4 when both β-catenin and E-cadherin were minimal. The half maximal dose-response for these effects was in the range 2–4 Gy. Recovery of colonic transport was concurrent with recovery of pericryptal smooth muscle actin and OB cadherin. The pan caspase inhibitor Z-Val-Ala-Asp.fluoromethylketone (1 mg/kg per day) had a small effect in conserving the pericryptal sheath myofibroblasts and sheath permeability but had no systemic therapeutic effects. CONCLUSIONS These data suggest that radiation damage to the colon may be initiated by mitochondrial events. Loss of crypt fluid absorption and increased permeability coincided with decreased intercellular adhesion between crypt epithelial cells and loss of pericryptal sheath barrier function.

Secretory diarrhoea: mechanisms and emerging therapies.

Diarrhoeal disease remains a major health burden worldwide. Secretory diarrhoeas are caused by certain bacterial and viral infections, inflammatory processes, drugs and genetic disorders. Fluid secretion across the intestinal epithelium in secretory diarrhoeas involves multiple ion and solute transporters, as well as activation of cyclic nucleotide and Ca2+ signalling pathways. In many secretory diarrhoeas, activation of Cl− channels in the apical membrane of enterocytes, including the cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl− channels, increases fluid secretion, while inhibition of Na+ transport reduces fluid absorption. Current treatment of diarrhoea includes replacement of fluid and electrolyte losses using oral rehydration solutions, and drugs targeting intestinal motility or fluid secretion. Therapeutics in the development pipeline target intestinal ion channels and transporters, regulatory proteins and cell surface receptors. This Review describes pathogenic mechanisms of secretory diarrhoea, current and emerging therapeutics, and the challenges in developing antidiarrhoeal therapeutics.