Altered Ion Transport Research Articles

Role of ion transporters in the bile acid-induced esophageal injury

Barrett's esophagus (BE) is considered to be the most severe complication of gastro-esophageal reflux disease (GERD), in which the prolonged, repetitive episodes of combined acidic and biliary reflux result in the replacement of the squamous esophageal lining by columnar epithelium. Therefore, the acid-extruding mechanisms of esophageal epithelial cells (EECs) may play an important role in the defense. Our aim was to identify the presence of acid/base transporters on EECs and to investigate the effect of bile acids on their expressions and functions. Human EEC lines (CP-A and CP-D) were acutely exposed to bile acid cocktail (BAC) and the changes in intracellular pH (pHi) and Ca(2+) concentration ([Ca(2+)]i) were measured by microfluorometry. mRNA and protein expression of ion transporters was investigated by RT-PCR, Western blot, and immunohistochemistry. We have identified the presence of a Na(+)/H(+) exchanger (NHE), Na(+)/HCO3 (-) cotransporter (NBC), and a Cl(-)-dependent HCO3 (-) secretory mechanism in CP-A and CP-D cells. Acute administration of BAC stimulated HCO3 (-) secretion in both cell lines and the NHE activity in CP-D cells by an inositol triphosphate-dependent calcium release. Chronic administration of BAC to EECs increased the expression of ion transporters compared with nontreated cells. A similar expression pattern was observed in biopsy samples from BE compared with normal epithelium. We have shown that acute administration of bile acids differently alters ion transport mechanisms of EECs, whereas chronic exposure to bile acids increases the expression of acid/base transporters. We speculate that these adaptive processes of EECs represent an important mucosal defense against the bile acid-induced epithelial injury.

Altered Ion Transport in Normal Human Bronchial Epithelial (NHBE) Cells Following Exposure to Metal Welding Particles

Inhalation of welding fumes is associated with adverse effects in the pulmonary system, including susceptibility to infection. Several types of welding processes occur, giving rise to fumes of different compositions. In this study we investigated the effects of particles derived from fumes generated from manual metal arc‐stainless steel (MMA‐SS) and gas metalarc‐mild steel (GMA‐MS) welding on ion transport in NHBE in culture. The toxicities and the composition of the particles generated during MMA‐SS and GMA‐MS processes are different. MMA‐SS particles are more soluble and contain Cr, Ni, Fe and Mn, whereas GMA‐MS particles are less soluble and contain Fe and Mn. Ion transport is critical to the maintenance of the airway surface liquid. We hypothesized that epithelial cells are targets of inhaled welding fumetoxicity. NHBE (Lonza) were cultured in submersion culture for 3 days followed by 21 d in air‐interface on permeable inserts; cells with transepithelial resistance (Rt) >1000 Ω·cm2were used. Generated MMA‐SS or GMA‐MS particles collected on filters were suspended in medium and applied (50 μl) to the apical chamber. The doses of both types of particles ranged from 0.0167 to166.7 μg/cm2. After 18 h of incubation the inserts were placed in Ussing chambers for the measurement of transepithelial potential difference (Vt), short circuit current (Isc) and Rt. The effects of fume exposure on Na+ and Cl− channels, the Na+,K+,2Cl−‐cotransporter and the Na+,K+‐pump, were evaluated by applying the following inhibitors, respectively: amiloride(A; apical; 3×10−5 M), NPPB (N; apical; 10−4 M), bumetanide (B; 10−4 M) and ouabain (O; basolateral; 10−4M). Changes in lactate dehydrogenase(LDH) levels in apical medium also were assessed after exposure. Following exposure, 0.0167–16.7 μg/cm2MMA increased basal Vt. Only16.7 μg/cm2 GMA‐MS increased basal Vt. MMA‐SS exposure potentiated Isc responses (decreases in Isc) to A and B, while not affecting those to N and O. Likewise, exposure to GMA‐MS potentiated Isc responses to A and B, but to a lesser degree than observed after MMA‐SS exposure. Variable effects on Rt were observed in response to A, B and O. Generally, MMA‐SS was more potent in altering responses to A and B than GMA‐MS. MMA‐SS evoked greater increases in LDH release than GMA‐MS, but bioelectric changes occurred in the absence of increased LDH release. Our findings indicate that both apical Na+ transport and basolateral Na+,K+,2Cl−‐cotransportin airway epithelium is affected by fume particle exposure. The greater effect of MMA‐MS compared to GMA‐MS might reflect the actions of soluble metals. Enhanced Na+ absorption and decreased airway surface liquid could compromise lung defenses against infection.Support or Funding InformationSupported by NIOSH

Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6h to diacetyl or 2,3-pentanedione vapors (25 or ≥60ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25ppm both flavorings reduced Na+ transport, without affecting Cl− transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18h after exposure. Concentrations (100–360ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60ppm, caused death of NHBEs 0h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro.

Interfacial engineering of solid electrolytes

The roles of interfaces in either blocking or enhancing ionic conduction in various types of solid electrolytes, including lithium, sodium, oxygen and other types of ion conductors as well as proton conductors, are critically reviewed. Two important fundamental interfacial phenomena, namely the formation of space charges and two-dimensional interfacial phases (complexions), can markedly alter ion transport along or across various types of interfaces, including grain and phase boundaries as well as free surfaces. Since the experiments and models of space charges have been well documented in literature, a new focus of this short review and viewpoint article is to propose and discuss an emerging opportunity of utilizing the formation and transition of interfacial phases to either alleviate the blocking effects or further enhance ionic conduction in solid electrolytes.

Pulmonary epithelial barrier function: some new players and mechanisms.

The pulmonary epithelium serves as a barrier to prevent access of the inspired luminal contents to the subepithelium. In addition, the epithelium dictates the initial responses of the lung to both infectious and noninfectious stimuli. One mechanism by which the epithelium does this is by coordinating transport of diffusible molecules across the epithelial barrier, both through the cell and between cells. In this review, we will discuss a few emerging paradigms of permeability changes through altered ion transport and paracellular regulation by which the epithelium gates its response to potentially detrimental luminal stimuli. This review is a summary of talks presented during a symposium in Experimental Biology geared toward novel and less recognized methods of epithelial barrier regulation. First, we will discuss mechanisms of dynamic regulation of cell-cell contacts in the context of repetitive exposure to inhaled infectious and noninfectious insults. In the second section, we will briefly discuss mechanisms of transcellular ion homeostasis specifically focused on the role of claudins and paracellular ion-channel regulation in chronic barrier dysfunction. In the next section, we will address transcellular ion transport and highlight the role of Trek-1 in epithelial responses to lung injury. In the final section, we will outline the role of epithelial growth receptor in barrier regulation in baseline, acute lung injury, and airway disease. We will then end with a summary of mechanisms of epithelial control as well as discuss emerging paradigms of the epithelium role in shifting between a structural element that maintains tight cell-cell adhesion to a cell that initiates and participates in immune responses.

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (I_SC) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The I_SC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca²⁺]_i) and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in I_SC, which was due to Cl^- secretion. The increase in I_SC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTR_inh-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated I_SC was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in I_SC in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca²⁺]_i. Conclusion: Nobiletin stimulated transepithelial Cl^- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl^- channels.

Interaction of Small Cationic Peptides with Intact Basement Membranes. A Study Using Intrinsic Optical Signals of Chick Retinas

Cationic peptides (polylysines and polyarginines) are being developed as drug delivery systems to nuclei. Therefore, a detailed description of tissue response changes upon the application of cationic peptides over intact basement membranes of excitable tissue is of interest in pharmacology. In this paper we examine the effects of two naturally occurring cationic peptides protamine (polyarginine) and crotamine (polylysine) on the optical profiles of retinal spreading depression waves (RSDs). This intrinsic optical signal (IOS), recorded non-invasively, provides information about dissipation of electrochemical gradients within the tissue and its metabolic consequences. Protamine at nanomolar range brought the tissue excitability to collapse without any signs of acute toxicity whereas crotamine, a known myotoxin from rattlesnake, decreased the tissue transparency and changed markedly the optical profiles of RSDs. Also, fluorescent crotamine was incorporated to Müller cells in a few minutes, suggesting a close membrane interaction. The optical changes brought about by crotamine were easily washed off. By contrast, the excitability collapse in presence of protamine lasted for at least two hours. we concluded that crotamine has fusogenic properties that alters ion transport in excitable tissue. Protamine effect seems to be similar to its effect on basement membrane of epithelium due to its property of making heteropolymers with heparan sulfate. The clinical syndrome expressed in mice after crotamine injection suggested excitotoxic CNS effects confirmed by the isolated retina experiments.

The CF-modifying gene EHF promotes p.Phe508del-CFTR residual function by altering protein glycosylation and trafficking in epithelial cells

The three-base-pair deletion c.1521_1523delCTT (p.Phe508del, F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most frequent disease-causing lesion in cystic fibrosis (CF). The CFTR gene encodes a chloride and bicarbonate channel at the apical membrane of epithelial cells. Altered ion transport of CFTR-expressing epithelia can be used to differentiate manifestations of the so-called CF basic defect. Recently, an 11p13 region has been described as a CF modifier by the North American CF Genetic Modifier Study Consortium. Selecting the epithelial-specific transcription factor EHF (ets homologous factor) as the likely candidate gene on 11p13, we have genotyped two intragenic microsatellites in EHF to replicate the 11p13 finding in the patient cohort of the European CF Twin and Sibling Study. We could observe an association of rare EHF haplotypes among homozygotes for c.1521_1523delCTT in CFTR, which exhibit a CF-untypical manifestation of the CF basic defect such as CFTR-mediated residual chloride secretion and low response to amiloride. We have reviewed transcriptome data obtained from intestinal epithelial samples of homozygotes for c.1521_1523delCTT in CFTR, which were stratified for their EHF genetic background. Transcripts that were upregulated among homozygotes for c.1521_1523delCTT in CFTR, who carry two rare EHF alleles, were enriched for genes that alter protein glycosylation and trafficking, both mechanisms being pivotal for the effective targeting of fully functional p.Phe508del-CFTR to the apical membrane of epithelial cells. We conclude that EHF modifies the CF phenotype by altering capabilities of the epithelial cell to correctly process the folding and trafficking of mutant p.Phe508del-CFTR.

Cadmium Transport in Epithelial Cells of a Crab: comparison between Gills and Hepatopancreas

Cadmium (Cd) is a non‐essential metal for crustaceans and is responsible for alterations in ion transport in a number of animals. Cd transport has not been studied in detail in crustaceans. Therefore, the present work was undertaken to characterize Cd transport in gill and hepatopancreatic cells of a crab, Ucides cordatus, found in contaminated mangrove regions. Cells were marked with a fluorescent probe (Fluo‐3) and the change in fluorescence was followed with the addition of Cd (0, 1.0 and 1.5 ìM), ATP (1.5 ìM) and different inhibitors. Results show that Cd enters gill cells and the fluorescence decreases to baseline values within few seconds. Cd transport for posterior gills is double that for anterior gills in terms of fluorescence change (1,600 against 800 ÄF × 5s−1 × 22.104 cells, respectively). ATP added together with Cd at 1.5 ìM causes a decrease in fluorescence, suggesting an efflux of intracellular Cd. For hepatopancreas, different cell types display different Cd uptake. Embryonic (E) and resorptive cells (R) showed the highest Cd uptake compared to the other cells, and the uptake was lower than that seen for gill cells (1,600 against 1,000 ÄF × 5s−1 × 22.104 cells, respectively). Results show that Cd enters gill and hepatopancreatic cells, and equilibrates intracellularly within 4–5 seconds. Moreover, Cd influx is followed by a rapid cell sequestration and a decrease of intracellular Cd in these crab cells.

Luminal [Na+] enhances Bacteroides thetaiotaomicron growth in vitro and in the NHE3−/− mouse terminal ileum

Microbial dysbiosis is known to play a key role in a variety of complex diseases such as IBD, diabetes, obesity and C. difficile‐associated disease. Little is known regarding the effect of epithelial ion transport processes on the intestinal microbiota. Na+/H+ exchanger isoform 3 (NHE3) plays an integral role in intestinal Na+ absorption and was used to examine region specific microbial dysbiosis. qRT‐PCR revealed that NHE3−/− mice exhibit bacterial overgrowth and microbial dysbiosis in both the lumen and mucosa‐associated bacteria with decreased Firmicutes and increased Bacteriodetes. The genus Bacteroides and the species Bacteroides thetaiotaomicron (B. theta) were significantly increased only in the NHE3−/− terminal ileum where a luminal [Na+] of 43±1 mM was found (WT was 33±3 mM). This correlated with the optimal [Na+] for B. theta growth in vitro,~44 mM Na+. Increased B. theta correlated with increased fut2 mRNA and fucosylation in the NHE3−/− terminal ileum. This study provides a first look at microbial dysbiosis due to altered ion transport. Our data shows the NHE3−/− mice exhibit region specific microbial dysbiosis with changes in B. theta, which correlate with luminal [Na+] and host epithelial changes. These data together show a mechanism for B. theta proliferation and contribution to microbial dysbiosis in the NHE3−/− mouse intestine. Supported by NIH DK079979 to RTW and DK050594 to GES.

Activation of Chloride Secretion by Isoflavone Genistein in Endometrial Epithelial Cells

Background /Aim: Genistein, the most active isoflavone found primarily in soybeans, alters ion transport functions in intestinal and airway epithelia. The present study aims to investigate the acute effects and mechanisms of action of genistein in immortalized porcine endometrial epithelial cells. Methods: Ussing chamber technique was used for transepithelial electrical measurements. Results: Genistein increased short-circuit currents (I_sc) which were inhibited by glibenclamide, NPPB, CFTRinh-172, DIDS or bumetanide, but not amiloride. In experiments with amphotericin B-permeabilized monolayers, genistein activated the apical Cl^- current and barium-sensitive basolateral K⁺ current while inhibiting the apical K⁺ current. Genistein failed to increase the I_sc in the presence of forskolin or IBMX, but did increase the I_sc in UTP. Pretreatment with genistein also abolished the increase in the I_sc when induced by forskolin, IBMX or UTP. However, Ca²⁺-chelating BAPTA-AM did not affect the genistein-induced increase in the I_sc. The genistein-stimulated I_sc was reduced by tyrosine kinase inhibitors, tyrphostin A23 or AG490. However, vanadate, a tyrosine phosphatase inhibitor, failed to inhibit the genistein response. Estrogen receptor antagonist ICI182,780 did not alter the genistein's action. Conclusion: The soy isoflavone, genistein, stimulates Cl^- secretion in endometrial epithelial cells possibly via a direct activation of CFTR which appears to be modulated through a tyrosine kinase-dependent pathway. The present findings may be of benefit for the therapeutic application of genistein in the treatment of electrolyte transport disorders in the epithelia.

PAR-2-mediated control of barrier function and motility differs between early and late phases of postinfectious gut dysfunction in the rat

Proteinase-activated receptor-2 (PAR-2) and mast cell (MC) mediators contribute to inflammatory and functional gastrointestinal disorders. We aimed to characterize jejunal PAR-2-mediated responses and the potential MC involvement in the early and late phases of a rat model of postinfectious gut dysfunction. Jejunal tissues of control and Trichinella spiralis-infected (14 and 30 days postinfection) rats, treated or not with the MC stabilizer, ketotifen, were used. Histopathology and immunostaining were used to characterize inflammation, PAR-2 expression, and mucosal and connective tissue MCs. Epithelial barrier function (hydroelectrolytic transport and permeability) and motility were assessed in vitro in basal conditions and after PAR-2 activation. Intestinal inflammation on day 14 postinfection (early phase) was significantly resolved by day 30 (late phase) although MC counts and epithelial permeability remained increased. PAR-2-mediated ion transport (Ussing chambers, in vitro) and epithelial surface PAR-2 expression were reduced in the early phase, with a trend toward normalization during the late phase. In control conditions, PAR-2 activation (organ bath) induced biphasic motor responses (relaxation followed by excitation). At 14 days postinfection, spontaneous contractility and PAR-2-mediated relaxations were enhanced; motor responses were normalized on day 30. Postinfectious changes in PAR-2 functions were not affected by ketotifen treatment. We concluded that, in the rat model of Trichinella spiralis infection, alterations of intestinal PAR-2 function and expression depend on the inflammatory phase considered. A lack of a ketotifen effect suggests no interplay between MCs and PAR-2-mediated motility and ion transport alterations. These observations question the role of MC mediators in PAR-2-modulating postinfectious gut dysfunction.

Zinc sulphate attenuates chloride secretion in Human colonic mucosae in vitro

Zinc's usefulness in the treatment of diarrhoea is well established as an addition to oral rehydration. Mechanisms of action of zinc have been explored in intestinal epithelia from rodents and in cell lines. The aim was to examine how zinc alters ion transport and signal transduction in human colon in vitro. Voltage clamped colonic sheets obtained at the time of surgical resection were used to quantify ion transport responses to established secretagogues. Nystatin permeabilisation was used to study basolaterally-sited ion channels. Direct actions of zinc were determined using preparations of colonic crypts isolated from human mucosal sheets. Electrophysiological measurements revealed zinc to be an inhibitor of electrogenic ion transport stimulated by forskolin, PGE2, histamine and carbachol in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effect on the epithelium. To further outline the mechanism of action, levels of secondary intracellular messengers (3’, 5’-cyclic adenosine monophosphate; cAMP) were determined in isolated colonic crypts, and were found to be reduced by zinc sulphate. Finally, indirect evidence from nystatin-permeabilised mucosae further suggested that zinc inhibits basolateral K+ channels, which are critical for transepithelial Cl− secretion linked to water flux. Anti-secretory, and therefore anti-diarrhoeal, actions of exogenous zinc are due, at least in part, to direct basolateral epithelial K+ channel inhibition.

Effects of diacetyl vapor exposure on human cultured airway epithelial cell ion transport

In August 2000, NIOSH investigated the development of bronchiolitis obliterans following inhalation of the butter flavoring, diacetyl, in employees at a popcorn manufacturing plant. Rats exposed to diacetyl vapor for 6 h developed diacetyl dose‐dependent epithelial damage of the upper airways 18 h after exposure. Nevertheless, diacetyl inhalation did not result in hyperreactivity to methacholine (MCh) both in vivo (inhaled MCh aerosol) and in vitro (isolated, perfused trachea), despite damage to the epithelium. In order to investigate the direct effects of diacetyl on airway epithelium, we examined ion transport of human air‐interface, cultured epithelial cells following diacetyl exposure. We hypothesized that altered epithelial ion transport may be among the earliest events associated with the onset of diacetyl toxicity. Using an exposure system designed for cultured cells, cells were exposed for 6 h to diacetyl vapors (100, 200, 300, 360 ppm). Eighteen hours after exposure, cells were placed into an Ussing chamber to measure transepithelial potential difference (Vt). The results demonstrated that, whereas inhalation of diacetyl had no effect in vivo or in vitro, all concentrations of diacetyl abolished Vt in cultured cells. Our results indicate that diacetyl causes substantial damage to airway epithelium in culture at exposure concentrations that do not alter reactivity of the airways in vivo or in vitro.

CCR7 deficiency causes diarrhea associated with altered ion transport in colonocytes in the absence of overt colitis.

The chemokine receptor CCR7 is a central regulator in the maintenance of cellular homeostasis of mucosal tissues. CCR7⁻/⁻ mice develop autoimmune gastritis and exocrinopathy accompanied by the formation of mucosal tertiary lymphoid follicles. Here we found that CCR7-deficient mice frequently suffered from chronic diarrhea linked with increased gastrointestinal motility and the development of severe anorectal prolapse. Enhanced formation of intestinal lymphoid follicles was associated with an elevated proportion of activated colonic T cells and increased production of the cytokine interleukin (IL)-1β. To uncover the pathomechanisms of diarrhea in CCR7⁻/⁻ mice, colonic epithelial barrier and ion channel activities were analyzed in Ussing chambers. Although overt acute colitis was absent, CCR7 deficiency resulted in reduced electrogenic sodium absorption and colonic chloride secretion. As it is known that IL-1β regulates epithelial sodium channel (ENaC) activity, these data imply a causal link between CCR7 expression, IL-1β level, and Na⁺ malabsorption owing to altered ENaC expression and diarrhea.

Intestinal Microbiome in NHE2/3 Null Mouse Models

Sodium/proton exchangers (SLC9s, NHEs) play an integral role in sodium absorption in the intestine. Studies using knockout mice showed that loss of NHE3, but not NHE2, results in diarrhea. The intestinal contentin NHE3−/− mice is significantly more alkaline than in WT littermates. Alkalinizationof the normally acidic microclimate immediately adjacent tothe brush-border surface layer may aid bacterialadhesion and facilitate microbial-epithelial interactionsand translocation. This study sought to determine the effect of altered intestinal ion transport in NHE2−/− and NHE3−/− mice on the luminal and mucosa-associated microbiota. Data using 16S ribosomal DNA bacterial primers in quantitative real time PCR (qRT-PCR) revealed increases in total bacteria in the terminal ileum and distal colon of NHE3−/− mice and no change in the total bacteria of NHE2−/− mice. Interestingly decreases in beta-Proteobacteria were observed in NHE3−/− terminal ileum, cecum, proximal colon and distal colon and in NHE2−/− cecum. In NHE3−/− mice, decreases in alpha-Proteobacteria were observed in proximal and distal colon, while in NHE2−/− mice alpha-Proteobacteria was found to increase in the distal colon. Finally, gamma-Proteobacteria was found to increase only in NHE2−/− proximal colon. These data suggest that differences in intestinal ion transport can result in changes in bacterial families which may increase the susceptibility of the host to intestinal inflammation and dysfunction. Supported by NIH DK079979 to RTW and DK050594 to GES.

P103 Oral contraceptive use does not affect CF disease severity

Several studies using a variety of in vitro models indicate that sex hormones such as oestrogen can alter ion transport across epithelial cells by either directly affecting CFTR or altering...

Altered ion transport by thyroid epithelia from CFTR−/− pigs suggests mechanisms for hypothyroidism in cystic fibrosis

Subclinical hypothyroidism has been linked to cystic fibrosis, and the cystic fibrosis transmembrane conductance regulator (CFTR) shown to be expressed in the thyroid. The thyroid epithelium secretes Cl⁻ and absorbs Na(+) in response to cAMP. Chloride secretion may provide a counter-ion for the SLC26A4 (pendrin)-mediated I⁻ secretion which is required for the first step of thyroid hormonogenesis, thyroglobulin iodination. In contrast, few models exist to explain a role for Na(+) absorption. Whether CFTR mediates the secretory Cl⁻ current in thyroid epithelium has not been directly addressed. We used thyroids from a novel pig CFTR(-/-) model, generated primary pig thyroid epithelial cell cultures (pThECs), analysed these cultures for preservation of thyroid-specific transcripts and proteins, and monitored the following parameters: (1) the Cl⁻ secretory response to the cAMP agonist, isoprenaline; and (2) the amiloride-sensitive Na(+) current. Baseline short-circuit current (I(sc)) did not differ between CFTR(+/+) and CFTR(-/-) cultures. Serosal isoprenaline increased I(sc) in CFTR(+/+), but not CFTR(-/-), monolayers. Compared with CFTR(+/+) thyroid cultures, amiloride-sensitive Na(+) absorption measured in CFTR(-/-) pThECs represented a greater fraction of the resting I(sc). However, levels of transcripts encoding epithelial sodium channel (ENaC) subunits did not differ between CFTR(+/+) and CFTR(-/-) pThECs. Immunoblot analysis verified ENaC subunit protein expression, but quantification indicated no difference in expression levels. Our studies definitively demonstrate that CFTR mediates cAMP-stimulated Cl⁻ secretion in a well-differentiated thyroid culture model and that knockout of CFTR promotes increased Na(+) absorption by a mechanism other than increased ENaC expression. These findings suggest several models for the mechanism of cystic fibrosis-associated hypothyroidism.

Lack of cystic fibrosis transmembrane conductance regulator in CD3+ lymphocytes leads to aberrant cytokine secretion and hyperinflammatory adaptive immune responses.

Cystic fibrosis (CF), the most common fatal monogenic disease in the United States, results from mutations in CF transmembrane conductance regulator (CFTR), a chloride channel. The mechanisms by which CFTR mutations cause lung disease in CF are not fully defined but may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr(-/-) mice mount an exaggerated IgE response toward Aspergillus fumigatus, with higher levels of IL-13 and IL-4, mimicking both the T helper cell type 2-biased immune responses seen in patients with CF. Herein, we demonstrate that these aberrations are primarily due to Cftr deficiency in lymphocytes rather than in the epithelium. Adoptive transfer experiments with CF splenocytes confer a higher IgE response to Aspergillus fumigatus compared with hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount T helper cell type 2 responses with high IL-13 and IL-4 was confirmed by in vitro antigen recall experiments. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T cell lineages developed higher IgE than their wild-type control littermates. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca(2+) flux in response to T cell receptor activation. This was accompanied by an increase in nuclear localization of the calcium-sensitive transcription factor, nuclear factor of activated T cell, which could drive the IL-13 response. In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. These findings implicate the lymphocyte population as a potentially important target for CF therapeutics.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K+ channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.