Inhibition Of Cl Research Articles

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

A fungus-derived purpactin A as an inhibitor of TMEM16A chloride channels and mucin secretion in airway epithelial cells

TMEM16A is a Ca2+-activated Cl- channel involved in mucus secretion in inflamed airways and proposed as a drug target for diseases associated with mucus hypersecretion including asthma. This study aimed to identify novel inhibitors of TMEM16A-mediated Cl- secretion in airway epithelial cells from a collection of compounds isolated from fungi indigenous in Thailand and examine its potential utility in mitigating airway mucus secretion using Calu-3 cells as a study model. Screening of > 400 fungal metabolites revealed purpactin A isolated from a soil-derived fungus Penicillium aculeatum PSU-RSPG105 as an inhibitor of TMEM16A-mediated Cl- transport with an IC50 value of ~2 µM. A consistent inhibitory effect of purpactin A on TMEM16A were observed regardless of TMEM16A activators or in the presence of an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a negative regulator of TMEM16A. In addition, purpactin A did not affect cell viability, epithelial barrier integrity and activities of membrane transport proteins essential for maintaining airway hydration including CFTR Cl- channels and apical BK K+ channels. Intriguingly, purpactin A prevented a Ca2+-induced mucin release in cytokine-treated airway cells. Taken together, purpactin A represents the first class of TMEM16A inhibitor derived from fungus, which may be beneficial for the treatment of diseases associated with mucus hypersecretion.

Synthesis and Characterization of Zinc Oxide and Zinc Oxide Doped with Chlorine Nanoparticles as Novel <i>α</i>-Amylase Inhibitors

In this study we used a chemical solution method from oxalic acid (OX. acid) and zinc acetate (ZnAc) to prepare Zinc Oxide nanoparticles (ZnONPs) and Zinc Oxide nanoparticles doped with Chlorine (Cl:ZnONPs). The characterizations (FTIR, X-ray, SEM, TEM) of ZnONPs and Cl:ZnONPs were determined. Amylase inhibitors of ZnONPs and Cl:ZnONPs also were determined. SEM indicated that the ZnONPs and Cl:ZnONPs have an average particle size of 46.65 - 74.64 nm. TEM images of the ZnONPs and Cl:ZnONPs showed the round shaped. Compounds b, d and e exhibited significant inhibitory activity against amylase enzyme (from 69.21 ± 1.44 to 76.32 ± 0.78), respectively, and were comparable with that of acarbose (86.32 ± 0.63) at 1000 μg, thereby, projecting ZnONPs and Cl:ZnONPs as α-amylase inhibitors.

Inhibition of CFTR-mediated intestinal chloride secretion by a fungus-derived arthropsolide A: Mechanism of action and anti-diarrheal efficacy

Secretory diarrhea is one of the most common types of diarrhea with high morbidity and mortality. Previous studies showed that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels alleviated fluid loss in secretory diarrheas. This study aimed to identify novel CFTR inhibitors from fungal metabolites and explore its underlying mechanisms and potential utility in secretory diarrheas. Electrophysiological analyses in human intestinal epithelial (T84) cells were performed to investigate the effect and mechanism of fungal metabolites on CFTR-mediated Cl- secretion. Anti-diarrheal efficacy and the effect of compound on fluid absorption were investigated in mouse closed-loop models. We found that the screening identified arthropsolide A, a fungal metabolite from an endophytic fungus Roussoella sp. PSU-H51, as an inhibitor of CFTR-mediated Cl- secretion in T84 cells (IC50 ~0.8 μM). Arthropsolide A inhibited both CFTR and cAMP-activated basolateral K+ channels. Arthropsolide A had no effect on Na+-K+ ATPase activity. Interestingly, the inhibitory effect of arthropsolide A on CFTR was attenuated by cell depolarization and AMPK inhibition independent of multi-drug resistance protein 4, phosphodiesterases, and protein phosphatases. Importantly, arthropsolide A suppressed cholera toxin (CT)-induced Cl- secretion in T84 cells and CT-induced intestinal fluid secretion in mice by ~75% without affecting intestinal fluid absorption. Taken together, arthropsolide A represents a novel class of fungal metabolites that acts as a potent CFTR inhibitor. Further development of this class of compounds may provide a therapy for secretory diarrheas.

β2 adrenoceptor signaling regulates ion transport in 16HBE14o- human airway epithelial cells.

We investigated the regulation of Cl- secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective β adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (ISC ), which was inhibited by the β adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the β2 adrenoceptor subtype, stimulated a similar increase in ISC , which was inhibited by the β2 adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl- channel (CaCC), but not K+ channel blockers, were able to inhibit the increase in ISC . "Trimultaneous" recording of ISC and intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ levels in 16HBE14o- epithelia confirmed that the ISC induced by isoprenaline or procaterol involved both cAMP and Ca2+ signaling. Our results demonstrate that β2 adrenoceptors regulate Cl- secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca2+ -dependent mechanisms, respectively.

A highly-selective chloride microelectrode based on a mercuracarborand anion carrier

The chloride gradient plays an important role in regulating cell volume, membrane potential, pH, secretion, and the reversal potential of inhibitory glycine and GABAA receptors. Measurement of intracellular chloride activity, {{boldsymbol{a}}}_{{boldsymbol{Cl}}}^{{boldsymbol{i}}}, using liquid membrane ion-selective microelectrodes (ISM), however, has been limited by the physiochemical properties of Cl− ionophores which have caused poor stability, drift, sluggish response times, and interference from other biologically relevant anions. Most importantly, intracellular {bf{HC}}{{bf{O}}}_{{bf{3}}}^{-} may be up to 4 times more abundant than Cl− (e.g. skeletal muscle) which places severe constraints on the required selectivity of a Cl− – sensing ISM. Previously, a sensitive and highly-selective Cl− sensor was developed in a polymeric membrane electrode using a trinuclear Hg(II) complex containing carborane-based ligands, [9]-mercuracarborand-3, or MC3 for short. Here, we have adapted the use of the MC3 anion carrier in a liquid membrane ion-selective microelectrode and show the MC3-ISM has a linear Nernstian response over a wide range of aCl (0.1 mM to 100 mM), is highly selective for Cl− over other biological anions or inhibitors of Cl− transport, and has a 10% to 90% settling time of 3 sec. Importantly, over the physiological range of aCl (1 mM to 100 mM) the potentiometric response of the MC3-ISM is insensitive to {bf{HC}}{{bf{O}}}_{{bf{3}}}^{-} or changes in pH. Finally, we demonstrate the biological application of an MC3-ISM by measuring intracellular aCl, and the response to an external Cl-free challenge, for an isolated skeletal muscle fiber.

A fungal metabolite zearalenone as a CFTR inhibitor and potential therapy of secretory diarrheas

Overstimulation of CFTR-mediated Cl− secretion plays an important role in the pathogenesis of secretory diarrheas, which remain an important global health problem. This study aimed to identify inhibitors of CFTR-mediated Cl− secretion from a library of fungus-derived compounds and to evaluate their pharmacological properties and anti-diarrheal utility. We identified zearalenone, 7′-dehydrozearalenone and 8′-hydroxyzearalenone isolated from the seagrass-derived fungus Fusarium sp. PSU-ES123 as inhibitors of CFTR-mediated Cl− secretion in human intestinal epithelial (T84) cells. Being the most potent fungal metabolite capable of inhibiting CFTR-mediated Cl− secretion, zearalenone reversibly inhibited CFTR Cl− channel activity in T84 cells with an IC50 of ∼0.5 μM. Functional and biochemical analyses and molecular docking studies indicate that zearalenone binds to the β-estradiol binding sites in the ATP-binding pockets on NBD1 and NBD2 of CFTR. Mechanisms of CFTR inhibition by zearalenone do not involve activation of phosphodiesterases, protein phosphatases, multidrug-resistance protein 4 and AMP-activated protein kinases. Importantly, zearalenone significantly inhibited cholera toxin (CT)-induced Cl− secretion in T84 cells and blocked CT-induced intestinal fluid secretion in mice. Collectively, our study indicates that zearalenone represents the first class of fungus-derived CFTR inhibitors. Further development of this class of compounds may provide an effective treatment of secretory diarrheas.

Electrophysiological properties of anion exchangers in the luminal membrane of guinea pig pancreatic duct cells

The pancreatic duct epithelium secretes the HCO3−-rich pancreatic juice. The HCO3− transport across the luminal membrane has been proposed to be mediated by SLC26A Cl−–HCO3− exchangers. To examine the electrophysiological properties of Cl−–HCO3− exchangers, we directly measured HCO3− conductance in the luminal membrane of the interlobular pancreatic duct cells from guinea pigs using an inside-out patch-clamp technique. Intracellular HCO3− increased the HCO3− conductance with a half-maximal effective concentration value of approximately 30 mM. The selectivity sequence based on permeability ratios was SCN− (1.4) > Cl− (1.2) = gluconate (1.1) = I− (1.1) = HCO3− (1.0) > methanesulfonate (0.6). The sequence of the relative conductance was HCO3− (1.0) > SCN− (0.7) = I− (0.7) > Cl− (0.5) = gluconate (0.4) > methanesulfonate (0.2). The current dependent on intracellular HCO3− was reduced by replacement of extracellular Cl− with gluconate or by H2DIDS, an inhibitor of Cl−–HCO3− exchangers. RT-PCR analysis revealed that the interlobular and main ducts expressed all SLC26A family members except Slc26a5 and Slc26a8. SLC26A1, SLC26A4, SLC26A6, and SLC26A10 were found to be localized to the luminal membrane of the guinea pig pancreatic duct by immunohistochemistry. These results demonstrate that these SLC26A Cl−–HCO3− exchangers may mediate the electrogenic HCO3− transport through the luminal membrane and may be involved in pancreatic secretion in guinea pig ducts.

Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance.

Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.

TRPM7 Is Involved in Volume Regulation in Salivary Glands

Under hypotonic conditions, the regulatory volume decrease (RVD) is essential to maintain physiological homeostasis and functions in diverse biological systems. Intracellular Ca2+ has been reported as an important mediator of this response, but the underlying Ca2+ mechanism responsible for RVD is still controversial. Here we investigate the role of Ca2+ in the RVD response using live-cell imaging, microspectrofluorimetry, and a patch-clamp technique. A typical RVD was observed in submandibular gland acinar cells after swelling in a hypotonic solution, whereas intracellular Ca2+ chelation completely inhibited the RVD response. The incidence and magnitude of the Ca2+ transient were proportional to the degree of hypotonicity of the extracellular medium, and there was a close relationship between intracellular Ca2+ concentration and the volumetric changes of the cells. Notably, this response was mediated by Ca2+-induced Ca2+ release, which is triggered by Ca2+ influx via stretch-activated TRPM7 channels. Furthermore, we detected the generation of Cl− currents in the swelling acinar cells upon hypotonic stress, and the current profile matched that of the Ca2+-activated Cl− currents. A specific inhibitor of Cl− currents also inhibited the RVD response. In conclusion, an intracellular Ca2+ increase in response to osmotically induced cell swelling plays a critical role in RVD in salivary gland acinar cells.

The ICl,swell inhibitor DCPIB blocks Kir channels that possess weak affinity for PIP2.

Inwardly rectifying K(+) (Kir) channels are important contributors to the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP2. Several modulators of the activity of Kir channels alter the apparent affinity of the channel to PIP2. Channels with high apparent affinity to PIP2 may not respond to a given modulator, but mutations that decrease such affinity can render the channel susceptible to modulation. Here, we identify a known inhibitor of the swelling-activated Cl(-) current, DCPIB, as an effective inhibitor of a number of Kir channels both in native cardiac cells and in heterologous expression systems. We show that the apparent affinity to PIP2 determines whether DCPIB will serve as an efficient blocker of Kir channels. These effects are consistent with a model in which DCPIB competes with PIP2 for a common binding site.

Enhanced nonsynaptic epileptiform activity in the dentate gyrus after kainate-induced status epilepticus

Understanding the mechanisms that influence brain excitability and synchronization provides hope that epileptic seizures can be controlled. In this scenario, non-synaptic mechanisms have a critical role in seizure activity. The contribution of ion transporters to the regulation of seizure-like activity has not been extensively studied. Here, we examined how non-synaptic epileptiform activity (NEA) in the CA1 and dentate gyrus (DG) regions of the hippocampal formation were affected by kainic acid (KA) administration. NEA enhancement in the DG and suppression in area CA1 were associated with increased NKCC1 expression in neurons and severe neuronal loss accompanied by marked glial proliferation, respectively. Twenty-four hours after KA, the DG exhibited intense microglial activation that was associated with reduced cell density in the infra-pyramidal lamina; however, cellular density recovered 7days after KA. Intense Ki67 immunoreactivity was observed in the subgranular proliferative zone of the DG, which indicates new neuron incorporation into the granule layer. In addition, bumetanide, a selective inhibitor of neuronal Cl− uptake mediated by NKCC1, was used to confirm that the NKCC1 increase effectively contributed to NEA changes in the DG. Furthermore, 7days after KA, prominent NKCC1 staining was identified in the axon initial segments of granule cells, at the exact site where action potentials are preferentially initiated, which endowed these neurons with increased excitability. Taken together, our data suggest a key role of NKCC1 in NEA in the DG.

Characterization of slow waves generated by myenteric interstitial cells of Cajal of the rabbit small intestine.

Slow waves (slow wavesICC) were recorded from myenteric interstitial cells of Cajal (ICC-MY) in situ in the rabbit small intestine, and their properties were compared with those of mouse small intestine. Rabbit slow wavesICC consisted of an upstroke depolarization followed by a distinct plateau component. Ni(2+) and nominally Ca(2+)-free solutions reduced the rate-of-rise and amplitude of the upstroke depolarization. Replacement of Ca(2+) with Sr(2+) enhanced the upstroke component but decreased the plateau component of rabbit slow wavesICC. In contrast, replacing Ca(2+) with Sr(2+) decreased both components of mouse slow wavesICC. The plateau component of rabbit slow wavesICC was inhibited in low-extracellular-Cl(-)-concentration (low-[Cl(-)]o) solutions and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl(-) channels, cyclopiazonic acid (CPA), an inhibitor of internal Ca(2+) pumps, or bumetanide, an inhibitor of Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Bumetanide also inhibited the plateau component of mouse slow wavesICC. NKCC1-like immunoreactivity was observed mainly in ICC-MY in the rabbit small intestine. Membrane depolarization with a high-K(+) solution reduced the upstroke component of rabbit slow wavesICC. In cells depolarized with elevated external K(+), DIDS, CPA, and bumetanide blocked slow wavesICC. These results suggest that the upstroke component of rabbit slow wavesICC is partially mediated by voltage-dependent Ca(2+) influx, whereas the plateau component is dependent on Ca(2+)-activated Cl(-) efflux. NKCC1 is likely to be responsible for Cl(-) accumulation in ICC-MY. The results also suggest that the mechanism of the upstroke component differs in rabbit and mouse slow wavesICC in the small intestine.

Oridonin: A small molecule inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) isolated from traditional Chinese medicine

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel regulating the transepithelial transport of electrolyte and water. In the recent years, CFTR chloride channel becomes the new molecular target of treating secretory diarrhea. The objective of this study is to find out a novel CFTR inhibitor from traditional Chinese medicine (TCM) and study on its pharmacological activity. About 34,000 factions of TCM extracts were screened by high throughput screening (HTS) in this research. We found that Rabdosia rubescens show a potent inhibition on CFTR. Under the bio-active analysis guidance, an ent-kaurane diterpenoid — oridonin (PubChem CID: 34378) was isolated from R. rubescens. A series of intensive studies showed that oridonin remarkably reduced iodide influx in wt-CFTR and ΔF508-CFTR FRT epithelial cells in a dose-dependent and irreversible way. Oridonin sharply blocked FSK-stimulated short-circuit current in both rats and mice intestine in vitro. In mouse closed-loop model, oridonin reduced cholera toxin-induced fluid secretion significantly over 6hours in vivo. Thus we concluded that oridonin is a new inhibitor of CFTR Cl− channel. It will be a good leading compound for developing the new drug of cholera toxin-induced secretory diarrhea.

P69 Sodium sulphide relaxation of rat uterus is related to calcium signaling

H2S was shown as an uterine relaxant. However, the signaling pathways, including ion channels regulated by H2S mediating relaxation in uterus are still unknown. The effects on contractility in response to sodium sulphide Na2S were examined on myometrial strips from virgin Wistar rats. Our results showed that Na2S induces concentration-dependent relaxations affecting amplitude as well as frequency of contractions. Activation of potassium channels, and in particular of KATP, was one of the primary mechanisms proposed, responsible for the relaxing effects of H2S. Sodium sulphide (20–200 × 10−6 M) inhibits myometrial contractility through a K-channel-independent mechanism. An inhibitor of 4,4 – inhibitor of Cl - / HCO 3 - exchanger and/or Cl− channel, DIDS, caused a significant rightward shift of the Na2S concentration–response curve. We performed experiments aimed at different Ca2+ concentrations, using spontaneous, calcium and KCl (15 mM and 75 mM) induced contractions, as well as pharmacological inhibitors of calcium channels and modulators, showing that Na2S induced relaxation is dependent on the precontractile agent used. Taken together, our results suggests that decreased frequency induced by Na2S could be a consequence of a alterated pacemaker cells which might be related for Ca2+ events originates from sarco endoplasmatic reticulum and/or mitochondria.

Effect of Chloride Channel Inhibitors on Cytosolic Ca2+ Levels and Ca2+-Activated K+ (Gardos) Channel Activity in Human Red Blood Cells

DIDS, NPPB, tannic acid (TA) and AO1 are widely used inhibitors of Cl(-) channels. Some Cl(-) channel inhibitors (NPPB, DIDS, niflumic acid) were shown to affect phosphatidylserine (PS) scrambling and, thus, the life span of human red blood cells (hRBCs). Since a number of publications suggest Ca(2+) dependence of PS scrambling, we explored whether inhibitors of Cl(-) channels (DIDS, NPPB) or of Ca(2+)-activated Cl(-) channels (DIDS, NPPB, TA, AO1) modified intracellular free Ca(2+) concentration ([Ca(2+)]i) and activity of Ca(2+)-activated K(+) (Gardos) channel in hRBCs. According to Fluo-3 fluorescence in flow cytometry, a short treatment (15 min, +37 °C) with Cl(-) channels inhibitors decreased [Ca(2+)]i in the following order: TA > AO1 > DIDS > NPPB. According to forward scatter, the decrease of [Ca(2+)]i was accompanied by a slight but significant increase in cell volume following DIDS, NPPB and AO1 treatments. TA treatment resulted in cell shrinkage. According to whole-cell patch-clamp experiments, TA activated and NPPB and AO1 inhibited Gardos channels. The Cl(-) channel blockers further modified the alterations of [Ca(2+)]i following ATP depletion (glucose deprivation, iodoacetic acid, 6-inosine), oxidative stress (1 mM t-BHP) and treatment with Ca(2+) ionophore ionomycin (1 μM). The ability of the Cl(-) channel inhibitors to modulate PS scrambling did not correlate with their influence on [Ca(2+)]i as TA and AO1 had a particularly strong decreasing effect on [Ca(2+)]i but at the same time enhanced PS exposure. In conclusion, Cl(-) channel inhibitors affect Gardos channels, influence Ca(2+) homeostasis and induce PS exposure of hRBCs by Ca(2+)-independent mechanisms.

Evaluation of an Electrochemical Method for the Analysis of Enzymatic Inhibition Reactions

An approximate equation for bioelectrocatalitic current was applied to an inhibition reaction analysis of bilirubin oxidase by anion (Cl(-), SCN(-), and F(-)) in order to assess the possibility of the electrochemical method for the analysis of enzymatic inhibition reactions. The approximate equation can be transformed into the Michaelis-Menten form, so that the bioelectrocatalitic current can be analyzed by the usual graphical manner, that is, Lineweaver-Burk, Hanes-Woolf, Dixon and Cornish-Bowden plots, if the rate of inhibition reaction is described as a simple Michaelis-Menten form. From the electrochemical assay, it was found that the inhibitor of Cl(-) and SCN(-) anions exhibit non-competitive inhibition while that of F(-) exhibits competitive inhibition, and their inhibition constants were 220, 45, and 22 mM, respectively. The results were essentially similar to those obtained from the conventional spectrophotometric assay.

Lipoxin A4 Stimulates Calcium-Activated Chloride Currents and Increases Airway Surface Liquid Height in Normal and Cystic Fibrosis Airway Epithelia

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl− secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA4 is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA4 are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA4 produced a rapid and transient increase in intracellular Ca2+. We have investigated, the effect of LXA4 on Cl− secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA4 stimulated a rapid intracellular Ca2+ increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA4 stimulated whole-cell Cl− currents which were inhibited by NPPB (calcium-activated Cl− channel inhibitor), BAPTA-AM (chelator of intracellular Ca2+) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA4 increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA4 effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl− secretion. The LXA4 stimulation of intracellular Ca2+, whole-cell Cl− currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA4 in the stimulation of intracellular Ca2+ signalling leading to Ca2+-activated Cl− secretion and enhanced ASL height in non-CF and CF bronchial epithelia.

Prolonged stimulation of pancreatic serous secretions by bile and sodium taurocholate in anaesthetized rats

There have been numerous reports that infusion of either natural bile or bile salts into the duodenum evokes a rapid increase in pancreatic secretion through the release of the hormone secretin from the duodenal mucosa. We have extended this observation by the demonstration of an additional late increase in secretion which persisted for many hours and have sought to identify the processes underlying this increase. In anaesthetised rats, infusion of 20 mM taurocholate into the duodenum caused a staircase-like increase in the weight of pancreatic secretion which extended over many hours during which, the HCO[Formula: see text] and protein output of the secretion showed only minimal changes. This effect was also reproduced with intra-duodenal infusion of natural bile which was inferred to act though its taurocholate content. Since the stimulatory action was also obtained with superfusion of taurocholate or natural bile onto the small intestine and by intravenous injection of taurocholate, it was concluded that taurocholate acted by being absorbed into the bloodstream and then by exerting a stimulatory action on the exocrine pancreas. This action was inhibited by puromycin (a protein synthesis inhibitor), by furosemide (a Na( + )/K( + )/2Cl(-) cotransporter inhibitor), though not by SITS (an inhibitor of Cl(-)/HCO[Formula: see text] exchange). The long lasting increase in pancreatic serous secretion would be consistent with the possible activation of gene transcription by taurocholate leading to increased activity of the Na( + )/K( + )/2Cl(-) cotransporter through which the acinar cells increased their secretions.

An Inhibitor of Na+/H+ Exchanger (NHE), Ethyl-Isopropyl Amiloride (EIPA), Diminishes Proliferation of MKN28 Human Gastric Cancer Cells by Decreasing the Cytosolic Cl- Concentration via DIDS-Sensitive Pathways

Background/Aims: Tumor cells produce a large amount of acidic metabolites due to their high metabolic condition. However, cytosolic pH (pH_c) of tumor cells is identical to or even slightly higher than that of normal cells. To maintain pH_c at a normal or higher level, tumor cells would have to have higher expression and/or activity of H⁺ transporting systems than normal cells. The purpose of the present study was to identify effects of ethyl-isopropyl amiloride (EIPA, an inhibitor of Na⁺/H⁺ exchanger (NHE)) on proliferation of human gastric cancer MKN28 cells. Methods: Effects of EIPA on proliferation, pH_c, [Cl^-]_c and expression of proteins regulating cell cycle and MAPKs were studied in MKN28 expressing NHE exposed to EIPA for 48 h. Results: EIPA suppressed proliferation of MKN28 cells by causing G₀/G₁ arrest without any significant effects on pH_c, but associated with reduction of [Cl^-]_c. Although EIPA alone had no effects on pH_c, EIPA co-applied with DIDS (an inhibitor of Cl^-/HCO₃^- exchangers; i.e., anion exchanger (AE) and Na+-driven Cl^-/HCO₃^- exchanger (NDCBE)) reduced pH_c, suggesting that DIDS-sensitive Cl^-/HCO₃^- transporters such as AE and/or NDCBE keep pH_c normal by stimulating HCO₃^- uptake coupled with Cl^- release under an NHE-inhibited condition. EIPA-induced lowered [Cl^-]_c up-regulated expression of p21associated with phosphorylation of MAPKs, suppressing proliferation associated with G₀/G₁ arrest. Conclusions: EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl^-]_c as a result from DIDS-sensitive Cl^-/HCO₃^- exchanger-mediated compensation for keeping pH_c normal under an NHE-inhibited condition. This is the first study revealing that an NHE inhibitor suppressed the proliferation of cancer cells by reducing [Cl^-]_c but not pH_c.