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Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A 1 receptors (A 1 Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K + conductance mediated by K ir 4.1/5.1 channels to mediate K + recycling and to set up a favorable driving force for Na + /K + exchange (47). Intercalated cells express the Cl - ClC-K2/b channel mediating transcellular Cl - reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on K ir 4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A 1 R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A 1 R agonist N 6 -cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and K ir 4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A 1 Rs. We propose that inhibition of transcellular Cl - reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.

CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of N -arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs. Computational docking to a CLC-Ka homology model has identified a BIM1 binding site on the extracellular face of the protein near the chloride permeation pathway in a region previously identified as a binding site for other less selective inhibitors. Results from site-directed mutagenesis experiments are consistent with predictions of this docking model. The residue at position 68 is 1 of only ∼20 extracellular residues that differ between CLC-Ka and CLC-Kb. Mutation of this residue in CLC-Ka and CLC-Kb (N68D and D68N, respectively) reverses the preference of BIM1 for CLC-Ka over CLC-Kb, thus showing the critical role of residue 68 in establishing BIM1 selectivity. Molecular docking studies together with results from structure-activity relationship studies with 19 BIM derivatives give insight into the increased selectivity of BIM1 compared with other inhibitors and identify strategies for further developing this class of compounds., Competing Interests: The authors declare no conflict of interest.

Activation of swelling-induced Cl - current (I Cl,swell ) during neonatal hypoxia-ischemia (HI) may induce brain damage. Hypoxic-ischemic brain injury causes chronic neurological morbidity in neonates as well as acute mortality. In this study, we investigated the role of I Cl,swell in hypoxic-ischemic brain injury using a selective blocker, 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB). In primary cultured cortical neurons perfusion of a 30% hypotonic solution activated I Cl,swell , which was completely blocked by the application of DCPIB (10 μmol/L). The role of I Cl,swell in neonatal hypoxic-ischemic brain injury in vivo was evaluated in a modified neonatal hypoxic-ischemic brain injury model. Before receiving the ischemic insult, the mouse pups were injected with DCPIB (10 mg/kg, ip). We found that pretreatment with DCPIB significantly reduced the brain damage assessed using TTC staining, Nissl staining and whole brain imaging, and improved the sensorimotor and vestibular recovery outcomes evaluated in neurobehavioural tests (i.e. geotaxis reflex, and cliff avoidance reflex). These results show that DCPIB has neuroprotective effects on neonatal hypoxic-ischemic brain injury, and that the I Cl,swell may serve as a therapeutic target for treatment of hypoxic-ischemic encephalopathy.

Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling-induced chloride current I Cl,swell . In this study, we investigated the effects of I Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I Cl,swell , DCPIB, potently reduced the I Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB-treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I Cl,swell may be a potential drug target for GBM., (© 2017 Wiley Periodicals, Inc.)

Intestinal Cl - secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl - secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl - secretion in T84 cell monolayers with IC 50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl - channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K + channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca 2+ -dependent Cl - secretion with IC 50 of ∼10 μM. FFA inhibited activities of Ca 2+ -activated Cl - channels and K Ca 3.1, a Ca 2+ -activated basolateral K + channels, but had no effect on activities of Na + -K + -Cl - cotransporters and Na + -K + ATPases. These results indicate that FFA inhibits both cAMP and Ca 2+ -dependent Cl - secretion by suppressing activities of both apical Cl - channels and basolateral K + channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas., (Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Main Conclusion: Potassium-permeable slow activating vacuolar channels (SV) and chloride-permeable channels in the vacuole of the liverwort Marchantia polymorpha were characterized in respect to calcium dependence, selectivity, and pharmacology. The patch-clamp method was used in the study of ion channel activity in the vacuoles from the liverwort Marchantia polymorpha. The whole-vacuole recordings allowed simultaneous observation of two types of currents-predominant slow activated currents recorded at positive voltages and fast activated currents recorded at negative voltages. Single-channel recordings carried out in the gradient of KCl indicated that slow activated currents were carried by potassium-permeable slowly activating vacuolar channels (SV) and fast activated currents-by chloride-permeable channels. Both types of the channels were dependent in an opposite way on calcium, since elimination of this ion from the cytoplasmic side caused inhibition of SV channels, but the open probability of chloride-permeable channels even increased. The dependence of the activity of both channels on different types of ion channel inhibitors was studied. SV channels exhibited different sensitivity to potassium channel inhibitors. These channels were insensitive to 3 mM Ba 2+ , but were blocked by 3 mM tetraethyl ammonium (TEA). Moreover, the activity of the channels was modified in a different way by calcium channel inhibitors. 200 µM Gd 3+ was an effective blocker, but 50 µM ruthenium red evoked bursts of the channel activity resulting in an increase in the open probability. Different effectiveness of anion channel inhibitors was observed in chloride-permeable channels. After the application of 100 µM Zn 2+ , a decrease in the open probability was recorded but the channels were still active. 50 µM DIDS was more effective, as it completely blocked the channels.

A prerequisite for tissue electrolyte homeostasis is highly regulated ion and water transport through kidney or intestinal epithelia. In the present work, we monitored changes in the cell and luminal volumes of type II Madin-Darby canine kidney (MDCK) cells grown in a 3D environment in response to drugs, or to changes in the composition of the basal extracellular fluid. Using fluorescent markers and high-resolution spinning disc confocal microscopy, we could show that lack of sodium and potassium ions in the basal fluid (tetramethylammonium chloride (TMACl) buffer) induces a rapid increase in the cell and luminal volumes. This transepithelial water flow could be regulated by inhibitors and agonists of chloride channels. Hence, the driving force for the transepithelial water flow is chloride secretion, stimulated by hyperpolarization. Chloride ion depletion of the basal fluid (using sodium gluconate buffer) induces a strong reduction in the lumen size, indicating reabsorption of water from the lumen to the basal side. Lumen size also decreased following depolarization of the cell interior by rendering the membrane permeable to potassium. Hence, MDCK cells are capable of both absorption and secretion of chloride ions and water; negative potential within the lumen supports secretion, while depolarizing conditions promote reabsorption.

Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice., (Copyright © 2016. Published by Elsevier B.V.)

Unlabelled: Polycystic kidney diseases are characterized by the development of numerous bilateral renal cysts that continuously enlarge resulting in a decline of kidney function due to compression of intact nephrons. Cyst growth is driven by transepithelial chloride secretion which depends on both intracellular cAMP and calcium. Mechanisms that are involved in the regulation of the underlying secretory pathways remain incompletely understood. Here we show that glucose concentration has a strong impact on cyst growth of renal tubular cells within a collagen matrix as well as in embryonic kidneys deficient or competent for Pkd1. Glucose-dependent cyst growth correlates with the transcriptional induction of the calcium-activated chloride channel anoctamin 1 (ANO1) and its increased expression in the apical membrane of cyst-forming cells. Inhibition of ANO1 with the specific inhibitor CaCCinh-AO1 significantly decreases glucose-dependent cyst growth in both models. Ussing chamber analyses revealed increased apical chloride secretion of renal tubular cells upon exposure to high glucose medium which can also be inhibited by the use of CaCCinh-AO1. These data suggest that glycemic control may help to reduce renal cyst growth in patients with polycystic kidney disease., Key Message: Renal cyst growth depends on glucose concentration in two in vitro cyst models. High glucose leads to upregulation of the calcium-activated chloride channel ANO1. High glucose promotes calcium-activated chloride secretion via ANO1. Glucose-dependent secretion can be inhibited by a specific inhibitor of ANO1.

Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors., (Copyright © 2015 the American Physiological Society.)

Eight paralogue members form the family of transmembrane channel-like (TMC) proteins that share considerable sequence homology to anoctamin 1 (Ano1, TMEM16A). Ano1 is a Ca(2+) activated Cl(-) channel that is related to head and neck cancer, often caused by human papilloma virus (HPV) infection. Mutations in TMC 6 and 8 (EVER1, EVER2) cause epidermodysplasia verruciformis. This rare skin disease is characterized by abnormal susceptibility to HPV infection and cancer. We found that in contrast to Ano1 the common paralogues TMC4-TMC8 did not produce Ca(2+) activated Cl(-) currents when expressed in HEK293 cells. On the contrary, TMC8 was found to be localized in the endoplasmic reticulum (ER), where it inhibited receptor mediated Ca(2+) release, activation of Ano1 and volume regulated LRRC8-related Cl(-) currents. Zn(2+) is co-released from the ER together with Ca(2+) and thereby further augments Ca(2+) store release. Because TMC8 is required to lower cytosolic Zn(2+) concentrations by the Zn(2+) transporter ZnT-1, we hypothesize that HPV infections and cancer caused by mutations in TMC8 are related to upregulated Zn(2+)/Ca(2+) signaling and activation of Ano1., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background and Aims: The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth., Methods: Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO3 were also performed., Key Results: Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ~30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO3 significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO3 did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion., Conclusions: These results suggest that Cl(-) plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl(-) uptake during IAA-induced growth is proposed in which uptake of K(+) and Cl(-) ions in concert with IAA-induced plasma membrane H(+)-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Adrenomedullin (ADM) may regulate seminal vesicle fluid secretion, and this may affect sperm quality. In this study, we investigated the effect of ADM on chloride secretion in the mouse seminal vesicle. The presence of ADM in mouse seminal vesicle was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with enzyme-linked assay for ADM. The effects of ADM on chloride secretion were studied by short-circuit current technique in a whole-mount preparation of mouse seminal vesicle in an Ussing chamber. The effects of specific ADM and calcitonin gene-related peptide (CGRP) receptor antagonists were investigated. Whether the ADM effect depended on the cAMP- and/or calcium-activated chloride channel was also studied using specific chloride channel blockers. The results showed that ADM was present in seminal vesicle epithelial cells. The major molecular species was precursor in the mouse seminal vesicle. ADM increased short-circuit current through the calcium-activated chloride channel in mouse seminal vesicle, and CGRP receptor was involved. We conclude that ADM may regulate chloride and fluid secretion from the seminal vesicle, which may affect the composition of the seminal plasma bathing the sperm and, hence, fertility., (© 2014 by the Society for the Study of Reproduction, Inc.)

Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

Background: Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl currents (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown., Methods and Results: In the present study, we found that the specific ICl,swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50 = 2.7 μmol/l) ICl,swell and inhibited (IC50 = 6.9 μmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl channel genes underlying ICl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to ICl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter ICl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution., Conclusion: Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.

Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1.

Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs(+) resistant, and was not affected by removal of external Na(+) or K(+). The current was inhibited with Cd(2+), and the reversal potential was sensitive to changes in [Cl(-)] on either side of the membrane in a manner consistent with a Cl(-) selective channel. Cl(-) channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl(-) current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl(-) current in rat PVs was shifted to negative potentials as [Cl(-)]i increased, (ii) the Cl(-) current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl(-) channel, and that the channel may play a functional role in norepinephrine-induced automaticity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Here, we describe an outward rectifying current in Xenopus tropicalis oocytes that we have called xtClC-or. The current has two components; the major component is voltage activated and independent of intracellular or extracellular Ca(2+), whereas the second is a smaller component that is Ca(2+) dependent. The properties of the Ca(2+)-independent current, such as voltage dependence and outward rectification, resemble those of ClC anion channels/transporters. This current is sensitive to NPPB and NFA, insensitive to 9AC and DIDS, and showed a whole-cell conductance sequence of SCN(-)>I(-)>Br(-)>CI(-). RT-PCR revealed the expression in oocytes of ClC-2 to ClC-7, and major reductions of current amplitudes were observed when a ClC-5 antisense oligonucleotide was injected into oocytes. The Ca(2+)-dependent component was abated after injection of 10mM BAPTA or EGTA, whereas 10mMMg(2+) inhibited the current to 26±3.1%. This component was blocked by 9-AC, NFA, and NPPB, whereas DIDS did not elicit any evident effect. The ion sequence selectivity was SCN=I(-)>Br(-)>Cl(-). To try to determine the molecular identity that gives rise to this component we assessed by RT-PCR the expression of the Ca(2+)-dependent Cl(-) channel TMEM16A, which was found to be present in the oocytes. However, injection of antisense TMEM16A oligonucleotides did not inhibit the transient outward current. This result fits well with the electrophysiological data. Together, these results suggest that ClC-5 is a major, but not the sole channel responsible for this outwardly rectifying Cl(-) current., (Copyright © 2013 Elsevier B.V. All rights reserved.)

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H(+)/Cl(-) exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.

The epithelium of the developing lung displays an evolving liquid transport phenotype, in which Cl(-) secretion during fetal life is rapidly switched to Na(+) absorption perinatally. However, the mechanisms underlying the homeostasis of the thin layer of liquid lining the postnatal pulmonary epithelium remain elusive. In particular, it remains unclear whether the stimulated clearance of excess alveolar liquid is mediated via transepithelial Cl(-) transport. Our study is a pharmacological analysis with the aim of addressing this issue, which is of major physiological significance in cases of pulmonary oedema from any cause. We measured the rate of transepithelial liquid movement (J(v)) with (125)I-albumin, in the in situ perfused adult rat lung. Transepithelial Cl(-) transport was studied with the use of the Cl(-) channel inhibitor NPPB in the resting state and during stimulation with the β(2)-adrenergic agonist terbutaline. The study of J(v) in these conditions revealed the following findings: (1) there is net absorption of excess of alveolar liquid in the resting, unstimulated state, which is predominantly amiloride sensitive; (2) inhibition of Cl(-) transport with NPPB in the resting state results in a 1.6-fold increase in net absorption of alveolar liquid; and (3) the terbutaline-stimulated net absorption of the excess liquid is enhanced by 2.8-fold in the presence of NPPB. Our results are suggestive of the functional presence of secretory, but not absorptive, Cl(-) mechanisms and show that transepithelial Cl(-) transport is not part of the mechanism underlying lung liquid clearance in response to β-adrenergic stimulation.

Resting skeletal muscle fibres have a large membrane Cl(-) conductance (G(Cl)) that dampens their excitability. Recently, however, muscle activity was shown to induce PKC-mediated reduction in G(Cl) in rat muscles of 40-90%. To examine the physiological significance of this PKC-mediated G(Cl) reduction for the function of muscles, this study explored effects of G(Cl) reductions on contractile endurance in isolated rat muscles. Contractile endurance was assessed from the ability of muscle to maintain force during prolonged stimulation under conditions when G(Cl) was manipulated by: (i) inhibition of PKC, (ii) reduction of solution Cl(-) or (iii) inhibition of ClC-1 Cl(-) channels using 9-anthracene-carboxylic acid (9-AC). Experiments showed that contractile endurance was optimally preserved by reductions in G(Cl) similar to what occurs in active muscle. Contrastingly, further G(Cl) reductions compromised the endurance. The experiments thus show a biphasic relationship between G(Cl) and contractile endurance in which partial G(Cl) reduction improves endurance while further G(Cl) reduction compromises endurance. Intracellular recordings of trains of action potentials suggest that this biphasic dependency of contractile endurance on G(Cl) reflects that lowering G(Cl) enhances muscle excitability but low G(Cl) also increases the depolarisation of muscle fibres during excitation and reduces their ability to re-accumulate K(+) lost during excitation. If G(Cl) becomes very low, the latter actions dominate causing reduced endurance. It is concluded that the PKC-mediated ClC-1 channel inhibition in active muscle reduces G(Cl) to a level that optimises contractile endurance during intense exercise.

Picrotoxin is a pore blocker that can differentiate ligand-gated inhibitory chloride channels. Even within one receptor type, such as the glycine receptor, picrotoxin block differs between subunits. The effect of subunit gating properties on block of the inhibitory glycine receptor (GlyR) was explored using heteromeric α subunit expression in voltage-clamped HEK293 cells. The α2 GlyR is more sensitive to picrotin block than the α1 GlyR, and this difference was used to explore whether mutations that interfered with gating of the α2 subunit would also interfere with picrotin block. Two mutations were used: one that decreased the glycine sensitivity of α2 by almost two log units and the other that was unresponsive to glycine. In both cases, the sensitivity to picrotin was essentially unaltered. The results indicated that α2 subunits can determine the picrotin sensitivity of α1α2-heteromeric receptors and that direct gating of the α2 subunit is not required for this picrotin inhibition.

Purpose: We investigated the role that the cystic fibrosis transmembrane conductance regulator (CFTR) may play in Cl(-) transport in the acinar and ductal epithelial cells of rabbit lacrimal gland (LG)., Methods: Primary cultured LG acinar cells were processed for whole-cell patch-clamp electrophysiological recording of Cl(-) currents by using perfusion media with high and low [Cl(-)], 10 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX), the non-specific Cl(-) channel blocker 4,4'-disothiocyanostilbene-2, 2' sulphonic acid (DIDS; 100 µM) and CFTRinh-172 (10 µM), a specific blocker for CFTR. Ex vivo live cell imaging of [Cl(-)] changes in duct cells was performed on freshly dissected LG duct with a multiphoton confocal laser scanning microscope using a Cl(-) sensitive fluorescence dye, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide., Results: Whole-cell patch-clamp studies demonstrated the presence of Cl(-) current in isolated acinar cells and revealed that this Cl(-) current was mediated by CFTR channel. Live cell imaging also showed the presence of CFTR-mediated Cl(-) transport across the plasma membrane of duct cells., Conclusions: Our previous data showed the presence of CFTR in all acinar and duct cells within the rabbit LG, with expression most prominent in the apical membranes of duct cells. The present study demonstrates that CFTR is actively involved in Cl(-) transport in both acinar cells and epithelial cells from duct segments, suggesting that CFTR may play a significant role in LG secretion.

It is accepted that K(+) and Cl(-) flows are coupled tightly in regulatory volume decrease (RVD). However, using self referencing microelectrodes, we proved that K(+) and Cl(-) transport mainly by channels in RVD was uncoupled in nasopharyngeal carcinoma CNE-2Z cells, with the transient K(+) efflux activated earlier and sustained Cl(-) efflux activated later. Hypotonic challenges decreased intracellular pH (pH(i)), and activated a proton pump-dependent H(+) efflux, resulting in a decline of extracellular pH (pH(o)). Modest decreases of pH(o) inhibited the volume-activated K(+) outflow and RVD, but not the Cl(-) outflow, while inhibition of H(+) efflux or increase of pH(o) buffer ability promoted K(+) efflux and RVD. The results suggest that the temporal dynamics of K(+) channel activities is different from that of Cl(-) channels in RVD, due to differential sensitivity of K(+) and Cl(-) channels to pH(o). H(+) efflux may play important roles in cell volume regulation, and may be a therapeutic target for human nasopharyngeal carcinoma., (Copyright © 2012. Published by Elsevier Inc.)

Acid-activated chloride currents have been reported in several cell types and may play important roles in regulation of cell function. However, the molecular identities of the channels that mediate the currents are not defined. In this study, activation of the acid-induced chloride current and the possible candidates of the acid-activated chloride channel were investigated in human nasopharyngeal carcinoma cells (CNE-2Z). A chloride current was activated when extracellular pH was reduced to 6.6 from 7.4. However, a further decrease of extracellular pH to 5.8 inhibited the current. The current was weakly outward-rectified and was suppressed by hypertonicity-induced cell shrinkage and by the chloride channel blockers 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB), tamoxifen, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS). The permeability sequence of the channel to anions was I(-) > Br(-) > Cl(-) > gluconate(-). Among the ClC chloride channels, ClC-3 and ClC-7 were strongly expressed in CNE-2Z cells. Knockdown of ClC-3 expression with ClC-3 small interfering (si)RNA prevented the activation of the acid-induced current, but silence of ClC-7 expression with ClC-7 siRNA did not significantly affect the current. The results suggest that the chloride channel mediating the acid-induced chloride current was volume sensitive. ClC-3 is a candidate of the channel proteins that mediate or regulate the acid-activated chloride current in nasopharyngeal carcinoma cells.

We studied the possibility of K+ and Cl- efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl- efflux; potassium channel inhibitor (tetraethylammonium chloride) partially reduced K+ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba2+ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K+ and Cl- release. An important role in these processes is played by NPPB-, TEA- and Ba(2+)-sensitive plasmalemma ion channels.

Volume-activated chloride channels have been studied by us extensively in human nasopharyngeal carcinoma cells. However, the chloride channels in the counterpart of the carcinoma cells have not been investigated. In this study, volume-activated chloride currents (I(cl,vol)) were characterized in normal fetal human nasopharyngeal epithelial cells using the whole-cell patch-clamp technique. Under isotonic conditions, nasopharyngeal epithelial cells displayed only a weak background current. Exposure to 47% hypotonic solution activated a volume-sensitive current. The reversal potential of the current was close to the calculated equilibrium potential for Cl(-). The peak values of the hypotonicity-activated current at +80 mV ranged from 0.82 to 2.71 nA in 23 cells. Further analysis indicated that the density of the hypotonicity-activated current in most cells (18/23) was smaller than 60 pA/pF. Only five cells presented a current larger than 60 pA/pF. The hypotonicity-activated current was independent of the exogenous ATP. Chloride channel inhibitors ATP, tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibited the current dramatically. The anion permeability of the hypotonicity-activated chloride channels was I(-) > Br(-) > Cl(-) > gluconate. Unexpectedly, in isotonic conditions, ATP (10 mM) activated an inward-rectified current, which had not been observed in the nasopharyngeal carcinoma cells. These results suggest that, under hypotonic challenges, fetal human nasopharyngeal epithelial cells can produce I(cl,vol), which might be involved in cell volume regulation., (© Springer Science+Business Media, LLC 2012)

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 G(0)/G(1) 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(-)/HCO(3)(-) exchangers; i.e., anion exchanger (AE) and Na+-driven Cl(-)/HCO(3)(-) exchanger (NDCBE)) reduced pH(c), suggesting that DIDS-sensitive Cl(-)/HCO(3)(-) transporters such as AE and/or NDCBE keep pH(c) normal by stimulating HCO(3)(-) 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 G(0)/G(1) 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(-)/HCO(3)(-) 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)., (Copyright © 2012 S. Karger AG, Basel.)

In this study, the activation mechanisms of the background chloride current and the role of the current in maintaining of basal cell volume were investigated in human nasopharyngeal carcinoma CNE-2Z cells. Under isotonic conditions, a background chloride current was recorded by the patch clamp technique. The current presented the properties similar to those of the volume-activated chloride current in the same cell line and was inhibited by chloride channel blockers or by cell shrinkage induced by hypertonic challenges. Extracellular applications of reactive blue 2, a purinergic receptor antagonist, suppressed the background chloride current in a concentration-dependent manner under isotonic conditions. Depletion of extracellular ATP with apyrase or inhibition of ATP release from cells by gadolinium chloride decreased the background current. Extracellular applications of micromolar concentrations of ATP activated a chloride current which was inhibited by chloride channel blockers and hypertonic solutions. Extracellular ATP could also reverse the action of gadolinium chloride. Transfection of CNE-2Z cells with ClC-3 siRNA knocked down expression of ClC-3 proteins, attenuated the background chloride current and prevented activation of the ATP-induced current. Furthermore, knockdown of ClC-3 expression or exposures of cells to ATP (10 mM), the chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, or reactive blue 2 increased cell volume under isotonic conditions. The results suggest that ClC-3 protein may be a main component of background chloride channels which can be activated under isotonic conditions by autocrine/paracrine ATP through purinergic receptor pathways; the background current is involved in maintenance of basal cell volume., (Copyright © 2010 Wiley-Liss, Inc.)

During anoxia/reoxygenation (A/R) injury, intracellular chloride ion concentration ([Cl(-)](i)) homeostasis may play a role in maintaining the normal physiological function of cardiomyocytes. Various chloride transport systems could have influenced the concentration of chloride ion, but what kinds of chloride transport systems could play an important role in cardiomyocytes subjected to A/R injury and its mechanism are unknown. The aim of our study was to clarify the contributions of various chloride transport systems to anoxia/reoxygenation in rat neonatal cardiac myocytes and further to investigate the involved mechanisms. Oxidative stress and redox-sensitive transcription factor (NF-kappaB) activation are believed to play an important role in the A/R injury. To assess whether oxidative stress and NF-kappaB involve [Cl(-)](i) changes resulting in cardiomyocytes injury, the anoxia-reoxygenation (A/R) injury model was successfully established and administered with inhibitors of various chloride transport systems. Administration with Cl(-)-substitution and Cl(-)/HCO(3) (-) exchange inhibitor(SITS) has been shown to produce a protective effect against A/R injury by decreasing [Cl(-)](i) concentration, lipid peroxidation (malondialdehyde (MDA)) levels, and NF-kappaB activity, and by increasing antioxidant enzyme (glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase(CAT)) activity. However, inhibitors for the Cl(-)-channel (9-AC) and Na(+)-K(+)-2Cl(-) co-transporter (bumetanide) had no effects. Our results indicate that Cl(-)/HCO(3) (-) exchange system plays an important role in the cardiocyte A/R injury by influencing [Cl(-)](i) concentration. The protective effects of SITS and Cl(-)-substitution on cardiomyocytes may be due to the attenuation of oxidative stress and inhibition of NF-kappaB activation.

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood-brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87-MG cells migration. Using the perforated patch-clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM-34 sensitive, intermediate-conductance calcium-activated K (IK(Ca) ) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS- and NPPB-sensitive Cl current displaying an outward rectifying instantaneous current-voltage relationship and a slow, voltage-dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IK(Ca) current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS-induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM-34 and DIDS on the FCS-induced U87-MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87-MG cell migration in the presence of FCS, and that their co-application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS-induced glioblastoma cell migration., (Copyright © 2010 Wiley-Liss, Inc.)

Objective: Volume-regulated Cl(-) channel (VRCC) plays a critical role in regulation of a variety of physiological functions. However, little is known whether VRCC is involved in atherosclerosis. In this study, we investigated the functions of VRCC during foam cell formation in macrophages., Methods and Results: Treatment of RAW264.7 cells with ox-LDL increased intracellular cholesterol content as well as cell volume. After ox-LDL treatment, the resting [Cl(-)](i) in isotonic solution was decreased. Hypotonic solution reduced [Cl(-)](i) and evoked volume-regulated Cl(-) current in all the cells, however, the swelling-induced reduction of [Cl(-)](i) and increase of Cl(-) current were more prominent in ox-LDL treated cells than that in control. The increases of volume-regulated Cl(-) movement positively correlated with the intracellular cholesterol content. Moreover, in peritoneal macrophages isolated from high-fat diet ApoE(-/-) mice, the swelling-induced Cl(-) movement and current were enhanced compared with those in control group, and their increments positively correlated with atherosclerotic plaque area. Finally, activation of VRCC by hypotonic medium significantly accelerated, whereas, inhibition of VRCC with Cl(-) channel blockers remarkably attenuated, ox-LDL-induced macrophage-derived foam cell formation., Conclusion: The activity of VRCC is augmented during macrophage-derived foam cell formation. Activation of VRCC accelerated, whereas, inhibition of VRCC attenuated, ox-LDL-induced lipid accumulation in macrophages, suggesting VRCC is involved in the regulation of foam cell formation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Adrenaline activates transient Cl(-) secretion and sustained K(+) secretion across isolated distal colonic mucosa of guinea-pigs. The Ca(2+)-activated Cl(-) channel inhibitor CaCCinh-A01 (30 μm) significantly reduced electrogenic K(+) secretion, detected as short-circuit current (I(sc)). This inhibition supported the cell model for K(+) secretion in which basolateral membrane Cl(-) channels provide an exit pathway for Cl(-) entering the cell via Na(+)-K(+)-2Cl(-) cotransporters. CaCCinh-A01 inhibited both I(sc) and transepithelial conductance in a concentration-dependent manner (IC(50) = 6.3 μm). Another Cl(-) channel inhibitor, GlyH-101, also reduced sustained adrenaline-activated I(sc) (IC(50) = 9.4 μm). Adrenaline activated whole-cell Cl(-) current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl(-) current was also inhibited by CaCCinh-A01 or GlyH-101. In contrast to K(+) secretion, CaCCinh-A01 augmented the electrogenic Cl(-) secretion activated by adrenaline as well as that activated by prostaglandin E(2). Synergistic Cl(-) secretion activated by cholinergic/prostaglandin E(2) stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca(2+)-activated Cl(-) channel protein Tmem16A was supported by RT-PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K(+) secretion and augmentation of Cl(-) secretion by CaCCinh-A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl(-) channels contributes to the production of electrogenic K(+) secretion and limits the rate of Cl(-) secretion. Maximal physiological Cl(-) secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl(-) channels.

In this study, an outwardly rectifying chloride channel was characterized in the trophoblastic cell line BeWo, a human hormone-synthesizing cell which displays many biochemical and morphological properties similar to those reported for the human cytotrophoblast. Ion channel activity was recorded in the cell attached and inside-out configurations with standard patch-clamp technology. In most of the BeWo cells studied, the channel under symmetrical N-methyl-d-glucamine (NMDG-Cl) concentration (Na(+) free solution) in both sides of the membrane exhibited spontaneous activity, an outwardly rectifying current/voltage relationship and single-channel conductances of 15 pS and 48 pS for inwards and outwards currents, respectively. The channel has a low permeability for gluconate with a relative permeability P(gluconate)/P(Cl) of 0.23, and a higher permeability to I(-). The open probability (Po) of the channel exhibited dependence with the applied membrane potential with greater activity at positive pulses. The channel activity was inhibited by the sulphonylurea hypoglycemic agent glibenclamide (50 μM) or by diphenylamine-2-carboxylate (DPC, 500 μM) added to the cytoplasmic side of the patch whereas conductances remained unchanged. The blockade with glibenclamide and DPC was independent of the applied membrane potential. All these results are characteristic of the outwardly rectifying Cl channel (ORCC) found in other types of cells. Neither Po, conductances nor reversal potential (Er) values were affected by the absence of intracellular Ca(2+), suggesting that the channel is not sensitive to Ca(2+)., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aims: We assessed regulation of volume-sensitive Cl(-) current (I(Cl,swell)) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes., Methods and Results: ET-1 elicited I(Cl,swell) under isosmotic conditions. Outwardly rectified Cl(-) current was blocked by the I(Cl,swell)-selective inhibitor DCPIB or osmotic shrinkage and involved ET(A) but not ET(B) receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I(Cl,swell) by osmotic swelling or angiotensin II (AngII) was suppressed by ET(A) blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I(Cl,swell). As expected, blockade of NOX suppressed ET-1-induced I(Cl,swell), but blockade of mitochondrial ROS production with rotenone also suppressed I(Cl,swell). I(Cl,swell) was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I(Cl,swell) was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade., Conclusion: ET-1-ET(A) signalling activated I(Cl,swell) via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET(A) receptors were downstream effectors when I(Cl,swell) was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I(Cl,swell) is likely to participate in multiple physiological and pathophysiological processes.

Channels and transporters of monovalent ions are increasingly suggested as putative anticarcinogenic targets. However, the mechanisms involved in modulation of proliferation by monovalent ions are poorly understood. Here, we investigated the role of K+, Na+, and Cl(-) ions for the proliferation of Ehrlich Lettre ascites (ELA) cells. We measured the intracellular concentration of each ion in G(0), G(1), and S phases of the cell cycle following synchronization by serum starvation and release. We show that intracellular concentrations and content of Na+ and Cl(-) were reduced in the G(0)-G(1) phase transition, followed by an increased content of both ions in S phase concomitant with water uptake. The effect of substituting extracellular monovalent ions was investigated by bromodeoxyuridine incorporation and showed marked reduction after Na+ and Cl(-) substitution. In spectrofluorometric measurements with the pH-sensitive dye BCECF, substitution of Na+ was observed to upregulate the activity of the Na+/H+ exchanger NHE1 as well as of Na+-independent acid extrusion mechanisms, facilitating intracellular pH (pH(i)) recovery after acid loading and increasing pH(i). Results using the potential sensitive dye DiBaC4(3) showed a reduced Cl(-) conductance in S compared with G(1) followed by transmembrane potential (E(m)) hyperpolarization in S. Cl(-) substitution by impermeable anions strongly inhibited proliferation and increased free, intracellular Ca2+ ([Ca2+]i), whereas a more permeable anion had little effect. Western blots showed reduced chloride intracellular channel CLIC1 and chloride channel ClC-2 expression in the plasma membrane in S compared with G(1). Our results suggest that Na+ regulates ELA cell proliferation by regulating intracellular pH while Cl(-) may regulate proliferation by fine-tuning of E(m) in S phase and altered Ca2+ signaling.

CLC-5 is a H(+)/Cl(-) exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent's disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H(+) transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H(+) binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN(-) is the main charge carrier: H(+) transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H(+) translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.

Recent evidence suggests a major role for ionic fluxes in apoptotic cell death and apoptotic volume decrease. Cerebellar granule neurons (CGN) undergo apoptosis when they are treated with staurosporine or camptothecin (CPT) or when cells are transferred from high extracellular potassium (25 mM KCl [K(+)](e), K25) to low potassium concentration (5 mM KCl [K(+)](e), K5). In this study we described that all three apoptotic conditions induced apoptotic volume decrease in CGN and that two different potassium channel blockers, cesium (Cs(+)) and tetraethylammonium (TEA(+)), prevented the apoptotic volume decrease, caspase-3 activation, nuclear condensation and cell death induced by K5 and CPT, but not by staurosporine. Cs(+) and TEA(+) also blocked membrane currents generated in K5 conditions in CGN. On the other hand, non specific Cl(-) channel blockers such as 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) prevented loss of cell volume induced by K5 or staurosporine. Only the Cl(-) channels blocker but not the K(+) channels blockers protected from staurosporine-induced death of CGN. These data suggest that ionic fluxes play a key role in the activation of the apoptotic volume decrease and apoptotic death of CGN, but the fine mechanism seems to depend on the apoptotic condition., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

We investigated the importance of pharmacologically blocking calcium-activated chloride (I(Cl(Ca))) and L-type calcium currents on isometric contractions of strips of D21 pregnant rat myometrial tissue, while simultaneously measuring the electrical activity of the tissue strips with extracellular contact electrodes. When measured with contact electrodes, the duration of the spiking activity directly reflects the duration of the tissue-level plateau potential. We correlated the number of spikes, durations of spiking activity, and the spiking frequencies with changes of the area under the force curves as a function of exposure to low doses of anthracene-9-carboxylate (9-AC, a non-specific Cl channel blocker), chlorotoxin (a specific I(Cl(Ca)) blocker) and nifedipine (an L-type calcium channel blocker). The area under the force curve was measured only during spiking electrical activity, thereby separating pharmacological effects on tissue relaxation from those that modulate force production. Blocking chloride channels reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes generated in each contraction. This was observed without a change in the frequency of spike production or a reduction of peak force. Nifedipine reduced impulse, shortened the duration of spiking activity, and reduced the number of spikes. In contrast to chloride channel blockade, nifedipine reduced maximum spike frequency and peak force. Taken together, our data suggest that blocking L-type calcium channels reduces impulse directly by reducing peak force, and indirectly by reducing activation of I(Cl(Ca)) , which shortens the duration of the contraction.

It was investigated whether azithromycin (AZM) stimulates chloride (Cl-) efflux from cystic fibrosis (CF) and non-CF airway epithelial cells, possibly secondary to up-regulation of the multidrug resistance protein (MDR). CF and non-CF human airway epithelial cell lines (CFBE and 16HBE) were treated with 0.4, 4, and 40 microg/mL AZM for 4 days. Cl- efflux was explored in the presence or absence of specific inhibitors of CFTR and alternative Cl- channels. Six CF patients received AZM (500 mg daily) for 6 months. The percentage of predicted forced vital capacity (FVC%), forced expiratory volume (FEV1%), and the number of acute exacerbations were compared before and after treatment. Nasal biopsies were taken before and after treatment, and mRNA expression of MDR and CFTR was determined by in situ hybridization. A significant dose-dependent increase of Cl- efflux from CFBE cells (but not from 16HBE cells) was observed after AZM treatment. A CFTR inhibitor significantly reduced AZM-stimulated Cl- efflux from CFBE cells. A significant improvement in FEV1%, and fewer exacerbations were observed. AZM treatment did not affect mRNA expression of MDR and CFTR. The stimulation of Cl- efflux could be part of the explanation for the clinical improvement seen among the patients.

Chloride channels represent a relatively under-explored target class for drug discovery as elucidation of their identity and physiological roles has lagged behind that of many other drug targets. Chloride channels are involved in a wide range of biological functions, including epithelial fluid secretion, cell-volume regulation, neuroexcitation, smooth-muscle contraction and acidification of intracellular organelles. Mutations in several chloride channels cause human diseases, including cystic fibrosis, macular degeneration, myotonia, kidney stones, renal salt wasting and hyperekplexia. Chloride-channel modulators have potential applications in the treatment of some of these disorders, as well as in secretory diarrhoeas, polycystic kidney disease, osteoporosis and hypertension. Modulators of GABA(A) (gamma-aminobutyric acid A) receptor chloride channels are in clinical use and several small-molecule chloride-channel modulators are in preclinical development and clinical trials. Here, we discuss the broad opportunities that remain in chloride-channel-based drug discovery.

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume-activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of approximately 75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(- )> Cl(- )> F(-)). Staurosporine (200 nM) did not block the activation of I(Cl,swell). The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 microM), SITS (200 microM), ATP (500 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 100 miccroM), and Suramin (10 microM) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(do) and fractional distance of the binding site (delta) of 334 microM and 47 %, 880 microM and 35 %, 2,100 microM and 49%, 188 microM and 27%, and 66.5 microM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I(Cl,swell) in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

In the present study, the intracellular regulatory pathways involved in the adrenalin-stimulated chloride secretion across T84 cells were investigated. Biphasic characteristics were observed in the Isc response to the basolateral addition of adrenalin (0.25 nM-100 microM). The biphasic response was almost abolished by removing ambient Cl(-). Chloride secretion was found to depend on the activities of basolaterally located Na+-K+-2Cl(-) cotransporters and K+ channels. The alpha-adrenoceptor antagonist phentolamine did not have any effect on either phase of adrenalin-induced Isc, while after pretreatment of the beta-adrenoceptor antagonist propranolol, the adrenalin-induced Isc was substantially abolished, suggesting the biphasic response may be mediated by the beta-adrenoceptor. Under whole cell patch-clamp conditions, T84 cells responded to adrenalin with a rise in inward current. The current, which exhibited a linear I-V relationship and time- and voltage-independent characteristics, was inhibited by the chloride channel blocker DPC and the reverse potential was close to the equilibrium potential for Cl(-) (0 mV), implying that the current was Cl(-) selective. When preloaded with a Ca2+-chelating agent, BAPTA/AM did not affect the Isc response to adrenalin, whereas the Isc was destroyed by pretreating the cells with an adenyl cyclase inhibitor, MDL12330A. These observations were further supported by the intracellular [cAMP] measurement experiment, indicating that adrenalin induced chloride secretion could be mediated by a beta-adrenoceptor only involving cAMP as an intracellular second messenger.

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl(-) concentration were measured to characterize the role of Ca(2+)-dependent Cl(-) channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl(-) exit (i.e., mediated by opening of Ca(2+)-dependent K(+) channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K(+) concentrations. The Ca(2+)-dependent secretagogue carbachol induced a stilbene-sensitive Cl(-) efflux, which was mimicked by the Ca(2+) ionophore ionomycin. Surprisingly, the activation of Ca(2+)-dependent Cl(-) efflux was resistant against blockers of classical Ca(2+) signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl(- )efflux. Vice versa, the NO synthase inhibitor N-omega-monomethyl-arginine (L: -NMMA) reduced the carbachol-induced Cl(- )efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca(2+)-dependent Cl(-) channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl(-) efflux. Consequently, the activation of apical Cl(-) channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.

Regulatory volume decrease (RVD) occurs after hypotonicity-caused cell swelling. RVD is caused by activation of ion channels and transporters, which cause effluxes of K(+), Cl(-), and H(2)O, leading to cell shrinkage. Recently, we showed that hypotonicity stimulated transepithelial Na(+) reabsorption via elevation of epithelial Na(+) channel (alpha-ENaC) expression in renal epithelia A6 cells in an RVD-dependent manner and that reduction of intracellular Cl(-) concentration ([Cl(-)](i)) stimulated the Na(+) reabsorption. These suggest that RVD would reveal its stimulatory action on the Na(+) reabsorption by reducing [Cl(-)](i). However, the reduction of [Cl(-)](i) during RVD has not been definitely analyzed due to technical difficulties involved in halide-sensitive fluorescent dyes. In the present study, we developed a new method for the measurement of [Cl(-)](i) change during RVD by using a high-resolution flow cytometer with a halide-specific fluorescent dye, N-(6-methoxyquinolyl) acetoethyl ester. The [Cl(-)](i) in A6 cells in an isotonic medium was 43.6 +/- 3.1 mM. After hypotonic shock (268 to 134 mosmol/kgH(2)O), a rapid increase of cell volume followed by RVD occurred. The RVD caused drastic diminution of [Cl(-)](i) from 43.6 to 10.8 mM. Under an RVD-blocked condition with NPPB (Cl(-) channel blocker) or quinine (K(+) channel blocker), we did not detect the reduction of [Cl(-)](i). Based on these observations, we conclude that one of the physiological significances of RVD is the reduction of [Cl(-)](i) and that RVD shows its action via reduction of [Cl(-)](i) acting as an intracellular signal regulating cellular physiological functions.

Changes in ionic composition of airway surface fluid may modulate airway epithelial functions. We tested the hypothesis that fluctuations of ambient ionic composition could affect airway epithelial Ca(2+) dynamics and Ca(2+)-dependent cellular functions, including NO release and PGE(2) production in vitro. The responses of intracellular Ca(2+) concentration ([Ca(2+)](i)) to changes in extracellular Cl(-) and Na(+ )concentrations ([Cl(-)](e), [Na(+)](e)) in the human bronchial epithelial cell line, 16HBE cells, were measured by the fura-2 method. The NO release to the medium after lowering [Cl(-)](e) was measured by an amperometric NO sensor. PGE(2) production was measured by radioimmunoassay. Changing to isotonic low [Cl(-)](e) solution by substitution with gluconate caused a sustained increase in [Ca(2+)](i) in a concentration-dependent manner, with the maximal [Ca(2+)](i) increase from the baseline level being 243 +/- 110 nM with Cl-free solution. The effect was not altered by thapsigargin but abolished by EGTA and by Cl channel blockers, including diphenylamine-2-carboxylate, disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and disodium cromoglycate. In contrast, the effect of reduction of [Na(+)](e) by substitution with N-methyl-D-glucamine(+) on [Ca(2+)](i) was less than that of reduction of [Cl(-)](e). The reduction of [Cl(-)](e) caused a concentration-dependent rise in NO contents in the medium and PGE(2) production. This release of NO was inhibited by EGTA but not by dexamethasone pretreatment. These results suggest that the decrease in ambient [Cl(-)] induces Ca(2+) mobilization probably through Ca(2+) influx, followed by the release of NO and PGE(2), thereby modulating various cellular functions.

It was the aim of the present study to investigate chloride secretion across the proximal colon of Cftr (TgH(neoim)1Hgu) congenic mice. Stripped epithelia were incubated in Ussing chambers and the electrophysiological data were compared between cystic fibrosis (CF) animals and wild type (WT) animals. In comparison with the control animals, all Cftr (TgH(neoim)1Hgu) congenic mice had a distinctly reduced basal chloride secretion and a reduced chloride secretion after stimulation with carbachol and forskolin. When comparing chloride secretion across the proximal colon between WT animals, all mice showed a comparable pattern of response to carbachol and forskolin but quantitative differences, BALB/c exhibiting the highest and HsdOla:MF1 exhibiting the lowest increase in Cl current. Likewise, all CF animals showed the same reaction pattern to carbachol and forskolin, but there was no distinct difference that lasted for the whole measurement. To investigate interferences between Ca- and cyclic adenosine monophosphate-activated pathways of Cl secretion in CF animals, we studied epithelia from CF/3CF/1F1 animals with a mixed background. In these animals, the levels of the carbachol or forskolin-induced chloride currents did not depend on the prestimulation with the respective other secretagogue. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, which blocks calcium-activated chloride channels, reduced the current response to carbachol by about 23%. This result, obtained in BALB/c-Cftr (TgH(neoim)1Hgu) mice, indicates that alternative chloride channels might be present in the proximal colon of these mice. In contrast, there was no evidence for alternative chloride conductances in BALB/c WT animals, but we cannot exclude that in WT mice a higher chloride secretion via Cftr-channels may have masked an alternative chloride secretion.

Purpose: To investigate the electrophysiology and mechanisms of chloride (Cl-) transport across the ciliary body-epithelium (CBE) of the porcine eye. The pig is widely believed to be a good model for studying human physiology. Current results strengthen our understanding of the physiology of aqueous humor formation (AHF)., Methods: Freshly isolated porcine CBE were maintained in modified Ussing-Zerahn-type chambers. The effects of the bathing anion substitution (Cl- and HCO3-) and transport inhibitors including bumetanide, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS), heptanol, and two chloride channel inhibitors, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and niflumic acid, on the electrical properties and transepithelial Cl- transport were investigated., Results: Viable porcine CBE preparations were maintained in vitro. A spontaneous transepithelial potential difference (PD) of approximately 1 mV was found across the CBE (aqueous side negative). The magnitudes of the PD and short-circuit current (I(sc)) were found to be dependent on both the bathing Cl- and HCO3- concentrations. In short-circuited conditions, a significant net Cl- transport (1.01 microEq x h(-1) x cm(-2); n = 109; P < 0.001) in the stromal-to-aqueous direction (J(net)Cl) was detected. The magnitude of the Cl- current carried by the J(net)Cl was approximately 2.2 times the measured I(sc), suggesting there was cation (e.g., Na+) transport along with Cl- and/or anion transport (e.g., HCO3-) in the opposite direction. Bilateral bumetanide (0.1 mM) reduced the J(net)Cl by approximately 56% while stromal DIDS (0.1 mM) produced no inhibition. Instead, aqueous DIDS (0.1 mM) triggered a sustained stimulation of both I(sc) and J(net)Cl. Even if bilateral DIDS was used at a higher concentration (1 mM), together with bilateral dimethylamiloride (DMA, 0.1 mM), no inhibition of the I(sc) was observed. Bilateral heptanol (3.5 mM) drastically reduced the I(sc) and J(net)Cl. NPPB (0.1 mM), a common chloride channel inhibitor, did not inhibit the J(net)Cl, whereas NFA (1.0 mM) virtually abolished it., Conclusions: In the porcine eye, active secretion of Cl- into aqueous was identified that may act as a driving force for AHF. The bumetanide-sensitive Na+/K+/2Cl- cotransporter (NKCC) clearly contributes to the Cl- uptake into the pigmented epithelium (PE), whereas the DIDS-sensitive Cl-/HCO3- anion exchanger (AE) may exert a minor role. The intercellular gap junctions couple the porcine ciliary bilayers and thus the transepithelial Cl- transport, as in other species. The Cl- channel/efflux pathway located in the nonpigmented epithelium (NPE) is niflumic acid-sensitive but NPPB-insensitive. We also hypothesize that the AE located on the NPE may regulate the activity of a putative Cl- channel on the basolateral membrane facing aqueous via modulation of the intracellular pH (pHi). This work reinforces the general consensus that active secretion of Cl- is the major driving force of AHF in mammalian eye and further substantiates the existence of species differences in the mechanism that accomplishes transepithelial Cl- transport.

The role of chloride ions in regulated secretion is well described but remains poorly characterised in the constitutive system. In the liver, newly synthesised proalbumin is transported to the trans Golgi network where it is converted to albumin by a furin protease and then immediately secreted. We used this acid-dependent hydrolysis and the measurement of specific protein secretion rates to examine the H+ and Cl- ion dependence of albumin synthesis and secretion, a major constitutive protein secretory event in all mammals. Using permeabilised primary rat hepatocytes we show that ordinarily chloride ions are essential for the processing of proalbumin to albumin. However Cl- is not required for transport which continues but releases solely proalbumin. Prior treatment of the cells with Tris (used as a membrane-permeable weak base to neutralise Golgi luminal pH) both eliminated the formation of albumin and very greatly reduced secretion. After washing out Tris, both authentic secretion and processing could be restarted if Cl-, ATP, GTP, cAMP, Ca2+ and cytosolic proteins were added. Hence a requirement for chloride in transport, in addition to processing, can be uncovered by first neutralising pH gradients. Furthermore, the chloride channel blocker DIDS (4,4-diisothiocyanostilbene 2,2-disulphonic acid) reversibly inhibited the constitutive secretory pathway. However, the total mass of proalbumin detectable in DIDS-treated cells fell to 36% of control while the fraction processed to albumin remained almost constant. This clearly dissociates a large part of the Cl- requirement of the constitutive protein secretory pathway from the function of known liver Golgi Cl- channels.