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Abstract: The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na+–K+–2Cl− cotransporters (NKCC1–2) and Na+–Cl− cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking and protein design, we comprehensively explored the sequence space recognized by the CCT domain. Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addition, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed through the use of yeast two-hybrid screening. [Copyright &y& Elsevier]

Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4′-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties. [ABSTRACT FROM AUTHOR]

Highlights: [•] Macroscopic (EIS) and microscopic (SECM) techniques were combined to study the flux of chlorides in cementitious membranes. [•] The transport of chlorides occurs mainly through pores of sub-micrometre equivalent diameter. [•] An explanation for the beneficial effect of the AC current in the transport of chlorides is given. [•] The SECM study evidences the dynamic behaviour of the transport of chlorides. [Copyright &y& Elsevier]

Guanylin (GN) and uroguanylin (UGN) are low-molecular-weight peptide hormones produced mainly in the intestinal mucosa in response to oral salt load. GN and UGN (guanylin peptides) induce secretion of electrolytes and water in both intestine and kidney. Thought to act as 'intestinal natriuretic factors', GN and UGN modulate renal salt secretion by both endocrine mechanisms (linking the digestive system and kidney) and paracrine/autocrine (intrarenal) mechanisms. The cellular function of GN and UGN in intestine and proximal tubule is mediated by guanylyl cyclase C (GC-C)-, cGMP-, and G protein-dependent pathways, whereas, in principal cells of the cortical collecting duct (CCD), these peptide hormones act via GC-C-independent signaling through phospholipase A2 (PLA2). The Cl-/HCO-3 exchanger pendrin (SLC26A4), encoded by the PDS gene, is expressed in non-α intercalated cells of the CCD. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. Our recent studies have provided evidence that pendrin-mediated anion exchange in the CCD is regulated at the transcriptional level by UGN. UGN exerts an inhibitory effect on the pendrin gene promoter likely via heat shock factor 1 (HSF1) action at a defined heat shock element (HSE) site. Recent studies have unraveled novel roles for guanylin peptides in several organ systems including involvement in appetite regulation, olfactory function, cell proliferation and differentiation, inflammation, and reproductive function. Both the guanylin system and pendrin have also been implicated in airway function. Future molecular research into the receptors and signal transduction pathways involved in the action of guanylin peptides and the pendrin anion exchanger in the kidney and other organs, and into the links between them, may facilitate discovery of new therapies for hypertension, heart failure, hepatic failure and other fluid retention syndromes, as well as for diverse diseases such as obesity, asthma, and cancer. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background /Aim: Genistein, the most active isoflavone found primarily in soybeans, alters ion transport functions in intestinal and airway epithelia. The present study aims to investigate the acute effects and mechanisms of action of genistein in immortalized porcine endometrial epithelial cells. Methods: Ussing chamber technique was used for transepithelial electrical measurements. Results: Genistein increased short-circuit currents (Isc) which were inhibited by glibenclamide, NPPB, CFTRinh-172, DIDS or bumetanide, but not amiloride. In experiments with amphotericin B-permeabilized monolayers, genistein activated the apical Cl- current and barium-sensitive basolateral K+ current while inhibiting the apical K+ current. Genistein failed to increase the Isc in the presence of forskolin or IBMX, but did increase the Isc in UTP. Pretreatment with genistein also abolished the increase in the Isc when induced by forskolin, IBMX or UTP. However, Ca2+-chelating BAPTA-AM did not affect the genistein-induced increase in the Isc. The genistein-stimulated Isc was reduced by tyrosine kinase inhibitors, tyrphostin A23 or AG490. However, vanadate, a tyrosine phosphatase inhibitor, failed to inhibit the genistein response. Estrogen receptor antagonist ICI182,780 did not alter the genistein's action. Conclusion: The soy isoflavone, genistein, stimulates Cl- secretion in endometrial epithelial cells possibly via a direct activation of CFTR which appears to be modulated through a tyrosine kinase-dependent pathway. The present findings may be of benefit for the therapeutic application of genistein in the treatment of electrolyte transport disorders in the epithelia. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

CLC-5 plays a critical role in the process of endocytosis in the proximal tubule of the kidney and mutations that alter protein function are the cause of Dent’s I disease. In this X-linked disorder impaired reabsorption results in the wasting of calcium and low molecular weight protein to the urine, kidney stones, and progressive renal failure. Several different ion-transporting and protein clustering roles have been proposed as the physiological function of CLC-5 in endosomal membranes. At the time of its discovery, nearly twenty years ago, it was understandably assumed to be a chloride channel similar to known members of the CLC family, such as CLC-1, suggesting that chloride transport by CLC-5 was critical for endosomal function. Since then CLC-5 was found instead to be a 2Cl-/H+ exchange transporter with voltage-dependent activity. Recent studies have determined that it is this coupled exchange of protons for chloride, and not just chloride transport, which is critical for endosomal and kidney function. This review discusses the recent ideas that describe how CLC-5 might function in endosomal membranes, the aspects that we still do not understand, and where controversies remain.

CLC-K channels belong to the CLC gene family, which comprises both Cl- channels and Cl-/H+ antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption ; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter’s syndrome); mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter’s syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 µM have been discovered. Niflumic acid (NFA) exhibits a complex interaction with CLC-K channels. Below ~ 1 mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca2+]ext. The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca2+ binding sites, whose occupation increases channel activity.The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that underlie NFA activation.

• Chloride transport in AAMs and influencing factors are reviewed. • Advantages and limitations of current testing methods are addressed. • Chloride resistance of AAMs is compared with that of OPC. • Roles of precursors, activators and admixtures are elucidated. • Types and ratios of precursors, activators and admixtures are suggested. The development of alkali-activated materials (AAMs) to replace ordinary Portland cement (OPC) under chloride-containing environments has been widely investigated by academia and industry for decades. Due to varied mix proportions and parameters applied, studies on the chloride resistance of AAMs are inconsistent and require to be systematically analysed. In this review, the existing knowledge about chloride transport in AAMs is systematically reviewed. Chloride transport mechanisms in AAMs and influencing parameters are discussed. Different testing methods are evaluated and the feasibility of AAMs to replace OPC is demonstrated. Roles of different precursors, activators and admixtures on the phase evolution, pore structure, chloride binding capacity and pore solution are discussed in detail. The types and ratios of precursors, activators and admixtures are recommended. Finally, the limitations of existing research and possible future research directions are proposed. [ABSTRACT FROM AUTHOR]

Development of improved durable, conductive, and selective anion exchange membranes (AEMs) are critical for electrodialysis. A series of three ABA triblock copolymers derived from polychloromethylstyrene- b -polyethylene- b -polychloromethylstyrene (PCMS- b -PE- b -PCMS), designated as large, 0.8:1, medium, 0.2:1, and small 0.1:1, block AEMs by their PCMS:PE block size were quaternized with methylpiperidine. The water uptake decreases from 100% for the large block AEM to 5% for the small block AEM. At this low water content in liquid water, the small block AEM shows complete ion dissociation, with similar diffusion (2.6 × 10−6 mol m−2 s−1) and E a (24 kJ/mol) as the larger block AEMs. However, a 34% increase in apparent permselectivity was achieved with the small block AEM. In electrodialysis, the small block AEM improved salt removal by 300% compared with a commercial AEM. Under further investigation, migration was shown to be enhanced by 600% in the small block AEM. The small block AEM has a λ = 5 in liquid water, which is too low to explain the chloride conductivity behavior. Furthermore, the permselectivity in the small block AEM is much higher than expected from its small-angle X-ray scattering morphology, indicating there must be a water-rich region of smaller dimensions where cation/anion pairs are fully solvated. [Display omitted] • A triblock copolymer AEM was developed with decreasing hydrophilic block lengths. • Small block has low water uptake and charge density, but full ion dissociation. • Small block achieves enhanced permselectivity and migrative flux. • Small block improves electrodialysis by nearly 300x compared to large block. [ABSTRACT FROM AUTHOR]

CLC proteins comprise Cl− channels and anion/H+ antiporters involved in several fundamental physiological processes. ClC-7 is a lysosomal Cl−/H+ antiporter that together with its beta subunit Ostm1 has a critical role in the ionic homeostasis of lysosomes and of the osteoclasts' resorption lacuna, although the specific underlying mechanism has so far remained elusive. Mutations in ClC-7 cause osteopetrosis, but also a form of lysosomal storage disease and neurodegeneration. Interestingly, both loss-of- and gain-of-function mutations of ClC-7 can be pathogenic, but the mechanistic implications of this finding are still unclear. This review will focus on the recent advances in our understanding of the biophysical properties of ClC-7 and of its role in human diseases with a focus on osteopetrosis and neurodegeneration. [ABSTRACT FROM AUTHOR]

Salinity and waterlogging (root-zone hypoxia) are abiotic stresses that often occur together on saltland. It is widely recognised that these two factors interact to increase Na+ and/or Cl[sup -] concentrations in shoots, which can have adverse effects on plant growth and survival. This review expands on this understanding, providing evidence that the adverse effects of the interaction are also associated with a disturbance to plant K+ homeostasis. This conclusion is based on a comparative analysis of changes in ion concentrations and growth reported in the literature between species (glycophytes vs halophytes) and within a single species (Hordeum marinum L.). Comparisons between species show that hypoxia under saline conditions causes simultaneous increases in Na+ and Cl[sup -] concentrations and decreases in K+ concentrations in shoots and that these changes can all be related to changes in shoot dry mass. Comparisons between accessions of a single species (Hordeum maritima L.) strengthen the argument, with increases in Na+ and decreases in K+ being related to decreases in shoot relative growth rate. Salinity and waterlogging (oxygen deficiency around the roots) are plant stresses that often occur together on saltland. This combination of stresses impacts on plant growth by decreasing the concentration of potassium and increasing the concentrations of sodium and chloride in shoots. The synchronicity between these changes in ion concentrations has important implications for our understanding of the mechanisms of ion regulation in plants. [ABSTRACT FROM AUTHOR]

Abstract: Silicon can alleviate salt damage to plants, although the mechanism(s) still remains to be elucidated. In this paper, we report the effect of silicon on chloride transport in rice (Oryza sativa L.) seedlings in saline conditions. In the absence of salinity, silicon enhanced the growth of shoots, but not roots in three cultivars (cv. GR4, IR36, and CSR10). Salinity reduced the growth of both shoots and roots in all three genotypes. In saline conditions, addition of silicon to the culture solution again improved the growth of shoots, but not of roots. Under these saline conditions, the concentrations of chloride in the shoot were markedly decreased by adding silicon and the ratio of K+/Cl− was significantly increased, while the concentration of chloride in the roots was unchanged. The decrease in chloride concentration in the shoot was correlated with the decrease in transpirational bypass flow in rice, as shown by the transport of the apoplastic tracer trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS). Addition of silicon increased the net photosynthetic rate, stomata conductance, and transpiration of salt-stressed plants in cv. IR36, indicating that the reduction of chloride (and sodium) uptake by silicon was not through a reduction in transpiration rate. Silicon addition also increased the instantaneous water use efficiency of salt-stressed plants, while it did not change the relative growth rate of shoots. The results suggest that silicon addition decreased transpirational bypass flow in the roots, and therefore decreased the transport of chloride to the shoot. [Copyright &y& Elsevier]

This paper aims to study the effect of measurement methods and cracking on chloride transport of concrete materials. Three kinds of measurement methods were carried out, including immersion test, rapid migration test and steady-state migration test. All of these measurements of chloride transport show that chloride ion diffusion coefficient decreased with the reduction of water to cement ratio. Results of the immersion test were less than that of rapid migration test and steady-state migration test. For the specimen of lower water to cement ratio, the external electrical field has little effect on chloride binding relatively. Compared with the results obtained by these different measurement methods, the lower water to cement ratio may cause smaller differences among these different methods. The external voltage can reduce chloride binding of concrete, and the higher electrical field made a strong impact on the chloride binding. Considering the effect of high voltage on the specimen, results indicate that results based on the steady-state migration test should be more reasonable. For cracked concrete, cracking can accelerate the chloride ion diffusion. [ABSTRACT FROM AUTHOR]

Understanding flow and transport in low-permeability media is very important in the context of nuclear waste disposal, oil and gas reservoirs and long term evolution of groundwater systems. In low-permeability media, transport by diffusion is often the most important mass transport process. This study investigates the effect of the heterogeneity of diffusion parameters on mass transport in low-permeability media. A geostatistical approach for integrating heterogeneity of diffusion parameters in groundwater flow and transport models is proposed and applied to the Toarcian argillites in France which are studied in the framework of feasibility of storing radioactive waste in deep clayey massifs. Stochastic fields of the diffusion parameters of the Toarcian argillites (France) are generated based on 64 measured values of diffusion coefficient and diffusion accessible porosity and used as input for a 3D local-scale groundwater flow and transport model. The chloride concentrations computed by these heterogeneous models are compared to the measured chloride concentrations and to concentrations calculated with a model in which the Toarcian argillites are subdivided into several homogeneous zones. The heterogeneous simulations result in a slightly better correspondence between measured and calculated values and have the additional advantage that the measured diffusion coefficient values in the Toarcian are perfectly honored in the model. This study shows that small-scale variability of diffusion parameters has a significant effect on solute concentrations and omitting this heterogeneity may be a problem in transport calculations in low-permeability media, depending on the specific setting and objectives of the study. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents a Fick-based chloride transport model, which is mathematically consistent with time dependent apparent diffusion coefficient and surface chloride for fly ash concrete. In the paper, the inconsistency in a simple close-formed solution used to predict chloride penetration through fly ash concrete in a previous study of other researchers is pointed out. The inconsistency can be seen by comparing chloride profiles calculated by the simple close-formed solution to those calculated by a finite difference program. The inconsistency is caused by the use of simplified diffusion coefficient and mathematically incompatible surface chloride models. To avoid the inconsistency, a chloride transport model is developed in this study. In developing the model, regression analysis to fit coefficients of the developed model is compared to the regression analysis results obtained from the experiment of the researchers. In the experiment of the researchers, the effect of water to binder ratio and the amount of fly ash replacement in concrete is considered. Furthermore, the chloride transport calculated from the developed model is validated by comparing to other experimental results. Finally, the sensitivity analysis of the model coefficients and some remarks on the developed model are presented. [Copyright &y& Elsevier]

In order to study the chloride ion transport performance in fly ash addition mortar, a new method, in which the fatigue loading and chloride diffusion are undertaken simultaneously, was developed. This method realizes coupling the fatigue damage process and the process of chloride transporting of fly ash mortar. The transport performance of chloride in fly ash mortar specimens was studied under different stress levels. Moreover, the effect of fly ash content on transport performance of chloride ion in mortar was investigated. AE (Acoustic Emission) and SEM were used to acquire the damage distribution of mortar specimens under action of fatigue load. The results show that the diffusion coefficient of chloride in mortar specimens increases with stress level of fatigue loading. The addition of fly ash can mitigate the penetration of chloride ion. The results of microcrack 3D location acquired by AE, accompanied with crack characterizing from SEM, indicate that the damage degree of mortar specimen increases with stress level of fatigue loading. Furthermore, higher damage degree of mortar leads to more the chloride ion content in the sample. [ABSTRACT FROM AUTHOR]

The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na+ channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na+ transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na+ homeostasis. To explore this process, we examined the coordinated effect of this protease on Na+ and Cl- transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current (Isc) was used to measure transport of these ions. The Isc exhibited an ENaC-dependent Na+ component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl- component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated IENaC but was without effects on ICFTR. Subtilisin an exogenous S8 protease that mimics endogenous furintype proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na+ and Cl- transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na+ and Cl- transport in these epithelia. [ABSTRACT FROM AUTHOR]

CLC-5 plays a critical role in the process of endocytosis in the proximal tubule of the kidney and mutations that alter protein function are the cause of Dent's I disease. In this X-linked disorder impaired reabsorption results in the wasting of calcium and low molecular weight protein to the urine, kidney stones, and progressive renal failure. Several different ion-transporting and protein clustering roles have been proposed as the physiological function of CLC-5 in endosomal membranes. At the time of its discovery, nearly 20 years ago, it was understandably assumed to be a chloride channel similar to known members of the CLC family, such as CLC-1, suggesting that chloride transport by CLC-5 was critical for endosomal function. Since then CLC-5 was found instead to be a 2Cl-/H+ exchange transporter with voltage-dependent activity. Recent studies have determined that it is this coupled exchange of protons for chloride, and not just chloride transport, which is critical for endosomal and kidney function. This review discusses the recent ideas that describe how CLC-5 might function in endosomal membranes, the aspects that we still do not understand, and where controversies remain. [ABSTRACT FROM AUTHOR]

Abstract: Cystic fibrosis is the most common life-threatening recessively inherited disease in Caucasians. Due to early provision of care in specialized reference centers and more comprehensive care, survival has improved over time. Despite great advances in supportive care and in our understanding of its pathophysiology, there is still no cure for the disease. Therapeutic strategies aimed at rescuing the abnormal protein are either being sought after or under investigation. This review highlights salient insights into pathophysiology and candidate molecules suitable for CFTR pharmacotherapy. Clinical trials using Ataluren, VX-809 and ivacaftor have provided encouraging data. Preclinical data with inhibitors of phosphodiesterase type 5, such as sildenafil and analogs, have highlighted their potential for CFTR pharmacotherapy. Because sildenafil and analogs are in clinical use for other clinical applications, research on this class of drugs might speed up the development of new therapies for CF. [Copyright &y& Elsevier]

Abstract: Seizures are thought to originate from a failure of inhibition to quell hyperactive neural circuits, but the nature of this failure remains unknown. Here we combine high-speed two-photon imaging with electrophysiological recordings to directly evaluate the interaction between populations of interneurons and principal cells during the onset of seizure-like activity in mouse hippocampal slices. Both calcium imaging and dual patch clamp recordings reveal that in vitro seizure-like events (SLEs) are preceded by pre-ictal bursts of activity in which interneurons predominate. Corresponding changes in intracellular chloride concentration were observed in pyramidal cells using the chloride indicator Clomeleon. These changes were measurable at SLE onset and became very large during the SLE. Pharmacological manipulation of GABAergic transmission, either by blocking GABAA receptors or by hyperpolarizing the GABAA reversal potential, converted SLEs to short interictal-like bursts. Together, our results support a model in which pre-ictal GABAA receptor-mediated chloride influx shifts EGABA to produce a positive feedback loop that contributes to the initiation of seizure activity. [Copyright &y& Elsevier]

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (Isc) in the distal colon with an EC50 value of 100 ng/ml and increased Isc in the proximal colon with an EC50 value of 49 ng/ml. In the distal colon, the PRL-induced decrease in Isc was not affected by Na+ channel blocker amiloride or Cl- channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K+ channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K+ channel blockers, Ba2+, tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRLinduced decrease in Isc was also inhibited by Na+-K+-2Cl- transporter inhibitor bumetanide, Ba2+, and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in Isc was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in Isc in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca2+ chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K+ secretion in the distal colon and activation of Cl- secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca2+ mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon. [ABSTRACT FROM AUTHOR]

The pendrin/ SLC26A4 Cl-/HCO3- exchanger, encoded by the PDS gene, is expressed in cortical collecting duct (CCD) non-A intercalated cells. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. The intestinal peptide uroguanylin (UGN) is produced in response to oral salt load and can function as an "intestinal natriuretic hormone." We aimed to investigate whether UGN modulates pendrin activity and to explore the molecular mechanisms responsible for this modulation. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. UGN decreased endogenous pendrin mRNA levels in HEK293 cells. A 4.2-kb human PDS (hPDS) promoter sequence and consecutive 5' deletion products were cloned into luciferase reporter vectors and transiently transfected into HEK293 cells. Exposure of transfected cells to UGN decreased hPDS promoter activity. This UGN-induced effect on the hPDS promoter occurred within a 52-bp region encompassing a single heat shock element (HSE). The effect of UGN on the promoter was abolished when the HSE located between nt -1119 and -1115 was absent or was mutated. Furthermore, treatment of HEK293 cells with heat shock factor 1 (HSF1) small interfering RNA (siRNA) reversed the UGNinduced decrease in endogenous PDS mRNA level. In conclusion, pendrin-mediated Cl-/HCO3- exchange in the renal tubule may be regulated transcriptionally by the peptide hormone UGN. UGN exerts its inhibitory activity on the hPDS promoter likely via HSF1 action at a defined HSE site. These data define a novel signaling pathway involved in the enterorenal axis controlling electrolyte and water homeostasis. [ABSTRACT FROM AUTHOR]

SLC26A3 is a Cl-/HCO3- exchanger that plays a major role in Cl- absorption from the intestine. Its mutation causes congenital chloridelosing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation. [ABSTRACT FROM AUTHOR]

Among the most prevalent and deadly primary brain tumors, high-grade gliomas evade complete surgical resection by diffuse invasion into surrounding brain parenchyma. Navigating through tight extracellular spaces requires invading glioma cells to alter their shape and volume. Cell volume changes are achieved through transmembrane transport of osmolytes along with obligated water. The sodium-potassium-chloride cotransporter isoform-1 (NKCC1) plays a pivotal role in this process, and previous work has demonstrated that NKCC1 inhibition compromises glioma invasion in vitro and in vivo by interfering with the required cell volume changes. In this study, we show that NKCC1 activity in gliomas requires the With-No-Lysine Kinase-3 (WNK3) kinase. Western blots of patient biopsies and patient-derived cell lines shows prominent expression of Ste-20-related, proline-alanine-rich kinase (SPAK), oxidative stress response kinase (OSR1), and WNK family members 1, 3, and 4. Of these, only WNK3 colocalized and coimmunoprecipitated with NKCC1 upon changes in cell volume. Stable knockdown of WNK3 using specific short hairpin RNA constructs completely abolished NKCC1 activity, as measured by the loss of bumetanide-sensitive cell volume regulation. Consequently, WNK3 knockdown cells showed a reduced ability to invade across Transwell barriers and lacked bumetanide-sensitive migration. This data indicates that WNK3 is an essential regulator of NKCC1 and that WNK3 activates NKCC1-mediated ion transport necessary for cell volume changes associated with cell invasion. [ABSTRACT FROM AUTHOR]

Abstract: Non-steady state migration (NSSM) test is commonly employed to evaluate chloride ion ingress into concretes. This paper quantifies the microstructural changes in concretes subjected to NSSM tests, induced by the chemical constitution of the cement and replacement materials. Electrical impedance spectroscopy (EIS) and equivalent electrical circuit modeling are used to obtain characteristic features of the concrete pore structure that aid in this quantification. A methodology is provided in this paper to convert the bulk resistances of the specimens under the action of any applied voltages to the ones corresponding to a 30V applied potential. The relationships between: (i) bulk resistance of the specimens before the NSSM test and the NSSM coefficient D nssm, (ii) resistance of the connected pores and the microstructural parameter ϕβ (product of porosity and pore connectivity), and (iii) bulk resistance after the NSSM test corresponding to a 30V potential and the chloride penetration depths, demonstrate the effect of chloride binding. The capacitance related to the pore–solid interface obtained from the equivalent circuit is used to indicate that the chloride binding products are formed at the interfaces and influence the tortuosity of the pore system. The experiments and the model suggest an average reduction of about 10% in ϕβ values after the NSSM test. [Copyright &y& Elsevier]

Background: Increasing epithelial chloride (Cl-) secretion in the upper airways represents a putative method for promoting mucociliary clearance through augmentation of airway surface liquid depth. Several naturally occurring flavonoid compounds, including quercetin, have shown the capacity to increase transepithelial Cl- transport. Quercetin exhibits well-known antioxidant and anti-inflammatory activity and is now recognized as a potent activator of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel activity in a fashion largely independent of cyclic adenosine monophosphate signaling. The present study investigates whether this compound activates Cl- secretion and ciliary beat frequency (CBF) in well-characterized culture models of sinonasal epithelium. Methods: Cystic fibrosis and non-cystic fibrosis primary human sinonasal epithelial (HSNE) and murine nasal septal epithelial (MNSE) cultures were studied for transepithelial ion transport in Ussing chambers under voltage clamp conditions and CBF was performed using pharmacologic manipulation. Results: Change in short circuit current (DeltaISC, expressed as microamperes per squared centimeter) in response to quercetin were significantly greater than controls in both MNSE (23.23 ± 5.44 versus 2.47 ± 1.62; p < 0.0001) and HSNE (-8.72 ± 1.88 versus -1.88 ± 0.66; p < 0.01) cultures. CBF was significantly increased in quercetin-treated cells (expressed as fold change over baseline) in wild type (1.65 ± 0.13 versus 1.23 ± 0.05 [control]; p < 0.01), but not CFTR-/- (1.65 ± 0.29 versus 1.48 ± 0.38; p = 0.23). Conclusion: Quercetin significantly increased transepithelial Cl- transport and CBF in MNSE and HSNE cultures. Future studies investigating quercetin as a means to promote mucociliary transport in individuals with rhinosinusitis are warranted. [ABSTRACT FROM AUTHOR]

Anion transport drives fluid into the airways and is essential for humidifying inspired air and supplying surface liquid for mucociliary transport. Despite the importance of airway secretion in diseases such as cystic fibrosis, the cellular mechanisms remain poorly understood, in part due to the small size and complicated structure of the submucosal glands that produce most of the fluid. The Calu-3 human lung adenocarcinoma cell line has become a popular model for studying airway secretion because it can be cultured as a flat sheet, expresses the cystic fibrosis transmembrane conductance regulator and several acinar cell markers, forms polarized monolayers with tight junctions, has robust cAMP-stimulated anion transport, and responds to secretagogues that regulate the glands in vivo. However, some properties of Calu-3 cells are less consistent with those of native tissue. In particular, Calu-3 monolayers do not secrete chloride when stimulated by forskolin under short-circuit conditions. Bicarbonate ions are thought to carry the short-circuit current ( I) and the drive secretion of alkaline fluid, in contrast to the neutral pH secretions that are produced by submucosal glands. Calu-3 cells also have abnormal chromosomes and characteristics of both serous and mucus cells. In this article, we discuss Calu-3 as a model in light of our ongoing studies, which suggest that Calu-3 monolayers resemble submucosal glands more closely than was previously thought. For example, we find that net HCO flux fully accounts for I as previously suggested but Cl is the main anion transported under physiological conditions. A novel, HCO-dependent mechanism of Cl transport is emerging which may explain secretion by Calu-3 and perhaps other epithelial cells. [ABSTRACT FROM AUTHOR]

Potassium (K) and chloride (Cl) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K and Cl, it remains unclear if they actively use K-coupled Cl cotransporters (KCC), as used in animals, to transport K and Cl. We have cloned an Oryza sativa cDNA encoding for a member of the cation-Cl cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K-Cl cotransporters than to other cation-Cl cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na, K, and Cl contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K and Cl than the latter but that they did not differ in terms of Na contents, suggesting OsCCC1 may be involved in K and Cl transport. Results from different tests indicated that the OsCCC1 plays a significant role in K and Cl homeostasis and rice plant development. [ABSTRACT FROM AUTHOR]

Abstract: This experimental work studied the influence of mortars used as external rendering on chloride transport in concrete. Prismatic concrete specimens were cast with dimensions of 80×80×80mm and water to binder ratio of 0.55. Three different mixtures of mortar were used to cover these specimens, after making a thin layer of spatter dash treatment. Reference concrete specimens were also cast. After a curing period in wet chamber and in laboratory environment, five of the six faces of the specimens were coated with epoxy resin to simulate unidirectional flux of chloride during the test. The specimens were subjected to natural diffusion tests for 49days and, afterwards, samples were extracted and analysed to obtain chloride profiles. Results show that mortar renderings directly influence chloride penetration into concrete and this influence is more pronounced for mortars with higher contents of cement and less porosity. This shows that, although mortars have higher porosity than concrete, they can provide an additional protection to concrete structures, delaying chloride penetration into bulk concrete. [ABSTRACT FROM AUTHOR]

ording to the two-stage hypothesis, primary saliva, a NaCl-rich plasma-like isotonic fluid is secreted by salivary acinar cells and its ionic composition becomes modified in the duct sytem. The ducts secrete K and HCO and reabsorb Na and Cl without any water movement, thus establishing a hypotonic final saliva. Salivary secretion depends on the coordinated action of several channels and transporters localized in the apical and basolateral membrane of acinar and duct cells. Early functional studies in perfused glands, followed by the molecular cloning of several transport proteins and the subsequent analysis of mutant mice, have greatly contributed to our understanding of salivary fluid and the electrolyte secretion process. With a few exceptions, most of the key channels and transporters involved in salivary secretion have now been identified and characterized. However, the picture that has emerged from all these studies is one of a complex molecular network characterized by redundancy for several transport proteins, compensatory mechanisms, and adaptive changes in health and disease. Current research is directed to the molecular interactions between the determinants and the ways in which they are regulated by extracellular signals and intracellular mediators. This review focuses on the functionally and molecularly best-characterized channels and transporters that are considered to be involved in transepithelial fluid and electrolyte transport in salivary glands. [ABSTRACT FROM AUTHOR]

Abstract: It is known that insect kinins increase diuresis and fluid secretion in the Aedes aegypti Malpighian tubule, causing a rapid drop of the transepithelial resistance and increasing chloride conductance from the hemolymph towards the tubule lumen. The tubule is composed of both principal and stellate cells. The main route for increased chloride influx upon kinin treatment is proposed to be paracellular, with septate junctions acquiring increased chloride selectivity and conductance. Therefore, kinin treatment renders the Ae. aegypti tubule a “leaky epithelium”, and under this model the kinin receptor is postulated to be expressed in principal cells. However, in another dipteran, the fruit fly Drosophila melanogaster, the main route for chloride transport is transcellular through stellate cells. In both the fruit fly and the mosquito Anopheles stephensi the kinin receptor has been immunolocalized in stellate cells, where it regulates transepithelial chloride permeability. Here we show that in Ae. aegypti, similarly, the stellate cells express the kinin receptor. This was confirmed through immunohistochemistry with two specific anti-kinin receptor antibodies and confocal analysis. The receptor is detected as a 75 kDa band in western blot. These results indicate that the currently accepted model for chloride transport must be re-evaluated in Ae. aegypti and suggest the kinin regulatory signals controlling intercellular junctions originate in the stellate cells. [ABSTRACT FROM AUTHOR]

The inhibitory transmitter GABA has been suggested to play an important role in infantile autistic syndrome (IAS), and extensive investigations suggest that excitatory actions of GABA in neurological disorders are because of a persistent increase of [Cl]. To test the effects of the chloride co-transporter NKCC1 diuretic compound Bumetanide that reduces [Cl] on IAS. Bumetanide was administered daily (1 mg daily) during a 3 -month period and clinical and biological tests made. We used 5 standard IAS severity tests - Childhood Autism Rating Scale, Aberrant Behaviour Checklist, Clinical Global Impressions; Repetitive and Restrictive Behaviour and the Regulation Disorder Evaluation Grid. We report a significant improvement in IAS with no side effects. Bumetanide decreases autistic behaviour with no side effects suggesting that diuretic agents may exert beneficial effects on IAS and that alterations of the actions of GABA may be efficient in IAS treatment calling for large scale randomized trials. [ABSTRACT FROM AUTHOR]

To reveal specific Cl transport activities in the symplastic pathway, uptake, long-distance transport and distribution of Cl have been investigated in the citrus rootstocks Carrizo citrange (CC, Cl includer) and Cleopatra mandarin (CM, Cl excluder). Using an external concentration of 4.5 m Cl, both species actively transported Cl to levels that exceeded the critical requirement concentration by one and two orders of magnitude in the excluder and the includer rootstocks, respectively. Both CC and CM modulated Cl influx according to the availability of the nutrient as uptake capacity was induced by Cl starvation, but inhibited after Cl resupply. Net Cl uptake was higher in the includer CC, an observation that correlated with a lower root-to-shoot transport capacity in the excluder CM. The patterns of tissue Cl accumulation indicated that chloride exclusion in the salt-tolerant rootstock CM was caused by a reduced net Cl loading into the root xylem. Genes CcCCC1, CcSLAH1 and CcICln1 putatively involved in the regulation of chloride transport were isolated and their expression analysed in response to both changes in the nutritional status of Cl and salt stress. The previously uncharacterized ICln gene exhibited a strong repression to Cl application in the excluder rootstock, suggesting a role in regulating Cl homeostasis in plants. [ABSTRACT FROM AUTHOR]

The family of CLC proteins comprises both Cl- channels and Cl-/H+ exchange transporters with varying degrees of voltage dependence. The human CLC-5 is an electrogenic voltage-dependent 2Cl-/ 1H+ exchanger that gives rise to strongly outwardly rectifying currents when expressed. We conducted whole-cell recordings from HEK293 cells transiently transfected with either wild-type CLC-5 or a permeation-deficient mutant, E268A. With E268A CLC-5 we recorded transient voltage-dependent currents that represent the gating currents associated with CLC-5 activation and had kinetics that could be described by voltage-dependent forward and reverse transition rates. In extracellular solutions rich in Cl- or Br-, CLC-5 exhibited a gating charge of 1.3, but this was reduced to 0.9 in solutions comprising the impermeant anions aspartate, methanesulfonate, sulfate, or HEPES. Extracellular ion depletion by local perfusion with isotonic mannitol failed to reduce the gating charge further. Lowering intracellular pH from 7.4 to 5.4 did not shift the voltage-dependence of the gating currents, but reducing and increasing intracellular Cl- shifted the charge-voltage relationship to more negative and positive potentials, respectively. Our data suggest that voltage sensing is an intrinsic property of the CLC-5 protein and that permeant anions, particularly Cl-, modulate a voltage-dependent transition to an activated state from which Cl-/H+ exchange can occur. [ABSTRACT FROM AUTHOR]

Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl– efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl– efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37°C. The effect of NAC on Cl– transport was measured by Cl– efflux measurements and by X-ray microanalysis. Cl– efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl– efflux with nearly 80% in CFBE cells. The intracellular Cl– concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl– efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients. [ABSTRACT FROM AUTHOR]

Abstract: This paper discusses the influence of rapid chloride transport test methods on the microstructure and the measured chloride penetration resistance of concretes containing fly ash or fine glass powder as partial cement replacement materials. Rapid chloride permeability (RCP), non-steady-state migration (NSSM), and steady state conduction (SSC) tests are performed on plain and modified concretes. The glass powder modified concretes demonstrate similar or lower RCP values as compared to the fly ash modified concretes of the same cement replacement level whereas the steady state conductivities are lower for the fly ash modified mixtures. The NSSM coefficients are lower for the fly ash modified concretes even when the initial conductivities are similar to those of plain or glass powder modified concretes. Chloride binding under the conditions of NSSM test, that influence the microstructure and the transport parameter, is quantified using thermal analysis and XRD patterns as well as an electrical circuit model for the impedance response. The resistance of connected pores (Rc ) extracted from the model adequately captures the changes in microstructure with time and with chloride transport. The changes in Rc between the start and finish of the NSSM test also indicate microstructural alteration in fly ash modified concretes. [Copyright &y& Elsevier]