Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3

Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pH(i)) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (similar to 7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least + 20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake

The cytotoxic T cell proteome and its shaping by the kinase mTOR

High-resolution mass spectrometry maps the cytotoxic T lymphocyte (CTL) proteome and the impact of mammalian target of rapamycin complex 1 (mTORC1) on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes and mTORC1 selectively repressed and promoted expression of subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for mTORC1 negative control of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) production in CTL. mTORC1 was shown to repress PtdIns(3,4,5)P(3) production and to determine the mTORC2 requirement for activation of the kinase Akt. Unbiased proteomic analysis thus provides a comprehensive understanding of CTL identity and mTORC1 control of CTL function

The glucokinase activator GKA50 causes an increase in cell volume and activation of volume-regulated anion channels in rat pancreatic β-cells

International audience► The paper examines the effects of a potential anti-diabetic drug glucokinase activator 50 (GKA50) on the function of insulin-secreting, pancreatic β-cells. ► GKA50 caused a sustained increase in β-cell volume, which was dependent on glucose metabolism. ► GKA50 stimulated a sustained depolarisation of the β-cell membrane potential by activating volume regulated anion channels (probably as a result of the increase in cell volume). ► The stimulatory effects of GKA50 on insulin secretion may at least partially be explained by cell swelling and activation of volume regulated anion channels leading to a depolarisation of membrane potential

HCO3- -dependent volume regulation in α-cells of the rat endocrine pancreas

Ion transport activity in pancreatic α-cells was assessed by studying cell volume regulation in response to anisotonic solutions. Cell volume was measured by a video imaging method, and cells were superfused with either 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-buffered or HCO(3)(−)-buffered solutions. α-Cells did not exhibit a regulatory volume increase (RVI) in response to cell shrinkage caused by hypertonic solutions. A RVI was observed, however, in cells that had first undergone a regulatory volume decrease (RVD), but only in HCO(3)(−)-buffered solutions. RVI was also observed in response to a HCO(3)(−)-buffered hypertonic solution in which the glucose concentration was increased from 4 to 20 mM. The post-RVD RVI and the glucose-induced RVI were both inhibited by 10 μM 5-(N-methyl-N-isobutyl) amiloride or 100 μM 2,2′-(1,2-ethenediyl) bis (5-isothio-cyanatobenzenesulfonic acid), but not by 10 μM benzamil nor 10 μM bumetanide. These data suggest that Na(+)–H(+) exchangers and Cl(−)–HCO(3)(−) exchangers contribute to volume regulation in α-cells

Opposing effects of tenidap on the volume-regulated anion channel and KATP channel activity in rat pancreatic β-cells

Tenidap (5-chloro-2-hydroxy-3-(thiophene-2-carbonyl)indole-1-carboxamide) is a non-steroidal anti-inflammatory and anti-rheumatic drug with several cellular actions including inhibition of anion transport processes. Since other anion transport inhibitors have been shown to inhibit activity of the volume-regulated anion channel (VRAC), the present study investigated the effects of tenidap on activity of this channel in pancreatic β-cells. Membrane potential, VRAC currents and input conductance were recorded from single rat β-cells in primary culture using perforated patch, conventional whole-cell and cell-attached configurations of the patch-clamp technique. Relative cell volume was measured using a video-imaging method. Tenidap (0.1 mM) was found to rapidly hyperpolarise the β-cell membrane potential and terminate glucose-induced electrical activity. This effect was associated with a pronounced outward current shift at a holding potential of - 65 mV. Tenidap was found to inhibit activity of the volume-regulated anion channel with IC50 values of 31 and 43 μM for outward and inward currents respectively. Tenidap also markedly increased β-cell input conductance, representing an activation of the KATP conductance. β-cell regulatory volume decrease following hypotonically-induced cell swelling was sensitive to inhibition by 50 μM tenidap. Tenidap is a potent inhibitor of the volume-regulated anion channel and KATP channel activator in rat pancreatic β-cells. These actions could at least in part explain the recently reported inhibitory actions of the drug on electrical and secretory activity in the β-cell, and could also underlie other pharmacological actions of the drug. © 2009 Elsevier B.V. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Expression of K+–Cl− cotransporters in the α-cells of rat endocrine pancreas

AbstractThe expression of K+–Cl− cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in α-cells, but not pancreatic β-cells nor δ-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both α-cell and β-cells. Exposure of isolated α-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 μM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in β-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of α-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on β-cell volume. In conclusion, KCCs are expressed in pancreatic α-cells and β-cells. However, they make a significant contribution to volume homeostasis only in α-cells

Stimulus-secretion coupling of hypotonicity-induced insulin release in BRIN-BD11 cells

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