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Roman Skryma - One of the best experts on this subject based on the ideXlab platform.
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volume regulated Chloride Conductance in the lncap human prostate cancer cell line
American Journal of Physiology-cell Physiology, 2000Co-Authors: Yaroslav Shuba, Natalia Prevarskaya, Loic Lemonnier, F Van Coppenolle, P G Kostyuk, Brigitte Mauroy, Roman SkrymaAbstract:Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoke...
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volume regulated Chloride Conductance in the lncap human prostate cancer cell line
American Journal of Physiology-cell Physiology, 2000Co-Authors: Yaroslav Shuba, Natalia Prevarskaya, Loic Lemonnier, P G Kostyuk, Brigitte Mauroy, F Van Coppenolle, Roman SkrymaAbstract:Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl(-) was the primary charge carrier (termed I(Cl,swell)). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br(-) approximately I(-) > Cl(-) > F(-) > methanesulfonate >> glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC Conductance was determined at 9.6 +/- 1.8 pS. Conventional Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) and DIDS (100 microM) inhibited whole cell I(Cl,swell) in a voltage-dependent manner, with the block decreasing from 39.6 +/- 9.7% and 71.0 +/- 11. 0% at +50 mV to 26.2 +/- 7.2% and 14.5 +/- 6.6% at -100 mV, respectively. Verapamil (50 microM), a standard Ca(2+) antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I(Cl,swell) (genistein with an IC(50) of 2.6 microM and lavendustin A by 60 +/- 14% at 1 microM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 microM) stimulated I(Cl,swell) by 54 +/- 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.
A S Verkman - One of the best experts on this subject based on the ideXlab platform.
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anchored pde4 regulates Chloride Conductance in wild type and δf508 cftr human airway epithelia
The FASEB Journal, 2014Co-Authors: Elise Blanchard, A S Verkman, Lorna Zlock, Anna Lao, Delphine Mika, Wan Namkung, Moses Xie, Colleen Scheitrum, Dieter C Gruenert, Walter E FinkbeinerAbstract:Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane Conductance regulator (CFTR) that impair its expression and/or Chloride channel function. Here, we provide evidence that type 4 cyclic nucleotide phosphodiesterases (PDE4s) are critical regulators of the cAMP/PKA-dependent activation of CFTR in primary human bronchial epithelial cells. In non-CF cells, PDE4 inhibition increased CFTR activity under basal conditions (ΔISC 7.1 μA/cm2) and after isoproterenol stimulation (increased ΔISC from 13.9 to 21.0 μA/cm2) and slowed the return of stimulated CFTR activity to basal levels by >3-fold. In cells homozygous for ΔF508-CFTR, the most common mutation found in CF, PDE4 inhibition alone produced minimal channel activation. However, PDE4 inhibition strongly amplified the effects of CFTR correctors, drugs that increase expression and membrane localization of CFTR, and/or CFTR potentiators, drugs that increase channel gating, to reach ∼25% of the Chloride Conductance o...
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impaired acidification in early endosomes of clc 5 deficient proximal tubule
Biochemical and Biophysical Research Communications, 2005Co-Authors: Mariko Harachikuma, Yinghong Wang, Sandra E Guggino, William B Guggino, A S VerkmanAbstract:Abstract ClC-5 Chloride channel deficiency causes proteinuria, hypercalciuria, and nephrolithiasis (Dent’s disease). Impaired endosomal acidification in proximal tubule caused by reduced Chloride Conductance is a proposed mechanism; however, functional analysis of ClC-5 in oocytes predicts low ClC-5 Chloride Conductance in endosomes because of their acid interior pH and positive potential. Here, endosomal pH and Chloride concentration were measured in proximal tubule cell cultures from wildtype vs. ClC-5 deficient mice using fluorescent sensors coupled to transferrin (early/recycling endosomes) or α2-macroglobulin (late endosomes). Initial pH in transferrin-labeled endosomes was ∼7.2, decreasing at 15 min to 6.0 vs. 6.5 in wildtype vs. ClC-5 deficient cells, respectively; corresponding endosomal Chloride concentration increased from ∼16 mM to 47 vs. 36 mM. In contrast, acidification and Chloride accumulation were not impaired in late endosomes or Golgi. Our results provide direct evidence for ClC-5 involvement in acidification of early endosomes in proximal tubule by a Chloride shunt mechanism.
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high affinity activators of cystic fibrosis transmembrane Conductance regulator cftr Chloride Conductance identified by high throughput screening
Journal of Biological Chemistry, 2002Co-Authors: L Vetrivel, Hong Yang, Nicoletta Pedemonte, Olga Zegarramoran, Luis J V Galietta, A S VerkmanAbstract:Abstract Cystic fibrosis (CF) is caused by mutations in the CF transmembrane Conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl− Conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 μmtogether with a low concentration of forskolin (0.5 μm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl− sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl− currents rapidly ( 10 μm). When added for 10 min, none of the compounds activated ΔPhe508-CFTR in transfected cells grown at 37 °C (with ΔPhe508-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 °C, tetrahydrocarbazol andN-phenyltriazine derivatives strongly stimulated Cl− Conductance with Kd < 1 μm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.
William B Guggino - One of the best experts on this subject based on the ideXlab platform.
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impaired acidification in early endosomes of clc 5 deficient proximal tubule
Biochemical and Biophysical Research Communications, 2005Co-Authors: Mariko Harachikuma, Yinghong Wang, Sandra E Guggino, William B Guggino, A S VerkmanAbstract:Abstract ClC-5 Chloride channel deficiency causes proteinuria, hypercalciuria, and nephrolithiasis (Dent’s disease). Impaired endosomal acidification in proximal tubule caused by reduced Chloride Conductance is a proposed mechanism; however, functional analysis of ClC-5 in oocytes predicts low ClC-5 Chloride Conductance in endosomes because of their acid interior pH and positive potential. Here, endosomal pH and Chloride concentration were measured in proximal tubule cell cultures from wildtype vs. ClC-5 deficient mice using fluorescent sensors coupled to transferrin (early/recycling endosomes) or α2-macroglobulin (late endosomes). Initial pH in transferrin-labeled endosomes was ∼7.2, decreasing at 15 min to 6.0 vs. 6.5 in wildtype vs. ClC-5 deficient cells, respectively; corresponding endosomal Chloride concentration increased from ∼16 mM to 47 vs. 36 mM. In contrast, acidification and Chloride accumulation were not impaired in late endosomes or Golgi. Our results provide direct evidence for ClC-5 involvement in acidification of early endosomes in proximal tubule by a Chloride shunt mechanism.
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Chloride channel and Chloride Conductance regulator domains of cftr the cystic fibrosis transmembrane Conductance regulator
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Erik M Schwiebert, Marcelo M Morales, Sreenivas Devidas, Marie E Egan, William B GugginoAbstract:CFTR is a cyclic AMP (cAMP)-activated Chloride (Cl−) channel and a regulator of outwardly rectifying Cl− channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl− channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3–1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl− efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl− channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted α-helices 5 and 6, forms an essential part of the Cl− channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl− channel and a Conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.
Yaroslav Shuba - One of the best experts on this subject based on the ideXlab platform.
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volume regulated Chloride Conductance in the lncap human prostate cancer cell line
American Journal of Physiology-cell Physiology, 2000Co-Authors: Yaroslav Shuba, Natalia Prevarskaya, Loic Lemonnier, F Van Coppenolle, P G Kostyuk, Brigitte Mauroy, Roman SkrymaAbstract:Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoke...
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volume regulated Chloride Conductance in the lncap human prostate cancer cell line
American Journal of Physiology-cell Physiology, 2000Co-Authors: Yaroslav Shuba, Natalia Prevarskaya, Loic Lemonnier, P G Kostyuk, Brigitte Mauroy, F Van Coppenolle, Roman SkrymaAbstract:Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl(-) was the primary charge carrier (termed I(Cl,swell)). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br(-) approximately I(-) > Cl(-) > F(-) > methanesulfonate >> glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC Conductance was determined at 9.6 +/- 1.8 pS. Conventional Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) and DIDS (100 microM) inhibited whole cell I(Cl,swell) in a voltage-dependent manner, with the block decreasing from 39.6 +/- 9.7% and 71.0 +/- 11. 0% at +50 mV to 26.2 +/- 7.2% and 14.5 +/- 6.6% at -100 mV, respectively. Verapamil (50 microM), a standard Ca(2+) antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I(Cl,swell) (genistein with an IC(50) of 2.6 microM and lavendustin A by 60 +/- 14% at 1 microM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 microM) stimulated I(Cl,swell) by 54 +/- 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.
Edward M. Blumenthal - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule
American Journal of Physiology-Cell Physiology, 2013Co-Authors: Edward M. BlumenthalAbstract:The Malpighian (renal) tubule of Drosophila melanogaster is a useful model for studying epithelial transport. The purpose of this study was to identify factors responsible for modulating transepithelial Chloride Conductance in isolated tubules. I have found that tyrosine and several of its metabolites cause an increase in Chloride Conductance. The most potent of these agonists is tyramine, which is active at low nanomolar concentrations; the pharmacology of this response matches that of the previously published cloned insect tyramine receptor. In addition, the tubule appears capable of synthesizing tyramine from applied tyrosine, as shown by direct measurement of tyrosine decarboxylase activity. Immunohistochemical staining of tubules with an antibody against tyramine indicates that the principal cells are the sites of tyramine production, whereas previous characterization of the regulation of Chloride Conductance suggests that tyramine acts on the stellate cells. This is the first demonstration of a physiological role for an insect tyramine receptor.
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characterization of transepithelial potential oscillations in the drosophila malpighian tubule
The Journal of Experimental Biology, 2001Co-Authors: Edward M. BlumenthalAbstract:SUMMARY The Malpighian tubule of Drosophila melanogaster is a useful model system for studying the regulation of epithelial ion transport. In acutely isolated tubules, the transepithelial potential (TEP) undergoes large oscillations in amplitude with a period of approximately 30s. The TEP oscillations are diminished by reductions in the peritubular Chloride concentration in a manner consistent with their being caused by fluctuations in Chloride Conductance. The oscillations are eliminated by pretreating tubules with the calcium chelator BAPTA-AM, although removal of peritubular calcium has no effect, suggesting that the oscillations are a result of either the release of calcium from intracellular stores or the entry of calcium from the tubule lumen. Transcripts encoding two calcium-release channels, the ryanodine receptor and the inositol trisphosphate receptor, are detectable in the tubule by reverse transcription–polymerase chain reaction. To identify the cell type responsible for the oscillations, tubules were treated with diuretic hormones known to alter calcium levels in each of the two cell types. Leucokinin-IV, which increases calcium levels in the stellate cells, suppressed the oscillations, whereas cardioacceleratory peptide 2b (CAP 2b ), which increases calcium levels in the principal cells, had no effect. These data are consistent with a model in which rhythmic changes in transepithelial Chloride Conductance, regulated by intracellular calcium levels in the stellate cells, cause the TEP oscillations.