The Experts below are selected from a list of 393 Experts worldwide ranked by ideXlab platform
Philippe Poujeol - One of the best experts on this subject based on the ideXlab platform.
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Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules.
Journal of General Physiology, 2003Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Florian Lesage, Chantal Poujeol, Nicolas Guy, Jacques Barhanin, Philippe PoujeolAbstract:Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM Clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.
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role of task2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules
The Journal of General Physiology, 2003Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Florian Lesage, Chantal Poujeol, Nicolas Guy, Jacques Barhanin, Philippe PoujeolAbstract:Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM Clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.
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Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein
The Journal of Membrane Biology, 2003Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Chantal Poujeol, Jacques Barhanin, N. Tonnerieux, Philippe PoujeolAbstract:Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 −/− mice. In wild-type PCT cells in primary culture, a Cl^− conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage ( I-V ) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^− > Br^− > Cl^− >> gluconate. This conductance was sensitive to 1 m M 4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 0.1 m M 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 m M diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K^+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 m M quinidine, 5 m M barium, and 10 µ M Clofilium but are insensitive to 1 m M tetraethylammonium (TEA), 10 n M charybdotoxin (CTX), and 10 µ M 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^− and K^+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.
Céline Chauvin - One of the best experts on this subject based on the ideXlab platform.
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high throughput drug screening identifies pazopanib and Clofilium tosylate as promising treatments for malignant rhabdoid tumors
Social Science Research Network, 2018Co-Authors: Céline Chauvin, Amaury Leruste, Didier Surdez, Aurianne Lescure, Zhi-yan Han, Elodie Anthony, Wilfrid Richer, Arnault Tauziedeespariat, Sakina Zaidi, Marieming AynaudAbstract:The poor prognosis of rhabdoid tumors (RT) justifies the search for new therapies. By a high‐throughput drug screening and subsequent in vitro confirmation of selected drugs, we identified as good SMARCB1‐dependent therapeutic candidates, broad inhibitors of tyrosine kinase receptors (RTK) such as pazopanib, and the potassium channel inhibitor Clofilium tosylate (CfT). Their combination significantly induced RT apoptotic cell death in vitro. A human phospho‐kinase antibody array revealed PDGFRα/β and FGFR2 as pazopanib targets, while these genes were the most highly expressed RTK in our primary tumor data set. Of note, combined genetic inhibition of at least two RTK was only partially able to recapitulate the effect of pazopanib, emphasizing the requirement of broad inhibition. We then showed that pazopanib treatment reduced tumor growth in vivo while its combination with CfT could at least recapitulate the efficiency of conventional chemotherapy. Altogether, these results may influence future clinical trials for RT.
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high throughput drug screening identifies pazopanib and Clofilium tosylate as promising treatments for malignant rhabdoid tumors
Cell Reports, 2017Co-Authors: Céline Chauvin, Amaury Leruste, Mamy Andrianteranagna, Didier Surdez, Aurianne Lescure, Zhi-yan Han, Elodie Anthony, Wilfrid Richer, Arnault Tauziedeespariat, Sylvain BaulandeAbstract:Summary Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor Clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro . In vivo , reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs.
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High-Throughput Drug Screening Identifies Pazopanib and Clofilium Tosylate as Promising Treatments for Malignant Rhabdoid Tumors
Elsevier, 2017Co-Authors: Céline Chauvin, Amaury Leruste, Arnault Tauziede-espariat, Mamy Andrianteranagna, Didier Surdez, Aurianne Lescure, Zhi-yan Han, Elodie Anthony, Wilfrid Richer, Sylvain BaulandeAbstract:Summary: Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor Clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs. : Rhabdoid tumors (RTs) are aggressive pediatric tumors characterized by SMARCB1 inactivation. Chauvin et al. identify two SMARCB1-dependent targeted therapies for RT: pazopanib, which inhibits PDGFR and FGFR2, and the potassium channel inhibitor Clofilium tosylate, which induces endoplasmic reticulum stress. Combining both drugs induces cell apoptosis and reduces PDX tumor growth. Keywords: rhabdoid tumors, SMARCB1, pazopanib, Clofilium tosylate, high-throughput drug screening, tyrosine kinase inhibitor
Herve Barriere - One of the best experts on this subject based on the ideXlab platform.
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The Journal of General Physiology
2013Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Florian Lesage, Chantal Poujeol, Nicolas Guy, Jacques BarhaninAbstract:abstract Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wildtype and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using �-galactosidase staining. TASK2 was only localized in PCT cells. K � currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K � currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 �M 293B, but blocked by 500 �M quinidine and 10 �M Clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K � currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K � channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules. key words
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Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules.
Journal of General Physiology, 2003Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Florian Lesage, Chantal Poujeol, Nicolas Guy, Jacques Barhanin, Philippe PoujeolAbstract:Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM Clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.
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role of task2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules
The Journal of General Physiology, 2003Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Florian Lesage, Chantal Poujeol, Nicolas Guy, Jacques Barhanin, Philippe PoujeolAbstract:Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM Clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.
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Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein
The Journal of Membrane Biology, 2003Co-Authors: Herve Barriere, Radia Belfodil, Isabelle Rubera, Michel Tauc, Chantal Poujeol, Jacques Barhanin, N. Tonnerieux, Philippe PoujeolAbstract:Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 −/− mice. In wild-type PCT cells in primary culture, a Cl^− conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage ( I-V ) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^− > Br^− > Cl^− >> gluconate. This conductance was sensitive to 1 m M 4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 0.1 m M 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 m M diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K^+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 m M quinidine, 5 m M barium, and 10 µ M Clofilium but are insensitive to 1 m M tetraethylammonium (TEA), 10 n M charybdotoxin (CTX), and 10 µ M 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^− and K^+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.
Giovanni Risato - One of the best experts on this subject based on the ideXlab platform.
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Efficient Clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish
Cell death & disease, 2021Co-Authors: Nicola Facchinello, Claudio Laquatra, Lisa Locatello, Giorgia Beffagna, Raquel Brañas Casas, Chiara Fornetto, Alberto Dinarello, Laura Martorano, Andrea Vettori, Giovanni RisatoAbstract:The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.
Claudia L. Treviño - One of the best experts on this subject based on the ideXlab platform.
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membrane hyperpolarization during human sperm capacitation
Molecular Human Reproduction, 2014Co-Authors: Ignacio Lopezgonzalez, Alberto Darszon, Celia M. Santi, Paulina Torresrodriguez, O Sanchezcarranza, A Solislopez, Claudia L. TreviñoAbstract:Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fer- tilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpo- larization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma mem- brane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca 2+ concentration, measured also by flow cytometry. We found that sperm membrane hyperpolar- ization was completely abolished in the presence of a high external K + concentration (60 mM), indicating the participation of K + channels. In order to identify, which of the potential K + channels were involved in this hyperpolarization, we used different K + channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and Clofilium (a more specific blocker for Slo3). All these K + channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K + currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to Clofilium (50 mM). Considered altogether, our data indicate that Slo1 and Slo3 could share the prepon- derant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.
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[Na+]i is decreased in SLO3 mutant sperm and in sperm treated with the SLO3 inhibitor Clofilium.
2013Co-Authors: Julio C. Chávez, José L. De La Vega-beltrán, Jessica Escoffier, Pablo E. Visconti, Claudia L. Treviño, Alberto Darszon, Lawrence Salkoff, Celia M. SantiAbstract:Cauda epididymal sperm from wild type or SLO3 mutant mice were recovered and loaded with CoroNaRed in media lacking BSA and HCO3− which does not support capacitation (Non Cap). After thirty minutes incubation, the sperm were washed once and resuspended in the same media or in media containing BSA and HCO3− (Cap) in the absence or in the presence of Clofilium (50 µM) (for wild-type sperm). After 1 hour incubation, PI was added and the sperm population analyzed by flow cytometry. A) SLO3 wild-type sperm: PI vs CoroNa Red two-dimensional dot plots of sperm incubated in non capacitating conditions (Non Cap), in media that support capacitation (Cap) or in media that support capacitation in the presence of Clofilium (Cap+Clofilium 50 µM). The left merged panel combined data from Non Cap and Cap dot plots, the right merged panel combined the Cap and the Cap+Clofilium dot plots. B) SLO3 mutant sperm: PI vs CoroNa Red two-dimensional dot plots of sperm incubated in non capacitating conditions (Non Cap) or in media that support capacitation (Cap). Live sperm populations in each case were then analyzed for their individual [Na+]i CoroNa Red fluorescence histograms (Non Cap and Cap); the merged panel combined both data. C) Summary for plots showed in A and B. The bars represent the mean n = 3 experiments ± S.E.M. NS indicates No statistical significance (P≥0.05), **indicates statistical significance (P≤0.01).
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Membrane potential measurements when SLO3 channel antagonists are present in capacitated wild-type and SLO3 mutant sperm; no significant changes in ion permeabilities are seen compared to non-capacitated sperm.
2013Co-Authors: Julio C. Chávez, José L. De La Vega-beltrán, Jessica Escoffier, Pablo E. Visconti, Claudia L. Treviño, Alberto Darszon, Lawrence Salkoff, Celia M. SantiAbstract:Curves shown are GHK fits to wild-type (A) and SLO3 mutant sperm (B). When SLO3 channels are blocked in wild-type sperm, with either Ba2+ (1 mM), or Clofilium (50 µM), no hyperpolarization is noted during capacitation and membrane permeability values are similar to those of non-capacitated wild-type sperm. Note that there is no predicted decrease in PNa in either wild-type sperm or SLO3 mutant subjected to capacitating conditions. SLO3 blocking agents may have a slight blocking effect on the residual PK in sperm. Permeability values predicted by the GHK equation are given in (C). The curves correspond to mean n = 3 experiments ± S.E.M. See table S3 for membrane potential values.