Marc Chanson - Academia.edu (original) (raw)

Papers by Marc Chanson

American Journal of …, Jan 1, 1996

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye an... more The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.

Pflügers Archiv : European journal of physiology, 1988

The electrical properties of single acinar cells isolated from rat pancreas were studied with the... more The electrical properties of single acinar cells isolated from rat pancreas were studied with the whole-cell tight-seal recording method. Under resting conditions, the relative permeabilities of Cl and K were PCl/PK approximately equal to 3. At 1 microM internal calcium, a Ca and voltage-dependent Cl conductance was activated. At 10 microM internal calcium, the major conductance was selective for cations. It was not voltage-dependent. Acetylcholine and cholecystokinin induced an increase of internal Ca which in turn activated either only a Cl conductance or both Cl and cationic conductances. The secretagogue-induced conductance was increased to a variable extent by depolarisation. The absence of K channels activated by internal calcium indicates that, in pancreatic acinar cells, the mechanism of fluid secretion differs from that observed in other exocrine glands.

American Journal of Physiology Cell Physiology, Apr 1, 1991

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is ... more Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to "oval" cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

Circulation Research

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends... more Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. L...

Circulation Research, 2001

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the car... more Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values ("lower conductance" and "main") were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT). (Circ Res. 2001;88:666-673.)

American Journal Of Pathology

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclea... more Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF15 cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF15 cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the ΔF508 CFTR cDNA, or the β-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF15 monolayers expressing β-galactosidase was similar to that seen across CF15 monolayers rescued with ...

Pflügers Archiv : European journal of physiology, 1988

The electrical properties of single acinar cells isolated from rat pancreas were studied with the... more The electrical properties of single acinar cells isolated from rat pancreas were studied with the whole-cell tight-seal recording method. Under resting conditions, the relative permeabilities of Cl and K were PCl/PK approximately equal to 3. At 1 microM internal calcium, a Ca and voltage-dependent Cl conductance was activated. At 10 microM internal calcium, the major conductance was selective for cations. It was not voltage-dependent. Acetylcholine and cholecystokinin induced an increase of internal Ca which in turn activated either only a Cl conductance or both Cl and cationic conductances. The secretagogue-induced conductance was increased to a variable extent by depolarisation. The absence of K channels activated by internal calcium indicates that, in pancreatic acinar cells, the mechanism of fluid secretion differs from that observed in other exocrine glands.

PLoS ONE, 2009

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic p... more The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2a) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2a. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-a. This was concomitant with increased IL-8 synthesis and cPLA2a activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-b-cyclodextrin induced further cPLA2a activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-a-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2a and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-a-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.

Thrombosis and Haemostasis, 2014

Ubiquitous reduction of the gap junction protein Connexin43 (Cx43) in mice provides beneficial ef... more Ubiquitous reduction of the gap junction protein Connexin43 (Cx43) in mice provides beneficial effects on progression and composition of atherosclerotic lesions. Cx43 is expressed in multiple atheroma-associated cells but its function in each cell type is not known. To examine specifically the role of Cx43 in immune cells, we have lethally irradiated low-density lipoprotein receptor-deficient mice and reconstituted with Cx43 +/+ , Cx43 +/or Cx43 -/haematopoietic fetal liver cells. Progression of atherosclerosis was significantly lower in aortic roots of Cx43 +/chimeras compared with Cx43 +/+ and Cx43 -/chimeras, and their plaques contained significantly less neutrophils. The relative proportion of circulating leukocytes was similar between the three groups. Interestingly, the chemoattraction of neutrophils, which did not express Cx43, was reduced in response to supernatant secreted by Cx43 +/macrophages in comparison with the ones of Cx43 +/+ and Cx43 -/macrophages. Cx43 +/macrophages did not differ from Cx43 +/+ and Cx43 -/macrophages in terms of M1/M2 polarisation but show modified gene expression for a variety chemokines and complement components. In conclusion, titration of Cx43 expression in bone marrow-derived macrophages reduces atherosclerotic plaque formation and chemoattraction of neutrophils to the lesions.

Pfl�gers Archiv European Journal of Physiology, 1995

The effects of 8-bromoguanosine 3': Y-cyclic monophosphate (8Br-cGMP), a membrane-permeant activa... more The effects of 8-bromoguanosine 3': Y-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHepl cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43transfected cells by 24.0 + 3.7% (mean + SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMR Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (yj) of about 20, 40~5 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the ~j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32p] into rat Cx43 was significantly altered by 8Br-cGMR whereas this incorporation of [32p] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHepl cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

Journal of Cystic Fibrosis, 2004

Polarized expression of ion channels is a prerequisite for vectorial electrolyte transport by epi... more Polarized expression of ion channels is a prerequisite for vectorial electrolyte transport by epithelial cells. Clchannels such as the cystic fibrosis transmembrane conductance regulator, CFTR, are mainly localized in the apical membrane of secretory epithelia while basolateral K + channels are believed to constitute the major driving force for ion movement. To independently assess the functional properties of the two separate membranes of polarized cells, various techniques have been developed. The most popular approaches to date are either to isolate the membrane of interest by mechanical force (excised inside-out membrane patch, see protocol by T.S. Scott-Ward et al.) or to remove one membrane by selective permeabilization (see protocol by H. Li and D.N. Sheppard). Both techniques separate the membranes and the ion channels from their intact physiological environment and thus give a very incomplete reflection of the native state. Impalement of membranes with microelectrodes is an alternative approach with the advantage that it is a relatively uninvasive technique.

Journal of Clinical Investigation, 2008

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used... more Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo. Nonstandard abbreviations used: BFU-E, burst-forming unit erythroid; CFU-E,

Journal of Cell Science, 2003

Journal of Cellular and Molecular Medicine, 2009

Acute lung injury (ALI) is attributable to a severe inflammation that is most commonly caused by ... more Acute lung injury (ALI) is attributable to a severe inflammation that is most commonly caused by sepsis, infection, trauma or acid aspiration. Despite optimal management, ALI remains a clinical syndrome related to a high mortality in both children and adults . Hallmarks of ALI are massive invasion of leukocytes and protein-rich oedema in the interstitial and intra-alveolar spaces leading to impaired gas exchange and increased pulmonary resistance . ALI develops rapidly across the lung vascular surface involving an entire lung or even both lungs , but the mechanism underlying the spatial expansion of inflammation is unexplained.

Biology of the Cell, 2002

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic infla... more Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-α signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-α to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-α signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-ΔTRL), TNF-α increased the amount of WtCFTR but not CFTR-ΔTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-ΔTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013

The American Journal of Pathology, 2001

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cel... more Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-kappaB (NF-kappaB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced maximal translocation of NF-kappaB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-alpha progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-alpha remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-alpha-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-alpha. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.

Acta Physiologica Scandinavica, 2003

American Journal of …, Jan 1, 1996

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye an... more The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.

Pflügers Archiv : European journal of physiology, 1988

The electrical properties of single acinar cells isolated from rat pancreas were studied with the... more The electrical properties of single acinar cells isolated from rat pancreas were studied with the whole-cell tight-seal recording method. Under resting conditions, the relative permeabilities of Cl and K were PCl/PK approximately equal to 3. At 1 microM internal calcium, a Ca and voltage-dependent Cl conductance was activated. At 10 microM internal calcium, the major conductance was selective for cations. It was not voltage-dependent. Acetylcholine and cholecystokinin induced an increase of internal Ca which in turn activated either only a Cl conductance or both Cl and cationic conductances. The secretagogue-induced conductance was increased to a variable extent by depolarisation. The absence of K channels activated by internal calcium indicates that, in pancreatic acinar cells, the mechanism of fluid secretion differs from that observed in other exocrine glands.

American Journal of Physiology Cell Physiology, Apr 1, 1991

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is ... more Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to "oval" cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

Circulation Research

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends... more Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. L...

Circulation Research, 2001

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the car... more Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values ("lower conductance" and "main") were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT). (Circ Res. 2001;88:666-673.)

American Journal Of Pathology

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclea... more Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF15 cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF15 cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the ΔF508 CFTR cDNA, or the β-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF15 monolayers expressing β-galactosidase was similar to that seen across CF15 monolayers rescued with ...

Pflügers Archiv : European journal of physiology, 1988

The electrical properties of single acinar cells isolated from rat pancreas were studied with the... more The electrical properties of single acinar cells isolated from rat pancreas were studied with the whole-cell tight-seal recording method. Under resting conditions, the relative permeabilities of Cl and K were PCl/PK approximately equal to 3. At 1 microM internal calcium, a Ca and voltage-dependent Cl conductance was activated. At 10 microM internal calcium, the major conductance was selective for cations. It was not voltage-dependent. Acetylcholine and cholecystokinin induced an increase of internal Ca which in turn activated either only a Cl conductance or both Cl and cationic conductances. The secretagogue-induced conductance was increased to a variable extent by depolarisation. The absence of K channels activated by internal calcium indicates that, in pancreatic acinar cells, the mechanism of fluid secretion differs from that observed in other exocrine glands.

PLoS ONE, 2009

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic p... more The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2a) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2a. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-a. This was concomitant with increased IL-8 synthesis and cPLA2a activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-b-cyclodextrin induced further cPLA2a activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-a-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2a and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-a-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.

Thrombosis and Haemostasis, 2014

Ubiquitous reduction of the gap junction protein Connexin43 (Cx43) in mice provides beneficial ef... more Ubiquitous reduction of the gap junction protein Connexin43 (Cx43) in mice provides beneficial effects on progression and composition of atherosclerotic lesions. Cx43 is expressed in multiple atheroma-associated cells but its function in each cell type is not known. To examine specifically the role of Cx43 in immune cells, we have lethally irradiated low-density lipoprotein receptor-deficient mice and reconstituted with Cx43 +/+ , Cx43 +/or Cx43 -/haematopoietic fetal liver cells. Progression of atherosclerosis was significantly lower in aortic roots of Cx43 +/chimeras compared with Cx43 +/+ and Cx43 -/chimeras, and their plaques contained significantly less neutrophils. The relative proportion of circulating leukocytes was similar between the three groups. Interestingly, the chemoattraction of neutrophils, which did not express Cx43, was reduced in response to supernatant secreted by Cx43 +/macrophages in comparison with the ones of Cx43 +/+ and Cx43 -/macrophages. Cx43 +/macrophages did not differ from Cx43 +/+ and Cx43 -/macrophages in terms of M1/M2 polarisation but show modified gene expression for a variety chemokines and complement components. In conclusion, titration of Cx43 expression in bone marrow-derived macrophages reduces atherosclerotic plaque formation and chemoattraction of neutrophils to the lesions.

Pfl�gers Archiv European Journal of Physiology, 1995

The effects of 8-bromoguanosine 3': Y-cyclic monophosphate (8Br-cGMP), a membrane-permeant activa... more The effects of 8-bromoguanosine 3': Y-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHepl cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43transfected cells by 24.0 + 3.7% (mean + SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMR Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (yj) of about 20, 40~5 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the ~j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32p] into rat Cx43 was significantly altered by 8Br-cGMR whereas this incorporation of [32p] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHepl cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.

Journal of Cystic Fibrosis, 2004

Polarized expression of ion channels is a prerequisite for vectorial electrolyte transport by epi... more Polarized expression of ion channels is a prerequisite for vectorial electrolyte transport by epithelial cells. Clchannels such as the cystic fibrosis transmembrane conductance regulator, CFTR, are mainly localized in the apical membrane of secretory epithelia while basolateral K + channels are believed to constitute the major driving force for ion movement. To independently assess the functional properties of the two separate membranes of polarized cells, various techniques have been developed. The most popular approaches to date are either to isolate the membrane of interest by mechanical force (excised inside-out membrane patch, see protocol by T.S. Scott-Ward et al.) or to remove one membrane by selective permeabilization (see protocol by H. Li and D.N. Sheppard). Both techniques separate the membranes and the ion channels from their intact physiological environment and thus give a very incomplete reflection of the native state. Impalement of membranes with microelectrodes is an alternative approach with the advantage that it is a relatively uninvasive technique.

Journal of Clinical Investigation, 2008

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used... more Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo. Nonstandard abbreviations used: BFU-E, burst-forming unit erythroid; CFU-E,

Journal of Cell Science, 2003

Journal of Cellular and Molecular Medicine, 2009

Acute lung injury (ALI) is attributable to a severe inflammation that is most commonly caused by ... more Acute lung injury (ALI) is attributable to a severe inflammation that is most commonly caused by sepsis, infection, trauma or acid aspiration. Despite optimal management, ALI remains a clinical syndrome related to a high mortality in both children and adults . Hallmarks of ALI are massive invasion of leukocytes and protein-rich oedema in the interstitial and intra-alveolar spaces leading to impaired gas exchange and increased pulmonary resistance . ALI develops rapidly across the lung vascular surface involving an entire lung or even both lungs , but the mechanism underlying the spatial expansion of inflammation is unexplained.

Biology of the Cell, 2002

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2008

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic infla... more Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause a chronic inflammatory response in the lung of patients with Cystic Fibrosis (CF). We have showed that TNF-α signaling through the Src family tyrosine kinases (SFKs) was defective as determined by an inability of TNF-α to regulate gap junctional communication (GJIC) in CF cells. Here, we sought to elucidate the mechanisms linking TNF-α signaling to the functions of CFTR at the molecular level. In a MDCKI epithelial cell model expressing wild-type (WtCFTR) or mutant CFTR lacking its PDZ-interacting motif (CFTR-ΔTRL), TNF-α increased the amount of WtCFTR but not CFTR-ΔTRL in detergent-resistant membrane microdomains (DRMs). This recruitment was modulated by SFK activity and associated with DRM localization of TNFR1 and c-Src. Activation of TNFR1 signaling also decreased GJIC and markedly stimulated IL-8 production in WtCFTR cells. In contrast, the absence of CFTR in DRMs was associated with abnormal TNFR1 signaling as revealed by no recruitment of TNFR1 and c-Src to lipid rafts in CFTR-ΔTRL cells and loss of regulation of GJIC and IL-8 secretion. These results suggest that localization of CFTR in lipid rafts in association with c-Src and TNFR1 provides a responsive signaling complex to regulate GJIC and cytokine signaling.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2013

The American Journal of Pathology, 2001

Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cel... more Airway inflammation is orchestrated by cell-cell interactions involving soluble mediators and cell adhesion molecules. Alterations in the coordination of the multicellular process of inflammation may play a major role in the chronic lung disease state of cystic fibrosis (CF). The aim of this study was to determine whether direct cell-cell interactions via gap junctional communication is affected during the inflammatory response of the airway epithelium. We have examined the strength of intercellular communication and the activation of nuclear factor-kappaB (NF-kappaB) in normal (non-CF) and CF human airway cell lines stimulated with tumor necrosis factor-alpha (TNF-alpha). TNF-alpha induced maximal translocation of NF-kappaB into the nucleus of non-CF as well as CF airway cells within 20 minutes. In non-CF cells, TNF-alpha progressively decreased the extent of intercellular communication. In contrast, gap junctional communication between CF cells exposed to TNF-alpha remained unaltered. CF results from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Interestingly, transfer of wild-type CFTR into CF cells by adenovirus-mediated infection was associated with the recovery of TNF-alpha-induced uncoupling. These results suggest that expression of functional CFTR is necessary for regulation of gap junctional communication by TNF-alpha. Gap junction channels close during the inflammatory response, therefore limiting the intercellular diffusion of signaling molecules, and thereby the recruitment of neighboring cells. Defects in this mechanism may contribute to the excessive inflammatory response of CF airway epithelium.

Acta Physiologica Scandinavica, 2003