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  • 11
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    Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells
    American Physiological Society ; 2009
    In:  American Journal of Physiology-Cell Physiology Vol. 296, No. 2 ( 2009-02), p. C285-C295
    Details
    In: American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 296, No. 2 ( 2009-02), p. C285-C295
    Abstract: The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5′-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 μM AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the γ1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp 380 to Ala 400 in COOH-terminal were essential for the interaction AMPK-γ1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-γ1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the γ1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-γ1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the γ1-subunit of AMPK.
    Type of Medium: Online Resource
    ISSN: 0363-6143 , 1522-1563
    URL: Article
    DOI: 10.1152/ajpcell.00418.2008
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2009
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  • 12
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    Regulation of ENaC and CFTR expression with K + channel modulators and effect on fluid absorption across alveolar epithelial cells
    American Physiological Society ; 2006
    In:  American Journal of Physiology-Lung Cellular and Molecular Physiology Vol. 291, No. 6 ( 2006-12), p. L1207-L1219
    Details
    In: American Journal of Physiology-Lung Cellular and Molecular Physiology, American Physiological Society, Vol. 291, No. 6 ( 2006-12), p. L1207-L1219
    Abstract: In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027–L1037, 2004), we identified an ATP-sensitive K + (K ATP ) channel in alveolar epithelial cells, formed by inwardly rectifying K + channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K ATP , voltage-dependent K + channel KvLQT1, and calcium-activated K + (K Ca ) channel modulators modified Na + and Cl − currents in alveolar monolayers. In addition, it was shown previously that a K ATP opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K + channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K ATP , KvLQT1, and K Ca channels identified by PCR. Glibenclamide and clofilium (K ATP and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na + current, and forskolin-activated Cl − currents, whereas pinacidil, a K ATP activator, increased them. Interestingly, K + inhibitors or membrane depolarization (induced by valinomycin in high-K + medium) decreased α-, β-, and γ-ENaC and CFTR mRNA. α-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na + transport through ENaC, we tested the impact of K + channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K + channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.
    Type of Medium: Online Resource
    ISSN: 1040-0605 , 1522-1504
    URL: Article
    DOI: 10.1152/ajplung.00376.2005
    Language: English
    Publisher: American Physiological Society
    Publication Date: 2006
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  • 13
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    DataSheet1.PDF
    Frontiers Media SA ; 2023
    In:  Frontiers in Physiology Vol. 13 ( 2023-1-9)
    Details
    In: Frontiers in Physiology, Frontiers Media SA, Vol. 13 ( 2023-1-9)
    Abstract: Alveolar ion and fluid absorption is essential for lung homeostasis in healthy conditions as well as for the resorption of lung edema, a key feature of acute respiratory distress syndrome. Liquid absorption is driven by active transepithelial sodium transport, through apical ENaC Na + channels and basolateral Na + /K + -ATPase. Our previous work unveiled that KvLQT1 K + channels also participate in the control of Na + /liquid absorption in alveolar epithelial cells. Our aim was to further investigate the function of KvLQT1 channels and their interplay with other channels/transporters involved in ion/liquid transport in vivo using adult wild-type (WT) and KvLQT1 knock-out (KO) mice under physiological conditions and after thiourea-induced lung edema. A slight but significant increase in water lung content (WLC) was observed in naïve KvLQT1-KO mice, relative to WT littermates, whereas lung function was generally preserved and histological structure unaltered. Following thiourea-induced lung edema, KvLQT1-KO did not worsen WLC or lung function. Similarly, lung edema was not aggravated by the administration of a KvLQT1 inhibitor (chromanol). However, KvLQT1 activation (R-L3) significantly reduced WLC in thiourea-challenged WT mice. The benefits of R-L3 were prevented in KO or chromanol-treated WT mice. Furthermore, R-L3 treatment had no effect on thiourea-induced endothelial barrier alteration but restored or enhanced the levels of epithelial alveolar AQP5, Na + /K + -ATPase, and ENaC expressions. Altogether, the results indicate the benefits of KvLQT1 activation in the resolution of lung edema, probably through the observed up-regulation of epithelial alveolar channels/transporters involved in ion/water transport.
    Type of Medium: Online Resource
    ISSN: 1664-042X
    URL: Article
    URL: Article
    DOI: 10.3389/fphys.2022.1069466
    DOI: 10.3389/fphys.2022.1069466.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
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  • 14
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    Presentation1.pdf
    Frontiers Media SA ; 2024
    In:  Frontiers in Physiology Vol. 15 ( 2024-2-20)
    Details
    In: Frontiers in Physiology, Frontiers Media SA, Vol. 15 ( 2024-2-20)
    Abstract: Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K + ) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo . The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.
    Type of Medium: Online Resource
    ISSN: 1664-042X
    URL: Article
    URL: Article
    DOI: 10.3389/fphys.2024.1345488
    DOI: 10.3389/fphys.2024.1345488.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2024
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  • 15
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    SARS-CoV-2 Spike Expression at the Surface of Infected Primary Human Airway Epithelial Cells
    MDPI AG ; 2021
    In:  Viruses Vol. 14, No. 1 ( 2021-12-21), p. 5-
    Details
    In: Viruses, MDPI AG, Vol. 14, No. 1 ( 2021-12-21), p. 5-
    Abstract: Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.
    Type of Medium: Online Resource
    ISSN: 1999-4915
    URL: Article
    DOI: 10.3390/v14010005
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
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  • 16
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    Potassium channels as operators of alveolar epithelial repair
    Wiley ; 2012
    In:  The FASEB Journal Vol. 26, No. S1 ( 2012-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 26, No. S1 ( 2012-04)
    Abstract: A common feature of inflammatory lung diseases such as acute lung injury and acute respiratory distress syndrome (ARDS) is extensive damage and remodeling of alveolar epithelium. A better understanding of determinants of alveolar regeneration is thus necessary to develop strategies able to restore alveolar integrity. Recent studies have shown that K + channels (KCh) are key components of cell proliferation and migration processes, necessary for tissues repair. We therefore postulated that KCh play a role in respiratory epithelial repair. Using a wound‐healing assay, we first showed that glibenclamide and clofilium, inhibitors of K ATP and KvLQT1 channels, decrease repair rates of A549 and primary alveolar ATII cell monolayers and their inhibitory effects are additive. The role of KvLQT1 and K ATP was then confirmed by a molecular approach using specific siRNAs. On the contrary, KvLQT1 and K ATP activators significantly stimulate the wound repair. We also evaluated the influence of KCh on cell growth and we showed that clofilium dose‐dependently inhibits alveolar cell growth and induces an accumulation of cells arrested in G0/G1 phase. In conclusion, we demonstrated that KCh, particularly KvLQT1 and K ATP , are involved in epithelial repair processes and should be identify as novel targets to promote alveolar repair necessary to ARDS resolution. Project funded by Canadian Institute of Health Research.
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v26.S1
    DOI: 10.1096/fasebj.26.1_supplement.56.1
    Language: English
    Publisher: Wiley
    Publication Date: 2012
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  • 17
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    Complementary roles of KCa3.1 channels and β1‐integrin in the regulation of alveolar epithelial repair
    Wiley ; 2013
    In:  The FASEB Journal Vol. 27, No. S1 ( 2013-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 27, No. S1 ( 2013-04)
    Abstract: A common feature of acute lung injury is extensive damage and remodeling of alveolar epithelium. It has been shown that epithelial regeneration and secondary lung edema resorption are crucial to patient recovery. We previously reported that KvLQT1 and K ATP K + channels regulated alveolar epithelial repair processes, but the role of another candidate, the KCa3.1, has never been evaluated. We first demonstrated that the wound‐healing rates were higher in primary rat alveolar cell monolayers cultured on a fibronectin‐collagen (FC) matrix, than on non‐coated supports. KCa3.1 inhibition, with TRAM‐34, or activation, with 1‐EBIO, respectively down‐ and up‐regulated the alveolar wound‐healing rates on FC matrix, but were without effect in absence of FC coating. Accordingly, single cell migration rates were also dependent, at least in part, of FC matrix coating and KCa3.1 activity. A possible relationship between KCa3.1 and migratory cell proteins such as the integrin, activated by fibronectin, was then assessed. Co‐immunoprecipitation experiments thus revealed a physical interaction between KCa3.1 and the β‐1 integrin subunit. Altogether, our data showed for the first time a complementary role of KCa3.1 channels, extracellular matrix and β‐1 integrin, in the regulation of alveolar repair after injury. Project funded by CIHR, RHN of FRQS, and FESP of Université de Montréal.
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v27.S1
    DOI: 10.1096/fasebj.27.1_supplement.1166.3
    Language: English
    Publisher: Wiley
    Publication Date: 2013
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  • 18
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    Acute Respiratory Distress Syndrome (ARDS): Are corticosteroids harmful in lung injury
    Wiley ; 2011
    In:  The FASEB Journal Vol. 25, No. S1 ( 2011-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 25, No. S1 ( 2011-04)
    Abstract: ARDS is a rapidly evolving lung injury that is linked with permeability oedema and excessive inflammation, leading to a severe oxygenation defect. Albeit there has been significant progress in survival, ARDS mortality remains ~ 40 %. Glucocorticoids therapy has been proposed to decrease the inflammatory response, but its usage remains controversial. The objective of this study was to evaluate the impact of dexamethasone treatment (dex; 0.5 mg/kg/d) on the in vivo evolution of bleomycin‐induced (bleo; 4 U/kg) lung injury in NMRI mouse. Treatment with dex did not decrease the mortality, the weight loss, the wet/dry ratio, the severity of epithelial injuries and the inflammatory infiltrating of neutrophils induced by bleo. To better evaluate the lack of response to dex, the impact of dex on the repair process of primary epithelial cells was studied. Our results show that dex inhibits the closure of mechanical wound. This effect of dex is not related to a default of proliferation, or an activation of apoptosis. This inhibitory effect of dex on wound closure is associated with an increase in cell adhesion suggesting that its effect could be related a modulation of the cytoskeleton. Our results indicate that dex does not decrease the severity of lung injury model and might slow its recovery by preventing proper repair of the alveolar epithelium. Supported by RSR‐FRSQ, FCFK, IRSC.
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v25.S1
    DOI: 10.1096/fasebj.25.1_supplement.865.5
    Language: English
    Publisher: Wiley
    Publication Date: 2011
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  • 19
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    KvLQT1 Function in Lung Physiology
    Wiley ; 2015
    In:  The FASEB Journal Vol. 29, No. S1 ( 2015-04)
    Details
    In: The FASEB Journal, Wiley, Vol. 29, No. S1 ( 2015-04)
    Abstract: Acute lung injury and its more severe form, acute respiratory distress syndrome (ARDS), are characterized by extensive alveolar injury and lung edema. It has been established that the resolution of ARDS parameters is highly dependent on alveolar regeneration and edema resorption. Because we previously demonstrated that KvLQT1 potassium channels are involved in alveolar repair processes and in the control of ion and liquid transports in vitro , we now decided to investigate their role in lung function and in the resolution of ARDS parameters in vivo , using a KvLQT1 knockout (KO) mice model. We first noted an absence of morphological difference in airway structures (trachea, bronchus and alveolus) between control (wild type, WT) and KvLQT1 KO mice. We then evaluated the impact of KvLQT1 extinction on the mechanical properties of the respiratory system using the flexiVent system (SCIREQ Inc.). Our results showed a significant increase of tissue damping and dynamic resistance of the respiratory system in KvLQT1 KO mice compared to WT. The role of KvLQT1 was then evaluated in a model of lung edema induced by thiourea, disrupting the integrity of endothelial/alveolar barrier. We observed a significant increase in the wet/dry ratio (W/D, index of lung edema) in both WT and KO mice. Interestingly, the thiourea‐induced W/D increase was partially prevented by a treatment with R‐L3, a KvLQT1 activator, in WT mice. However, this beneficial effect of R‐L3 was lost in KO mice. Our results show that the activation of KvLQT1 K + channels could play a role in the resolution of ARDS parameters, especially pulmonary edema resorption. Funded by CIHR, NSERC, FRQS.
    Type of Medium: Online Resource
    ISSN: 0892-6638 , 1530-6860
    URL: Issue
    URL: Article
    DOI: 10.1096/fsb2.v29.S1
    DOI: 10.1096/fasebj.29.1_supplement.927.15
    Language: English
    Publisher: Wiley
    Publication Date: 2015
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  • 20
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    Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue
    European Respiratory Society (ERS) ; 2012
    In:  European Respiratory Journal Vol. 40, No. 6 ( 2012-12), p. 1390-1400
    Details
    In: European Respiratory Journal, European Respiratory Society (ERS), Vol. 40, No. 6 ( 2012-12), p. 1390-1400
    Type of Medium: Online Resource
    ISSN: 0903-1936 , 1399-3003
    URL: Article
    DOI: 10.1183/09031936.00221711
    Language: English
    Publisher: European Respiratory Society (ERS)
    Publication Date: 2012
    detail.hit.zdb_id: 639359-7
    detail.hit.zdb_id: 1499101-9
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