Frédérique Mies

Epithelial Na+ channel stimulation by n-3 fatty acids requires proximity to a membrane-bound A-kinase-anchoring protein complexed with protein kinase A and phosphodiesterase

Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/min to 1.3 pmol/mg of protein/min. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC β subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase.

Halothane Directly Modifies Na+ and K+ Channel Activities in Cultured Human Alveolar Epithelial Cells

During inhalational anesthesia, halogenated gases are in direct contact with the alveolar epithelium, in which they may affect transepithelial ion and fluid transport. The effects of halogenated gases in vivo on epithelial Na+ and K+ channels, which participate in alveolar liquid clearance, remain unclear. In the present study, the effects of halothane (1, 2, and 4% atm) on ion-channel function in cultured human alveolar cells were investigated using the patch-clamp technique. After exposure to 4% halothane, amiloride-sensitive whole-cell inward currents increased by 84 ± 22%, whereas tetraethylammonium-sensitive outward currents decreased by 63 ± 7%. These effects, which occurred within 30 s, remained for 30-min periods of exposure to the gas, were concentration-dependent, and were reversible upon washout. Pretreatment with amiloride prevented 90 ± 7% of the increase in inward currents without change in outward currents, consistent with an activation of amiloride-sensitive epithelial sodium channels. Tetraethylammonium obliterated 90 ± 9% of the effect of halothane on outward currents, without change in inward currents, indicating inhibition of Ca2+-activated K+ channels. These channels were identified in excised patches to be small-conductance Ca2+-activated K+ channels. These effects of halothane were not modified after the inhibition of cytosolic phospholipase A2 by aristolochic acid. Exposure of the cells to either trypsin or to low Na+ completely prevented the increase in amiloride-sensitive currents induced by halothane, suggesting a release of Na+ channels self-inhibition. Thus, halothane modifies differentially and independently Na+ and K+ permeabilities in human alveolar cells.

Opposing Effects of Bone Morphogenetic Protein-2 and Endothelin-1 on Lung Fibroblast Chloride Currents

Alteration in the control of bone morphogenetic protein (BMP)-regulated genes and increased expression of endothelin (ET)-1 are both believed to play important roles in the still incompletely understood pathobiology of pulmonary vascular remodeling and fibrosis. Recent studies have drawn attention to the contribution of adventitial fibroblast activation in these phenomena. Because chloride channels are involved in the control of physiological function of fibroblasts, we hypothesized that these channels are differentially regulated by BMPs and ET. We measured chloride ion currents by whole-cell path-clamping in cultured primary human pulmonary fibroblasts. The application of BMP2 prevented activation of these currents by hypotonic challenge in a time- and dose-dependent manner, partially via protein kinase C signaling. Maximal inhibition was observed after 45-minute incubation of cells in the presence of 10 ng/ml of BMP2. ET-1 did not activate chloride channels acutely; however, prolonged treatment of cells with ET-1 (100 nM, 2 h) induced the appearance of lysophosphatidic acid-activated chloride currents (a marker of differentiated myofibroblasts), and this induction could be effectively blocked by BMP2 pretreatment (10 ng/ml). BMP2 also prevented stimulation of α-smooth muscle actin gene expression and cell migration of fibroblasts induced by ET-1. We conclude that ET-1 and BMP2 have opposing effects on chloride channel activity in human fibroblasts. This is a potentially relevant mechanism involved in pulmonary vascular remodeling and fibrosis.

Electrophysiological Characterization of Rat Type II Pneumocytes in Situ

Optimal aeration of the lungs is dependent on an alveolar fluid clearance, a process that is governed by Na+ and Cl- transport. However, the specific contribution of various ion channels in different alveolar cell types under basal or stimulated conditions is not exactly known. We established a novel functional model of rat lung slices suitable for nystatin-perforated whole-cell patch-clamp experiments. Lung slices retained a majority of live cells for up to 72 hours. Type II pneumocytes in situ had a mean capacitance of 8.8 +/- 2.5 pF and a resting membrane potential of -4.4 +/- 1.9 mV. Bath replacement of Na+ with NMDG+ decreased inward whole-cell currents by 70%, 21% and 52% of which were sensitive to 10 microM and 1 mM of amiloride, respectively. Exposure of slices to 0.5 microM dexamethasone for 1 hour did not affect ion currents, while chronic exposure (0.5 microM, 24-72 h) induced an increase in both total Na+-entry currents and amiloride-sensitive currents. Under acute exposure to 100 microM cpt-cAMP, Type II cells in situ rapidly hyperpolarized by 25-30 mV, due to activation of whole-cell Cl- currents sensitive to 0.1 mM of 5-Nitro-2-(3-phenylpropylamino)benzoic acid. In addition, in the presence of cpt-cAMP, total sodium currents and currents sensitive to 10 microM amiloride increased by 32% and 70%, respectively. Thus, in Type II pneumocytes in situ: (1) amiloride-sensitive sodium channels contribute to only half of total Na+-entry and are stimulated by chronic exposure to glucocorticoids; (2) acute increase in cellular cAMP content simultaneously stimulates the entry of Cl- and Na+ ions.

Possible role of lysophosphatidic acid in rat model of hypoxic pulmonary vascular remodeling

Pulmonary hypertension is characterized by cellular and structural changes in the vascular wall of pulmonary arteries. We hypothesized that lysophosphatidic acid (LPA), a bioactive lipid, is implicated in this vascular remodeling in a rat model of hypoxic pulmonary hypertension. Exposure of Wistar rats to 10% O2 for 3 weeks induced an increase in the mean serum levels of LPA, to 40.9 (log-detransformed standard deviations: 23.4–71.7) μM versus 21.6 (11.0–42.3) μM in a matched control animal group (P = 0.037). We also observed perivascular LPA immunohistochemical staining in lungs of hypoxic rats colocalized with the secreted lysophospholipase D autotaxin (ATX). Moreover, ATX colocalized with mast cell tryptase, suggesting implication of these cells in perivascular LPA production. Hypoxic rat lungs expressed more ATX transcripts (2.4-fold) and more transcripts of proteins implicated in cell migration: β2 integrin (1.74-fold), intracellular adhesion molecule 1 (ICAM-1; 1.84-fold), and αM integrin (2.70-fold). Serum from the hypoxic group of animals had significantly higher chemoattractant properties toward rat primary lung fibroblasts, and this increase in cell migration could be prevented by the LPA receptor 1 and 3 antagonists. LPA also increased adhesive properties of human pulmonary artery endothelial cells as well as those of human peripheral blood mononuclear cells, via the activation of LPA receptor 1 or 3 followed by the stimulation of gene expression of ICAM-1, β−1, E-selectin, and vascular cell adhesion molecule integrins. In conclusion, chronic hypoxia increases circulating and tissue levels of LPA, which might induce fibroblast migration and recruitment of mononuclear cells in pulmonary vasculature, both of which contribute to pulmonary vascular remodeling.

Hydrogen Peroxide and Sodium Transport in the Lung and Kidney.

Renal and lung epithelial cells are exposed to some significant concentrations of H2O2. In urine it may reach 100 μM, while in the epithelial lining fluid in the lung it is estimated to be in micromolar to tens-micromolar range. Hydrogen peroxide has a stimulatory action on the epithelial sodium channel (ENaC) single-channel activity. It also increases stability of the channel at the membrane and slows down the transcription of the ENaC subunits. The expression and the activity of the channel may be inhibited in some other, likely higher, oxidative states of the cell. This review discusses the role and the origin of H2O2 in the lung and kidney. Concentration-dependent effects of hydrogen peroxide on ENaC and the mechanisms of its action have been summarized. This review also describes outlooks for future investigations linking oxidative stress, epithelial sodium transport, and lung and kidney function.

Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPγS revealed a class of binding sites with an apparent K d value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Gαi-3, Gα0, Gαs, and Gαq were not increased after aldosterone. The patterns of small M r G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [α-32P]GTP, in control membranes, showed either no labeling or a faint band of M r 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of M r 59.5 kDa.The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPgammaS revealed a class of binding sites with an apparent Kd value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Galphai-3, Galpha0, Galphas, and Galphaq were not increased after aldosterone. The patterns of small Mr G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [alpha-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of Mr 59.5 kDa.

Rosmarinic acid potentiates carnosic acid induced apoptosis in lung fibroblasts

Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA) and rosmarinic acid (RA) was reported to cure bleomycin-(BLM)-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF) viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively) treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy) partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vitamine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts.

Human bone morphogenetic protein-7 does not counteract aristolochic acid-induced renal toxicity

Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)‐7 in several models of renal fibrosis, we investigated the putative effect of rhBMP‐7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo. In vitro, the toxicity of AA on renal tubular cells was demonstrated by an increase in vimentin as well as a decrease in β‐catenin expressions, reflecting a dedifferentiation process. Increased fibronectin and interleukin‐6 levels were measured in the supernatants. Enhanced α‐SMA mRNA levels associated to decreased E‐cadherin mRNA levels were also measured. Incubation with rhBMP‐7 only prevented the increase in vimentin and the decrease in β‐catenin expressions. In vivo, in a rat model of AA nephropathy, severe tubulointerstitial lesions induced by AA after 10 and 35 days (collagen IV deposition and tubular atrophy), were not prevented by the rhBMP‐7 treatment. Similarly, rhBMP‐7 did not ameliorate the significant increase in urinary concentrations of transforming growth factor‐β. In summary, our in vitro data demonstrated a poor beneficial effect of rhBMP‐7 to reverse cell toxicity while, in vivo, there was no beneficial effect of rhBMP‐7. Therefore, further investigations are needed to confirm the exact role of BMP‐7 in progressive chronic kidney disease. Copyright

DUOX1-mediated hydrogen peroxide release regulates sodium transport in H441 bronchiolar epithelial cells

Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase‐1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H2O2 is instrumental for the sodium channel upregulation observed in H441 cells.