André Dagenais

Transforming Growth Factor-β1 Decreases Expression of the Epithelial Sodium Channel αENaC and Alveolar Epithelial Vectorial Sodium and Fluid Transport via an ERK1/2-dependent Mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-β1 (TGF-β1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-β1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-β1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-β1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-β1 also significantly decreased αENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the αENaC promoter in lung epithelial cells. The inhibitory effect of TGF-β1 on sodium uptake and αENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-β1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-β1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane αENaC expression mediated through an ERK1/2-dependent inhibition of the αENaC promoter activity.

Treatment of adult respiratory distress syndrome: plea for rescue therapy of the alveolar epithelium

Although much has been learned about the mechanisms leading to acute lung injury, mortality—which is mainly related to sepsis or associated non-pulmonary organ dysfunction1—remains high (around 50%) in patients with adult respiratory distress syndrome (ARDS).2-4 Many new therapeutic approaches aiming to control the inflammatory response accompanying ARDS have been evaluated.5 However, these treatments have had no impact on the mortality stemming from the disease.5 The lack of success with these new interventions is probably multifactorial.6 One possible explanation is that the appropriate patient population had not been enrolled for study.7 In this regard, it is also probably unrealistic to hope that a single treatment will modify the evolution of all ARDS patients who represent a heterogeneous population with very different severities of lung injury. Thus, it is unlikely that the efficacious treatment of patients with mild lung injury will be as efficient in patients with severe lung injury. Most of the treatments tested recently were targeted to control the inflammatory response.5 Although the development of lung injury is mainly dependent on aggression of endothelial cells by inflammatory cells,8 its severity and recovery also depend on epithelial cell function.9 In fact, the predominant pathological finding in acute lung injury is diffuse alveolar epithelial damage.10 11 Furthermore, physiologically it has been shown that the structure and function of the alveolar epithelium are important determinants of lung injury.12Finally, the alveolar epithelium is also the site of alveolar fluid reabsorption and plays a major role in the development of lung fibrosis associated with ARDS.13-15 Treatments aimed at improving epithelial function might therefore become one of the key elements to accelerate recovery and decrease the mortality of patients with ARDS. In this review we will emphasise the importance of modulating two …

Impact of β-adrenergic agonist on Na+ channel and Na+-K+-ATPase expression in alveolar type II cells

It has been shown that short-term (hours) treatment with beta-adrenergic agonists can stimulate lung liquid clearance via augmented Na+ transport across alveolar epithelial cells. This increase in Na+ transport with short-term beta-agonist treatment has been explained by activation of the Na+ channel or Na+-K+-ATPase by cAMP. However, because the effect of sustained stimulation (days) with beta-adrenergic agonists on the Na+ transport mechanism is unknown, we examined this question in cultured rat alveolar type II cells. Na+-K+-ATPase activity was increased in these cells by 10(-4) M terbutaline in an exposure time-dependent manner over 7 days in culture. This increased activity was also associated with an elevation in transepithelial current that was inhibited by amiloride. The enzymes activity was also augmented by continuous treatment with dibutyryl-cAMP (DBcAMP) for 5 days. This increase in Na+-K+-ATPase activity by 10(-4) M terbutaline was associated with an increased expression of alpha1-Na+-K+-ATPase mRNA and protein. beta-Adrenergic agonist treatment also enhanced the expression of the alpha-subunit of the epithelial Na+ channel (ENaC). These increases in gene expression were inhibited by propranolol. Amiloride also suppressed this long-term effect of terbutaline and DBcAMP on Na+-K+-ATPase activity. In conclusion, beta-adrenergic agonists enhance the gene expression of Na+-K+-ATPase, which results in an increased quantity and activity of the enzyme. This heightened expression is also associated with augmented ENaC expression. Although the cAMP system is involved, the inhibition of enhanced enzyme activity with amiloride suggests that increased Na+ entry at the apical surface plays a role in this process.

The alpha subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression.

Sodium reabsorption by the amiloride-sensitive sodium channel of epithelial cells plays a crucial role in the management of ionic composition and fluid volume in the body. In the respiratory system, sodium transport is involved in the clearance of pulmonary edema and of liquid secreted during fetal life at birth. We have cloned a partial cDNA of the α subunit of the mouse amiloride-sensitive sodium channel (αmENaC). In the region of comparison, the mouse α subunit shows 92% identity at the DNA level and 95% identity at the amino acid level with the rat sequence. The kidneys, lungs, and distal colon are major sites of expression of a 3.5-kb αmENaC mRNA. During mouse development, αmENaC transcripts appear late during gestation (d 17.5) and are expressed continuously thereafter. In the distal colon, a short 1.2-kb mRNA deleted of the 5′ part of the transcript is detected during gestation and is replaced gradually by the mature 3.5-kb transcript after birth. αmENaC and α1 Na+-K+-ATPase mRNAs have an expression profile that is modulated similarly during development for a given tissue. The expression ofαmENaC transcripts increases transiently in the lungs at birth(2.5-fold), as for α1 Na+-K+-ATPase mRNAs(1.5-fold), suggesting that the expression of several components of the sodium transport system is modulated in the lungs at that time. In the kidney, there is no significant increase of αmENaC and α1 Na+-K+-ATPase mRNAs in newborns.

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

BackgroundThe intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.MethodsPseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.ResultsThe infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.ConclusionsThese results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.

The regulation of amiloride-sensitive epithelial sodium channels by tumor necrosis factor-alpha in injured lungs and alveolar type II cells

Alveolar liquid clearance, which mainly depends on sodium transport in alveolar epithelial cells, is an important mechanism by which excess water in the alveoli is reabsorbed during the resolution of pulmonary edema. In this study, we examined the regulation of epithelial sodium channel (ENaC), the main contributor to sodium transport, during acute lung injury and the direct impact of tumor necrosis factor-alpha (TNF-alpha), one of the important cytokines in acute lung injury, on the ENaC regulation. During the development of pulmonary edema, the increases in the number of neutrophils and the levels of TNF-alpha in blood and bronchoalveolar lavage were seen. In parallel, the mRNA expression of the alpha-, beta- and gamma-ENaC subunits in the whole lung tissue was inhibited to 72.0, 47.8 and 53.9%, respectively. The direct exposure of rat alveolar type II cells to TNF-alpha inhibited the mRNA expression of alpha- and gamma-ENaC to 64.0 and 78.0%, but not that of the beta-ENaC. TNF-alpha also inhibited the ENaC function as indicated by the reduction of amiloride-sensitive current (control 4.4, TNF-alpha 1.9 microA/cm(2)). These data suggest that TNF-alpha may affect the pathophysiology of acute lung injury and pulmonary edema through the inhibition of alveolar liquid clearance and sodium transport.

Modulation of epithelial sodium channel activity by lipopolysaccharide in alveolar type II cells: involvement of purinergic signaling

Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na(+) channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a approximately 60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y(2) purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.

Detection in BHK cells of a precursor form for lamin A

Lamins are structural proteins found in the fibrous lamina underlining the nuclear envelope. In vitro translation of polyadenylated RNA or polysomes followed by immunoprecipitation with a serum raised against BHK nuclear matrix proteins showed that lamin A (72 kD) is synthesized as a high molecular weight precursor (74 kD) (Laliberté et al., J Cell Biol 98 (1984) 980) [23]. We have thus investigated the presence in BHK cells of this putative precursor by in vivo labelling with [35S]methionine and immunoprecipitation of lamin proteins. Short labelling times, ranging from 5 to 60 min reveal the presence of the 74 kD protein. Pulse-chase experiments indicate that the half-life of the precursor is about 60 min. On two-dimensional gel, the 74 kD protein is resolved in a cluster of isovariants between pH 7.4 and 6.6, which are generally slightly more alkaline than their counterparts in lamin A. These results indicate that lamin A is synthesized as a precursor of 74 kD; the long half-life further suggests that pre-lamin A might accumulate in some sort of cellular pool before undergoing post-transcriptional modification(s) to give the mature form of lamin A.

In situ localization of the major capsid protein during lytic infection by herpes simplex virus.

The intracellular localization of the major capsid protein (ICP5) of herpes simplex virus was studied during virogenesis. Except for a brief period at the onset of synthesis, this protein was found almost exclusively inside the nucleus. Its localization was not at random since 80% was tightly bound to the nuclear matrix as early as 4 h after infection. Discrete modifications of the fluorescence pattern occurred in an orderly fashion during the progression of the infection. Immunoelectron microscopic studies using Protein A-gold labelling demonstrated that this protein is synthesized on cytoskeleton-bound polyribosomes and accumulates in the central part of the nucleus where formation of viral capsids occurs; no gold particles were found in association with the peripheral chromatin or with the nucleolus.