Lipopolysaccharide modifies amiloride-sensitive Na+ transport processes across human airway cells: role of mitogen-activated protein kinases ERK 1/2 and 5

Bacterial lipopolysaccharides (LPS) are potent inducers of proinflammatory signaling pathways via the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), causing changes in the processes that control lung fluid homeostasis and contributing to the pathogenesis of lung disease. In human H441 airway epithelial cells, incubation of cells with 15 µg ml−1 LPS caused a significant reduction in amiloride-sensitive Isc from 15 ± 2 to 8 ± 2 µA cm−2 (p = 0.01, n = 13) and a shift in IC50 amiloride of currents from 6.8 × 10−7 to 6.4 × 10−6 M. This effect was associated with a decrease in the activity of 5 pS, highly Na+ selective, amiloride-sensitive <1 µM channels (HSC) and an increase in the activity of ∼18 pS, nonselective, amiloride-sensitive >10 µM cation channels (NSC) in the apical membrane. LPS decreased αENaC mRNA and protein abundance, inferring that LPS inhibited αENaC gene expression. This correlated with the decrease in HSC activity, indicating that these channels, but not NSCs, were comprised of at least αENaC protein. LPS increased NF-κB DNA binding activity and phosphorylation of extracellular signal-related kinase (ERK)1/2, but decreased phosphorylation of ERK5 in H441 cells. Pretreatment of monolayers with PD98059 (20 µM) inhibited ERK1/2 phosphorylation, promoted phosphorylation of ERK5, increased αENaC protein abundance, and reversed the effect of LPS on Isc and the shift in amiloride sensitivity. Inhibitors of NF-κB activation were without effect. Taken together, our data indicate that LPS acts via ERK signaling pathways to decrease αENaC transcription, reducing HSC/ENaC channel abundance, activity, and transepithelial Na+ transport in H441 airway epithelial cells

Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes

A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis

A novel vasopressin-induced transcript promotes MAP kinase activation and ENaC downregulation

In the principal cell of the renal collecting duct, vasopressin regulates the expression of a gene network responsible for sodium and water reabsorption through the regulation of the water channel and the epithelial sodium channel (ENaC). We have recently identified a novel vasopressin-induced transcript (VIT32) that encodes for a 142 amino acid vasopressin-induced protein (VIP32), which has no homology with any protein of known function. The Xenopus oocyte expression system revealed two functions: (i) when injected alone, VIT32 cRNA rapidly induces oocyte meiotic maturation through the activation of the maturation promoting factor, the amphibian homolog of the universal M phase trigger Cdc2/cyclin; and (ii) when co-injected with the ENaC, VIT32 cRNA selectively downregulates channel activity, but not channel cell surface expression. In the kidney principal cell, VIP32 may be involved in the downregulation of transepithelial sodium transport observed within a few hours after vasopressin treatment. VIP32 belongs to a novel gene family ubiquitously expressed in oocyte and somatic cells that may be involved in G to M transition and cell cycling

Post-anaesthesia pulmonary complications after use of muscle relaxants (POPULAR): a multicentre, prospective observational study

Background: Results from retrospective studies suggest that use of neuromuscular blocking agents during general anaesthesia might be linked to postoperative pulmonary complications. We therefore aimed to assess whether the use of neuromuscular blocking agents is associated with postoperative pulmonary complications. Methods: We did a multicentre, prospective observational cohort study. Patients were recruited from 211 hospitals in 28 European countries. We included patients (aged ≥18 years) who received general anaesthesia for any in-hospital procedure except cardiac surgery. Patient characteristics, surgical and anaesthetic details, and chart review at discharge were prospectively collected over 2 weeks. Additionally, each patient underwent postoperative physical examination within 3 days of surgery to check for adverse pulmonary events. The study outcome was the incidence of postoperative pulmonary complications from the end of surgery up to postoperative day 28. Logistic regression analyses were adjusted for surgical factors and patients' preoperative physical status, providing adjusted odds ratios (ORadj) and adjusted absolute risk reduction (ARRadj). This study is registered with ClinicalTrials.gov, number NCT01865513. Findings: Between June 16, 2014, and April 29, 2015, data from 22 803 patients were collected. The use of neuromuscular blocking agents was associated with an increased incidence of postoperative pulmonary complications in patients who had undergone general anaesthesia (1658 [7·6%] of 21 694); ORadj 1·86, 95% CI 1·53–2·26; ARRadj −4·4%, 95% CI −5·5 to −3·2). Only 2·3% of high-risk surgical patients and those with adverse respiratory profiles were anaesthetised without neuromuscular blocking agents. The use of neuromuscular monitoring (ORadj 1·31, 95% CI 1·15–1·49; ARRadj −2·6%, 95% CI −3·9 to −1·4) and the administration of reversal agents (1·23, 1·07–1·41; −1·9%, −3·2 to −0·7) were not associated with a decreased risk of postoperative pulmonary complications. Neither the choice of sugammadex instead of neostigmine for reversal (ORadj 1·03, 95% CI 0·85–1·25; ARRadj −0·3%, 95% CI −2·4 to 1·5) nor extubation at a train-of-four ratio of 0·9 or more (1·03, 0·82–1·31; −0·4%, −3·5 to 2·2) was associated with better pulmonary outcomes. Interpretation: We showed that the use of neuromuscular blocking drugs in general anaesthesia is associated with an increased risk of postoperative pulmonary complications. Anaesthetists must balance the potential benefits of neuromuscular blockade against the increased risk of postoperative pulmonary complications. Funding: European Society of Anaesthesiology