Essential Structural Features of TNF-α Lectin-like Domain Derived Peptides for Activation of Amiloride-Sensitive Sodium Current in A549 Cells

Hazemi, Parastoo; Tzotzos, Susan; Fischer, Bernhard; Andavan, Gowri Shankar Bagavananthem; Fischer, Hendrik; Pietschmann, Helmut; Lucas, Rudolf; Lemmens‐Gruber, Rosa

doi:10.1021/jm100767p

Cited by 26 publications

(61 citation statements)

References 34 publications

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“…We demonstrate that PKC-a inhibitors are able to blunt PLY-mediated arginase activation in vitro and can prevent PLY-induced pulmonary endothelial hyperpermeability in mice in vivo. We also observed a potent protective effect of the TNFderived tonoplast intrinsic protein (TIP) peptide (21)(22)(23)(24), which was shown to activate amiloride-sensitive sodium transport in alveolar epithelial and microvascular endothelial cells (24,25), the latter of which express the a subunit of the epithelial sodium channel ENaC (26,27). The TIP peptide was previously shown to protect from hyperpermeability induced by the cholesterol-binding pore-forming toxin listeriolysin in human lung microvascular endothelial cell (HL-MVEC) monolayers in an amiloride-sensitive and PKC-a-dependent manner (28).…”

mentioning

confidence: 86%

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Lucas

Yang

Gorshkov

et al. 2012

Am J Respir Cell Mol Biol

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Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-a activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-a inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-a activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI) 1/2 /arginase II (AII) 2/2 C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI 1/1 /AII 2/2 counterparts, indicating an important role for arginase I in PLYinduced endothelial hyperpermeability. These results identify PKC-a and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.Keywords: PKC; arginase; pneumococcus; pneumolysin; TNF Although severe pneumonia remains the leading cause of mortality worldwide in children aged less than 5 years (1), community-acquired pneumonia represents a major cause of morbidity and mortality mainly in elderly patients (2). Despite the use of potent antibiotics and aggressive intensive care support, the fatality rate associated with Streptococcus pneumoniae, accounting for 45% of all cases of community-acquired pneumonia, is approximately 20% (1, 2). Pulmonary permeability edema, a major complication of severe pneumonia characterized by endothelial hyperpermeability, can occur days after initiation of antibiotics therapy when tissues are sterile and the pneumonia is clearing and correlates with the presence of the bacterial virulence factor pneumolysin (PLY) (3, 4). This cytoplasmic hemolytic protein is released during bacterial lysis, as occurs after treatment with b-lactam antibiotics (5). PLYinduced lung injury was suggested to result from direct pneumotoxic effects on the alveolar-capillary barrier rather than from resident or recruited phagocytic cells (6).Upon binding of PLY to cholesterol in cell membranes, oligo...

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mentioning

confidence: 86%

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Lucas

Yang

Gorshkov

et al. 2012

Am J Respir Cell Mol Biol

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“…TNF-a, AP301, or AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] was added to the pipette solution in concentrations corresponding to their respective EC 50 values (8 and 60 nM) (Hazemi et al, 2010). For single-channel current measurements, 10 mM TEA was also added to the pipette solution to block the potassium channel, which has a large amplitude compared with the sodium channel.…”

Section: Electrophysiologymentioning

confidence: 99%

“…See Hazemi et al (2010) for structural details. The test compounds were studied at a concentration range of 3.5-240 nM.…”

Section: Test Compounds and Chemicalsmentioning

confidence: 99%

“…We previously showed that AP301 activates amiloride-sensitive current in A549 cells (Hazemi et al, 2010), a human lung AP301-Induced Activation of hENaC adenocarcinoma cell line endogenously expressing ENaC. The A549 cell line is a widely accepted cell model of ENaC research (Lazrak et al, 2000).…”

Section: Ap301mentioning

confidence: 99%

“…The lectin-like domain of tumor necrosis factor (TIP) and the TIP peptide, a cyclic peptide mimicking this domain (Lucas et al, 1994), effect ALF reabsorption due to their capacity to enhance amiloride-sensitive Na 1 current in alveolar epithelial cells (Fukuda et al, 2001;Elia et al, 2003;Braun et al, 2005;Vadász et al, 2008;Hamacher et al, 2010;Hazemi et al, 2010). The edema-reducing effect of the lectin-like domain involves binding to specific oligosaccharides such as N,N-diacetylchitobiose and branched trimannoses (Hribar et al, 1999;Braun et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

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Mechanism of Action of Novel Lung Edema Therapeutic AP301 by Activation of the Epithelial Sodium Channel

et al. 2013

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AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na 1 channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (a, b, g, and d) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. abg-or dbg-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).

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Glycosylation-dependent activation of epithelial sodium channel by solnatide

Shabbir

Tzotzos

Bedak

et al. 2015

Biochemical Pharmacology

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Essential Structural Features of TNF-α Lectin-like Domain Derived Peptides for Activation of Amiloride-Sensitive Sodium Current in A549 Cells

Cited by 26 publications

References 34 publications

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Protein Kinase C-α and Arginase I Mediate Pneumolysin-Induced Pulmonary Endothelial Hyperpermeability

Mechanism of Action of Novel Lung Edema Therapeutic AP301 by Activation of the Epithelial Sodium Channel

Glycosylation-dependent activation of epithelial sodium channel by solnatide

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