bluuebunny/crossref_metadata_2025 huggingface dataset:
Perinatal regulation of the ClC-2 chloride channel in lung is mediated by Sp1 and Sp3
Authors: Chu Shijian , Blaisdell Carol J. , Liu Min-Zhi M. , Zeitlin Pamela L.
Published: December 2017

Mechanisms responsible for regulation of pulmonary epithelial chloride-channel expression in the perinatal period are under investigation to better understand normal lung development and airway disease pathogenesis. The ClC-2 epithelial chloride channel is regulated by changes in pH and volume and is most abundant in lung during fetal development. In this study, we identify and sequence the ClC-2 promoter, which is GC rich and lacks a TATA box. By construction of a series of promoter-luciferase constructs, a 67-bp GC box-containing sequence in the promoter is shown to be critical to ClC-2 expression in primary and immortalized fetal lung epithelial cells. Electrophoretic mobility shift assays and antibody supershifts demonstrate that the Sp1 and Sp3 transcription factors are expressed in fetal lung nuclei and interact with the GC box sequences in the promoter. Immunoblotting techniques demonstrate that Sp1 and Sp3 are perinatally downregulated in the lung with the same temporal sequence as ClC-2 downregulation. This work suggests that Sp1 and Sp3 activate ClC-2 gene transcription and that reduction in Sp1 and Sp3 at birth explains perinatal downregulation of ClC-2 in the lung.
Filaments of surfactant protein A specifically interact with corrugated surfaces of phospholipid membranes
Authors: Palaniyar Nades , Ridsdale Ross A. , Hearn Stephen A. , Heng Yew Meng , Ottensmeyer F. Peter , Possmayer Fred , Harauz George
Published: December 2017

Pulmonary surfactant, a mixture of lipids and surfactant proteins (SPs), plays an important role in respiration and gas exchange. SP-A, the major SP, exists as an octadecamer that can self-associate to form elongated protein filaments in vitro. We have studied here the association of purified bovine SP-A with lipid vesicle bilayers in vitro with negative staining with uranyl acetate and transmission electron microscopy. Native bovine surfactant was also examined by transmission electron microscopy of thinly sectioned embedded material. Lipid vesicles made from dipalmitoylphosphatidylcholine and egg phosphatidylcholine (1:1 wt/wt) generally showed a smooth surface morphology, but some large vesicles showed a corrugated one. On the smooth-surfaced vesicles, SP-As primarily interacted in the form of separate octadecamers or as multidirectional protein networks. On the surfaces of the striated vesicles, SP-As primarily formed regularly spaced unidirectional filaments. The mean spacing between adjacent striations and between adjacent filaments was 49 nm. The striated surfaces were not essential for the formation of filaments but appeared to stabilize them. In native surfactant preparations, SP-A was detected in the dense layers. This latter arrangement of the lipid bilayer-associated SP-As supported the potential relevance of the in vitro structures to the in vivo situation.
Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator
Authors: Bradbury Neil A. , Clark John A. , Watkins Simon C. , Widnell Christopher C. , Smith H. Skipper , Bridges Robert J.
Published: December 2017

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.
PKC role in mechanically induced Ca2+waves and ATP-induced Ca2+oscillations in airway epithelial cells
Authors: Woodruff Michael L. , Chaban Victor V. , Worley Christopher M. , Dirksen Ellen R.
Published: December 2017

Mechanical stimulation of airway epithelial cells generates the Ca2+mobilization messenger inositol 1,4,5-trisphosphate and the protein kinase (PK) C activator diacylglycerol. Inositol 1,4,5-trisphosphate diffuses through gap junctions to mediate intercellular communication of the mechanical stimulus (a “Ca2+wave”); the role that diacylglycerol-activated PKC might play in the response is unknown. Using primary cultures of rabbit tracheal cells, we show that 12- O-tetradecanoylphorbol 13-acetate- or 1,2-dioctanyl- sn-glycerol-induced activation of PKC slows the Ca2+wave, decreases the amplitude of induced intracellular free Ca2+concentration ([Ca2+]i) increases, and decreases the number of affected cells. The PKC inhibitors bisindolylmaleimide and Gö 6976 slowed the spread of the wave but did not change the number of affected cells. We show that ATP-induced [Ca2+]iincreases and oscillations, responses independent of intercellular communication, were inhibited by PKC activators. Bisindolylmaleimide decreased the amplitude of ATP-induced [Ca2+]iincreases and blocked oscillations, suggesting that PKC has an initial positive effect on Ca2+mobilization and then mediates feedback inhibition. PKC activators also reduced the [Ca2+]iincrease that followed thapsigargin treatment, indicating a PKC effect associated with the Ca2+release mechanism.
Regulation of lung manganese superoxide dismutase: species variation in response to lipopolysaccharide
Authors: Dirami Ghenima , Massaro Donald , Clerch Linda Biadasz
Published: December 2017

Lipopolysaccharide (LPS) treatment increases survival of rats, but not of mice, during hyperoxia. Manganese superoxide dismutase (Mn SOD) in the lung plays a critical role in LPS-induced tolerance to hyperoxia in rats. Therefore, we now compared the response of lung Mn SOD with treatment of mice and rats with LPS. LPS treatment of rats increased Mn SOD activity and protein concentration, did not change its specific activity, increased Mn SOD mRNA concentration 35-fold, and elevated Mn SOD synthesis 50% without changing general protein synthesis. LPS treatment of mice did not alter any of these parameters except for a 16-fold increase in Mn SOD mRNA concentration. Mn SOD translational efficiency (synthesis/mRNA concentration) was diminished 93% in rat lung and 76% in mouse lung by treatment with LPS. However, the absolute translational efficiency was twofold higher in lungs of LPS-treated rats than in lungs of LPS-treated mice. The failure of LPS to raise Mn SOD activity in mouse lungs is due, at least in part, to a smaller increase in Mn SOD mRNA and lower translational efficiency in LPS-treated mice than in LPS-treated rats.
Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin orPseudomonas aeruginosa
Authors: Skerrett Shawn J. , Martin Thomas R. , Chi Emil Y. , Peschon Jacques J. , Mohler Kendall M. , Wilson Christopher B.
Published: December 2017

To determine the roles of the type 1 tumor necrosis factor (TNF) receptor (TNFR1) in lung inflammation and antibacterial defense, we exposed transgenic mice lacking TNFR1 [TNFR1(−/−)] and wild-type control mice to aerosolized lipopolysaccharide or Pseudomonas aeruginosa. After LPS, bronchoalveolar lavage fluid (BALF) from TNFR1(−/−) mice contained fewer neutrophils and less macrophage inflammatory protein-2 than BALF from control mice. TNF-α, interleukin-1β, and total protein levels in BALF as well as tissue intercellular adhesion molecule-1 expression did not differ between the two groups. In contrast, lung inflammation and bacterial clearance after infection were augmented in TNFR1(−/−) mice. BALF from infected TNFR1(−/−) mice contained more neutrophils and TNF-α and less interleukin-1β and macrophage inflammatory protein-2 than that from control mice, but protein levels were similarly elevated in both groups. Lung inflammation and bacterial clearance were also augmented in mice lacking both TNF receptors. Thus TNFR1 facilitates neutrophil recruitment after inhalation of lipopolysaccharide, in part by augmenting chemokine induction. In contrast, TNFR1 attenuates lung inflammation in response to live bacteria but does not contribute to increased lung permeability and is not required for the elimination of P. aeruginosa.
Superficial buffer barrier and preferentially directed release of Ca2+in canine airway smooth muscle
Authors: Janssen Luke J. , Betti Pierre A. , Netherton Stuart J. , Walters Denise K.
Published: December 2017

We examined cytosolic concentration of Ca2+([Ca2+]i) in canine airway smooth muscle using fura 2 fluorimetry (global changes in [Ca2+]i), membrane currents (subsarcolemmal [Ca2+]i), and contractions (deep cytosolic [Ca2+]i). Acetylcholine (10−4M) elicited fluorimetric, electrophysiological, and mechanical responses. Caffeine (5 mM), ryanodine (0.1–30 μM), and 4-chloro-3-ethylphenol (0.1–0.3 mM), all of which trigger Ca2+-induced Ca2+release, evoked Ca2+transients and membrane currents but not contractions. The sarcoplasmic reticulum (SR) Ca2+-pump inhibitor cyclopiazonic acid (CPA; 10 μM) evoked Ca2+transients and contractions but not membrane currents. Caffeine occluded the response to CPA, whereas CPA occluded the response to acetylcholine. Finally, KCl contractions were augmented by CPA, ryanodine, or saturation of the SR and reduced when SR filling state was decreased before exposure to KCl. We conclude that 1) the SR forms a superficial buffer barrier dividing the cytosol into functionally distinct compartments in which [Ca2+]iis regulated independently; 2) Ca2+-induced Ca2+release is preferentially directed toward the sarcolemma; and 3) there is no evidence for multiple, pharmacologically distinct Ca2+pools.
Inhibition of β-adrenergic-dependent alveolar epithelial clearance by oxidant mechanisms after hemorrhagic shock
Authors: Modelska K. , Matthay M. A. , Brown L. A. S. , Deutch E. , Lu L. N. , Pittet J. F.
Published: December 2017

Endogenous release of catecholamines is an important mechanism that can prevent alveolar flooding after brief but severe hemorrhagic shock. The objective of this study was to determine whether this catecholamine-dependent mechanism upregulates alveolar liquid clearance after prolonged hemorrhagic shock. Rats were hemorrhaged to a mean arterial pressure of 30–35 mmHg for 60 min and then resuscitated with a 4% albumin solution. Alveolar liquid clearance was measured 5 h later as the concentration of protein in the distal air spaces over 1 h after instillation of a 5% albumin solution into one lung. There was no upregulation of alveolar liquid clearance after prolonged hemorrhagic shock and fluid resuscitation despite a significant increase in plasma epinephrine levels. The intravenous or intra-alveolar administration of exogenous catecholamines did not upregulate alveolar liquid clearance. In contrast, catecholamine-mediated upregulation of alveolar liquid clearance was restored either by depletion of neutrophils with vinblastine, by the normalization of the concentration of reduced glutathione in the alveolar epithelial lining fluid by N-acetylcysteine, or by the inhibition of the conversion from xanthine dehydrogenase to xanthine oxidase. These experiments provide the first in vivo evidence that a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the upregulation of alveolar fluid clearance by catecholamines in the absence of a major alteration in paracellular permeability to protein after prolonged hemorrhagic shock.
Differential expression of VEGF mRNA splice variants in newborn and adult hyperoxic lung injury
Authors: Watkins Richard H. , D’Angio Carl T. , Ryan Rita M. , Patel Alka , Maniscalco William M.
Published: December 2017

Lung development and repair of hyperoxic injury require closely regulated growth and regeneration of alveolar capillaries. Vascular endothelial growth factor (VEGF), a mitogen for endothelial cells, is expressed by alveolar epithelial cells. Alternative splicing of VEGF mRNA results in isoforms of varying mitogenicity and solubility. We examined changes in the proportions of the VEGF splice variant mRNAs in rabbit lung development and in control, oxygen-injured, and recovering newborn and adult rabbit lungs. The proportion of the 189-amino acid VEGF mRNA, which codes for an isoform that binds to the extracellular matrix, increased fivefold during development (from 8% of total VEGF message at 22 days gestation to 40% in 10-day newborn lungs; P < 0.001). During neonatal oxygen injury, its expression declined from 38 to 8% of VEGF message ( P < 0.002) and returned to the control value in recovery. A similar pattern was observed in adults. VEGF protein in lung lavage fluid increased slightly during hyperoxia, declined to barely detectable levels at the 50% lethal dose time point, and increased 10-fold (newborn) or up to 40-fold (adult) in recovering animals. We conclude that alternative splicing may have important roles in the regulation of VEGF activity in developing and injured lungs.
A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor
Authors: Kaplan Feige , Ledoux Pierre , Kassamali Ferhat Q. , Gagnon Stéphane , Post Martin , Koehler David , Deimling Julie , Sweezey Neil B.
Published: December 2017

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.
NO causes perinatal pulmonary vasodilation through K+-channel activation and intracellular Ca2+release
Authors: Saqueton Connie B. , Miller Robert B. , Porter Valerie A. , Milla Carlos E. , Cornfield David N.
Published: December 2017

Evidence suggests that nitric oxide (NO) causes perinatal pulmonary vasodilation through K+-channel activation. We hypothesized that this effect worked through cGMP-dependent kinase-mediated activation of Ca2+-activated K+channel that requires release of intracellular Ca2+from a ryanodine-sensitive store. We studied the effects of 1) K+-channel blockade with tetraethylammonium, 4-aminopyridine, a voltage-dependent K+-channel blocker, or glibenclamide, an ATP-sensitive K+-channel blocker; 2) cyclic nucleotide-sensitive kinase blockade with either KT-5823, a guanylate-sensitive kinase blocker, or H-89, an adenylate-sensitive kinase blocker; and 3) blockade of intracellular Ca2+release with ryanodine on NO-induced pulmonary vasodilation in acutely prepared late-gestation fetal lambs. N-nitro-l-arginine, a competitive inhibitor of endothelium-derived NO synthase, was infused into the left pulmonary artery, and tracheotomy was placed. The animals were ventilated with 100% oxygen for 20 min, followed by ventilation with 100% oxygen and inhaled NO at 20 parts/million (ppm) for 20 min. This represents the control period. In separate protocols, the animals received an intrapulmonary infusion of the different blockers and were ventilated as above. Tetraethylammonium ( n = 6 animals) and KT-5823 ( n = 4 animals) attenuated the response, whereas ryanodine ( n = 5 animals) blocked NO-induced perinatal pulmonary vasodilation. 4-Aminopyridine ( n = 5 animals), glibenclamide ( n = 5 animals), and H-89 ( n = 4 animals) did not affect NO-induced pulmonary vasodilation. We conclude that NO causes perinatal pulmonary vasodilation through cGMP-dependent kinase-mediated activation of Ca2+-activated K+channels and release of Ca2+from ryanodine-sensitive stores.
Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60src
Authors: Garcia Joe G. N. , Verin Alexander D. , Schaphorst Kane , Siddiqui Rafat , Patterson Carolyn E. , Csortos Csilla , Natarajan Viswanathan
Published: December 2017

Inflammatory diseases of the lung are characterized by increases in vascular permeability and enhanced leukocyte infiltration, reflecting compromise of the endothelial cell (EC) barrier. We examined potential molecular mechanisms that underlie these alterations and assessed the effects of diperoxovanadate (DPV), a potent tyrosine kinase activator and phosphatase inhibitor, on EC contractile events. Confocal immunofluorescent microscopy confirmed dramatic increases in stress-fiber formation and colocalization of EC myosin light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent with activation of the endothelial contractile apparatus. DPV produced significant time-dependent increases in MLC phosphorylation that were significantly attenuated but not abolished by EC MLCK inhibition with KT-5926. Pretreatment with the Rho GTPase-inhibitory C3exotoxin completely abolished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC phosphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic time-dependent tyrosine phosphorylation of the kinase in association with increased MLCK activity and a stable association of MLCK with the p85 actin-binding protein cortactin and p60src. Translocation of immunoreactive cortactin from the cytosol to the cytoskeleton was noted after DPV in concert with cortactin tyrosine phosphorylation. These studies indicate that DPV activates the endothelial contractile apparatus in a Rho GTPase-dependent fashion and suggests that p60src-induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.
Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function
Authors: Stevens Troy , Creighton Judy , Thompson W. Joseph
Published: December 2017

Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells (PAECs); however, the mechanisms responsible for this intrinsic feature of PMVECs are unknown. Because cAMP improves endothelial barrier function, we hypothesized that differences in enzyme regulation of cAMP synthesis and/or degradation uniquely establish an elevated content in PMVECs. PMVECs possessed 20% higher basal cAMP concentrations than did PAECs; however, increased content was accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs, responsiveness to β-adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and responsiveness to phosphodiesterase inhibition (rolipram) was increased compared with those in PAECs. Although both types of endothelial cells express calcium-inhibited adenylyl cyclase, constitutive PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at elevated levels compared with PAECs, which likely contribute to enhanced microvascular barrier function.
Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1β
Authors: Hakonarson Hakon , Carter Carrie , Maskeri Neil , Hodinka Richard , Grunstein Michael M.
Published: December 2017

An important interplay exists between specific viral respiratory pathogens, most commonly rhinovirus (RV), and altered airway responsiveness in the development and exacerbations of asthma. Given that RV infection reportedly induces the release of various cytokines in different cell types and that the reported effects of RV on airway smooth muscle (ASM) responsiveness are highly comparable to those obtained in ASM exposed to the proinflammatory cytokine interleukin (IL)-1β, this study examined whether RV (serotype 16)-mediated pertubations in ASM responsiveness are mechanistically coupled to altered induced expression and action of IL-1β in RV-exposed isolated rabbit and human ASM tissue and cultured cells. Relative to control tissues, ASM inoculated with RV exhibited significantly increased maximal isometric contractility to ACh ( P < 0.01) and attenuated relaxation to isoproterenol ( P < 0.005). In extended studies, we found that 1) the RV-induced changes in ASM responsiveness were ablated by pretreating the tissues with the IL-1 recombinant human receptor antagonist; 2) in contrast to their respective controls, RV-inoculated ASM tissue and cultured cells exhibited progressively induced expression of IL-1β mRNA and elaboration of IL-1β protein at 6 and 24 h after viral exposure; and 3) the latter effect of RV was inhibited in the presence of a monoclonal antibody to intercellular adhesion molecule-1, the endogenous receptor for most RV. Collectively, these observations provide new evidence demonstrating that “pro-asthmatic-like” pertubations in agonist responsiveness elicited in RV-exposed ASM are largely attributed to the induced autologous expression and autocrine action of IL-1β in the virus-infected ASM.
Mechanical strain-induced posttranscriptional regulation of fibronectin production in fetal lung cells
Authors: Mourgeon Eric , Xu Jing , Tanswell A. Keith , Liu Mingyao , Post Martin
Published: December 2017

We have shown that intermittent mechanical strain, simulating fetal breathing movements, stimulated fetal rat lung cell proliferation. Because normal lung growth requires proper coordination between cell proliferation and extracellular matrix remodeling, we investigated the effect of strain on fibronectin metabolism. Organotypic cultures of fetal rat lung cells, subjected to intermittent strain, showed increased fibronectin content in the culture media. Fibronectin-degrading activity in media from strained cells was similar to that of static cultures. Northern analysis revealed that strain inhibited fibronectin mRNA accumulation seen during static culture. Synthesis of fibronectin, determined by metabolic labeling, was increased by strain despite lower mRNA levels or presence of actinomycin D. This increase was not mediated via a rapamycin-sensitive mechanism. Strain stimulated prelabeled fibronectin secretion even in the presence of cycloheximide. These results suggest that strain differentially regulates fibronectin production of fetal lung cells at the transcriptional and posttranscriptional levels. Mechanical strain increases soluble fibronectin content by stimulating its synthesis and secretion without increasing fibronectin message levels.
Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells
Authors: Vepa Suryanarayana , Scribner William M. , Parinandi Narasimham L. , English Denis , Garcia Joe G. N. , Natarajan Viswanathan
Published: December 2017

Reactive oxygen species (ROS) are implicated in the pathophysiology of several vascular disorders including atherosclerosis. Although the mechanism(s) of ROS-induced vascular damage remains unclear, there is increasing evidence for ROS-mediated modulation of signal transduction pathways. Exposure of bovine pulmonary artery endothelial cells to hydrogen peroxide (H2O2) enhanced tyrosine phosphorylation of 60- to 80- and 110- to 130-kDa cellular proteins, which were determined by immunoprecipitation with specific antibodies focal adhesion kinase (p125FAK) and paxillin (p68). Brief exposure of cells to a relatively high concentration of H2O2(1 mM) resulted in a time- and dose-dependent tyrosine phosphorylation of FAK, which reached maximum levels within 10 min (290% of basal levels). Cytoskeletal reorganization as evidenced by the appearance of actin stress fibers preceded H2O2-induced tyrosine phosphorylation of FAK, and the microfilament disruptor cytochalasin D also attenuated the tyrosine phosphorylation of FAK. Treatment of BPAECs with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM attenuated H2O2-induced increases in intracellular Ca2+but did not show any consistent effect on H2O2-induced tyrosine phosphorylation of FAK. Several tyrosine kinase inhibitors, including genistein, herbimycin, and tyrphostin, had no detectable effect on tyrosine phosphorylation of FAK but attenuated the H2O2-induction of mitogen-activated protein kinase activity. We conclude that H2O2-induced increases in FAK tyrosine phosphorylation may be important in H2O2-mediated endothelial cell activation.
ERK activation protects against DNA damage and apoptosis in hyperoxic rat AEC2
Authors: Buckley S. , Driscoll B. , Barsky L. , Weinberg K. , Anderson K. , Warburton D.
Published: December 2017

The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L714–L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-d-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1β-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-d-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 μM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.
Efficient killing of inhaled bacteria in ΔF508 mice: role of airway surface liquid composition
Authors: McCray Paul B. , Zabner Joseph , Jia Hong Peng , Welsh Michael J. , Thorne Peter S.
Published: December 2017

Cystic fibrosis mice have been generated by gene targeting but show little lung disease without repeated exposure to bacteria. We asked if murine mucosal defenses and airway surface liquid (ASL) Cl−were altered by the ΔF508 cystic fibrosis transmembrane conductance regulator mutation. Naive ΔF508 −/− and +/− mice showed no pulmonary inflammation and after inhaled Pseudomonas aeruginosa had similar inflammatory responses and bacterial clearance rates. We therefore investigated components of the innate immune system. Bronchoalveolar lavage fluid from mice killed Escherichia coli, and the microbicidal activity was inhibited by NaCl. Because β-defensins are salt-sensitive epithelial products, we looked for pulmonary β-defensin expression. A mouse homolog of human β-defensin-1 (termed “MBD-1”) was identified; the mRNA was expressed in the lung. Using a radiotracer technique, ASL volume and Cl−concentration ([Cl−]) were measured in cultured tracheal epithelia from normal and ΔF508 −/− mice. The estimated ASL volume was similar for both groups. There were no differences in ASL [Cl−] in ΔF508 −/− and normal mice (13.8 ± 2.6 vs. 17.8 ± 5.6 meq/l). Because ASL [Cl−] is low in normal and mutant mice, salt-sensitive antimicrobial factors, including MBD-1, may be normally active.
Na+-K+-ATPase gene regulation by glucocorticoids in a fetal lung epithelial cell line
Authors: Chalaka Sridar , Ingbar David H. , Sharma Renuka , Zhau Zhong , Wendt Christine H.
Published: December 2017

The Na+pump, Na+-K+-ATPase, along with the Na+channel is essential for the removal of alveolar solute and fluid perinatally. Because Na+-pump mRNA and activity increase before birth and maternal glucocorticoids (GCs) influence Na+-K+-ATPase mRNA expression in fetal rat lung, we hypothesized that GCs increased Na+-K+-ATPase gene expression in a fetal lung epithelial cell line. After 24 h of exposure, dexamethasone increased the steady-state levels of Na+-K+-ATPase α1and β1mRNA in a fetal rat lung epithelial cell line in a dose-dependent fashion (10−7to 10−5M). The maximal increase in mRNA levels was 3.8-fold for α1and 2.8-fold for β1. The increase in mRNA was detected as early as 6 h for the β1-subunit and 18 h for the α1-subunit, and both peaked at 24 h. This gene upregulation was not due to increased mRNA stability based on mRNA half-life determination after actinomycin D inhibition. Transfection experiments with α1and β1promoter-reporter constructs demonstrated 3.2 ± 0.5- and 2.6 ± 0.4-fold increases, respectively, in promoter activity, consistent with transcriptional activation of the promoter-reporter construct. These findings, increased promoter activity with no change in stability, indicate that GCs increased Na+-K+-ATPase transcription in a fetal lung epithelial cell line.
α-Thrombin stimulates contraction of human bronchial rings by activation of protease-activated receptors
Authors: Hauck R. W. , Schulz C. , Schömig A. , Hoffman R. K. , Panettieri R. A.
Published: December 2017

In a variety of diseases, inflammation causes microvascular leakage and activates thrombin. Evidence suggests that thrombin increases cytosolic calcium and stimulates human airway smooth muscle (ASM) cell proliferation. The receptor subtypes, however, that mediate the effects of thrombin on ASM cell growth or calcium mobilization remain unknown. In this study, we postulate that thrombin, which activates specific protease-activated receptors (PARs), also stimulates contraction of isolated human bronchial rings. With the use of intact human bronchial rings, α-thrombin (1–20 U/ml) increased bronchial tone to 19 ± 3% of basal tone ( P = 0.008; n = 5 experiments) and represents 20 ± 8% of the maximum carbachol response. The EC50for thrombin-induced force generation was 12.2 U/ml (95% confidence interval 9.9–15.3 U/ml) and was not altered in bronchial rings that had the epithelium removed. In parallel experiments, a specific thrombin receptor-activating peptide (TRAP-14; 0.1–100 μmol/l) increased isometric tension to levels (14 ± 2%; P = 0.0005; n = 5 experiments) comparable to those rings stimulated with thrombin. To characterize the receptors that mediate thrombin effects on human ASM, the expression of PARs in cultured human ASM cells was analyzed by RT-PCR analysis with specific primers for PARs. In these cells, PAR1 (thrombin receptor), PAR2, and PAR3 were expressed at comparable levels. In other experiments using immunocytochemical staining with specific antibodies to PAR1 and PAR2, we showed that ASM in bronchial rings and cultured ASM cells express PAR1 and PAR2 proteins. Taken together, these studies suggest that α-thrombin, in a receptor-specific and dose-dependent manner, induces contraction of bronchial rings in vitro. In addition, cultured human ASM cells express mRNA of PAR1, PAR2, and PAR3 and express PAR1 and PAR2 protein. Further studies are needed to determine whether α-thrombin plays a role in stimulating bronchoconstriction in inflammatory airway diseases such as asthma and bronchiolitis obliterans.
EVE and beyond, retro and prospective insights
Authors: Rabinovitch Marlene
Published: December 2017

Our work has focused on the discovery that an endogenous vascular elastase (EVE) plays a pivotal role in the vascular changes associated with the development and progression of pulmonary hypertension. Recent studies have identified serum factors that stimulate transcription of this enzyme and have elucidated a signal transduction process involving activation of the mitogen-activated protein kinase pathway and nuclear expression of the transcription factor AML1. Proteases release and activate growth factors that are bound to the extracellular matrix and also induce, in a β3-integrin-dependent manner, the transcription of the gene for the matrix glycoprotein tenascin. Tenascin alters smooth muscle cell shape and facilitates the proliferative response to growth factors by clustering and activating growth factor receptors. In addition, breakdown products of elastin, elastin peptides, can upregulate the production of fibronectin, a glycoprotein that is critical to smooth muscle cell migration. The mechanisms regulating enhanced fibronectin production have recently been successfully targeted to prevent the development of intimal lesions.
Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation
Authors: Krymskaya Vera P. , Penn Raymond B. , Orsini Michael J. , Scott Pamela H. , Plevin Robin J. , Walker Trevor R. , Eszterhas Andrew J. , Amrani Yassine , Chilvers Edwin R. , Panettieri Reynold A.
Published: December 2017

Hypertrophy and hyperplasia of airway smooth muscle (ASM) are important pathological features that contribute to airflow obstruction in chronic severe asthma. Despite considerable research effort, the cellular mechanisms that modulate ASM growth remain unknown. Recent evidence suggests that mitogen-induced activation of phosphoinositide (PI)-specific phospholipase C (PLC) and PI-dependent calcium mobilization are neither sufficient nor necessary to stimulate human ASM proliferation. In this study, we identify phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM proliferation. Pretreatment of human ASM with the PtdIns 3-kinase inhibitors wortmannin and LY-294002 significantly reduced thrombin- and epidermal growth factor (EGF)-induced DNA synthesis (IC50∼10 nM and ∼3 μM, respectively). In separate experiments, wortmannin and LY-294002 markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase (pp70S6k) activation induced by stimulation of human ASM cells with EGF and thrombin but had no effect on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase (MAPK) activation. The specificity of wortmannin and LY-294002 was further suggested by the demonstrated inability of these compounds to alter thrombin-induced calcium transients, total PI hydrolysis, or basal cAMP levels. Transient expression of constitutively active PtdIns 3-kinase (p110*) activated pp70S6k, whereas a dominant-negative PtdIns 3-kinase (Δp85) blocked EGF- and thrombin-stimulated pp70S6kactivity. Collectively, these data suggest that activation of PtdIns 3-kinase is required for the mitogenic effect of EGF and thrombin in human ASM cells. Further investigation of the role of PtdIns 3-kinase may offer new therapeutic approaches in the treatment of diseases characterized by smooth muscle cell hyperplasia such as asthma and chronic bronchitis.
Regulation of alveolar macrophage-T cell interactions during Th1-type sarcoid inflammatory process
Authors: Agostini Carlo , Trentin Livio , Perin Alessandra , Facco Monica , Siviero Marta , Piazza Francesco , Basso Umberto , Adami Fausto , Zambello Renato , Semenzato Gianpietro
Published: December 2017

The accessory function of antigen-presenting cells depends on the presence of a number of costimulatory molecules, including members of the B7 family (CD80 and CD86) and the CD5 coligand CD72. The aim of this study was to evaluate the regulation of T cell-antigen-presenting cell costimulatory pathways in the lung of patients with a typical Th1-type reaction, i.e., sarcoidosis. Although normal alveolar macrophages (AMs) did not bear or bore low levels of costimulatory molecules, AMs from sarcoid patients with CD4 T-cell alveolitis upmodulated CD80, CD86, and CD72 and expressed high levels of interleukin (IL)-15; lymphocytes accounting for T-cell alveolitis expressed Th1-type cytokines [interferon (IFN)-γ and/or IL-2] and bore high levels of CD5 and CD28 but not of CD152 molecules. In vitro stimulation of AMs with Th1-related cytokines (IL-15 and IFN-γ) upregulated the expression of CD80 and CD86 molecules. However, stimulation with IL-15 induced the expression of Th1-type cytokines (IFN-γ) and CD28 on sarcoid T cells, suggesting a role for this macrophage-derived cytokine in the activation of the sarcoid T-cell pool. The hypothesis that CD80 and CD86 molecules regulate the sarcoid T-cell response was confirmed by the evidence that AMs induced a strong proliferation of T cells that was inhibited by pretreatment with CD80 and CD86 monoclonal antibodies. To account for these data, it is proposed that locally released cytokines provide AMs with accessory properties that contribute to the development of sarcoid T-cell alveolitis.
Effects of TNF-α and IL-1β on iron metabolism by A549 cells and influence on cytotoxicity
Authors: Smirnov Igor M. , Bailey Kirstin , Flowers Carol H. , Garrigues Ned W. , Wesselius Lewis J.
Published: December 2017

Extracellular iron, which is predominantly bound by transferrin, is present in low concentrations within alveolar structures, and concentrations are increased in various pulmonary disorders. Iron accumulation by cells can promote oxidative injury. However, the synthesis of ferritin stimulated by metal exposure for intracellular iron storage is normally protective. The cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β may alter iron metabolism by alveolar cells. In this study, we assessed the effects of TNF-α and IL-1β on iron metabolism with a cell line with properties of type 2 alveolar epithelial cells (A549) exposed to non-transferrin-bound (NTBI; FeSO4) or transferrin-bound (TBI) iron. In addition, we assessed the cytotoxicity of these exposures by measuring the cell accumulation of malondialdehyde (MDA), a product of lipid peroxidation, and cell death (MTT assay and lactate dehydrogenase release). A549 cells treated with NTBI or TBI in concentrations up to 40 μM accumulated iron and synthesized predominantly L-type ferritin without accumulation of MDA or cell death. Treatment of A549 cells with TNF-α (20 ng) or IL-1β (20 ng) decreased cell transferrin-receptor expression and induced synthesis of H-type ferritin. TNF-α and IL-1β decreased the uptake of TBI; however, the uptake of NTBI was increased. Both cytokines enhanced total ferritin synthesis (H plus L types) in response to iron treatments due to enhanced synthesis of H-type ferritin. Coexposure to TNF-α and NTBI, but not to TBI, induced MDA accumulation and greater cytotoxicity (MTT and lactate dehydrogenase release) than TNF-α alone. These findings indicate that TNF-α and IL-1β modulate iron uptake by A549 cells, with differing effects on TBI and NTBI, as well as on H-ferritin synthesis. Enhanced iron uptake induced by TNF-α and NTBI was also associated with increased cytotoxicity to A549 cells.
Ablation of the SERCA3 gene alters epithelium-dependent relaxation in mouse tracheal smooth muscle
Authors: Kao James , Fortner Christopher N. , Liu Lynne H. , Shull Gary E. , Paul Richard J.
Published: December 2017

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3), an isoform of the intracellular Ca2+pump that has been shown to mediate endothelium-dependent relaxation of vascular smooth muscle, is also expressed in tracheal epithelium. To determine its possible role in regulation of airway mechanical function, we compared tracheal contractility in gene-targeted mice deficient in SERCA3 (SERCA3−) with that in wild-type tracheae. Cumulative addition of ACh elicited concentration-dependent increases in isometric force (ED50= 2 μM, maximum force = 8 mN/mm2) that were identical in SERCA3−and wild-type tracheae. After ACh stimulation, substance P (SP) elicited a transient relaxation (42.6 ± 3.2%, n = 28) in both tracheae. However, the rate of relaxation was significantly ( P < 0.04, n = 9) more rapid in the wild-type [half-time ( t½) = 34.3 s] than in the SERCA3−( t½= 61.6 s) trachea. The SP relaxation was reduced by rubbing the trachea, indicative of epithelial cell involvement. This was verified using a perfused trachea preparation. SP in the outside medium had no effect, whereas SP in the perfusate bathing the epithelial side elicited a relaxation. Nitric oxide synthase inhibition (0.2 mM Nω-nitro-l-arginine) reduced the SP relaxation by 36.5 ± 12.5%, whereas the SP effect was abolished by eicosanoid inhibition (10 μM indomethacin). ATP also elicited an epithelium-dependent relaxation similar to SP but with a more rapid relaxation in the SERCA3−trachea than in the wild-type trachea. Our results indicate that SERCA3 gene ablation does not directly affect smooth muscle, which is consistent with the distribution of the isoform, but suggest that SERCA3 plays a role in epithelial cell modulation of airway smooth muscle function.
Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion
Authors: Lee Sheu-Ling , Wang Wei-Wei , Finlay Geraldine A. , Fanburg Barry L.
Published: December 2017

Our previous studies have shown that, through an active transport process, serotonin (5-HT) rapidly elevates[Formula: see text] formation, stimulates protein phosphorylation, and enhances proliferation of bovine pulmonary artery smooth muscle cells (SMCs). We presently show that 1 μM 5-HT also rapidly elevates phosphorylation and activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) 1 and ERK2 of SMCs, and the enhanced phosphorylation is blocked by the antioxidants Tiron, N-acetyl-l-cysteine (NAC), and Ginkgo biloba extract. Inhibition of MAP kinase with PD-98059 failed to block enhanced[Formula: see text] formation by 5-HT. Chinese hamster lung fibroblasts (CCL-39 cells), which demonstrate both 5-HT transporter and receptor activity, showed a similar response to 5-HT (i.e., enhanced mitogenesis, [Formula: see text]formation, and ERK1 and ERK2 phosphorylation and activation). Unlike SMCs, they also responded to 5-HT receptor agonists. We conclude that downstream signaling of MAP kinase is a generalized cellular response to 5-HT that occurs secondary to[Formula: see text] formation and may be initiated by either the 5-HT transporter or receptor depending on the cell type.
Asbestos exposure upregulates the adhesion of pleural leukocytes to pleural mesothelial cells via VCAM-1
Authors: Choe Nonghoon , Zhang Jun , Iwagaki Akitaka , Tanaka Shogo , Hemenway David R. , Kagan Elliott
Published: December 2017

This study was designed to assess the effects of in vitro and in vivo asbestos exposure on the adhesion of rat pleural leukocytes (RPLs) labeled with the fluorochrome calcein AM to rat pleural mesothelial cells (RPMCs). Exposure of RPMCs for 24 h to either crocidolite or chrysotile fibers (1.25–10 μg/cm2) increased the adhesion of RPLs to RPMCs in a dose-dependent fashion, an effect that was potentiated by interleukin-1β. These findings were not observed with nonfibrogenic carbonyl iron particles. Crocidolite and chrysotile plus interleukin-1β also upregulated vascular cell adhesion molecule-1 mRNA and protein expression in RPMCs, and the binding of RPL to asbestos-treated RPMCs was abrogated by anti-vascular cell adhesion molecule-1 antibody. PRLs exposed by intermittent inhalation to crocidolite for 2 wk manifested significantly greater binding to RPMCs than did RPLs from sham-exposed animals. The ability of asbestos fibers to upregulate RPL adhesion to RPMCs may play a role in the induction and/or potentiation of asbestos-induced pleural injury.
Mechanical ventilation of rat lung: effect on surfactant forms
Authors: Savov Jordan , Silbajoris R. , Young S. L.
Published: December 2017

Mechanical ventilation of the lung could affect surfactant turnover by alteration of its secretion, recycling, and degradation. In vitro studies of surfactant subfractions recoverable from lavage fluid have led to predictions about surfactant physiology in vivo that include morphological transformations. We used electron microscopy to study in situ lipid forms in alveoli of rat lungs after two ventilation strategies [15 min at pressures (cmH2O) of 20/0 or 20/10]. In control animals, 4% of the lipid profile area in the surface lining layer was myelin figures (MF), 14% was tubular myelin, 37% was vesicular forms (VF), and the remainder (45%) was hypophase. Compared with controls, the length-normalized sum of the lipid forms and the hypophase was two times as great in the lungs of the 20/0 group. MF were threefold higher in the 20/0 group and fivefold higher in the 20/10 group. VF doubled after ventilation at 20/0, but VF were the same as control after ventilation at 20/10. The results showed that a ventilation pattern of 20/0 compared with that of 20/10 group was associated with a significantly larger VF, suggesting an increased net production of these surfactant forms during a large tidal volume breathing pattern. These morphological results are consistent with published results using physical methods of fractionating lung lavage.