bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanical strain increases velocity and extent of shortening in cultured airway smooth muscle cells
Authors: Smith Paul G. , Roy Chaity , Dreger Jamie , Brozovich Frank
Published: December 2017

Abnormal mechanical stress on lung tissue is associated with increased mass and contractility of airway smooth muscle (ASM). We have reported that cultured ASM cells subjected to cyclic strain exhibit increased myosin light chain kinase (MLCK) and stress filaments. Increased MLCK may increase contractile velocity, whereas increased stress filaments could impede cell shortening by increasing the cell’s internal load. To study strain-induced changes in cell contractility, the time course of shortening of individual cells exposed to 90 mM KCl was recorded. Length vs. time plots revealed significantly greater maximal velocity of shortening in strain cells than control (no strain). This correlated with an increase in MLCK and myosin light chain phosphorylation measured in strain cells in separate experiments. The extent of cell shortening tended to be greater in the strain cells so that increased impedance to shortening was not detected. Mechanical stress may therefore increase the contractility of ASM by increasing the content of MLCK.
15-HETE-substituted diglycerides selectively regulate PKC isotypes in human tracheal epithelial cells
Authors: Alpert Stephen E. , Walenga Ronald W. , Mandal Atashi , Bourbon Nicole , Kester Mark
Published: December 2017

Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273–281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 μM 15-[3H]-HETE or 15-[14C]HETE for 1–2 h were stimulated with 1 nM to 1 μM platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 ± 5% decrease after 1 μM PAF) without a concomitant release of free intra- or extracellular 15-HETE.14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major14C band, identified as sn-1,2-15-[14C]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes α, βI, βII, δ, ε, and ζ. In vitro, cell-generated sn-1,2-15-[14C]HETE-DG selectively activated immunoprecipitated PKC-α and inhibited diacylglycerol-induced activation of PKC-α, -δ, -βI, and -βII. Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.
Endotoxin-specific NF-κB activation in pulmonary epithelial cells harboring adenovirus E1A
Authors: Keicho N. , Higashimoto Y. , Bondy G. P. , Elliott W. M. , Hogg J. C. , Hayashi S.
Published: December 2017

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-κB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-κB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c- jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-κB induction in these cells.
Hypoxia downregulates tropoelastin gene expression in rat lung fibroblasts by pretranslational mechanisms
Authors: Berk John L. , Massoomi Nima , Hatch Christine , Goldstein Ronald H.
Published: December 2017

Elastolytic lung injury disrupts cell barriers, flooding alveoli and producing regional hypoxia. Abnormal O2tensions may alter repair of damaged elastin fibers. To determine the effect of hypoxia on extravascular elastin formation, we isolated rat lung fibroblasts and cultured them under a variety of O2conditions. Hypoxia downregulated tropoelastin mRNA in a dose- and time-related fashion while upregulating glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The changes in tropoelastin gene expression were not due to cell toxicity as measured by chromium release and cell proliferation studies. Neither cycloheximide nor actinomycin D abrogated this effect. Hypoxia induced early decreases in tropoelastin mRNA stability; minor suppression of gene transcription occurred later. When returned to 21% O2, tropoelastin mRNA recovered to control levels in part by upregulating tropoelastin gene transcription. Taken together, these data indicate that hypoxia regulates tropoelastin gene expression and may alter repair of acutely injured lung.
SP-A enhances viral clearance and inhibits inflammation after pulmonary adenoviral infection
Authors: Harrod Kevin S. , Trapnell Bruce C. , Otake Kazuhisa , Korfhagen Thomas R. , Whitsett Jeffrey A.
Published: December 2017

Surfactant protein A (SP-A) is a member of the collectin family of host defense molecules expressed primarily in the epithelial cells of the lung. To determine the role of SP-A in pulmonary adenoviral infection, SP-A-deficient (SP-A −/−) mice were intratracheally infected with a replication-deficient recombinant adenovirus, Av1Luc1. Lung inflammation was markedly increased in SP-A −/− compared with SP-A +/+ mice and was associated with increased hemorrhage and epithelial cell injury. Polymorphonuclear cells in bronchoalveolar lavage fluid (BALF) were increased in SP-A −/− mice after administration of adenovirus. Coadministration of adenovirus and purified human SP-A ameliorated adenoviral-induced lung inflammation in SP-A −/− mice. Concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β were increased in BALF of SP-A −/− mice. Likewise, TNF-α, IL-6, macrophage inflammatory protein (MIP)-1α, monocyte chemotactic protein-1, and MIP-2 mRNAs were increased in lung homogenates from SP-A −/− mice 6 and 24 h after viral administration. Clearance of adenoviral DNA from the lung and uptake of fluorescent-labeled adenovirus by alveolar macrophages were decreased in SP-A −/− mice. SP-A enhances viral clearance and inhibits lung inflammation during pulmonary adenoviral infection, providing support for the importance of SP-A in antiviral host defense.
Inhaled nitric oxide protects against hyperoxia-induced apoptosis in rat lungs
Authors: Howlett Clare E. , Hutchison James S. , Veinot John P. , Chiu Aaron , Merchant Pradeep , Fliss Henry
Published: December 2017

Inhaled nitric oxide (NO), frequently administered in combination with hyperoxic gas mixtures, was recently shown to protect against the injurious consequences of prolonged hyperoxia. We investigated the possibility that this protective effect is attributable to the ability of NO to block pulmonary apoptosis. We show that rats exposed to 100% O2for 60 h develop severe lung injury consisting of pronounced vascular leak and alveolar apoptosis as inferred from the presence of positive terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA ladders in agarose gels and a decrease in constitutive procaspase-3 levels. However, the inclusion of NO (20 parts/million) in the hyperoxic gas mixture significantly attenuated both the vascular leak and apoptosis. NO reversed the hyperoxia-associated changes in the activity of the redox-sensitive transcription factors nuclear factor-κB, activator protein-1, and Sp1 after 24 h, lowered intercellular adhesion molecule-1 levels, and increased glutathione content. We therefore show, for the first time, that NO can protect against both hyperoxia-induced apoptosis and inflammation. The data suggest that this protection may occur at the transcriptional and caspase-activation levels.
PBA increases CFTR expression but at high doses inhibits Cl−secretion in Calu-3 airway epithelial cells
Authors: Loffing Johannes , Moyer Bryan D. , Reynolds Donna , Stanton Bruce A.
Published: December 2017

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl−secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl−secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na+-K+-ATPase. PBA reduced CFTR Cl−currents across the apical membrane but had no effect on Na+-K+-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl−secretion by inhibiting CFTR Cl−currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05–2 mM) had no effect on cAMP-stimulated Cl−secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl−secretion. However, concentrations >5 mM might reduce transepithelial Cl−secretion by serous cells in submucosal glands in individuals expressing wt CFTR.
Lung matrix incorporation of plasma fibronectin reduces vascular permeability in postsurgical bacteremia
Authors: Resnikoff Michael , Brien Thomas , Vincent Peter A. , Rotundo Robert F. , Lewis Edward , McKeown-Longo Paula J. , Saba Thomas M.
Published: December 2017

Plasma fibronectin (pFN) can incorporate into the lung extracellular matrix (ECM) as well as enhance hepatic cell phagocytic removal of bloodborne microparticulate debris that can contribute to lung vascular injury. Treatment of human pFN (hFN) with N-ethylmaleimide (NEM) blocks its ECM incorporation but not its ability to augment phagocytosis. Using hFN purified from fresh human plasma cryoprecipitate, we compared the effect of NEM-treated hFN versus normal hFN on lung transvascular protein clearance (TVPC) in postoperative bacteremic sheep to determine whether the ability of hFN to attenuate the increase in lung endothelial permeability required its ECM incorporation. Sheep with lung lymph fistulas were infused with a sublethal dose of Pseudomonas aeruginosa (5 × 108) 48 h after surgery. In the first study, sheep received either FN-rich human cryoprecipitate, FN-deficient cryoprecipitate, FN purified from cryoprecipitate (hFN), FN-deficient cryoprecipitate reconstituted with purified hFN, or the sterile saline diluent. In the second study, sheep received either 200 mg of purified hFN ( group I), 200 mg of NEM-treated hFN ( group II), or the saline diluent ( group III). In the first study, the increase in TVPC after bacterial challenge was attenuated by FN-rich cryoprecipitate, hFN, or reconstituted FN-deficient cryoprecipitate ( P < 0.05) but not by saline and FN-deficient cryoprecipitate. In the second study, TVPC increased by 2 h ( P < 0.05) and peaked over 4–8 h ( P < 0.05) at 380–420% above baseline in postoperative bacteremic sheep given the diluent ( group III). In contrast, intravenous infusion of hFN, but not of NEM-treated hFN, significantly ( P < 0.05) attenuated this increase of lung protein clearance. Thus the ability for the intravenously infused purified pFN to attenuate the increase in lung endothelial protein permeability in sheep during postsurgical bacteremia appears to require its ECM incorporation into the interstitial ECM of the lung.
Inhibition of PARS attenuates endotoxin-induced dysfunction of pulmonary vasorelaxation
Authors: Pulido Edward J. , Shames Brian D. , Selzman Craig H. , Barton Hazel A. , Banerjee Anirban , Bensard Denis D. , McIntyre Robert C.
Published: December 2017

Endotoxin (Etx) causes excessive activation of the nuclear repair enzyme poly(ADP-ribose) synthase (PARS), which depletes cellular energy stores and leads to vascular dysfunction. We hypothesized that PARS inhibition would attenuate injury to mechanisms of pulmonary vasorelaxation in acute lung injury. The purpose of this study was to determine the effect of in vivo PARS inhibition on Etx-induced dysfunction of pulmonary vasorelaxation. Rats received intraperitoneal saline or Etx ( Salmonella typhimurium; 20 mg/kg) and one of the PARS inhibitors, 3-aminobenzamide (3-AB; 10 mg/kg) or nicotinamide (Nic; 200 mg/kg), 90 min later. After 6 h, concentration-response curves were determined in isolated pulmonary arterial rings. Etx impaired endothelium-dependent (response to ACh and calcium ionophore) and -independent (sodium nitroprusside) cGMP-mediated vasorelaxation. 3-AB and Nic attenuated Etx-induced impairment of endothelium-dependent and -independent pulmonary vasorelaxation. 3-AB and Nic had no effect on Etx-induced increases in lung myeloperoxidase activity and edema. Lung ATP decreased after Etx but was maintained by 3-AB and Nic. Pulmonary arterial PARS activity increased fivefold after Etx, which 3-AB and Nic prevented. The beneficial effects were not observed with benzoic acid, a structural analog of 3-AB that does not inhibit PARS. Our results suggest that PARS inhibition with 3-AB or Nic improves pulmonary vasorelaxation and preserves lung ATP levels in acute lung injury.
Nitric oxide-induced reduction of lung cell and whole lung thioredoxin expression is regulated by NF-κB
Authors: Zhang Jianliang , Velsor Leonard W. , Patel Jawaharlal M. , Postlethwait Edward M. , Block Edward R.
Published: December 2017

We examined whether nitric oxide (NO)-induced inhibition of thioredoxin (Thx) expression is regulated by a mechanism mediated by a transcription factor, i.e., nuclear factor-κB (NF-κB), in cultured porcine pulmonary artery endothelial cells (PAEC) and in mouse lungs. Western blot analysis revealed that IκB-α content was reduced by 20 and 60% in PAEC exposed to 8.5 ppm NO for 2 and 24 h, respectively. NO exposure also caused significant reductions of cytosol fraction p65 and p52 content in PAEC. The nuclear fraction p65 and p52 contents were significantly reduced only in PAEC exposed to NO for 24 h. Exposure to NO resulted in a 50% reduction of p52 mRNA but not of the IκB-α subunit. DNA binding activity of the oligonucleotide encoding the NF-κB sequence in the Thxgene was significantly reduced in PAEC exposed to NO for 24 h. Exposure of mice to 10 ppm NO for 24 h resulted in a significant reduction of lung Thx and IκB-α mRNA and protein expression and in the oligonucleotide encoding Thx and NF-κB/DNA binding. These results 1) demonstrate that the effects of NO exposure on Thx expression in PAEC are comparable to those observed in intact lung and 2) suggest that reduced expression of the NF-κB subunit, leading to reduced NF-κB/DNA binding, is associated with the loss of Thx expression in PAEC and in intact mouse lungs.
Respiratory syncytial virus upregulates expression of the substance P receptor in rat lungs
Authors: Piedimonte Giovanni , Rodriguez Maria M. , King Katherine A. , McLean Stafford , Jiang Xiaobo
Published: December 2017

Respiratory syncytial virus (RSV) is a major respiratory pathogen in infants. The first goal of this study was to determine whether the infection following endotracheal inoculation of RSV in Fischer 344 rats results in increased inflammatory responses to substance P (SP) either released by capsaicin from sensory nerves or injected into the circulation. Five days after inoculation, the extravasation of Evans blue-labeled albumin after capsaicin or SP was significantly greater in RSV-infected airways than in pathogen-free controls. The peptide-degrading activity of the regulatory enzyme neutral endopeptidase was unaffected by RSV. However, SP(NK1) receptor mRNA levels increased fivefold in RSV-infected lungs, and the density of SP binding sites in the bronchial mucosa increased threefold. These data suggest that RSV makes the airways abnormally susceptible to the proinflammatory effects of SP by upregulating SP(NK1) receptor gene expression, thereby increasing the density of these receptors on target cells. This effect may contribute to the inflammatory reaction to the virus and could be a target for the therapy of RSV disease and its sequelae.
Spatial and temporal aspects of calcium sparks in porcine tracheal smooth muscle cells
Authors: Pabelick Christina M. , Prakash Y. S. , Kannan Mathur S. , Sieck Gary C.
Published: December 2017

Spontaneous, localized intracellular Ca2+concentration ([Ca2+]i) transients (Ca2+sparks) in skeletal, cardiac, and smooth muscle cells are thought to represent Ca2+release through ryanodine-receptor (RyR) channels. In porcine tracheal smooth muscle (TSM) cells, ACh induces propagating [Ca2+]ioscillations that also represent Ca2+release through RyR channels. We used real-time confocal imaging to examine the spatial and temporal relationships of Ca2+sparks to propagating [Ca2+]ioscillations in TSM cells. Ca2+sparks within an intracellular region displayed different spatial Ca2+distributions with every occurrence. The amplitudes of Ca2+sparks within a region were approximately integer multiples of the smallest response. However, across different regions, the attributes of Ca2+sparks varied considerably. Individual sparks were often grouped together and coupled across adjacent regions. Fusion of individual sparks produced large local elevations in [Ca2+]ithat occasionally triggered a propagating [Ca2+]iwave. The incidence of sparks was increased by ryanodine and caffeine but was unaffected by removal of extracellular Ca2+. Exposure to ACh triggered repetitive, propagating [Ca2+]ioscillations that always originated from foci with a high spark incidence. The [Ca2+]ioscillations disappeared with the removal of ACh, and Ca2+sparks reappeared. We conclude that agonist-induced [Ca2+]ioscillations represent a spatial and temporal integration of local Ca2+-release events through RyR channels in TSM cells.
Structural requirements for intracellular targeting of SP-C proprotein
Authors: Russo Scott J. , Wang Wenjing , Lomax Catherine A. , Beers Michael F.
Published: December 2017

Rat surfactant protein (SP) C is synthesized as a 194-amino acid proprotein that is proteolytically processed to a 35-amino acid mature form in subcellular compartments distal to the medial Golgi compartment. To identify domains of SP-C proprotein (proSP-C) necessary for endoplasmic reticulum translocation and for targeting to cytosolic processing compartments, we characterized expression patterns of heterologous SP-C fusion proteins in A549 lung epithelial cells and in the rat pheochromocytoma cell line PC-12. cDNA constructs were produced; these constructs encoded fusion proteins consisting of enhanced green fluorescent protein (EGFP) and wild-type proSP-C (EGFP/SP-C1–194), mature SP-C (EGFP/SP-C24–59), or progressive deletions of the NH2- or COOH-terminal flanking domains. By fluorescence microscopy, EGFP/SP-C1–194transfected into A549 cells was translocated and expressed in acidic cytoplasmic vesicles. By deletional analysis, a functional signal peptide was mapped to the domain Phe24to His59, whereas a motif for targeting to cytosolic vesicular compartments was localized to the NH2flanking domain Met10to Gln23. Truncations of the distal COOH terminus were retained in the endoplasmic reticulum/Golgi compartment; however, the COOH flanking region alone was insufficient for targeting. In PC-12 cells, EGFP/SP-C1–194was expressed in peripheral cytosolic vesicles, whereas EGFP/SP-C24–194and EGFP/SP-C24–59were each translocated but not targeted. We conclude that two domains in the proSP-C sequence are required for targeting: mature SP-C (Phe24to Leu58) contains a functional signal sequence active in epithelial and nonepithelial cells, whereas Met10to Gln23, but not the COOH flanking peptide, is required for targeting to late vesicular compartments.
Nucleotide-induced PMN adhesion to cultured epithelial cells: possible role of MUC1 mucin
Authors: Rounds Sharon , Likar Laura L. , Harrington Elizabeth O. , Kim K. Chul , Smeglin Anthony , Heins Karen , Parks Nancy
Published: December 2017

Accumulation of intraluminal polymorphonuclear leukocytes (PMN) is a hallmark of inflammatory diseases of the airways. Extracellular nucleotides stimulate PMN adhesion to human main pulmonary artery endothelial cells (HPAEC) by a purinoceptor-mediated mechanism. We investigated the effects of nucleotides on adhesion of freshly isolated human PMN to cultured human tracheobronchial epithelial cells (HBEC). We found that extracellular ATP and UTP were much less effective in stimulating PMN adhesion to HBEC compared with HPAEC, whereas the bacterial chemotactic peptide N-formyl-Met-Leu-Phe stimulated PMN adhesion to both cell types to an equal degree. We investigated several mechanisms that might account for decreased nucleotide-induced PMN adhesion to HBEC. The ectonucleotidase-resistant ATP analog adenosine 5′- O-(3-thiotriphosphate) was also ineffective in stimulating PMN adhesion to HBEC, indicating that degradation of ATP by ectonucleotidase(s) was not responsible for altered PMN adhesion. HBEC responded to ATP and UTP with increased intracellular calcium, indicating that these cells are capable of purinoceptor-mediated responses. We found that ATP and UTP also did not stimulate PMN adhesion to Chinese hamster ovary (CHO) cells, which had been stably transfected with the gene for hamster Muc1, a cell-associated mucin. However, ATP and UTP did stimulate adhesion of PMN to nontransfected CHO cells. These results suggested that MUC1 mucin modulates PMN adhesion to epithelium. We found that cultured HBEC expressed more mRNA and protein for MUC1 mucin than did HPAEC. We conclude that extracellular nucleotides are less effective in stimulating PMN adhesion to epithelial cells than to endothelial cells and that overexpression of hamster Muc1 mucin inhibits nucleotide-induced PMN adhesion to CHO cells.
Surfactant lipid uptake and metabolism by neonatal and adult type II pneumocytes
Authors: Griese Matthias , Beck Julia , Feuerhake Friedrich
Published: December 2017

Animal experiments suggest developmental changes in surfactant homeostasis. The uptake and metabolism of [3H]dipalmitoylphosphatidylcholine-labeled liposomes with a surfactant-like composition were evaluated in type II cells isolated from rats of different postnatal ages. The early part of the uptake process (0–60 min) was more rapid and reached higher levels in cells from 2-day-old rats than in those from 7-day-old, 14-day-old, or adult rats. Temperature independence of this initial phase, differences in response to trypsin-EDTA or neuraminidase treatment, and the dependency of increased neonatal uptake on the presence of phosphatidylglycerol in liposomes suggested binding as a major mechanism of cell-lipid interaction. Although a two to three times larger amount of lipid was associated with neonatal cells, the metabolism of phosphatidylcholine, indicated by a decrease in label in phosphatidylcholine and an accompanying increase in sphingomyelin, was significantly smaller in 2-day-old than in adult cells. These studies support the hypothesis that neonatal and adult cells may have differences in the interaction with alveolar phospholipids and in the metabolism of phosphatidylcholine.
Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals
Authors: Wu Weidong , Graves Lee M. , Jaspers Ilona , Devlin Robert B. , Reed William , Samet James M.
Published: December 2017

We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms responsible for metal-induced activation of ERK, we examined the effect of noncytotoxic exposures to As, Cu, V, or Zn on the kinases upstream of ERK in the epidermal growth factor (EGF) receptor signaling pathway. Western blotting using phospho-specific ERK1/2 antibody demonstrated the selective MEK1/2 inhibitor PD-98059 blocked metal-induced phosphorylation of ERK1/2. Meanwhile, Western blotting using a phospho-specific MEK1/2 antibody showed that these metals induce a rapid phosphorylation of MEK1/2. Kinase activity assays confirmed the activation of MEK1/2 by metal treatment. Immunoprecipitation studies demonstrated that As, Cu, V, or Zn induces EGF receptor phosphorylation. Furthermore, the EGF receptor-specific tyrosine kinase inhibitor (PD-153035) significantly blocked the phosphorylation of MEK1/2 initiated by metals. Interestingly, we observed low levels of Raf-1 activity that were not increased by metal exposure in these cells through kinase activity assay. Finally, transfection assays showed that MEK1/2 inhibition could inhibit trans-activation of Elk1, a transcription factor in the ERK pathway, in BEAS cells exposed to metals. Together, these data demonstrate that As, Cu, V, and Zn can activate the EGF receptor signaling pathway in BEAS cells and suggest that this mechanism may be involved in pulmonary responses to metal inhalation.
Fluid absorption related to ion transport in human airway epithelial spheroids
Authors: Pedersen Peter Steen , Holstein-Rathlou Niels-Henrik , Larsen Per Leganger , Qvortrup Klaus , Frederiksen Ole
Published: December 2017

Airway epithelium explants from cystic fibrosis (CF) patients and non-CF subjects formed monolayered spheres, with the apical ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. With the use of two microelectrodes, transepithelial potential difference and changes in potential difference in response to passage of current pulses were recorded, and epithelial resistance and the equivalent short-circuit current were calculated. Non-CF control potential difference and short-circuit current values were significantly lower than the CF values, and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 μl ⋅ cm−2 ⋅ h−1), 2) amiloride inhibited fluid absorption to a lower residual level in non-CF than in CF spheroids, 3) Cl−-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5) osmotic water permeabilities were equal in non-CF and CF spheroids (∼27 × 10−7cm ⋅ s−1 ⋅ atm−1).
Regulation of connective tissue growth factor expression by prostaglandin E2
Authors: Ricupero Dennis A. , Rishikof David C. , Kuang Ping-Ping , Poliks Christine F. , Goldstein Ronald H.
Published: December 2017

Transforming growth factor-β (TGF-β) stimulates α1(I) collagen mRNA synthesis in human lung fibroblasts through a mechanism that is partially sensitive to cycloheximide and that may involve synthesis of connective tissue growth factor (CTGF). Northern blot analyses indicate that TGF-β stimulates time- and dose-dependent increases in CTGF mRNA. In TGF-β-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold above baseline) occur at 6 h. The TGF-β-stimulated increase in CTGF mRNA was not blocked by cycloheximide. Nuclear run-on analysis indicates that TGF-β increases the CTGF transcription rate. The TGF-β-stimulated increases in CTGF transcription and steady-state levels of CTGF mRNA are attenuated in prostaglandin E2(PGE2)-treated fibroblasts. PGE2fails to attenuate luciferase activity induced by TGF-β in fibroblasts transfected with the TGF-β-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE2and insulin regulate α1(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of α1(I) collagen mRNA levels by TGF-β and PGE2may function through both CTGF-dependent and CTGF-independent mechanisms.
Vascular inflammation inhibits gene transfer to the pulmonary circulation in vivo
Authors: Tyler Robert C. , Fagan Karen A. , Unfer Robert C. , Gorman Cornelia , McClarrion Molly , Bullock Clayton , Rodman David M.
Published: December 2017

We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase ( CAT) or human placental alkaline phosphatase ( hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAPreporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 ± 8 and 86 ± 6% at 7 and 21 days, respectively, after monocrotaline ( P < 0.05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR ( TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury.
TGF-β3-null mutation does not abrogate fetal lung maturation in vivo by glucocorticoids
Authors: Shi Wei , Heisterkamp Nora , Groffen John , Zhao Jingsong , Warburton David , Kaartinen Vesa
Published: December 2017

Newborn transforming growth factor (TGF)-β3-null mutant mice exhibit defects of palatogenesis and pulmonary development. Glucocorticoids, which play a central role in fetal lung maturation, have been postulated to mediate their stimulatory effects on tropoelastin mRNA expression through TGF-β3 in cultured lung fibroblasts. In the present study, we analyzed the abnormally developed lungs in TGF-β3-null mutant mice and compared the effects of glucocorticoids on gene expression and lung morphology between TGF-β3 knockout and wild-type mice. Lungs of TGF-β3-null mutant mice on embryonic day 18.5 did not form normal saccular structures and had a thick mesenchyme between terminal air spaces. Moreover, the number of surfactant protein C-positive cells was decreased in TGF-β3-null mutant lungs. Interestingly, glucocorticoids were able to promote lung maturation and increased expression of both tropoelastin and fibronectin but decreased the relative number of surfactant protein C-positive cells in fetal lungs of both genotypes. This finding provides direct evidence that glucocorticoid signaling in the lung can use alternative pathways and can exert its effect without the presence of TGF-β3.
Alveolar sodium and liquid transport in mice
Authors: Icard Philippe , Saumon Georges
Published: December 2017

We have developed a simple isolated lung preparation for measurement of liquid and solute fluxes across mouse alveolar epithelium. Liquid instilled into air spaces was absorbed at the rate ( J w) of 3.7 ± 0.32 ml ⋅ h−1 ⋅ g dry lung wt−1. J w was significantly depressed by ouabain ( P< 0.001) and amiloride ( P < 0.001). Omission of glucose from the instillate or addition of the Na+-glucose cotransport inhibitor phloridzin did not affect J w. However, the low epithelial lining fluid glucose concentration (one-third that of plasma), the larger-than-mannitol permeability of methyl-α-d-glucopyranoside, and the presence of Na+-glucose cotransporter SGLT1 mRNA in mouse lung tissue suggest that there is a Na+-glucose cotransporter in the mouse alveolar-airway barrier. Isoproterenol stimulated J w (6.5 ± 0.45 ml ⋅ h−1 ⋅ g dry lung wt−1; P < 0.001), and this effect was blocked by amiloride, benzamil, ouabain, and the specific β2-adrenergic antagonist ICI-118551 but not by atenolol. Similar stimulation was obtained with terbutaline (6.4 ± 0.46 ml ⋅ h−1 ⋅ g dry lung wt−1). Na+ unidirectional fluxes out of air spaces varied in agreement with J w changes. Thus alveolar liquid absorption in mice follows Na+ transport via the amiloride-sensitive pathway, with little contribution from Na+-glucose cotransport, and is stimulated by β2-adrenergic agonists.
Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung
Authors: Parker Thomas A. , le Cras Timothy D. , Kinsella John P. , Abman Steven H.
Published: December 2017

Endothelial nitric oxide (NO) synthase (eNOS) produces NO, which contributes to vascular reactivity in the fetal lung. Pulmonary vasoreactivity develops during late gestation in the ovine fetal lung, during the period of rapid capillary and alveolar growth. Although eNOS expression peaks near birth in the fetal rat, lung capillary and distal air space development occur much later than in the fetal lamb. To determine whether lung eNOS expression in the lamb differs from the timing and pattern reported in the rat, we measured eNOS mRNA and protein by Northern and Western blot analyses and NOS activity by the arginine-to-citrulline conversion assay in lung tissue from fetal, newborn, and maternal sheep. Cellular localization of eNOS expression was determined by immunohistochemistry. eNOS mRNA, protein, and activity were detected in samples from all ages, and eNOS was expressed predominantly in the vascular endothelium. Lung eNOS mRNA expression increases from low levels at 70 days gestation to peak at 113 days and remains high for the rest of fetal life. Newborn eNOS mRNA expression does not change from fetal levels but is lower in the adult ewe. Lung eNOS protein expression in the fetus rises and peaks at 118 days gestation but decreases before birth. eNOS protein expression rises in the newborn period but is lower in the adult. Lung NOS activity also peaks at 118 days gestation in the fetus before falling in late gestation and remaining low in the newborn and adult. We conclude that the pattern of lung eNOS expression in the sheep differs from that in the rat and may reflect species-related differences in lung development. We speculate that the rise in fetal lung eNOS may contribute to the marked lung growth and angiogenesis that occurs during the same period of time.
TNF-α stimulates alveolar liquid clearance during intestinal ischemia-reperfusion in rats
Authors: Börjesson Anna , Norlin Andreas , Wang Xiangdong , Andersson Roland , Folkesson Hans G.
Published: December 2017

Intestinal ischemia-reperfusion commonly occurs in critically ill patients and may lead to the development of remote organ injury, frequently involving the lungs. In the present study, alveolar liquid clearance was studied in ventilated, anesthetized rats subjected to 45 min of intestinal ischemia followed by 3 h of reperfusion. An isosmolar 5% albumin solution was instilled into the lungs, and alveolar liquid clearance was measured from the increase in alveolar protein concentration as water was reabsorbed over 45 min. Intestinal ischemia-reperfusion resulted in a 76% increase in alveolar liquid clearance compared with the control value ( P< 0.05). The stimulated alveolar liquid clearance seen after intestinal ischemia-reperfusion was not inhibited by propranolol, indicating stimulation through a noncatecholamine-dependent pathway. Intestinal ischemia-reperfusion did not result in increased intracellular cAMP levels. Amiloride inhibited similar fractions in animals subjected to ischemia-reperfusion and control animals. Administration of a neutralizing polyclonal anti-tumor necrosis factor-α antibody before induction of intestinal ischemia completely inhibited the increased alveolar liquid clearance observed after intestinal ischemia-reperfusion. In conclusion, our results suggest that intestinal ischemia-reperfusion in rats leads to stimulation of alveolar liquid clearance and that this stimulation is mediated, at least in part, by a tumor necrosis factor-α-dependent mechanism.
Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins
Authors: van Wetering Sandra , van der Linden Abraham C. , van Sterkenburg Marianne A. J. A. , de Boer Willem I. , Kuijpers Astrid L. A. , Schalkwijk Joost , Hiemstra Pieter S.
Published: December 2017

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release α-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of α1-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of α1-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.
Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells
Authors: Heberlein Wolf , Wodopia Ralf , Bärtsch Peter , Mairbäurl Heimo
Published: December 2017

In oxygen-sensitive excitable cells, responses to hypoxia are initiated by membrane depolarization due to closing of the K channels that is thought to be mediated by a decrease in reactive oxygen species (ROS). Because the mechanisms of hypoxic inhibition of ion transport of alveolar epithelial cells (Planes C, Friedlander G, Loiseau A, Amiel C, and Clerici C. Am J Physiol Lung Cell Mol Physiol 271: L70–L78, 1996; Mairbäurl H, Wodopia R, Eckes S, Schulz S, and Bärtsch P. Am J Physiol Lung Cell Mol Physiol 273: L797–L806, 1997) are not yet understood, we tested the possible involvement of a hypoxia-induced change in ROS that might control transport activity. Transport was measured as86Rb and22Na uptake in A549 cells exposed to normoxia, hyperoxia, or hypoxia together with ROS donors and scavengers. H2O2< 1 mM did not affect transport, whereas 1 mM H2O2activated22Na uptake (+200%) but inhibited86Rb uptake (−30%). Also hyperoxia, aminotriazole plus menadione, and diethyldithiocarbamate inhibited86Rb uptake. N-acetyl-l-cysteine, diphenyleneiodonium, and tetramethylpiperidine- N-oxyl, used to reduce ROS, inhibited86Rb uptake, thus mimicking the hypoxic effects, whereas deferoxamine, superoxide dismutase, and catalase were ineffective. Also, hypoxic effects on ion transport were not prevented in the presence of H2O2, diethyldithiocarbamate, and N-acetyl-l-cysteine. These results indicate that ion transport of A549 cells is significantly affected by decreasing or increasing cellular ROS levels and that it is possible that certain species of ROS might mediate the hypoxic effects on ion transport of alveolar epithelial cells.
Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells
Authors: Zhao Dong-Mei , Xue Hai-Hui , Chida Kingo , Suda Takafumi , Oki Yutaka , Kanai Miho , Uchida Chiharu , Ichiyama Arata , Nakamura Hirotoshi
Published: December 2017

ATP induced a biphasic increase in the intracellular Ca2+concentration ([Ca2+]i), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca2+]ispike but only in the presence of extracellular calcium ([Formula: see text]). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5) P3] formation and UTP-induced [Ca2+]ispike, implying that EM perturbs Ca2+influx from the extracellular space rather than Ca2+release from intracellular Ca2+stores via the G protein-phospholipase C-Ins(1,4,5) P3pathway. A verapamil-sensitive, KCl-induced increase in [Ca2+]iand the Ca2+influx activated by Ca2+store depletion were insensitive to EM. 3′- O-(4-benzoylbenzoyl)-ATP evoked an[Formula: see text]-dependent [Ca2+]iresponse even in the presence of verapamil or the absence of extracellular Na+, and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X4as well as P2Y2, P2Y4, and P2Y6are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X4and P2Y2/P2Y4subtypes contribute to the ATP-induced [Ca2+]ispike and 2) EM selectively inhibits Ca2+influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.
Developmental changes in endothelin expression and activity in the ovine fetal lung
Authors: Dunbar Ivy D. , le Cras Timothy D. , Parker Thomas A. , Zenge Jeanne P. , Jakkula Malathi , Markham Neil E. , Kinsella John P. , Abman Steven H.
Published: December 2017

Mechanisms that regulate endothelin (ET) in the perinatal lung are complex and poorly understood, especially with regard to the role of ET before and after birth. We hypothesized that the ET system is developmentally regulated and that the balance of ETAand ETBreceptor activity favors vasoconstriction. To test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and adult sheep. Lung preproET-1 mRNA levels, tissue ET peptide levels, and cellular localization of ET-1 expression were determined by Northern blot analysis, peptide assay, and immunohistochemistry in distal lung tissue from fetal lambs between 70 and 140 days (term = 145 days), newborn lambs, and ewes. Lung mRNA expression for the ETAand ETBreceptors was also measured at these ages. We found that preproET-1 mRNA expression increased from 113 to 130 days gestation. Whole lung ET protein content was highest at 130 days gestation but decreased before birth in the fetal lamb lung. Immunolocalization of ET-1 protein showed expression of ET-1 in the vasculature and bronchial epithelium at all gestational ages. ETAreceptor mRNA expression and ETBreceptor mRNA increased from 90 to 125 and 135 days gestation. To determine changes in activity of the ETAand ETBreceptors, we studied the effect of selective antagonists to the ETAor ETBreceptors at 120, 130, and 140 days of fetal gestation. ETAreceptor-mediated vasoconstriction increased from 120 to 140 days, whereas blockade of the ETBreceptor did not change basal fetal pulmonary vascular tone at any age examined. We conclude that the ET system is developmentally regulated and that the increase in ETAreceptor gene expression correlates with the onset of the vasodilator response to ETAreceptor blockade. Although ETBreceptor gene expression increases during late gestation, the balance of ET receptor activity favors vasoconstriction under basal conditions. We speculate that changes in ET receptor activity play important roles in regulation of pulmonary vascular tone in the ovine fetus.
Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1
Authors: Tchekneva Elena , Lawrence Mayme L. , Meyrick Barbara
Published: December 2017

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes sustained constriction of the pulmonary artery and modulates normal vascular tone. Endothelial cells were thought to be the major source of ET-1, but recent studies show that vascular smooth muscle cells (SMCs) are also capable of its synthesis. We examined the ET-1 and endothelin-converting enzyme-1 (ECE-1) system in cells cultured from two adjacent layers, subendothelial (L1) and inner medial (L2), of normal sheep main pulmonary artery and the response of this system to exogenous ET-1 and transforming growth factor-β1 (TGF-β1). End points include assessment of preproET-1 (ppET-1) and ECE-1 gene coexpression, measurement of intracellular and released ET-1, and ECE-1 activity. RT-PCR analysis revealed that ppET-1 and ECE-1 transcripts were greater in L1 than in L2 cells. The L1 cells also synthesized (L1, 3.2 ± 0.1; L2, 1.2 ± 0.1 fmol/106cells) and released (L1, 9.2 ± 0.5; L2, 2.3 ±0.1 fmol/ml) greater amounts of ET-1 than L2 cells. The L2 cells internalized exogenous ET-1 in a dose-dependent manner (EC508 nmol/l) and were more responsive to exogenous ET-1 than L1 cells, showing upregulation of both the ppET-1 and ECE genes. TGF-β1 downregulated ET-1-stimulated ppET-1 and ECE-1 transcripts but only in L2 cells. In addition, L1 cells showed greater ECE-1 activity than L2 cells, and in both, the activity was sensitive to the metalloprotease inhibitor phosphoramidon. We conclude that the ET-1 system in L1 and L2 cells is distinct. The data suggest that the two cell types have diverse functions in the arterial wall; the L1 cells, like endothelial cells, provide a local source of ET-1; and since the L2 cells are more responsive to exogenous ET-1, they are likely to affect normal pulmonary vascular tone.