bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperoxia inhibits proliferation of Mv1Lu epithelial cells independent of TGF-β signaling
Authors: Rancourt Raymond C. , Staversky Rhonda J. , Keng Peter C. , O'reilly Michael A.
Published: December 2017

High concentrations of O2inhibit epithelial cell proliferation that resumes on recovery in room air. To determine whether growth arrest is mediated by transforming growth factor-β (TGF-β), changes in cell proliferation during exposure to hyperoxia were assessed in the mink lung epithelial cell line Mv1Lu and the clonal variant R1B, which is deficient for the type I TGF-β receptor. Mv1Lu cells treated with TGF-β accumulated in the G1phase of the cell cycle as determined by propidium iodide staining, whereas proliferation of R1B cells was unaffected by TGF-β. In contrast, hyperoxia inhibited proliferation of both cell lines within 24 h of exposure through an accumulation in the S phase. Mv1Lu cells treated with TGF-β and exposed to hyperoxia accumulated in the G1phase, suggesting that TGF-β can inhibit the S phase accumulation observed with hyperoxia alone. Cyclin A was detected in cultures exposed to room air or growth arrested by hyperoxia while decreasing in cells growth arrested in the G1phase by TGF-β. Finally, hyperoxia failed to activate a TGF-β-dependent transcriptional reporter in both Mv1Lu and R1B cells. These findings reveal that simple growth arrest by hyperoxia involves a defect in S phase progression that is independent of TGF-β signaling.
Human neutrophil elastase releases two pools of mucinlike glycoconjugate from tracheal submucosal gland cells
Authors: Dwyer Terry M. , Farley Jerry M.
Published: December 2017

Neutrophil elastase can contribute to the pathogenesis of increased airway reactivity and excess mucus secretion in many pulmonary diseases. Ten nanomolar human neutrophil elastase (HNE) effectively empties airway serous cells, raising the question of why HNE is not equally effective at emptying mucous cells of their stored mucin because total release of mucin granules is not seen in postmortem examination of even the most severe disease. To better resolve the mucus secretagogue action of HNE, we measured secretion of mucinlike glycoconjugates (MGCs) released from freshly isolated swine tracheal submucosal gland cells in fractions of the superfusate acquired every 2 min. Six to fifty nanomolar HNE released a fixed quantity of MGCs at an increasing rate with increasing concentrations of enzyme, an action consistent with the release of cell surface mucinlike molecules. The polycation poly-l-lysine (1 μg/ml) released a similar transient of MGCs. A steady-state doubling of MGC rate of release was seen as long as 100 nM HNE was present, but this stimulus represented less than a 1% release of stored MGCs/min and was consistent with release of mucin vesicles from cell stores. Both actions of HNE were inhibited by the specific inhibitors L-680833 and DMP-777 but not by 30 μM erythromycin. Therefore, HNE release of MGCs from tracheal submucosal glands is limited by both the fixed quantity of the MGCs in the transient pool and by the small steady-state response to the higher concentrations of enzyme.
Phorbol ester-induced U-937 differentiation: effects on integrin α5gene transcription
Authors: Boles Bonnie K. , Ritzenthaler Jeffrey , Birkenmeier Thomas , Roman Jesse
Published: December 2017

Lung injury is accompanied by increased deposition of fibronectin (FN) matrices. Activated monocytic cells recruited to sites of lung injury express integrin receptors for FN that mediate their interaction with this matrix. One such integrin, α5β1, mediates many of the biological effects of FN, and its expression may be important for immune cell function at sites of lung injury. Herein, we examine the expression of α5β1in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) in the human promonocytic cell line U-937. We demonstrate that PMA enhanced the adherence of U-937 cells to FN by increasing the expression of both the α5- and β1-subunit mRNAs and the surface expression of the protein. In U-937 cells transfected with an α5promoter-reporter gene, we found that PMA induced the transcription of the α5gene by acting on very specific promoter sequences other than activator protein-1 in a protein kinase C-dependent manner. Lipopolysaccharide had a similar effect. Modulation of α5β1expression may be important for regulation of monocytic cell function in lung inflammation after injury.
Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia
Authors: Braems Geert A. , Yao Li-Juan , Inchley Kevin , Brickenden Anne , Han Victor K. M. , Grolla Allen , Challis John R. G. , Possmayer Fred
Published: December 2017

cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126– 130 and 134– 136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126–130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.
Protein kinase C-α mediates endothelial barrier dysfunction induced by TNF-α
Authors: Ferro Thomas , Neumann Paul , Gertzberg Nancy , Clements Richard , Johnson Arnold
Published: December 2017

We tested the hypothesis that protein kinase C-α (PKC-α) mediates tumor necrosis factor-α (TNF-α)-induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM lysates were analyzed for PKC-α mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-α (1,000 U/ml) for 4 h resulted in increases in 1) PKC-α protein, 2) cytoskeletal-associated PKC-α, 3) PKC-α activity, and 4) permeability to albumin. The TNF-α-induced increase in PKC-α protein, PKC-α activity, and permeability was prevented by a 4-h pretreatment with PKC-α antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-α-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-α/β, 100 μM) and calphostin (an inhibitor of the classic and novel PKC isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-α still prevented barrier dysfunction induced by 4 h of TNF-α treatment. The data indicate that prolonged activation of PKC-α, maintained by a translation-dependent pool of PKC-α protein, mediates TNF-α-induced increases in endothelial permeability in PEM.
Intrinsic ICAM-1/LFA-1 activation mediates altered responsiveness of atopic asthmatic airway smooth muscle
Authors: Grunstein Michael M. , Hakonarson Hakon , Maskeri Neil , Kim Cecilia , Chuang Sing
Published: December 2017

Cell adhesion molecules (CAMs) have been importantly implicated in the pathobiology of the airway responses in allergic asthma, including inflammatory cell recruitment into the lungs and altered bronchial responsiveness. To elucidate the mechanism of CAM-related mediation of altered airway responsiveness in the atopic asthmatic state, the expressions and actions of intercellular adhesion molecule-1 (ICAM-1) and its counterreceptor ligand lymphocyte function-associated antigen-1 (LFA-1; i.e., CD11a/CD18) were examined in isolated rabbit airway smooth muscle (ASM) tissues and cultured human ASM cells passively sensitized with sera from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with a monoclonal blocking antibody (MAb) to either ICAM-1 or CD11a, whereas a MAb directed against the related β2-integrin Mac-1 had no effect. Moreover, relative to control tissues, atopic asthmatic sensitized ASM cells displayed an autologously upregulated mRNA and cell surface expression of ICAM-1, whereas constitutive expression of CD11a was unaltered. Extended studies further demonstrated that 1) the enhanced expression and release of soluble ICAM-1 by atopic sensitized ASM cells was prevented when cells were pretreated with an interleukin (IL)-5-receptor-α blocking antibody and 2) administration of exogenous IL-5 to naive (nonsensitized) ASM cells induced a pronounced soluble ICAM-1 release from the cells. Collectively, these observations provide new evidence demonstrating that activation of the CAM counterreceptor ligands ICAM-1 and LFA-1, both of which are endogenously expressed in ASM cells, elicits autologously upregulated IL-5 release and associated changes in ICAM-1 expression and agonist responsiveness in atopic asthmatic sensitized ASM.
Determinants of surfactant function in acute lung injury and early recovery
Authors: Mora R. , Arold S. , Marzan Y. , Suki B. , Ingenito E. P.
Published: December 2017

Relationships between lung function and surfactant function and composition were examined during the evolution of acute lung injury in guinea pigs. Lung mechanics and gas exchange were assessed 12, 24, or 48 h after exposure to nebulized lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) fluid was processed for phospholipid and protein contents and surfactant protein (SP) A and SP-B levels; surfactant function was measured by pulsating bubble surfactometry. Lung elastance, tissue resistance, and arterial-alveolar gradient were moderately elevated by 12 h after LPS exposure and continued to increase over the first 24 h but began to recover between 24 and 48 h. Similarly, the absolute amount of 30,000 g pelleted SP-A and SP-B, the phospholipid content of BAL fluid, and surfactant function declined over the first 24 h after exposure, with recovery between 24 and 48 h. BAL fluid total protein content increased steadily over the first 48 h after LPS nebulization. In this model of acute lung injury, the intra-alveolar repletion of surfactant components in early recovery led to improved surfactant function despite the presence of potentially inhibitory plasma proteins.
Role of Ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction
Authors: Verin Alexander D. , Liu Feng , Bogatcheva Natalia , Borbiev Talaibek , Hershenson Marc B. , Wang Peiyi , Garcia Joe G. N.
Published: December 2017

The treatment of endothelial cell monolayers with phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C (PKC) activator, leads to disruption of endothelial cell monolayer integrity and intercellular gap formation. Selective inhibition of PKC (with bisindolylmaleimide) and extracellular signal-regulated kinases (ERKs; with PD-98059, olomoucine, or ERK antisense oligonucleotides) significantly attenuated PMA-induced reductions in transmonolayer electrical resistance consistent with PKC- and ERK-mediated endothelial cell barrier regulation. An inhibitor of the dual-specificity ERK kinase (MEK), PD-98059, completely abolished PMA-induced ERK activation. PMA also produced significant time-dependent increases in the activity of Raf-1, a Ser/Thr kinase known to activate MEK (∼6-fold increase over basal level). Similarly, PMA increased the activity of Ras, which binds and activates Raf-1 (∼80% increase over basal level). The Ras inhibitor farnesyltransferase inhibitor III (100 μM for 3 h) completely abolished PMA-induced Raf-1 activation. Taken together, these data suggest that the sequential activation of Ras, Raf-1, and MEK are involved in PKC-dependent endothelial cell barrier regulation.
Hypoxia reduces atrial natriuretic peptide clearance receptor gene expression in ANP knockout mice
Authors: Sun Ju-Zhong , Chen Shi-Juan , Li Guohong , Chen Yiu-Fai
Published: December 2017

We tested the hypotheses that hypoxic exposure is associated with exacerbated pulmonary hypertension and right ventricular (RV) enlargement, reduced atrial natriuretic peptide (ANP) clearance receptor (NPR-C) expression, and enhanced B-type natriuretic peptide (BNP) expression in the absence of ANP. Male wild-type [ANP(+/+)], heterozygous [ANP(+/−)], and homozygous [ANP(−/−)] mice were studied after a 5-wk hypoxic exposure (10% O2). Hypoxia increased RV ANP mRNA and plasma ANP levels only in ANP(+/+) and ANP(+/−) mice. Hypoxia-induced increases in RV pressure were significantly greater in ANP(−/−) than in ANP(+/+) or ANP(+/−) mice (104 ± 17 vs. 45 ± 10 and 63 ± 7%, respectively) as were increases in RV mass (38 ± 4 vs. 26 ± 5 and 29 ± 4%, respectively). NPR-C mRNA levels were greatly reduced in the kidney, lung, and brain by hypoxia in all three genotypes. RV BNP mRNA and lung and kidney cGMP levels were increased in hypoxic mice. These findings indicate that disrupted ANP expression worsens hypoxic pulmonary hypertension and RV enlargement but does not alter hypoxia-induced decreases in NPR-C and suggest that compensatory increases in BNP expression occur in the absence of ANP.
Adherence of adoptively transferred alloreactive Th1 cells in lung: partial dependence on LFA-1 and ICAM-1
Authors: Dixon Anne E. , Mandac Janis B. , Martin Paul J. , Hackman Robert C. , Madtes David K. , Clark Joan G.
Published: December 2017

T helper type 1 (Th1) cells are important effectors in a number of immune-mediated lung diseases. We recently described a murine model of lung injury induced by adoptive transfer of cloned alloreactive Th1 cells. To investigate mechanisms that result in injury to the lung, we studied the in vivo distribution of51Cr-labeled Th1 cells. One hour after intravenous administration, >85% of injected radioactivity was left in the lung, and at 24 h, 40% of radioactivity was left in the lung. Adherence of Th1 cells in the lung was significantly inhibited by neutralizing antibody to lymphocyte function-associated antigen-1. Th1 cell adherence also was decreased in lungs of mice deficient in intercellular adhesion molecule-1 (ICAM-1). Th1 cell transfer further induced expression of ICAM-1 and vascular cell adhesion molecule-1 in the lung. Vascular cell adhesion molecule-1-immunoreactive protein was markedly induced in lung endothelium by alloreactive Th1 cells. These findings indicate that Th1 cells localize in normal lung by a mechanism involving lymphocyte function-associated antigen-1 and ICAM-1. Alloreactive cells further induce endothelial adhesion molecules that may facilitate recruitment of inflammatory cells to the lung and amplify Th1 cell-induced lung injury.
Inhibition of angiogenesis decreases alveolarization in the developing rat lung
Authors: Jakkula Malathi , Le Cras Timothy D. , Gebb Sarah , Hirth K. Peter , Tuder Rubin M. , Voelkel Norbert F. , Abman Steven H.
Published: December 2017

To determine whether angiogenesis is necessary for normal alveolarization, we studied the effects of two antiangiogenic agents, thalidomide and fumagillin, on alveolarization during a critical period of lung growth in infant rats. Newborn rats were treated with daily injections of fumagillin, thalidomide, or vehicle during the first 2 wk of life. Compared with control treatment, fumagillin and thalidomide treatment reduced lung weight-to-body weight ratio and pulmonary arterial density by 20 and 36%, respectively, and reduced alveolarization by 22%. Because these drugs potentially have nonspecific effects on lung growth, we also studied the effects of Su-5416, an inhibitor of the vascular endothelial growth factor receptor known as kinase insert domain-containing receptor/fetal liver kinase (KDR/flk)-1. As observed with the other antiangiogenic agents, Su-5416 treatment decreased alveolarization and arterial density. We conclude that treatment with three different antiangiogenic agents attenuated lung vascular growth and reduced alveolarization in the infant rat. We speculate that angiogenesis is necessary for alveolarization during normal lung development and that injury to the developing pulmonary circulation during a critical period of lung growth can contribute to lung hypoplasia.
Interleukin-1β augments in vitro alveolar epithelial repair
Authors: Geiser Thomas , Jarreau Pierre-Henri , Atabai Kamran , Matthay Michael A.
Published: December 2017

Biologically active interleukin (IL)-1β is present in the pulmonary edema fluid obtained from patients with acute lung injury and has been implicated as an important early mediator of nonpulmonary epithelial wound repair. Therefore, we tested the hypothesis that IL-1β would enhance wound repair in cultured monolayers from rat alveolar epithelial type II cells. IL-1β (20 ng/ml) increased the rate of in vitro alveolar epithelial repair by 118 ± 11% compared with that in serum-free medium control cells ( P < 0.01). IL-1β induced cell spreading and migration at the edge of the wound but not proliferation. Neutralizing antibodies to epidermal growth factor (EGF) and transforming growth factor-α or inhibition of the EGF receptor by tyrphostin AG-1478 or genistein inhibited IL-1β-induced alveolar epithelial repair, indicating that IL-1β enhances in vitro alveolar epithelial repair by an EGF- or transforming growth factor-α-dependent mechanism. Moreover, the mitogen-activated protein kinase pathway is involved in IL-1β-induced alveolar epithelial repair because inhibition of extracellular signal-regulated kinase activation by PD-98059 inhibited IL-1β-induced alveolar epithelial repair. In conclusion, IL-1β augments in vitro alveolar epithelial repair, indicating a possible novel role for IL-1β in the early repair process of the alveolar epithelium in acute lung injury.
Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke
Authors: Takeyama Kiyoshi , Jung Birgit , Shim Jae Jeong , Burgel Pierre-Regis , Dao-Pick Trang , Ueki Iris F. , Protin Ursula , Kroschel Peer , Nadel Jay A.
Published: December 2017

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.
Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase
Authors: Suliman Hagir B. , Ryan Lisa K. , Bishop Lisa , Folz Rodney J.
Published: December 2017

Reactive oxygen and nitrogen species such as superoxide and nitric oxide are released into the extracellular spaces by inflammatory and airway epithelial cells. These molecules may exacerbate lung injury after influenza virus pneumonia. We hypothesized that enhanced expression of extracellular superoxide dismutase (EC SOD) in mouse airways would attenuate the pathological effects of influenza pneumonia. We compared the pathogenic effects of a nonlethal primary infection with mouse-adapted Hong Kong influenza A/68 virus in transgenic (TG) EC SOD mice versus non-TG (wild-type) littermates. Compared with wild-type mice, EC SOD TG mice showed less lung injury and inflammation as measured by significant blunting of interferon-γ induction, reduced cell count and total protein in bronchoalveolar lavage fluid, reduced levels of lung nitrite/nitrate nitrotyrosine, and markedly reduced lung pathology. These results demonstrate that enhancing EC SOD in the conducting and distal airways of the lung minimizes influenza-induced lung injury by both ameliorating inflammation and attenuating oxidative stress.
Effect of glycosaminoglycan degradation on lung tissue viscoelasticity
Authors: Al Jamal Rehab , Roughley Peter J. , Ludwig Mara S.
Published: December 2017

We tested the hypothesis that matrix glycosaminoglycans contribute to lung tissue viscoelasticity. We exposed lung parenchymal strips to specific degradative enzymes (chondroitinase ABC, heparitinase I, and hyaluronidase) and determined whether the mechanical properties of the tissue were affected. Subpleural parenchymal strips were obtained from Sprague-Dawley rats and suspended in a Krebs-filled organ bath. One end of the strip was attached to a force transducer and the other to a servo-controlled lever arm that effected sinusoidal oscillations. Recordings of tension and length at different amplitudes and frequencies of oscillation were recorded before and after enzyme exposure. Resistance, dynamic elastance, and hysteresivity were estimated by fitting the equation of motion to changes in tension and length. Quasi-static stress-strain curves were also obtained. Exposure to chondroitinase and heparitinase I caused significant increases in hysteresivity, no decrement in resistance, and similar decreases in dynamic elastance relative to control strips exposed to Krebs solution only. Conversely, measures of static elastance were different in treated versus control strips. Hyaluronidase treatment did not alter any of the mechanical measures. These data demonstrate that digestion of chondroitin sulfate and heparan sulfate alters the mechanical behavior of lung parenchymal tissues.
Prostaglandin E2regulates wound closure in airway epithelium
Authors: Savla Ushma , Appel Heidi J. , Sporn Peter H. S. , Waters Christopher M.
Published: December 2017

Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E2(PGE2) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE2stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.
PAF mediates cigarette smoke-induced goblet cell metaplasia in guinea pig airways
Authors: Komori Masashi , Inoue Hiromasa , Matsumoto Koichiro , Koto Hiroshi , Fukuyama Satoru , Aizawa Hisamichi , Hara Nobuyuki
Published: December 2017

Goblet cell metaplasia is an important morphological feature in the airways of patients with chronic airway diseases; however, the precise mechanisms that cause this feature are unknown. We investigated the role of endogenous platelet-activating factor (PAF) in airway goblet cell metaplasia induced by cigarette smoke in vivo. Guinea pigs were exposed repeatedly to cigarette smoke for 14 consecutive days. The number of goblet cells in each trachea was determined with Alcian blue-periodic acid-Schiff staining. Differential cell counts and PAF levels in bronchoalveolar lavage fluid were also evaluated. Cigarette smoke exposure significantly increased the number of goblet cells. Eosinophils, neutrophils, and PAF levels in bronchoalveolar lavage fluid were also significantly increased after cigarette smoke. Treatment with a specific PAF receptor antagonist, E-6123, significantly attenuated the increases in the number of airway goblet cells, eosinophils, and neutrophils observed after cigarette smoke exposure. These results suggest that endogenous PAF may play a key role in goblet cell metaplasia induced by cigarette smoke and that potential roles exist for inhibitors of PAF receptor in the treatment of hypersecretory airway diseases.
EDHF contributes to strain-related differences in pulmonary arterial relaxation in rats
Authors: Karamsetty M. R. , Nakashima J. M. , Ou L.-C. , Klinger J. R. , Hill N. S.
Published: December 2017

Pulmonary arteries from the Madison (M) strain relax more in response to acetylcholine (ACh) than those from the Hilltop (H) strain of Sprague-Dawley rats. We hypothesized that differences in endothelial nitric oxide (NO) synthase (eNOS) expression and function, metabolism of ACh by cholinesterases, release of prostacyclin, or endothelium-derived hyperpolarizing factor(s) (EDHF) from the endothelium would explain the differences in the relaxation response to ACh in isolated pulmonary arteries. eNOS mRNA and protein levels as well as the NO-dependent relaxation responses to thapsigargin in phenylephrine (10−6 M)-precontracted pulmonary arteries from the M and H strains were identical. The greater relaxation response to ACh in M compared with H rats was also observed with carbachol, a cholinesterase-resistant analog of ACh, a response that was not modified by pretreatment with meclofenamate (10−5M). N ω-nitro-l-arginine (10−4 M) completely abolished carbachol-induced relaxation in H rat pulmonary arteries but not in M rat pulmonary arteries. Precontraction with KCl (20 mM) blunted the relaxation response to carbachol in M rat pulmonary arteries and eliminated differences between the M and H rat pulmonary arteries. NO-independent relaxation present in the M rat pulmonary arteries was significantly reduced by 17-octadecynoic acid (2 μM) and was completely abolished by charybdotoxin plus apamin (100 nM each). These findings suggest that EDHF, but not NO, contributes to the strain-related differences in pulmonary artery reactivity. Also, EDHF may be a metabolite of cytochrome P-450 that activates Ca2+-dependent K+ channels.
Differential regulation of Cl−transport proteins by PKC in Calu-3 cells
Authors: Liedtke Carole M. , Cody Derek , Cole Thomas S.
Published: December 2017

Cl−transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl−transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-ε)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-δ). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-ε decreased the amount of PKC-ε and reduced cAMP-dependent activation of CFTR but not α1-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-δ did not affect CFTR function but did block α1-adrenergic activation of NKCC1 and reduce PKC-δ mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.
Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis
Authors: Shi Wei , Zhao Jingsong , Anderson Kathryn D. , Warburton David
Published: December 2017

Bone morphogenetic protein-4 (BMP-4) is a key morphogen for embryonic lung development that is expressed at high levels in the peripheral epithelium, but the mechanisms that modulate BMP-4 function in early mouse lung branching morphogenesis are unclear. Here, we studied the BMP-4 antagonist Gremlin, which is a member of the DAN family of BMP antagonists that can bind and block BMP-2/4 activity. The expression level of gremlin in embryonic mouse lungs is highest in the early embryonic pseudoglandular stage [embryonic days (E) 11.5–14.5] and is reduced during fetal lung maturation (E18.5 to postnatal day 1). In situ hybridization indicates that gremlin is diffusely expressed in peripheral lung mesenchyme and epithelium, but relatively high epithelial expression occurs in branching buds at E11.5 and in large airways after E16.5. In E11.5 lung organ culture, we found that exogenous BMP-4 dramatically enhanced peripheral lung epithelial branching morphogenesis, whereas reduction of endogenous gremlin expression with antisense oligonucleotides achieved the same gain-of-function phenotype as exogenous BMP-4, including increased epithelial cell proliferation and surfactant protein C expression. On the other hand, adenoviral overexpression of gremlin blocked the stimulatory effects of exogenous BMP-4. Therefore, our data support the hypothesis that Gremlin is a physiologically negative regulator of BMP-4 in lung branching morphogenesis.
Bafilomycin A1inhibits rhinovirus infection in human airway epithelium: effects on endosome and ICAM-1
Authors: Suzuki Tomoko , Yamaya Mutsuo , Sekizawa Kiyohisa , Hosoda Masayoshi , Yamada Norihiro , Ishizuka Satoshi , Nakayama Katsutoshi , Yanai Masaru , Numazaki Yoshio , Sasaki Hidetada
Published: December 2017

To examine the effects of bafilomycin A1, a blocker of vacuolar H+-ATPase, on rhinovirus (RV) infection in the airway epithelium, primary cultures of human tracheal epithelial cells were infected with RV14. Viral infection was confirmed by showing that viral RNA in the infected cells and the viral titers in the supernatants of infected cells increased with time. RV14 infection upregulated the production of cytokines and mRNA of intercellular adhesion molecule (ICAM)-1 in epithelial cells. Bafilomycin A1reduced the viral titers of RV14 and inhibited the production of cytokines and ICAM-1 before and after RV14 infection. Bafilomycin A1reduced susceptibility of epithelial cells to RV14 infection. RV14 increased activated nuclear factor-κB in the cells, and bafilomycin A1reduced the activated nuclear factor-κB. Bafilomycin A1decreased the number of acidic endosomes in the epithelial cells. These results suggest that bafilomycin A1may inhibit infection by RV14 by not only blocking RV RNA entry into the endosomes but also reducing ICAM-1 expression in the epithelial cells. Bafilomycin A1may therefore modulate airway inflammation after RV infection.
Pathophysiological analysis of combined burn and smoke inhalation injuries in sheep
Authors: Soejima Kazutaka , Schmalstieg Frank C. , Sakurai Hiroyuki , Traber Lillian D. , Traber Daniel L.
Published: December 2017

We investigated the pathophysiological alterations seen with combined burn and smoke inhalation injuries by focusing on pulmonary vascular permeability and cardiopulmonary function compared with those seen with either burn or smoke inhalation injury alone. To estimate the effect of factors other than injury, the experiments were also performed with no injury in the same experimental setting. Lung edema was most severe in the combined injury group. Our study revealed that burn injury does not affect protein leakage from the pulmonary microvasculature, even when burn is associated with smoke inhalation injury. The severity of lung edema seen with the combined injury is mainly due to augmentation of pulmonary microvascular permeability to fluid, not to protein. Cardiac dysfunction after the combined injury consisted of at least two phases. An initial depression was mostly related to hypovolemia due to burn injury. It was improved by a large amount of fluid resuscitation. The later phase, which was indicated to be a myocardial contractile dysfunction independent of the Starling equation, seemed to be correlated with smoke inhalation injury.
Pulmonary reduction of an intravascular redox polymer
Authors: Audi Said H. , Bongard Robert D. , Okamoto Yoshiyuki , Merker Marilyn P. , Roerig David L. , Dawson Christopher A.
Published: December 2017

Pulmonary endothelial cells in culture reduce external electron acceptors via transplasma membrane electron transport (TPMET). In studying endothelial TPMET in intact lungs, it is difficult to exclude intracellular reduction and reducing agents released by the lung. Therefore, we evaluated the role of endothelial TPMET in the reduction of a cell-impermeant redox polymer, toluidine blue O polyacrylamide (TBOP+), in intact rat lungs. When added to the perfusate recirculating through the lungs, the venous effluent TBOP+concentration decreased to an equilibrium level reflecting TBOP+reduction and autooxidation of its reduced (TBOPH) form. Adding superoxide dismutase (SOD) to the perfusate increased the equilibrium TBOP+concentration. Kinetic analysis indicated that the SOD effect could be attributed to elimination of the superoxide product of TBOPH autooxidation rather than of superoxide released by the lungs, and experiments with lung-conditioned perfusate excluded release of other TBOP+reductants in sufficient quantities to cause significant TBOP+reduction. Thus the results indicate that TBOP+reduction is via TPMET and support the utility of TBOP+and the kinetic model for investigating TPMET mechanisms and their adaptations to physiological and pathophysiological stresses in the intact lung.
Na+transport in normal and CF human bronchial epithelial cells is inhibited by BAY 39-9437
Authors: Bridges Robert J. , Newton Ben B. , Pilewski Joseph M. , Devor Daniel C. , Poll Christopher T. , Hall Rod L.
Published: December 2017

To test the hypothesis that Na+transport in human bronchial epithelial (HBE) cells is regulated by a protease-mediated mechanism, we investigated the effects of BAY 39-9437, a recombinant Kunitz-type serine protease inhibitor, on amiloride-sensitive short-circuit current of normal [non-cystic fibrosis (CF) cells] and CF HBE cells. Mucosal treatment of non-CF and CF HBE cells with BAY 39-9437 decreased the short-circuit current, with a half-life of ∼45 min. At 90 min, BAY 39-9437 (470 nM) reduced Na+transport by ∼70%. The inhibitory effect of BAY 39-9437 was concentration dependent, with a half-maximal inhibitory concentration of ∼25 nM. Na+transport was restored to control levels, with a half-life of ∼15 min, on washout of BAY 39-9437. In addition, trypsin (1 μM) rapidly reversed the inhibitory effect of BAY 39-9437. These data indicate that Na+transport in HBE cells is activated by a BAY 39-9437-inhibitable, endogenously expressed serine protease. BAY 39-9437 inhibition of this serine protease maybe of therapeutic potential for the treatment of Na+hyperabsorption in CF.
Lack of amiloride-sensitive transport across alveolar and respiratory epithelium of iNOS(−/−) mice in vivo
Authors: Hardiman Karin M. , Lindsey J. Russell , Matalon Sadis
Published: December 2017

The extent to which endogenously generated nitric oxide alters Na+transport across the mammalian alveolar epithelium in vivo has not been documented. Herein we measured alveolar fluid clearance and nasal potential differences in mice lacking the inducible form of nitric oxide synthase [iNOS; iNOS(−/−)] and their corresponding wild-type controls [iNOS(+/+)]. Alveolar fluid clearance values in iNOS(+/+) and iNOS(−/−) anesthetized mice with normal oxygenation and acid-base balance were ∼30% of instilled fluid/30 min. In both groups of mice, fluid absorption was dependent on vectorial Na+movement. Amiloride (1.5 mM) decreased alveolar fluid clearance in iNOS(+/+) mice by 61%, whereas forskolin (50 μM) increased alveolar fluid clearance by 55% by stimulating amiloride-insensitive pathways. Neither agent altered alveolar fluid clearance in iNOS(−/−) mice. Hyperoxia upregulated iNOS expression in iNOS(+/+) mice and decreased their amiloride-sensitive component of alveolar fluid clearance but had no effect on the corresponding values in iNOS(−/−) mice. Nasal potential difference measurements were consistent with alveolar fluid clearance in that both groups of mice had similar baseline values, which were amiloride sensitive in the iNOS(+/+) but not in the iNOS(−/−) mice. These data suggest that nitric oxide produced by iNOS under basal conditions plays an important role in regulating amiloride-sensitive Na+channels in alveolar and airway epithelia.
Augmented K+currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis
Authors: Krick Stefanie , Platoshyn Oleksandr , McDaniel Sharon S. , Rubin Lewis J. , Yuan Jason X.-J.
Published: December 2017

The balance between apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMCs) is important in maintaining normal pulmonary vascular structure. Activity of voltage-gated K+(KV) channels has been demonstrated to regulate cell apoptosis and proliferation. Treatment of PASMCs with staurosporine (ST) induced apoptosis in PASMCs, augmented KVcurrent [ IK(V)], and induced mitochondrial membrane depolarization. High K+(40 mM) negligibly affected the ST-induced mitochondrial membrane depolarization but inhibited the ST-induced IK(V)increase and apoptosis. Blockade of KVchannels with 4-aminopyridine diminished IK(V)and markedly decreased the ST-mediated apoptosis. Furthermore, the ST-induced apoptosis was preceded by the increase in IK(V). These results indicate that ST induces PASMC apoptosis by activation of plasmalemmal KVchannels and mitochondrial membrane depolarization. The increased IK(V)would result in an apoptotic volume decrease due to a loss of cytosolic K+and induce apoptosis. The mitochondrial membrane depolarization would cause cytochrome c release, activate the cytosolic caspases, and induce apoptosis. Inhibition of KVchannels would thus attenuate PASMC apoptosis.
Mechanisms of cytokine effects on G protein-coupled receptor-mediated signaling in airway smooth muscle
Authors: Pascual Rodolfo M. , Billington Charlotte K. , Hall Ian P. , Panettieri Reynold A. , Fish James E. , Peters Stephen P. , Penn Raymond B.
Published: December 2017

Numerous in vitro and in vivo studies have implicated the cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) as mediators of airway inflammation and therefore potentially important substances in the pathogenesis of asthma. In this study, we examined the mechanisms by which IL-1β and TNF-α affect inhibition of cell growth, G protein-coupled receptor (GPCR) desensitization, and the recently reported adenylyl cyclase sensitization in human airway smooth muscle (HASM) cultures. Our findings demonstrate that adenylyl cyclase sensitization is independent of cytokine-mediated cyclooxygenase type 2 (COX-2) and prostaglandin E2(PGE2) induction, whereas COX-2 induction appears to be required for both growth inhibition and GPCR desensitization. However, GPCR desensitization was highly dependent on the presence of EGF during chronic treatment with cytokines, which could be explained by a synergistic effect of EGF on cytokine-mediated COX-2 and PGE2induction. Interestingly, various agents (including inhibitors of p42/p44 and p38 mitogen-activated protein kinase signaling) were significantly more effective in inhibiting cytokine-mediated PGE2induction, GPCR desensitization, and cell growth inhibition than in inhibiting COX-2 induction. These data demonstrate disparity in the requirement and sufficiency of COX-2 induction in promoting different functional effects of IL-1β and TNF-α in HASM.
Molecular cloning and expression of pulmonary lipid phosphate phosphohydrolases
Authors: Nanjundan Meera , Possmayer Fred
Published: December 2017

Pulmonary lipid phosphate phosphohydrolase (LPP) was shown previously to hydrolyze phosphatidic acid and lysophosphatidic acid in purified rat lung plasma membranes. To better investigate the nature of pulmonary LPP isoforms and their role in the lung, LPPs were cloned by RT-PCR from both adult rat lung and type II cell RNA. The RT-PCR generated LPP1 (849 bp), up to three LPP1 variants, and LPP3 (936 bp) cDNAs. The three LPP1 variants include LPP1a (852 bp) and two novel isoforms, LPP1b (697 bp) and LPP1c (1004 bp). The pulmonary LPP1 and LPP3 isoforms are essentially identical to the previously cloned rat liver and intestinal LPPs, respectively, and the LPP1a isoform has 80% sequence identity to the human homolog. The LPP2 isoform was not detected in lung by RT-PCR. Northern analyses revealed that the mRNAs for LPP1 and LPP3 increase in fetal rat lung in late gestation to day 1 after birth. These mRNAs decrease somewhat during the neonatal period but increase slightly during postnatal development. Expression of LPP1, LPP1a, and LPP3 cDNAs in HEK 293 cells established that they encode functional LPP. In contrast, the novel isoforms LPP1b and LPP1c contain frameshifts that would result in premature termination, producing putative catalytically inactive polypeptides of 30 and 76 amino acids, respectively. Further investigation of the LPP1b isoform revealed that it was present across a variety of tissues, although at lower levels than LPP1/1a. Transient mammalian expression of LPP1b failed to increase phosphatidate phosphohydrolase activity in HEK 293 cells.
Altered lung gene expression in CCSP-null mice suggests immunoregulatory roles for Clara cells
Authors: Watson T. M. , Reynolds S. D. , Mango G. W. , Boe I.-M. , Lund J. , Stripp B. R.
Published: December 2017

Clara cell secretory protein (CCSP) is one of the most abundant proteins present in airway lining fluid of mammals. In an effort to elucidate the function of CCSP, we established CCSP-null [CCSP(−/−)] mice and demonstrated altered sensitivity to various environmental agents including oxidant pollutants and microorganisms. Although CCSP deficiency itself may be central to the observed changes in environmental susceptibility, altered lung gene expression associated with CCSP deficiency may contribute to the observed phenotype. To determine whether CCSP deficiency results in altered lung gene expression, high-density cDNA microarrays were used to profile gene expression in the total lung RNA of wild-type and CCSP(−/−) mice. Genes that were differentially expressed between wild-type and CCSP(−/−) mice included a previously nonannotated expressed sequence tag (EST W82219 ) and immunoglobulin A (IgA), both of which were elevated with CCSP deficiency. mRNA expression of EST W82219 and IgA was localized in the lungs of wild-type and CCSP(−/−) mice to airway Clara cells and peribronchial lymphoid tissues, respectively. We conclude that CCSP deficiency is associated with 1) altered gene expression in Clara cells of the conducting airway epithelium and 2) alterations to peribronchial B lymphocytes. These findings identify new roles for Clara cells and their secretions in airway homeostasis.
Crystal accumulation and very high short-circuit currents in rabbit urinary bladder
Authors: Loo D. D. , Diamond J. M.
Published: December 2017

We describe a condition in rabbits characterized by CaCO3 crystal accumulation and very high short-circuit current (Isc) in the urinary bladder. The incidence of the condition was high in rabbits kept for 2 mo or more in the vivarium. The crystal mass in the bladder increased with age or time until it occupied up to one-third of the bladder volume and occasionally aggregated as stones, and the urine attained a sludgy consistency. In animals with sludgy urine, the urine excreted had a crystal content 20 times lower than that of urine contained in the bladder at time of death, implying retention of crystals in the bladder. Kidney stones were not detected in rabbits with bladder crystals, and bladder crystals were absent in rabbits with kidney stones induced experimentally by uremia. In old rabbits with sludgy urine the Isc, a measure of Na+ transport, was up to 46 microA/cm2 and averaged 12 microA/cm2, seven times the Isc of rabbits with nonsludgy urine. The increased Isc was entirely amiloride sensitive. Noise analysis showed it to arise entirely from increased channel density, without change in single-channel current. With one possible exception, we could not find bacterial infection or abnormalities in plasma aldosterone, GFR, or urinary [Na+], [Ca2+], pH, or osmolality that could explain the condition. The exception is that some unidentified component accounting for half the osmolality of nonsludgy urine is absent or at low concentration in sludgy urine. It remains unknown why the condition develops and whether CaCO3 crystals cause high Isc or vice versa or whether both result from a third factor.
Glomerular response to verapamil by isolated spontaneously hypertensive rat kidney
Authors: Steele T. H. , Challoner-Hue L.
Published: December 2017

We investigated the possibility that altered cell calcium regulation may affect function of isolated Kyoto spontaneously hypertensive rat (SHR) kidneys as compared with kidneys from Wistar-Kyoto control (WKY) rats. The kidneys were perfused at 120 and 160 mmHg. At 120 mmHg, SHR glomerular filtration rate (GFR) was 0.24 +/- 0.04 compared with WKY GFR of 0.70 +/- 0.10 ml/min (P = 0.001). At 160 mmHg, SHR GFR was 0.48 +/- 0.05 compared with WKY GFR of 1.09 +/- 0.05 ml/min (P less than 0.001). At 120 mmHg, addition of norepinephrine increased renal vascular resistance (RVR) by 50% and decreased SHR GFR by 27% and WKY GFR by 57% (P = 0.04). At 160 mmHg, norepinephrine elicited similar changes. Addition of verapamil, 5-10 microM, in the presence of norepinephrine returned RVR to 100-110% of control. With verapamil at 120 mmHg, SHR GFR increased to 0.84 +/- 0.23 ml/min, a value 3.5 times that of control (P = 0.03). In contrast, WKY GFR in the presence of norepinephrine and verapamil was 0.97 +/- 0.07 ml/min, unchanged from control (P = 0.07). At 160 mmHg, norepinephrine and verapamil also failed to increase WKY GFR above control (P = 0.4) but increased SHR GFR to 52% above control (P = 0.03). Isolated SHR kidneys exhibited exaggerated GFR responses to verapamil but not to norepinephrine. Abnormal cell calcium regulation may underlie the marked decrease in GFR when SHR kidneys are perfused acutely at normotensive perfusion pressures.