bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of substance P on permeability of airway epithelial cells in culture
Authors: Yu X. Y. , Undem B. J. , Spannhake E. W.
Published: December 2017

Although substance P (SP) has been shown to mediate microvascular leakage in response to various stimuli, some data suggest that, in contrast, SP may play a protective role in the maintenance of airway epithelial integrity. To investigate the effect of SP on epithelial barrier function, we measured paracellular mannitol flux and the transepithelial potential difference (PD) of human bronchial epithelial (HBE) and canine bronchial epithelial (CBE) cells. Incubation of confluent cell cultures with SP had no effect on baseline flux. However, pretreatment inhibited the flux-enhancing effects of 0.5 ppm ozone by 50% in HBE cells and 40% in CBE cells and inhibited the ozone-induced decrease in PD in CBE cells by 54%. SP-afforded protection was reduced by the neurokinin (NK)-1 receptor antagonist CP-96,345.NK1 and NK3 receptor agonists also inhibited ozone-induced permeability, whereas an NK2 receptor agonist was without significant effect. These data indicate that SP exerts a protective effect on bronchial epithelial barrier function under conditions of challenge, which appears to be mediated in large part through NK1 receptors.
Expression of fibronectin mRNA splice variants by rabbit lung in vivo and by alveolar type II cells in vitro
Authors: Maniscalco W. M. , Watkins R. H. , Campbell M. H.
Published: December 2017

Fibronectin (FN) is a multidomain glycoprotein with putative functions in tissue development and repair. In repair of alveolar injury, FN may promote the transition of type II epithelial cells to type I epithelial cells. Alternative splicing of FN mRNA, including the EIIIA and EIIIB exons, results in protein isoforms that have cell, tissue, and developmental specificity. The present work found that FN mRNA with the EIIIA exon was in fetal, adult, and oxidant-injured lung. The EIIIB splice variant, however, was restricted to fetal lung and adult lung recovering from oxidant injury. Because alveolar type II cells in vitro express FN, we examined the splice variants in two conditions that induce FN [transforming growth factor-beta 1 (TGF-beta 1) treatment and time in culture]. TGF-beta 1 increased both EIIIA and EIIIB mRNA abundance by 10-fold. Increased EIIIA isoform immunostaining was also noted. Type II cells that spontaneously express FN at 72 h in vitro had increased EIIIA and EIIIB mRNA and increased immunostaining for EIIIA. Nuclear runoff showed induction of FN gene transcription at 72 h in vitro. Together, these data show differential FN splice variant expression in lung, with EIIIB mRNA restricted to fetal and recovering oxidant-injured lung. Furthermore, the transition of type II cells to a type I-like cell is accompanied by increased FN gene transcription and induction of both EIIIA and EIIIB mRNA.
Keratinocyte growth factor stimulates CFTR-independent fluid secretion in the fetal lung in vitro
Authors: Zhou L. , Graeff R. W. , McCray P. B. , Simonet W. S. , Whitsett J. A.
Published: December 2017

Keratinocyte growth factor (KGF) caused cystic dilation of mouse fetal lung explants in vitro, markedly increasing the luminal volume of lung buds and disrupting branching morphogenesis. Effects of KGF were dose dependent, were detected within 4 h of treatment, and were blocked by cycloheximide but not by actinomycin D, indicating that de novo protein synthesis mediated the response. Effects of KGF were inhibited by bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, and ouabain, an inhibitor of the Na(+)-K(+)-ATPase. KGF stimulated fluid secretion equally in lung buds from cystic fibrosis transmembrane conductance regulators (CFTR) -/- and wild-type embryos, indicating that the effects were mediated by CFTR-independent Cl- transport. Microelectrode studies demonstrated that, whereas KGF did not acutely alter the transepithelial potential difference (PD) across the respiratory epithelium, the PD decreased while luminal volume increased during chronic exposure. KGF inhibited expression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) mRNA, suggesting that KGF may inhibit Na+ absorption, which may contribute to KGF-induced fluid accumulation. KGF-induced fluid accumulation is driven by CFTR-independent Cl- transport and associated with decreased expression of alpha-ENaC.
Hydroxylation of salicylate in lungs of Fischer 344 rats: effects of aging and ozone exposure
Authors: Liu L. , Kumarathasan P. , Guenette J. , Vincent R.
Published: December 2017

The formation of 2,3-dihydroxybenzoic acid (DHBA) from salicylate was measured in the lungs and plasma to assay for the .OH in juvenile (2 mo), adult (9 mo), and senescent (24 mo) Fischer 344 male rats exposed to clean air, 1 part/million (ppm) ozone, or 2 ppm ozone for 2 h. Similar rates of distribution of salicylic acid in plasma and to the lungs were observed among air control animals of all age groups. Levels of 2,3-DHBA were about twice as high in the lungs of air control senescent rats compared with juvenile and adult rats (P < 0.05). Exposure to ozone resulted in 1.5- to 2-fold elevation of 2,3-DHBA in lungs and plasma of all age groups (P < 0.05), whereas levels of 2,5-DHBA were not changed significantly. There was no effect of age on the magnitude of 2,3-DHBA increase in the lungs or plasma after ozone exposure. The postulated source of .OH is chemical reduction of H2O2, which could be generated from increased age-dependent endogenous oxidant production or the age-independent reaction of ozone or macrophage-derived oxidants with the surfactant lining.
Lipopolysaccharide-induced goblet cell hypersecretion in the guinea pig trachea: inhibition by macrolides
Authors: Tamaoki J. , Takeyama K. , Yamawaki I. , Kondo M. , Konno K.
Published: December 2017

We studied the effects of macrolides on lipopolysaccharide (LPS)-induced airway goblet cell secretion in the guinea pig trachea. The goblet cell secretion was assessed in histological sections of the tracheal mucosa stained with alcian blue and periodic acid Schiff by arbitrarily determining mucus score, which is inversely related to the magnitude of mucus discharge. Inhalation of Escherichia coli LPS (5 mg/kg) caused a time-dependent decrease in mucus score, with the maximal response being from 542 +/- 49 to 92 +/- 20 arbitrary units (P < 0.001) after 3 h, which was accompanied by an increase in the number of neutrophils in the tracheal mucosa. The LPS-induced mucus discharge was inhibited by oral clarithromycin and erythromycin in a dose-dependent manner (5 and 10 mg/kg), whereas amoxicillin and cefaclor had no effect. Each dose of clarithromycin and erythromycin, but not amoxicillin or cefaclor, likewise attenuated the LPS-induced recruitment of neutrophils. These results suggest that LPS stimulates goblet cell secretion and neutrophil accumulation in the airways and that macrolides may be of value in protecting against neutrophil-associated airway hypersecretion.
Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells
Authors: Aoshiba K. , Rennard S. I. , Spurzem J. R.
Published: December 2017

Apoptosis is an important process maintaining cell number and tissue structure. To determine whether cell-extracellular matrix (ECM) and cell-cell interactions modulate apoptosis in bronchial epithelium, we cultured human bronchial epithelial cells in different conditions and evaluated the cells for apoptosis. We found that plating cells in conditions that prevent cell-ECM adhesion induced apoptosis. Plating cells on type I collagen, fibronectin, and biosynthesized matrix prevented apoptosis, due at least in part to integrin-mediated adhesion. When cells were cultured at high density but under conditions preventing cell-substratum adhesion, aggregation occurred. Apoptosis was inversely correlated with aggregation. Cell-cell adhesion in these conditions was mediated at least partly by integrins containing alpha v. Cell aggregation was not associated with activation of a signaling pathway that is usually activated by cell-ECM adhesion, phosphorylation of focal adhesion kinase, but was associated with Bcl-2 protein expression, consistent with the concept that Bcl-2 protects against apoptosis. We conclude that both cell-ECM and cell-cell interactions, likely mediated in part by integrins, modulate apoptosis in bronchial epithelium.
Mouse strain differences in ozone dosimetry and body temperature changes
Authors: Slade R. , Watkinson W. P. , Hatch G. E.
Published: December 2017

Strain differences in susceptibility to inhaled ozone (O3) have been observed in mice, with C57BL/6J (B6) mice reported to be more sensitive than C3H/HEJ (C3) mice when exposed to equal concentrations of O3. To determine whether differences in the delivered dose of O3 to the lung could help explain these differences, C3 and B6 mice were exposed to 18O-labeled ozone (18O3), and the resulting 18O concentrations in pulmonary tissues were monitored as an indicator of O3 delivered dose. Body core temperatures (Tco) of similarly treated mice were measured during O3 exposures (using surgically implanted temperature probes) in an effort to correlate lung O3 dose to changes in basal metabolism. Immediately after exposure to 18O3, C3 mice had 46% less 18O (per mg dry wt) in lungs and 61% less in tracheas than B6 mice. Nasal 18O tended to be lower in the C3 mice, but these differences were not significant. Although both strains responded to the O3 exposure with significant decreases in Tco, C3 mice had a 70% greater mean temperature x time product decrease during the exposure than B6 mice. These results suggest that the strain differences in O3 susceptibility may be due to differences in O3 dose to the lung, which may be related to differences in the ability of the mice to lower their Tco in response to O3 exposure.
Role of membrane potential in hypoxic inhibition of L-arginine uptake by lung endothelial cells
Authors: Zharikov S. I. , Herrera H. , Block E. R.
Published: December 2017

System y+ accounts for the majority of L-arginine transport by pulmonary artery endothelial cells (PAEC). Given that membrane potential is a driving force for transport via system y+, we examined the hypothesis that hypoxia inhibits this transport by decreasing membrane potential. Porcine PAEC or plasma membrane vesicles derived from these cells were exposed to normoxia (room air-5% CO2) or hypoxia (0% O2-95% N2-5% CO2). After exposure, L-[3H]arginine transport and/or accumulation of the lipophilic cation [3H]tetraphenylphosphonium, a quantitative sensor of changes in cell membrane potential, were measured. Hypoxia caused reversible time-dependent decrease in L-arginine transport and membrane potential in PAEC and in plasma membrane vesicles. Comparable decreases in membrane potential and L-arginine transport by PAEC were also observed after depolarization induced by KCl or ouabain. Hyperpolarization, induced by valinomycin, increased membrane potential and L-arginine transport in PAEC and plasma membrane vesicles. Valinomycin also prevented the hypoxia-mediated decreases in membrane potential and L-arginine transport in PAEC. These results indicate that hypoxia-induced plasma membrane depolarization is responsible for reduced L-arginine transport by system y+ in hypoxic porcine PAEC.
Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells
Authors: Zhang X. L. , Danto S. I. , Borok Z. , Eber J. T. , Martin-Vasallo P. , Lubman R. L.
Published: December 2017

The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be glycosylated differently from kidney and other tissues. These differences appear to be due to organ- or cell-specific posttranslational processing of the beta 1-subunit and may result in altered regulation of sodium pumps in lung compared with other epithelia.
Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production
Authors: Murakami K. , Okajima K. , Uchiba M. , Johno M. , Nakagaki T. , Okabe H. , Takatsuki K.
Published: December 2017

We investigated the effect of activated protein C (APC) on pulmonary vascular injury and the increase in tumor necrosis factor (TNF) levels in lipopolysaccharide (LPS)-treated rats to determine whether APC reduces LPS-induced endothelial damage by inhibiting cytokine production. Intravenously administered LPS (5 mg/kg) induced pulmonary vascular injury, as indicated by an increase in the lung wet-to-dry weight ratio. LPS-induced pulmonary vascular injury was prevented by APC but not by active site-blocked factor Xa [dansyl glutamyl-glycyl-arginyl chloromethyl detone-treated activated factor X (DEGR-Xa)], a selective inhibitor of thrombin generation, or inactivated APC [diisopropyl fluorophosphate-treated APC (DIP-APC)]. APC, but not DEGR-Xa or DIP-APC, significantly inhibited the LPS-induced increase in the plasma level of TNF. APC significantly inhibited the production of TNF by LPS-stimulated monocytes in a dose-dependent fashion in vitro, but DIP-APC did not. APC did not inhibit the functions of activated neutrophils in vitro. These findings suggest that APC prevented LPS-induced pulmonary vascular injury by inhibiting TNF production by monocytes and not via its anticoagulant activity. The serine protease activity of APC appears to be essential for inhibition of TNF production.
Muscarinic-induced mucin secretion and intracellular signaling by hamster tracheal goblet cells
Authors: Steel D. M. , Hanrahan J. W.
Published: December 2017

Hamster tracheal epithelial cell cultures were used to investigate muscarinic regulation of high-molecular-weight glycoconjugate (HMWG) secretion by airway goblet cells. HMWG were radiolabeled with N-acetyl-D-[1-(3)H]glucosamine, precipitated with trichloroacetic acid and phosphotungstic acid, and counted by liquid scintillation. Carbachol (100 microM) increased HMWG secretion (166.6 +/- 18.7%, P < 0.001, n = 20), and this response was blocked by the muscarinic receptor antagonist atropine. Ca2+ may not be essential for carbachol response since 1) carbachol-activated secretion was not inhibited by chelating extracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by reducing both extracellular and intracellular Ca2+ with BAPTA-acetoxymethyl ester in low-Ca2+ medium; 2) the carbachol response was only partially blocked in low-Ca2+ medium; and 3) calcium ionophore did not stimulate HMWG secretion. However, carbachol-stimulated secretion was abolished by pertussis toxin (PTX), indicating the involvement of a PTX-sensitive guanine nucleotide-binding regulatory protein (G protein), and by the protein kinase C (PKC) inhibitor chelerythrine chloride. Furthermore, carbachol-stimulated secretion was not inhibited by overnight incubation with phorbol 12-myristate 13-acetate. In conclusion, carbachol-stimulated secretion of HMWG appears to be coupled to a PTX-sensitive G protein and requires the activation of a phorbol ester-insensitive PKC isoform.
Role of acidic Ca2+-independent phospholipase A2 in synthesis of lung dipalmitoyl phosphatidylcholine
Authors: Fisher A. B. , Dodia C.
Published: December 2017

Dipalmitoyl phosphatidylcholine (deltaPC) synthesis by lung epithelium occurs in part by a deacylation/reacylation pathway utilizing phospholipase A2 (PLA2) and an acyl transferase. The role of acidic Ca2+-independent PLA2 (aiPLA2) in this pathway was investigated using a transition-state analog enzyme inhibitor [1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33)]. Granular pneumocytes were isolated from rat lung with elastase and were maintained in primary culture for 24 h on microporous membranes in the presence of radiolabeled choline or free fatty acids (palmitate plus oleate). Disaturated phosphatidylcholine (DSPC) was determined by osmication chromatography. Incorporation (nmol/mg protein) into DSPC at 24 h incubation was 11.9 +/- 0.2 for [3H]choline and 12.1 +/- 0.04 for [3H]palmitate. In the presence of 3 mol% MJ33, incorporation of [3H] choline and [3H]palmitate was decreased by 37 and 69%, respectively, and DSPC pool size (microg/mg cell protein) decreased by 9% (P < 0.05). A similar decrease in radiolabel incorporation was observed with 2 h of incubation. The presence of p-bromophenacyl bromide (20 microm) had a significantly smaller effect that was additive with that of MJ33. After 24 h of labeling and 4 h of chase with unlabeled substrate, there was a significant decrease of radiolabel in DSPC that was inhibited by MJ33. Under all experimental conditions, MJ33 resulted in either no change or a modest increase of radiolabel in the cellular unsaturated PC fraction. These results indicate that aiPLA2 has a major role in DSPC synthesis by granular pneumocytes.
Pharmacological heterogeneity of constrictions mediated by endothelin receptors in rat pulmonary arteries
Authors: Higashi T. , Ishizaki T. , Shigemori K. , Nakai T. , Miyabo S. , Inui T. , Yamamura T.
Published: December 2017

The endothelin (ET) receptors mediating rat pulmonary arterial constrictions were investigated. ET-1 and ET-3 constricted both isolated intrapulmonary artery (IPA) and extrapulmonary artery (EPA), with ET-1 having a potency approximately 10 times that of ET-3. The ET(B) selective agonist IRL-1620 produced constriction of only IPA. The ET(B) selective antagonist BQ-788 suppressed the ET-1-induced constriction of IPA only. The ET(A) selective antagonist BQ-123 more effectively antagonized the ET-1-induced constriction of EPA than that of IPA. The combination of BQ-123 and BQ-788 increased the antagonistic response to ET-1 in IPA but not in EPA. Then, large constriction remained in IPA and EPA. The receptor nonselective antagonist PD-145065 almost completely inhibited the ET-1-induced constriction of IPA and EPA. BQ-123 completely inhibited the ET-3-induced constriction of EPA; however, it partially suppressed that of IPA. The combination of BQ-123 and BQ-788 completely inhibited the ET-3-induced constriction of IPA. These results demonstrate that the ET-1-induced constrictions are mediated by both ET(A) and ET(B) in IPA and by ET(A) in EPA. Because of differences in sensitivity to ET receptor antagonists for ET-1- and ET-3-induced constrictions, pharmacological heterogeneity of ET(A) is suggested. Additionally, endothelial denudation affected the ET-3-induced constriction of EPA, but not of IPA, and it didn't affect the response to ET-1. This suggests that the vasodilatory effect of endothelium on ET-3-induced vasoconstrictions varies depending on pulmonary vascular regions.
Thrombin inhibits myosin light chain dephosphorylation in endothelial cells
Authors: Shasby D. M. , Stevens T. , Ries D. , Moy A. B. , Kamath J. M. , Kamath A. M. , Shasby S. S.
Published: December 2017

Histamine and thrombin increase myosin light-chain kinase-mediated phosphorylation of myosin light chain (MLC) in human umbilical vein endothelial cells (HUVEC). The increase in MLC phosphorylation caused by thrombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesized that some of the longer duration of the increase in MLC phosphorylation caused by thrombin was because of inhibition of myosin dephosphorylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A protein phosphatases, caused a time-dependent increase in MLC phosphorylation in unstimulated HUVEC. As thrombin-stimulated phosphorylation approached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no increase at the peak of thrombin stimulation. In HUVEC in which cell calcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coinciding with the peak of thrombin stimulation. However, when phosphatase activity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC not only by increasing cell calcium but also by inhibiting calyculin A-sensitive dephosphorylation of MLC.
Stabilization of lung surfactant particles against conversion by a cycling interface
Authors: Hall S. B. , Hyde R. W. , Kahn M. C.
Published: December 2017

The large active particles of pulmonary surfactant are depleted in patients with acute respiratory distress syndrome and in animal models of this disorder. We studied in vitro conversion of large to small particles, separated by differential sedimentation, to determine how factors lavaged from rabbits injured by intravenous oleic acid would affect conversion. In half-filled test tubes rotated end over end, samples from injured animals increased the recovery of large particles from 40 +/- 6% of uncycled samples for controls to 62 +/- 21%. We hypothesized that proteins in the injured samples, and perhaps also the proteinase inhibitors used previously to block conversion (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222-230, 1990), stabilized surfactant particles by limiting access to the cycling interface. Hemoglobin, neutrophil elastase, and alpha1-antiproteinase (alpha1-PI) oxidized to eliminate its antiproteinase activity all stabilized large particles against conversion. Hemoglobin was most effective, increasing recovery from 18 +/- 5% for controls to 86 +/- 5% with 0.4 mg/ml hemoglobin. Native alpha1-PI had no effect on conversion. Our results suggest that acceleration of normal conversion is unlikely to explain the depletion of large particles in injured lungs. They also suggest that conversion of surfactant particles separated by differential sedimentation requires no proteinase susceptible to inhibition by alpha1-PI. They provide an alternate hypothesis related to interfacial effects rather than proteinase inhibition for the previously reported effect of alpha1-PI on conversion of particles separated according to density.
SP-A enhances phagocytosis of Klebsiella by interaction with capsular polysaccharides and alveolar macrophages
Authors: Kabha K. , Schmegner J. , Keisari Y. , Parolis H. , Schlepper-Schaeffer J. , Ofek I.
Published: December 2017

We found that surfactant protein A (SP-A) enhances phagocytosis of Klebsiella pneumoniae K21a but not of K2 serotypes by alveolar macrophages. SP-A interacted with the capsule of K21a (containing Man alpha1 Man sequences) as shown by SP-A-induced agglutination of the bacteria, by binding of SP-A-coated particles onto the bacterial surface, and by binding of SP-A to immobilized parent K21a strain and recombinant strains that switched their capsule from K2 to K21a. In contrast, only marginal binding of SP-A to K2 parent strain (lacking this sequence) could be detected. Furthermore, binding of capsular polysaccharide of K21a to immobilized SP-A was inhibited by mannan but not by lipopolysaccharide and K2 capsular polysaccharide. SP-A-treated macrophages bound increased numbers of parent K21a strain and recombinant strains of K21a capsule type but considerably less parent K2 strain. SP-A also enhanced killing of K21a strains by macrophages. The enhanced binding of K21a by macrophages pretreated with SP-A was inhibited by mannan, suggesting that binding is mediated by the mannose receptor on macrophages. We conclude that SP-A increases phagocytosis of the Klebsiella by two mechanisms, one of which is by serving as an opsonin, which binds to the capsular polysaccharides of the bacteria and potentially to SP-A receptors on the macrophages, and the other by activating the macrophages, resulting in increased activity of the mannose receptor.
Effect of anion transport inhibition on mucus secretion by airway submucosal glands
Authors: Inglis S. K. , Corboz M. R. , Taylor A. E. , Ballard S. T.
Published: December 2017

To model the airway glandular defect in cystic fibrosis (CF), the effect of anion secretion blockers on submucosal gland mucus secretion was investigated. Porcine distal bronchi were isolated, pretreated with a Cl- secretion blocker (bumetanide) and/or a combination of blockers to inhibit HCO3- secretion (dimethylamiloride, acetazolamide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), and then treated with acetylcholine (ACh), a glandular liquid and mucus secretagogue. Bronchi were then fixed, sectioned, and stained for mucins. Each gland duct was ranked for mucin content from zero (no mucin) to five (duct completely occluded with mucin). Untreated bronchi, bronchi treated only with ACh, and ACh-treated bronchi that received either bumetanide or the HCO3- secretion blockers all exhibited low gland duct mucin content (1.18 +/- 0.34, 0.59 +/- 0.07, 0.65 +/- 0.03, and 0.83 +/- 0.11, respectively). However, pretreatment with both Cl- and HCO3- secretion blockers before ACh addition resulted in substantial and significant ductal mucus accumulation (3.57 +/- 0.22). In situ videomicroscopy studies of intact airways confirmed these results. Thus inhibition of the anion (and presumably liquid) secretion response to ACh leads to mucus obstruction of submucosal gland ducts that resembles the early pathological changes observed in CF.
Cytokines regulate membrane-bound leukocyte elastase on neutrophils: a novel mechanism for effector activity
Authors: Owen C. A. , Campbell M. A. , Boukedes S. S. , Campbell E. J.
Published: December 2017

Membrane-bound leukocyte elastase activity on neutrophils may have potent proinflammatory effects. Herein, we report the effects of tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), N-formyl-leucyl-methionyl-phenylalanine (fMLP), and interleukin-8 (IL-8) on membrane-bound elastase expression. TNF-alpha or PAF alone induced only approximately two- to threefold increases in membrane-bound elastase but exhibited marked dose- and time-dependent priming effects for subsequent stimulation with fMLP or IL-8 (up to 20-fold increases in membrane-bound human leukocyte elastase compared with unstimulated cells). Optimally PAF-primed and fMLP-stimulated cells expressed 1.105 +/- 0.25 (SD) x 10(-17) mol [6.65 +/- 1.51 (SD) x 10(6) molecules] membrane-bound elastase activity/cell or approximately 12% of the content of unstimulated cells. Elastase binds to the cell surface by a charge-dependent mechanism since 1) incubation of cells with cationic molecules abrogated agonist-induced upregulation of membrane-bound elastase and 2) elastase was progressively eluted from the cell surface by solutions with increasing ionic strength. Thus interactions between proinflammatory mediators strikingly upregulate membrane-bound elastase on neutrophils, which may promote inflammatory responses and/or contribute to tissue injury.
Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats
Authors: Yue G. , Matalon S.
Published: December 2017

We instilled 4 ml isotonic fluid containing trace amounts of fluorescently labeled dextran (molecular mass 150 kDa) in the lungs of rats exposed to either 85% O(2) for 7 days or to 85% O(2) for 7 days and 100% O(2) for 3 days. We withdrew the fluid every hour for a 3-h period and calculated alveolar fluid clearance (AFC) from changes in dextran concentration. Postinstillation (3 h), AFC values in the control and the two hyperoxic groups were 51 +/- 1, 63 +/- 2, and 62 +/- 3 (SE), respectively (%instilled volume; n > or = 5; P < 0.05). Addition of either 1 mM amiloride or N-ethyl-N-isopropyl amiloride (EIPA) in the instillate decreased the AFC values in all groups 3 h later to approximately 30% of instilled volume. Instillation of phenamil, an irreversible blocker of epithelial Na+ channels into the lungs of rats exposed to 85% O(2) for 7 days and 100% O(2) for 2 days, resulted in a significant increase of their extravascular lung fluid volumes 24 h later. These results demonstrate the existence of EIPA-inhibitable Na+ channels in alveolar epithelial cells in vivo and indicate that an increase in Na+ transport plays an important role in limiting the amount of alveolar edema in O(2)-damaged lungs.
Effect of hyperoxia on adhesion molecule expression in human endothelial cells and neutrophils
Authors: Suzuki Y. , Aoki T. , Takeuchi O. , Nishio K. , Suzuki K. , Miyata A. , Oyamada Y. , Takasugi T. , Mori M. , Fujita H. , Yamaguchi K.
Published: December 2017

To investigate the pathogenesis of pulmonary oxygen toxicity, we examined the effect of hyperoxia on adhesion molecule expression in cultured human pulmonary artery endothelial cells (HPAEC) and human umbilical vein endothelial cells (HUVEC). Endothelial cell monolayers were exposed to either hyperoxic (90% O(2)-5% CO(2)) or normoxic (21% O(2)-5% CO(2)) conditions for various periods. The level of intercellular adhesion molecule (ICAM)-1 expression had increased in hyperoxia-exposed HPAEC and HUVEC at 48 h (194 +/- 38 and 233 +/- 56%, respectively; P < 0.001) and at 72 h (200 +/- 43 and 223 +/- 52%, respectively; P < 0.001) compared with normoxic conditions. These hyperoxia-induced ICAM-1 expressions were dose dependently attenuated by a protein kinase C inhibitor (H-7). In contrast, the levels of P-selectin and E-selectin expression in HPAEC and HUVEC were unchanged. The levels of ICAM-1 mRNA and the numbers of adherent neutrophils were increased in HPAEC and HUVEC at 48 and 72 h of hyperoxia. On the other hand, hyperoxia caused neutrophil H(2)O(2) production without affecting the level of CD11/CD18 expression. These results suggest that increased ICAM-1 expression in endothelial cells plays an important role in neutrophil accumulation during hyperoxia.
Effects of hyperoxia on type II cell Na-K-ATPase function and expression
Authors: Carter E. P. , Wangensteen O. D. , O'Grady S. M. , Ingbar D. H.
Published: December 2017

Alveolar fluid is resorbed using active Na+ transport primarily through basolateral sodium-potassium-adenosinetriphosphatase (Na-K-ATPase) and apical Na+ channels that are particularly dense on the alveolar type II (ATII) epithelial cells. During lung injury with pulmonary edema, continued or accelerated Na+ and fluid resorption is critical for a favorable outcome. However, little is known of how ATII cell Na+ transport is affected during injury. These experiments examined the effects of acute lung injury on ATII cell Na-K-ATPase activity and expression using an established model of rats exposed to 100% O(2) for 60 h. Na-K-ATPase activity of ATII cells isolated immediately after exposure was assessed by ouabain-sensitive (86)Rb+ uptake in intact cells and by ouabain-sensitive P(i) production by cell membranes. In the presence of 1 mM ouabain, ouabain-sensitive Rb+ uptake was not different between normoxic and hyperoxic cells, but the apparent Na-K-ATPase maximal velocity (Vmax) of hyperoxic cell membranes was 75 +/- 8% of normoxic membranes (P < 0.05). On Western blots of ATII cell membranes, alpha1-subunit protein significantly decreased with hyperoxia (35 +/- 9% of normoxia; P < 0.05), whereas the amounts of the beta-subunit were unchanged (P > 0.05). On Northern blots of ATII cell total RNA, steady-state levels of both the alpha1- and beta1-subunit mRNA increased after hyperoxia (alpha1 = 2.5 +/- 1.3-fold; beta1 = 4.6 +/- 2.5-fold). Thus despite hyperoxic decreases in Na-K-ATPase Vmax and the amount of alpha1-protein, Rb+ uptake by Na-K-ATPase in intact cells was unchanged. The mRNA levels, protein amounts, and enzyme activity did not respond in parallel to hyperoxic injury, and the activity in intact cells correlated best with the amounts of the beta-subunit, the limiting component in de novo pump assembly in many tissues.
Separation of human alveolar macrophages by flow cytometry
Authors: Maus U. , Rosseau S. , Seeger W. , Lohmeyer J.
Published: December 2017

Alveolar macrophages (AM), which represent the major resident population of immunocompetent cells in the lower respiratory tract, have been implicated in the pathogenesis of acute lung injury in view of their exceptional capacity to release a large array of inflammatory mediators. The ex vivo analysis of these cells, accessible to bronchoalveolar lavage (BAL) is hampered by the fact that, under conditions of respiratory failure, the AM pool is heavily expanded by polymorphonuclear neutrophils (PMN), which necessitates separation of these cell populations. In the present study, we describe a flow cytometric approach to sort human AM obtained from BAL samples of both healthy volunteers (n = 10) and patients with severe pneumonia demanding mechanical ventilation (n = 10), using forward scatter and high autofluorescence characteristics to discriminate AM from PMN and lymphocytes. This technique yielded highly purified AM populations (>95%) as evidenced by morphological analysis, cytochemistry, and CD71 and CD14 expression of the sorted cells. The flow sorting process, per se, did not induce the expression of the acute-phase cytokine tumor necrosis factor-alpha (TNF-alpha) in control AM as determined by reverse transcriptase-polymerase chain reaction. Unstimulated and lipopolysaccharide-induced TNF-alpha protein secretion were comparable in sorted and unsorted AM as demonstrated by enzyme-linked immunosorbent assay. We suggest flow sorting of viable human AM as an efficient and nonperturbing separation technique to yield highly purified cell populations especially from PMN-rich BAL fluids of critically ill patients.
Effects of phospholipase A2, 12-lipoxygenase, and cyclooxygenase inhibitors in the feline pulmonary bed
Authors: Kaye A. D. , Nossaman B. D. , Smith D. E. , Ibrahim I. N. , Imig J. D. , Kadowitz P. J.
Published: December 2017

The effects of phosphonofluoridic acid, methyl-5,8,11,14-eicosatetraenyl ester (MAFP), a phospholipase A2 inhibitor, on pressor responses to angiotensin II (ANG II), norepinephrine (NE), serotonin (5-HT), the calcium channel opener BAY K 8644, and the thromboxane A2 mimic U-46619 were studied in the pulmonary vascular bed of the intact-chest cat. Under conditions of constant lobar blood flow, injections of ANG II, NE, 5-HT, U-46619, and BAY K 8644 into the lobar arterial perfusion circuit caused dose-related increases in lobar arterial pressure that were reproducible with respect to time. Infusion of MAFP into the perfused lobar artery at doses of 100 to 300 microg/kg for 10 min significantly reduced vasoconstrictor responses to ANG II; however, the phospholipase A2 inhibitor did not alter vasoconstrictor responses to BAY K 8644, 5-HT, NE, or U-46619. The combination of the higher dose of the phospholipase A2 inhibitor MAFP with the phospholipase C inhibitor U-73122 significantly affected vasoconstrictor responses to ANG II, NE, and 5-HT but not to BAY K 8644. In a separate series of animals, the effects of a lipoxygenase inhibitor, baicalein, were investigated. Infusion of baicalein into the perfused lobar artery at doses of 100 microg/kg for 10 min significantly reduced vasoconstrictor responses to ANG II but not the vasoconstrictor responses to BAY K 8644, 5-HT, NE, or U-46619. In a final series of experiments, the effects of a cyclooxygenase inhibitor, meclofenamate, were investigated, and intravenous injection of the meclofenamate at a dose of 2.5 mg/kg did not significantly affect vasoconstrictor responses to ANG II, 5-HT, BAY K 8644, NE, or U-46619. These data provide support for the hypothesis that pulmonary vasopressor responses to ANG II are mediated, in part, by the activation of phospholipase A2, phospholipase C, and the lipoxygenase pathway in the pulmonary vascular bed of the cat.
Increased cAMP levels in stimulated neutrophils inhibit their adhesion to human bronchial epithelial cells
Authors: Bloemen P. G. , van den Tweel M. C. , Henricks P. A. , Engels F. , Kester M. H. , van de Loo P. G. , Blomjous F. J. , Nijkamp F. P.
Published: December 2017

Bronchial epithelial cells express the intercellular adhesion molecule-1 that mediates binding of activated neutrophils via interaction with Mac-1 and/or leukocyte function-associated antigen-1. In this study, we examined whether increased intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) affected neutrophil adhesion to the human bronchial epithelial cells. It was found that the N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated neutrophil adhesion was concentration dependently inhibited when the cAMP analogs dibutyryl adenosine 3',5'-cyclic monophosphate or 8-bromoadenosine 3',5'-cyclic monophosphate were present. The beta-adrenergic receptor agonists isoprenaline and salmeterol, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), were also able to inhibit the fMLP-stimulated adhesion of neutrophils to bronchial epithelial cells. These agonists in combination with IBMX significantly increased the intracellular cAMP level in both neutrophils and epithelial cells. Preincubation of neutrophils with the long-acting beta2-adrenergic receptor agonist salmeterol (in the presence of IBMX) inhibited their fMLP-stimulated adhesion to epithelial cells, whereas pretreatment of epithelial cells did not influence the adhesion process. When ethanol-fixed epithelium was used, salmeterol pretreatment also diminished the adhesion of stimulated neutrophils. Moreover, combinations of salmeterol or isoprenaline with IBMX inhibited fMLP-upregulated Mac-1 expression. Therefore, we conclude from these data that elevation of intracellular cAMP in the neutrophil inhibits stimulated neutrophil adhesion to bronchial epithelial cells via Mac-1.
Nitric oxide inhibits ACh-induced intracellular calcium oscillations in porcine tracheal smooth muscle
Authors: Prakash Y. S. , Kannan M. S. , Sieck G. C.
Published: December 2017

With real-time confocal microscopy, the effect of three nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and diethylamine NO adduct, on the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh) was examined. ACh initiated propagating [Ca2+]i oscillations in TSM cells, which were inhibited by NO donors. 8-Bromoguanosine 3',5'-cyclic monophosphate slowed the frequency of [Ca2+]i oscillations but did not completely inhibit oscillations, suggesting that the effects of NO donors are only partially mediated via guanosine 3',5'-cyclic monophosphate-dependent mechanisms. After preexposure to NO donors, ACh induced a small biphasic [Ca2+]i response that was blocked by nifedipine, suggesting a lack of effect on Ca2+ influx through voltage-gated channels. In addition, NO donors did not inhibit Ca2+ influx induced by BAY K 8644. The [Ca2+]i response to caffeine was inhibited by NO donors, indicating inhibition of sarcoplasmic reticulum (SR) Ca2+ release. When Ca2+ influx and SR Ca2+ reuptake were blocked, basal [Ca2+]i increased, and this was inhibited by NO donors, suggesting enhanced Ca2+ efflux. These results indicate that NO donors inhibit [Ca2+]i oscillations by blocking SR Ca2+ release and enhancing Ca2+ extrusion.
Sickle erythrocytes induce prostacyclin and thromboxane synthesis by isolated perfused rat lungs
Authors: Ibe B. O. , Morris J. , Kurantsin-Mills J. , Raj J. U.
Published: December 2017

The role of eicosanoids in the pathogenesis of acute or chronic lung syndrome in sickle cell disease is unknown. We investigated the synthesis of prostacyclin (PGI2), thromboxane (Tx) A2, and prostaglandin (PG) E2 by three groups of isolated rat lungs perfused with buffer (GPBS), normal (HbAA), and sickle (HbSS) erythrocyte suspensions. Isolated lungs were perfused at a constant pressure and flow rate (Q) of 40 ml x kg(-1) x min(-1) with GPBS or 7% erythrocyte suspensions for 15 min. Autologous platelet-rich plasma (PRP) was added, and perfusion was continued for 15 min and then at two times Q for another 15 min. Perfusate samples were assayed for the specific eicosanoids. Perfusate level of PGI2 in GPBS lungs was the least among the three groups. However, the PGI2 level in HbSS lungs was 90% higher than from HbAA lungs after 15 min of perfusion and was 180% higher on perfusion with PRP. Additionally, coperfusion of erythrocytes and PRP augmented perfusate levels of TxA2 and PGE2 over 1,000% more in HbSS than HbAAlungs. These data show that HbSS erythrocytes increased perfusate levels of the eicosanoids, suggesting increased synthesis, perhaps due to aberrant erythrocyte-endothelium interactions.
Pulmonary endothelial thiazine uptake: separation of cell surface reduction from intracellular reoxidation
Authors: Merker M. P. , Bongard R. D. , Linehan J. H. , Okamoto Y. , Vyprachticky D. , Brantmeier B. M. , Roerig D. L. , Dawson C. A.
Published: December 2017

The objective of this study was to further evaluate the hypothesis that the accumulation of thiazine dyes, such as methylene blue, by cultured bovine pulmonary arterial endothelial cells involves reduction on the cell surface, followed by diffusion of the lipophilic reduced form of the dye into the cells and intracellular reoxidation to the relatively membrane-impermeant hydrophilic form. The specific question addressed was whether inhibition of methylene blue uptake by cyanide and azide is via inhibition of extracellular reduction or inhibition of intracellular reoxidation. We used the cell membrane-impermeant ferricyanide ion as a secondary electron acceptor to measure the extracellular reduction of methylene blue independently from its uptake by the cells. In addition, toluidine blue O, incorporated into an acrylamide polymer so that it could not permeate the cells in either its reduced or oxidized forms, was used to examine the effects of cyanide and azide on the extracellular reduction. Microscopic observations of the effect of the inhibitors on the intracellular accumulation of methylene blue were also made. The results indicate that the reduction and intracellular sequestration are separate processes and that, in doses that inhibited intracellular reoxidation, and therefore uptake and sequestration, neither cyanide nor azide had an inhibitory effect on extracellular reduction. The intracellular distribution of the observable oxidized form of the dye was consistent with oxidation of the reduced dye within subcellular organelles. The demonstration that extracellular reduction and intracellular sequestration are separate events is consistent with the hypothesized sequence of events.
Pulmonary mechanical and immunologic dysfunction in a murine model of AIDS
Authors: Hartsfield C. L. , Lipke D. , Lai Y. L. , Cohen D. A. , Gillespie M. N.
Published: December 2017

Human immunodeficiency virus-infected patients occasionally exhibit alveolar septal wall thickening and decreases in gas diffusion capacity, but the mechanism underlying these abnormalities is unknown. The present study evaluated septal wall thickness and gas exchange properties in a murine model of the acquired immunodeficiency syndrome and determined whether there were alterations in lung lymphocyte deposition and activation that could contribute to changes in respiratory structure and function. Although alveolar septal wall thickness did not differ from control at 1, 2, and 4 wk postimmunosuppressive virus infection, at 8 wk after infection, septal wall thickness was substantially increased. Immunohistochemical evaluation at this time revealed marked increases in the septal wall deposition of fibronectin and collagen type IV. Pulmonary function tests on anesthetized mice with virus-induced septal wall thickening demonstrated that, although total lung capacity, compliance, and functional residual capacity were unaltered, diffusion capacity for carbon monoxide was significantly impaired. A diffuse nonspecific interstitial pneumonitis was present in lungs of immunodeficient mice, and flow cytometry indicated that both lymphocytes and macrophages were activated. Reverse transcriptase-polymerase chain reaction analysis of lung lymphocytes demonstrated enhanced mRNA expression for several cytokines known to affect lung structure. These results show that impaired gas exchange occurs in a murine model of acquired immunodeficiency syndrome and suggest that such alterations may be mediated by elaboration of cytokines from activated lung lymphocytes and macrophages.
RNA in polysomes is an inhibitor of manganese superoxide dismutase RNA-binding protein activity
Authors: Chung D. J. , Clerch L. B.
Published: December 2017

A redox-sensitive protein in rat lung binds to the 3'-untranslated region (3'-UTR) of manganese superoxide dismutase (Mn-SOD) mRNA; the activity of this Mn-SOD RNA-binding protein (Mn-SOD-BP) is greater in 12,000-g supernatant fractions (S12) from neonates than in S12 from adults (H. Fazzone, A. Wangner, and L. B. Clerch. J. Clin. Invest. 92: 1278-1281, 1993). To determine the mechanism underlying this developmental difference, lung subcellular fractions were tested for their effect on Mn-SOD-BP activity. Protein in the 130,000-g supernatant (S130) of lung extracts bound the 3'-UTR. However, the developmental difference in binding was not present in S130. The 130,000-g pellet (P130) did not bind the 3'-UTR; rather, it contained an inhibitor of Mn-SOD-BP activity. Addition of P130 to S130 decreased RNA binding in a dose-dependent manner. Furthermore, adult P130 was a more potent inhibitor of RNA-binding activity than neonatal P130. These data indicate that the developmental difference in Mn-SOD-BP activity is due, in part, to an inhibitor in P130. Biochemical characterization revealed that the inhibitor is an RNA that may participate in the posttranscriptional control of Mn-SOD gene expression.