bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of extracellular matrix synthesis by TNF-α and TGF-β1 in type II cells exposed to coal dust
Authors: Lee Yu-Chen , Rannels D. Eugene
Published: December 2017

Type II pulmonary epithelial cells respond to anthracite coal dust PSOC 867 with increased synthesis of extracellular matrix (ECM) components. Alveolar macrophages modulate this response by pathways that may involve soluble mediators, including tumor necrosis factor-α (TNF-α) or transforming growth factor-β1 (TGF-β1). The effects of TNF-α (10 ng/ml) and/or TGF-β1 (2 ng/ml) were thus investigated in dust-exposed primary type II cell cultures. In control day 1 or day 3 cultures, TNF-α and/or TGF-β1 had little or no effect on the synthesis of type II cellular proteins, independent of whether the cells were exposed to dust. With PSOC 867 exposure, where ECM protein synthesis is elevated, TNF-α and TGF-β1 further increased both the absolute and relative rates of ECM synthesis on day 3 but had little effect on day 1. Each mediator increased expression of fibronectin mRNA, as well as of ECM fibronectin content, in a manner qualitatively similar to their effects on synthesis. Thus TNF-α and TGF-β1 modulate both ECM synthesis and fibronectin content in coal dust-exposed type II cell cultures.
Alveolar type II-like cells release G-CSF as neutrophil chemotactic activity
Authors: Koyama Sekiya , Sato Etsuro , Masubuchi Takeshi , Takamizawa Akemi , Kubo Keishi , Nagai Sonoko , Izumi Takateru
Published: December 2017

We evaluated the potential of A549 cells, an alveolar type II epithelial cell line, to release granulocyte colony-stimulating factor (G-CSF), in addition to interleukin (IL)-8 and leukotriene B4, as neutrophil chemotactic activity (NCA). Human recombinant IL-1β stimulated A549 cells to release NCA in a time- and dose-dependent fashion. The released NCA was blocked by mouse anti-human G-CSF polyclonal antibody. Molecular-sieve column chromatography revealed that IL-1β induced the release of a 19- to 20-kDa chemotactic mass that was inhibited by anti-human G-CSF antibody. IL-1β stimulated the release of G-CSF in a dose-dependent fashion, but the time-dependent profile of G-CSF showed that the concentration of G-CSF declined after 48 h. Tumor necrosis factor (TNF)-α, Escherichia coli lipopolysaccharide (LPS), and bradykinin (BK) stimulated A549 cells to release NCA that was inhibited by anti-G-CSF antibody. The release of G-CSF in response to TNF-α, LPS, and BK was significantly increased. The similar concentrations of human recombinant G-CSF (10–1,000 pg/ml) as in the supernatant fluid induced neutrophil chemotaxis. G-CSF mRNA was expressed time and dose dependently at 4 h and declined after 4 h in response to IL-1β as evaluated by RT-PCR. The expression of G-CSF mRNA was also observed by TNF-α, LPS, and BK stimulation. These data suggest that type II alveolar epithelial cells may produce G-CSF as NCA and may participate in the regulation of leukocyte extravasation.
Sulfation of extracellular matrices modifies growth factor effects on type II cells on laminin substrata
Authors: Sannes Philip L. , Khosla Jody , Li Cheng-Ming , Pagan Ines
Published: December 2017

The alveolar basement membrane contains a variety of extracellular matrix (ECM) molecules, including laminin and sulfated glycosaminoglycans of proteoglycans. These mixtures exist within microdomains of differing levels of sulfate, which may specifically interact to be key determinants of the known capacity of the type II cell to respond to certain growth factors. Isolated type II cells were exposed to either acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), or keratinocyte growth factor (KGF; FGF-7) on culture wells precoated with laminin alone or in combination with chondroitin sulfate (CS), high-molecular-weight heparin, or their desulfated forms. Desulfated heparin significantly elevated FGF-1- and FGF-2-stimulated DNA synthesis, whereas desulfated CS and N-desulfated heparin elevated FGF-7-stimulated DNA synthesis by type II cells on laminin substrata. When FGF-1 was mixed into the various test matrix substrata, DNA synthesis was significantly increased in all cases. These results demonstrated that decreased levels of sulfate in ECM substrata act to upregulate responses to heparin-binding growth factors by alveolar epithelial cells on laminin substrata.
ET-1 modulates KCa-channel activity and arterial tension in normoxic and hypoxic human pulmonary vasculature
Authors: Peng Wei , Michael John R. , Hoidal John R. , Karwande S. V. , Farrukh Imad S.
Published: December 2017

The molecular mechanisms by which endothelin (ET)-1 induces pulmonary hypertension are poorly understood. We investigated the effects of ET-1 on outward K+currents of normoxic and chronically hypoxic human pulmonary arterial (PA) smooth muscle cells (HPSMCs). In normoxic HPSMCs, ET-1 has dual effects. In intact cells, 5 nM ET-1 activates the large-conductance and Ca2+-activated K+(KCa)-channel current [ IK(Ca)] by increasing intracellular Ca2+concentration, whereas it directly inhibits IK(Ca)in isolated membrane patches. At a higher concentration (10 nM), ET-1-induced IK(Ca)inhibition predominates. In hypoxic HPSMCs, ET-1 at 5 nM significantly reduces IK(Ca). The ETA-receptor antagonist BQ-123 reverses the ET-1-induced decrease in IK(Ca). Chronic BQ-123 treatment also prevents the hypoxia-induced decrease in IK(Ca). In PA rings obtained from human organ donors, ET-1 causes a concentration-dependent increase in tension. The ET-1-mediated increase in tension is reversed by a KCa-channel agonist. The increase in tension at the highest concentration studied (9 nM) was more pronounced in PA rings obtained from patients with chronic obstructive pulmonary disease. These results imply that an ET-1-induced decrease in IK(Ca)contributes to chronic hypoxia-induced pulmonary hypertension.
Role of G proteins in agonist-induced Ca2+sensitization of tracheal smooth muscle
Authors: Croxton Thomas L. , Lande Boris , Hirshman Carol A.
Published: December 2017

Increased sensitivity to intracellular Ca2+concentration ([Ca2+]) is an important mechanism for agonist-induced contraction of airway smooth muscle, but the signal transduction pathways involved are uncertain. We studied Ca2+sensitization with acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth muscle by measuring contractions at a constant [Ca2+] in strips permeabilized with α-toxin or β-escin. The peptide inhibitor G protein antagonist 2A (GP Ant-2A), which has selectivity for Gqover Gi, inhibited contractile responses to ET-1, ACh, and guanosine 5′- O-(3-thiotriphosphate) (GTPγS), but the proportional inhibition of ACh responses was less than that of ET-1. Pretreatment with pertussis toxin reduced ACh contractions but had no effect on those of ET-1 or GTPγS. Clostridium botulinum C3 exoenzyme, which inactivates Rho family monomeric G proteins, caused similar reductions in contractile responses to ACh, ET-1, and GTPγS. Farnesyltransferase inhibition, which inhibits Ras G proteins, reduced responses to ET-1. We conclude that the heterotrimeric G proteins Gqand Giboth contribute to Ca2+sensitization by ACh, whereas ET-1 responses involve Gqbut not Gi. Both Gqand Gipathways likely involve Rho family small G proteins. A Ras-mediated pathway also contributes to Ca2+sensitization by ET-1 in airway smooth muscle.
Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung
Authors: Yu Aimee Y. , Frid Maria G. , Shimoda Larissa A. , Wiener Charles M. , Stenmark Kurt , Semenza Gregg L.
Published: December 2017

Hypoxia-inducible factor (HIF)-1 is a basic helix-loop-helix transcription factor that transactivates genes encoding proteins that participate in homeostatic responses to hypoxia. Several of these downstream gene products, such as erythropoietin, vascular endothelial growth factor, heme oxygenase-1, and inducible nitric oxide synthase, may contribute to the pathogenesis of pulmonary hypertension. Previous studies demonstrated increased HIF-1 mRNA levels in rats and mice subjected to hypoxia. In this study, we have demonstrated spatial, temporal, and O2-dependent expression of HIF-1 protein. Immunoblot analysis revealed hypoxic induction of HIF-1 in all cultured pulmonary cell types assayed, including those derived from pulmonary arterial endothelium and smooth muscle, bronchial epithelium, alveolar macrophages, alveolar epithelium, and microvascular endothelium. In contrast to all other cell types, pulmonary arterial smooth muscle cells expressed HIF-1 under nonhypoxic conditions. Immunohistochemistry and immunoblot analysis of ferret lungs demonstrated pulmonary expression of HIF-1 in vivo. HIF-1 protein expression was induced maximally when lungs were ventilated with 0 or 1% O2for 4 h. On reoxygenation, HIF-1 was rapidly degraded, with a half-life of <1 min. These findings demonstrate that HIF-1 expression is tightly coupled to O2concentration in vivo and are consistent with the involvement of HIF-1 in the physiological and pathophysiological responses to hypoxia in the lung.
Regulation of ciliary beat frequency by both PKA and PKG in bovine airway epithelial cells
Authors: Wyatt T. A. , Spurzem J. R. , May K. , Sisson J. H.
Published: December 2017

Ciliary beating is required for the maintenance of lung mucociliary transport. We investigated the role of cyclic nucleotide-dependent protein kinases in stimulating ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). cAMP-dependent protein kinase (PKA) activity and cGMP-dependent protein kinase (PKG) activity were distinguished after DEAE-Sephacel chromatography of BBEC extracts. cAMP levels and PKA activity are increased in BBECs stimulated with 0.01–1 mM isoproterenol, with a corresponding increase in CBF. cGMP levels and PKG activity are increased in BBECs stimulated with 0.1–10 μM sodium nitroprusside, with a corresponding increase in CBF. Direct protein kinase-activating analogs of cAMP and cGMP (dibutyryl cAMP and 8-bromo-cGMP, respectively) also activate their specific kinases and stimulate CBF. Preincubation of BBECs with inhibitors of PKA or PKG [KT-5720 or Rp-8-( p-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphothioate] results in the inhibition of specific kinase activity as well as in the inhibition of CBF. These studies suggest that the activation of either PKA or PKG can lead to the stimulation of CBF in bovine airway epithelium.
Substance P and capsaicin release prostaglandin E2from rat intrapulmonary bronchi
Authors: Szarek John L. , Spurlock Beverly , Gruetter Carl A. , Lemke Sally
Published: December 2017

We hypothesized that substance P and capsaicin would cause the release of prostaglandin E2(PGE2) from intrapulmonary bronchi isolated from Sprague-Dawley rats. Substance P (1 μM) caused the release of PGE2, measured with enzyme immunoassay, from the isolated airway segments; PGE2release was inhibited by the neurokinin (NK)1-receptor antagonist, RP-67580, by inhibition of cyclooxygenase with meclofenamate, and by removal of the epithelium. The release of PGE2caused by capsaicin (1 μM) was similar in magnitude to that caused by substance P. The capsaicin-induced release of PGE2was inhibited by desensitization of sensory nerves with capsaicin and by RP-67580, meclofenamate, and epithelial denudation. We conclude that activation of NK1receptors on epithelium causes release of PGE2, which most likely represents the ultimate mediator of airway smooth muscle relaxation, produced by exogenous neuropeptides and by activation of the sensory nerve inhibitory system. Epithelial damage, such as that seen in asthmatic airways, would disrupt this protective system in the lungs, which could contribute to the development of airway disease.
Role of plasmalemmal caveolae in signal transduction
Authors: Shaul Philip W. , Anderson Richard G. W.
Published: December 2017

Caveolae are specialized plasmalemmal microdomains originally studied in numerous cell types for their involvement in the transcytosis of macromolecules. They are enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4°C. Once the identification of the marker protein caveolin made it possible to purify this specialized membrane domain, it was discovered that caveolae also contain a variety of signal transduction molecules. This includes G protein-coupled receptors, G proteins and adenylyl cyclase, molecules involved in the regulation of intracellular calcium homeostasis, and their effectors including the endothelial isoform of nitric oxide synthase, multiple components of the tyrosine kinase-mitogen-activated protein kinase pathway, and numerous lipid signaling molecules. More recent work has indicated that caveolae further serve to compartmentalize, modulate, and integrate signaling events at the cell surface. This specialized plasmalemmal domain warrants direct consideration in future investigations of both normal and pathological signal transduction in pulmonary cell types.
Nitric oxide synthesis by tracheal smooth muscle cells by a nitric oxide synthase-independent pathway
Authors: Jia Yanlin , Zacour Mary , Tolloczko Barbara , Martin James G.
Published: December 2017

Nitric oxide (NO) is known to be synthesized from l-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of CN bonds of compounds containing a -C(NH2)NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10−2to 10−4M), a compound containing -C(NH2)NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereasl-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10−7to 10−4M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors Nω-nitro-l-arginine (10−3M) and NG-monomethyl-l-arginine (10−3M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.
Clara cell secretory protein decreases lung inflammation after acute virus infection
Authors: Harrod Kevin S. , Mounday Amber D. , Stripp Barry R. , Whitsett Jeffrey A.
Published: December 2017

Clara cell secretory protein (CCSP) is an abundant 10-kDa polypeptide synthesized and secreted primarily by nonciliated bronchiolar epithelial cells in the mammalian lung. To determine the potential role of CCSP in pulmonary inflammation after acute viral infection, CCSP gene-targeted {CCSP-deficient [CCSP(−/−)]} mice were exposed to a recombinant E1- and E3-deficient adenoviral vector, Av1Luc1, intratracheally. Lung inflammation was markedly increased in CCSP(−/−) mice compared with wild-type control mice and was associated with an increased number of polymorphonuclear cell infiltrates and epithelial cell injury in both conducting airways and alveolar regions. Histological evidence of pulmonary inflammation in CCSP(−/−) mice was associated with increased production of cytokine (interleukin-1β and -6 and tumor necrosis factor-α) mRNA and protein, as well as chemokine (macrophage inflammatory protein-1α and -2 and monocyte chemoattractant protein-1) mRNA expression within the lung in response to adenoviral infection. Adenoviral-mediated gene transfer was decreased in CCSP(−/−) mice relative to wild-type mice as measured by luciferase enzyme activity in lung homogenates. The present study suggests that CCSP is involved in modulating lung inflammation during viral infection and supports a role for CCSP in lung host defense.
A1adenosine receptor-mediated Ins(1,4,5)P3generation in allergic rabbit airway smooth muscle
Authors: Abebe Worku , Mustafa S. Jamal
Published: December 2017

The signal transduction pathway for A1adenosine receptor in airway smooth muscle from allergic rabbits was studied by investigating the effect of the selective A1adenosine-receptor agonist N6-cyclopentyladenosine (CPA) on tissue levels of inositol 1,4,5-trisphosphate [Ins(1,4,5) P3] measured by protein binding assay. CPA caused a rapid, transient, and concentration-dependent elevation of Ins(1,4,5) P3in airways from allergic rabbits. The agonist also produced a concentration-dependent contraction of the airway preparations from these animals. Both the Ins(1,4,5) P3and contractile responses generated by CPA were attenuated by the phospholipase C (PLC) inhibitor U-73122, indicating the coupling of these responses to PLC. The CPA-induced Ins(1,4,5) P3production observed in the allergic rabbit tissues was also inhibited by the adenosine-receptor antagonist 8-( p-sulfophenyl)-theophylline, suggesting that the effect was mediated by A1adenosine receptors. On the other hand, the A2adenosine-receptor agonist CGS-21680 was ineffective in altering the tissue concentration of Ins(1,4,5) P3, indicating that A2adenosine receptors may not be involved in the activation of PLC in the allergic rabbit airway smooth muscle. In this preparation, the Gi-Goinhibitor pertussis toxin (PTX) attenuated the CPA-induced Ins(1,4,5) P3accumulation, providing evidence that the generation of Ins(1,4,5) P3by A1adenosine-receptor stimulation is coupled to a PTX-sensitive G protein(s). The results suggest that activation of A1adenosine receptors in allergic rabbit airway smooth muscle causes the production of Ins(1,4,5) P3via a PTX-sensitive G protein-coupled PLC, and this signaling mechanism may be involved, at least in part, in the generation of contractile responses. It is hypothesized that this process may contribute to adenosine-induced bronchoconstriction in allergic asthma.
Role of wall tension in hypoxic responses of isolated rat pulmonary arteries
Authors: Ozaki Masami , Marshall Carol , Amaki Yoshikiyo , Marshall Bryan E.
Published: December 2017

The changes in force developed during 40-min exposures to hypoxia (37 ± 1 mmHg) were recorded in large (0.84 ± 0.02-mm-diameter) and small (0.39 ± 0.01-mm-diameter) intrapulmonary arteries during combinations of mechanical wall stretch tensions (passive + active myogenic components), equivalent to transmural vascular pressures of 5, 15, 30, 50, and 100 mmHg, and active (vasoconstriction) tensions, stimulated by PGF2α in doses of 0, 25, 50, and 75% effective concentrations. Constriction was observed in all arteries during the first minute; however, at any active tension, the pattern of the subsequent response was a function of the stretch tension. At 5, 15, and 30 mmHg, the constriction decreased slightly at 5 min and then increased again to remain constrictor throughout. At 50 and 100 mmHg, the initial constriction was followed by persistent dilation. Hypoxic constrictor responses, most resembling those observed in lungs in vivo and in vitro, were observed when the mechanical stretch wall tension was equivalent to 15 or 30 mmHg and the dose of PGF2α was 25 or 50% effective concentration. These observations reconcile many apparently contradictory results reported previously.
Exercise effects on IFN-β expression and viral replication in lung macrophages after HSV-1 infection
Authors: Kohut M. L. , Davis J. M. , Jackson D. A. , Jani P. , Ghaffar A. , Mayer E. P. , Essig D. A.
Published: December 2017

Mice exercised to fatigue and exposed to herpes simplex virus type 1 (HSV-1) exhibit greater mortality than control mice. In this study, we examined lung macrophage resistance to HSV-1 after exercise in terms of both viral replication and interferon (IFN)-β production. We utilized the reverse transcriptase-rapid polymerase chain reaction to measure the IFN-β mRNA content in alveolar macrophages. IFN release was measured with a bioassay, and viral replication within the macrophage was assessed by plaque titration. Exercised (Ex) mice ran on a treadmill until fatigue while control (Con) mice remained in lanes above the treadmill. After exercise, alveolar macrophages were removed and incubated with HSV-1. Alveolar macrophage IFN-β mRNA was greater in Ex than in Con mice. Culture supernatant from infected macrophages showed a higher degree of IFN release and a higher number of infectious viral particles in Ex vs. Con mice. It is likely that the increase in IFN-β mRNA occurs in response to a higher degree of viral replication. These results suggest that macrophages from Ex mice are less resistant to infection with HSV-1.
Acetylcholine-induced liquid secretion by bronchial epithelium: role of Cl− and HCO 3 − transport
Authors: Trout Laura , Gatzy John T. , Ballard Stephen T.
Published: December 2017

Inhibitors of Cl− and[Formula: see text] secretion reduce acetylcholine-induced liquid, but not mucin, secretion by bronchial submucosal glands [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L372–L377, 1997]. The present study quantified contributions of Cl− and[Formula: see text] transport to volume and composition of acetylcholine-induced liquid secretion by airway epithelium. When distal bronchi were excised from 33 pigs and treated with 10 μM acetylcholine, the airways secreted 13.4 ± 0.7 μl ⋅ cm−2 ⋅ h−1. Bumetanide (10 μM) pretreatment reduced acetylcholine-induced liquid and Cl− secretion rates by ∼70%, but [Formula: see text] secretion fell by only 40%. Dimethylamiloride (DMA; 100 μM) pretreatment reduced Cl− secretion rates by ∼15%, but[Formula: see text] secretion fell 47%. DMA alone had little effect on liquid secretion. When airways were pretreated with both bumetanide and DMA, acetylcholine-induced liquid secretion was nearly abolished. We conclude that about three-fourths of acetylcholine-induced liquid secretion in distal bronchi is dependent on Cl− secretion. Most of the remaining response is driven by[Formula: see text] secretion. We speculate that the principal source of this liquid is submucosal glands. Crossover inhibition of bumetanide on [Formula: see text]secretion and DMA on Cl−secretion implies modulation of anion secretion secondary to changes in cell electrolyte composition.
cAMP activation of chloride and fluid secretion across the rabbit alveolar epithelium
Authors: Nielsen Vance G. , Duvall Michael D. , Baird Manuel S. , Matalon Sadis
Published: December 2017

Active Na+ transport by alveolar epithelial cells has been demonstrated to contribute significantly to alveolar fluid clearance. However, the contribution of transepithelial Cl− movement to the reabsorption of isosmotic fluid across the alveolar epithelium in vivo has not been elucidated. We hypothesized that Cl− transport could be increased across the alveolar epithelium in vivo and across cultured alveolar type II cells by agents that increase intracellular cAMP (e.g., forskolin). In studies where 5% albumin in sodium methanesulfonate (a Cl−-free solution) was administered into the lung, forskolin administration significantly increased intracellular influx of Cl− and fluid into the alveolar space. In vitro studies with cultured rabbit alveolar type II cell monolayers in Ussing chambers demonstrated that elevations in intracellular cAMP increase short-circuit current by increasing both Cl−secretion and Na+ reabsorption. The cystic fibrosis transmembrane conductance regulator channel blocker glibenclamide and the loop diuretic bumetanide partially decreased the forskolin-induced increase in short-circuit current. These data may explain the failure of agonist that stimulated intracellular cAMP to increase alveolar fluid clearance in the rabbit. Moreover, the data suggest that in the event Na+absorptive pathways are damaged, transepithelial Cl− secretion and the consequent intra-alveolar fluid influx may be upregulated.
Time course of neutrophil and macrophage elastinolytic activities in cigarette smoke-induced emphysema
Authors: Ofulue A. Felix , Ko Mary , Abboud Raja T.
Published: December 2017

The aim of this study was to compare the time course of neutrophil and macrophage elastinolytic potentials in the lungs of rats exposed daily to cigarette smoke inhalation for 1–6 mo in relation to the onset and progression of cigarette smoke-induced emphysema. Normal room air-exposed rats served as controls. Morphometric data of lung histological sections showed evidence of emphysema lesions in the lungs of smoke-exposed rats at month 2 and continuing to month 6. Data of total and differential cell counts in bronchoalveolar lavage fluid and collagenase-dissociated lung showed an increased number of lung neutrophils at month 1 of smoke exposure, but this was reduced to control levels at months 2– 6. In contrast, an increased number of lung macrophages was evident in the smoke-exposed rats at month 2 of exposure and continued to month 6. Data of the elastinolytic activities of the neutrophils and macrophages, determined in [3H]elastin-coated culture wells, showed that the elastinolytic activity of lung neutrophils in the smoke-exposed rats was similar to that of the control air-exposed rats at months 1– 6 of exposure. In contrast, the elastinolytic activity of lung macrophages in the smoke-exposed rats was increased at month 2 of exposure and remained increased at month 6. Excessive in vivo lung elastin breakdown (judged by increased levels of elastin-derived peptides and desmosine in lavage fluid, determined immunologically) was observed in the smoke-exposed rats at months 2– 6 of exposure. These data indicate that the time course of increased macrophage-directed elastinolytic activity in the lung, not that of neutrophils, is more closely associated with the evolution of cigarette smoke-induced emphysema.
Effects of maternal vitamin A status on fetal heart and lung: changes in expression of key developmental genes
Authors: Antipatis Christos , Ashworth Cheryl J. , Grant George , Lea Richard G. , Hay Susan M. , Rees William D.
Published: December 2017

Vitamin A is required during pregnancy for fetal lung development. These experiments monitored fetal lung morphology in normal and vitamin A-deficient rats. The expression of elastin and the growth arrest-specific gene 6 ( gas6) in fetal and neonatal hearts and lungs was assessed by Northern blotting. In normal-fed rats, elastin and gas6 were expressed in the fetal lung and heart from day 19 of gestation up to day 2 postnatally. Maternal vitamin A deficiency altered fetal lung development. On day 20, the bronchial passageways were less developed and showed reduced staining for elastic fibers, and in the neonates, the relative air space and the size of the sacculi were reduced. In the fetal lung, the mRNAs for elastin and gas6 were reduced to 56 and 68% of the control values, respectively. In the fetal heart, the mRNA for elastin was reduced to 64% of the control value, whereas gas6 was increased twofold. In the neonate, there was no change in elastin expression in the lung or heart, but gas6 expression in the heart was increased twofold. These results suggest that, in the pregnant rat, vitamin A deficiency may retard fetal lung development or influence the differentiation of critical cell lines. The changes in elastin and gas6 expression may be used to identify the cell types affected.
Ischemia-reperfusion lung injury in rabbits: mechanisms of injury and protection
Authors: Sakuma Tsutomu , Takahashi Keiji , Ohya Nobuo , Kajikawa Osamu , Martin Thomas R. , Albertine Kurt H. , Matthay Michael A. ,   (With the Technical Assistance of Oscar O. Osorio)
Published: December 2017

To study the mechanisms responsible for ischemia-reperfusion lung injury, we developed an anesthetized rabbit model in which the effects of lung deflation, lung inflation, alveolar gas composition, hypothermia, and neutrophils on reperfusion pulmonary edema could be studied. Rabbits were anesthetized and ventilated, and the left pulmonary hilum was clamped for either 2 or 4 h. Next, the left lung was reperfused and ventilated with 100% oxygen. As indexes of lung injury, we measured arterial oxygenation, extravascular lung water, and the influx of a vascular protein (131I-labeled albumin) into the extravascular space of the lungs. The principal results were that 1) all rabbits with the deflation of the lung during ischemia for 4 h died of fulminant pulmonary edema within 1 h of reperfusion; 2) inflation of the ischemic lung with either 100% oxygen, air, or 100% nitrogen prevented the reperfusion lung injury; 3) hypothermia at 6–8°C also prevented the reperfusion lung injury; 4) although circulating neutrophils declined during reperfusion lung injury, there was no increase in interleukin-8 levels in the plasma or the pulmonary edema fluid, and, furthermore, neutrophil depletion did not prevent the reperfusion injury; and 5) ultrastructural studies demonstrated injury to both the lung endothelium and the alveolar epithelium after reperfusion in deflated lungs, whereas the inflated lungs had no detectable injury. In summary, ischemia-reperfusion injury to the rabbit lung can be prevented by either hypothermia or lung inflation with either air, oxygen, or nitrogen.
Mediators of alkalosis-induced relaxation in pulmonary arteries from normoxic and chronically hypoxic piglets
Authors: Gordon John B. , Halla Ted R. , Fike Candice D. , Madden Jane A.
Published: December 2017

Alkalosis-induced relaxation was measured in precontracted arterial rings from 1-wk-old piglets exposed to normoxia or to 3 days of chronic hypoxia. In normoxic piglet arteries, alkalosis-induced relaxation was blunted in arteries without functional endothelium and in arteries treated with nitric oxide synthase or guanylate cyclase inhibitors but not in arteries treated with cyclooxygenase inhibitors or Ca2+- and ATP-dependent K+-channel inhibitors. Inhibition of voltage-dependent K+channels with 10−3M 4-aminopyridine also failed to block alkalosis-induced relaxation. 4-Aminopyridine at 10−2M did block the response, but this may have been due to sustained vascular smooth muscle depolarization. Arteries from hypoxic piglets exhibited greater contraction to the thromboxane mimetic U-46619, decreased endothelium-dependent relaxation, and blunted alkalosis-induced relaxation. The residual relaxation was eliminated by nitric oxide synthase but not by cyclooxygenase or voltage-dependent K+-channel inhibition. Alkalosis-induced relaxation of newborn piglet pulmonary arteries appears to be mediated by the nitric oxide-cGMP pathway and is attenuated after 3 days of hypoxia, likely because of decreased nitric oxide activity.
Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes
Authors: Halayko Andrew J. , Camoretti-Mercado Blanca , Forsythe Sean M. , Vieira Joaquim E. , Mitchell Richard W. , Wylam Mark E. , Hershenson Marc B. , Solway Julian
Published: December 2017

We tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Passage 1 or 2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways emerged. One-sixth of the cells developed an elongated morphology and aligned into bundles. Elongated myocytes contained cables of contractile myofilaments, dense bodies, gap junctions, and membrane caveoli, ultrastructural features of contractile SM in tissue. These cells immunostained intensely for SM α-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific actin-binding protein), and Western analysis of culture lysates disclosed 1.8 (SM α-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases during serum deprivation. Immunoreactive M3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. ACh (10−3 M) induced a marked shortening (59.7 ± 14.4% of original length) in 62% of elongated myocytes made semiadherent by gentle proteolytic digestion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cultured airway myocytes that did not elongate during serum deprivation instead became short and flattened, lost immunoreactivity for contractile proteins, lacked the M3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate that prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.
Neutrophil elastase and elastase-rich cystic fibrosis sputum degranulate human eosinophils in vitro
Authors: Liu Hong , Lazarus Stephen C. , Caughey George H. , Fahy John V.
Published: December 2017

Neutrophils, eosinophils, and their proinflammatory constituents are important mediators of airway disease, and high levels of neutrophil proteases and eosinophil cationic protein (ECP) are found in sputum from patients with cystic fibrosis (CF). To investigate whether neutrophil proteases or CF sputum causes eosinophil degranulation, purified eosinophils from atopic asthmatic subjects were incubated for 2 h with neutrophil elastase, cathepsin G, and CF sputum, and the release of ECP was measured. We found that the percent release of ECP was higher after incubation with neutrophil elastase (10−5M) than with a buffer control [6.1 ± 0.8 (SE) vs. 1.7 ± 0.1%; P < 0.003] and represented >50% of the release caused by positive controls [Ca2+ionophore A-23187 (5 × 10−6M) or serum-coated Sephadex beads]. The release of ECP after incubation with cathepsin G (2.3 ± 0.2%) and CF sputum (6.2 ± 2.0%) was also significantly higher than that with a buffer control ( P < 0.05). Neutralization of free elastase activity with α1-proteinase inhibitor reduced the mean percent degranulation of eosinophils by neutrophil elastase by 50% ( P = 0.0004) and by CF sputum by 75% ( P = 0.02). Preincubation of eosinophils with cytochalasin B (10 mg/ml) and depletion of the incubation medium of Ca2+also significantly attenuated degranulation of eosinophils incubated with purified free neutrophil elastase or CF sputum ( P < 0.05). We conclude that neutrophil proteases, especially neutrophil elastase, and elastase-rich CF sputum cause degranulation of eosinophils in a mechanism partially dependent on Ca2+and actin filaments.
cGMP modulation of Ca2+sensitivity in airway smooth muscle
Authors: Jones Keith A. , Wong Gilbert Y. , Jankowski Christopher J. , Akao Masaki , Warner David O.
Published: December 2017

A β-escin-permeabilized canine tracheal smooth muscle preparation was used to test the hypothesis that cGMP decreases Ca2+sensitivity in airway smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+sensitivity and not by inhibiting Ca2+/calmodulin activation of the contractile proteins. 8-Bromo-cGMP (100 μM) had no effect on the free Ca2+concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 μM ACh plus 10 μM GTP, 8-bromo-cGMP (100 μM) caused a rightward shift of the free Ca2+concentration-response curve, significantly increasing the EC50for free Ca2+from 0.35 ± 0.03 to 0.75 ± 0.06 μM; this effect of 8-bromo-cGMP was concentration dependent from 1 to 100 μM. 8-Bromo-cGMP (100 μM) decreased the level of regulatory myosin light chain (rMLC) phosphorylation for a given cytosolic Ca2+concentration but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. These findings suggest that cGMP decreases Ca2+sensitivity in canine tracheal smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that modulate the relationship between cytosolic Ca2+concentration and rMLC phosphorylation.
Segmental regulation of pulmonary vascular permeability by store-operated Ca2+entry
Authors: Chetham Paul M. , Babál Pavel , Bridges James P. , Moore Timothy M. , Stevens Troy
Published: December 2017

An intact endothelial cell barrier maintains normal gas exchange in the lung, and inflammatory conditions result in barrier disruption that produces life-threatening hypoxemia. Activation of store-operated Ca2+(SOC) entry increases the capillary filtration coefficient ( Kf,c) in the isolated rat lung; however, activation of SOC entry does not promote permeability in cultured rat pulmonary microvascular endothelial cells. Therefore, current studies tested whether activation of SOC entry increases macro- and/or microvascular permeability in the intact rat lung circulation. Activation of SOC entry by the administration of thapsigargin induced perivascular edema in pre- and postcapillary vessels, with apparent sparing of the microcirculation as evaluated by light microscopy. Scanning and transmission electron microscopy revealed that the leak was due to gaps in vessels ≥ 100 μm, consistent with the idea that activation of SOC entry influences macrovascular but not microvascular endothelial cell shape. In contrast, ischemia and reperfusion induced microvascular endothelial cell disruption independent of Ca2+entry, which similarly increased Kf,c. These data suggest that 1) activation of SOC entry is sufficient to promote macrovascular barrier disruption and 2) unique mechanisms regulate pulmonary micro- and macrovascular endothelial barrier functions.
Ion transport in epithelial spheroids derived from human airway cells
Authors: Pedersen Peter Steen , Frederiksen Ole , Holstein-Rathlou Niels-Henrik , Larsen Per Leganger , Qvortrup Klaus
Published: December 2017

In the present study, we describe a novel three-dimensional airway epithelial explant preparation and demonstrate its use for ion transport studies by electrophysiological technique. Suspension cultures of sheets of epithelial cells released by protease treatment from cystic fibrosis (CF) and non-CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+absorption followed by water. Transformation to a Cl− secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model for the airway surface epithelium and is suitable for studies of transport mechanisms and regulation.
Potential identification of the O2-sensitive K+ current in a human neuroepithelial body-derived cell line
Authors: O’Kelly I. , Stephens R. H. , Peers C. , Kemp P. J.
Published: December 2017

Whole cell recording of H-146 cells revealed that the outward K+ current was completely inhibited by quinidine (IC50 ∼17 μM). In contrast, maximal concentrations of 4-aminopyridine (4-AP; ≥10 mM) reversibly blocked only ∼60% (IC50 ∼1.52 mM). Ten millimolar 4-AP had no effect on the inhibition by hypoxia, which reduced current density from ∼27 to ∼13 pA/pF, whereas 1 mM quinidine abolished the hypoxic effect. In current clamp, 10 mM 4-AP depolarized the cell by ∼18 mV and hypoxia caused further reversible depolarization of ∼4 mV. One millimolar quinidine collapsed the membrane potential and abrogated any further hypoxic depolarization. RT-PCR revealed expression of the acid-sensitive, twin P domain K+ channel TASK but not of TWIK, TREK, or the known hypoxia-sensitive Kv2.1, which was confirmed by sequencing and further PCR with primers to the coding region of TASK. However, a reduction in extracellular pH had no effect on K+ current. Thus, although the current more closely resembles TWIK than TASK pharmacologically, structurally the reverse appears to be true. This suggests that a novel acid-insensitive channel related to TASK may be responsible for the hypoxia-sensitive K+ current of these cells.
K+-channel blockade inhibits shear stress-induced pulmonary vasodilation in the ovine fetus
Authors: Storme Laurent , Rairigh Robyn L. , Parker Thomas A. , Cornfield David N. , Kinsella John P. , Abman Steven H.
Published: December 2017

To determine whether K+-channel activation mediates shear stress-induced pulmonary vasodilation in the fetus, we studied the hemodynamic effects of K+-channel blockers on basal pulmonary vascular resistance and on the pulmonary vascular response to partial compression of the ductus arteriosus (DA) in chronically prepared late-gestation fetal lambs (128–132 days gestation). Study drugs included tetraethylammonium (TEA; Ca2+-dependent K+-channel blocker), glibenclamide (Glib; ATP-dependent K+-channel blocker), charybdotoxin (CTX; preferential high-conductance Ca2+-dependent K+-channel blocker), apamin (Apa; low-conductance Ca2+-dependent K+-channel blocker), and 4-aminopyridine (4-AP; voltage-dependent K+-channel blocker). Catheters were inserted in the left pulmonary artery (LPA) for selective drug infusion and in the main pulmonary artery, aorta, and left atrium to measure pressure. An inflatable vascular occluder was placed around the DA. LPA flow was measured with an ultrasonic flow transducer. Animals were treated with saline, high- or low-dose TEA, Glib, Apa, CTX, CTX plus Apa, or 4-AP injected into the LPA. DA compression caused a time-related decrease in pulmonary vascular resistance in the control, Glib, Apa, CTX, CTX plus Apa, and low-dose TEA groups but not in the high-dose TEA and 4-AP groups. These data suggest that pharmacological blockade of Ca2+- and voltage-dependent K+-channel activity but not of low-conductance Ca2+- and ATP-dependent K+-channel activity attenuates shear stress-induced fetal pulmonary vasodilation.
O3-induced inflammation in prepregnant, pregnant, and lactating rats correlates with O3 dose estimated by18O
Authors: Gunnison Albert F. , Hatch Gary E.
Published: December 2017

Previous studies have shown that rats late in pregnancy and throughout lactation are more susceptible to ozone (O3)-induced pulmonary inflammation than are prepregnant (virgin) or postlactating rats. The major aim of the present study was to determine whether these differences in response intensity could be accounted for by the O3 dose to the lower region of the lung. The relative O3 dose to the lower lung of groups of pregnant, lactating, and virgin female rats was estimated by measuring the incorporation of the18O isotope into low-speed (cells) and high-speed (surfactant) pellets of bronchoalveolar lavage fluid immediately after acute exposure to 0.5–1.1 parts/million18O3. The polymorphonuclear leukocyte (PMN) and protein inflammatory responses were established 20 h after acute exposure of identical physiological groups to 0.5–1.1 parts/million16O3(common isotope). A single regression of PMN inflammation data against surfactant 18O concentration for all physiological groups gave a linear relationship, indicating direct proportionality of PMN inflammation with this estimate of relative dose to the lower lung regardless of physiological status. This implies that the chemical species that react with surfactant molecules, i.e., O3 or its metabolites, are the same as or proportional to those chemical species responsible for initiating PMN inflammation. Additional experiments showed that lung tissue ascorbic acid concentration was significantly lower in pregnant and lactating rats than in virgin female rats. Although a causative relationship cannot be assumed, the deficit in tissue ascorbic acid concentration in pregnant and lactating rats compared with virgin female rats is consistent with their greater responsiveness and higher relative surfactant O3 dose.
Nitric oxide synthase isoform expression in the developing lung epithelium
Authors: Sherman Todd S. , Chen Zhong , Yuhanna Ivan S. , Lau Kim S. , Margraf Linda R. , Shaul Philip W.
Published: December 2017

Nitric oxide (NO), generated by NO synthase (NOS), is an important mediator of physiological processes in the airway and lung parenchyma, and there is evidence that the pulmonary expression of the endothelial isoform of NOS (eNOS) is developmentally regulated. The purpose of the present study was to delineate the cellular distribution of expression of eNOS in the developing respiratory epithelium and to compare it with inducible (iNOS) and neuronal (nNOS) NOS. Immunohistochemistry was performed on fetal (125–135 days gestation, term 144 days), newborn (2–4 wk), and maternal sheep lungs. In fetal lung, eNOS expression was evident in bronchial and proximal bronchiolar epithelia but was absent in terminal and respiratory bronchioles and alveolar epithelium. Similar to eNOS, iNOS was detected in bronchial and proximal bronchiolar epithelia but not in alveolar epithelium. However, iNOS was also detected in terminal and respiratory bronchioles. nNOS was found in epithelium at all levels including the alveolar wall. iNOS and nNOS were also detected in airway and vascular smooth muscle. The cellular distribution of all three isoforms was similar in fetal, newborn, and adult lungs. Findings in the epithelium were confirmed by isoform-specific reverse transcription-polymerase chain reaction assays and NADPH diaphorase histochemistry. Thus the three NOS isoforms are commonly expressed in proximal lung epithelium and are differentially expressed in distal lung epithelium. All three isoforms may be important sources of epithelium-derived NO throughout lung development.
Effects and interactions of opioids on plasma exudation induced by cigarette smoke in guinea pig bronchi
Authors: Lei Yu-Hong , Rogers Duncan F.
Published: December 2017

The effects of opioids on cigarette smoke-induced plasma exudation were investigated in vivo in the main bronchi of anesthetized guinea pigs, with Evans blue dye as a plasma marker. Acute inhalation of cigarette smoke increased plasma exudation by 216% above air control values. Morphine, 0.1–10 mg/kg but not 30 mg/kg, inhibited the exudation but had no significant effect on substance P-induced exudation. Both 10 and 30 mg/kg of morphine increased exudation in air control animals, an effect inhibited by antihistamines but not by a tachykinin neurokinin type 1-receptor antagonist. Naloxone inhibited all morphine responses. Cigarette smoke-induced plasma exudation was inhibited by a μ-opioid-receptor agonist (DAMGO) but not by agonists at δ (DPDPE)- or κ (U-50488H)-receptors. None of these agonists affected exudation in air control animals. DPDPE prevented the inhibition by DAMGO of cigarette smoke-induced plasma exudation, and the combination of DAMGO and DPDPE increased exudation in air control animals. Prevention of inhibition and the combination-induced increase were inhibited by antihistamines or the mast cell-stabilizing drug sodium cromoglycate. U-50488H did not alter the response to either DAMGO or DPDPE. We conclude that, in guinea pig main bronchi in vivo, μ-opioid-receptor agonists inhibit cigarette smoke-induced plasma exudation via a prejunctional mechanism. Plasma exudation induced by μ- and δ-receptor interactions is due to endogenous histamine release from mast cells.