bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of surfactant proteins A and B by TNF-α and phorbol ester independent of NF-κB
Authors: Pryhuber Gloria S. , Khalak Rubia , Zhao Qian
Published: December 2017

Acute lung inflammation is complicated by altered pulmonary surfactant phospholipid and protein composition. The proinflammatory cytokine tumor necrosis factor-α (TNF-α) and the phorbol ester 12- O-tetradecanoyl phorbol-13-acetate (TPA) inhibit expression of surfactant-associated proteins A and B (SP-A and SP-B), both important for normal surfactant function. The transcription factor nuclear factor-κB (NF-κB) frequently mediates regulation of gene expression by TPA and TNF-α. In the present study, electrophoretic mobility shift assays (EMSAs) and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, were utilized to determine the role of NF-κB activation in TPA and TNF-α inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline (PTX), which inhibits TNF-α cellular effects without preventing NF-κB activation, was also tested. By EMSA, TPA and TNF-α increased nuclear NF-κB binding activity in temporally distinct patterns. PDTC decreased TPA- and TNF-α-induced NF-κB binding activity but did not limit their inhibition of SP-A and SP-B mRNAs. PDTC independently decreased both SP-A and SP-B mRNAs. PTX partially reversed TNF-α- but not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-κB binding. The effects of PDTC and PTX on NF-κB and the surfactant proteins suggest that NF-κB activation does not mediate TPA or TNF-α inhibition of SP-A and SP-B mRNA accumulation.
Changes in pulmonary expression of hexokinase and glucose transporter mRNAs in rats adapted to hyperoxia
Authors: Allen Corrie B. , Guo Xiao-Ling , White Carl W.
Published: December 2017

Impairment of lung aconitase activity, citric acid cycle, and mitochondrial respiration by hyperoxia necessitates the elevation of glycolysis for energy production and of pentose shunt activity for reducing equivalents. The molecular mechanisms that allow increased glucose utilization are unknown. Adult male and female rats were adapted to sublethal hyperoxia, equivalent to 83% oxygen at sea level, or air for 7 days. Lung RNA and protein increased in hyperoxia (197 and 57%, respectively), whereas total DNA was unchanged. In hyperoxia, lung total hexokinase (HK) activity increased threefold, and mRNAs for HK-II and -III were specifically upregulated. HK-I mRNA was unchanged. mRNAs for HK-II and -III gradually increased during the first 72 h in hyperoxia. HK-II mRNA was significantly elevated at 72 h, preceding changes in lung cell populations. Although virtually absent in air, HK-II activity was highly expressed in hyperoxia. Among lung glucose transporters, specific expression of mRNAs for GLUT-4 (insulin dependent) and sodium-glucose cotransporter-1 was decreased, whereas that for GLUT-1 was minimally changed. Adaptation to hyperoxia involves coordinated changes in gene expression for the proteins regulating pulmonary glucose transport.
Quinones increase γ-glutamyl transpeptidase expression by multiple mechanisms in rat lung epithelial cells
Authors: Liu Rui-Ming , Shi Michael Ming , Giulivi Cecilia , Forman Henry Jay
Published: December 2017

γ-Glutamyl transpeptidase (GGT) plays an important role in glutathione (GSH) metabolism. GGT expression is increased in oxidant-challenged cells; however, the signaling mechanisms involved are uncertain. The present study used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone that continuously produced H2O2in rat lung epithelial L2 cells. It was found that DMNQ increased GGT mRNA content by increasing transcription, as measured by nuclear run-on. This was accompanied by increased GGT specific activity. Cycloheximide, a protein synthesis inhibitor, blocked neither the increased GGT mRNA content nor the increased GGT transcription rate caused by DMNQ, suggesting that increased GGT transcription was a direct rather than secondary response. Previous data from this laboratory (R.-M. Liu, H. Hu, T. W. Robison, and H. J. Forman. Am. J. Respir. Cell Mol. Biol.14: 186–191, 1996) showed that tert-butylhydroquinone (TBHQ) increased GGT mRNA content by increasing its stability. TBHQ differs markedly from DMNQ in terms of its conjugation with GSH and H2O2generation. Together, the data suggest that quinones upregulate GGT through multiple mechanisms, increased transcription and posttranscriptional modulation, which are apparently mediated through generation of reactive oxygen species and GSH conjugate formation, respectively.
A human forkhead/winged-helix transcription factor expressed in developing pulmonary and renal epithelium
Authors: Pelletier Glenn J. , Brody Steven L. , Liapis Helen , White Robert A. , Hackett Brian P.
Published: December 2017

Members of the forkhead/winged-helix transcription factor family play crucial roles during vertebrate development. A human hepatocyte nuclear factor/forkhead homolog (HFH)-4 cDNA encoding a 421-amino acid protein was isolated from a human fetal lung cDNA library. By Southern blot analysis of human-rodent somatic cell hybrid genomic DNA, the human HFH-4 gene localizes to chromosome 17q23-qter. This is the locus of another forkhead/winged-helix gene, the interleukin enhancer binding factor gene. RNA blot analysis revealed a 2.5-kilobase human HFH-4 transcript in fetal lung, kidney, and brain as well as in adult reproductive tissues, lung, and brain. By in situ hybridization, HFH-4 expression is associated with differentiation of the proximal pulmonary epithelium, starting during the pseudoglandular stage of human lung development. During human renal morphogenesis, HFH-4 is expressed in the developing epithelial cells of the ureteric duct, glomerulus, and epithelial vesicles. The unique pattern of HFH-4 expression during human fetal development suggests a role for this forkhead/winged-helix factor during pulmonary and renal epithelial development.
Constitutive nitric oxide production by rat alveolar macrophages
Authors: Miles P. R. , Bowman L. , Rengasamy A. , Huffman L.
Published: December 2017

Results from previous studies suggest that alveolar macrophages must be exposed to inflammatory stimuli to produce nitric oxide (⋅ NO). In this study, we report that naive unstimulated rat alveolar macrophages do produce ⋅ NO and attempt to characterize this process. Western blot analysis demonstrates that the enzyme responsible is an endothelial nitric oxide synthase (eNOS). No brain or inducible NOS can be detected. The rate of ⋅ NO production is ∼0.07 nmol ⋅ 106cells−1⋅ h−1, an amount that is less than that produced by the eNOS found in alveolar type II or endothelial cells. Alveolar macrophage ⋅ NO formation is increased in the presence of extracellularl-arginine, incubation medium containing magnesium and no calcium, a calcium ionophore (A-23187), or methacholine. ⋅ NO production is inhibited by NG-nitro-l-arginine methyl ester (l-NAME) but not by NG-nitro-l-arginine,l- N5-(1-iminomethyl)ornithine hydrochloride, or aminoguanidine. Incubation with ATP, ADP, or histamine also inhibits ⋅ NO formation. Some of these properties are similar to and some are different from properties of eNOS in other cell types. Cellular ⋅ NO levels do not appear to be related to ATP or lactate content. Alveolar macrophage production of ⋅ NO can be increased approximately threefold in the presence of lung surfactant or its major component, dipalmitoyl phosphatidylcholine (DPPC). The DPPC-induced increase in ⋅ NO formation is time and concentration dependent, can be completely inhibited by l-NAME, and does not appear to be related to the degradation of DPPC by alveolar macrophages. These results demonstrate that unstimulated alveolar macrophages produce ⋅ NO via an eNOS and that lung surfactant increases ⋅ NO formation. This latter effect may be important in maintaining an anti-inflammatory state in vivo.
Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide
Authors: Ding Jin Wen , Dickie John , O’Brodovich Hugh , Shintani Yutaka , Rafii Bijan , Hackam David , Marunaka Yoshinori , Rotstein Ori D.
Published: December 2017

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( Isc) without affecting total Iscor monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.
Evidence that Calu-3 human airway cells secrete bicarbonate
Authors: Lee Michael C. , Penland Christopher M. , Widdicombe Jonathan H. , Wine Jeffrey J.
Published: December 2017

The Calu-3 cell line is being investigated as a model for human submucosal gland serous cells. In a previous investigation of basal short-circuit current ( I sc) in Calu-3 cells, high levels of bumetanide-insensitive basal I sc (∼60 μA/cm2) were measured in cells grown at an air interface. Basal I sc was reduced only 7% by bumetanide, and the largest component of basal I sc required both Cl− and[Formula: see text] in the bathing solutions. Because I sc could be partially inhibited by basolateral 4,4′-dinitrostilbene-2,2′-disulfonic acid and because the only known apical exit pathway for anions is the cystic fibrosis transmembrane conductance regulator, which has a relatively poor conductance for [Formula: see text], it was concluded that most basal I sc is[Formula: see text]-dependent Cl− secretion [M. Singh, M. Krouse, S. Moon, and J. J. Wine. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L690–L698, 1997]. We have now measured isotopic fluxes of36Cl−and22Na+across short-circuited Calu-3 cells and found that virtually none of the basal I sc is Cl− secretion or Na+ absorption. Thus, in contrast to the earlier report, we conclude that the major component of basal I sc is[Formula: see text] secretion. Stimulation recruits primarily Cl− secretion, as previously proposed.
H2O2causes endothelial barrier dysfunction without disrupting the arginine-nitric oxide pathway
Authors: Gupta Mahesh P. , Steinberg Helmut O. , Hart C. Michael
Published: December 2017

We have previously demonstrated that nitric oxide (⋅ NO) donors attenuate and that inhibition of endogenous nitric oxide synthase (NOS) enhances hydrogen peroxide (H2O2)-mediated porcine pulmonary artery endothelial cell (PAEC) injury. The current study investigates the hypothesis that oxidant-mediated inhibition of NOS contributes to PAEC injury. PAEC barrier function, measured as the transmonolayer clearance of albumin, was significantly impaired by H2O2(10–100 μM) in the absence of cytotoxicity. Treatment with H2O2did not alter NOS activity, measured as the conversion of [3H]arginine to [3H]citrulline in PAEC lysates, either immediately after treatment with 0–250 μM H2O2for 30 min or for up to 120 min after treatment with 100 μM H2O2. H2O2had little effect on NOS activity in intact PAECs, measured as 1) the formation of [3H]citrulline in [3H]arginine-loaded PAECs, 2) PAEC guanosine 3′,5′-cyclic monophosphate content, and 3) PAEC ⋅ NO release to the culture media. These results indicate that the arginine-⋅ NO pathway remains intact after exposure to oxidant conditions sufficient to promote functional derangements of vascular endothelial cells.
Myosin heavy chain gene expression changes in the diaphragm of patients with chronic lung hyperinflation
Authors: Mercadier Jean-Jacques , Schwartz Ketty , Schiaffino Stefano , Wisnewsky Claudine , Ausoni Simonetta , Heimburger Michèle , Marrash Rolana , Pariente René , Aubier Michel
Published: December 2017

In striated muscle, chronic increases in workload result in changes in myosin phenotype. The aim of this study was to determine whether such changes occur in the diaphragm of patients with severe chronic obstructive pulmonary disease, a situation characterized by a chronic increase in respiratory load and lung volume. Diaphragm biopsies were obtained from 22 patients who underwent thoracic surgery. Myosin was characterized with electrophoresis in nondenaturing conditions, SDS-glycerol PAGE, and Western blotting with monoclonal antibodies specific for slow and fast myosin heavy chain (MHC) isoforms. Flow volume curves, total lung capacity, and functional residual capacity were measured before surgery in 20 patients. We found that the human diaphragm is composed of at least four myosin isoforms, one slow and three fast, resulting from the combination of three MHC species. Chronic overload was associated with an increase in the slow β-MHC species at the expense of the fast species (β-MHC, 78.2 ± 4.6 and 50.0 ± 6.5% in emphysematous and control patients, respectively; P < 0.005). Linear correlations were found between β-MHC percentage and forced expiratory volume in 1 s ( r = −0.52; P < 0.02), total lung capacity ( r = 0.44; P < 0.05), and functional residual capacity ( r = 0.65; P < 0.003). The human adult diaphragm is composed of a balanced proportion of slow and fast myosin isoforms. In patients with chronic obstructive pulmonary disease, the proportion of fast myosins decreases, whereas that of slow myosin increases. This increase appears to be closely related to lung hyperinflation and may reflect an adaptation of the diaphragm to the new functional requirements.
Increased lung preproET-1 and decreased ETB-receptor gene expression in fetal pulmonary hypertension
Authors: Dunbar Ivy D. , Le Cras Timothy D. , Horan Marilee P. , Abman Steven H.
Published: December 2017

Endothelin (ET)-1, a potent vasoconstrictor and smooth muscle mitogen, is produced from its precursor, preproET-1, by endothelin-converting enzyme (ECE)-1 activity. ET-1 may bind to two receptors, ETAand ETB, that mediate vasoconstriction and vasodilation in the ovine fetal lung, respectively. ET-1 contributes to high pulmonary vascular resistance in experimental perinatal pulmonary hypertension induced by ligation of the ductus arteriosus in the fetal lamb. Physiological studies in this model have demonstrated enhanced ETA- and diminished ETB-receptor activities and a threefold increase in lung immunoreactive ET-1 protein content. We hypothesized that increased ET production and an imbalance in receptor expression would favor vasoconstriction and smooth muscle cell hypertrophy in pulmonary hypertension and may be partially due to alterations in gene expression. To test this hypothesis, we studied lung mRNA expression of preproET-1, ECE-1, and the ETAand ETBreceptors in normal and hypertensive fetal lambs. Total RNA was isolated from whole lung tissue in normal late-gestation fetuses (135 ± 3 days; 147 days = term) and from animals with pulmonary hypertension after ductus arteriosus ligation for 8 days (134 ± 4 days). Ductus arteriosus ligation increased right ventricular hypertrophy [control 0.56 ± 0.02 vs. hypertension 0.85 ± 0.05; right ventricle/(left ventricle + septum); P < 0.05]. Northern blot analysis was performed using cDNA probes and was normalized to the signal for 18S rRNA. We found a 71 ± 24% increase in steady-state preproET-1 mRNA ( P < 0.05) and a 62 ± 5% decrease in ETBmRNA ( P < 0.05) expression in ductus arteriosus ligation. ECE-1 and ETA-receptor mRNA expression did not change. We conclude that chronic intrauterine pulmonary hypertension after ductus arteriosus ligation increases steady-state preproET-1 mRNA and decreases ETB-receptor mRNA without changing ECE-1 mRNA or ETA-receptor mRNA expression. These findings suggest that increased ET-1 production and decreased ETB-receptor expression may contribute to increased vasoconstrictor tone in this experimental model of neonatal pulmonary hypertension.
Effect of tyrosine kinase inhibition on surfactant protein A gene expression during human lung development
Authors: Klein Jonathan M. , Dewild Louis J. , McCarthy Troy A.
Published: December 2017

Epidermal growth factor (EGF) stimulates surfactant protein (SP) A synthesis in human fetal lung explants. Ligand binding to the EGF receptor stimulates an intrinsic receptor tyrosine kinase with subsequent activation of second messengers. We hypothesized that inhibition of EGF-receptor tyrosine kinase activity would block SP-A expression in spontaneously differentiating cultured human fetal lung tissue. Midtrimester fetal lung explants were exposed for 4 days to genistein (a broad-range inhibitor of tyrosine kinases) and tyrphostin AG-1478 (a specific inhibitor of EGF-receptor tyrosine kinase). Genistein significantly decreased SP-A and SP-A mRNA levels without affecting either tissue viability or the morphological differentiation of alveolar type II cells. Tyrphostin AG-1478 also decreased SP-A content and SP-A mRNA levels in cultured fetal lung explants. Treatment with EGF could not overcome the inhibitory effects of either genistein or tyrphostin on SP-A; however, only tyrphostin inhibited EGF-receptor tyrosine phosphorylation. We conclude that specific inhibition of EGF-receptor tyrosine kinase with tyrphostin AG-1478 blocks the expression of SP-A during spontaneous differentiation of cultured human fetal lung tissue. Furthermore, exposure to genistein also decreases SP-A expression and blocks the effects of EGF in human fetal lung tissue without inhibiting EGF-receptor tyrosine phosphorylation. These findings support the importance of tyrosine kinase-dependent signal transduction pathways in the regulation of SP-A during human fetal lung development.
PKC activation is required by EGF-stimulated Na+-H+exchanger in human pleural mesothelial cells
Authors: Liaw Yuang-Shuang , Yang Pan-Chyr , Yu Chong-Jen , Kuo Sow-Hsong , Luh Kwen-Tay , Lin Yuh-Jeng , Wu Mei-Lin
Published: December 2017

Epidermal growth factor (EGF) stimulates the Na+-H+exchanger, leading to enhanced cell proliferation. In human pleural mesothelial cells (PMCs), the intracellular signaling mechanism mediating the EGF-induced stimulation of the Na+-H+exchanger has not yet been identified. Using a pH-sensitive fluorescent probe, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, to measure changes in intracellular pH (pHi), we found that 1) EGF and 12- O-tetradecanoylphorbol 13-acetate (TPA; a phorbol ester) both stimulate the ethylisopropyl amiloride-sensitive Na+-H+exchanger; 2) TPA-induced alkalosis can be blocked by protein kinase C (PKC) inhibitors (chelerythrine and staurosporine) or by PKC downregulation, indicating that PKC activation is involved in the stimulation of the Na+-H+exchanger. However, TPA-induced alkalosis is not blocked by tyrosine kinase inhibitors; and 3) the stimulatory effect of EGF on the Na+-H+exchanger acts via stimulation of tyrosine kinase-receptor activity because it is inhibited by tyrosine kinase inhibitors (genistein, lavendustin A, and herbimycin A). It also involves PKC activation because EGF-induced alkalosis was blocked by PKC inhibitors. These results suggest that PKC activation is one of the downstream signals for EGF-induced activation of the Na+-H+exchanger in primary cultures of human pleural mesothelial cells.
Carbachol-induced actin reorganization involves Giactivation of Rho in human airway smooth muscle cells
Authors: Togashi Hideaki , Emala Charles W. , Hall Ian P. , Hirshman Carol A.
Published: December 2017

To determine whether M2muscarinic receptors are linked to the monomeric G protein Rho, we studied the effect of carbachol on actin reorganization (stress fiber formation) in cultured human airway smooth muscle cells that expressed mainly M2muscarinic receptors by dual- fluorescence labeling of filamentous (F) and monomeric (G) actin. F-actin was labeled with FITC-labeled phalloidin, and G-actin was labeled with Texas Red-labeled DNase I. Carbachol stimulation induced stress fiber formation (increased F-actin staining) in the cells and increased the F- to G-actin ratio 3.6 ± 0.4-fold (mean ± SE; n = 5 experiments). Preincubation with pertussis toxin, Clostridium C3 exoenzyme, or tyrosine kinase inhibitors reduced the carbachol-induced increase in stress fiber formation and significantly decreased the F- to G-actin ratio, whereas a mitogen-activated protein kinase inhibitor, a phosphatidylinositol 3-kinase inhibitor, and a protein kinase C inhibitor were without effect. This study demonstrates that in cultured human airway smooth muscle cells, muscarinic-receptor activation induces stress fiber formation via a pathway involving a pertussis-sensitive G protein, Rho proteins, and tyrosine phosphorylation.
Exogenous NO enhances hydrogen peroxide-mediated neutrophil adherence to cultured endothelial cells
Authors: Okayama Naotsuka , Ichikawa Hiroshi , Coe Laura , Itoh Makoto , Alexander J. Steven
Published: December 2017

One important aspect of oxidant injury is the enhancement of neutrophil-endothelial adhesion by oxidants such as hydrogen peroxide. Recent studies suggest that nitric oxide (NO) can limit oxidant-mediated tissue injury, since inhibitors of endogenous NO synthesis often promote neutrophil-endothelial adhesion. However, less is known about the direct role of exogenous NO in modulating proadhesive effects of oxidants. The objective of this study was to examine how an NO donor modifies hydrogen peroxide-mediated adhesion of neutrophils to cultured endothelial cells. Human umbilical vein endothelial cell monolayers were exposed for 30 min to 0–0.1 mM hydrogen peroxide with or without the NO donor spermine-NONOate (SNO; 0–0.5 mM), and the adhesion of51Cr-labeled polymorphonuclear neutrophils (PMNs) was measured in a static adhesion assay. PMN adherence was not altered by either peroxide (up to 0.1 mM) or SNO (up to 0.5 mM) alone but was significantly increased by over 300% by coadministration of both 0.1 mM peroxide and 0.5 mM SNO. This increase in adhesion with these two agents was correlated with an increase in the presentation of surface P-selectin but not intercellular adhesion molecule-1. Both PMN adhesion and P-selectin presentation were blocked by 0.1 mM desferrioxamine (an iron chelator) and 1 mM methionine (an oxy-radical scavenger). WEB-2086, a platelet-activating factor-receptor antagonist (10 μM), also prevented PMN adhesion but not P-selectin expression. An antibody directed against either P-selectin or intercellular adhesion molecule-1 also blocked adhesion. These data indicate that NO may actually exacerbate rather than protect against the inflammatory effects of peroxide in some models of inflammation through the synthesis of platelet-activating factor and the mobilization of P-selectin.
Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide
Authors: Sheehy A. Macduff , Burson Michael A. , Black Stephen M.
Published: December 2017

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy for pulmonary hypertension, suggesting that inhaled NO may downregulate basal NO production. However, the exact mechanism of this rebound pulmonary hypertension remains unclear. The objectives of these studies were to determine the effect of NO exposure on endothelial NO synthase (eNOS) gene expression, enzyme activity, and posttranslational modification in cultured pulmonary arterial endothelial cells. Sodium nitroprusside (SNP) treatment had no effect on eNOS mRNA or protein levels but did produce a significant decrease in enzyme activity. Furthermore, although SNP treatment induced protein kinase C (PKC)-dependent eNOS phosphorylation, blockade of PKC activity did not protect against the effects of SNP. When the xanthine oxidase inhibitor allopurinol or the superoxide scavenger 4,5-dihydroxy-1-benzene-disulfonic acid were coincubated with SNP, the inhibitory effects on eNOS activity could be partially alleviated. Also, the levels of superoxide were found to be elevated 4.5-fold when cultured pulmonary arterial endothelial cells were exposed to the NO donor spermine/NO. This suggests that NO can stimulate xanthine oxidase to cause an increase in cellular superoxide generation. A reaction between NO and superoxide would produce peroxynitrite, which could then react with the eNOS protein, resulting in enzyme inactivation. This mechanism may explain, at least in part, how NO produces NOS inhibition in vivo and may delineate, in part, the mechanism of rebound pulmonary hypertension after withdrawal of inhaled NO.
Transcriptional regulation of tropoelastin expression in rat lung fibroblasts: changes with age and hyperoxia
Authors: Bruce Margaret C. , Honaker Catherine E.
Published: December 2017

Elastic fibers are thought to provide structural support for secondary septa as the lung undergoes the transition from the saccular to the alveolar stage. The synthesis of the soluble precursor of elastin, tropoelastin, occurs during a finite developmental period. We have investigated the developmental regulation of tropoelastin gene transcription and mRNA expression in fetal and postnatal rat lung fibroblasts and have assessed the changes in tropoelastin gene expression caused by hyperoxic exposure during secondary septal development. With the use of an RT-PCR assay and intron-specific primers to detect heterogeneous nuclear RNA (hnRNA) and intron-spanning primers to detect mRNA in freshly isolated rat lung fibroblasts, tropoelastin gene expression was found to be upregulated late in gestation. From days 18 to 21 of gestation, there was a 4.5-fold increase in tropoelastin hnRNA ( P < 0.0001) and a 6-fold increase in mRNA ( P = 0.002). After birth, tropoelastin expression was downregulated. Signals decreased from fetal day 21 to postnatal day 2 for both tropoelastin hnRNA ( P = 0.021) and mRNA ( P = 0.043). Tropoelastin hnRNA decreased further from days 2 to 6 ( P= 0.04). Both tropoelastin hnRNA and mRNA were again upregulated during alveolarization from days 9 to 11, indicating that, once upregulated, transcription of the tropoelastin gene is not constant but varies with fetal and postnatal age. Exposure to >95% oxygen, when initiated on postnatal day 2 or 3 and continued until day 11, significantly diminished the developmental increase in tropoelastin hnRNA ( P < 0.005) and mRNA ( P < 0.05) normally seen on days 9– 11, indicating that the postnatal upregulation of tropoelastin gene expression is inhibited by hyperoxic exposure in the early postnatal period.
Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria
Authors: Hartshorn Kevan L. , Crouch Erika , White Mitchell R. , Colamussi Maria L. , Kakkanatt Anand , Tauber Benjamin , Shepherd Virginia , Sastry Kedarnath N.
Published: December 2017

The collectins are a class of collagenous lectin proteins present in serum and pulmonary secretions [pulmonary surfactant protein (SP) A and SP-D] that are believed to participate in innate immune responses to various pathogens. With the use of flow cytometric and fluorescent-microscopic assays, SP-A and SP-D were shown to increase calcium-dependent neutrophil uptake of Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. Evidence is provided that the collectins enhanced bacterial uptake through a mechanism that involved both bacterial aggregation and direct actions on neutrophils. The degree of multimerization of SP-D preparations was a critical determinant of both aggregating activity and potency in enhancing bacterial uptake. The mechanisms of opsonizing activity of SP-D and SP-A differed in important respects from those of opsonizing antibodies. These results provide the first evidence that surfactant collectins may promote neutrophil-mediated clearance of bacteria in the lung independently of opsonizing antibody.
Regulation of amiloride-sensitive sodium absorption in murine airway epithelium by C-type natriuretic peptide
Authors: Kelley Thomas J. , Cotton Calvin U. , Drumm Mitchell L.
Published: December 2017

We have previously shown that C-type natriuretic peptide (CNP), a guanylate cyclase agonist, can stimulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion in murine airway epithelial cells via protein kinase (PK) A activation through the inhibition of cGMP-inhibited phosphodiesterases. In this paper, we show that CNP is also capable of reducing amiloride-sensitive sodium absorption in murine airway epithelium through a cGMP-dependent mechanism that is separate from the CFTR regulatory signaling pathway. Both murine tracheal and nasal tissues exhibit sensitivity to amiloride-sensitive sodium regulation by exogenously added CNP. CNP depolarized the nasal transepithelial potential difference by 6.3 ± 0.5 mV, whereas the cGMP-inhibited phosphodiesterase inhibitor milrinone actually hyperpolarized the nasal transepithelial potential difference by 2.0 ± 1.2 mV in mice homozygous for a CFTR stop mutation [CFTR(−/−)]. Inhibition of guanylate cyclase activity and PKG activity in normal mice resulted in an increase in amiloride-sensitive sodium absorption, suggesting that tonic regulation of amiloride-sensitive sodium absorption is in part due to basal cGMP levels and PKG activity.
Glucocorticoid receptor mRNA and protein in fetal rat lung in vivo: modulation by glucocorticoid and androgen
Authors: Sweezey Neil B. , Ghibu F. , Gagnon S. , Schotman E. , Hamid Q.
Published: December 2017

Pulmonary glucocorticoid receptor (GR) is essential to timely preparation for the onset of breathing air at birth. We have previously used primary culture of late-gestation fetal rat lung cells to demonstrate differential regulation of GR by glucocorticoid depending on cell type. In this study, we hypothesized that the action of glucocorticoid on GR mRNA expression and protein elaboration in lung cells might be modulated by interactions present in vivo but not in primary culture. Given that male sex hormone (androgen) has an inhibitory effect on antenatal lung development, we also postulated that androgen would decrease antenatal lung GR. We report that antenatal maternal injection of the glucocorticoid dexamethasone (1 mg/kg) enhanced fetal lung cellular levels of GR mRNA and protein as assessed by in situ hybridization and immunocytochemistry (ICC), respectively. ICC was performed using polyclonal rabbit anti-human antibody that reacts with rat GR whether bound to ligand or not and does not interfere with GR binding to DNA. Levels of GR mRNA and protein were enhanced in cells throughout all areas of the lung tissue, suggesting that interactions occurring in intact tissue may override the previously reported direct inhibition by glucocorticoid of GR protein elaboration in isolated fetal rat lung epithelial cells. Furthermore, antenatal administration of the androgen 5α-dihydrotestosterone (0.2 mg/kg) reduced tissue levels of GR mRNA and protein, consistent with androgenic inhibition of antenatal lung development by decreasing GR. We conclude that glucocorticoids and androgens exert opposite effects on fetal lung GR.
Functional and pathological effects of prolonged hyperoxia in neonatal mice
Authors: Warner Barbara B. , Stuart Lorie A. , Papes Richard A. , Wispé Jonathan R.
Published: December 2017

Bronchopulmonary dysplasia (BPD) commonly develops in premature infants. An improved understanding of the pathophysiology of BPD requires better models. In this study, neonatal FVB/N mice were exposed to room air or 85% oxygen for 28 days. Neonatal hyperoxia resulted in decreased alveolar septation, increased terminal air space size, and increased lung fibrosis. These changes were evident after 7 days and more pronounced by 28 days. Decreased alveolarization was preceded by decreased proliferation of lung cells. After 3 days of hyperoxia, cell proliferation was decreased compared with room air littermates. Cell proliferation continued to be decreased in the first 2 wk but normalized by 4 wk. Hyperoxia caused an increased number of inflammatory cells in lung tissue and in lung lavage fluid. Analysis of lung tissue RNA by RT-PCR showed that hyperoxia increased expression of the proinflammatory cytokines interleukin-1α and macrophage inflammatory protein-1α. Prolonged neonatal hyperoxia caused functional changes, decreasing lung volume and pulmonary compliance. We conclude that prolonged exposure of neonatal mice to hyperoxia creates a lesion that is very similar to human BPD and suggests that altered cell proliferation may be important in the pathogenesis of chronic neonatal lung disease.
Characterization of a mitogen-activated protein kinase fromPneumocystis carinii
Authors: Thomas Charles F. , Kottom Theodore J. , Leof Edward B. , Limper Andrew H.
Published: December 2017

The pathogenic fungus Pneumocystis carinii causes severe pneumonia in patients with impaired immunity, particularly patients with acquired immunodeficiency syndrome. The life cycle of P. carinii is poorly understood, and the inability to continuously culture P. carinii is a major limitation in understanding its cell biology. In fungi homologous to P. carinii, pheromone mating factors signal through a mitogen-activated protein kinase (MAPK) signal transduction cascade, resulting in mitotic cell cycle arrest and entry into a pathway of conjugation, cellular differentiation, and proliferation. Using degenerate PCR and library screening, we have identified a MAPK cDNA in P. carinii that is highly homologous to fungal MAPKs involved in the pheromone mating signal transduction cascade, and we demonstrate MAPK activity in P. carinii lysates with a specific antiserum derived from the translated P. carinii MAPK cDNA sequence.
Increased lung inflation induces gene expression after pneumonectomy
Authors: Gilbert Kirk A. , Rannels D. Eugene
Published: December 2017

Rapid hyperplastic growth of the remaining lung is initiated by partial pneumonectomy in many mammalian species. The response restores normal tissue structure and function. Although physiological control of compensatory lung growth is documented, little is known about the molecular mechanisms that underlie the process. The aim of this study was to investigate the role of mechanical signals in the induction of immediate-early gene (IEG) expression after pneumonectomy. Expression of c-fos and junB increased nine- and fourfold, respectively, in the right lung within 30 min after left pneumonectomy in rats. In contrast, changes in expression of c-jun and c-myc were not observed. When isolated lungs were subjected to elevated airway pressures in vitro, expression of c-fos and junB was induced in a time- and dose-dependent manner similar to that observed in vivo. Similarly, in vitro lung perfusion induced c-fos and junB expression in the absence of increasing lung inflation. These results support the premise that rapid changes in IEG expression after pneumonectomy are initiated by mechanical signaling in the remaining lung. Elevated IEG expression may contribute to initiation of compensatory lung growth.
Quantitative comparison of C-X-C chemokines produced by endotoxin-stimulated human alveolar macrophages
Authors: Goodman Richard B. , Strieter Robert M. , Frevert Charles W. , Cummings C. James , Tekamp-Olson Patricia , Kunkel Steven L. , Walz Alfred , Martin Thomas R.
Published: December 2017

The C-X-C chemokines are a structurally related and functionally redundant family of proteins with neutrophil chemotactic activity. Many of the C-X-C chemokines are produced by endotoxin-stimulated alveolar macrophages (AMs), but knowledge of their relative quantities and their relative contributions to the total chemotactic activity released from these cells is incomplete. Human AMs were stimulated with or without Escherichia coli endotoxin for 2, 4, 8, and 24 h. The mRNA sequences of interleukin (IL)-8, the 78-amino acid epithelial cell-derived neutrophil activator (ENA-78), growth-related protein (GRO) α, GROβ, and GROγ were cloned by PCR and identified by sequence analysis. The relative mRNA quantities were compared by Northern analysis, and IL-8 was found to predominate. Similarly, IL-8 protein concentrations in the cell supernatants were consistently higher than either the ENA-78 or GRO concentration, and by 24 h, IL-8 concentrations were 10-fold higher than those of the other C-X-C chemokines. Blocking polyclonal antibodies to IL-8 substantially reduced the chemotactic activity in the AM supernatants, whereas antibodies to ENA-78 and GRO had little or no effect. We conclude that IL-8 is the predominant C-X-C chemokine and the dominant neutrophil chemoattractant accumulating in 24-h supernatants of lipopolysaccharide-stimulated human AMs. These studies provide insight into potentially effective strategies of interrupting AM-derived inflammatory signals in the lungs.
Retinoic acid-receptor activation of SP-B gene transcription in respiratory epithelial cells
Authors: Yan Cong , Ghaffari Manely , Whitsett Jeffrey A. , Zeng Xin , Sever Zvjezdana , Lin Sui
Published: December 2017

Retinoids are known to play important roles in organ development of the lung. Retinoids exert their activity by modulating the expression of numerous genes, generally influencing gene transcription, in target cells. In the present work, the mechanism by which retinoic acid (RA) regulates surfactant protein (SP) B expression was assessed in vitro. RA (9- cis-RA) enhanced SP-B mRNA in pulmonary adenocarcinoma cells (H441 cells) and increased transcriptional activity of the SP-B promoter in both H441 and mouse lung epithelial cells (MLE-15). Cotransfection of H441 cells with retinoid nuclear receptor (RAR)-α, -β, and -γ and retinoid X receptor (RXR)-γ further increased the response of the SP-B promoter to RA. Treatment of H441 cells with RA increased immunostaining for the SP-B proprotein and increased the number of cells in which the SP-B proprotein was detected. An RA responsive element mediating RA stimulation of the human SP-B promoter was identified. RAR-α and -γ and RXR-α but not RAR-β or RXR-β and -γ were detected by immunohistochemical analysis of H441 cells. RA, by activating RAR activity, stimulated the transcription and synthesis of SP-B in pulmonary adenocarcinoma cells.
Characteristics of surfactant from SP-A-deficient mice
Authors: Ikegami Machiko , Korfhagen Thomas R. , Whitsett Jeffrey A. , Bruno Michael D. , Wert Susan E. , Wada Kazuko , Jobe Alan H.
Published: December 2017

Mice that are surfactant protein (SP) A deficient [SP-A(−/−)] have no apparent abnormalities in lung function. To understand the contributions of SP-A to surfactant, the biophysical properties and functional characteristics of surfactant from normal [SP-A(+/+)] and SP-A(−/−) mice were evaluated. SP-A-deficient surfactant had a lower buoyant density, a lower percentage of large-aggregate forms, an increased rate of conversion from large-aggregate to small-aggregate forms with surface area cycling, increased sensitivity to inhibition of minimum surface tension by plasma protein, and no tubular myelin by electron microscopy. Nevertheless, large-aggregate surfactants from SP-A(−/−) and SP-A(+/+) mice had similar adsorption rates and improved the lung volume of surfactant-deficient preterm rabbits similarly. Pulmonary edema and death caused by N-nitroso- N-methylurethane-induced lung injury were not different in SP-A(−/−) and SP-A(+/+) mice. The clearance of125I-labeled SP-A from lungs of SP-A(−/−) mice was slightly slower than from SP-A(+/+) mice. Although the absence of SP-A changed the structure and in vitro properties of surfactant, the in vivo function of surfactant in SP-A(−/−) mice was not changed under the conditions of these experiments.
Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules
Authors: Takeyama Kiyoshi , Agustí Carlos , Ueki Iris , Lausier James , Cardell Lars Olaf , Nadel Jay A.
Published: December 2017

We examined the effect of the neutrophil chemoattractants interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine on goblet cell (GC) degranulation in guinea pigs. Chemoattractants caused time-dependent neutrophil recruitment and GC degranulation in vivo. NPC 15669 (an inhibitor of leukocyte infiltration) prevented both responses, implicating neutrophils. ICI 200,355 (an inhibitor of neutrophil elastase and proteinase-3) or secretory leukocyte protease inhibitor (an inhibitor of elastase but not of proteinase-3) abolished IL-8-induced GC degranulation, implicating elastase. Incubating tracheal segments with IL-8 plus neutrophils caused GC degranulation in vitro, an effect due to activation of the neutrophils themselves (and not an effect present in the supernatant). Chemoattractant increased surface staining of elastase and the cleavage of elastase-specific fluorogenic substrate by neutrophils. Pretreatment with anti-intercellular adhesion molecule-1, anti-CD18, or anti-CD11b antibody inhibited the chemoattractant-induced GC degranulation in vitro, implicating adhesion molecules. These studies suggest that chemoattractants cause neutrophil-dependent GC degranulation involving adhesive interactions between cells, with elastase activity occurring at the cell interface, causing GC secretion. The findings, reproduced in human airways, suggest novel methods of therapeutic intervention.
Changes in PKC isoforms in human alveolar macrophages compared with blood monocytes
Authors: Monick Martha M. , Carter A. Brent , Gudmundsson Gunnar , Geist Lois J. , Hunninghake Gary W.
Published: December 2017

Alveolar macrophages play an important role in host defense and in other types of inflammatory processes in the lung. These cells exhibit many alterations in function compared with their precursor cells, blood monocytes. To evaluate a potential mechanism for these differences in function, we evaluated expression of protein kinase C (PKC) isoforms. We found an increase in Ca2+-dependent PKC isoforms in monocytes compared with alveolar macrophages. We also found differential expression of the Ca2+-independent isoforms in alveolar macrophages compared with monocytes. One consequence of the activation of PKC can be increased expression of mitogen-activated protein (MAP) kinase pathways. Therefore, we also evaluated activation of the MAP kinase extracellular signal-regulated kinase (ERK) 2 by the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA activated ERK2 kinase in both alveolar macrophages and monocytes; however, monocytes consistently showed a significantly greater activation of ERK2 kinase by PMA compared with alveolar macrophages. Another known consequence of the activation of PKC and subsequent activation of ERK kinase is activation of the transcription factor activator protein-1 (AP-1). We evaluated the activation of AP-1 by PMA in both monocytes and macrophages. We found very little detectable activation of AP-1, as assessed in a gel shift assay, in alveolar macrophages, whereas monocytes showed a substantial activation of AP-1 by PMA. These studies show that the differential expression of PKC isoforms in alveolar macrophages and blood monocytes is associated with important functional alterations in the cells.
Glucocorticoid hormones downregulate histidine decarboxylase mRNA and enzyme activity in rat lung
Authors: Zahnow Cynthia A. , Panula Pertti , Yamatodani Atsushi , Millhorn David E.
Published: December 2017

Histidine decarboxylase (HDC) is the primary enzyme regulating histamine biosynthesis. Histamine contributes to the pathogenesis of chronic inflammatory disorders such as asthma. Because glucocorticoids are effective in the treatment of asthma, we examined the effects of 6 h of exogenously administered dexamethasone (0.5–3,000 μg/kg ip), corticosterone (0.2–200 mg/kg ip), or endogenously elevated corticosterone (via exposure of rats to 10% oxygen) on HDC expression in the rat lung. HDC transcripts were decreased ∼73% with dexamethasone treatment, 57% with corticosterone treatment, and 50% with exposure to 10% oxygen. Likewise, HDC enzyme activity was decreased 80% by treatment with dexamethasone and corticosterone and 60% by exposure to 10% oxygen. Adrenalectomy prevented the decreases in HDC mRNA and enzyme activity observed in rats exposed to 10% oxygen, suggesting that the adrenal gland is necessary for the mediation of hypoxic effects on HDC gene expression. These results demonstrate that corticosteroids initiate a process that leads to the decrease of HDC mRNA levels and enzyme activity in rat lung.
Direct modulation of tracheal Cl−-channel activity by 5,6- and 11,12-EET
Authors: Salvail Dany , Dumoulin Marc , Rousseau Eric
Published: December 2017

Using microelectrode potential measurements, we tested the involvement of Cl− conductances in the hyperpolarization induced by 5,6- and 11,12-epoxyeicosatrienoic acid (EET) in airway smooth muscle (ASM) cells. 5,6-EET and 11,12-EET (0.75 μM) caused −5.4 ± 1.1- and −3.34 ± 0.95-mV hyperpolarizations, respectively, of rabbit tracheal cells (from a resting membrane potential of −53.25 ± 0.44 mV), with significant residual repolarizations remaining after the Ca2+-activated K+ channels had been blocked by 10 nM iberiotoxin. In bilayer reconstitution experiments, we demonstrated that the EETs directly inhibit a Ca2+-insensitive Cl− channel from bovine ASM; 1 μM 5,6-EET and 1.5 μM 11,12-EET lowered the unitary current amplitude by 40 ( n = 6 experiments) and 44.7% ( n = 4 experiments), respectively. Concentration-dependent decreases in channel open probability were observed, with estimated IC50 values of 0.26 μM for 5,6- and 1.15 μM for 11,12-EET. Furthermore, pharmacomechanical tension measurements showed that both regioisomers induced significant bronchorelaxations in epithelium-denuded ASM strips. These results suggest that 5,6- and 11,12-EET can act in ASM as epithelium-derived hyperpolarizing factors.
Effect of hypoxic exposure on Na+/H+antiport activity, isoform expression, and localization in endothelial cells
Authors: Cutaia M. V. , Parks N. , Centracchio J. , Rounds S. , Yip K. P. , Sun A. M.
Published: December 2017

Little is known about the effects of prolonged hypoxic exposure on membrane ion transport activity. The Na+/H+antiport is an ion transport site that regulates intracellular pH in mammalian cells. We determined the effect of prolonged hypoxic exposure on human pulmonary arterial endothelial cell antiport activity, gene expression, and localization. Monolayers were incubated under hypoxic or normoxic conditions for 72 h. Antiport activity was determined as the rate of recovery from intracellular acidosis. Antiport isoform identification and gene expression were determined with RT-PCR and Northern and Western blots. Antiport localization and F-actin cytoskeleton organization were defined with immunofluorescent staining. Prolonged hypoxic exposure decreased antiport activity, with no change in cell viability compared with normoxic control cells. One antiport isoform [Na+/H+exchanger isoform (NHE) 1] that was localized to the basolateral cell surface was present in human pulmonary arterial endothelial cells. Hypoxic exposure had no effect on NHE1 mRNA transcript expression, but NHE1 protein expression was upregulated. Immunofluorescent staining demonstrated a significant alteration of the F-actin cytoskeleton after hypoxic exposure but no change in NHE1 localization. These results demonstrate that the decrease in NHE1 activity after prolonged hypoxic exposure is not related to altered gene expression. The change in NHE1 activity may have important consequences for vascular function.