bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and function of the mouse surfactant protein B gene
Authors: Bruno M. A. , Bohinski R. J. , Carter J. E. , Foss K. A. , Whitsett J. A.
Published: December 2017

The mouse surfactant protein B (SP-B) gene was isolated from a DBA/2J EMBL3 genomic library, and the nucleotide sequence and transcriptional start site was determined. The SP-B gene was comprised of 11 exons and 10 introns and spanned 9,882 bases. Repetitive elements of the B1, B2, and R families and several other motifs were located within nontranslated regions of the gene. The 5' flanking region of the gene was utilized to express a bacterial chloramphenicol acetyl transferase reporter gene in mouse and human pulmonary adenocarcinoma cell lines. Transcriptionally active regions of the promoter were identified that determined lung epithelial cell-specific gene expression. Regions of the gene promoter shared between the mouse and human SP-B gene were used to locate putative transcription factor binding sites for thyroid transcription factor-1 and hepatocyte nuclear factor-3. The 5' flanking region of the murine SP-B contains cell-specific cis-active elements that regulate SP-B expression in the respiratory epithelium.
The role of alpha 1-antitrypsin in the control of extracellular surfactant metabolism
Authors: Gross N. J. , Bublys V. , D'Anza J. , Brown C. L.
Published: December 2017

Alveolar surfactant exists in several structural forms that are believed to be secular products of the secreted form, lamellar bodies. The conversion of tubular myelin to the small vesicular form appears to require the action of a novel serine protease, surfactant convertase. As the in vitro activity of this enzyme is quite sensitive to inhibition by the serine protease inhibitor alpha 1-antitrypsin, a normal constituent of the alveolar fluid lining layer, we explored the possibility that the alveolar level of alpha 1-antitrypsin might affect the rate of subtype conversion in vivo. When the alveolar alpha 1-antitrypsin level was augmented by tracheal instillation of exogenous alpha 1-antitrypsin, the newly synthesized surfactant phospholipids of spontaneously breathing mice accumulated in the heavy subtype, and virtually none was found in the light subtype form up to 72 h later. An in vivo turnover study suggested that when the alveolar alpha 1-antitrypsin level was raised by the same means, the flux of surfactant through the light (small vesicular) compartment was virtually shut off, despite the availability of abundant amounts of its precursor, heavy subtype (tubular myelin). Finally, mouse lungs that were lavaged to remove resident surfactant and mechanically ventilated for 1-5 h released surfactant that progressed through heavy and light subtype compartments as in vivo. But in mice whose lung alpha 1-antitrypsin might affect the metabolism of alveolar surfactant in addition to its well-known role in lung defense.
Blockade of eosinophil migration by 5-lipoxygenase and cyclooxygenase inhibition in explanted guinea pig trachealis
Authors: Munoz N. M. , Leff A. R.
Published: December 2017

We studied the effects of 5-lipoxygenase inhibition with A63162 and cyclooxygenase inhibition with indomethacin (INDO) on 1) eosinophil chemotaxis and 2) airway narrowing caused by 10(-6) M formyl-Met-Leu-Phe (fMLP) in tracheal explants from guinea pigs. Airway narrowing was assessed by calibrated micrometry, and eosinophil migration from the lamina propria was expressed as number of eosinophils contained per 1 cm tracheal segment. After 120 min, treatment with fMLP caused an increase in luminal eosinophils from 6,804 +/- 1,786 to 303,347 +/- 75,609 cells (P < 0.001); airway diameter narrowed by 20.4 +/- 1.4%. In six preparations, A63162 inhibited airway narrowing caused by fMLP by 54.9 +/- 6.1%; INDO had a similar effect on airway diameter. However, maximal inhibition of eosinophil migration was greater after 10(-6) M A63162 (38,393 +/- 7,434 cells; P < 0.001 vs. fMLP alone) than after treatment with 10(-5) M INDO (123,547 +/- 19,499 cells; P < 0.05). We demonstrate a method that permits simultaneous measurements of eosinophil migration and airway smooth muscle contraction in a guinea pig tracheal explant preparation. Our data suggest that eosinophil chemotaxis and changes in internal airway diameter are caused by activation of both 5-lipoxygenase and cyclooxygenase pathways and that cell migration is independent of the physical consequences of airway smooth muscle contraction.
Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development
Authors: Pierce R. A. , Mariencheck W. I. , Sandefur S. , Crouch E. C. , Parks W. C.
Published: December 2017

The production of elastin, an essential extracellular matrix protein of terminal airway interstitium, occurs mostly during early development. Because glucocorticoids influence airway maturation, we studied the effect of dexamethasone (Dex) on tropoelastin expression during fetal lung development. Timed-pregnant rats were treated with Dex (1 mg/kg daily), and fetal lungs were collected 3 days later at 17, 19, and 21 days of gestation. Dex treatment resulted in about a threefold increase in tropoelastin mRNA levels at 19 days concomitant with accelerated airway development. By in situ hybridization, Dex treatment increased the number of tropoelastin-expressing cells and the level of tropoelastin mRNA per cell. In organ culture, Dex increased lung tropoelastin expression and augmented cortisol stimulation of tropoelastin expression. In fetal pulmonary artery smooth muscle cells, 10(-8) M Dex upregulated tropoelastin mRNA expression and increased tropoelastin promoter-chloramphenicol acetyl transferase activity in transient transfections. These data indicate that pharmacologically administered glucocorticoids transcriptionally upregulate fetal lung tropoelastin expression and suggest that steroid hormones may be important regulators of elastin production in vivo.
NO and H2O2 mechanisms of guanylate cyclase activation in oxygen-dependent responses of rat pulmonary circulation
Authors: Monaco J. A. , Burke-Wolin T.
Published: December 2017

Pulmonary hypoxic vasoconstriction appears to have both endothelium-dependent and -independent regulatory pathways. We have previously described a mechanism of guanylate cyclase activation in isolated pulmonary arteries that is smooth muscle contained and oxygen tension dependent. In this study we examine this mechanism, involving H2O2 metabolism by catalase, and its relationship to endothelial-derived nitric oxide in the regulation of pulmonary artery pressure (PAP) by oxygen tension. Using probes selective for these two distinct mechanisms of guanylate cyclase activation, we found in the isolated buffer-perfused rat lung that 100 microM nitro-L-arginine (NLA), an inhibitor of NO formation, increased baseline PAP from 4.8 +/- 0.6 to 6.0 +/- 0.6 mmHg and hypoxic PAP from 6.8 +/- 0.8 to 8.56 +/- 0.6 mmHg. Aminotriazole (AT), an inhibitor of H2O2 metabolism by catalase, also increased PAP from 4.5 +/- 0.9 to 6.1 +/- 2.0 mmHg (P < or = 0.05) and hypoxic PAP from 6.0 +/- 1.7 to 8.7 +/- 2.7 mmHg (P < or = 0.05). Additionally, while NLA did not affect the vasodilation that occurs upon reoxygenation, AT inhibited the immediate response to reoxygenation. In the presence of both NLA and AT, baseline PAP increased from 4.25 +/- 0.8 to 9.9 +/- 0.92 mmHg (P < or = 0.05), but hypoxia did not significantly increase PAP and the reoxygenation response was inhibited. These data suggest that both NO and H2O2-catalase mechanisms contribute to a similar degree to maintain low PAP under normoxic conditions. The removal of either mediator may contribute to hypoxic vasoconstriction.
Modulation of Ca2+ influx by a mediator released from human tracheal epithelial cells exposed to ozone in vitro
Authors: Qu Q. S. , Chen L. C.
Published: December 2017

Intracellular free Ca2+ ([Ca2+]i) plays a vital role both in maintaining normal cellular function and in cell killing. Few studies have been published regarding its role in ozone (O3)-induced health effects. This study investigated the effect and mechanism of O3 exposure on [Ca2+]i in human tracheal epithelial (HTE) cells. HTE cells grown on Costar Transwell inserts with a liquid-gas interface were exposed to 0, 0.05, 0.1, 0.2 and 0.4 ppm O3 at 37 degrees C for 1 h. After exposure, [Ca2+]i was measured using the fluorescent dye Fluo 3. O3 at 0.4 ppm produced a significant increase in [Ca2+]i, and the increases in [Ca2+]i were blocked by verapamil and 8-(diethylamino)-octyl-3,4,5,-trimethoxybenzoate (TMB-8). These results suggest that the O3-induced [Ca2+]i elevation may involve both Ca2+ release from internal stores and Ca2+ influx across the plasma membrane. Furthermore, both buffer and cell lysate of HTE cells exposed to 0.4 ppm O3 caused a rapid increase in [Ca2+]i of THP-1 human phagocytic monocytes, but the buffer and lysate from air exposed cells did not. These results suggest that O3 exposure causes HTE cells to release a diffusible mediator from the empty Ca(2+)-storing organelle and may be responsible for the sustained and persistent [Ca2+]i elevation in HTE cells exposed to 0.4 ppm O3.
Variability in distribution and populations of gap junctions in ferret trachea during postnatal development
Authors: Carson J. L. , Collier A. M. , Hu S. C. , McLachlan J. B.
Published: December 2017

Immunocytochemical probes have been used to characterize gap junction distribution in the postnatal ferret trachea by epifluorescence and by laser scanning confocal and electron microscopy. A battery of antibodies directed against fragments of different connexins localized beta 1- and beta 2-gap junction antigens (connexins 32 and 26, respectively) at the intercellular borders of the superficial epithelium while alpha 1-gap junction antigen (connexin 43) was localized to the loose connective tissues. Gap junction labeling in the superficial epithelium declined in the first weeks of life but persisted in the developing submucosal glands to the weanling stage. Localization of the alpha- and beta-antigens was specific for connective tissues and epithelial layers, respectively. These observations suggest that communication competence is an important component of early development in the mammalian airways.
Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung
Authors: Ichikawa S. , Sreedharan S. P. , Owen R. L. , Goetzl E. J.
Published: December 2017

Peptidergic nerves in the respiratory tract release vasoactive intestinal peptide (VIP) and substance P (SP), which mediate physiological and immune functions. Antipeptide antibodies to type I VIP receptor (VIPR) and NK-1-type SP receptor (SPR) were used to identify these receptors in normal rat lungs. VIPRs and SPRs were detected on airway epithelium from the trachea to the respiratory bronchioles but not in alveoli, submucosal glands, or pulmonary smooth muscle, except for that of some pulmonary veins. VIPRs also were expressed on macrophages around capillaries, in tracheal and bronchial connective tissue, in alveolar walls, and in the subintima of pulmonary veins and some arterioles. The absence of receptors from airway smooth muscle and submucosal glands implies that mediation of some known effects of SP and VIP may be epithelial or macrophage dependent. Other types of VIPRs and SPRs on airway glands and smooth muscle may transduce direct effects. The similar localization of VIPRs and SPRs in rat lung suggests that VIP and SP may coordinately regulate some pulmonary functions.
Characterization of endothelin receptors in newborn piglet lung
Authors: Perreault T. , Baribeau J.
Published: December 2017

Endothelins (ET-1, ET-2, and ET-3) cause dilation and constriction as a result of binding to different ET receptors. ETA receptor is responsible for the vasoconstrictor response, while ETB receptors lead to vasodilation (ETB1) or vasoconstriction (ETB2). Although the effects of ETs have been described in the neonatal pulmonary vasculature, ET receptors have not been characterized extensively. Therefore, in newborn piglets we aimed to characterize ET receptors by studying 1) in isolated perfused lungs the effects of ET-1, ET-3, and the ETB receptor agonists sarafotoxin S6c (S6c) and BQ-3020 on perfusion pressure with or without an ETA antagonist, BQ-123, or an ETB1 antagonist, RES-701-1, and 2) the concentration-dependence of ET-1 and ET-3 on their binding to microsomes from arteries and veins of piglet lungs. ET-1, ET-3, S6c, and BQ-3020 cause an early-onset dilation followed by a late-onset constriction. The dilator response to ET-3 is blunted by RES-701-1 (P < 0.005), while the inhibition of the dilator response of ET-1 almost reaches significance (P = 0.06). BQ-123 inhibits incompletely (P < 0.05) the constrictor response to ET-1, while it does not alter the response to ET-3. This suggests that constriction may follow binding to ETA as well as ETB2 receptors. Binding studies reveal that ET receptors are abundant in pulmonary vessels. ETA receptors are predominant, but ETB1 and likely ETB2 receptors are also present. Also, receptor affinities are higher in veins than in arteries.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of the promoter of human pulmonary surfactant protein B gene
Authors: Venkatesh V. C. , Planer B. C. , Schwartz M. , Vanderbilt J. N. , White R. T. , Ballard P. L.
Published: December 2017

Pulmonary surfactant protein B (SP-B) is required for normal surfactant function and for survival at birth. To further study SP-B gene expression, we sequenced genomic clones and examined promoter activity of SP-B DNA fragments by transient transfection. A plasmid construct containing human SP-B fragment -1039/+431 linked to chloramphenicol acetyltransferase (CAT) reporter gene was readily expressed in H441 cells, which are derived from a human lung adenocarcinoma, but was < 4% as active in Hep G2, HeLa, and Calu 6 cell lines. SP-B promoter activity in H441 cells was orientation dependent and increased by linked Rous sarcoma virus (RSV) enhancer and was stronger than for thymidine kinase (tk) and RSV promoters. Deletional mapping of the 5' flanking region with exonuclease III suggested nonspecific negative (-811/-1039)- and positive (-453/-641)-control regions and a cell-specific enhancer region at -208 to -54. When a fragment from -403 to -35 base pairs (bp) was placed upstream or downstream of tkCAT, in either orientation, expression in H441 cells but not other cell lines was increased 4- to 28-fold relative to tkCAT. Deletional analysis of the 3' terminus indicated a requirement for at least 7 bp 3' of the transcription start site. Promoter activity was strongly inhibited in a dose-dependent fashion by phorbol ester, with responsiveness mapped to bp -208/-54, but was not responsive to glucocorticoid.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanical strain-enhanced fetal lung cell proliferation is mediated by phospholipase C and D and protein kinase C
Authors: Liu M. , Xu J. , Liu J. , Kraw M. E. , Tanswell A. K. , Post M.
Published: December 2017

The signaling pathways by which intermittent strain (60 cycles/min, 15 min/h) regulates proliferation of mixed fetal rat lung cell in vitro have been investigated. Adenosine 3',5'-cyclic monophosphate (cAMP) content and cAMP-dependent protein kinase (PKA) activity were not affected by strain. The stimulatory effect of strain on DNA synthesis was also not influenced by the cyclic nucleotide-dependent protein kinase inhibitors H-8 or HA-1004, the adenylate cyclase inhibitor SQ-22536, or a PKA inhibitor and cAMP antagonist, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). In contrast, intracellular concentrations of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), were dramatically increased after a short period of strain. This increase in second messengers was accompanied by an increased tyrosine phosphorylation of phospholipase C-gamma 1. Phospholipase D activity was also increased by strain. Mechanical strain elicited a shift in the subcellular distribution of PKC activity from cytosol to membranes shortly after the onset of strain. The specific activity of PKC in the membranes increased 6- to 10-fold within 5-15 min and remained increased throughout a 48-h period of intermittent strain. Strain-induced PKC activation and DNA synthesis were blocked by the PKC inhibitors H-7, staurosporine, and calphostin C, as well as by the phospholipase C inhibitor U-73,122. We conclude that mechanical strain of mixed fetal rat lung cells activates phospholipid turnover via phospholipases, followed by PKC activation, which then triggers the downstream events that lead to cell proliferation.
Effects of NADPH oxidase inhibitors on hypoxic vasoconstriction in buffer-perfused rabbit lungs
Authors: Grimminger F. , Weissmann N. , Spriestersbach R. , Becker E. , Rosseau S. , Seeger W.
Published: December 2017

The involvement of NADPH oxidase in hypoxic pulmonary vasoconstriction (HPV) was investigated in buffer-perfused rabbit lungs, employing the inhibitors diphenyleneiodonium (DPI) and apocynin. Responses to the vasoconstrictors U-46619 and angiotensin II (ANG II) were used to test specificity. Lung nitric oxide (NO) generation was assessed by on-line monitoring of NO exhalation (chemiluminescence), and the efficacy of DPI and apocynin on the NADPH oxidase-dependent O2- generation was quantified in alveolar macrophages by fluorescent-activated cell sorter technique. In a concentration range between 1 and 5 mM, apocynin inhibited macrophage respiratory burst and HPV but similarly suppressed U-46619-induced vasoconstrictor responses. DPI inhibited macrophage O2- generation in concentrations > or = 0.5 microM. At doses between 0.5 and 1.5 microM, DPI blocked lung NO generation, thereby increasing HPV. At higher doses (4 microM), in contrast, DPI fully blocked the hypoxia-induced pressor responses, whereas the vasoconstrictor responses to U-46619 and [Asn1, Val5] ANG II were not diminished. In the presence of NG-monomethyl-L-arginine, used to block lung NO generation throughout, DPI exhibited only the monophasic selective inhibition of HPV. We conclude that apocynin lacks specificity for HPV, but DPI, in addition to inhibiting lung NO generation, causes selective blockade of the hypoxia-induced vasoconstriction. This finding supports the hypothesis that an NADPH oxidase is involved in hypoxia sensing or specific signal transduction events underlying HPV.
Hypoxia induces capillary network formation in cultured bovine pulmonary microvessel endothelial cells
Authors: Phillips P. G. , Birnby L. M. , Narendran A.
Published: December 2017

The development of new vessels (angiogenesis) is essential to wound healing. The center of a wound space is hypoxic, a condition that has been shown to stimulate angiogenesis in animal models of coronary artery occlusion. Because the mechanisms involved in this complex process are difficult to study in situ, an in vitro model would provide a useful complement to in vivo studies. This laboratory has developed and characterized calf pulmonary microvessel endothelial cell (PMVEC) cultures and an in vitro model system of angiogenesis using collagen three-dimensional gels that permit migration of cells into vessel networks. This system was used to study the direct effect of normoxia (20% O2) or hypoxia (5% O2) on PMVEC ability to undergo angiogenesis in vitro. Major changes leading to formation of capillary-like networks occurred during the first 3 days of hypoxic exposure only and included restructuring of actin filament networks, focal changes in distribution of basic fibroblast growth factor, and orientation and migration of cell tracts into a collagen gel matrix to form vessel networks.
Developmental changes in intracellular Ca2+ response of carotid chemoreceptor cells to hypoxia
Authors: Sterni L. M. , Bamford O. S. , Tomares S. M. , Montrose M. H. , Carroll J. L.
Published: December 2017

The carotid chemoreceptor response to hypoxia is weak just after birth and increases during postnatal development. The mechanisms underlying chemoreceptor maturation are unknown. We tested the hypothesis that carotid chemoreceptor maturation occurs at the glomus cell level by measuring intracellular calcium ([Ca2+]i) mobilization in response to hypoxia, anoxia, and NaCN in freshly dissociated cells from newborn vs. adult rabbit carotid bodies. Cells were loaded with fura 2 and superfused at 37 degrees C with balanced salt solution equilibrated with 5% CO2. [Ca2+]i mobilization in response to 3-min challenges of hypoxia (PO2 approximately 15 mmHg), anoxia (PO2 approximately 0 mmHg), and NaCN (1 mM) was measured using a digital imaging microscope. The fluorescence intensity ratio was used to calculate [Ca2+]i. Peak [Ca2+]i responses to all three challenges were three- to fivefold greater in glomus cells from adult compared with newborn carotid chemoreceptors. In addition, the average normoxic [Ca2+]i baseline was approximately threefold higher in the adult glomus cells. These results suggest that carotid chemoreceptor glomus cell sensitivity to natural stimuli, as reflected by the [Ca2+]i response, depends on the level of postnatal maturity.
Rat mesothelial and tracheal epithelial cells show equal DNA sensitivity to hydrogen peroxide-induced oxidant injury
Authors: Churg A. , Keeling B. , Gilks B. , Porter S. , Olive P.
Published: December 2017

To study the relative sensitivity of rat tracheal epithelial and mesothelial cell DNA to oxidant damage, we used the comet assay, a gel microelectrophoresis method that allows visual determination of DNA strand breaks on a cell-by-cell basis, to evaluate damage after hydrogen peroxide exposure. By both a qualitative and a quantitative assay, tracheal epithelial mesothelial cells demonstrated a similar dose-response increase in the number of cells showing strand breaks and the number of breaks per cell after exposure to increasing concentrations of hydrogen peroxide; but even at the highest concentration, some cells failed to show damage. By contrast, 100% of cultured V79 lung fibroblasts showed evidence of damage. Catalase and deferoxamine largely prevented the formation of strand breaks, while superoxide dismutase was not protective. To evaluate DNA repair, cells were exposed to 10 microM hydrogen peroxide for 10 min, washed, and maintained in culture medium; by 2 h the proportion of mesothelial and epithelial cells showing comets had returned to control levels for both cell types. Both cell types also showed a similar pattern of increasing damage after continuous exposure to 10 microM hydrogen peroxide for periods up to 2 h. We conclude that, in this system, 1) mesothelial and tracheobronchial epithelial cells show a similar pattern of DNA injury and repair after hydrogen peroxide exposure; 2) hydrogen peroxide damages DNA of both cell types via a mechanism probably related to the iron-catalyzed formation of hydroxyl radical; and 3) both types of cells appear to be heterogeneous in their sensitivity to oxidant damage, with some cells showing extreme resistance to such damage.
Cigarette smoking, emphysema, and damage to α1-proteinase inhibitor
Authors: Evans Mark D. , Pryor William A.
Published: December 2017

Pages L593–L611: Mark D. Evans and William A. Pryor. Invited Review, “Cigarette smoking, emphysema, and damage to α1-proteinase inhibitor.” On p. L600, Fig. 4 B should appear as shown below, with S (sulfur) and not C (carbon) in the formula for methionine oxidation to the sulfoxide.
Modulation of mast cell functions by in vitro ozone exposure
Authors: Peden D. B. , Dailey L.
Published: December 2017

Exposure to ozone has been reported to cause increased immediate bronchial reactivity to inhaled allergen in asthmatics. The purpose of these studies was to determine whether ozone induces either spontaneous physiological degranulation or enhanced immunoglobulin E (IgE)-mediated degranulation of mast cells, thus accounting for the in vivo effects noted in asthmatics. A rat mast cell line (RBL-2H3) was exposed to different levels of ozone (0.1, 0.3, 0.5, and 1.0 ppm), covered by different amounts of buffer, and both cytotoxic and nontoxic exposure conditions were determined. In addition to cytotoxicity, spontaneous release of granule products and prostaglandin D2 (PGD2) associated with ozone exposure were assessed. RBL-2H3 cells were also exposed to ozone under noncytotoxic conditions followed by stimulation with alpha-IgE to cross-link membrane-bound IgE and A23187 so that the effect of ozone on stimulated degranulation could be examined. Only exposure conditions associated with cytotoxicity were associated with spontaneous release of mast cell serotonin, indicating no physiologic degranulation due to ozone exposure. Data presented herein also demonstrate that ozone substantially inhibited both IgE- and A23187-induced degranulation. Neither catalase nor superoxide dismutase protected cells from the inhibitory effect of ozone, indicating that ozone does not act through generation of H2O2 or superoxide. Additionally, ozone caused a modest increase in spontaneous PGD2 generation only under cytotoxic conditions. Thus ozone appears to inhibit mast cell degranulation after IgE- or A23187-mediated stimulation and causes direct release of mast cell granule products and PGD2 only under conditions associated with membrane cytotoxicity.
Mechanical strain increases contractile enzyme activity in cultured airway smooth muscle cells
Authors: Smith P. G. , Tokui T. , Ikebe M.
Published: December 2017

Smooth muscle hypertrophy is often found in tissue subjected to abnormal physical stress. To determine if physical stress (strain) per se could increase the contractile potential of airway smooth muscle (ASM), we compared cultured ASM cells subjected to strain to control cells (no strain) for rates of 1) myosin light chain kinase (MLCK)-mediated myosin light chain (LC20) phosphorylation, 2) actin-activated myosin ATPase, and 3) myosin light chain phosphatase-mediated myosin dephosphorylation. Lysates from strained cells showed increases in both LC20 phosphorylation activity and actomyosin ATPase activity but decreased rates of phosphatase-dependent myosin dephosphorylation. The increased LC20 phosphorylation activity and ATPase activity of the strained cells were accompanied by increases in cellular content of MLCK and myosin, respectively, compared with control. Because the cultured ASM cells exposed to strain expressed higher MLCK activity and actomyosin ATPase activity but lower myosin light chain phosphatase activity, these data suggest that physical stress in part determines ASM potential for contractile state.
Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome
Authors: Kim D. K. , Fukuda T. , Thompson B. T. , Cockrill B. , Hales C. , Bonventre J. V.
Published: December 2017

The role of phospholipase A2 (PLA2) in lung injury in humans is unclear. Previous studies have failed to identify an increase in PLA2 activity in bronchoalveolar lavage fluids (BALF) of patients with the adult respiratory distress syndrome (ARDS). In this study, increased phospholipase A2 (PLA2) activity was detected in BALF from patients with ARDS. PLA2 levels in BALF correlated positively with lung injury score in patients with lung disease. BALF PLA2 activity in patients with ARDS was resolved into heparin binding and nonbinding activities. Both PLA2 activities were increased in BALF of ARDS patients. The PLA2 activity that bound to heparin was identified as a group II PLA2 by its chromatographic characteristics, its inhibition by dithiothreitol, its substrate specificity, and its approximate molecular mass of 14 kDa. The second PLA2 activity was further purified and found to require Ca2+ at a concentration > 2 x 10(-4) M for activity. This form of PLA2 exhibited a neutral and broad pH optimum (pH 6.0-8.0) and hydrolyzed both phosphatidylethanolamine and phosphatidylcholine effectively. Its apparent molecular mass was estimated to be 80–90 kDa. Neither anti-pancreatic PLA2 antiserum nor anti-pig spleen cytosolic 100-kDa PLA2 antiserum immunoprecipitated the enzymatic activity. Thus at least two forms of PLA2 are increased in activity in BALF of patients with ARDS, a group II PLA2 and a biochemically and immunochemically form distinct from group I, group II, and cytosolic PLA2. Increased lung PLA2 activity may be important for the pathophysiology of ARDS.
Disparate cytokine regulation of ICAM-1 in rat alveolar epithelial cells and pulmonary endothelial cells in vitro
Authors: Barton W. W. , Wilcoxen S. , Christensen P. J. , Paine R.
Published: December 2017

Intercellular adhesion molecule-1 (ICAM-1) is expressed at high levels on type I alveolar epithelial cells in the normal lung and is induced in vitro as type II cells spread in primary culture. In contrast, in most nonhematopoetic cells ICAM-1 expression is induced in response to inflammatory cytokines. We have formed the hypothesis that the signals that control ICAM-1 expression in alveolar epithelial cells are fundamentally different from those controlling expression in most other cells. To test this hypothesis, we have investigated the influence of inflammatory cytokines on ICAM-1 expression in isolated type II cells that have spread in culture and compared this response to that of rat pulmonary artery endothelial cells (RPAEC). ICAM-1 protein, determined both by a cell-based enzyme-linked immunosorbent assay and by Western blot analysis, and mRNA were minimally expressed in unstimulated RPAEC but were significantly induced in a time- and dose-dependent manner by treatment with tumor necrosis factor-alpha, interleukin-1 beta, or interferon-gamma. In contrast, these cytokines did not influence the constitutive high level ICAM-1 protein expression in alveolar epithelial cells and only minimally affected steady-state mRNA levels. ICAM-1 mRNA half-life, measured in the presence of actinomycin D, was relatively long at 7 h in alveolar epithelial cells and 4 h in RPAEC. The striking lack of response of ICAM-1 expression by alveolar epithelial cells to inflammatory cytokines is in contrast to virtually all other epithelial cells studied to date and supports the hypothesis that ICAM-1 expression by these cells is a function of cellular differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Purinergic regulation of ion transport across nonciliated bronchiolar epithelial (Clara) cells
Authors: Van Scott M. R. , Chinet T. C. , Burnette A. D. , Paradiso A. M.
Published: December 2017

Previous studies demonstrated that elevation of intracellular calcium concentration ([Ca2+]i) increased electrogenic anion transport by bronchiolar epithelia. Extracellular nucleotides were shown to elevate [Ca2+]i and transepithelial short-circuit current (Isc) in proximal airways epithelia. In this study purine and pyrimidine nucleotides were investigated for their ability to regulate ion transport by rabbit nonciliated bronchiolar epithelial (Clara) cells in culture. ATP in the apical bath induced a concentration-dependent transient increase in [Ca2+]i and Isc. Mean effective doses (ED50) of the responses were 10(-7) M and 10(-6) M, respectively. Transepithelial resistance (Rt) decreased. The peak changes in Isc and Rt were 7.8 +/- 1.2 microA/cm2 and -59 +/- 14 omega.cm2 (n = 26, basal Isc = 47.4 +/- 4.3 microA/cm2 and Rt = 428 +/- 40 omega.cm2). Some preparations exhibited a small residual increase in Isc after the initial response, but the change was not statistically significant (delta Isc = 1.7 +/- 1.2 microA/cm2, n = 18). Addition of ATP to the basolateral bath had no detectable effects. Purinoceptor agonists were used to characterize the receptors mediating the change in Isc. UTP and ATP gamma S increased Isc and inhibited subsequent stimulation by ATP. ADP, ADP beta S, 2-methylthio-ATP, and alpha, beta-methylene-ATP had negligible effects on the peak delta Isc and subsequent stimulation by ATP. The ionic mechanism underlying the ATP-induced increase in Isc was investigated with the use of specific ion-transport inhibitors and by ion substitution.(ABSTRACT TRUNCATED AT 250 WORDS)
Bradykinin stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity
Authors: Koyama S. , Rennard S. I. , Robbins R. A.
Published: December 2017

In the present investigation, we evaluated the potential of bradykinin (BK), histamine, and serotonin to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) from bronchial epithelial cells (BEC). BK significantly stimulated BEC to release NCA and MCA in a dose- and time-dependent manner. Histamine weakly but significantly induced the release of both NCA and MCA in a similar fashion. Serotonin did not stimulate BEC. Checkerboard analysis showed that the NCA and MCA released in response to BK were chemotactic. Molecular-sieve column chromatography by Sephadex G-75 revealed that BK induced a single low-molecular-weight peak (approximately 400 Da) for both NCA and MCA. The releases of NCA and MCA in response to BK and histamine were inhibited by lipoxygenase inhibitors (P < 0.01). The released NCA was inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01) and was slightly inhibited by platelet-activating factor receptor antagonist. LTB4 was increased in BK-stimulated BEC supernatant (P < 0.01). BK B2-receptor antagonist attenuated the release of NCA and MCA. These data suggest that BK and histamine may stimulate BEC to release NCA and MCA and may modulate neutrophil and monocyte recruitment into the airways in patients with asthma.
Optimization of cationic lipid-mediated gene transfer to airway epithelia
Authors: Fasbender A. J. , Zabner J. , Welsh M. J.
Published: December 2017

The use of cationic lipids for gene transfer to airway epithelia has shown promise in in vitro and in vivo studies. However, previous studies have used a wide variety of different lipid preparations and different formulations. Few studies have been designed to optimize the variables involved in transfection, and none have been focused on airway epithelia. Therefore we examined variables that affect cationic lipid-mediated transfection of HeLa cells and of airway epithelial cells grown on permeable filter supports at the air-liquid interface. To quantitate expression of cDNA, we assayed expression of luciferase. We found that the ratio of DNA to lipid was an important variable that determined transfection efficiency. In both HeLa and airway epithelial cells, the optimum charge ratio of cationic lipid to anionic DNA was approximately 1.25, consistent with the notion that a positively charged complex facilitates interaction with the negatively charged cell membrane. After testing a series of readily available cationic lipids, we found that 1,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE)/dioleoyl phosphatidylethandamine (DOPE) appeared to show good efficacy. The concentration of DNA and cationic lipid also played an important role: in HeLa cells the optimum concentration of cationic lipid was approximately 5 microM and in airway epithelial cells was approximately 15 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of poly(ADP-ribose) polymerase preserves surfactant synthesis after hydrogen peroxide exposure
Authors: Hudak B. B. , Tufariello J. , Sokolowski J. , Maloney C. , Holm B. A.
Published: December 2017

Exposure to hydrogen peroxide (H2O2) decreases phosphatidylcholine (PC) synthesis in rabbit type II pneumocytes. Activation of poly(ADP-ribose) polymerase (PARP) may play a role in this process. Exposure of type II pneumocytes to H2O2 resulted in a 53% decrease in the rate of incorporation of [3H]choline into PC (P < 0.001). Cell NAD and ATP levels were decreased by 52% (P < 0.001) and 39% (P < 0.01), respectively, without significant changes in cell viability. Exposure to H2O2 also resulted in a 52% (P < 0.05) increase in the activity of PARP. Preincubation of type II cells with inhibitors of PARP (nicotinamide; 3-aminobenzamide) before H2O2 exposure prevented the increase in PARP activity, and blocked the decreases in ATP, NAD, and rate of PC synthesis. These results suggest that the energy depletion associated with activation of PARP contributes to the effects of oxidant stress on type II cell metabolic function and may be ameliorated by pharmacological agents in vitro.
Genetic control of susceptibility to ozone-induced changes in mouse tracheal electrophysiology
Authors: Takahashi M. , Kleeberger S. R. , Croxton T. L.
Published: December 2017

Genetic factors influence the responses of humans and rodents to ozone (O3) inhalation. We previously demonstrated differential O3-induced decreases of tracheal potential (VT) in C57BL/6J (B6) and C3H/HeJ (C3) strain mice. To characterize the genetic basis of this strain-specific response, we measured VT in progeny of B6 and C3 strain mice and in six additional inbred strains of mice 6 h after O3 exposures (2 ppm x 3 h). First filial generation (F1) mice and second generation backcrosses with the resistant parent were uniformly resistant. The distribution of VT in second generation backcrosses with the susceptible parent resembled that of a population composed of resistant and susceptible mice in a 1:1 ratio. These data suggested simple autosomal recessive inheritance of susceptibility. However, overlapping distributions prevented statistical confirmation of that hypothesis. Strain screening revealed a susceptible phenotype in 129/J, A/J, B6, C3HeB/FeJ, and SJL/J and a resistant phenotype in AKR/J, C3, and CBA/J inbred mouse strains. Because this pattern of susceptibility to changes in VT differs from that of susceptibility to lung inflammation, the genetic factors that determine these two responses to acute O3 are not identical.
Airways of a hyperresponsive rat strain show decreased relaxant responses to sodium nitroprusside
Authors: Jia Y. , Xu L. , Heisler S. , Martin J. G.
Published: December 2017

The aim of the current studies was to investigate the possibility that a decreased relaxant response to nitric oxide (NO) might contribute to strain-related differences in airway responsiveness in the rat. Isolated tracheal rings from hyperresponsive. Fisher rats were confirmed to be more responsive to carbachol [mean effective concentration (EC50) = 2.45 x 10(-7) M] than those from Lewis (EC50 = 3.60 x 10(-7) M, P < 0.03) and ACI (EC50 = 3.85 x 10(-7) M, P < 0.01) rats. Sodium nitroprusside (SNP), a NO donor, caused relaxation of the carbachol (10(-6) M) contracted tracheal rings, but the half-maximal inhibition concentration (IC50) SNP in Fisher rats (5.60 x 10(-6) M) was significantly higher than in Lewis (1.34 x 10(-6) M, P < 0.001) and ACI rats (1.13 x 10(-6) M, P < 0.0005). The inhibitory effect of SNP on airway responsiveness to inhaled methacholine (MCh) in vivo was also less pronounced for Fisher than Lewis rats. SNP induced an accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in cultured tracheal smooth muscle cells (TSM). Fisher TSM produced less cGMP on exposure to SNP compared with TSM from ACI (P < 0.01) and Lewis (P < 0.0001) rats. A decreased guanylyl cyclase activity may account for the impaired relaxant effect of SNP in Fisher rats.
Collagen is a survival factor against LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells
Authors: Hoyt D. G. , Mannix R. J. , Rusnak J. M. , Pitt B. R. , Lazo J. S.
Published: December 2017

Lipopolysaccharide (LPS) causes direct pulmonary endothelial injury that can precipitate cell death. We investigated the ability of LPS to produce apoptosis in sheep pulmonary artery endothelial cells (SPAEC) grown in monolayer on plastic or collagen. When SPAEC were grown on plastic, LPS (100 ng/ml) caused internucleosomal DNA fragmentation (IDF) to 180- to 200-base pair ladders after 4 h. Higher-order chromatin damage, producing 50-kilobase DNA fragments, occurred within 2 h. Significant DNA strand breaks were seen in attached cells within 1 h incubation with > or = 1 ng LPS/ml, using in situ labeling by break extension (ISBE). DNA strand breakage in attached cells peaked after 2 h and remained elevated after 4 h. Detachment of SPAEC from the monolayer did not begin until 4 h. SPAEC cultured on collagen were protected from LPS-induced apoptosis; DNA damage measured by IDF, high-molecular-weight DNA fragmentation, and ISBE were suppressed. The protective effect of collagen was not due to inactivation of LPS. Thus LPS-induced apoptosis occurs in SPAEC after genotoxic damage and this process is suppressed by the extracellular matrix.
Antisense oligonucleotides for PDGF-B and its receptor inhibit mechanical strain-induced fetal lung cell growth
Authors: Liu M. , Liu J. , Buch S. , Tanswell A. K. , Post M.
Published: December 2017

An intermittent mechanical strain regimen, which simulates fetal breathing movements, has been shown to enhance DNA synthesis and cell division of fetal rat lung cells. The signaling mechanism through which the physical stimulus is transduced is unknown. Herein, we report that mechanical strain (5% elongation, 60 cycles/min) of fetal lung cells, cultured in a three-dimensional environment provided by Gelfoam sponges, increased the mRNA levels of platelet-derived growth factor B (PDGF-B) and beta-receptor (PDGF-beta-R) within 5 min of the onset of strain. Both PDGF-BB and PDGF-beta-R proteins were increased after a 24-h intermittent strain (15 min/h). Phosphorothioate antisense PDGF-B oligonucleotides (ON) at 15 microM abolished the strain-enhanced DNA synthesis and cell growth. Scrambled PDGF-B ON had no such effect. A neutralizing PDGF-BB antibody (10 micrograms/ml) also attenuated strain-induced DNA synthesis. Furthermore, the strain-induced stimulatory effect on DNA synthesis of fetal lung cells was blocked by tyrphostin 9 (1 microM), a PDGF receptor-associated tyrosine kinase inhibitor, but not by its inactive structural analogue tyrphostin 1. Antisense but not sense PDGF-beta-R ON (10 microM) also abrogated the strain-enhanced DNA synthesis. These results suggest that physical forces such as fetal breathing movements regulate fetal lung cell growth by controlling PDGF-B and PDGF-beta-R gene expression.
Inhibition of glycolysis by 2-DG increases [Ca2+]i in pulmonary arterial smooth muscle cells
Authors: Bright R. T. , Salvaterra C. G. , Rubin L. J. , Yuan X. J.
Published: December 2017

Inhibition of glycolysis depolarizes single pulmonary arterial smooth muscle cells (PASMC) and potentiates hypoxic pulmonary vasoconstriction (HPV) in isolated perfused rat lungs. Whether glycolytic inhibition causes an increase in the intracellular Ca2+ concentration ([Ca2+]i) in PASMC was determined in this study. [Ca2+]i was measured in primary cultured rat PASMC using the Ca2+ -sensitive fluorescent indicator, fura 2, and quantitative fluorescence microscopy. Extracellular application of the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), significantly and reversibly increased [Ca2+]i in PASMC. Removal of extracellular Ca2+ and application of the Ca2+ channel blocker, verapamil (10 microM), attenuated, but did not eliminate, the 2-DG-induced rise in [Ca2+]i. In the absence of extracellular Ca2+, however, depletion of inositol triphosphate-sensitive intracellular Ca2+ stores by 10 microM cyclopiazonic acid (CPA) completely abolished the 2-DG-induced increase in [Ca2+]i. The data suggest that 2-DG-induced increases in [Ca2+]i result from both Ca2+ influx through the verapamil-sensitive voltage-gated Ca2+ channels and Ca2+ release from the CPA-sensitive intracellular Ca2+ stores.
Correlation of expression of transcription factor C/EBP alpha and surfactant protein genes in lung cells
Authors: Li F. , Rosenberg E. , Smith C. I. , Notarfrancesco K. , Reisher S. R. , Shuman H. , Feinstein S. I.
Published: December 2017

C/EBP alpha is a transcription factor which can stimulate expression of genes in lipid-metabolizing epithelial cells. We have detected an increase in mRNA for C/EBP alpha in lungs of fetal rats between days 18 and 20 of gestation, correlating with events occurring during the maturation of the surfactant system, such as an increase in the amount of surfactant protein A mRNA. We have found that C/EBP alpha mRNA levels are substantially enriched in type II alveolar epithelial cells purified from adult lung and that the C/EBP alpha protein is present in type II cell nuclei. When the type II cells are removed from the lung and purified, the protein is rapidly lost. However, both surfactant protein gene expression and C/EBP alpha reappear when cells are plated on Matrigel. Levels of C/EBP alpha mRNA from purified cells decline much more slowly than the protein and are still detectable 48 h after cells have been plated on standard tissue culture plastic. We have also detected the C/EBP alpha protein in nuclear extracts of NCI-H441, a lung-derived cell line that expresses surfactant proteins A and B, but not in A549, a lung-derived cell line which does not express the surfactant proteins. Our data suggest that C/EBP alpha is involved in the development and maintenance of the surfactant system in lung type II cells.
Glutathione homeostasis in alveolar epithelial cells in vitro and lung in vivo under oxidative stress
Authors: Rahman I. , Li X. Y. , Donaldson K. , Harrison D. J. , MacNee W.
Published: December 2017

We studied the acute effects of cigarette smoke condensate (CSC), H2O2, and tumor necrosis factor (TNF)-alpha on the glutathione (GSH) redox system in a human type II epithelial cell line (A549) in vitro. CSC, in vitro and in vivo after intratracheal instillation of CSC in the rat, produced a depletion of intracellular soluble GSH, concomitant with GSH-conjugate formation, without significant elevation of oxidized GSH (GSSG), protein-GSH mixed disulfides (PrSSG), nor any GSH efflux from the cells. By contrast, H2O2 (500 microM) after 5-min exposure to A549 cells caused significant depletion of intracellular GSH associated with an efflux of GSSG and a significant increase in the formation of PrSSG. TNF-alpha, in concentrations of 100 U/ml and 1,000 U/ml, produced a significant depletion of GSH in A549 cells after 4- and 24-h exposure, with an associated elevation of GSSG. The activities of glutathione peroxidase, gamma-glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase were significantly decreased in epithelial cells and in rat lungs after CSC exposure, without change in glutathione S-transferase and glutathione reductase activities. By contrast, H2O2 and TNF-alpha did not alter these enzyme activities in epithelial cells. Thus GSH depletion and alteration in enzyme activities in alveolar epithelial cells by CSC, H2O2, and TNF-alpha occur by different mechanisms.
Hydrogen peroxide contracts human airways in vitro: role of epithelium
Authors: Rabe K. F. , Dent G. , Magnussen H.
Published: December 2017

The effects of hydrogen peroxide (H2O2) on human airway smooth muscle tone were determined in vitro. Treatment with H2O2 led to transient concentration-related contractions in the organ bath, amounting to 118 +/- 14 mg (mean +/- SE; n = 12) at 1 mM H2O2, and to greater and more prolonged contractions under superfusion conditions, amounting to 451 +/- 71 mg (n = 17) at 1 mM H2O2. Epithelial removal augmented the response to H2O2 in both systems. Addition of catalase (500 U/ml) abolished the effects of H2O2. Pretreatment of superfused tissues with indomethacin (3 microM) shifted the concentration-effect curve to H2O2 rightward and almost abolished the response to 1 mM H2O2 in epithelium-intact preparations (n = 16; P < 0.05); the response in epithelium-denuded tissues was also significantly inhibited (n = 16; P < 0.05). Pretreatment of the tissues with the TP prostanoid-receptor antagonist GR-32191B (1 microM) also inhibited the contractile effect of H2O2 in epithelium-intact and -denuded tissues. In separate experiments, H2O2 resulted in concentration-related generation of prostaglandin (PG) D2 from isolated airway preparations. The amount of PGD2 released was not different in tissues with intact epithelium compared with those without (n = 9; NS). We conclude that H2O2 exerts on isolated human airways a contractile effect that is augmented by epithelium removal and is largely mediated by prostanoids. The source of PGD2 does not appear to be the epithelium, which we suggest serves mainly as a barrier against H2O2-mediated bronchoconstriction.