bluuebunny/crossref_metadata_2025 huggingface dataset:
Deoxyglucose and reduced glutathione mimic effects of hypoxia on K+ and Ca2+ conductances in pulmonary artery cells
Authors: Yuan X. J. , Tod M. L. , Rubin L. J. , Blaustein M. P.
Published: December 2017

Hypoxia-induced pulmonary vasoconstriction (HPV) is triggered by a rise in cytosolic Ca2+ concentration ([Ca2+]i) that is partially controlled by voltage-gated Ca2+ channels. Hypoxia inhibits voltage-gated K+ (KV) channels in pulmonary artery (PA) myocytes. This depolarizes the cells, opens voltage-gated Ca2+ channels, thereby increases [Ca2+]i, and initiates HPV. In intact animals and isolated perfused lungs, metabolic inhibitors and reducing agents augment HPV. We compared the effects of hypoxia with the glycolysis inhibitor, 2-deoxy-D-glucose (2-DOG), and the reducing agent, reduced glutathione (GSH), on voltage-gated steady-state K+ currents (IK,ss) and membrane potential (Em) in cultured rat pulmonary and mesenteric arterial (MA) smooth muscle cells. Bath application of 10 mM 2-DOG (glucose-free) or 5-10 mM GSH reversibly reduced IK,ss by 25-35% in PA myocytes, with 5 mM ATP present in the pipette solution. Neither hypoxia nor 2-DOG significantly affected IK,ss in MA myocytes, but GSH did reduce IK,ss in these cells. Furthermore, hypoxia, 2-DOG, and GSH depolarized PA cells in the absence as well as in the presence of external Ca2+. Hypoxia, 2-DOG, and GSH also evoked action potentials on the top of the steady depolarization in 36-50% of PA myocytes but not in any MA myocytes; removal of external Ca2+ abolished the action potentials without affecting the steady depolarization. These effects were comparable to those produced by 4-aminopyridine (5-10 mM), a blocker of KV channels. This implies that the action potentials are attributable to Ca2+ influx through voltage-gated Ca2+ channels opened by the steady depolarization due to KV channel inhibition. In the presence of 2-DOG or GSH, hypoxia had no further effect on IK,ss or Em in PA cells; this implies that hypoxia, 2-DOG, and GSH all block the same K+ channels. The data suggest that 1) the hypoxia-induced decrease of IK,ss and the resultant depolarization in PA myocytes may be related to a local decrease of intracellular ATP level and/or a change in redox status of the membrane or cytosol and 2) extracellular Ca(2+)-dependent action potentials may be responsible for at least part of the increase in [Ca2+]i during HPV. Similarities between the effects of hypoxia, 2-DOG, and GSH on IK,ss and Em in PA myocytes, along with the dissimilar responses of PA and MA myocytes, suggest that a common mechanism may underlie the responses of PA cells to these treatments.
Hyperoxia inhibits proliferation of cultured rat tracheal smooth muscle cells
Authors: Absher M. , Makrides W. , Shapiro P. , Evans J. N.
Published: December 2017

Exposure of the lung to elevated oxygen leads to structural and cellular injury followed by extensive tissue remodeling. In vitro models utilizing isolated cells exposed to hyperoxic conditions or exogenously added oxidants may be injurious or stimulatory depending on the specific cell type and level and duration of exposure. In the present study, proliferation of cultured rat tracheal smooth muscle cells was inhibited by oxygen concentrations of 40 and 70% compared with a "normoxic" concentration of 21%. Exposure to 70% oxygen had a hypertrophic effect on the cells, as indicated by increased cellular protein content, whereas cells exposed to 21% oxygen did not show increased protein content. Exogenously added oxidant, H2O2, resulted in complete inhibition of growth of tracheal smooth muscle cells at concentrations > 3 microM. Much higher concentrations of H2O2 were required to inhibit proliferation of vascular smooth muscle cells and rat lung fibroblasts. The heightened sensitivity of airway smooth muscle cells to oxygen and oxidants may be an important factor in the early events following hyperoxia-induced lung injury.
Modulation of thrombin and thrombin receptor peptide mitogenicity by human lung mast cell tryptase
Authors: Hartmann T. , Ruoss S. J. , Caughey G. H.
Published: December 2017

In previous studies, mast cell tryptase acted as a potent mitogen for fibroblasts from human lung and rodent embryonic tissue but failed to stimulate growth of cultured rat aortic vascular smooth muscle cells (VSMC). The current study shows that tryptase inhibits DNA synthesis in VSMC stimulated by thrombin. However, it does not affect the stimulation of DNA synthesis by the synthetic thrombin receptor peptide Ser-Phe-Phe-Leu-Arg-Asn-Pro (SFFLRNP), which mimics the amino-terminus of thrombin receptor proteolytically activated by thrombin. Nor does tryptase alter the mitogenic response of VSMC to purified growth factors, such as platelet-derived growth factor (PDGF). These data suggest that tryptase inhibits thrombin-induced DNA synthesis without interfering with intracellular mitogenic signaling pathways activated by thrombin or other growth factors. This study further suggests that tryptase neither cleaves nor inactivates thrombin. Therefore, inhibition of thrombin's mitogenic effects by tryptase is not mediated by destruction of thrombin itself. The inhibition by tryptase of thrombin-induced DNA synthesis in VSMC contrasts with the stimulatory effect of tryptase on fibroblasts, in which synergy is observed with thrombin, with thrombin receptor peptide and with other growth factors. These data provide in vitro evidence that mast cell tryptase interferes with thrombin-stimulated vascular smooth muscle growth and suggest that tryptase is a multifunctional growth factor whose actions are cell specific.
Regulation of Na and Cl transport in sheep distal airways
Authors: Al-Bazzaz F. J.
Published: December 2017

To investigate ion transport function of distal airways mucosa we dissected and isolated segments of sheep bronchioles (outside diameter 664 +/- 10 microns, means +/- SE). Both ends of a segment were cannulated with glass micropipettes, and the preparation was placed in a bath and perfused with oxygenated Krebs-Henseleit solution at 37 degrees C. Luminal amiloride (0.1 mM) reduced potential difference (PD) from 3.06 +/- 0.68 mV to 1.08 +/- 0.24 mV (n = 5, P < 0.02). Submucosal ouabain (0.1 mM) decreased PD from 1.82 +/- 0.38 to 0.07 +/- 0.02 mV (n = 5, P < 0.009). Submucosal bumetanide (0.1 mM) caused a decline in PD from 3.44 +/- 0.98 to 2.75 +/- 0.71 mV (n = 19, P < 0.03). Exposure of the lumen to Cl channel blocker diphenylamine-2-carboxylate (1 mM) depolarized the bronchiole from a PD of 4.46 +/- 1.7 mV to 2.50 +/- 0.97 mV (n = 7, P < 0.03). Submucosal adenosine 3',5'-cyclic monophosphate (cAMP) analogue, 8-(4-chlorophenylthio)-cAMP (0.1 mM), raised PD from 3.45 +/- 1.08 to 3.82 +/- 1.24 mV (n = 10, P < 0.05). In eight experiments submucosal 0.1 mM carbachol, which elevates cytosolic Ca, resulted in rapid hyperpolarization (P < 0.02) followed by amiloride-inhibitable slow depolarization (P < 0.05). The data provide evidence for the presence in the sheep bronchiolar epithelium of active Na absorption that depends on a basolaterally located Na-K-ATPase ion pump. Increased cytosolic Ca probably inhibits Na absorption. The epithelium also has a Cl secretory process through apical Cl channels.
Lung lymphatics cast from the airspace
Authors: Hainis K. D. , Sznajder J. I. , Schraufnagel D. E.
Published: December 2017

Lymphatics are important in the resolution of pulmonary edema, but which lymphatics drain alveolar fluid and how they change during lung injury and edema is uncertain. To study this question 16 rats were exposed to 85% O2 for 7 days. At 0, 3, 7, and 14 days after removal from the hyperoxic chamber, the lungs of the rats were cast by instilling methyl methacrylate into the trachea. The lungs of four similar room-air breathing rats served as controls. Tissue was taken for light microscopy and the casts were examined for lymphatic filling with a scanning electron microscope. Rats exposed to hyperoxia had diffuse damage and extensive edema. On removal from hyperoxia (day 0), 29% of the rat bronchioles had saccular lymphatic casts around them and 6% of bronchioles were surrounded by these lymphatics. Twenty-five percent of bronchioles had conduit lymphatic casts. Fourteen percent of arteries had lymphatic casts around them. All were different from the rats kept in room air (P < 0.0001). Rats exposed to hyperoxia had lymphatics on the pleural surface, near alveoli and alveolar ducts, and around veins. The peribronchial and periarterial saccular lymphatics formed separate groups with communicating conduit lymphatics. The perivenous lymphatics had their own separate conduit lymphatics. Fourteen days after returning to ambient air, the lymphatics were similar to those of control animals. In this model, airway casting allows three-dimensional analysis of the lung lymphatics. It shows that lymphatic compartments expand during hyperoxic lung injury and that peribronchial and perivascular saccular lymphatics connect to conduit lymphatics of the bronchoalveolar bundle.
Phagocytosis of viable Candida albicans by alveolar macrophages: flow cytometric quantification
Authors: Rosseau S. , Seeger W. , Pralle H. , Lohmeyer J.
Published: December 2017

The phagocytic capacity of blood leukocytes may be assessed by flow cytometric techniques using fluorochrome-labeled particles including viable microorganisms. Application of this approach to alveolar macrophages (AM) is hampered or even rendered impossible by the strong autofluorescence of this cell type, superimposing the fluorescence intensity of the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodafluor 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intracellularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion laser, and the emission peak at 633 nm was used for quantification of dye-associated fluorescence. Rabbit and human AM were labeled with fluorescein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodies. After coincubation of macrophages and yeast, 4% paraformaldehyde plus 0.5% EDTA in phosphate-buffered saline was used to stop the phagocytic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dual (green and red) fluorescence. The percentage of yeast-positive AM and red fluorescence intensity of phagocytosing macrophages were quantified. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment was completely inhibited by N-ethylmaleimide and iodoacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Activation of protein kinase C mediates altered pulmonary vasoreactivity induced by tumor necrosis factor-alpha
Authors: Serfilippi G. , Ferro T. J. , Johnson A.
Published: December 2017

We postulated that tumor necrosis factor-alpha (TNF) "primes" the lung for the development of pulmonary vasoconstriction and edema by activating protein kinase C (PKC). Guinea pigs were injected with TNF (1.6 x 10(5) U/kg i.p.), and the lungs were isolated 4 h later. Compared with controls, TNF pretreatment resulted in greater increases in pulmonary vascular resistance and pressure and lung weight, in response to the thromboxane A2 mimetic, U-46619 (122 pmol/min). Treatment with TNF resulted in 1) pulmonary arterial endothelial PKC activation, 2) increased lung polymorphonuclear neutrophil (PMN) sequestration, 3) increased levels of superoxide radical (O2.) in lung effluent, and 4) decreased nitrite levels (NO2-, oxidation product of nitric oxide) in lung effluent. Intraperitoneal treatment with calphostin C (3 microM, 15 min prior to treatment with TNF) prevented the effects of TNF on 1) PKC activation, 2) the hemodynamic responses to U-46619, and 3) the levels of NO2- and O2(.). PKC activation does not mediate TNF-induced lung sequestration of PMN, since calphostin C had no effect on lung myeloperoxidase activity. The data suggest that PKC activation mediates TNF-induced 1) increases in O2., 2) decreases in NO2-, and 3) increases in vasoreactivity and edema in response to U-46619.
Inhibition of lung calcium-independent phospholipase A2 by surfactant protein A
Authors: Fisher A. B. , Dodia C. , Chander A.
Published: December 2017

The effect of lung surfactant protein A (SP-A) on lung phospholipase A2 (PLA2) activity was investigated. SP-A was purified from bovine surfactant obtained by lung lavage. PLA2 was assayed using radiolabeled 1,2-dipalmitoyl phosphatidylcholine (DPPC) in surfactant-like unilamellar liposomes with Ca(2+)-free acidic (pH 4) or 10 mM Ca2+, alkaline (pH 8.5) buffer. SP-A significantly inhibited Ca(2+)-independent acidic PLA2 of rat lung homogenate or isolated lamellar bodies but had no effect on the Ca(2+)-dependent alkaline enzyme. Lamellar body PLA2 was inhibited by 50% with 0.25 micrograms SP-A/microgram lamellar body protein. Similar inhibition by SP-A was observed when 1-palmitoyl,2-oleoyl PC (POPC) was the substrate. Binding assay showed binding of 125I-labeled SP-A to DPPC but not to POPC, indicating that removal of substrate was not the mechanism for inhibition of the enzyme by SP-A. Chemical reduction or alkylation of SP-A abolished its inhibitory effect on PLA2 activity. Inactivation of endogenous SP-A in isolated lamellar bodies or surfactant increased Ca(2+)-independent PLA2 activity in these fractions. The presence of SP-A in liposomes stimulated the uptake of DPPC by isolated granular pneumocytes in primary culture but significantly inhibited its degradation. These results indicate that the Ca(2+)-independent acidic PLA2 has a role in the metabolism of internalized surfactant phospholipid and that SP-A can modulate the activity of this enzyme.
Expression of TGF-alpha, EGF, and EGF receptor in fetal rat lung
Authors: Strandjord T. P. , Clark J. G. , Madtes D. K.
Published: December 2017

To define the distribution of transforming growth factor-alpha (TGF-alpha) and its relationship to epidermal growth factor (EGF) and EGF receptor in lung development and to determine whether epithelial cells produce TGF-alpha, we studied the expression of TGF-alpha, EGF, and their receptor in late-gestation fetal rat lung and in cultured fetal rat lung cells. TGF-alpha, EGF, and EGF receptor were colocalized in epithelial and smooth muscle cells of bronchioles and bronchi and in epithelial cells of saccules. Epithelial cells cultured from late-gestation fetal rat lung transcribe TGF-alpha and EGF receptor mRNA and produce TGF-alpha and EGF receptor proteins. Cultured fibroblasts contained EGF receptor mRNA, but no detectable TGF-alpha mRNA. These results demonstrate fetal lung epithelial cells are a source for TGF-alpha and suggest that TGF-alpha might act through an autocrine or paracrine mechanism with epithelial and mesenchymal cells. The colocalization of TGF-alpha and EGF suggests that these growth factors might act in parallel in lung development.
11 beta-hydroxysteroid dehydrogenase activity in human lung cells and transcription regulation by glucocorticoids
Authors: Page N. , Warriar N. , Govindan M. V.
Published: December 2017

Selectivity to aldosterone (Aldo) in mineralocorticoid target tissues has been suggested to be due to the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). This enzyme inactivates the endogenous glucocorticoid cortisol, thus permitting the unhindered access of Aldo to the mineralocorticoid receptor. The 11 beta-HSD activity was measured by the conversion of cortisol to cortisone and vice versa. Concomitant treatment of the cells with either cortisone or cortisol in the presence of the glycyrrhetinic acid derivative carbenoxolone (CBX) blocked both activities of 11 beta-HSD. Dexamethasone and Aldo activated the transcription of transiently transfected mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene in LU-19 cells. The transcription activation by cortisol was synergized by concomitant treatment of the transfectants with CBX. Transactivation with Aldo was inhibited by spironolactone. The enzyme 11 beta-HSD in LU-19 cells is similar to the cloned liver isoform and catalyzes both reduction and dehydrogenation.
Donor lung preservation: effect of cold preservation fluids on cultured pulmonary endothelial cells
Authors: Hall S. M. , Komai H. , Reader J. , Haworth S. G.
Published: December 2017

Pulmonary arterial (PA) endothelial cell morphology changes during cold preservation. In the present study, the efficacy of University of Wisconsin solution (UW), UW solution without colloid (modified UW), Euro-Collins (EC), Marshall's solution (MS), and medium 199 + 10% fetal calf serum [culture medium (CM)] in maintaining and regaining the cytoskeleton of cultured porcine PA endothelial cells kept at 4 degrees C and then rewarmed was compared. Features studied were actin, microtubules, vinculin, and talin, using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting; permeability of the cell sheet; wound healing; and phagocytic capacity. When cooled, microtubules depolymerized in all fluids but EC and MS reduced depolymerization of actin. Permeability decreased at 4 degrees C (P <0.05), and wound healing and phagocytosis ceased. When rewarmed after EC, UW, and CM preservation, wound healing and phagocytosis started within 15 min and proceeded normally. Permeability returned to normal but was excessive following UW preservation. Microtubule repolymerization was fastest following UW preservation, and actin filament repolymerization was fastest after EC preservation. Thus the type of preservation fluid used influenced the rate of loss and recovery of specific cytoskeletal components, with EC giving the fastest structural and functional recovery.
Interferon-gamma regulation of interleukin 6 in monocytic cells
Authors: Biondillo D. E. , Konicek S. A. , Iwamoto G. K.
Published: December 2017

The regulation of interleukin 6 (IL-6) is dependent on many factors that include numerous stimuli such as lipopolysaccharide (LPS), viruses, and other cytokines. These studies demonstrate the ability of interferon-gamma (IFN-gamma) to significantly enhance IL-6 mRNA and protein production in LPS-stimulated monocytes and THP-1 cells. IL-6 protein production was increased sevenfold in LPS-stimulated cells with the addition of IFN-gamma. IL-6 mRNA production was increased in a dose-dependent fashion up to 15-fold in LPS-stimulated cells with the addition of IFN-gamma. The enhanced production of IL-6 mRNA that occurs with the addition of IFN-gamma to LPS-stimulated monocytic cells was due to increased transcription of IL-6 mRNA. The ability of IFN-gamma to enhance IL-6 production may play an important role in many inflammatory processes.
Regulation of CTP:choline-phosphate cytidylyltransferase by polyunsaturated n-3 fatty acids
Authors: Mallampalli R. K. , Salome R. G. , Spector A. A.
Published: December 2017

Disaturated phosphatidylcholine (DSPC) is the most distinctive surface-active lipid in pulmonary surfactant. The feeding of docosahexanoic acid (DHA) 22:6 n-3 has recently been described to elevate the levels of DSPC in rodent lung. The purpose of the present study was to determine the mechanisms by which this n-3 fatty acid might regulate CTP:choline-phosphate cytidylyltransferase, a key enzyme required for phosphatidylcholine (PC) synthesis. Cytidylyltransferase exists in lung cytosol as a large lipid-associated aggregate (H form) which is active, and as an inactive, low-molecular-weight species (L form). Fatty acids in vitro stimulate and aggregate the inactive L form to the active H form. Short-term (2-h) and long-term (24-h) exposure of fetal lung explants to DHA (150 microM) stimulated choline incorporation into PC by 54 and 64%, respectively. The fatty acid also enhanced DSPC synthesis by 88%. These changes were associated with an increase in the activity of cytidylyltransferase by 63% after addition of DHA to the explant medium. In vitro, DHA (50 microM) stimulated L form nearly 15-fold and appeared to be a more potent activator and aggregator of the enzyme than either linoleic 18:2 n-6 or arachidonic 20:4 n-6 acids. The effect of DHA on L-form activation was comparable, however, with other members of the n-3 family. Kinetic studies revealed that DHA increased the maximum velocity of enzyme reaction for cytidylyltransferase, although it did not alter the Michaelis constant of the enzyme for CTP. These observations provide in vitro evidence that n-3 fatty acids may play an important role in the regulation of surfactant PC biosynthesis.
Oxidant stress decreases Na+/H+ antiport activity in bovine pulmonary artery endothelial cells
Authors: Cutaia M. , Parks N.
Published: December 2017

We determined the effects of oxidant stress by the use of tert-butyl hydroperoxide (t-BOOH) on Na+/H+ exchange in pulmonary artery endothelial cells. Monolayers were exposed to the hydroperoxide, followed by measurement of intracellular pH and the rate of recovery from acidosis by utilizing the pH-sensitive probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein. t-BOOH (0.4 mM) decreased the rate of acid recovery after a 2-h exposure without evidence of overt cytotoxicity (51Cr-release assay). Glutathione repletion (N-acetyl-L-cysteine) abolished the effect of the hydroperoxide. Lowering intracellular glutathione with buthionine sulfoximine decreased the acid recovery rate at a dose of t-BOOH (0.04 mM) that was not normally associated with a change in this parameter. Preincubation with vitamin E had no protective effect. Dithiothreitol abolished the effect of the hydroperoxide, suggesting oxidation of protein sulfhydryl groups as a mechanism for the altered kinetics of acid recovery. There was no difference in cell buffering capacity between control and treated monolayers. The findings suggest that the decrease in Na+/H+ antiport activity in this model of oxidant stress represents an early perturbation of membrane function and illustrate the role of the glutathione redox system in maintaining the functional integrity of the Na+/H+ antiport in these cells.
Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia
Authors: Xue C. , Rengasamy A. , Le Cras T. D. , Koberna P. A. , Dailey G. C. , Johns R. A.
Published: December 2017

Expression and localization of nitric oxide synthase (NOS) in the lungs of chronically hypoxic and normoxic rats were studied using both immunohistochemistry and NADPH diaphorase (NADPH-d) staining techniques. In the normoxic and in the hypoxic rat, NOS was detected by both methods in the endothelium of large pulmonary vessels and in the epithelium of bronchi and bronchioli. NOS expression was not detected in the endothelium of normoxic pulmonary resistance vessels but was prominently expressed in the endothelium of these vessels after 2-4 wk of chronic hypoxia. In contrast to small pulmonary vessels, the endothelium of small bronchial vessels exhibited NOS immunostaining in both normoxic and hypoxic lungs. Hypoxia was also found to induce de novo NOS expression in the smooth muscle of large and small pulmonary vessels and in bronchial smooth muscle. NOS enzyme activity in lung homogenates was assessed by [3H]arginine to [3H]citrulline conversion. The activity of soluble NOS, but not particulate NOS, was increased in the hypoxic lungs. These results demonstrate chronic hypoxia-induced upregulation of NOS protein expression and activity in the rat lung, suggesting a potentially important role of nitric oxide in adaptation of the pulmonary circulation to chronic hypoxia. The lack of immunostaining in small pulmonary resistance vessels is also consistent with physiological studies suggesting that NO may not be involved in the mechanism for maintaining the normally low pulmonary vascular resistance.
Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model
Authors: Kheradmand F. , Folkesson H. G. , Shum L. , Derynk R. , Pytela R. , Matthay M. A.
Published: December 2017

Alveolar epithelial type II cells are essential for regenerating an intact alveolar barrier after destruction of type I cells in vivo. The first objective of these experimental studies was to develop an in vitro model to quantify alveolar epithelial cell wound repair. The second objective was to investigate mechanisms of alveolar epithelial cell wound healing by studying the effects of serum and transforming growth factor-alpha (TGF-alpha) on wound closure. Primary cultures of rat alveolar type II cells were prepared by standard methods and grown to form confluent monolayers in 48 h. Then a wound was made by denuding an area (mean initial area of 2.1 +/- 0.6 mm2) of the monolayer. Re-epithelialization of the denuded area over time in the presence or absence of serum was measured using quantitative measurements from time-lapse video microscopy. The half time of wound healing was significantly enhanced in the presence of serum compared with serum-free conditions (2.4 +/- 0.2 vs. 17.4 +/- 0.8 h, P < 0.001). We then tested the hypothesis that TGF-alpha is an important growth factor for stimulating wound repair of alveolar epithelial cells. Exogenous addition of TGF-alpha in serum-free medium resulted in a significantly more rapid wound closure, and, furthermore, the addition of a monoclonal antibody to TGF-alpha in the presence of serum significantly decreased fourfold the rate of wound closure. Measurement of internuclear cell distance confirmed that both cell motility and cell spreading were responsible for closure of the wound. These data demonstrate that 1) the mechanisms of alveolar cell repair can be studied in vitro and that 2) TGF-alpha is a potent growth factor that enhances in vitro alveolar epithelial cell wound closure.
Insulin-like growth factor axis in airway smooth muscle cells
Authors: Noveral J. P. , Bhala A. , Hintz R. L. , Grunstein M. M. , Cohen P.
Published: December 2017

Insulin-like growth factors (IGFs) mediate cell proliferation and differentiation and bind with high affinities and specificities to IGF receptors and IGF-binding proteins (IGFBPs). We examined the roles of these three groups of proteins in cultured rabbit airway smooth muscle (ASM) cells. Affinity cross-linking of IGF-I and IGF-II to membranes of ASM cells revealed type 1 and type 2 IGF receptors. Western ligand blot analysis of ASM cell-conditioned medium revealed the presence of a single IGFBP band that precipitated with an antibody specific to IGFBP-2. ASM cells secreted radioimmunoassayable IGF-II; however, no IGF-I was detected under the same conditions. Two molecular weight forms of IGF-II were produced by the ASM cells. Exposure of cells to 1,000 ng/ml of IGF-I stimulated them to proliferate to 230 +/- 9.7% of their respective controls. Exposure to 1,000 ng/ml of IGF-II was approximately 40% as effective as exposure to 1,000 ng/ml of IGF-I. Both IGF-I and IGF-II exhibited binding to the type 1 IGF receptor. In summary, IGFs are mitogens for cultured rabbit ASM cells, and their actions are most likely mediated through the type 1 IGF receptor. The ASM cells secrete IGF-II and IGFBP-2, and the latter could modulate the actions of the IGFs in these cells.
Properties of a superoxide anion-generating microsomal NADH oxidoreductase, a potential pulmonary artery PO2 sensor
Authors: Mohazzab K. M. , Wolin M. S.
Published: December 2017

In this study, we describe properties of a microsomal NADH oxidoreductase that is a potential PO2-dependent source of vasoactive reactive O2 species in the calf pulmonary artery. Microsomes show an NADH-dependent production of superoxide anion (O2-.), as detected by lucigenin-elicited chemiluminescence, a superoxide dismutase inhibited reduction of nitro blue tetrazolium (NBT) and 2,6-dichlorophenol-indophenol, and O2 consumption. The microsomal production of O2-. was modulated by physiologically relevant levels of NADH and PO2, and O2-. production was reduced by inhibitors of NADH-dependent microsomal electron transport. Microsomes catalyzed an NADH-mediated reduction of several electron acceptor dyes, cytochrome c (rotenone insensitive) and methemoglobin. On reduction with dithionite, a cytochrome with an absorbance at approximately 558 nm was observed. Arterial O2-. levels (chemiluminescence) were also reduced by NBT and microsomal electron transport inhibitors. In pulmonary arteries, NBT selectively inhibited PO2 and lactate elicited changes in force generation, presumably by trapping O2-. and preventing H2O2 formation. Thus these studies are consistent with an involvement of O2-.-derived H2O2 generation via a microsomal NADH-cytochrome b558 electron transport system in calf pulmonary artery smooth muscle PO2 and lactate-elicited tone responses.
Glucocorticosteroids increase beta 2-adrenergic receptor transcription in human lung
Authors: Mak J. C. , Nishikawa M. , Barnes P. J.
Published: December 2017

beta 2-Adrenergic receptors (beta 2R) are widely distributed and mediate a wide range of cellular responses in lung. Because glucocorticosteroids increase expression of beta 2R in cell lines, we have investigated the effects of glucocorticoids on the beta 2R mRNA level and the number of beta 2R in human peripheral lung in vitro. Incubation of lung tissues with dexamethasone (Dex) elevated both beta 2R mRNA level (as measured by Northern blot analysis) and beta 2R number (as measured by [125I]iodocyanopindolol binding). The increased accumulation of beta 2R mRNA could be detected at 15 min (1.27 +/- 0.1-fold) and the maximal accumulation occurred at 2 h (2.73 +/- 0.5-fold). The Dex-induced increase in beta 2R mRNA returned to the control level by 17 h. The increase in beta 2R number (1.58 +/- 0.2-fold) was slower, reaching a maximum between 17 and 24 h. Dex increased beta 2R mRNA in a time- and concentration-dependent manner that was abolished by the steroid receptor antagonist mifepristone (RU-38486 or RU-486). The stability of beta 2R mRNA was unchanged by Dex, and a nuclear run-on assay revealed that Dex approximately doubled the transcriptional rate of the beta 2R gene. These observations suggest that glucocorticoids act on steroid receptors to increase beta 2R expression by increasing the rate of beta 2R gene transcription.
Neural and myogenic effects of cyclooxygenase products on canine bronchial smooth muscle
Authors: Abela A. P. , Daniel E. E.
Published: December 2017

In canine bronchi bathed in 10(-6) M indomethacin (IDM), prostaglandin (PG) E2 inhibited electrical field stimulation (EFS)- and acetylcholine (ACh)-mediated contractions and excitatory junction potentials (EJP) in a concentration-dependent manner without altering the resting membrane potential. EFS-induced EJPs were abolished at 10(-7) M PGE2, which shifted responses to ACh 10-fold rightward. Thus PGE2 predominantly inhibited the release of ACh and secondarily decreased smooth muscle response to ACh. U-46619, an analogue of thromboxane A2 (TxA2), initiated tetrodotoxin- and atropine-insensitive contractions in a concentration-dependent manner. U-46619 (10(-9) M) did not alter significantly EFS- or ACh-stimulated contractions and potentiated EFS amplitude of EJPs without depolarizing muscle cells. Either prejunctional activation of ACh release by TxA2 or postjunctional potentiation of the response to ACh can explain these findings. U-46619 (<or = 10(-8) M) depolarized the membrane potential, initiating oscillations accompanied by a large contraction. Addition of 10(-8) M nitrendipine, but not tetraethylammonium (25 mM), blocked the oscillations selectively. Other prostanoids (PGD2, PGI2, and PGF2 alpha) had no significant effects on canine bronchi. In the absence of IDM, PGE2 accumulated, EFS contractions decreased with time, and EJPs disappeared. We conclude that in canine bronchi PGE2 predominantly inhibits ACh release and endogenous PGE2 acts similarly, whereas TxA2 excites, probably at postjunctional sites.
Human SP-A: then and now
Authors: Floros J. , Karinch A. M.
Published: December 2017

In the short span of ten years, our understanding of human surfactant-associated protein A (SP-A) has advanced rapidly at both the level of the protein and the level of the gene. In the period 1984–1988, the protein was biochemically characterized and two SP-A precursors were identified. The molecular characterization was begun with the publication of an SP-A genomic sequence and sequences of two SP-A cDNAs, suggesting the presence of two SP-A genes. In the period 1991–1992, an SP-A pseudogene, a second SP-A genomic sequence, and an SP-A allelic variant were described. Since that time, a picture of increasing complexity has emerged from studies of the two SP-A genes. This complexity includes alternative splicing of 5' untranslated exons, allelic variants of both SP-A genes, and sequence heterogeneity within the 3' untranslated region. The challenge for the future will be to discover the physiological significance of the genetic complexity of human SP-A.
Role of intracellular pH in relaxation of porcine tracheal smooth muscle by respiratory gases
Authors: Croxton T. L. , Lande B. , Hirshman C. A.
Published: December 2017

Hypercapnia and hypoxia both relax airway smooth muscle, but the mechanisms responsible are poorly understood. Because hypercapnia and hypoxia can each decrease intracellular pH (pHi) and acidosis can inhibit Ca2+ channels, we hypothesized that decreased pHi mediates relaxation of trachealis muscle by each of these respiratory gases. To examine the relationship between pHi and tone, we measured isometric tension, bath pH, and fluorescence intensity (540 nm) in porcine tracheal smooth muscle strips loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and excited alternately with 440- and 500-nm light. Strips equilibrated in Krebs-Henseleit solution bubbled with 95% O2-5% CO2 were contracted with carbachol and then relaxed with either 95% N2-5% CO2 or 93% O2-7% CO2. The ratio of fluorescence intensity at 500 nm to 440 nm was calibrated vs. pHi with use of nigericin. Baseline pHi was 7.19 +/- 0.03 (n = 13). Hypoxia decreased active tension by approximately 60% but did not change pHi. Hypercapnia induced decreases in tension that were associated with substantial decreases in pHi. Thus, decreased pHi does not mediate hypoxic relaxation, but the relaxation during physiologically relevant increases in CO2 concentration is associated with significant cellular acidification.
Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle
Authors: Kume H. , Mikawa K. , Takagi K. , Kotlikoff M. I.
Published: December 2017

We have examined the functional consequences of G protein coupling to calcium-activated potassium (KCa) channels using isometric tension records from guinea pig tracheal smooth muscle. After incubation with 1 microgram/ml pertussis toxin (PTX) for 6 h, the contraction response to 1 microM methacholine (MCh) was suppressed by 31.7 +/- 5.0% (n = 10). Similarly, the contraction was inhibited by 29.1 +/- 5.0% (n = 6) after application of 0.1 microM AF-DX 116, an M2-selective muscarinic receptor antagonist. Cholera toxin (CTX, 2.0 micrograms/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), also suppressed contraction by 43.9 +/- 3.3% (n = 11). The inhibitory effects of PTX, AF-DX 116, or CTX were reversed in the presence of 100 nM charybdotoxin (ChTX), a selective KCa channel inhibitor. These findings suggest that disruption of inhibitory coupling between muscarinic receptor and KCa channels mediated by PTX-sensitive G proteins, or KCa channel activation induced by Gs/adenylyl cyclase-linked processes, antagonizes muscarinic contraction. The isoproterenol concentration-inhibition curves for precontracted trachea (1 microM MCh) were shifted to the left after perfusion with PTX or AF-DX 116, and the leftward shift of the curve was blocked by ChTX. Thus direct or indirect regulation of KCa channels mediated by the inhibitory guanine nucleotide binding protein (Gi) and Gs may play a functionally important role in the mechanical antagonism by the two receptor agonists.
Insulin modulation of bronchial epithelial cell fibronectin in vitro
Authors: Romberger D. J. , Pladsen P. , Claassen L. , Yoshida M. , Beckmann J. D. , Rennard S. I.
Published: December 2017

Fibronectin (Fn) is involved in the migration of epithelial cells in re-epithelialization of wounds. Epithelial cell-derived Fn is particularly potent as a chemotactic factor for bronchial epithelial cells (BECs) in vitro. Thus modulation of airway epithelial cell Fn may be a key aspect of airway repair. Insulin is both an important growth factor and known chemotactic factor for cultured BECs. We postulated that insulin may modulate Fn production of cultured BECs. We examined this hypothesis utilizing bovine BECs in culture with serum-free media with and without insulin. BECs grown in media without insulin released more Fn into culture supernatants and contained more Fn in cell layers than cells grown with insulin. Labeling of cells with [35S]methionine demonstrated an increase in new protein production and Fn mRNA expression was increased. Increased Fn in BEC cultures without insulin was associated with an increase in active transforming growth factor-beta (TGF-beta) release as measured by a standard bioassay. Increased BEC Fn in cultures without insulin was partially inhibited by exposure of cultures to TGF-beta antibody. Thus insulin appears to modulate BEC Fn production in vitro in part through a TGF-beta-dependent mechanism. Insulin may be involved in airway repair mechanisms through modulation of epithelial cell Fn production.
Response of cultured human pulmonary artery endothelial cells to endotoxin
Authors: Meyrick B. , Berry L. C. , Christman B. W.
Published: December 2017

Endotoxemia is the leading cause of the adult respiratory distress syndrome. The effects of endotoxin on pulmonary endothelium, both in vivo and in culture, are diverse and complicated, and vary between species and cellular origin. Species such as sheep and cows are particularly sensitive to endotoxin, whereas rats and mice are more resistant. Studies using cultured pulmonary endothelial cells confirm these findings. Such species variations lead us to question whether human pulmonary artery endothelial cells (HPAEC) are directly affected by endotoxin. The present study examined the effects of endotoxin on HPAEC. Cells were exposed to endotoxin (0.001-10 micrograms/ml) for 24 h and were examined by phase-contrast microscopy, and measurements were made of lactate dehydrogenase, prostacyclin, and prostaglandin E2 release in the cell-free supernatant. In the presence of serum, endotoxin doses as small as 0.01 microgram/ml resulted in endothelial retraction and pyknosis compared with controls (P < 0.05). Exposure to 10 micrograms/ml of endotoxin resulted in a significant increase in the number of pyknotic cells (P < 0.05), and lactate dehydrogenase release paralleled this finding. Endotoxin also resulted in a gradual increase in prostaglandin E2 release, reaching significance at 1 and 10 micrograms/ml of endotoxin (P < 0.05). A similar trend was noted for prostacyclin release. We conclude that the direct cytotoxic effects elicited by endotoxin on HPAEC may contribute to the onset of pulmonary edema in patients with adult respiratory distress syndrome.
Selective downregulation of ANP-clearance-receptor gene expression in lung of rats adapted to hypoxia
Authors: Li H. , Oparil S. , Meng Q. C. , Elton T. S. , Chen Y. F.
Published: December 2017

To test the hypothesis that expression of atrial natriuretic peptide (ANP)-receptor genes is modified to provide a compensatory mechanism against hypoxic pulmonary hypertension, steady state mRNA levels for the ANP-A receptor (or guanylate cyclase-A; ANPAR), ANP-B receptor (or guanylate cyclase-B; ANPBR), and ANP-clearance receptor (ANPCR) were quantitated by Northern blot and slot-blot analysis in lung, kidney, spleen, and liver of hypoxia-adapted rats and air controls. Exposure of rats to short-term (48 h) and chronic (4 wk) hypoxia (10% O2, 1 atm) did not affect lung ANPAR-mRNA levels. Lung ANPBR-mRNA levels were unchanged by short-term hypoxia but selectively increased (approximately twofold) by chronic hypoxia. ANPCR-mRNA levels were selectively and significantly downregulated by 48-h and 4-wk hypoxia in lung but were unchanged or upregulated in other tissues. Lung ANPCR gene transcription, assessed by nuclear-runoff analysis, was decreased by hypoxia. These data support the conclusion that altered pulmonary ANP-receptor gene expression modulates the development of hypoxic pulmonary hypertension.
Specific binding of endothelin-1 to canine tracheal epithelial cells in culture
Authors: Ninomiya H. , Yu X. Y. , Uchida Y. , Hasegawa S. , Spannhake E. W.
Published: December 2017

We have studied the binding of endothelin-1 (ET-1) to cultured canine tracheal epithelial cells. A single specific binding site for 125I-labeled ET-1 was identified with an apparent dissociation constant (Kd) of 0.2 nM, maximal binding sites (Bmax) of 6.7 x 10(3) sites/cell, and half-maximal inhibition (IC50) of 0.3 nM during a 2-h incubation period. The binding of 125I-ET-1 to these cells was inhibited by the presence of unlabeled ET-1, ET-2, or BQ-123, whereas ET-3 and sarafotoxin S6c did not compete for this binding site. These binding characteristics are consistent with those of the ETA receptor. At 37 degrees C, specific binding continuously increased over 18 h, while at 4 degrees C, it reached a plateau by 2 h. The increase in binding at 37 degrees C was not associated with DNA synthesis but was dependent upon protein synthesis, suggesting that epithelial binding sites were produced continuously under these incubation conditions. Our results indicate that canine tracheal epithelial cells possess specific binding sites for ET-1 with characteristics similar to those of the ETA receptor subtype. Because these cells are demonstrated to both release and bind ET-1, the results further suggest that ET-1 is involved in paracrine and/or autocrine control mechanisms in the airway epithelium.
Chloride secretion across distal airway epithelium: relationship to submucosal gland distribution
Authors: Ballard S. T. , Fountain J. D. , Inglis S. K. , Corboz M. R. , Taylor A. E.
Published: December 2017

To evaluate the possible relationship between submucosal glands and transepithelial Cl- secretion, we compared the bioelectric properties of two distal airway regions: bronchioles, which contain few submucosal glands, and small bronchi, which contain numerous glands. Intact distal bronchi were dissected from the lungs of 4-8 wk old pigs and cannulated with micropipettes and perfused. Transepithelial potential difference (PD), short-circuit current (Isc), and barrier resistance (Rm) in distal bronchi, determined by cable analysis, were -3.0 +/- 0.4 mV, 50.7 +/- 5.9 microA/cm2, and 59.6 +/- 5.9 omega.cm2, respectively (means +/- SE). Bumetanide (10 microM), which blocks Cl- secretion through inhibition of Na(+)-K(+)-2Cl- cotransport, reduced Isc in distal bronchi by 30.0 +/- 5.5 microA/cm2 (59.0% of the total Isc). By comparison, a previous study of porcine distal airways [S.T. Ballard and A.E. Taylor, Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L79-L84, 1994] determined that bumetamide-sensitive Isc in bronchioles was 2.6 +/- 1.4 microA/cm2 (only 9.0% of the total Isc). Submucosal gland duct openings to the luminal surface were identified microscopically and counted in representative fields. In eight bronchioles, 6.8 +/- 4.4 gland duct openings/cm2 of airway surface were observed, whereas seven distal bronchi contained 916.8 +/- 84.0 gland duct openings/cm2, over a 100-fold difference. These data suggest that a direct relationship may exist between the magnitude of active transepithelial Cl- secretion and the presence of submucosal glands in normal distal airways.
Evidence of Na-K-Cl cotransport in airway smooth muscle
Authors: Rhoden K. J. , Douglas J. S.
Published: December 2017

The presence of the Na-K-Cl cotransporter in airway smooth muscle was investigated by measuring 86Rb+ (as a marker for K+) and 36Cl- fluxes in the guinea pig trachealis. K+ uptake consisted of 1) a bumetanide- and furosemide-sensitive component, 2) a ouabain-sensitive component, and 3) a bumetanide- and ouabain-insensitive component. Bumetanide and furosemide inhibited K+ uptake with IC50s of 0.18 and 5.6 microM, respectively. Bumetanide-sensitive K+ uptake was reduced by the isosmotic replacement of extracellular Na+ and Cl- with choline and I-, respectively. Bumetanide caused a reduction in Cl- uptake, and the ratio of bumetanide-sensitive K+ to Cl-uptake was approximately 1:1.75. Bumetanide caused a decrease in 86Rb+ efflux, suggesting that the Na-K-Cl cotransporter mediates both K+ influx and efflux in airway smooth muscle. Ouabain caused an increase in bumetanide-sensitive 86Rb+ efflux from the guinea pig trachealis, which was prevented by exposure to a low-Na+ medium, suggesting that Na-K pump inhibition stimulates outward Na-K-Cl cotransport as a result of an increase in intracellular Na+ content.
Human CC10 gene expression in airway epithelium and subchromosomal locus suggest linkage to airway disease
Authors: Hay J. G. , Danel C. , Chu C. S. , Crystal R. G.
Published: December 2017

The CC10 gene encodes the Clara cell 10-kDa protein, which is expressed in airway epithelial cells. Quantification of CC10 gene expression in freshly isolated human proximal airway epithelial cells demonstrated very high mRNA levels, approximately fivefold greater than gamma-actin mRNA, and in situ hybridization localized CC10 mRNA to nonciliated airway epithelial cells. Sequence analysis demonstrated that the human CC10 gene is comprised of three short exons separated by a long first and short second intron, and with a 5' flanking region typical of a regulated gene. Three Alu repeats were observed in intron 1 and one in intron 2. Two polymorphic regions within the introns were identified. First, a microsatellite was localized 5' to the third Alu repeat in intron 1 with a variable number of 4- and 5-base pair (bp) repeats and a heterozygosity of 0.71. Second, in 3% of the 168 chromosomes examined, there was the insertion of a human-specific Alu repeat in intron 2, 45 bp 3' to the exon 2-intron junction. In three Centre d'Etude du Polymorphisme Humain families, meiotic breakpoint analysis using these two polymorphic loci localized the CC10 gene to 11 p12-q13 between markers D11S16 and D11S97, a region recently linked to atopy and to the beta-subunit of the high-affinity immunoglobulin E receptor. The observations in the present study of high-level expression of the CC10 gene in the epithelium of conducting airways and a subchromosomal localization of the gene to a region potentially linked to inflammatory airway disease, together with the reported anti-inflammatory and immune-modulating properties of the protein, suggest the CC10 gene product may be important in modulating inflammation within the airways. If so, the highly heterozygous microsatellite described in the present study should facilitate analysis of a possible linkage of the CC10 gene with an inherited susceptibility to asthma.