bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of agonist- and cell-mediated responses in airway microsections by computerized videomicrometry
Authors: Galens S. , Munoz N. M. , Rabe K. F. , Herrnreiter A. , Mayer D. , Morton B. , McAllister K. , Leff A. R.
Published: December 2017

The objective of this investigation was to develop a method for real-time measurement of changes in luminal area in microexplants of airways during pharmacological and physiological interventions. After guinea pigs were killed, tracheal rings (1- to 2-mm thick) were excised and placed in 300-microliters chambers. The area of the airway lumen was calculated as pixel number with the use of computerized videomicrometry. In 29 epithelium-intact airways, 10(-3) M acetylcholine (ACh) caused decrease in luminal area of 38.1 +/- 2.80% (P < 0.001 vs. 10(-9) M). Spontaneous tone also was demonstrated in 34 preparations from 4 guinea pigs; decrease in area of 17.0 +/- 1.45% after 60-min incubation in buffer alone was blocked completely by 10(-5) M indomethacin (P = 0.01). Luminal narrowing caused by < or = 10(-6) M ACh was reversed completely by 10(-6) M albuterol (P = 0.002). Addition of 100,000 activated human eosinophils caused 24.7 +/- 4.41% decrease in luminal area vs. 7.24 +/- 5.51% for nonactivated cells (P = 0.048). We demonstrate a real-time method for the assessment of auxotonic changes in airway caliber that utilizes microsections of explanted airways and permits the use of extremely small numbers of isolated cells to achieve physiological activation. Concentration-response characteristics and spontaneous tone are similar to those of large chamber preparations, and narrowing is reversed by beta 2-adrenoceptor activation.
Influence of protein kinase C inhibitors on vasoconstrictor responses in the pulmonary vascular bed of cat and rat
Authors: Kaye A. D. , Nossaman B. D. , Ibrahim I. N. , Feng C. J. , Kadowitz P. J.
Published: December 2017

The effects of staurosporine and calphostin C, two different protein kinase C (PKC) inhibitors, on pressor responses were studied in the pulmonary vascular bed of the intact chest anesthetized cat and the isolated rat lung. Under conditions of constant lobar blood flow in the cat, injections of the angiotensin peptides, norepinephrine (NE), serotonin, and U-46619 into the lobar arterial perfusion circuit caused dose-related increases in lobar arterial pressure and responses were reproducible with respect to time. Infusion of staurosporine into the perfused lobar artery at 1-2 micrograms/kg for 10 min reduced the pressor response to the angiotensin peptides and to NE; however, staurosporine did not alter pressor responses to serotonin or to the thromboxane mimic U-46619. In a separate series of experiments, the effects of calphostin C were investigated and infusion of the PKC inhibitor into the perfused lobar artery at 1-5 micrograms/kg for 10 min also reduced pressor responses to the angiotensin peptides and to NE and did not alter pressor responses to serotonin or to U-46619. In the isolated rat lung, the inhibitory effects of staurosporine on pulmonary pressor responses were investigated and injections of angiotensin II, NE, and serotonin produced dose-related increases in pulmonary arterial perfusion pressure that were decreased after administration of 20 micrograms ia of the PKC inhibitor staurosporine. (ABSTRACT TRUNCATED AT 250 WORDS)
An age-related difference in hyperoxia lethality: role of lung antioxidant defense mechanisms
Authors: Canada A. T. , Herman L. A. , Young S. L.
Published: December 2017

The role of animal age in the lethal response to > 98% oxygen has been extensively studied, with the observation that neonatal rats were resistant while mature animals were sensitive. Antioxidant enzymes increased during the oxygen exposure in neonatal but not in mature rats, suggesting they were important in the age-related toxicity difference. Because no studies had compared the response of mature and old rats to hyperoxia, we exposed Fischer 344 rats, aged 2 and 27 mo, to > 98% oxygen. Unexpectedly, the old rats lived significantly longer than young, 114 and 65 h, respectively. No histopathological differences were found to explain the results. Of the antioxidants, only glutathione peroxidase (GPx) activity was higher in the lungs of nonexposed old rats. Superoxide dismutase (SOD) was higher in the young, results opposite those expected if SOD was important in the lethality difference. No antioxidant induction occurred in the old oxygen-exposed rats. These results suggest that although there may be a role for GPx, mechanisms in addition to antioxidant protection and inflammation are likely responsible for the age-related difference in hyperoxia lethality.
Ethanol stimulates apparent nitric oxide-dependent ciliary beat frequency in bovine airway epithelial cells
Authors: Sisson J. H.
Published: December 2017

The mucociliary apparatus of the lung provides an important host-defense function by clearing the upper airway of inhaled particles and infectious microorganisms. Because lung host defenses are impaired in alcoholics, we hypothesized that ethanol would decrease ciliary motility in airway epithelium. Ciliary beat frequency (CBF) was measured by videomicroscopy in primary cultures of ciliated bovine bronchial epithelial cells (BBECs). Ethanol rapidly stimulated ciliary motility in a time-dependent fashion with concentrations as low as 10 mM. No detectable decreases in ciliary motility were noted until ethanol concentrations exceeded 1,000 mM. Because many substances stimulate ciliary motility by releasing nitric oxide (NO) via upregulation of nitric oxide synthase (NOS), we preincubated ciliated BBECs with a stereospecific NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA). L-NMMA completely blocked ethanol-induced stimulation of CBF, which could be subsequently restored by adding either L-arginine or sodium nitroprusside, which is a direct NO donor. These results indicate that ethanol, at clinically relevant concentrations, stimulates the release of NO by airway epithelium that upregulates ciliary motility. The rapidity of this response suggests upregulation of the constitutive NOS, known to be present in airway epithelium, and may explain the increases in mucociliary clearance observed in previous studies of ethanol ingestion in animals and in humans. These data also suggest a novel signal transduction pathway, the NO/NOS system, by which ethanol may exert some of its diverse biologic effects.
Extracellular nucleotides stimulate leukocyte adherence to cultured pulmonary artery endothelial cells
Authors: Dawicki D. D. , McGowan-Jordan J. , Bullard S. , Pond S. , Rounds S.
Published: December 2017

Adenosine, ATP, and various nucleotides were examined for their effects on the adherence of leukocytes to bovine pulmonary artery endothelial cells. Extracellular ATP enhanced adherence of HL-60 cells and human neutrophils to endothelial cells in a dose-dependent fashion. Maximal adherence occurred after 15 min coincubation of ATP and HL-60 cells or neutrophils with endothelial cells. ATP stimulation was mediated by direct effects on both HL-60 cells and endothelial cells. The potency profile of various nucleotides was ATP = 2-MeSATP > beta,gamma-CH2ATP, indicative of a P2y receptor. Interestingly, UTP was as potent as ATP in stimulating HL-60 cell adherence, suggesting the presence of a pyrimidine nucleotide receptor. Photoaffinity labeling of endothelial cells with 8-Az-[alpha-32P]ATP showed the presence of two ATP binding proteins of 48 and 87 kDa. ATP and 2-MeSATP inhibited binding by both proteins. Labeling of the 87-kDa protein was inhibited by beta,gamma-CH2ATP, whereas UTP blocked binding by the 48-kDa protein. Thus photoaffinity labeling experiments support the proposal that endothelial cells possess two ATP receptors, one of which is a P2u nucleotide receptor. These findings show that extracellular nucleotides enhance leukocyte adherence to endothelial cells. Nucleotide release into the extracellular space may be one mechanism of exacerbating vascular cell injury relevant to conditions such as adult respiratory distress syndrome and septic shock.
Glucocorticoid stimulation of fatty-acid synthase gene transcription in fetal lung: antagonism by retinoic acid
Authors: Xu Z. X. , Viviano C. J. , Rooney S. A.
Published: December 2017

Glucocorticoid hormones are known to stimulate the rate of fatty acid biosynthesis and to increase the activity and mRNA level of fatty-acid synthase (FAS) in late gestation fetal lung. We have now examined the effect of dexamethasone on FAS transcription in fetal rat lung. Explants of 19-day fetal rat lung cultured for 48 h in serum-free medium were exposed to dexamethasone (10(-7) M) for various time periods. Nuclei were isolated, and the rate of [32P]UTP incorporation into FAS and gamma-actin RNA transcripts was measured by transcription-elongation assay. Dexamethasone increased FAS transcription but had no effect on that of actin. The maximum effect of the hormone, approximately threefold increase, was observed 1–2 h after addition of the hormone but was still apparent up to 48 h. FAS transcription but not that of actin was inhibited by cycloheximide and puromycin in both control and dexamethasone-treated cultures. However, the stimulatory effect of the hormone was not significantly reduced by the inhibitors. Retinoic acid antagonized the stimulatory effects of dexamethasone on FAS activity, mRNA content as measured by Northern analysis, mass as measured by Western blotting, and rate of transcription. The effect of retinoic acid was dependent on concentration in the relatively narrow range of 5 x 10(-6) to 5 x 10(-4) M. These data show that glucocorticoids stimulate transcription of the FAS gene in late gestation fetal rat lung, that normal transcription of the FAS gene is dependent on ongoing protein synthesis, and that glucocorticoid stimulation of FAS gene expression is antagonized by retinoic acid.
Degradation of surfactant lipids and surfactant protein A by alveolar macrophages in vitro
Authors: Wright J. R. , Youmans D. C.
Published: December 2017

Pulmonary surfactant is synthesized and secreted into the airspaces by the alveolar type II cell. After it is secreted, surfactant undergoes a series of poorly understood transformations resulting in formation of a surface tension-reducing surface at the air-liquid interface. The by-products of the surface film and/or other products of surfactant metabolism are eventually cleared from the alveolar space. Both the alveolar type II cell and the macrophage are thought to be involved in surfactant clearance and have been shown to internalize surfactant lipid in vitro. The goal of the current investigation was to characterize further and to quantitate the role of the macrophage in surfactant clearance by investigating the uptake and metabolism of surfactant lipids and surfactant protein A (SP-A) by macrophages in vitro. SP-A enhanced the uptake of lipids by macrophages in a time-, temperature-, and concentration-dependent manner. In contrast, neither of the collagen-like proteins SP-D or C1q enhanced the uptake. Phosphatidylcholine was rapidly degraded by macrophages and the degradation occurred both in the presence and absence of SP-A. In addition, macrophages degrade SP-A by a process that is time- and temperature-dependent. These results and calculations of uptake and degradation rates suggest that macrophages may contribute significantly to the process of surfactant clearance.
Effect of nitric oxide and cell redox status on the regulation of endothelial cell xanthine dehydrogenase
Authors: Hassoun P. M. , Yu F. S. , Zulueta J. J. , White A. C. , Lanzillo J. J.
Published: December 2017

We have previously reported that endothelial cell (EC) xanthine dehydrogenase/xanthine oxidase (XD/XO) activity correlates inversely with the O2 tension to which the cells are exposed. Whether this effect is related to the production of reactive O2 species is unclear. We exposed bovine pulmonary artery EC to various conditions that altered the redox status of the cells: 1) hypoxia (3% O2) and normoxia (20% O2); 2) menadione (MEN), known to generate O2 radicals; 3) catalase (CAT) and reduced glutathione (GSH), which detoxify H2O2; and 4) various NO-generating systems. Changes in intracellular XO and XO + XD activities were correlated with rates of extracellular H2O2 release from the same cells. Conditions that decreased extracellular H2O2 release (hypoxia, CAT, and GSH) produced significant and parallel increases in intracellular XO and XO + XD activities in a time-dependent fashion. MEN treatment increased extracellular release of H2O2 and subsequently reduced intracellular XO and XO + XD activities. NO-generating agents did not change extracellular release of H2O2 but significantly reduced XO and XO + XD activities. The latter effect was prevented by reduced hemoglobin. Scavengers of hydroxyl radicals reversed the inhibition of XO and XO + XD activities produced by MEN but not that produced by NO. While NO significantly inhibited XD/XO activity from rat epididymal fat pad, it did not affect XD/XO mRNA expression in these cells. We conclude that intracellular XD/XO activity is sensitive to changes in oxidant-generating and protective systems. Inhibition of XD/XO activity by NO may be mediated through direct binding of NO to the enzyme iron-sulfur moiety or to its sulfhydryl groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Interleukin-8 and monocyte chemoattractant protein-1 mRNAs in oxygen-injured rabbit lung
Authors: D'Angio C. T. , Sinkin R. A. , LoMonaco M. B. , Finkelstein J. N.
Published: December 2017

The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are neutrophil and monocyte attractants, respectively. We hypothesized that IL-8 and MCP-1 mRNA expression in alveolar macrophages (AM) lavaged from rabbit lung would be increased by oxygen exposure, which is known to induce inflammation. Adult rabbits were exposed to > 95% oxygen for up to 64 h and allowed to recover in room air for up to 72 h before killing and pulmonary lavage. Numbers of lavageable polymorphonuclear cells (PMN) and AM rose during the exposure protocol. Quantitative in situ hybridization with 3H-labeled cRNA probes showed both IL-8 and MCP-1 mRNA expression in AM during oxygen exposure, with peak levels of IL-8 mRNA at 56-h oxygen exposure and of MCP-1 mRNA at 64-h oxygen exposure with 24-h room air recovery. IL-8 mRNA was present in PMN between 48-h oxygen exposure and 64-h oxygen exposure with 24-h room air recovery. MCP-1 mRNA was not expressed in PMN. This pattern of chemokine mRNA expression emphasizes the importance of inflammatory cells as effectors in the pulmonary response to oxygen exposure.
Cytokine-binding proteins in the lung
Authors: Bonner J. C. , Brody A. R.
Published: December 2017

Numerous cytokines and growth factors signal the normal processes of tissue maintenance and remodeling in the lung, yet the aberrant expression of these peptide mediators is involved in a variety of pulmonary diseases. Furthermore, several different binding proteins function in controlling the extracellular levels of many of these cytokines in the lung. For example, a variety of cytokines and growth factors bind to and are regulated by the ubiquitous proteinase inhibitor, alpha 2-macroglobulin. The insulin-like growth factors are controlled by a specific class of six different insulin-like growth factor binding proteins. The transforming growth factor-beta family and fibroblast growth factors interact with extracellular matrix proteins. Several growth factor receptors are shed into the extracellular milieu where they retain a functional binding domain and thereby act as specific binding proteins. Cytokine-binding proteins appear to have a diversity of functions and may serve as extracellular cytokine reservoirs, protective shields against proteolytic degradation of cytokines, modifiers of cytokine-induced biological activity, or as clearance avenues for cytokines. The wide spectrum of cytokine-regulating molecules is important in cell-cell communications under normal conditions, whereas cytokine-binding protein dysfunction could contribute to a number of pulmonary diseases.
Antigen depolarizes guinea pig bronchial parasympathetic ganglion neurons by activation of histamine H1 receptors
Authors: Myers A. C. , Undem B. J.
Published: December 2017

Studies were carried out to evaluate the mechanism by which neurotransmission through airway parasympathetic ganglia may be modulated during immediate hypersensitivity reactions. Guinea pigs were passively sensitized by injection of guinea pig serum containing high-titer anti-ovalbumin antibodies. Intracellular recordings were obtained from intrinsic parasympathetic ganglion neurons from the right mainstem bronchus in vitro. Ovalbumin (10 micrograms/ml) elicited a membrane potential depolarization and changes in membrane resistance in bronchial ganglion neurons from passively sensitized guinea pigs. Histamine mimicked the depolarizing effect of ovalbumin in a concentration-dependent manner (0.1–10 microM) and caused a transient increase and decrease in membrane resistance. Pyrilamine, a histamine H1-receptor antagonist, inhibited the histamine-induced membrane depolarization and decrease in resistance. By contrast, blocking histamine H2 and H3 receptors did not inhibit histamine-induced depolarization. Pyrilamine also reduced the antigen-induced depolarization of ganglion neurons, demonstrating a role for histamine H1 receptors in this response. The data provide evidence that the antigen-induced depolarization of airway ganglion neurons is secondary to an antigen-antibody interaction on intrinsic mast cells and the consequential effect of histamine on H1 receptors. These studies demonstrate that histamine released during an immediate hypersensitivity reaction has direct effects on airway parasympathetic nerves.
Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers
Authors: Patterson C. E. , Stasek J. E. , Schaphorst K. L. , Davis H. W. , Garcia J. G.
Published: December 2017

We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.
Beta 2-adrenoceptor activation augments acetylcholine release from tracheal parasympathetic nerves
Authors: Zhang X. Y. , Olszewski M. A. , Robinson N. E.
Published: December 2017

We determined the effects of isoproterenol (Iso) on parasympathetic neurotransmission in isolated equine trachealis strips by comparing the effects of Iso on the contractile response to electrical field stimulation (EFS) and acetylcholine (ACh), as well as by measuring EFS-induced ACh release. The interaction of Iso with muscarinic receptors and endogenous nitric oxide was also investigated. ACh release was measured by high-performance liquid chromatography with electrochemical detection. Iso (10(-7) M or greater) caused significantly more inhibition of EFS- than of ACh-induced contraction, an observation usually interpreted as evidence of prejunctional inhibition of ACh release. However, the latter conclusion was not supported by measurement of ACh release. Iso concentration dependently augmented ACh release, which was reversed by the beta 2-adrenoceptor antagonist ICI-118,551 but not by the beta 1-adrenoceptor antagonist CGP-20,712A. Our results indicate that activation of beta 2-adrenoceptors augments ACh release. Moreover, the comparison of inhibitory effects on EFS- and ACh-induced contraction does not provide correct information about the prejunctional actions of beta-agonists. ACh release was increased more by atropine (10(-7) M) than by Iso (10(-6) M), indicating the predominance of prejunctional inhibitory muscarinic autoreceptors over excitatory beta 2-adrenoceptors. Additionally, we found that inhibition of nitric oxide synthase by NG-nitro-L-arginine did not affect either the cholinergic contractile response or ACh release in both the absence and presence of Iso.
Urokinase receptor in human malignant mesothelioma cells: role in tumor cell mitogenesis and proteolysis
Authors: Shetty S. , Kumar A. , Johnson A. , Pueblitz S. , Idell S.
Published: December 2017

Urokinase (uPA) interacts with its receptor (uPAR) to promote proteolysis and tumor migration, functions of potential importance in the pathogenesis of malignant mesothelioma. Immunohistochemistry of human malignant mesothelioma tissue and mesothelioma cells (MS-1) showed that mesothelioma cells express uPAR. We isolated uPAR from MS-1 cells by metabolic labeling and showed that it could be induced by phorbol myristate acetate (PMA), lipopolysaccharide (LPS), a transforming growth factor-beta (TGF-beta) or tumor necrosis factor-alpha (TNF-alpha). Experiments with MS-1 cells showed that uPA binding was saturable, specific, and reversible with a mean dissociation constant (Kd) of 5.4 +/- 1.1 nM. Binding was inhibited by a blocking antibody to uPAR and by the uPA amino-terminal fragment (ATF), but not by low molecular weight uPA. uPAR expression was regulated transcriptionally and translationally; antisense oligonucleotides blocked expression of uPAR protein. Plasminogen activator inhibitor-1 (PAI-1) inhibited PA activity of preformed uPA/uPAR complexes and increased cycling of the receptor from the cell surface. Stimulation of subconfluent MS-1 cells by high molecular weight or recombinant uPA, but not ATF or low molecular weight fragment, caused concentration-dependent incorporation of [3H]thymidine. These data indicate a novel mechanism by which malignant mesothelioma cells localize pericellular proteolysis and concurrently regulate tumor cell proliferation.
Differential effects of in vivo ethanol on LPS-induced TNF and nitric oxide production in the lung
Authors: Kolls J. K. , Xie J. , Lei D. , Greenberg S. , Summer W. R. , Nelson S.
Published: December 2017

Alcohol (EtOH) has been shown to suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) generation and tumor necrosis factor (TNF) production in the lung in vivo. We have previously reported that EtOH suppressed gene expression for inducible nitric oxide synthase (iNOS) with a subsequent decrease in release of reactive nitrogen intermediates by alveolar macrophages and recruited lung neutrophils. We hypothesized that a similar mechanism may be involved in EtOH-induced suppression of LPS-stimulated TNF production. In contrast to what we found with iNOS, EtOH had no effect on TNF mRNA in alveolar macrophages or recruited lung neutrophils. However, immunoreactive and bioactive TNF was reduced by 72%. EtOH treatment resulted in an increased level of the membrane-bound 26-kDa form of TNF, which suggested that proteolytic cleavage of this prohormone was affected by EtOH. Experiments with t-butyl alcohol, a tertiary alcohol that is not metabolized to acetaldehyde, yielded similar results. Thus EtOH appears to be the active substance in suppression of TNF in the lung in vivo. Pretreatment with intratracheal interferon-gamma 24 h before intratracheal LPS increased TNF bioactivity partly due to increased TNF mRNA and by increasing TNF processing, as evidenced by a decrease in the 26-kDa TNF prohormone and an increase in immunoreactive and bioactive TNF.
Pathways for uptake of fluorescently labeled liposomes by alveolar type II cells in culture
Authors: Muller W. J. , Zen K. , Fisher A. B. , Shuman H.
Published: December 2017

Isolated alveolar type II cells are shown in real time to internalize 1-palmitoyl-2-[12-[(7 nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl] phosphatidyl choline (C12-NBD-PC)-labeled liposomes in an intact manner with the use of fluorescence microscopy. Cells isolated from rat lungs, cultured on plastic for 24 h, are exposed at 37 degrees C to liposomes on the microscope stage, using a microperfusion system. Liposomes composed of a self-quenching concentration of fluorophore (25 mol% C12-NBD-PC) do not dequench at the level of the plasma membrane; rather, they appear to fuse with intracellular lipids primarily in vesicular structures consistent with lamellar bodies. Temperature and energy dependence of this uptake mechanism is demonstrated by the lack of uptake of maximally fluorescent liposomes (15 mol% C12-NBD-PC) at 4 degrees C or when depleted of ATP. Inhibition of the coated-pit endocytic pathway by hypertonic media reduces fluorescent lipid uptake by 50%, suggesting a separate, clathrin-independent endocytic pathway for intact internalization of liposomes by type II cells. Uptake is inhibited by cytochalasin D by 49%, suggesting a dependence on action, and by 88% with combined cytochalasin D and hypertonicity, or nearly the same level as ATP depletion. The evidence is consistent with the existence of two separate endocytic pathways for lipids in type II cells: one clathrin dependent and the other actin dependent.
Migration and proliferation of guinea pig and human airway epithelial cells in response to tachykinins
Authors: Kim J. S. , Rabe K. F. , Magnussen H. , Green J. M. , White S. R.
Published: December 2017

Restoration of the epithelial lining of a damaged airway is a necessary component of airway repair. Tachykinins, including substance P (SP) and neurokinin A (NKA), are localized to sensory nerves within the airway mucosa. These tachykinins regulate several airway functions, but their role in the repair of the epithelium has not been explored. To determine whether tachykinins stimulate migration and proliferation of airway epithelial cells, guinea pig tracheal epithelial (GPTE) and human bronchial epithelial (HBE) cells were grown in primary culture for 4-5 days. Epithelial cell migration was assessed in a blindwell chemotaxis chamber, and proliferation was determined by immunohistochemistry after incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). Both GPTE and HBE cells migrated after stimulation with 10(-11) M NKA [23.0 +/- 3.6 vs. 5.4 +/- 1.2 cells per 10 high-power fields (hpf), P < 0.001, n = 8 for GPTE cells; 18.4 +/- 2.3 vs. 3.8 +/- 0.5 cells per 10 hpf for control, P < 0.001, n = 4 for HBE cells]. Migration was stimulated within 2 h, was maximal after 6 h, and was attenuated substantially by the neurokinin 2 (NKA)-receptor antagonist SR-48968. NKA-stimulated migration was both chemokinetic and chemotactic, and it could be blocked by inhibition of protein synthesis with cyclohexamide, inhibition of microtubular function with colchicine, or inhibition of actin microfilament elongation with cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinetic analysis of cytosolic pH regulation in alveolar macrophages: V-ATPase-mediated responses to a weak acid
Authors: Bidani A. , Heming T. A.
Published: December 2017

Three different mechanisms interact to control the cytosolic pH (pHi) of alveolar macrophages (M phi), namely, plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase), Na+/H+ exchange, and Na(+)-independent HCO3-/Cl- exchange. To investigate the activity of plasmalemmal V-ATPase in alveolar M phi, we developed a nonlinear mathematical model of pHi regulation that incorporates the biophysical determinants of pHi and the fluxes of individual acid-base equivalents. The model was used to analyze the acid-base responses of rabbit alveolar M phi to a weak acid (propionic acid) under conditions that favored V-ATPase-mediated effects (presence of 1 mM amiloride and nominal absence of CO2). The pHi was measured using the fluorescent probe, 2',7'-biscarboxethyl-5,6-carboxyfluorescein. M phi exposure to propionic acid caused a rapid fall in pHi. Recovery of pHi after acid loading varied directly with the magnitude of the acid load. Mathematical analyses showed that pHi recovery was hindered by persistent influx of propionic acid driven, in turn, by transporter-mediated H+ extrusion and propionate efflux. Eventually, a new steady state was established in which propionate and H+ were cycled out of the M phi and propionic acid was recycled into the cell. As a consequence, model predictions of the rate of V-ATPase-mediated H+ efflux were almost twice that estimated from experimental determinations of the initial rate of pHi recovery.
Characterization of zinc uptake and its regulation by arachidonic acid in fetal type II pneumocytes
Authors: Ong T. J. , Kemp P. J. , Oliver R. E. , McArdle H. J.
Published: December 2017

In freshly isolated fetal guinea pig type II pneumocytes, zinc uptake is time and temperature dependent. Two pathways of uptake exist, resulting in a rapid phase that reaches a steady state within 30 s and a slower linear phase that does not attain a steady state within 60 min. Both processes exhibit saturation kinetics. The rapid phase has a maximal zinc uptake of 60.7 +/- 9.3 pmol.10(6) cells-1.30 s-1 and an apparent affinity (Kt) of 13.7 +/- 5.4 microM. The maximum velocity of uptake (Vmax) of the slower phase is 24.6 +/- 1.9 pmol.10(6) cells-1.min-1 with a Kt of 22.0 +/- 3.6 microM. Epinephrine, terbutaline, dibutyryl adenosine 3',5'-cyclic monophosphate, and dexamethasone have no significant effect on zinc uptake, while arachidonic acid (AA) stimulates. Dose-response data of AA-stimulated zinc uptake gives an apparent K0.5 of 0.42 +/- 0.01 microM and a Hill coefficient of 1. The maximal uptake in the rapid phase is significantly increased to 146.8 +/- 12.4 pmol.10(6) cells-1.30 s-1 and in the slow phase, the Vmax for zinc uptake is also significantly increased to 33.0 +/- 1.8 pmol.10(6) cells-1.min-1 by 10 microM AA. However, the Kt values in both processes remain unchanged after AA stimulation. The effect is not mediated by either leukotrienes or prostaglandins but can be mimicked by other unsaturated fatty acids.
Ionomycin and PDBU increase MDCK monolayer permeability independently of myosin light chain phosphorylation
Authors: Shasby D. M. , Kamath J. M. , Moy A. B. , Shasby S. S.
Published: December 2017

It has been hypothesized that modulation of epithelial paracellular permeability may be mediated by initiation of contraction of a band of actin and myosin located at the tight junction. Phosphorylation of myosin light chain (MLC) is an important determinant of actomyosin contraction. We asked if ionomycin (iono) and phorbol 12,13-dibutyrate (PDBU), which increase paracellular permeability of Madin-Darby canine kidney (MDCK) cell monolayers, increased MLC phosphorylation in MDCK cells. MDCK cell MLC was constitutively phosphorylated by myosin light chain kinase (MLCK), and after PDBU and iono > 99% of MLC continued to be phosphorylated by MLCK. Neither iono or PDBU, nor the combination of iono and PDBU, increased MLC phosphorylation. In contrast, the phosphatase inhibitor okadaic acid did increase MLC phosphorylation. Adenosine 3',5'-cyclic monophosphate (cAMP) and forskolin decreased MLC phosphorylation in control MDCK cells and in cells exposed to iono and PDBU. In contrast, cAMP and forskolin did not blunt the decrease in transepithelial resistance caused by iono and PDBU. Iono and PDBU increase MDCK monolayer permeability independently of an increase in MLC phosphorylation.
Effects of hypoxia on MnSOD expression in mouse lung
Authors: Russell W. J. , Ho Y. S. , Parish G. , Jackson R. M.
Published: December 2017

Mitochondrial manganese-containing SOD (MnSOD) is located at the primary site of O2 metabolism, and its expression may be regulated by changes in O2 level. We hypothesized that lung MnSOD expression and promoter activity would decrease in response to hypoxia. We tested effects of hypoxia (10% O2 at sea level for 7 days) on chloramphenicol acetyltransferase (CAT) reporter and MnSOD gene expression in transgenic mice. The transgene consisted of a 3.3-kb portion of the rat MnSOD gene 5' flanking region coupled to a CAT reporter gene. Lung MnSOD activity in male (but not female) mice decreased significantly after hypoxia exposure. The decrease in MnSOD enzymatic activity in male mice was specific. Neither total SOD nor glucose-6-phosphate dehydrogenase (G-6-PDH) activity decreased significantly in hypoxia. CAT protein expression decreased in transgenic males exposed to hypoxia, while CAT protein expression in hypoxic transgenic females remained comparable with controls. The mRNA for both the native MnSOD and the MnSOD-CAT reporter genes remained constant after hypoxia, as did CuZnSOD and G-6-PDH mRNAs.
gamma-Glutamyl transpeptidase is a polarized alveolar epithelial membrane protein
Authors: Ingbar D. H. , Hepler K. , Dowin R. , Jacobsen E. , Dunitz J. M. , Nici L. , Jamieson J. D.
Published: December 2017

In many diseases the lung is injured by oxidants. gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme on the apical plasma membrane of many epithelial cells that protects against oxidants by replenishing intracellular glutathione. We sought to localize GGT within rat lungs in vivo and in cultured alveolar epithelial cells. In the adult rat lung, indirect immunofluorescence (IF) with a polyclonal antibody to triton-solubilized GGT revealed linear staining outlining the alveoli. Immunoelectron microscopy (IEM) localized the protein on the apical surface of the alveolar epithelial cells, but more densely on type I cells than type II cells, as well as on the apical surface of some ciliated bronchial cells. On Western blots of whole lung and isolated type II cell membrane proteins, the antibody predominantly recognized a broad protein band of 110-120 kDa, consistent with the uncleaved, glycosylated form of GGT. Over time in culture, isolated rat type II cells had increasing immunoreactivity on Western blots and indirect IF but decreasing enzyme activity. At 2 days in culture, confocal laser scanning microscopy demonstrated that GGT was polarized to the apical surface of nonconfluent type II cells. Thus GGT is a polarized apical membrane protein in type I and II cells, suggesting a role in the metabolic functions of these cells. The increased immunoreactive GGT of cultured type II cells is consistent with their acquisition of properties similar to type I cells, but the lack of correlation between immunoreactive protein and enzyme activity awaits explanation.
Differential display RT-PCR for identifying novel gene expression in the lung
Authors: Sunday M. E.
Published: December 2017

The unique identity of each cell is the result of differential gene expression. A new strategy for differential cDNA screening introduced by Liang and Pardee utilizes anchored oligo-dT primers and random 5' oligonucleotide 10-mers to carry out polymerase chain reaction (PCR) on reverse-transcribed RNA from different cell populations. The amplified cDNAs are displayed on a standard sequencing gel as 100-500 bands per lane on the resulting autoradiograms and comparisons are drawn between the different cell populations. The major advantages of this method over previous differential screening approaches are its high sensitivity, the small amounts of tissue required, and the ability to carry out rapid mRNA analyses using total RNA and to test multiple tissues in parallel. Its limitations are the need for many primer combinations for adequate representation of mRNAs and the large number of bands displayed. A screening strategy should include multiple positive and negative control samples and Northern blots to identify cDNAs differentially expressed. This approach should facilitate the identification of many novel genes expressed in a variety of physiological and pathological conditions.
Tachykinin regulation of airway smooth muscle cell proliferation
Authors: Noveral J. P. , Grunstein M. M.
Published: December 2017

The tachykinins, substance P (SP) and neurokinins A (NKA) and B (NKB), have been identified in the respiratory tract and implicated in mediating neurogenic inflammation of the airways. To the extent that these neuropeptides may be involved in the pathogenesis of asthma, a condition associated with hyperplasia of airway smooth muscle (ASM), we examined the mitogenic effects and mechanisms of action of tachykinins in cultured rabbit ASM cells. SP was found to elicit dose-dependent (10(-14) to 10(-4) M) stimulation of ASM cell proliferation, with a mean (+/- SE) maximal increase in cell number of 169 +/- 6.1% of control. In contrast, NKA and NKB had little and no effect on ASM cell growth, respectively. Because SP is nonselective in its binding to the tachykinin receptors, to identify the specific NK receptor subtype(s) mediating the promitogenic action of SP, in separate studies we found that 1) the NK1-receptor-specific agonist, [beta-Ala4, Sar9, Met(O2)11]SP-(4-11) induced stimulation of ASM cell growth similar in magnitude to that elicited by SP; 2) in contrast, neither the NK1- nor NK2-receptor-specific agonists, [beta-Ala8]NKA-(4-10) and [MePhe7]NKB, respectively, had any effect on ASM cell growth; and 3) the promitogenic action of SP was inhibited by the NK1-receptor antagonist, GR-82,334. Moreover, in extended experiments, we found that the phospholipase C and phospholipase A2 inhibitors, neomycin and quinacrine, respectively, each inhibited SP-induced ASM cell proliferation by approximately 45%. Collectively, these observations provide new evidence that the tachykinin SP induces ASM cell proliferation, and that this action is mediated by transmembrane signaling coupled to selective activation of the NK1 receptor.
Bronchial epithelial cells regulate fibroblast proliferation
Authors: Nakamura Y. , Tate L. , Ertl R. F. , Kawamoto M. , Mio T. , Adachi Y. , Romberger D. J. , Koizumi S. , Gossman G. , Robbins R. A. , et al.
Published: December 2017

Chronic bronchitis frequently leads to irreversible airway obstruction. Alteration of airway architecture with abnormal airway connective tissue is thought to play an important role in this process. We hypothesized that the epithelial cells that line the airways modulate the development of peribronchial fibrosis and fixed airway obstruction by directing fibroblast proliferation. To assess this, we examined stimulatory activities for human lung fibroblast proliferation in bovine bronchial epithelial cell-conditioned medium. The conditioned medium stimulated the proliferation of fibroblasts in a serum-free culture system in a concentration-dependent manner. The fibroblast growth stimulatory activity was heterogenous, with molecular masses of > 50 and approximately 10 kDa. Bronchial epithelial cell-conditioned medium also contained fibroblast growth inhibitory factors, including both transforming growth factor (TGF)-beta and, based on indomethacin sensitivity, cyclooxygenase products. TGF-beta appeared to contribute to the morphological change of fibroblasts induced by the conditioned medium. Co-culture of human lung fibroblasts with bronchial epithelial cells resulted in a stimulation of fibroblast proliferation. In summary, airway epithelial cells appear to regulate fibroblast proliferation and may play a role in peribronchial fibrosis in chronic bronchitis.
Effects of sodium concentration on human neutrophil bactericidal functions
Authors: Mizgerd J. P. , Kobzik L. , Warner A. E. , Brain J. D.
Published: December 2017

What are the ionic requirements for neutrophil (PMN) function and how might altered electrolyte concentrations contribute to airway disease? The in vitro killing of Pseudomonas aeruginosa by human peripheral white blood cells (WBCs) was progressively compromised Na+ concentration was lowered from 124 to 62 mM; at 62 mM Na+, bactericidal activity was 28.8 +/- 7.4% (SE) of normal. In contrast, Cl- concentration affected killing only when lowered to 8 mM. We examined phagocytosis and oxidative metabolism in response to P. aeruginosa or particles opsonized with either immunoglobulin G (IgG) or complement (C'). Phagocytosis of P. aeruginosa and of IgG-coated particles was Na(+)-dependent (31.2 +/- 3.1 and 58.6 +/- 14.2% of normal, respectively, at 62 mM Na+). However, no effect on uptake of C'-coated particles was observed, and the respiratory burst at 70 mM Na+ was normal regardless of stimuli. Thus low Na+ concentration compromises select PMN functions. These results may help explain why airways of cystic fibrosis (CF) patients become colonized with bacteria such as P. aeruginosa. Perhaps the low concentration of Na+ reported for some CF respiratory secretions inhibits bactericidal functions of PMNs, predisposing these patients to airway infections.
Pulmonary cytochrome P-450 monooxygenase system and Clara cell differentiation in rats
Authors: Ji C. M. , Cardoso W. V. , Gebremichael A. , Philpot R. M. , Buckpitt A. R. , Plopper C. G. , Pinkerton K. E.
Published: December 2017

Because a number of studies suggest that the developmental expression of cytochrome P-450s (CYP) in Clara cells is species specific, this study was designed to compare the developmental patterns of the isoform CYP2B and NADPH reductase protein expression and CYP2B activity with the time course of smooth endoplasmic reticulum (SER) formation in Clara cells of rat lung. Pulmonary CYP2B activity measured as pentoxyresorufin O-dealkylation in lung homogenates was not detectable before 7 days postnatal age, but was detectable at adult levels at 50 days postnatal age. In Clara cells, CYP2B and NADPH reductase were detected immunohistochemically at 4 days postnatal age and at adult levels at 10 days postnatal age. The volume density of SER in Clara cells of terminal bronchioles measured morphometrically increased significantly with postnatal age. We conclude that in the rat 1) CYP2B and NADPH reductase distribution and CYP2B activity are age dependent; 2) the increase in Clara cell SER precedes the expression of CYP2B protein; 3) cellular appearance of CYP2B protein precedes CYP activity; and 4) SER appearance and P-450 protein expression do not occur uniformly in differentiating Clara cells, even within the same bronchiole.
Glutathione S-transferases in tracheobronchial epithelium
Authors: Reddy P. M. , Tu C. P. , Wu R.
Published: December 2017

The purpose of this study is to characterize glutathione S-transferase (GST) gene expression in airway epithelium both in vivo and in vitro. Immunohistochemical staining of nonhuman primate lungs of well-controlled healthy animals reveals the presence of alpha- and pi-class GST isoenzymes in ciliated bronchial epithelium. The stain of mu-GST antibody is either very low or absent in some of these monkey lungs. We observed that primary tracheobronchial epithelial (TBE) cells isolated from human and monkey pulmonary tissues maintain a relatively high level of GST enzymatic activity in culture, compared with various immortalized human TBE cell lines and other nonpulmonary cell lines. Northern blot analysis demonstrated the presence of mu-, pi-, and microsomal-GST messages but not the alpha-class message in cultures of primary TBE cells as well as in various human TBE cell lines. The expression of mu- and pi-class GST genes can be further regulated in culture by various environmental factors; however, most of these regulating factors are associated with TBE cell differentiation in culture. For instance, vitamin A treatment, which was shown to enhance mucous cell differentiation in vitro, stimulated the message levels of mu- and pi-class GST. Furthermore, plating cells on collagen gel substrata, which also enhanced mucous cell differentiation in culture, instead of plastic culture surface, enhanced total GST enzymatic activity by eightfold, and this enhancement is related to an increase in the expression of the pi-class GST gene. These results demonstrated that GST genes are differentially expressed and regulated by various environmental factors in primary TBE cells and various cell lines, and the regulation is correlated to the mucous cell differentiation in culture.
Phosphocholine reverses inhibition of pulmonary surfactant adsorption caused by C-reactive protein
Authors: McEachren T. M. , Keough K. M.
Published: December 2017

The influence of the acute inflammatory phase protein human C-reactive protein (CRP) on the adsorption of porcine pulmonary surfactant from a subphase into an air-water interface has been investigated. CRP was shown to detract from the ability of surfactant to rapidly adsorb to the air-water interface at a molar ratio of 0.03:1 (protein:phospholipid) (weight ratio, 0.5:1). On a weight basis, CRP was found to be more effective than fibrinogen at reducing the adsorption rate of surfactant. The effect of CRP required the presence of calcium and was reversed by the addition of phosphocholine in a concentration-dependent manner. The inhibition of surfactant adsorption by CRP was effectively eliminated by the addition of phosphocholine at a molar ratio of 300:1 (phosphocholine:CRP), but it was not diminished by the addition of identical molar ratios of o-phosphoethanolamine or DL-alpha-glycerophosphate at the same molar ratios. These data suggest that the potent inhibition of surfactant adsorption by CRP is primarily a result of a specific interaction between CRP and the phosphocholine headgroup of surfactant lipids in the subphase and that it can be reversed by the water-soluble CRP ligand, phosphocholine.
Neutrophils enhance removal of ozone-injured alveolar epithelial cells in vitro
Authors: Cheek J. M. , McDonald R. J. , Rapalyea L. , Tarkington B. K. , Hyde D. M.
Published: December 2017

After acute exposure to oxidant gases in vivo, migration and accumulation of inflammatory cells in pulmonary epithelium coincides with epithelial cell necrosis. The present study was designed to test quantitatively the hypothesis that quiescent neutrophils enhance the removal of oxidant-injured pulmonary epithelial cells after exposure to ozone in vitro. Primary isolated rat alveolar type II cells were cultured as monolayers, using serum-free medium. After exposure to 0.1-0.5 ppm ozone for 0.5 h, apical sides of monolayers were administered either fresh nutrient medium only or medium containing quiescent human neutrophils. Monolayer bioelectric properties and cellular uptake of vital dye were recorded from 5 to 48 h after ozone exposure. Ozone dose-dependent increases in monolayer permeability were associated with proportionally higher numbers of injured epithelial cells. However, the direction and magnitude of neutrophil effects on monolayer permeability after ozone exposure were dependent on ozone concentration. Furthermore, neutrophil-treated monolayers exposed to 0.1 ppm ozone had significantly fewer attached cells positive for uptake of vital dye relative to monolayers exposed to the low level of ozone only; this effect was ablated with increasing ozone concentration. These data suggest that at high levels of ozone neutrophils may exacerbate injury to oxidant-impaired epithelial cells, whereas the presence of neutrophils after exposure to ambient concentrations of ozone may expedite the restoration of epithelial barrier function. We conclude that, by enhancing the removal of injured cells, neutrophils may facilitate the repair of centriacinar epithelium after ozone exposure in vivo.