bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous nitric oxide limits cytokine-induced damage of murine lung epithelial cells
Authors: Burke-Gaffney A. , Hellewell P. G.
Published: December 2017

This study investigated whether endogenous nitric oxide (NO) limits cytokine-induced damage to the murine lung epithelial cell line LA-4. NO production was assessed as nitrite using the Griess reaction, and cell damage was assessed using ethidium homodimer-1. Cytotoxicity was first detected after a 24-h incubation with a combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma (cytomix). Nitrite production increased to 78.0 +/- 0.5 nmol/10(6) cells at 24 h. Coincubation of LA-4 with cytomix and NO synthase inhibitors, aminoguanidine (3-1,000 microM) and N(G)-monomethyl-L-arginine (10-1,000 microM), but not N(G)-monomethyl-D-arginine, or a soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one, reduced cytomix-induced nitrite production and increased cytotoxicity up to twofold (24 h). Removal of L-arginine from the medium increased damage; reintroduction of 1,000 microM L-arginine, but not D-arginine, reversed this. In aminoguanidine-treated cells, replacement of NO with an NO donor, S-nitrosoglutathione (30 microM), reversed, in part, the cell damage observed in aminoguanidine/cytomix-treated cells. These results suggest that endogenous NO limits cytokine-induced lung epithelial damage.
Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis
Authors: D'Angio C. T. , Finkelstein J. N. , Lomonaco M. B. , Paxhia A. , Wright S. A. , Baggs R. B. , Notter R. H. , Ryan R. M.
Published: December 2017

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.
cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia
Authors: Illek B. , Yankaskas J. R. , Machen T. E.
Published: December 2017

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.
ET-1 induces mitogenesis in ovine airway smooth muscle cells via ETA and ETB receptors
Authors: Carratu P. , Scuri M. , Styblo J. L. , Wanner A. , Glassberg M. K.
Published: December 2017

The proliferation of airway smooth muscle cells is a characteristic feature of asthma. Endothelin (ET)-1, a member of a family of three isopeptides (ET-1, ET-2, and ET-3), functions as a spasmogen and mitogen for airway smooth muscle cells. Two types of ET receptors have been identified in mammalian species (ETA and ETB). Because the respective roles of ETA and ETB receptors in ET-1-induced mitogenesis are not known, we determined the effect of two selective ETA and ETB antagonists (BQ-610 and BQ-788) on ET-1-induced mitogenesis of cultured ovine airway smooth muscle cells. Both BQ-610 and BQ-788 inhibited ET-1-induced mitogenesis in a concentration-dependent manner, with BQ-788 exhibiting more potent antagonism [half-maximal inhibitory concentration (IC50) = 3.5 nM, slope of 0.49] compared with BQ-610 (IC50 = 20 nM, slope of 0.27). The combined ETA-ETB antagonist, bosentan, also inhibited ET-1-induced mitogenesis (IC50 = 20 nM, slope of 0.60). The effects of BQ-788 and bosentan appear to be mediated via the same receptor (ETB), as their slopes are comparable. These observations suggest that both receptor subtypes are utilized in ET-1-induced proliferation of ovine airway smooth muscle. ET receptor expression may be important in the increase in airway smooth muscle mass seen in the airways of patients with bronchial asthma.
RANTES inhibits HIV-1 replication in human peripheral blood monocytes and alveolar macrophages
Authors: Coffey M. J. , Woffendin C. , Phare S. M. , Strieter R. M. , Markovitz D. M.
Published: December 2017

Infection with human immunodeficiency virus (HIV)-1 most often leads to the development of acquired immune deficiency syndrome, which may manifest with opportunistic infections, many of which occur in the lung. Mononuclear phagocytes infected by HIV-1, being relatively resistant to its cytopathic effects, potentially act as a reservoir for the virus. The alveolar macrophage (AM), a differentiated lung tissue macrophage, is readily infected by HIV-1, after which the virus becomes relatively dormant. C-C chemokines, secreted by CD8 T lymphocytes and other cells, are known to suppress HIV replication in lymphocytes. In view of this observation, and the relative increase in CD8+ T lymphocytes during HIV-1 disease, particularly in the lung, we hypothesized that C-C chemokines might play a key role in suppressing HIV-1 replication in AM. We examined the effect of the C-C chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and regulated on activation normal T cell expressed and secreted (RANTES) singly and in combination on HIV-1 replication in peripheral blood monocytes (PBM) and AM infected in vitro. Our findings indicate that RANTES suppresses HIV-1 replication, as measured by reverse transcriptase activity, in PBM (41.3 +/- 15.2% of control, n = 3, P < 0.05) and AM (30.3 +/- 7.8% of control, n = 3, P < 0.05) in a dose-dependent manner. The other C-C chemokines had no significant effect singly (MIP-1 alpha PBM: 64.8 +/- 21.9%; AM: 115.0 +/- 2.4% of control; MIP-1 beta PBM: 68 +/- 19.6; AM: 63.3 +/- 26.2% of control) but modestly decreased HIV replication when incubated in addition to RANTES (24.5 +/- 6.5% of control). These observations suggest that RANTES plays a key role in modulating HIV-1 replication in mononuclear phagocytes in the blood and lung, and this may have therapeutic implications for prevention and/or treatment of HIV disease.
A new role for an old molecule: serotonin as a mitogen
Authors: Fanburg B. L. , Lee S. L.
Published: December 2017

Serotonin (5-hydroxytryptamine) has been known for the last half century to influence vasoactivity and to participate in neurotransmission. More recently, this compound has been recognized to cause proliferation of a variety of cells in culture, including those of vascular smooth muscle. Furthermore, the proliferative effect is synergistic with that of more conventional growth-producing polypeptides. A hypertrophic, as well as a proliferative response, has been shown to occur in some smooth muscle cells. Whether the mitogenic effect is initiated through a cell surface receptor or a serotonin transporter, or both depending on the cell type, is currently unresolved. Most evidence indicates that cellular cyclic nucleotides play an important role in the intracellular signaling process for growth regulation by serotonin, and newer studies point to protein phosphorylation pathways as being important in the mitogenic response. It has been proposed that, through these signaling pathways, serotonin plays an important role in remodeling of both the pulmonary and systemic circulations.
Human pulmonary arteries dilate to 20-HETE, an endogenous eicosanoid of lung tissue
Authors: Birks E. K. , Bousamra M. , Presberg K. , Marsh J. A. , Effros R. M. , Jacobs E. R.
Published: December 2017

We investigated the effect of 20-hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite of the cytochrome P-450 (cP450) 4A pathway, on human pulmonary arterial tone. 20-HETE elicited a dose-dependent and indomethacin-inhibitable vasodilation of isolated small pulmonary arteries. Whole lung microsomes metabolized [24C]arachidonic acid into 20-HETE and a variety of leukotrienes, epoxyeicosatrienoic acids, and prostanoids. Indomethacin blocked formation of prostanoids without effects on the conversion of arachidonate into 20-HETE, 20-HETE was converted by lung microsomes into prostanoids, raising the possibility that 20-HETE may be metabolized by cyclooxygenase enzymes in vascular tissue to a vasodilatory compound. Western blots probed with a polyclonal antibody to cP450 4A identified a protein of approximately 50 kDa immunologically similar to the cP450 4A in rat liver. We conclude that small arteries from human lungs dilate upon exposure to 20-HETE in a cyclooxygenase-dependent manner and that the proteins and enzymatic activity required to synthesize this product are present in lungs. Our observations suggest that cP450 enzyme products could be endogenous modulators of pulmonary vascular tone.
Glucocorticoid receptor signaling in a bronchial epithelial cell line
Authors: LeVan T. D. , Behr F. D. , Adkins K. K. , Miesfeld R. L. , Bloom J. W.
Published: December 2017

Glucocorticoids are an effective anti-inflammatory therapy for the treatment of asthma. The anti-inflammatory effects of glucocorticoids may be due to the inhibition of transcription factors that regulate cytokine synthesis. Because of the potential role of the bronchial epithelium in asthmatic inflammation and the possibility that this cell may be the main target of inhaled glucocorticoids, we have characterized glucocorticoid receptors (GR) and GR signaling in the human bronchial epithelial cell line BEAS-2B. Western blot analysis and radioligand binding studies demonstrated that BEAS-2B cells have functional GR that bind to dexamethasone (Dex) (dissociation constant = 5.6 nM and maximal density of binding sites = 228 +/- 3.3 fmol/mg protein). GR were activated by Dex as assessed using a glucocorticoid-responsive reporter plasmid. Transfection of BEAS-2B cells with an activator protein-1 (AP-1) reporter construct followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment resulted in a fivefold induction of reporter gene activity. Transfection with a nuclear factor (NF)-kappa B reporter construct followed by tumor necrosis factor-alpha (TNF-alpha) treatment resulted in a 10-fold induction of reporter gene activity. Dex (10(-7) M) markedly repressed both the induced AP-1 and NF-kappa B activity. The GR antagonist RU-486 inhibited the repressive effect of Dex on TNF-alpha-induced NF-kappa B activity by 81% but only counteracted the repressive effect of Dex on TPA-induced AP-1 activity by 43%. These studies demonstrate that cross-signaling between AP-1 and NF-kappa B with GR may explain the anti-inflammatory properties of glucocorticoids in airway epithelial cells.
Effects of cAMP on serotonin evoked calcium transients in cultured rat airway smooth muscle cells
Authors: Tolloczko B. , Jia Y. L. , Martin J. G.
Published: December 2017

Agents increasing intracellular adenosine 3',5'-cyclic monophosphate (cAMP) cause relaxation of airway smooth muscle. However, the mechanisms of their action are not fully understood. We investigated the role of cAMP in the modulation of intracellular Ca2+ concentration ([Ca2+]i) transients evoked by serotonin (5-HT) in cultured rat tracheal smooth muscle (TSM) cells. Forskolin (10(-7) M) caused a significant elevation of intracellular cAMP and a 60% relaxation of tracheal rings contracted with 5-HT but did not affect [Ca2+]i in TSM cells. Forskolin (10(-5) M) completely relaxed tracheal rings and significantly decreased [Ca2+]i during the sustained phase of the 5-HT response. Forskolin-induced relaxation was attenuated by the cAMP-dependent protein kinase A (PKA) inhibitor Rp diastereomer of cAMP (Rp-cAMPS; 10(-4) M) and by the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor [Rp isomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, 10(-4) M]. The effects of forskolin on [Ca2+]i were not altered by the PKA inhibitor but were abolished by the PKG inhibitor and thapsigargin. These results indicate that, in rat TSM, the relaxant effects of high concentrations of cAMP may be mediated, at least in part, by facilitating the sequestration of Ca2+ into intracellular stores by a mechanism involving PKG.
Bronchoalveolar macrophage CD14 expression: shift between membrane-associated and soluble pools
Authors: Hasday J. D. , Dubin W. , Mongovin S. , Goldblum S. E. , Swoveland P. , Leturcq D. J. , Moriarty A. M. , Bleecker E. R. , Martin T. R.
Published: December 2017

The bacterial endotoxin [lipopolysaccharide (LPS)]-binding protein CD14 modulates the host response to LPS, but membrane-associated and soluble forms of the molecule exert different biological effects. CD14 anchored to the mononuclear phagocyte membrane (mCD14) enhances response to LPS. Soluble CD14 (sCD14) may block LPS stimulation of CD14-bearing cells while supporting LPS presentation to non-CD14-bearing cells. We analyzed cell mCD14 and sCD14 expression in simultaneously collected human bronchoalveolar macrophages (BAM) and peripheral blood monocytes (PBM). Expression of mCD14 in freshly isolated BAM was only 9% as high as in PBM. Levels of sCD14 in 48 h in BAM culture supernatants were 19% as high as in PBM cultures. Interleukin (IL)-6 increased CD14 expression in both BAM and PBM but exerted different effects on CD14 distribution in these cell types. IL-6 increased only sCD14 release (2.5-fold) in BAM while increasing only mCD14 expression (2.5-fold) in PBM. IL-4 reduced both mCD14 (> 40%) and sCD14 (> 60%) expression in both cell types. We speculate that the balance between sCD14 and mCD14 expression influences the response to aspirated or inhaled LPS in the bronchoalveolar compartment. Cytokine expression and monocyte recruitment may influence this process by modulating CD14 expression.
Surfactant protein A regulates cytokine production in the monocytic cell line THP-1
Authors: Kremlev S. G. , Umstead T. M. , Phelps D. S.
Published: December 2017

Surfactant lipids inhibit cytokine production by immune cells, and surfactant protein A (SP-A) stimulates it. By enzyme-linked immunosorbent assay and mRNA blotting, we studied proinflammatory cytokine production by the monocytic cell line THP-1. SP-A caused increases in tumor necrosis factor (TNF)-alpha within 1 h, peaking at 4 h and then declining. Interleukin (IL)-1 beta increased and stayed elevated for 24 h. SP-A stimulated IL-8 also, peaking at 4 h, rapidly declining, and peaking again at 24 h. SP-A-dependent changes were detected for IL-6, but at higher SP-A doses. mRNA levels for TNF-alpha and IL-1 beta increased in response to SP-A, peaking within 2 h. The increases in TNF-alpha mRNA and protein induced by SP-A were inhibited by surfactant lipids. For IL-1 beta and IL-8, the lipids either had no inhibitory influence or inhibited less than for TNF-alpha. This suggests that the ability of macrophages to participate in inflammatory reactions is enhanced by SP-A alone or by mixtures of lipids and SP-A containing more SP-A than in normal surfactant, as occurs in many conditions leading to inflammation.
Nitric oxide donors inhibit spontaneous depolarizations by L-type Ca2+ currents in alveolar epithelial cells
Authors: Schobersberger W. , Friedrich F. , Hoffmann G. , Volkl H. , Dietl P.
Published: December 2017

L2 cells, a cloned pneumocyte-derived cell line, express voltage-dependent L-type Ca2+ channels, causing transient depolarizing spikes of the membrane potential (Vm) [P. Dietl, T. Haller, B. Wirleitner, H. Volkl, F. Friedrich, and J. Striessing. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L873-L883, 1995]. In this study, we examined the effect of nitric oxide (NO)- and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent cell signaling on the activity of L-type Ca2+ channels. Using conventional microelectrodes, spontaneous depolarizations (SD) of Vm by activation of these channels are regularly seen in the presence of 10 mM bath Sr2+. The NO donors sodium nitroprusside (SNP; 1 mM), 3-morpholinosydnonimine (SIN-1; 100 microM), as well as S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 10 microM) caused a significant reduction of the frequency of Sr(2+)-induced SD. These effects were completely reversed by 6-anilino-5,8-quinolinequinone (10 microM), an inhibitor of the soluble guanylyl cyclase, and could be mimicked by 8-bromoguanosine 3'5'-cyclic monophosphate (8-BrcGMP; 100 microM). Perforated patch-clamp experiments revealed that 8-BrcGMP exerted a significant decrease of the depolarization-induced L-type Sr2+ current in the majority of tested cells. Consistent with the dependency of these NO-mediated effects on cGMP, incubation of L2 cells with SNP, SIN-1, and SNAP lead to a pronounced increase of cellular cGMP concentration. We conclude that the NO donors inhibit the activity of L-type Ca2+ channels in L2 cells via a cGMP-dependent pathway. In the alveoli, this might occur under conditions associated with the release of NO.
Atrial natriuretic peptide accounts for increased cGMP in hypoxia-induced hypertensive rat lungs
Authors: Muramatsu M. , Tyler R. C. , Gutkowska J. , Klinger J. R. , Hill N. S. , Rodman D. M. , McMurtry I. F.
Published: December 2017

Perfusate levels of nitric oxide (NO)-containing compounds and guanosine 3',5'-cyclic monophosphate (cGMP) are increased in hypoxia-induced hypertensive rat lungs. To test if increased cGMP was due to NO stimulation of soluble guanylate cyclase (sGC), we examined effects of inhibition of NO synthase with N omega-nitro-L-arginine (L-NNA) on perfusate accumulation of cGMP in physiological salt solution (PSS)-perfused hypertensive lungs isolated from rats exposed for 3-4 wk to hypobaric hypoxia. Because 200 microM L-NNA did not reduce cGMP, we next examined inhibitors of other pathways of stimulation of either sGC or particulate GC (pGC). Neither 5 microM Zn-protophorphyrin, an inhibitor of CO production by heme oxygenase, nor 10 mM aminotriazole, an inhibitor of H2O2 metabolism by catalase, reduced perfusate cGMP. However, an antiserum to atrial natriuretic peptide (ANP; 100 microliters antiserum/30 ml PSS), to inhibit ANP activation of pGC, completely prevented accumulation of the nucleotide. ANP antiserum was also more effective than L-NNA in reducing lung tissue cGMP. In contrast, L-NNA but not ANP antiserum increased resting vascular tone. These results suggested that whereas ANP determined perfusate and tissue levels of cGMP, NO regulated vascular tone. To test if perfusate cGMP reflected ANP stimulation of pGC in endothelial rather than smooth muscle cells, we examined effects of 10 microM Zaprinast, an inhibitor of cGMP hydrolysis in smooth muscle but not endothelial cells, and found no increase of cGMP in hypertensive lungs. ANP levels were not elevated in hypertensive lungs, and it is unclear by what mechanism the ANP-stimulated activity of pGC is increased in hypertensive pulmonary vascular endothelial cells.
Nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells
Authors: Gupta M. P. , Evanoff V. , Hart C. M.
Published: December 2017

To examine the role of nitric oxide (.NO) in vascular endothelial cell injury, cultured porcine pulmonary artery endothelial cells (PAEC) were treated with H2O2 (100-500 microM) for 30 min in the presence or absence of the .NO donors (+/-)S-nitroso-N-acetylpenicillamine (SNAP) or diethylamine nitric oxide (DEANO). H2O2 caused dose-dependent PAEC cytotoxicity detected 2 h after H2O2 treatment as the release of lactate dehydrogenase. SNAP (100 microM) and DEANO (100 microM) attenuated H2O2-induced cytotoxicity if present during H2O2 treatment. In contrast, restricting treatment with .NO donors to periods before (30 min) or after (2 h) incubation with H2O2 did not prevent PAEC injury. Furthermore, the .NO synthase inhibitor NG-nitro-L-arginine methyl ester (1 mM) sensitized PAEC to H2O2-induced injury. SNAP also attenuated H2O2-induced PAEC lipid peroxidation even if restricted to periods before or after exposure to H2O2. Thus, although .NO effectively attenuated H2O2-mediated PAEC lipid peroxidation and cytotoxicity, these effects were clearly dissociated, suggesting that the antiperoxidative effects of .NO are not sufficient to account for its cytoprotective properties.
Interferon-gamma regulation of Clara cell gene expression: in vivo and in vitro
Authors: Magdaleno S. M. , Wang G. , Jackson K. J. , Ray M. K. , Welty S. , Costa R. H. , DeMayo F. J.
Published: December 2017

This report demonstrates that Clara cell 10-kDa protein (CC10) mRNA levels are regulated by interferon-gamma (IFN-gamma). An analysis of total lung RNA from mice given IFN-gamma intratracheally showed increased levels of CC10 mRNA compared to control animals but no significant increases in surfactant proteins B and C. These results were confirmed in a Clara cell line, mtCC1-2, generated from the lungs of a transgenic mouse expressing the SV40 large T antigen under the control of a Clara cell-specific promoter. Significant increases in mtCC1-2 CC10 mRNA levels were observed in a time- and a dose-dependent manner. The expression of transacting factors hepatocyte nuclear factors 3 alpha and 3 beta (HNF-3 alpha and HNF-3 beta) were also analyzed, and a transient increase in the expression of HNF-3 beta but not HNF-3 alpha was detected. Deoxyribonuclease I footprint analysis identified a signal transducer and activator of transcription (STAT) binding site (at nucleotides -293 to -284 of CC10) adjacent to two thyroid transcription factor-1 (TTF-1) binding sites, suggesting a potential interaction between STAT1 and TTF-1. This report reinforces the hypothesis that CC10 functions as an anti-inflammatory protein and that increases in CC10 protein may provide additional protection from inflammation and disease in the lung.
Cyclin D1 is required for S phase traversal in bovine tracheal myocytes
Authors: Xiong W. , Pestell R. G. , Watanabe G. , Li J. , Rosner M. R. , Hershenson M. B.
Published: December 2017

We examined the role of cyclin D1 in cultured bovine tracheal myocyte mitogenesis. Immunoblots using a polyclonal antibody against cyclin D1 showed the appearance of this protein 4 h after treatment with platelet-derived growth factor (PDGF), a potent mitogen for these cells. Immunoblots utilizing an antibody against the 110-kDa retinoblastoma protein (Rb), a downstream phosphorylation target of the cyclin D1/cyclin-dependent kinase 4 (cdk4) complex, showed reduced electrophoretic mobility of this protein as early as 8 h after PDGF treatment, suggesting phosphorylation of Rb by the cyclin D1/cdk4 dimer in vivo. Epidermal growth factor (EGF), a nonmitogen, failed to induce either cyclin D1 synthesis or Rb phosphorylation. PDGF treatment of cells transiently transfected with the full-length cyclin D1 promoter subcloned into a luciferase reporter increased luciferase activity almost threefold, demonstrating transcriptional activation of the cyclin D1 promoter with mitogenic stimulation. Finally, microinjection of individual myocytes with an affinity-purified antibody against cyclin D1 reduced the percentage of cells traversing S phase after serum stimulation, as assessed by fractional bromodeoxyuridine labeling (isotype control antibody, 0.74 +/- 0.10; anti-cyclin D1, 0.22 +/- 0.04; P = 0.0001). We conclude that 1) mitogenic stimulation of cultured bovine tracheal myocytes by PDGF induces cyclin D1 transcriptional activation and protein expression, 2) cyclin D1 expression is accompanied by Rb phosphorylation, which is evidence of increased cyclin D1-associated kinase activity in vivo, and 3) microinjection of anti-cyclin D1 antibodies inhibits cellular DNA synthesis, which is evidence that cyclin D1 is required for airway smooth muscle S phase traversal.
Alveolar neutrophils in endotoxin-induced and bacteria-induced acute lung injury in rats
Authors: Delclaux C. , Rezaiguia-Delclaux S. , Delacourt C. , Brun-Buisson C. , Lafuma C. , Harf A.
Published: December 2017

Polymorphonuclear neutrophils (PMNs) are thought to play a major role in the pathogenesis of adult respiratory distress syndrome. Because the alveolar epithelium is a decisive factor in alveolo-capillary wall permeability, a toxic effect of emigrated PMNs in alveolar spaces is conceivable. We evaluated alveolar PMN function in two rat models of acute lung injury induced by alveolar instillation of endotoxin [lipopolysaccharide (LPS)] or live Pseudomonas aeruginosa (PYO). Alveolar PMNs were isolated from bronchoalveolar lavage fluid 4 and 24 h after the challenge. Hypoxemia was assessed based on the ratio arterial partial pressure of O2 (PaO2)/fraction of inspired O2 (FIO2) during mechanical ventilation. The severity of lung injury in the two models was clearly different, since PaO2/FIO2 were approximately 400 mmHg in PYO- and LPS-induced injuries, respectively. Both contrast, alveolar neutrophil influx, unstimulated oxygen metabolite production, and proteinase (elastase, gelatinase B) secretions of ex vivo alveolar PMNs were not larger in the PYO model. Thus the difference in severity was not associated with variations in alveolar neutrophil recruitment or activation. Moreover, gelatinase and leukocyte elastase activities were absent in bronchoalveolar fluid, indicating effective antiproteinase defense in alveolar spaces. We conclude that alveolar neutrophils are not sufficient to create severe respiratory failure.
ATP-activated basolateral Na+/H+ exchange in human normal and cystic fibrosis airway epithelium
Authors: Paradiso A. M.
Published: December 2017

This study investigated the distribution (apical/basolateral membrane) of Na+/H+ exchange in human nasal epithelial cell monolayers from cystic fibrosis (CF) and non-CF individuals. Monolayers of non-CF and CF preparations were loaded with the pH probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein, and intracellular pH (pH1) was measured with a microfluorimeter. In nominally HCO3(-)-free NaCl Ringer solution, basal pHi values in non-CF and CF monolayers were essentially identical (approximately 7.1). When cells were acid loaded (NH4+ prepulse), subsequent alkalinization of non-CF and CF cell monolayers required serosal Na+ and was blocked by serosal, but not mucosal, amiloride (500 microM). In the presence of extracellular Na+, initial rates (delta pHi/min) of recovery from an acid load in non-CF and CF preparations were statistically identical (approximately 0.08). Pretreatment of monolayers with ATP (100 microM) or phorbol 12-myristate 13-acetate (PMA; 100 nM) caused initial rates of recovery to increase by twofold over control values in both preparations. These data provide evidence in support for an ATP- and PMA-activated Na+/H+ exchanger located at the basolateral surface of airway epithelia.
Distinct effects of SP-A and SP-B on endocytosis of SP-C by pulmonary epithelial cells
Authors: Horowitz A. D. , Moussavian B. , Han E. D. , Baatz J. E. , Whitsett J. A.
Published: December 2017

Binding and endocytosis of surfactant protein (SP) C were assessed in a mouse pulmonary adenocarcinoma cell line, MLE-12, and in isolated rat pulmonary type II epithelial cells. Binding and uptake of SP-C were detected using fluorescently labeled SP-C and dinitrophenyl-labeled SP-C (DNP-SP-C). Endocytosis of DNP-SP-C was visualized by immunocytochemistry and light microscopy. Endocytosis of DNP-SP-C occurred in MLE-12 cells, pulmonary type II epithelial cells, and NIH/3T3 cells, indicating that uptake of SP-C does not have an absolute requirement for a cell-specific receptor. After 30-60 min at 37 degrees C, DNP-SP-C was concentrated in large intracellular bodies in MLE-12 cells. Endocytosis of DNP-SP-C by MLE-12 cells or type II epithelial cells was decreased by SP-B or SP-B and SP-A together. SP-A alone did not inhibit DNP-SP-C uptake. Endocytosis of DNP-SP-C was inhibited by a 10-fold excess of lipid vesicles containing SP-C but not by a 10-fold excess of lipid alone. The inhibitory effect of SP-B on SP-C uptake may play a role in maintaining surface-active material at the air-liquid interface.
IL-1 beta inhibits ET-1 production by ATII cells in vitro: evidence for involvement of cyclooxygenase 2 pathway
Authors: Odoux C. , Crestani B. , Lebrun G. , Rolland C. , Aubin P. , Fournier T. , Fiet J. , Aubier M.
Published: December 2017

The aim of this study was to characterize the effect of alveolar macrophage (AM) secretory products on endothelin (ET)-1 production by rat alveolar type II (ATII) cells in primary culture. We quantified preproendothelin (ppET)-1 mRNA by Northern blot and ET-1 concentrations in cell supernatants by enzyme-linked immunosorbent assay. Conditioned medium (CM) from rat adherent AM decreased the ppET-1 mRNA levels in ATII cells and reduced ET-1 concentrations in cell culture supernatants. This effect was mediated by interleukin (IL)-1 beta as shown by pretreatment of CM with an anti-IL-1 beta neutralizing antiserum. IL-1 beta effect was dependent on protein synthesis, was partially prevented with indomethacin, and was totally prevented with dexamethasone. Specific inhibition of cyclooxygenase 2 activity completely reversed the effect of IL-1 beta. We conclude that rat AM inhibit ET-1 production by rat ATII cells in vitro through IL-1 beta secretion. The IL-1 beta-mediated inhibition is dependent on the cyclooxygenase 2 pathway. Downregulation of ET-1 production by activated AM could limit the intra-alveolar burden of this profibrogenic peptide and thus could prevent fibrosis development.
Ca(2+)-inhibitable adenylyl cyclase and pulmonary microvascular permeability
Authors: Chetham P. M. , Guldemeester H. A. , Mons N. , Brough G. H. , Bridges J. P. , Thompson W. J. , Stevens T.
Published: December 2017

Intracellular mechanisms responsible for endothelial cell disruption are unknown, although either elevated cytosolic Ca2+ ([Ca2+]i) or decreased adenosine 3',5'-cyclic monophosphate (cAMP) promotes permeability. Recent identification that Ca(2+)-inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]i and cAMP in macrovascular endothelial cells provided a possible mechanism of development of permeability. However, these data utilized an in vitro model; lacking was evidence supporting 1) expression of Ca(2+)-inhibitable adenylyl cyclase in pulmonary microvascular endothelium and 2) Ca2+ inhibition of adenylyl cyclase and cAMP content as a paradigm for inflammatory mediator-induced permeability in the intact circulation. We therefore addressed these issues in microvascular endothelial cells derived from rat lung and in an isolated perfused rat lung preparation. Results demonstrate expression of a Ca(2+)-inhibitable adenylyl cyclase in microvascular endothelial cells. Furthermore, data suggest that Ca2+ inhibition of adenylyl cyclase is necessary for development of microvascular permeability in the intact circulation. We conclude Ca2+ inhibition of cAMP represents a critical step in genesis of microvascular permeability in the intact pulmonary circulation.
Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo
Authors: Pilewski J. M. , Latoche J. D. , Arcasoy S. M. , Albelda S. M.
Published: December 2017

Airway epithelium is subject to injury during inflammation and exposure to a variety of inhaled and infectious agents. Little is known about the expression of integrins during human airway epithelial regeneration and differentiation after injury. We therefore characterized integrin subunit expression after mechanical injury in an in vivo xenograft model of human bronchial epithelium. On the migrating cells at the edges of surface epithelial wounds, there was increased expression of the alpha v-, beta 5-, beta 6-, and alpha 5-integrin subunits. During the later phase of repair, the increased expression of alpha v-, beta 5-, and beta 6-subunits persisted, but the expression of the beta 8-subunits was restricted to basal cells. In addition, there was a redistribution of the alpha 2- and alpha 6-collagen/laminin-binding integrins to suprabasal epithelial layers. There was no expression of the beta 3- or alpha 4-integrin subunit on reparative epithelium. A similar upregulation of alpha v-, beta 5-, and beta 6-integrin receptor subunits was observed in areas of undifferentiated airway from cystic fibrosis patients. Injured epithelium was found to be significantly more susceptible to gene transfer with a recombinant adenovirus, suggesting that the increased integrin expression has implications for the acquisition of adenovirus infections and for lung-directed gene therapy.
SEB is cytotoxic and alters EC barrier function through protein tyrosine phosphorylation in vitro
Authors: Campbell W. N. , Fitzpatrick M. , Ding X. , Jett M. , Gemski P. , Goldblum S. E.
Published: December 2017

We studied whether Staphylococcal enterotoxin B (SEB) has direct effects on endothelial cells (EC) in the absence of effector cells or their products. Bovine or human pulmonary artery EC were grown to confluence on filters mounted in chemotaxis chambers. Barrier function was assessed by placing [14C]bovine serum albumin in the chamber and sampling the lower well for 14C activity. SEB exposures induced a significant (P < 0.001) dose- and time-dependent increase in albumin flux across both bovine and human EC monolayers. Albumin flux was temperature dependent, and cycloheximide pretreatment of the monolayers did not block the SEB-induced increase in permeability. Preincubation of SEB with trypsin or anti-SEB antibody significantly (P < 0.0001) reduced the effect, whereas pretreatment with polymyxin B did not. SEB at > or = 10 micrograms/ml significantly (P < 0.03) increased EC injury as measured by 51Cr release in a dose- and time-dependent manner. Herbimycin and genistein, inhibitors of protein tyrosine kinases, each protected against SEB-induced cytotoxicity, barrier dysfunction, and intercellular gap formation. We conclude that SEB perturbs endothelial barrier function and viability in the absence of effector cells or their mediators.
Fas expression in pulmonary alveolar type II cells
Authors: Fine A. , Anderson N. L. , Rothstein T. L. , Williams M. C. , Gochuico B. R.
Published: December 2017

Fas, a type I membrane receptor protein, transduces a signal culminating in apoptosis after binding to the Fas ligand. Information regarding the expression of Fas in nonlymphoid tissues, although limited, suggests a role for Fas in epithelial progenitor cell populations. In this paper, we provide several lines of evidence indicating that the progenitor cell of the alveolus, the type II cell, displays restricted expression of Fas. We found 1) Fas gene expression in RNA derived from fresh isolates of primary rat type II cells; 2) restriction of Fas expression to a subset of alveolar type II cells by in situ hybridization and immunohistochemistry of the normal mouse lung; 3) induction of apoptosis in a mouse lung type II epithelial cell line (MLE) after activation of Fas; and 4) induction of apoptosis in a subpopulation of type II cells after the intratracheal instillation of an activating anti-Fas antibody in mice. These findings suggest that Fas-dependent apoptosis is involved in regulating turnover of the alveolar epithelium.
Maintenance of the differentiated type II cell phenotype by culture with an apical air surface
Authors: Dobbs L. G. , Pian M. S. , Maglio M. , Dumars S. , Allen L.
Published: December 2017

The study of differentiated functions of alveolar type II cells has been hampered because of the lack of good in vitro systems. We report that culture of type II cells on collagen gels with an apical surface exposed to air promotes expression of differentiated type II cell characteristics. Cells cultured in this manner are cuboidal, contain lamellar bodies, and produce tubular myelin; in addition, they secrete phosphatidylcholine in response to exogenous ATP. Cultures contain mRNA for surfactant proteins A, B, and C and surfactant proteins A, B, and D. In contrast, when type II cells are cultured with an apical surface exposed to liquid rather than to air, the cells are squamous, do not express surfactant proteins or their respective mRNA, and do not contain lamellar bodies or produce tubular myelin. Type II cells cultured on plastic for 7 days, which no longer express mRNA for surfactant proteins, can be induced to express these mRNA by changing culture conditions to that of an air surface. The culture system described in this paper should be useful for studies of surfactant metabolism, regulation of alveolar epithelial phenotypic expression, and the processing of transiently expressed transgenes.
Early increase in expression of surfactant protein A gene in type II cells from silica-treated rats
Authors: Viviano C. J. , Rooney S. A.
Published: December 2017

Silica is known to cause an increase in lung surfactant and to promote type II cell hypertrophy and hyperplasia. Two populations of type II cells can be isolated from silica-treated rats: type IIA cells that are similar to normal type II cells and type IIB cells that are larger, contain more surfactant phospholipids, and have increased rates of phospholipid biosynthesis. As much less is known about the influence of silica on the amounts of surfactant proteins (SPs) in type II cells, we examined expression of the genes for all four SPs in types IIA and IIB cells isolated from rats 1, 3, and 7 days after a single intratracheal injection of silica. There was a rapid increase in expression of the SP-A gene in type II cells from the silica-treated animals. SP-A mRNA content was 8- to 10-fold greater in types IIA and IIB cells isolated 1 day after silica injection than in type II cells from saline-injected animals. SP-A mRNA levels were also elevated in the cells isolated on days 3 and 7 after silica injection, but the extent of the increase was less than in the cells isolated on day 1 and declined with time after injection. SP-B, SP-C, and SP-D mRNA levels were 2.5- to 4-fold greater in type IIA cells on day 3 after silica injection than in control type II cells. However, those mRNA levels were not significantly increased in the type IIA cells isolated on days 1 and 7 or in type IIB cells at any time point. These data show that silica causes a rapid and substantial increase in expression of the SP-A gene in type II cells.
Ionic regulation of proton chemical (pH) and electrical gradients in lung lamellar bodies
Authors: Wadsworth S. J. , Spitzer A. R. , Chander A.
Published: December 2017

This study investigated the pH (chemical) and electrical gradients in lamellar bodies, the acidic surfactant-secreting organelles of lung epithelial type II cells, by following the uptake of a weak fluorescent base, quinacrine, and a membrane potential-sensitive dye, bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol (oxonol V). In isolated lung lamellar bodies, the ATP-dependent uptake of both agents could be inhibited by bafilomycin A1, a reportedly specific inhibitor of vacuolar-type H(+)-ATPase (V-ATPase) and could be dissipated by a protonophore, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, suggesting that the V-ATPase generates an electropositive interior. A closely linked uptake of Cl- neutralizes the positive electrical potential and increases the proton pump activity. The uptake of quinacrine, but not oxonol V, was decreased by Na+. This effect of Na+ could be prevented by dimethylamiloride, suggesting the presence of electroneutral Na+/H+ exchanger in lamellar body membranes. The initial rates of quinacrine and oxonol V uptake were increased by bumetanide, but only in the presence of Na+, K+, and Cl-, suggesting that the lamellar bodies also contain an outwardly directed electroneutral Na(+)-K(+)-2Cl- cotransporter. Thus three ion transporters, H(+)-translocating V-ATPase, Na+/H+ exchanger, and Na(+)-K(+)-2Cl- cotransporter, appear to determine the chemical and electrical gradients across the lamellar body membrane.
Inhibitory effects of Gs on desensitization of beta-adrenergic receptors in tracheal smooth muscle
Authors: Kume H. , Takagi K.
Published: December 2017

We examined the reduction of mechanical responsiveness to beta-agonists after continuous or repeated exposure to agonists in guinea pig tracheal smooth muscle, using an isometric tension record. The inhibitory action of 0.1 microM isoproterenol (Iso) on contraction induced by 1 microM methacholine (MCh) was suppressed markedly after incubation with 0.0003-3 microM Iso for 1 h. The effects of Iso on MCh-induced contraction gradually decreased after repeated application of these agents at intervals of 1 h. When Iso was perfused cumulatively to tissues repeatedly precontracted by MCh, the effects of Iso were reduced at the second application. This reduced responsiveness to beta-agonists was not mimicked by forskolin and did not occur after preexposure of the strips to 2 micrograms/ml cholera toxin for 6 h. We concluded that this reduced responsiveness to beta-agonists is homologous desensitization and that the irreversible activation of the stimulatory G proteins, Gs, may play an important role in prevention of the reduced responsiveness to beta-agonists.
Upregulation of substance P receptors in angiogenesis associated with chronic airway inflammation in rats
Authors: Baluk P. , Bowden J. J. , Lefevre P. M. , McDonald D. M.
Published: December 2017

In rat airways, substance P released from sensory nerves induces plasma leakage via neurokinin-1 (NK1) receptors on endothelial cells. In pathogen-free rats, both leakage and endothelial NK1 receptors are most abundant in postcapillary venules. In Mycoplasma pulmonis-infected rats, extensive angiogenesis occurs in the tracheal mucosa. The capillary-sized (< 10 microns in diameter) angiogenic blood vessels are abnormally sensitive to substance P. The aim of this study was to determine whether increased expression of NK1 receptors contributes to this abnormal sensitivity. Fischer 344 rats were infected with M. pulmonis and were challenged with substance P (5 micrograms/kg i.v.), and then plasma leakage in the tracheal mucosa was measured by extravasation of Monastral blue (30 mg/kg i.v.). NK1 receptors on endothelial cells were localized by immunohistochemistry. Five minutes after substance P, NK1 receptor-immunoreactive endosomes were five times more abundant in endothelial cells of angiogenic capillaries in M. pulmonis-infected rats than in corresponding capillaries in pathogen-free controls (17.1 +/- 2.3 vs. 3.5 +/- 0.4 endosomes/100 micron 2 of endothelial surface). Endosomes were slightly more abundant in postcapillary venules 15-35 microns in diameter in infected rats (23.0 +/- 0.6 vs. 19.2 +/- 0.7 endosomes/100 micron 2). Similarly, after substance P, angiogenic capillaries had much more Monastral blue labeling (area density: 18.8 +/- 1.5 vs. 2.9 +/- 0.5% of vessel wall), whereas postcapillary venules had about the same amount of labeling (36.0 +/- 3.7 vs. 34.1 +/- 1.8%). We conclude that increased expression of NK1 receptors, which are internalized into endosomes after ligand binding, contributes to the abnormal sensitivity of endothelial cells of angiogenic blood vessels to substance P in the airways of M. pulmonis-infected rats.
Chronic carbachol pretreatment decreases adenylyl cyclase activity in airway smooth muscle
Authors: Schears G. , Clancy J. , Hirshman C. A. , Emala C. W.
Published: December 2017

In airway smooth muscle, the regulation of adenylyl cyclase, the enzyme that synthesizes adenosine 3',5'-cyclic monophosphate, is under dual regulation by G protein-coupled receptors. It is unknown if chronic activation of muscarinic receptors in airway smooth muscle alters the stimulatory adenylyl cyclase cascade to decrease airway relaxation. Bovine airway smooth muscle pretreated with carbachol for 18 h, but not for 30 min or 2 h, showed decreased adenylyl cyclase activity under basal conditions and in response to isoproterenol, prostaglandin E1, GTP, and forskolin. The quantity of beta-adrenergic receptors or of Gi alpha proteins was unaffected by carbachol pretreatment. The effect of carbachol pretreatment was blocked by the inclusion of atropine or the protein kinase C (PKC) inhibitor staurosporine. These results suggest that chronic but not acute agonist pretreatment of muscarinic receptors decreases in adenylyl cyclase stimulation at a site distal to receptors and that this effect is mediated by the chronic activation of PKC via the M3 muscarinic receptor.
Fibronectin supports bronchial epithelial cell adhesion and survival in the absence of growth factors
Authors: Aoshiba K. , Rennard S. I. , Spurzem J. R.
Published: December 2017

Cell-extracellular matrix interactions support the ability of cells to migrate into areas of inflammation and injury. The present study evaluated the ability of different matrix proteins to support bronchial epithelial cell attachment and survival. Collagens were able to support attachment and survival of normal cultured human bronchial epithelial cells but only in the presence of added soluble growth factors such as insulin, epidermal growth factor, platelet-derived growth factor, and bovine pituitary extract. In contrast, fibronectin was able to support attachment and survival of normal human bronchial epithelial cells in growth factor-deficient medium. In addition, fibronectin, in the absence of added growth factors, was able to induce integrin clustering, focal adhesion formation, and phosphorylation of focal adhesion kinase. A 120-kDa chymotryptic fragment of fibronectin containing the Arg-Gly-Asp peptide sequence was able to reproduce the effects of the whole fibronectin molecule. This study supports the concept that fibronectin has specialized roles in injury and repair.