bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxic contraction of contractile interstitial cells isolated from bovine lung
Authors: Fukui M. , Yasui H. , Watanabe K. , Fujimoto T. , Kakuma T. , Yoshida R. , Ohi M. , Kuno K.
Published: December 2017

Although contractile interstitial cells (CIC) in the alveolar septum have been suggested to be involved in hypoxic pulmonary vasoconstriction (HPV), direct demonstration of cellular contraction under hypoxia has been lacking. To achieve this, we purified CIC from collagenase-dissociated bovine lung cells and examined the response of these cells to hypoxia. Prostaglandin (PG) F synthase served as a marker of CIC, and the isolated PGF synthase-positive cells were shown to preserve the ultrastructural features characteristic of CIC, most notably bundles of microfilaments. Isolated CIC seeded onto collagen gel disks became embedded and formed a lattice network with collagen fibrils. Exposure of these CIC-bearing gels to hypoxia (PO2 = 20-40 Torr) evoked a reversible reduction in gel volume, as assessed by measuring the surface area of the gel disks photographically. Thus CIC were shown to contract under hypoxia, providing the supportive evidence for the involvement of CIC in HPV.
Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability
Authors: Siflinger-Birnboim A. , Lum H. , Del Vecchio P. J. , Malik A. B.
Published: December 2017

We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to 125I-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+]i) were monitored in BMVEC monolayers loaded with the Ca(2+)-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 microM) produced a rise in [Ca2+]i within 10 s that was reduced by the addition of EGTA to the medium. Uptake of 45Ca2+ from the extracellular medium increased in the presence of H2O2 (100 microM) compared with control monolayers, suggesting that the H2O2-induced rise in [Ca2+]i is partly the result of extracellular Ca2+ influx. The effects of [Ca2+]i on endothelial permeability were addressed by pretreatment of BMVEC monolayers with BAPTA-AM (3-5 microM), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an approximately 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 microM), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+]i produced by H2O2 is a critical determinant of the increase in endothelial permeability.
Glucocorticoid-induced tropoelastin expression is mediated via transforming growth factor-beta 3
Authors: Yee W. , Wang J. , Liu J. , Tseu I. , Kuliszewski M. , Post M.
Published: December 2017

Glucocorticoids play a central role in fetal lung maturation. As fibroblasts have been shown to be an important target cell for glucocorticoids in fetal lung, we have recently cloned several cDNAs representing genes induced by cortisol in fetal rat lung fibroblasts. Approximately 30% of the cDNAs were identified as transforming growth factor (TGF)-beta 3. Here, we selected and sequenced another cDNA. Analysis of DNA sequence homology indicated that this cDNA encodes the rat tropoelastin (TE). Using this cDNA as a TE probe, we confirmed that TE mRNA was expressed in a developmental, organ- and cell type-specific fashion. Exogenous glucocorticoids indeed increased the number of TE transcripts in fetal lung fibroblasts. Blockage of endogenous TGF-beta 3 production with antisense TGF-beta 3 oligonucleotides abrogated the stimulatory effect of cortisol on TE mRNA production by fetal lung fibroblasts. Also, neutralizing antibodies to TGF-beta 3 blocked the cortisol-stimulated TE mRNA expression. Fetal lung fibroblasts expressed both TGF-beta type I and II receptors. Cortisol did not influence the expression of either receptor mRNA. Antisense TGF-beta type I receptor oligonucleotides inhibited cortisol-induced TE mRNA expression. Cortisol activated the transcription of stable transfected cDNA for TGF-beta 3 under the control of glucocorticoid-inducible long terminal repeat (LTR) promoter in RFL-6 fibroblasts. Induction of TGF-beta 3 in the transfectants was accompanied by a marked increase in TE mRNA. These findings suggest that glucocorticoids mediate their stimulatory effect on TE mRNA expression in fetal lung fibroblasts via an autocrine action of glucocorticoid-induced TGF-beta 3.
Protein kinase C in intracellular pH regulation in alveolar type II cells
Authors: Wadsworth S. , Wu A. M. , Spitzer A. , Chander A.
Published: December 2017

The Na+/H+ exchanger and Na(+)-HCO3- cotransporter have been implicated in regulation of intracellular pH (pHi) in alveolar type II cells. This study demonstrates that activation of protein kinase C (PKC) stimulates both of these ion transporters in type II cells. Treatment of type II cells with 80 nM phorbol 12-myristate 13-acetate (PMA) increased the resting pHi in a time-dependent manner. Compared with control cells, the rates of recovery from an acid load increased with PMA treatment, reaching a maximum at 15 min, and returned to control levels by 3 h. The PMA-stimulated changes in recovery rate were sensitive to H-7, a PKC inhibitor. For PMA treatment up to 2 h, these recoveries were also sensitive to dimethylamiloride (DMA), an inhibitor of Na+/H+ exchanger activity, and to HCO3-, suggesting activation of both the Na+/H+ exchanger and the Na(+)-HCO3- cotransporter. After prolonged (3 h) treatment with PMA, however, the recovery was insensitive to DMA but was sensitive to HCO3-, suggesting that the Na+/H+ exchanger was no longer active and that most of the recovery was mediated by the Na(+)-HCO3- cotransporter. PMA treatment also altered the Na+ kinetics of the recovery from an acid load with respect to the Michaelis constant (Km) and maximal ion flux (Vmax), suggesting protein modifications of each transporter. We suggest that PKC activation in type II cells results in acute and long-term changes in pHi regulatory mechanisms mediated by the Na+/H+ exchanger and by the Na(+)-HCO3- cotransporter.
Role of calmodulin and myosin light-chain kinase in lung ischemia-reperfusion injury
Authors: Khimenko P. L. , Moore T. M. , Wilson P. S. , Taylor A. E.
Published: December 2017

It is generally accepted that microvascular permeability is controlled by intercellular endothelial cell gap size. This process is controlled in endothelial cell monolayers and peripheral blood vessels by calmodulin (CaM)-dependent myosin light-chain kinase (MLCK), which phosphorylates MLC20 with subsequent actin-myosin interaction. In the present study both CaM and MLCK blockers were studied during ischemia-reperfusion (I/R)-induced injury in isolated buffer-perfused rat lungs. The effects of a calcium ionophore (CaI) were tested in isolated intact rat lungs to compare the effects of increasing intracellular Ca2+ to I/R-induced damage. Because protein kinase C (PKC) could also be a mediator of I/R injury, a PKC inhibitor was studied in lungs subjected to either I/R or CaI. In lungs subjected to I/R alone, a fivefold increase in microvascular permeability occurred after 30 min of reperfusion (P < 0.001), and a tenfold increase was present after an additional 60 min of reperfusion (P < 0.01). Pretreatment of the I/R lungs with a CaM inhibitor (trifluoperazine, 100 microM) or with a MLCK inhibitor (ML-7,500 nM) blocked the microvascular damage at both 30 and 90 min of reperfusion. When the CaM inhibitor was introduced into the venous reservoir after 46 min of reperfusion, after the microvascular damage was present, no further increase in microvascular permeability occurred. Pretreatment of the lungs with a PKC inhibitor (staurosporine, 100 nM) did not alter the magnitude of the increased microvascular permeability produced by I/R or the time course of the damage. The calcium ionophore A23187 (7.5 microM) caused increases in Kfc values similar to those produced by I/R. Pretreatment of A23187-treated lungs with a CaM inhibitor produced no protective effect on the microvascular injury at 30 min after administration. Pretreatment of the CaI-challenged lungs with staurosporine significantly increased the microvascular barrier injury at 30 min compared with that occurring with I/R. When a beta-adrenergic receptor agonist (isoproterenol, 10 microM) was introduced to the lung after CaI-induced damage had occurred, no further increase in microvascular permeability was observed, and a trend toward reversal of injury occurred. We conclude from these studies that CaM/MLCK/MLC20 system is involved in our model of I/R-induced rat lung injury but is not involved in lung injury associated with Ca2+ entering the cell.
Prostaglandin G/H synthase-2 is the constitutive and dominant isoform in cultured human lung epithelial cells
Authors: Asano K. , Lilly C. M. , Drazen J. M.
Published: December 2017

Two isoforms of prostaglandin G/H synthase (PGHS; prostaglandin endoperoxide synthase, cyclooxygenase) have been identified; PGHS-1 is expressed constitutively in most tissues, whereas PGHS-2 is thought to be induced by various proinflammatory cytokines and growth factors. In this study, we determined which isoform of PGHS mRNA, protein, and activity was present constitutively in A549 (a human lung adenocarcinoma cell line) and in untransformed (normal human bronchial epithelial or NHBE) and transformed (16HBE4o-) human bronchial epithelial cells. Two PGHS-2-specific inhibitors, NS-398 and L-745, 337, blocked the release of prostaglandin E2 from A549 cells with mean inhibitory concentrations of 5 and 18 nM, respectively, but did not inhibit its release from human bronchial smooth muscle cells (BSMC) at a concentration of 10 microM. Northern and immunoblot analysis demonstrated that BSMC expressed PGHS-1 mRNA and protein constitutively, whereas epithelial cells expressed PGHS-2 mRNA and protein constitutively with either undetectable (A549, 16HBE4o-) or very low levels (NHBE) of PGHS-1. We conclude that PGHS-2 is the dominant PGHS isoform in unstimulated and stimulated lung epithelial cells in culture.
In vivo exposure to NO2 reduces TNF and IL-6 production by endotoxin-stimulated alveolar macrophages
Authors: Erroi A. , Pagani P. , Sironi M. , Salmona M.
Published: December 2017

Exposure to NO2 appears to affect lung defense mechanisms. We exposed rats to 10 ppm of NO2 for 24 h or 7 days and studied the production of tumor necrosis factor (TNF), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) by alveolar macrophages after endotoxin stimulation. TNF and IL-6 production was significantly decreased (four-to sixfold) in the cell lysate of alveolar macrophages isolated from rats exposed to NO2. In parallel, PGE2 production was significantly increased in the same samples and in the bronchoalveolar lavage fluid. Northern blot analysis of the two cytokines indicated a reduction of the mRNA content. We also studied the expression of the TNF receptor type 1 (TNF-R1), known to neutralize TNF activity in its soluble form, and found that expression of the mRNA was increased after endotoxin stimulation. We can conclude that rats exposed to NO2 produce less TNF and IL-6 and that this might be related to increased PGE2 production and increased expression of TNF-R1.
Hypoxia impairs nitrovasodilator-induced pulmonary vasodilation: role of Na-K-ATPase activity
Authors: Tamaoki J. , Tagaya E. , Yamawaki I. , Konno K.
Published: December 2017

To elucidate the effect of hypoxia on nitrovasodilator-induced pulmonary vasodilation, we studied canine pulmonary arterial rings under isometric conditions in vitro. Exposure to hypoxia inhibited the relaxant responses of KCl-contracted tissues to sodium nitroprusside (SNP), so that the maximal relaxation (Emax) and the negative logarithm of molar concentration required to produce 50% relaxation (pD2) were decreased from 92 +/- 7 to 62 +/- 5% and from 5.8 +/- 0.2 to 4.7 +/- 0.3, respectively (means +/- SE, P < 0.01 for each). This effect was likewise observed when 8-bromoguanosine-3',5'-cyclic monophosphate was used as a relaxant. The impairment of SNP-induced relaxation of endothelium-denuded rings under hypoxia was abolished by ouabain or K(+)-free solution. Incubation with SNPincreased intracellular cGMP contents in a dose dependent manner, an effect that was not altered by hypoxia. SNP also increased ouabain-sensitive 86Rb uptake, and this effect was inhibited by hypoxia. These results suggest that hypoxia reduces nitrovasodilator-induced relaxation of pulmonary artery, probably through an inhibition of cGMP-dependent sarcolemmal Na-K-adenosine triphosphatase activity.
Hepatocyte growth factor is a mitogen for alveolar type II cells in rat lavage fluid
Authors: Mason R. J. , McCormick-Shannon K. , Rubin J. S. , Nakamura T. , Leslie C. C.
Published: December 2017

Proliferation of type II cells is required for maintenance of the alveolar epithelium and for restoration after lung injury. Although various known growth factors have been reported to stimulate type II cell proliferation in vitro, there is very little knowledge on which growth factors are present in the lung in vivo. We have previously reported that rat lavage fluid contains a mitogen(s) for type II cells, and this study was de signed to identify the growth factor(s) in this biological fluid for type II cells. The mitogenic activity was purified by sequential chromatography on blue Sepharose and heparin Sepharose. Hepatocyte growth factor (HGF) and acidic fibroblast growth factor by Western analysis. The amount of HGF recovered by lavage was approximately 6 ng/rat. By a use of neutralizing antibodies for different growth factors, HGF was found to be responsible for most of the stimulatory activity for rat type II cells in the partially purified lavage fluid. In addition to HGF, rat lavage fluid also contained potent mitogenic activity for fibroblasts. Finally, we have demonstrated that much of the mitogenic activity in salt extracts of human lung is HGF. We conclude that HGF is found in rat lavage fluid and is possibly an important mitogen for adult type II cells in vivo.
Dynamics of TGF-beta 3 peptide activity during rat alveolar epithelial cell proliferative recovery from acute hyperoxia
Authors: Buckley S. , Bui K. C. , Hussain M. , Warburton D.
Published: December 2017

Hyperoxia causes a reproducible pattern of lung injury and recovery, characterized by proliferation of type II alveolar epithelial cells (AEC2) during the recovery phase. We measured TGF-beta peptide production by AEC2 and macrophages from lungs of adult male rats exposed to 100% oxygen for 48 h and then allowed to recover for up to 72 h in room air. TGF-beta peptide activity levels were measured using the PAI-1 promoter-luciferase mink lung epithelial cell assay and characterized with peptide specific inhibitory antibodies. Control AEC2 produced 997 +/- 54 pg active TGF-beta x 10(6) cells-1.24h-1 (mean +/- SD), of which > 70% was TGF-beta 3, while cultured macrophages produced 58 +/- 17 pg active TGF-beta x 10(6) macrophages-1.24 h-1, > 80% of which was TGF-beta 1. During hyperoxia and recovery, active TGF-beta 3 production by AEC2 decreased by 75%, with a nadir at 24 h recovery (P < 0.005). In contrast, TGF-beta peptide activity increased from undetectable levels in lung lavage from control rats to a peak of 1,470 +/- 743 pg/rat after 48 h oxygen exposure and 24 h recovery, while lavaged macrophage TGF-beta production in culture also increased threefold to a peak of 150 +/- 5 pg. 10(6) cells-1. 24 h-1 after 48 h oxygen exposure (P < 0.005). The nadir of active TGF-beta 3 production by AEC2 coincided with the peak of the AEC2 proliferative phase of repair as determined by BrdU incorporation and FACS analysis of freshly isolated AEC2. We conclude that active TGF-beta 3 production by AEC2 is dynamically downregulated during the proliferative phase of recovery from acute hyperoxic injury in rat. We speculate that decreased autocrine negative regulation of AEC2 proliferation by TGF-beta 3 may facilitate AEC2 proliferation during recovery from acute hyperoxic injury.
Inhibition of Na-K-ATPase activity after prolonged hypoxia in an alveolar epithelial cell line
Authors: Planes C. , Friedlander G. , Loiseau A. , Amiel C. , Clerici C.
Published: December 2017

Exposure to alveolar hypoxia may induce acute pulmonary edema. Because the vectorial sodium transport by alveolar epithelium represents an important mechanism for alveolar edema clearance, we examined whether hypoxia affects Na-K-ATPase activity in cultured SV40-transformed rat alveolar type II cells (SV40 ATII cells). Hypoxic exposures (O or 5% O2 for at least 12 h) induced a time- and O2 concentration-dependent decrease in ouabain-sensitive rubidium (osRb) influx. Neither the sensitivity of Rb influx to ouabain nor the maximum velocity of the enzyme measured on crude cell homogenates was affected by hypoxia. The osRb influx decrease was independent of hypoxia-induced ATP depletion. Na-K-ATPase inhibition was most likely related to impaired calcium homeostasis, because 1) calcium influx was increased in hypoxic cells, 2) hypoxia-induced osRb influx decrease was completely prevented by nifedipine (10-5 M), and 3) osRb influx decreased in normoxic cells incubated with ionomycin (10-6 M, 15 min). Furthermore, hypoxia-induced Na-K-ATPase impairment might be due, at least in part, to the endogenous release by hypoxic cells of a lipidic factor in extracellular medium, because incubation of normoxic cells with hypoxic cells conditioned medium (CM), or with the lipidic subphase from hypoxic cells CM, also induced a partial decrease in osRb influx. This decrease was associated with increased calcium influx into normoxic cells and was suppressed either by the removal of external calcium or by nifedipine, suggesting that the lipidic factor exerted its inhibitory action on Na-K-ATPase via an enhancement of calcium entry. These results indicate that prolonged hypoxic exposure impairs Na-K-ATPase activity in SV40 ATII cells and may therefore decrease the vectorial sodium transport by alveolar epithelium.
Maximal PDGF-induced lung fibroblast chemotaxis requires PDGF receptor-alpha
Authors: Osornio-Vargas A. R. , Lindroos P. M. , Coin P. G. , Badgett A. , Hernandez-Rodriguez N. A. , Bonner J. C.
Published: December 2017

Alteration of the platelet-derived growth factor (PDGF) receptor system could be important in enhancing the mitogenic and chemotactic potential of lung fibroblasts during pulmonary fibrogenesis. We previously reported that interleukin-1 beta (IL-1 beta) upregulates the PDGF receptor-alpha (PDGFR-alpha) gene, and in this study we sought to establish the importance of the PDGFR-alpha relative to the PDGFR-beta in mediating a chemotactic response to PDGF-AA, -AB, and -BB. Pretreatment of fibroblasts for 24 h with IL-1 beta increased chemotaxis to all three PDGF isoforms. IL-1 beta pretreatment markedly increased the maximal number of 125I-labeled PDGF-AA binding sites but did not change the number of 125I-PDGF-AB or PDGF-BB sites. However, IL-1 beta increased 125I-PDGFR-AB affinity twofold. Neomycin (5 mM) was used as a PDGFR-alpha antagonist and completely blocked 125I-PDGF-AA binding and PDGF-AA-induced chemotaxis. The binding affinity of 125I-PDGF-AB and 125I-PDGF-BB was increased two-to threefold by neomycin, and chemotaxis to PDGF-AB and PDGF-BB was enhanced. These results define a role for the PDGFR-alpha as a regulatory receptor subtype that is necessary for PDGF isoforms to exert maximal chemotaxis.
Transforming growth factor-alpha increases alveolar liquid clearance in anesthetized ventilated rats
Authors: Folkesson H. G. , Pittet J. F. , Nitenberg G. , Matthay M. A.
Published: December 2017

The effect of transforming growth factor-alpha (TGF-alpha) on alveolar liquid clearance was examined in ventilated, anesthetized rats. An isosmolar Ringer lactate solution with 10, 50, or 200 ng/ml TGF-alpha and 125I-labeled albumin as the alveolar protein tracer was instilled into the right lower lung lobe; the rats were studied for 1 and 4 h. Compared with control rats, addition of 50 ng/ml TGF-alpha to the instilled fluid increased alveolar liquid clearance by 47% over 1 h and by 66% over 4 h (P < 0.05). This increase was similar to the 50% increase in alveolar liquid clearance over 1 h in rats instilled with a beta-adrenergic agonist, salmeterol (28). There was a dose-dependent effect of TGF-alpha (10, 50, 200 ng/ml) on alveolar liquid clearance. The combination of both TGF-alpha and salmeterol did not have an additive effect on alveolar liquid clearance. The TGF-alpha-stimulated increase in alveolar liquid clearance was inhibited by amiloride (10(-4) M), indicating that the increase in clearance depended on increased Na+ uptake across the alveolar epithelium. There was only a twofold increase in intracellular cAMP levels in isolated rat alveolar epithelial type II cells after stimulation with TGF-alpha. In contrast, beta-adrenergic agonist treatment increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels more than tenfold. Genistein (10(-6) M), a tyrosine protein kinase inhibitor, inhibited the TGF-alpha-stimulated increase in alveolar liquid clearance. In summary, TGF-alpha can stimulate in vivo alveolar liquid clearance at a rate similar to beta-adrenergic stimulation by increasing Na+ uptake by alveolar epithelial type II cells. However, the effect may be mediated by a non-cAMP dependent mechanism. Because genistein blocked the increase in alveolar fluid clearance, the signal transduction may involve genistein-dependent phosphorylation.
Expression of a calmodulin inhibitor peptide in progenitor alveolar type II cells disrupts lung development
Authors: Wang J. , Campos B. , Kaetzel M. A. , Dedman J. R.
Published: December 2017

Calmodulin (CaM) is a major intracellular Ca2+ mediator protein involved in cell growth and differentiation. To evaluate calmodulin function in lung, it was necessary to construct a gene that encodes a high-affinity calmodulin binding peptide, since chemically synthesized calmodulin inhibitors lack binding and targeting specificity. This calmodulin inhibitor peptide gene was targeted to type II epithelial cells in transgenic mice using the human surfactant protein C promoter. Neutralization of calmodulin function in progenitor type II epithelial pneumocytes alters epithelial cell growth and differentiation, which prevents branching morphogenesis of the bronchial tree. Newborn transgenic animals have undeveloped lungs. This study indicates that type II lung epithelial cells require functional CaM for proliferation and development. The targeting of specific inhibitor peptides to a single lung cell type is an approach to evaluate the role of calmodulin, the ubiquitous calcium-dependent regulator protein, in lung development and disease.
A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells
Authors: Young P. P. , Mendelson C. R.
Published: December 2017

The human has two genes encoding surfactant protein-A (SP-A), termed SP-A1 and SP-A2; the SP-A2 gene is more highly regulated by cAMP and during fetal development than is SP-A1. In this study, by use of primary cultures of human type II cells transfected with fusion genes containing various amounts of SP-A2 5'-flanking DNA linked to human growth hormone (hGH) structural gene, as reporter, we found that -296 bp of SP-A2 upstream sequence is sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types. By use of competitive EMSA, we observed that nuclear proteins isolated from midtrimester human fetal lung tissue bind specifically to a cAMP response element (CRE)-like sequence, TGACCTTA, at -242 bp, which we have termed CRESP-A2. Binding activity of CRESP-A2 for nuclear proteins from human fetal lung tissue before culture was manifest as two complexes of different mobilities and equivalent intensity. By contrast, upon differentiation of the human fetal lung in culture in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), the higher mobility complex was decreased to undetectable levels. By UV cross-linking analysis, using nuclear extracts from midgestation human fetal lung before culture and radiolabeled CRESP-A2 as a probe, we observed binding of proteins of approximately 50, 36, and 30 kDa. When nuclear extracts from human fetal lung cultured in the presence of DBcAMP were analyzed, binding of only the 50- and 36-kDa proteins was apparent. On the other hand, when the canonical CRE (TGACGTCA) known to bind the transcription factor CREB (M(r) approximately 43,000) was used as a probe, binding of only a approximately 43-kDa protein was evident using nuclear extracts from human fetal lung before and after culture. In type II cells transfected with an SP-A2(-296):hGH fusion gene in which CRESP-A2 was mutated, there was a marked reduction of basal and cAMP-stimulated fusion gene expression. These findings indicate that CRESP-A2 serves an important role in mediating basal and cAMP-inducible expression of the human SP-A2 gene in type II cells, that the fetal lung nuclear proteins bound to CRESP-A2 differ from those bound to the canonical palindromic CRE, and that changes in the complex of nuclear proteins bound to CRESP-A2 accompany induction of SP-A gene expression.
Localization of epithelial sodium channel subunit mRNAs in adult rat lung by in situ hybridization
Authors: Matsushita K. , McCray P. B. , Sigmund R. D. , Welsh M. J. , Stokes J. B.
Published: December 2017

The transport of Na+ through amiloride-sensitive sodium channels (ENaC) plays a major role in the absorption of fluid across the pulmonary epithelium. The proteins forming the ENaC channel are encoded by three genes in the rat (alpha-, beta-, and gamma-rENaC). According to Northern blot, all three subunit mRNAs were expressed in adult rat lung. Each subunit was expressed as a single transcript of approximately 3.7, 2.2, and 3.2 kb for alpha-, beta-, and gamma-rENaC, respectively. To localize the alpha-, beta-, and gamma-rENaC subunit mRNAs, we used in situ hybridization. Frozen and paraffin-embedded tissues were hybridized with sense and antisense 35S-labeled riboprobes. The alpha-rENaC mRNA was most abundant and was expressed diffusely in epithelia of the trachea, bronchi, bronchioles, and alveoli. At the alveolar level, alpha-rENaC was expressed in type II cells. The beta- and gamma-rENaC mRNAs were most abundant in the bronchial and bronchiolar epithelia. All three subunits were expressed in the renal cortical collecting duct in a pattern similar to that previously reported by other investigators. Thus the rENaC subunit mRNAs are expressed in regions of the lung where functional Na+ absorption is found. These results are consistent with an important role for ENaC in the absorption of Na+ and fluid across the pulmonary epithelium in all regions of the lung.
Specificity and uniqueness of endothelial cell stress responses
Authors: Tucci M. , McDonald R. J. , Aaronson R. , Graven K. K. , Farber H. W.
Published: December 2017

The mammalian response to cellular stresses often involves upregulation of certain stress proteins. This response is usually neither cell nor stress specific and sometimes results in cross-protection to other stresses. Endothelial cell (EC) hypoxia-associated proteins (HAP) are a unique set of stress proteins upregulated by exposure to environmental hypoxia. In the present study, the specificity of stress protein upregulation was assessed and any potential cross-protection was evaluated using DNA strand break analysis. EC cultured in 21% or 3% oxygen were exposed to single and combined cellular stresses (0% oxygen, reoxygenation, glucose deprivation, sodium arsenite, heat, or hydrogen peroxide). Although EC can upregulate various stress proteins, the HAP are specifically upregulated only with hypoxia and offer no cross-protection against other cellular stresses. Moreover, induction of other stress proteins does not alter the induction of the HAP or the effects of hypoxia in cultured EC. Thus EC display a unique specificity in regard to the stimulus for upregulation of stress proteins and are distinct from other cell types thus far examined.
Ion transport, ciliary activity, and mechanosensitivity of sinusal mucosa: an in vitro study
Authors: Leuba D. , De Ribaupierre Y. , Kucera P.
Published: December 2017

The hypothesis that relative movement between respiratory epithelium and surrounding fluid modulates both ciliary activity and mucosal secretion-absorption properties was tested. Fresh human (HM) and bovine sinusal mucosae (BM) were mounted in transparent chambers perfused with defined media at 36 degrees C. The fluid advanced along the ciliated surface at 15-30 microns/s. Transepithelial transport of electrolytes was studied by using voltage-clamp technique. Ciliary beating frequency (CBF) was recorded by using laser reflectometry. Average transmucosal potential difference (apical side negative) and short-circuit current (Isc) were -0.5 mV and 17 microA.cm-2 for HM and -1.4 mV and 28 microA.cm-2 for BM. Average CBF was 900 beats/min. Ionic pathways included basolateral Na,K-ATPase, K+ channels and symports for Na-Cl and Na-glucose, and apical channels for Na+ (absorption) and for Cl- and K+ (secretion). Increase of fluid velocity up to 300 microns/s induced significant increase of both Isc (63%) and CBF (29%). Such adaptations of transepithelial fluid transport and ciliary activity to hydrodynamic conditions might reflect a mechanism of coordination between the secretion of mucus electrolytes and mucociliary clearance.
TNF-alpha-induced inhibition of PC synthesis by human type II pneumocytes is sequentially mediated by PGE2 and NO
Authors: Vara E. , Arias-Diaz J. , Garcia C. , Hernandez J. , Balibrea J. L.
Published: December 2017

Tumor necrosis factor-alpha (TNF-alpha)-induced inhibition of surfactant synthesis participates in the pathogenesis of the acute respiratory distress syndrome. We examined the ability of human type II pneumocytes to produce nitric oxide (NO) in the presence of TNF-alpha as well as the role of NO and prostaglandin (PG) E2 in the transduction of the cytokine signal. Multiple organ donors were used as a source of lung tissue. After 24-h preculture, type II pneumocytes were cultured for 18 h in the presence or absence of additives. The D-[U-14C] glucose incorporation into phosphatidylcholine (PC) was selectively inhibited by TNF-alpha, PGE2, sodium nitroprusside (SNP), or 8-bromoguanosine 3',5'-cyclic monophosphate. The effect of TNF-alpha was attenuated by indomethacin, N omega-nitro-L-arginine methyl ester (NAME), or methylene blue (MB). The effect of PGE2 was attenuated by NAME, while that of SNP was reversed by MB but not by indomethacin. TNF-alpha induced an increase in PGE2 and guanosine 3',5'-cyclic monophosphate cell content and in the NO release to the medium. NAME did not affect PGE2 production, while indomethacin blunted NO generation. Our results suggest that NO generation, secondary to PGE2 production, is responsible for the TNF-alpha-induced inhibition of PC synthesis by human type II pneumocytes.
Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D
Authors: Natarajan V. , Vepa S. , Verma R. S. , Scribner W. M.
Published: December 2017

Oxidant-induced activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) is independent of protein kinase C and calcium. In the present study, the effects of tyrosine kinase and protein tyrosine phosphatase (PTPase) inhibitors on hydrogen peroxide (H2O2)-induced PLD activation and protein tyrosine phosphorylation were examined in BPAEC. Pretreatment of BPAEC with putative tyrosine kinase inhibitors genistein, tyrphostin, and herbimycin attenuated H2O2 (1 mM)-induced PLD activation. The inhibitory effect of the tyrosine kinase inhibitors was highly specific for H2O2-induced modulation and showed no effect on PLD activation mediated by 12-O-tetradecanoylphorbol 13-acetate or bradykinin. Furthermore, addition of H2O2 increased in a time-dependent manner tyrosine phosphorylation of several proteins (17-200 kDa), as determined by immunoblot analysis with antiphosphotyrosine antibodies. H2O2-mediated protein tyrosine phosphorylation preceded PLD activation, and a good correlation was observed on the effect of genistein in H2O2-induced PLD activation and protein tyrosine phosphorylation. Addition of vanadate, a phosphotyrosine phosphatase inhibitor, synergistically increased both PLD activation and protein tyrosine phosphorylation mediated by H2O2. Moreover, vanadate by itself had minimal effect on basal PLD activity in BPAEC; however, at 10 microM vanadate, an increase in protein tyrosine phosphorylation was observed. In addition to vanadate, phenylarsine oxide and diamide potentiated H2O2-induced PLD activation. These results suggest that tyrosine kinase activation may be involved in H2O2-induced PLD activation in vascular endothelial cells.
Altered vasoreactivity in lungs isolated from rats exposed to nitric oxide gas
Authors: Oka M. , Ohnishi M. , Takahashi H. , Soma S. , Hasunuma K. , Sato K. , Kira S.
Published: December 2017

A rebound phenomenon (severe arterial desaturation and pulmonary vasoconstriction) has been observed in some patients with pulmonary hypertension after sudden discontinuation of nitric oxide (NO) inhalation therapy. The mechanism responsible for this phenomenon is unknown. It has recently been reported that NO synthase (NOS) can be inhibited by NO as a negative feedback mechanism. We therefore hypothesized that this rebound phenomenon might be attributable to reduced endogenous NO production due to inhibition of pulmonary endothelial NOS (eNOS) activity by inhaled NO. To test this hypothesis, vasoreactivities were compared in isolated perfused lungs from rats exposed to 40 ppm NO gas and room air for 2 days. The pressor responses to angiotensin II and hypoxia of lungs from rats exposed to NO were markedly potentiated and their depressor response to bradykinin was considerably impaired, whereas that to sodium nitroprusside was preserved, compared with the responses of those exposed to room air. This altered pulmonary vasoreactivity was also induced by lower concentrations of inhaled NO (as low as 1 ppm) and was completely reversed by discontinuation of NO inhalation for several (< 8) hours. The expression of eNOS mRNA by lungs isolated from rats exposed to 40 ppm NO and room air for 2 days did not differ. These results suggest that pulmonary endogenous NO production is reversibly reduced after relatively short-term NO inhalation, which probably inhibits eNOS activity directly. We speculate that reduced production of pulmonary endogenous NO by exogenous (inhaled) NO could be the mechanism responsible for this rebound phenomenon.
Dexamethasone suppresses mucus production and MUC-2 and MUC-5AC gene expression by NCI-H292 cells
Authors: Kai H. , Yoshitake K. , Hisatsune A. , Kido T. , Isohama Y. , Takahama K. , Miyata T.
Published: December 2017

Excessive production of airway mucus is a characteristic feature of many chronic inflammatory lung diseases. Although current pharmacological approaches to excessive mucus production are limited, glucocorticoids appear to be the most effective among a few useful drugs. The exact evidence for the effectiveness of glucocorticoids on mucus production has not been fully elucidated to date. The purpose of this study is to clarify the effect of dexamethasone on mucus production and mucin gene expression in a human pulmonary mucoepidermoid carcinoma cell line (NCI-H292). NCI-H292 cells produced hyaluronidase-resistant high-molecular-weight glycoconjugates (HMWG), which elute in the void volume on Sepharose CL-4B column chromatography. Dexamethasone significantly suppressed the basal production of [3H]glucosamine-or [3H]serine-labeled HMWG in NCI-H292 cells. In Northern blot analysis, dexamethasone attenuated steady-state mRNA levels of MUC-2 and MUC-5AC mucin genes. These data indicate that dexamethasone suppresses the basal production of HMWG and decreases steady-state mRNA levels of mucin genes in airway mucus-producing cancer cells.
Sensitivity of lungs of aging Fischer 344 rats to ozone: assessment by bronchoalveolar lavage
Authors: Vincent R. , Vu D. , Hatch G. , Poon R. , Dreher K. , Guenette J. , Bjarnason S. , Potvin M. , Norwood J. , McMullen E.
Published: December 2017

Biological effects indicators in bronchoalveolar lavage fluid were studied in Fischer 344 rats of different ages after exposure to 0.4-0.8 ppm ozone for periods of 2-6 h on a single day or on 4 consecutive days. The magnitude of alveolar protein transudation induced by ozone was not different between age groups, but the interindividual variability of protein changes was higher in senescent (24-mo-old) rats. By comparison to juvenile (2-mo-old) and adult (9-mo-old) rats, senescent animals had higher increases of interleukin-6 (up to 10-fold higher) and N-acetyl-beta-D-glucosaminidase (NAGA; 2-fold higher) in lung lavage after ozone. Ascorbic acid was lower in lungs of senescent rats (one-half of juvenile values), and acute ozone exposure brought a further decrease in lung ascorbate. Whereas alveolar protein transudation was attenuated after ozone exposure on 4 days, persistent elevation of NAGA in senescent rats suggested only partial adaptation. Injection of endotoxin did not modify the patterns of effects. Incorporation of 18O-ozone into macrophages and surfactant was not different between age groups, indicating that the magnified biological responses in senescent rats were not dominated by differences in internal dose of ozone. The results indicate that senescent rats respond differently than juvenile and adult rats to lung injury.
IL-10 enhances resolution of pulmonary inflammation in vivo by promoting apoptosis of neutrophils
Authors: Cox G.
Published: December 2017

Interleukin-10 (IL-10) has been shown to be protective in models of sepsis. This protection is mediated in part by inhibition of monokine-dependent processes. Because IL-10 can act on other cells to regulate inflammatory events, and because we have previously shown that clearance of inflammation is an active process, we examined whether IL-10 could regulate processes of resolution during pulmonary inflammation induced by lipopolysaccharide (LPS) challenge. Administration of 1 microgram of IL-10 with 6 micrograms LPS intratracheally to rats did not alter the time of onset or the magnitude of the initial response, as assessed by bronchoalveolar lavage (BAL) neutrophilia. However, the extent of the neutrophilia was markedly reduced at 18 h, and longer, after challenge. During ex vivo culture of cells obtained by BAL, neutrophils died by apoptosis and were engulfed by macrophages. Clearance of neutrophils was more rapid in the cultured BAL of rats treated with IL-10. In separate experiments, IL-10 did not reduce survival rates of untreated human neutrophils, but did inhibit LPS-induced increases in survival in a dose-dependent fashion. Thus IL-10 did not modulate the onset of, or peak of, neutrophil accumulation in response to LPS but did promote the clearance of recruited neutrophils in vivo. The mechanism of this anti-inflammatory action may be through the prevention of stimulated increases in neutrophil survival.
Human alveolar macrophages prevent apoptosis in polymorphonuclear leukocytes
Authors: Herlihy J. P. , Vermeulen M. W. , Hales C. A.
Published: December 2017

Polymorphonuclear leukocytes (PMN) are recruited to the lungs to defend against injury and infection. However, PMN undergo apoptosis, thereby losing functional ability within hours, and die with lysis soon thereafter unless they receive specific signals preventing this phenomenon. We hypothesized that alveolar macrophages (AM) could provide these signals. Therefore AM, obtained through bronchoalveolar lavage of healthy volunteers (n = 9), were cultured for 24 h, after which the AM conditioned media (AM-CM) were removed. Freshly isolated PMN, which showed no apoptosis, were suspended in AM-CM, as well as in unconditioned media (UM), and followed over 48 h for apoptosis and survival. In eight of nine patients, AM-CM contained tumor necrosis factor (TNF), which modestly delayed AM apoptosis so that the percentage of PMN apoptotic at 24 h was 77 +/- 6% in AM-CM compared with 91 +/- 2% in UM (P < 0.05). In one patient, urticaria developed early in the lavage, and this subject's AM-CM profoundly prevented apoptosis of PMN (to 10% at 24 h). PMN survival in this patient was similarly enhanced, so that at 48 h of culture it was 60%, compared with 45 +/- 8% in AM-CM and 30 +/- 6% in UM (P < 0.05 UM vs. AM-CM). Granulocyte/macrophage colony-stimulating factor (GM-CSF), in addition to TNF-alpha, partly accounted for this medium's activity. Thus AM can delay apoptosis in PMN through production of TNF-alpha and in some cases by GM-CSF. When activated in vivo by conditions such as an allergic reaction, AM can rapidly and profoundly suppress PMN apoptosis.
Effects of organotypic culture on parasympathetic innervation of guinea pig trachealis
Authors: Canning B. J. , Undem B. J. , Karakousis P. C. , Dey R. D.
Published: December 2017

Nonadrenergic, noncholinergic (NANC) relaxations of airway smooth muscle are thought to be mediated by vasoactive intestinal peptide (VIP) and nitric oxide (NO). Previous studies of the parasympathetic innervation of guinea pig trachealis suggest that the ganglion neurons mediating NANC relaxations but not cholinergic contractions are associated with the esophagus. In this study, the location of the neurons mediating these responses and their neurochemical phenotype was further assessed. Guinea pig tracheas maintained in organotypic culture for 2 days with the adjacent esophagus intact displayed cholinergic contractions and NANC relaxations to electrical field stimulation (EFS) as well as VIP and NO synthase (NOS) nerve fiber densities that were similar to those of control tracheas. By contrast, in tracheas cultured without the esophagus, NANC relaxations to EFS were not observed, and VIP and NOS nerve fiber densities were reduced > 80%. EFS-induced cholinergic contractions were unaffected by esophagus removal. These results provide further evidence that NANC relaxations are mediated by VIP and NO coreleased from noncholinergic parasympathetic nerve endings derived from neurons intrinsic to the esophagus.
Association of ICAM-1 with the cytoskeleton in rat alveolar epithelial cells in primary culture
Authors: Barton W. W. , Wilcoxen S. E. , Christensen P. J. , Paine R.
Published: December 2017

Intercellular adhesion molecule-1 ICAM-1) is a transmembrane adhesion protein that is expressed constitutively on the apical surface of type I cells in vivo and on type II cells in vitro as they spread in culture, assuming type I cell-like characteristics. To investigate the possible interaction of ICAM-1 with the alveolar epithelial cell cytoskeleton, rat type II cells in primary culture were extracted with nonionic detergent, and residual ICAM-1 associated with the cytoskeletal remnants was determined using immunofluorescence microscopy, immunoprecipitation, and cell-based enzyme-linked immunosorbent assay. A large fraction of alveolar epithelial cell ICAM-1 remained associated with the cytoskeleton after detergent extraction, whereas two other transmembrane molecules, transferrin receptor and class II major histocompatibility complex, were completely removed. ICAM-1 was redistributed on the cell surface after the disruption of actin filaments with cytochalasin B, suggesting interaction with the actin cytoskeleton. In contrast, ICAM-1 was completely detergent soluble in rat pulmonary artery endothelial cells, human umbilical vein endothelial cells, and rat alveolar macrophages. The association of ICAM-1 with the alveolar epithelial cell cytoskeleton was not altered after stimulation with inflammatory cytokines. However, detergent resistant ICAM-1 was significantly increased after crosslinking of ICAM-1 on the cell surface, suggesting that this cytoskeletal association may be modulated by interactions of alveolar epithelial cells with inflammatory cells. The association of ICAM-1 with the cytoskeleton in alveolar epithelial cells may provide a fixed intermediary between mobile inflammatory cells and the alveolar surface.
Protein movement across cultured guinea pig trachea: specificity and effect of transcytosis inhibitors
Authors: Deffebach M. E. , Bryan C. J. , Hoy C. M.
Published: December 2017

Airway surface liquid (ASL) is a complex fluid with solutes including electrolytes, lipids, mucins, and proteins. The proximal airways are absorptive for most solutes, including proteins. We investigated the process of protein movement across confluent primary cultures of guinea pig trachea grown on filters using fluorescent-labeled bovine serum albumin (BSA), ovalbumin (OA), and 70-kDa dextran (Dex). We found marked asymmetry of BSA and OA transepithelial flux, with apical-to-basolateral flux (JA-->B) 10 times greater than the opposite direction (JB-->A) for both proteins. The apparent permeability for Dex was the same as that for proteins in the basolateral-to-apical direction and showed no asymmetry. Increasing concentrations of unlabeled BSA, OA, or transferrin inhibited JA-->B for both BSA and OA without affecting Dex movement. Cooling reduced JA-->B for BSA without affecting JB-->A. Monensin and nocodazole each reduced JA-->B for BSA and OA without affecting JB-->A. Monensin eliminated all asymmetry for BSA movement. Brefeldin A did not affect JA-->B for either protein but did increase JB-->A for BSA. Treatment with the protease inhibitors increased JA-->B for BSA. Western immunoblotting demonstrated immunologically intact protein in the downstream compartment. We conclude that there is transcytosis of proteins across cultured trachea epithelium in the apical-to-basolateral direction, which is monensin sensitive, involves microtubules, is not dependent on proteolysis, and is not protein species specific. This process may be important for maintenance of the ASL, and defects in this process may contribute to the abnormally thickened airway secretion seen in airway diseases.
Evidence for functional tachykinin NK1 receptors on human isolated small bronchi
Authors: Naline E. , Molimard M. , Regoli D. , Emonds-Alt X. , Bellamy J. F. , Advenier C.
Published: December 2017

On human small isolated bronchi (diameter < 1 mm), but not on larger bronchi (diameter 3-5 mm), substance P (SP) and specific tachykinin SP-preferring neurokinin (NK1) receptor agonists inverted question mark[beta Ala4, Sar9, Met(O2)11]SP-(4-11), [Sar9, Met(O2)11]SP, [Arg6,Sar9,Met(O2)11]SP-(6-11), and septide; 10(-10) to 10(-6) M] produced a concentration-dependent contraction that occurred at low concentrations (pD2 values of 7.79-8.33) and was characterized by a low intrinsic activity [maximal effect (Emax) of 38-45% of Emax induced by 3 mM acetylcholine, in a noncumulative manner]. Comparison of cumulative and noncumulative concentration-response curves to SP and NK1 receptor agonists suggest rapid receptor desensitization. The SP (10(-8) M)-induced contraction was inhibited by tachykinin NK1 receptor antagonists (rank order of potency: SR-140333 > CP-96,345 > RP-67580) but not by the tachykinin NK2 receptor antagonist SR-48968. Indomethacin (10(-6) M) abolished the SP-induced contraction. Our results suggest that tachykinin NK1 receptors are present on human small bronchi and that their stimulation induces a prostanoid-dependent contraction. The small isolated bronchus is an interesting model of human tissue to test NK1 receptor antagonists.