bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation
Authors: Fagan Karen A. , Tyler Robert C. , Sato Koichi , Fouty Brian W. , Morris Kenneth G. , Huang Paul L. , McMurtry Ivan F. , Rodman David M.
Published: December 2017

Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 ± 65% control, P < 0.02, mean ± SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 ± 1.2 mmHg, P< 0.001) and, to a lesser extent, in iNOS-deficient (37.2 ± 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 ± 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 ± 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.
Differential expression of stress proteins in nonhuman primate lung and conducting airway after ozone exposure
Authors: Wu Reen , Zhao Yu Hua , Plopper Charles G. , Chang Mary Mann-Jong , Chmiel Ken , Cross John J. , Weir Alison , Last Jerold A. , Tarkington Brian
Published: December 2017

The presence of seven stress proteins including various heat shock proteins [27-kDa (HSP27), 60-kDa (HSP60), 70-kDa (HSP70) and its constitutive form HSC70, and 90-kDa (HSP90) HSPs] and two glucose-regulated proteins [75-kDa (GRP75) and 78-kDa (GRP78) GRPs] in ozone-exposed lungs of nonhuman primates and in cultured tracheobronchial epithelial cells was examined immunohistochemically by various monoclonal antibodies. Heat treatment (42°C) resulted in increased HSP70, HSP60, and HSP27 and slightly increased HSC70 and GRP75 but no increase in GRP78 in primary cultures of monkey tracheobronchial epithelial cells. Ozone exposure did not elevate the expression of these HSPs and GRPs. All of these HSPs including HSP90, which was undetectable in vitro, were suppressed in vivo in monkey respiratory epithelial cells after ozone exposure. Both GRP75 and GRP78 were very low in control cells, and ozone exposure in vivo significantly elevated these proteins. These results suggest that the stress mechanism exerted on pulmonary epithelial cells by ozone is quite different from that induced by heat. Furthermore, differences between in vitro and in vivo with regard to activation of HSPs and GRPs suggest a secondary mechanism in vivo, perhaps related to inflammatory response after ozone exposure.
Increased group IV cytosolic phospholipase A2activity in lungs of sheep after smoke inhalation injury
Authors: Fukuda Taeko , Kim Dae Kyong , Chin Mi-Reyoung , Hales Charles A. , Bonventre Joseph V.
Published: December 2017

Increased phospholipase A2(PLA2) activity was measured in cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a cotton smoke toxin. Three peaks of PLA2activity were identified by heparin-Sepharose chromatography. The heparin-nonbinding PLA2activity was twofold higher in the extracts from lungs exposed to smoke than in normal lungs. This activity was identified as the group IV 85-kDa cytosolic PLA2(cPLA2). The activities of the forms of PLA2that bound to heparin did not change after smoke exposure. Those activities showed a pH optimum of 9.0, required a millimolar Ca2+concentration for full activity, and were inhibited by 5 mM dithiothreitol. One activity eluted at an NaCl concentration typical for group Ib and V PLA2and had the expected substrate specificity. The other form of lung PLA2that bound heparin was a group II PLA2. Lung myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA2was identified in sheep neutrophils. With 30 breaths of smoke exposure, there was an increase in cPLA2activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts of protein. In conclusion, smoke induces increases in resident lung cell cPLA2activity that is likely responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.
Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro
Authors: Sato Etsuro , Simpson Keith L. , Grisham Matthew B. , Koyama Sekiya , Robbins Richard A.
Published: December 2017

Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), can nitrate tyrosine residues, resulting in compromised protein function. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that attracts monocytes and has a tyrosine residue critical for function. We hypothesized that peroxynitrite would alter MCP-1 activity. Peroxynitrite attenuated MCP-1-induced monocyte chemotactic activity (MCA) in a dose-dependent manner ( P < 0.05) but did not attenuate leukotriene B4or complement-activated serum MCA. The reducing agents dithionite, deferoxamine, and dithiothreitol reversed the MCA inhibition by peroxynitrite, and exogenous l-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, an NO donor, or superoxide generated by xanthine and xanthine oxidase did not show an inhibitory effect on MCA induced by MCP-1. The peroxynitrite generator 3-morpholinosydnonimine caused a concentration-dependent inhibition of MCA by MCP-1. Peroxynitrite reduced MCP-1 binding to monocytes and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite, with subsequent inhibition of MCP-1 binding to monocytes, and suggest that peroxynitrite may play a role in regulation of MCP-1-induced monocyte chemotaxis.
Participation of urokinase-type plasminogen activator receptor in the clearance of fibrin from the lung
Authors: Hattori Noboru , Sisson Thomas H. , Xu Yin , Desai Tushar J. , Simon Richard H.
Published: December 2017

In vitro studies have demonstrated that the binding of urokinase-type plasminogen activator (uPA) to its cell surface receptor (uPAR) greatly accelerates plasminogen activation. However, the role of uPAR in clearing abnormal fibrin deposits from the lung is uncertain. Knowing that uPA binding to uPAR is species specific, we used adenoviral vectors to transfer human or murine uPA genes into human or mouse epithelial cells in vitro and to mouse lungs in vivo. By measuring degradation of fluorescein-labeled fibrin, we found that uPA lysed fibrin matrices more efficiently when expressed in cells of the same species. A monoclonal antibody that blocks the binding of human uPA to human uPAR suppressed fibrin degradation by human cells expressing human uPA but not murine uPA. Importantly, 3 days after intratracheal delivery of the vectors, mice receiving murine uPA transgenes degraded fibrin matrices formed within their air spaces more efficiently than animals transduced with human uPA genes. These results show that uPA bound to uPAR increases the efficiency of fibrinolysis on epithelial cell surfaces in a biologically relevant fashion.
Chronic hypoxia abolished the postnatal increase in carotid body type I cell sensitivity to hypoxia
Authors: Sterni L. M. , Bamford O. S. , Wasicko M. J. , Carroll J. L.
Published: December 2017

The O2sensitivity of carotid chemoreceptor type I cells is low just after birth and increases with postnatal age. Chronic hypoxia during postnatal maturation blunts ventilatory and carotid chemoreceptor neural responses to hypoxia, but the mechanism remains unknown. We tested the hypothesis that chronic hypoxia from birth impairs the postnatal increase in type I cell O2sensitivity by comparing intracellular Ca2+concentration ([Ca2+]i) responses to graded hypoxia in type I cell clusters from rats born and reared in room air or 12% O2. [Ca2+]ilevels at 0, 1, 5, and 21% O2, as well as with 40 mM K+, were measured at 3, 11, and 18 days of age with use of fura 2 in freshly isolated cells. The [Ca2+]iresponse to elevated CO2/low pH was measured at 11 days. Chronic hypoxia from birth abolished the normal developmental increase in the type I cell [Ca2+]iresponse to hypoxia. Effects of chronic hypoxia on development of [Ca2+]iresponses to elevated K+were small, and [Ca2+]iresponses to CO2remained unaffected. Impairment of type I cell maturation was partially reversible on return to normoxic conditions. These results indicate that chronic hypoxia severely impairs the postnatal development of carotid chemoreceptor O2sensitivity at the cellular level and leaves responses to other stimuli largely intact.
Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fosand apoptosis
Authors: Zanella Christine L. , Timblin Cynthia R. , Cummins Andrew , Jung Michael , Goldberg Jonathan , Raabe Rachel , Tritton Thomas R. , Mossman Brooke T.
Published: December 2017

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c- fos/c- jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR ( P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c- fos but not of c- jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c- fos and the development of apoptosis. The ability to block asbestos-induced elevations in c- fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.
cAMP stimulates Na+transport in rat fetal pneumocyte: involvement of a PTK- but not a PKA-dependent pathway
Authors: Niisato Naomi , Ito Yasushi , Marunaka Yoshinori
Published: December 2017

To study a cAMP-mediated signaling pathway in the regulation of amiloride-sensitive Na+transport in rat fetal distal lung epithelial cells, we measured an amiloride-sensitive short-circuit current (Na+transport). Forskolin, which increases the cytosolic cAMP concentration, stimulated the Na+transport. Forskolin also activated cAMP-dependent protein kinase (PKA). A β-adrenergic agonist and cAMP mimicked the forskolin action. PKA inhibitors KT-5720, H-8, and myristoylated PKA-inhibitory peptide amide-(14—22) did not influence the forskolin action. These results suggest that forskolin stimulates Na+transport through a PKA-independent pathway. Furthermore, forskolin increased tyrosine phosphorylation of ∼70- to 80-, ∼97-, and ∼110- to 120-kDa proteins. Protein tyrosine kinase (PTK) inhibitors (tyrphostin A23 and genistein) abolished the forskolin action. Moreover, 5-nitro-2-(3-phenylpropylamino)benzoate (a Cl−-channel blocker) prevented the stimulatory action of forskolin on Na+transport via abolishment of the forskolin-induced cell shrinkage and tyrosine phosphorylation. Based on these results, we conclude that forskolin (and cAMP) stimulates Na+transport in a PTK-dependent but not a PKA-dependent pathway by causing cell shrinkage, which activates PTK in rat fetal distal lung epithelial cells.
Augmentation of eosinophil degranulation and LTC4secretion by integrin-mediated endothelial cell adhesion
Authors: Muñoz Nilda M. , Hamann Kimm J. , Rabe Klaus F. , Sano Hiroyuki , Zhu Xiangdong , Leff Alan R.
Published: December 2017

We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) C4and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 ± 15.3 (BSA control) to 778 ± 225 ng/106cells for eosinophils exposed to interleukin-1α-treated HUVECs ( P < 0.05) and also caused a twofold increase in stimulated LTC4secretion ( P < 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of LTC4from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to α4- or β2-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and LTC4and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion.
Hydrogen peroxide decreases Ca2+sensitivity in airway smooth muscle by inhibiting rMLC phosphorylation
Authors: Lorenz Robert R. , Warner David O. , Jones Keith A.
Published: December 2017

The purpose of this study was to determine the mechanism by which hydrogen peroxide (H2O2), an important inflammatory mediator, relaxes canine tracheal smooth muscle (CTSM). H2O2caused concentration-dependent relaxations of CTSM strips contracted with ACh or isotonic KCl [EC50of 0.24 ± 0.04 (SE) and 0.23 ± 0.04 mM, respectively]. Indomethacin (10 μM) decreased the sensitivity of both KCl- and ACh-contracted strips to H2O2. H2O2increased intracellular cAMP levels, an increase that was abolished by indomethacin. H2O2did not affect intracellular cGMP levels. In strips treated with indomethacin and contracted with ACh or isotonic KCl, H2O2-evoked relaxations were accompanied by increases in intracellular Ca2+concentration and decreases in regulatory myosin light chain phosphorylation. We conclude that H2O2decreases Ca2+sensitivity in CTSM by decreasing regulatory myosin light chain phosphorylation through inhibition of myosin light chain kinase and/or activation of smooth muscle protein phosphatases.
Bombesin-like peptides and receptors in normal fetal baboon lung: roles in lung growth and maturation
Authors: Emanuel R. L. , Torday J. S. , Mu Q. , Asokananthan N. , Sikorski K. A. , Sunday M. E.
Published: December 2017

Previously, we have shown that bombesin-like peptide (BLP) promotes fetal lung development in rodents and humans but mediates postnatal lung injury in hyperoxic baboons. The present study analyzed the normal ontogeny of BLP and BLP receptors as well as the effects of BLP on cultured normal fetal baboon lungs. Transcripts encoding gastrin-releasing peptide (GRP), a pulmonary BLP, were detectable on gestational day 60(ED60), peaked on approximately ED90, and then declined before term (ED180). Numbers of BLP-immunopositive neuroendocrine cells peaked from ED80 to ED125 and declined by ED160, preceding GRP-receptor mRNAs detected from ED125 until birth. BLP (0.1–10 nM) stimulated type II cell differentiation in organ cultures as assessed by [3H]choline incorporation into surfactant phospholipids, electron microscopy, and increased surfactant protein (SP) A- and/or SP-C-immunopositive cells and SP-A mRNA. BLP also induced neuroendocrine differentiation on ED60. Cell proliferation was induced by GRP, peaking on ED90. Similarly, blocking BLP degradation stimulated lung growth and maturation, which was completely reversed by a BLP-specific antagonist. The dissociation between GRP and GRP-receptor gene expression during ontogeny suggests that novel BLP receptors and/or peptides might be implicated in these responses.
Attenuation of acute lung injury in transgenic mice expressing human transforming growth factor-α
Authors: Hardie William D. , Prows Daniel R. , Leikauf George D. , Korfhagen Thomas R.
Published: December 2017

Transforming growth factor-α (TGF-α) is produced in the lung in experimental and human lung diseases; however, its physiological actions after lung injury are not understood. To determine the influence of TGF-α on acute lung injury, transgenic mouse lines expressing differing levels of human TGF-α in distal pulmonary epithelial cells under control of the surfactant protein C gene promoter were generated. TGF-α transgenic and nontransgenic control mice were exposed to polytetrafluoroethylene (PTFE; Teflon) fumes to induce acute lung injury. Length of survival of four separate TGF-α transgenic mouse lines was significantly longer than that of nontransgenic control mice, and survival correlated with the levels of TGF-α expression in the lung. The transgenic line expressing the highest level of TGF-α (line 28) and nontransgenic control mice were then compared at time intervals of 2, 4, and 6 h of PTFE exposure for differences in pulmonary function, lung histology, bronchoalveolar lavage fluid protein and cell differential, and lung homogenate proinflammatory cytokines. Line 28 TGF-α transgenic mice demonstrated reduced histological changes, decreased bronchoalveolar lavage fluid total protein and neutrophils, and delayed alterations in pulmonary function measures of airway obstruction compared with those in nontransgenic control mice. Both line 28 and nontransgenic control mice had similar increases in interleukin-1β protein levels in lung homogenates. In contrast, interleukin-6 and macrophage inflammatory protein-2 levels were significantly reduced in line 28 transgenic mice compared with those in nontransgenic control mice. In the transgenic mouse model, TGF-α protects against PTFE-induced acute lung injury, at least in part, by attenuating the inflammatory response.
Hyperthermia enhances cytomegalovirus regulation of HIV-1 and TNF-α gene expression
Authors: Iwamoto Gary K. , Ainsworth Audrey M. , Moseley Pope L.
Published: December 2017

The immediate-early (IE) genes of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Reactivation of human immunodeficiency virus (HIV-1) may be stimulated by a variety of factors including other viruses and inflammatory cytokines. These studies examine the role of hyperthermia and CMV in the regulation of HIV-1 and tumor necrosis factor (TNF)-α. THP-1 cells were transfected with the CMV IE genes. HIV-1 and TNF-α transcription were assessed with chloramphenicol acetyltransferase promoter constructs. Hyperthermia sufficient to stimulate production of heat shock proteins was used to stimulate the cells. Hyperthermia significantly enhances the effect of CMV IE gene products on the expression of HIV-1 and TNF-α. The increases in HIV-1 transcription appear to be in part due to increases in TNF-α. Heat shock proteins induced by hyperthermia may play an important role in the viral regulation of monocytic function by CMV.
Interleukin-11 attenuates pulmonary inflammation and vasomotor dysfunction in endotoxin-induced lung injury
Authors: Sheridan Brett C. , Dinarello Charles A. , Meldrum Daniel R. , Fullerton David A. , Selzman Craig H. , McIntyre Robert C.
Published: December 2017

Interleukin (IL)-11, like other members of the gp130 receptor class, possesses anti-inflammatory properties. We hypothesized that IL-11 pretreatment would attenuate endotoxin [lipopolysaccharide (LPS)]-induced lung inflammation and diminish injury to endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation that require cGMP in Sprague-Dawley rats. LPS (20 mg/kg ip) increased lung tumor necrosis factor (TNF)-α compared with the saline control (0.7 ± 0.15 ng/g lung wet wt for control vs. 3.5 ± 0.09 ng/g lung wet wt for LPS; P < 0.05). IL-11 (200 mg/kg ip) injected 10 min before LPS administration attenuated the LPS-induced lung TNF-α levels (1.6 ± 0.91 ng/g lung wet wt; P < 0.05 vs. LPS). IL-11 also diminished LPS-induced lung neutrophil sequestration as assessed by myeloperoxidase units (2.1 ± 0.25 U/g lung wet wt for saline and 15.6 ± 2.02 U/g lung wet wt for LPS vs. 7.07 ± 1.65 U/g lung wet wt for LPS plus IL-11; P < 0.05). Similarly, TNF-α binding protein (175 mg/kg) attenuated LPS-induced myeloperoxidase activity (6.04 ± 0.14 U/g lung wet wt; P < 0.05). Both IL-11 and TNF-α binding protein similarly attenuated LPS-induced endothelium-dependent vasomotor dysfunction with improved relaxation responses to 10−7and 10−6M acetylcholine and A-23187 in phenylephrine-preconstricted isolated pulmonary artery rings ( P < 0.05 vs. LPS). Endothelium-independent relaxation responses to sodium nitroprusside were also improved after LPS at 10−6M ( P < 0.05 vs. LPS). Moreover, IL-11 decreased endotoxin-induced mortality in CF1 mice from 90 to 50% ( P ≤ 0.05 vs. LPS). Therefore, IL-11 prevents LPS-induced lung TNF-α production, neutrophil sequestration, and pulmonary vasomotor dysfunction. We conclude that IL-11 possesses anti-inflammatory activity that protects against LPS-induced lung injury and lethality.
Interleukin-10 inhibits pulmonary NF-κB activation and lung injury induced by hepatic ischemia-reperfusion
Authors: Yoshidome Hiroyuki , Kato Atsushi , Edwards Michael J. , Lentsch Alex B.
Published: December 2017

Hepatic ischemia and reperfusion cause local and remote organ injury. This injury culminates from an integrated cascade of proinflammatory cytokines, chemokines, and adhesion molecules, many of which are regulated by the transcription factor nuclear factor-κB (NF-κB). The anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to have inhibitory effects on NF-κB. The objective of the current study was to determine whether IL-10 could suppress pulmonary NF-κB activation and ensuing lung injury induced by hepatic ischemia-reperfusion. C57BL/6 mice underwent partial hepatic ischemia with or without intravenous administration of IL-10. Hepatic ischemia-reperfusion resulted in pulmonary NF-κB activation, increased mRNA expression of tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-2 (MIP-2), as well as increased pulmonary neutrophil accumulation and lung edema. Administration of IL-10 suppressed lung NF-κB activation, reduced TNF-α and MIP-2 mRNA expression, and decreased pulmonary neutrophil recruitment and lung injury. The data suggest that IL-10 protects against hepatic ischemia and reperfusion-induced lung injury by inhibiting lung NF-κB activation and the resulting pulmonary production of proinflammatory mediators.
Production of superoxide and TNF-α from alveolar macrophages is blunted by glycine
Authors: Wheeler Michael D. , Thurman Ronald G.
Published: December 2017

Glycine blunts lipopolysaccharide (LPS)-induced increases in intracellular calcium concentration ([Ca2+]i) and tumor necrosis factor-α (TNF-α) production by Kupffer cells through a glycine-gated chloride channel. Alveolar macrophages, which have a similar origin as Kupffer cells, play a significant role in the pathogenesis of several lung diseases including asthma, endotoxemia, and acute inflammation due to inhaled bacterial particles and dusts. Therefore, studies were designed here to test the hypothesis that alveolar macrophages could be inactivated by glycine via a glycine-gated chloride channel. The ability of glycine to prevent endotoxin [lipopolysaccharide (LPS)]-induced increases in [Ca2+]iand subsequent production of superoxide and TNF-α in alveolar macrophages was examined. LPS caused a transient increase in intracellular calcium to nearly 200 nM, with EC50values slightly greater than 25 ng/ml. Glycine, in a dose-dependent manner, blunted the increase in [Ca2+]i, with an IC50less than 100 μM. Like the glycine-gated chloride channel in the central nervous system, the effects of glycine on [Ca2+]iwere both strychnine sensitive and chloride dependent. Glycine also caused a dose-dependent influx of radiolabeled chloride with EC50values near 10 μM, a phenomenon which was also inhibited by strychnine (1 μM). LPS-induced superoxide production was also blunted in a dose-dependent manner by glycine and was reduced ∼50% with 10 μM glycine. Moreover, TNF-α production was also inhibited by glycine and also required nearly 10 μM glycine for half-inhibition. These data provide strong pharmacological evidence that alveolar macrophages contain glycine-gated chloride channels and that their activation is protective against the LPS-induced increase in [Ca2+]iand subsequent production of toxic radicals and cytokines.
Effects of aqueous extracts of PM10filters from the Utah Valley on human airway epithelial cells
Authors: Frampton Mark W. , Ghio Andrew J. , Samet James M. , Carson Johnny L. , Carter Jacqueline D. , Devlin Robert B.
Published: December 2017

We hypothesized that the reduction in hospital respiratory admissions in the Utah Valley during closure of a local steel mill in 1986–1987 was attributable in part to decreased toxicity of ambient air particles. Sampling filters for particulate matter < 10 μm (PM10) were obtained from a Utah Valley monitoring station for the year before ( year 1), during ( year 2), and after ( year 3) the steel mill closure. Aqueous extracts of the filters were analyzed for metal content and oxidant production and added to cultures of human respiratory epithelial (BEAS-2B) cells for 2 or 24 h. Year 2 dust contained the lowest concentrations of soluble iron, copper, and zinc and showed the least oxidant generation. Only dust from year 3 caused cytotoxicity (by microscopy and lactate dehydrogenase release) at 500 μg/ml. Year 1 and year 3, but not year 2, dust induced expression of interleukin-6 and -8 in a dose-response fashion. The effects of ambient air particles on human respiratory epithelial cells vary significantly with time and metal concentrations.
Mechanical strain modulates maximal phosphatidylinositol turnover in airway smooth muscle
Authors: An Steven S. , Hai Chi-Ming
Published: December 2017

Mechanical strain regulates the maximal level of myosin light chain phosphorylation mediated by muscarinic activation in airway smooth muscle. Accordingly, we tested the hypothesis that mechanical strain regulates maximal phosphatidylinositol (PI) turnover ( V max) coupled to muscarinic receptors in bovine tracheal smooth muscle. We found that PI turnover was not significantly length dependent in unstimulated tissues. However, carbachol-induced PI turnover was linearly dependent on muscle length at both 1 and 100 μM. The observed linear length dependence of PI turnover at maximal carbachol concentration (100 μM) suggests that mechanical strain regulates V max. When carbachol concentration-PI turnover relationships were measured at optimal length and at 20% optimal length, the results could be explained by changes in V max alone. To determine whether the length-dependent step is upstream from heterotrimeric G proteins, we investigated the length dependence of fluoroaluminate-induced PI turnover. The results indicate that fluoroaluminate-induced PI turnover remained significantly length dependent at maximal concentration. These findings together suggest that regulating functional units of G proteins and/or phospholipase C enzymes may be the primary mechanism of mechanosensitive modulation in airway smooth muscle.
Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease
Authors: Rahman Irfan , MacNee William
Published: December 2017

Glutathione (GSH), a ubiquitous tripeptide thiol, is a vital intra- and extracellular protective antioxidant in the lungs. The rate-limiting enzyme in GSH synthesis is γ-glutamylcysteine synthetase (γ-GCS). The promoter (5′-flanking) region of the human γ-GCS heavy and light subunits are regulated by activator protein-1 and antioxidant response elements. Both GSH and γ -GCS expression are modulated by oxidants, phenolic antioxidants, and inflammatory and anti-inflammatory agents in lung cells. γ-GCS is regulated at both the transcriptional and posttranscriptional levels. GSH plays a key role in maintaining oxidant-induced lung epithelial cell function and also in the control of proinflammatory processes. Alterations in alveolar and lung GSH metabolism are widely recognized as a central feature of many inflammatory lung diseases including chronic obstructive pulmonary disease (COPD). Cigarette smoking, the major factor in the pathogenesis of COPD, increases GSH in the lung epithelial lining fluid of chronic smokers, whereas in acute smoking, the levels are depleted. These changes in GSH may result from altered gene expression of γ -GCS in the lungs. The mechanism of regulation of GSH in the epithelial lining fluid in the lungs of smokers and patients with COPD is not known. Knowledge of the mechanisms of GSH regulation in the lungs could lead to the development of novel therapies based on the pharmacological or genetic manipulation of the production of this important antioxidant in lung inflammation and injury. This review outlines 1) the regulation of cellular GSH levels and γ -GCS expression under oxidative stress and 2) the evidence for lung oxidant stress and the potential role of GSH in the pathogenesis of COPD.
Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs
Authors: Tan Rosemarie C. , Ikegami Machiko , Jobe Alan H. , Yao Li Yuan , Possmayer Fred , Ballard Philip L.
Published: December 2017

Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1–4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h (≥2-fold); mRNA levels in fetuses delivered 1–3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational.
Smoke extract stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activities
Authors: Sato Etsuro , Koyama Sekiya , Takamizawa Akemi , Masubuchi Takeshi , Kubo Keishi , Robbins Richard A. , Nagai Sonoko , Izumi Takateru
Published: December 2017

Accumulation of monocytes and neutrophils and fibrous distortion of the airway are characteristics of airway disease secondary to smoking. The presence of inflammatory cells and fibrosis correlate, and, therefore, we postulated that lung fibroblasts might release chemotactic activity for neutrophils and monocytes in response to smoke extract. To test this hypothesis, human fetal lung (HFL1) fibroblasts were cultured, and the supernatant fluid was evaluated for neutrophil (NCA) and monocyte (MCA) chemotactic activities with a blind well chamber technique. HFL1 fibroblasts released chemotactic activity in response to smoke extract in a dose- and time-dependent manner ( P < 0.05). Checkerboard analysis showed that the activity was predominantly chemotactic. Partial characterization of the released chemotactic activity revealed that the activity was partly heat labile, trypsin sensitive, and ethyl acetate extractable. Lipoxygenase inhibitors and cycloheximide inhibited the release of both NCA and MCA. Molecular-sieve chromatography revealed that NCA and MCA were heterogeneous. NCA was inhibited by anti-human interleukin (IL)-8 and anti-granulocyte colony-stimulating factor antibodies and a leukotriene (LT) B4-receptor antagonist. Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) and anti-monocyte chemoattractant protein (MCP)-1 antibodies and an LTB4-receptor antagonist inhibited MCA. Immunoreactive IL-8, granulocyte colony-stimulating factor, GM-CSF, and MCP-1 significantly increased in culture supernatant fluid in response to smoke extract. Finally, smoke extract augmented the expression of mRNAs of IL-8, GM-CSF, and MCP-1. These data demonstrate that lung fibroblasts release NCA and MCA in response to smoke extract and suggest that lung fibroblasts may modulate the inflammatory cell recruitment into the lung.
SP-B refining of pulmonary surfactant phospholipid films
Authors: Nag Kaushik , Munro James G. , Inchley Kevin , Schürch Samuel , Petersen Nils O. , Possmayer Fred
Published: December 2017

Pulmonary surfactant stabilizes the alveoli by lining the air-fluid interface with films that reduce surface tension to near 0 mN/m (γmin). Surfactant protein B (SP-B) enhances the surface activity of surfactant phospholipids. A captive bubble tensiometer (CBT) was used to study the properties of adsorbed films of dipalmitoylphosphatidylcholine (DPPC) with acidic 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (POPG) or neutral 1-palmitoyl-2-oleoyl- sn-glycerol-3-phosphocholine with (7:3) and without 1% dimeric SP-B. SP-B enhanced the adsorption rate of DPPC-containing neutral or acidic lipid suspensions (1 mg/ml) to a similar extent. Quasi-static cycling of these films revealed that SP-B significantly decreased the film area reduction required to reach γminfor the acidic but not for the neutral system. The results obtained with DPPC-phosphatidylglycerol (PG)-SP-B were consistent with selective DPPC adsorption into the surface monolayer during film formation. Film area reduction required to reach γminwith this system (with and without calcium) approached that of pure DPPC, suggesting selective DPPC insertion and PG squeeze-out. Dynamic cycling of such films showed that larger film area reductions were required to reach γminfor the neutral than for acidic system, even after 20 cycles. Fluorescence microscopy of solvent-spread DPPC-POPG-SP-B planar films revealed highly condensed structures at ∼25 mN/m, although no specific PG phase-segregated structures could be identified. The study suggests that specific interactions of SP-B with acidic phospholipids of surfactant may be involved in the generation and maintenance of DPPC-rich films in the alveoli.
Inhaled NO reaches distal vasculatures to inhibit endothelium- but not leukocyte-dependent cell adhesion
Authors: Fox-Robichaud Alison , Payne Derrice , Kubes Paul
Published: December 2017

Nitric oxide (NO), in addition to being a potent vasodilator, also prevents leukocyte adhesion in the microvasculature. Based on the antiadhesive properties of NO and work suggesting that NO is transported by proteins in the circulation, we tested the possibility that inhaled NO could impart antiadhesive effects in peripheral microvessels. We also determined the underlying mechanisms of actions. Three well-established models that induce local microvascular changes (either endothelium or leukocyte) were used. Hydrogen peroxide (H2O2; 100 μM) was superfused onto the cat mesentery to induce an endothelium-derived, P-selectin- and platelet-activating factor-dependent, oxidant-dependent leukocyte recruitment. In a second series of experiments, the cat mesentery was superfused with histamine (100 μM) to induce rapid endothelium-derived, P-selectin- and platelet-activating factor-dependent, oxidant-independent leukocyte recruitment. Finally, in a third series of experiments to target the leukocyte (but not the endothelium) directly in the periphery, the chemotactic molecule leukotriene B4(20 nM) was superfused onto the cat mesentery. The above experiments were performed with and without cats breathing NO (80 parts/million). Intravital microscopy was used to visualize the mesenteric microcirculation. Inhaled NO reduced the increased leukocyte rolling and adhesion associated with H2O2superfusion of the feline mesentery via a cGMP-dependent mechanism. In contrast, inhaled NO had no effect on the histamine-induced increase in leukocyte rolling flux but partially inhibited the subsequent adhesion. The leukocyte chemotactic mediator leukotriene B4induced a significant increase in leukocyte adhesion, but NO inhalation did not impair this chemotactically induced leukocyte recruitment. These data suggest that inhaled NO can reach the endothelium in the distal microvasculature and alter the response to an oxidative and a nonoxidative activator of endothelium but imparts no antiadhesive effect directly on circulating leukocytes.
Protein kinase C is involved in enhanced airway smooth muscle cell growth in hyperresponsive rats
Authors: Zacour M. E. , Martin J. G.
Published: December 2017

Fischer rat airway smooth muscle (ASM) models two potential risk factors for asthma: hyperresponsiveness to contractile agonists and to growth stimuli. The aim of this study was to identify the mechanisms responsible for enhanced ASM mitogenic response in Fischer rats compared with the control Lewis strain. The enhanced Fischer ASM cell growth response to fetal bovine serum (FBS) could not be accounted for by phospholipase C, mitogen-activated protein kinases, or tyrosine kinase activities as assessed by pharmacological inhibition and Western blotting. In contrast, depletion of phorbol ester-sensitive isoforms of the serine/threonine kinase protein kinase C (PKC) removed the difference in growth response between the rat strains. Additionally, FBS selectively induced serine/threonine phosphorylation of a 115-kDa protein in Fischer ASM cells. Enhanced activation of PKC-βI and decreased activation of PKC-δ in Fischer compared with Lewis cells following FBS stimulation were suggested by Western blotting of membrane and cytosolic fractions. The data are consistent with a role for PKC in the enhanced ASM cell growth of hyperresponsive rats.
Influence of growth hormone on the lung growth response to tracheal obstruction in fetal sheep
Authors: Nardo L. , Young I. R. , Hooper S. B.
Published: December 2017

Obstructing the fetal trachea is a potent stimulus for fetal lung growth, but little is known about the factors that regulate this process. Our aim was to determine the role of growth hormone (GH) in regulating the increase in lung growth induced by obstruction of the trachea in fetal sheep. Twenty chronically catheterized fetal sheep, nine of which were hypophysectomized, were divided into four experimental groups: 1) control group ( n = 4), 2) a group in which the fetal trachea was obstructed for 3 days (3-day obstructed; n = 6), 3) a 3-day obstructed group in which the pituitary was removed [hypophysectomized (HX)] and the fetus was given maintenance infusions of ACTH, thyroxine, and human GH (hGH; HX hGH 3-day obstructed; n = 5), and 4) a HX 3-day obstructed group in which the fetus was given maintenance infusions of ACTH and thyroxine ( n = 5). Tracheal obstruction significantly increased fetal lung liquid volumes from 37.2 ± 3.2 ml/kg in control fetuses to 75.6 ± 9.0 ml/kg in 3-day obstructed fetuses, and the presence or absence of GH did not affect this increase. Similarly, the presence or absence of GH did not affect the increase in lung weight or protein content induced by 3 days of tracheal obstruction. However, in the absence of GH, 3 days of tracheal obstruction failed to increase total lung DNA content above unobstructed control values (107.9 ± 5.3 and 94.1 ± 7.0 mg/kg for control and HX 3-day obstructed groups, respectively). In contrast, 3 days of tracheal obstruction increased total lung DNA content to a similar extent in fetuses with an intact pituitary and HX fetuses that received GH replacement (126.0 ± 4.4 and 126.7 ± 4.0 mg/kg for 3-day obstructed and HX hGH 3-day obstructed groups, respectively). These data indicate that the absence of GH either abolishes or delays the acceleration in cell division caused by an increase in fetal lung expansion.
Transcriptional regulation and structural organization of the human cytosolic phospholipase A2gene
Authors: Dolan-O'Keefe Maureen , Chow Virginia , Monnier Joan , Visner Gary A. , Nick Harry S.
Published: December 2017

Cytokines are established regulators of the arachidonic acid cascade in lung cells. The levels of various arachidonic metabolites distinguish the normal and pathogenic states of the human lung. Arachidonyl-selective cytosolic phospholipase A2(cPLA2) is ubiquitously present in human lung and is most likely the rate-limiting step in eicosanoid generation. We therefore studied the regulation of this pivotal gene in human lung fibroblasts and epithelial cells by proinflammatory cytokines. We demonstrate a dose- and time-dependent induction of human cPLA2mRNA by interleukin-1β, tumor necrosis factor-α, and interferon-γ as well as the abrogation of this induction by glucocorticoids. Nuclear runoff studies demonstrate that de novo transcription of the cPLA2gene is required for cytokine induction. We have characterized the human cPLA2gene, which is encoded by 18 exons and spans in excess of 137 kb. Deletion analysis of a 3.4-kb fragment of the human promoter identified two regions responsible for basal expression of the cPLA2gene. Conversely, a CA-dinucleotide repeat in the proximal promoter appears to repress overall promoter activity. Understanding the molecular mechanisms associated with cytokine-dependent expression of the cPLA2gene should provide further insight into regulating the level of proinflammatory mediators in pulmonary diseases.
Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells
Authors: Järvinen Kristiina , Pietarinen-Runtti Petra , Linnainmaa Kaija , Raivio Kari O. , Krejsa Cecile M. , Kavanagh Terrance , Kinnula Vuokko L.
Published: December 2017

The development of drug resistance of tumors is multifactorial and still poorly understood. Some cytotoxic drugs generate free radicals, and, therefore, antioxidant enzymes may contribute to drug resistance. We investigated the levels of manganese superoxide dismutase (Mn SOD), its inducibility, and its protective role against tumor necrosis factor-α and cytotoxic drugs (cisplatin, epirubicin, methotrexate, and vindesin) in human pleural mesothelioma (M14K) and pulmonary adenocarcinoma (A549) cells. We also studied other major antioxidant mechanisms in relation to oxidant and drug resistance of these cells. A549 cells were more resistant than M14K cells toward both oxidants (hydrogen peroxide and menadione) and all the cytotoxic drugs tested. M14K cells contained higher basal Mn SOD activity than A549 cells (28.3 ± 3.4 vs. 1.8 ± 0.3 U/mg protein), and Mn SOD activity was significantly induced by tumor necrosis factor-α only in A549 cells (+524%), but the induction did not offer any protection during subsequent oxidant or drug exposure. Mn SOD was not induced significantly in either of these cell lines by any of the cytotoxic drugs (0.007–2 μM, 48 h) tested when assessed by Northern blotting, Western blotting, or specific activity. A549 cells contained higher catalase activity than M14K cells (7.6 ± 1.3 vs. 3.6 ± 0.5 nmol O2⋅ min−1⋅ mg protein−1). They also contained twofold higher levels of glutathione and higher immunoreactivity of the heavy subunit of γ-glutamylcysteine synthetase than M14K cells. Experiments with inhibitors of γ-glutamylcysteine synthetase and catalase supported our conclusion that mechanisms associated with glutathione contribute to the drug resistance of these cells.
Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages
Authors: Spech Robert W. , Wisniowski Paul , Kachel Diane L. , Wright Jo Rae , Martin William J.
Published: December 2017

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a51Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 ± 2.5, whereas the addition of rat SP-A (5 μg/ml) significantly reduced the cytotoxic index to 16.6 ± 1.2 ( P < 0.001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody ord-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.
Neonatal dexamethasone treatment increases the risk for pulmonary hypertension in adult rats
Authors: le Cras Timothy D. , Markham Neil E. , Morris Kenneth G. , Ahrens Charles R. , McMurtry Ivan F. , Abman Steven H.
Published: December 2017

Dexamethasone (Dex) treatment during a critical period of lung development causes lung hypoplasia in infant rats. However, the effects of Dex on the pulmonary circulation are unknown. To determine whether Dex increases the risk for development of pulmonary hypertension, we treated newborn Sprague-Dawley rats with Dex (0.25 μg/day, days 3– 13). Litters were divided equally between Dex-treated and vehicle control (ethanol) rats. Rats were raised in either room air until 10 wk of age (normoxic groups) or room air until 7 wk of age and then in a hypoxia chamber (inspired O2 fraction = 0.10; hypoxic groups) for 3 wk to induce pulmonary hypertension. Compared with vehicle control rats, Dex treatment of neonatal rats reduced alveolarization (by 42%; P< 0.05) and barium-filled pulmonary artery counts (by 37%; P< 0.05) in 10-wk-old adults. Pulmonary arterial pressure and the ratio of right ventricle to left ventricle plus septum weights (RV/LV+S) were higher in 10-wk-old Dex-treated normoxic rats compared with those in normoxic control rats (by 16 and 16% respectively; P < 0.05). Small pulmonary arteries of adult normoxic Dex-treated rats showed increased vessel wall thickness compared with that in control rats (by 15%; P < 0.05). After 3 wk of hypoxia, RV/LV+S values were 36% higher in rats treated with Dex in the neonatal period compared with those in hypoxic control rats ( P < 0.05). RV/LV+S was 42% higher in hypoxic control rats compared with those in normoxic control rats ( P < 0.05). We conclude that Dex treatment of neonatal rats caused sustained lung hypoplasia and increased pulmonary arterial pressures and augmented the severity of hypoxia-induced pulmonary hypertension in adult rats.