bluuebunny/crossref_metadata_2025 huggingface dataset:
Antisense inhibition of 85-kDa cPLA2 blocks arachidonic acid release from airway epithelial cells
Authors: Wu T. , Levine S. J. , Cowan M. , Logun C. , Angus C. W. , Shelhamer J. H.
Published: December 2017

Inflammatory cytokines play a critical role in the initiation and perpetuation of inflammation. Several cytokines are known to increase the production of arachidonic acid (AA) metabolites, which may mediate cytokine-induced acute and chronic inflammation. Although cytokines upregulate phospholipase A2 (PLA2) in several target cells, the contribution of individual PLA2 to cytokine-induced AA release and eicosanoid production remains unclear because of the existence of various forms of cellular PLA2. To examine the role of 85-kDa cytosolic PLA2 (cPLA2) in cytokine-induced AA release, a system was developed to inhibit the expression of cPLA2 in a human bronchial epithelial cell line (BEAS-2B cells) by antisense RNA. Cells stably expressing antisense cPLA2 exhibited decreased cPLA2 protein levels as well as decreased cPLA2 activity assayed in vitro. The effects of cytokines interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha) on the release of prelabeled [3H]AA were then tested in cells stably transfected with vector alone as well as cells transfected with cPLA2 antisense plasmid. IFN-gamma (300 U/ml), TNF-alpha (20 ng/ml), and IL-1 alpha (20 ng/ml) all induced a significantly increased release of prelabeled [3H]AA after 15 min to 2 h of treatment in control cells, and their effects were significantly reduced in cells transfected with cPLA2 antisense vector. These results demonstrate a critical role of cPLA2 in inflammatory cytokine-induced AA metabolism.
Alveolar type II cell cNOS activity and ATP levels are increased by lung surfactant or DPPC vesicles
Authors: Miles P. R. , Bowman L. , Rengasamy A. , Huffman L.
Published: December 2017

In a previous study, we reported that nitric oxide (.NO) affects surfactant synthesis and ATP levels in alveolar type II cells and suggested that there is constitutive nitric oxide synthase (cNOS) activity in the cells. In the present study, we performed experiments to confirm further the presence of cNOS and to determine the effects of lung surfactant on type II cell .NO and ATP levels. The supernatant from freshly isolated cells contains .NO (0.26 +/- 0.08 nmol/10(6) cells). During incubation, the cells produce additional .NO at a rate of approximately 0.3 nmol.10(5) cells-1.h-1. .NO formation is inhibited by 28-46% by three inhibitors of cNOS and inducible NOS (iNOS), NG-monomethyl-L-arginine (L-NMMA), L-N5-(1-iminoethyl)ornithine hydrochloride, and NG-nitro-L-arginine methyl ester, but a specific inhibitor of iNOS, aminoguanidine, has no effect. The production of .NO is reduced in Ca(2+)-free medium, is stimulated by the Ca2+ ionophore A-23187, and is independent of extracellular L-arginine. One known type of cNOS, endothelial NOS (eNOS), can be detected in the cells by using Western blot analysis. Incubation of the cells with lung surfactant leads to a relatively rapid (approximately 15 min), concentration-dependent increase in .NO formation that reaches levels as high as 238 +/- 14% of control. The surfactant effects appear to be caused by its major component, dipalmitoyl phosphatidylcholine (DPPC). Exposure of type II cells to DPPC results in maximal increases in .NO formation, ATP content, and O2 consumption, which are 268 +/- 32, 234 +/- 24, and 131 +/- 6% of control, respectively. The DPPC-induced increases in .NO, ATP, and O2 consumption are inhibited by L-NMMA. These results confirm the presence of type II cell cNOS and suggest that it may have a role in the cellular processing of lung surfactant.
Intracellular calcium responses in CF/T43 cells: calcium pools and influx pathways
Authors: Harris R. A. , Pon D. J. , Katz S. , Hanrahan J. W.
Published: December 2017

We studied the intracellular free Ca2+ concentration ([Ca2+]i) response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine (100 microM; duration approximately 10 min) biphasically increased [Ca2+]i, with a rapid initial peak (30-45 s) followed by a smaller second peak that lasted for several minutes before returning to baseline. Neither peak specifically depended on Ca2+ influx. Exposure to bradykinin (10 microM) elicited a single peak that lasted 3-3.5 min before returning to baseline. Bradykinin increased intracellular inositol 1,4,5-trisphosphate (IP3), which peaked and returned to baseline within 150 s. Histamine also increased IP3 monophasically, but the peak was brief (< 20 s). Both phases of the Ca2+ response to histamine exhibited similar responsiveness to histamine concentration and sensitivity to antagonists. Cimetidine or thioperamide (1 mM) had no effect on the second peak. Pyrilamine blocked the second peak at concentrations similar to those required to block the initial peak. Activation of the second peak was observed at a threshold concentration of 1 microM comparable with the threshold of the initial peak. Neither adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, nor cyclic ADP (cADP)-ribose altered the second phase of the histamine response.
Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants
Authors: McGowan S. E. , Doro M. M. , Jackson S. K.
Published: December 2017

During late gestation, the lungs of rats contain retinyl esters, but their concentration decreases considerably at the time of birth. The regulation of the acquisition and utilization of these stored retinoids remains poorly understood, although it has been hypothesized that they are involved in surfactant production and alveolar septal formation. Previous investigations demonstrated that exogenous retinoic acid increases elastin production in cultured neonatal lung fibroblasts and increases the number of alveoli when it is administered to neonatal rats. It has been hypothesized that these pulmonary stores of retinyl esters may regulate the perinatal expression of various genes in the lung, including elastin. To test this hypothesis, inhibitors of retinoid metabolism were used to reduce the flux of retinyl esters to retinoic acid, and the effects of this maneuver on elastin gene expression were analyzed. Inhibitors of alcohol and aldehyde dehydrogenases and of retinyl ester hydrolases decreased the steady-state level of tropoelastin mRNA without reducing alpha 1(I) procollagen mRNA. The magnitude of the effects of the inhibitors was retinol dependent and was significantly reduced in lung tissue that was obtained from vitamin A-deficient fetuses. These findings suggest that the late gestational pulmonary stores of retinoids may increase elastin gene expression during the fetal and early postnatal life in the rat.
Effects of reactive metabolites of oxygen and nitrogen on gelatinase A activity
Authors: Owens M. W. , Milligan S. A. , Jourd'heuil D. , Grisham M. B.
Published: December 2017

The regulation of matrix metalloproteinase activity is crucial for maintaining the proper balance of tissue remodeling vs. injury. Metalloproteinase proenzymes are activated when the active site zinc is exposed via a cysteine switch mechanism. Peroxynitrite, the product generated from the interaction between nitric oxide and superoxide, has been shown to release zinc from zinc-thiolate groups, suggesting that it might alter metalloproteinase activity. This study examined the effects of nitric oxide and superoxide generators on gelatinase A activity. Results showed that nitric oxide alone had no effect on gelatinase A activity relative to control, whereas superoxide-derived metabolites increased activity. The simultaneous generation of both nitric oxide and superoxide caused an inhibition of gelatinase A activity. This inhibition was reversed by the addition of hemoglobin, superoxide dismutase, or sodium urate, suggesting that peroxynitrite and/or peroxynitrous acid caused the inhibition. Authentic peroxynitrite also inhibited gelatinase A activity. We postulate that the relative fluxes of nitric oxide and superoxide at sites of inflammation may modulate metalloproteinase activity and thus affect matrix protein metabolism.
Upregulation of acidic fibroblast growth factor during development of experimental lung fibrosis
Authors: Barrios R. , Pardo A. , Ramos C. , Montano M. , Ramirez R. , Selman M.
Published: December 2017

Fibroblast proliferation and extracellular matrix accumulation are crucial in the pathogenesis of lung fibrosis. Fibroblast growth factor (FGF)-1 participates in both processes, but its role in lung fibrogenesis has not been evaluated. We analyzed the expression of FGF-1 and of FGF receptor (FGFR) in a model of lung fibrosis induced in rats with paraquat plus hyperoxia. Experimental and control animals were killed at 48 h and 2, 4, and 8 wk, and the lungs were studied by in situ hybridization, immunohistochemistry, and Northern blot. In normal lungs, scattered macrophages contained FGF-1. In contrast, at all times examined, the injured lungs exhibited FGF-1 transcript and the immunoreactive protein, mainly in alveolar epithelial cells and macrophages. In advanced fibrotic lesions, fibroblasts also appeared stained. Northern blot corroborated the upregulation of FGF-1 mRNA. FGFR was not observed in normal lungs, whereas it was strongly increased in the damaged lungs and was virtually immunolocalized in the same cell types as the corresponding ligand. These findings suggest that FGF-1 and FGFR are actively synthesized during the development of pulmonary fibrosis.
Preferential uptake of small-aggregate fraction of pulmonary surfactant in vitro
Authors: Horowitz A. D. , Kurak K. , Moussavian B. , Whitsett J. A. , Wert S. E. , Hull W. M. , McNanie J. , Ikegami M.
Published: December 2017

Homeostasis of pulmonary surfactant requires metabolic clearance of surfactant forms with decreased surface activity. Rabbit pulmonary surfactant was labeled in vivo with rhodamine-labeled dipalmitoylphosphatidylethanolamine (R-DPPE), isolated, and fractionated into large- and small-aggregate subfractions by differential centrifugation. Endocytosis of large (LA)- and small (SA)-aggregate surfactant by a mouse lung epithelial cell line (MLE-12) was evaluated in vitro by epifluorescence microscopy. More SA than LA surfactant was taken up by MLE-12 cells. Endocytosis of SA and LA surfactant was inhibited by preincubation of the subfractions with surfactant protein A and 3.3 mM Ca2+. The difference in uptake between SA and LA surfactant was lost for reconstituted organic extracts of the subfractions. Much of the difference in uptake of SA and LA surfactant may be attributed to the greater concentration of surfactant protein A in LA surfactant.
Hypoxia compared with normoxia alters the effects of nitric oxide in ischemia-reperfusion lung injury
Authors: Huang Y. C. , Fisher P. W. , Nozik-Grayck E. , Piantadosi C. A.
Published: December 2017

Because both the biosynthesis of nitric oxide (NO.) and its metabolic fate are related to molecular O2, we hypothesized that hypoxia would alter the effects of NO. during ischemia-reperfusion (IR) in the lung. In this study, buffer-perfused lungs from rabbits underwent either normoxic IR (AI), in which lungs were ventilated with 21% O2 during ischemia and reperfusion, or hypoxic IR (NI), in which lungs were ventilated with 95% N2 during ischemia followed by reoxygenation with 21% O2. Lung weight gain (WG) and pulmonary artery pressure (Ppa) were monitored continuously, and microvascular pressure (Pmv) was measured after reperfusion to calculate pulmonary vascular resistance. We found that both AI and NI produced acute lung injury, as shown by increased WG and Ppa during reperfusion. In AI, where perfusate PO2 was > 100 mmHg, the administration of the NO. synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) before ischemia worsened WG and Ppa. Pmv also increased, suggesting a hydrostatic mechanism involved in edema formation. The effects of L-NAME could be attenuated by giving L-arginine and exogenous NO. donors before ischemia or before reperfusion. Partial protection was also provided by superoxide dismutase. In contrast, lung injury in NI at perfusate PO2 of 25-30 mmHg was attenuated by L-NAME; this effect could be reversed by L-arginine. Exogenous NO. donors given either before ischemia or before reperfusion, however, did not increase lung injury. NO. production was measured by quantifying the total nitrogen oxides (NOx) accumulating in the perfusate. The average rate of NOx accumulation was greater in AI than in NI. We conclude that hypoxia prevented the protective effects of NO on AI lung injury. The effects of hypoxia may be related to lower NO. production relative to oxidant stress during IR and/or altered metabolic fates of NO.-mediated production of peroxynitrite by hypoxic ischemia.
N-acetylcysteine does not protect against type II cell injury after prolonged exposure to hyperoxia in rats
Authors: van Klaveren R. J. , Dinsdale D. , Pype J. L. , Demedts M. , Nemery B.
Published: December 2017

Although the antioxidant properties of N-acetylcysteine (NAC) in vitro are widely accepted, the efficacy of NAC in the prevention of O2 toxicity in vivo is poorly documented. The aim of our study was to investigate the presumed protective effect of NAC on hyperoxic lung injury, focusing on gamma-glutamyltransferase (gamma-GT) activity and glutathione (GSH) levels in lung tissue, epithelial lining fluid (ELF), and isolated rat type II cells immediately after their isolation and 48 h later when kept in culture in normoxia. Thirty-four male Wistar rats were divided in three groups (n = 10-14) and were exposed to air or to 60 or 85% O2 for 7 days. One-half of the rats in each group received 200 mg/kg NAC intraperitoneally one time per day from 3 days before exposure until the end of the experiment, and the other one-half received the vehicle. In the 85% O2-exposed animals, NAC led to more respiratory distress and weight loss. NAC did not prevent the rise in bronchoalveolar lavage lactate dehydrogenase and alkaline phosphatase, but it did prevent the rise in calculated ELF volume. NAC decreased GSH levels (1.4-fold) and gamma-GT activity (1.8-fold) in the air-exposed type II cells. In the 60% O2-exposed group, no effects of NAC were seen (except for a decrease in gamma-GT mRNA expression), but, in the 85% O2-exposed group, NAC gave rise to higher GSH (2.6-fold) and higher gamma-GT activity (2.9-fold) in the ELF and lower GSH (6.9-fold) and higher gamma-GT activity (3.6-fold) in the type II cells. Even in culture, GSH levels remained 1.5-fold lower than in the cells from the air-exposed animals and 2-fold lower than in the cells from the 85% O2-exposed animals. There was increased DNA damage (as assessed by thymidine incorporation) and apoptosis after hyperoxia, especially after 60% O2, and this effect was amplified after NAC treatment. Although protective at the endothelial side, NAC treatment led to adverse effects at the epithelial side, despite, or probably because of, restoration of the ELF GSH levels in the presence of high O2 levels. Because NAC is rapidly metabolized to cysteine, it is plausible that the effects of NAC are manifested through the toxic effects of cysteine.
Adenosine receptor-mediated relaxation of rabbit airway smooth muscle: a role for nitric oxide
Authors: Ali S. , Metzger W. J. , Olanrewaju H. A. , Mustafa S. J.
Published: December 2017

In this study, we investigated the relaxant effect of adenosine receptor agonists on KCl-precontracted airway smooth muscle from rabbits and characterized the type of receptor involved in bronchorelaxation in the presence and absence of epithelium. We further defined the role of epithelium-derived relaxing factor, i.e., nitric oxide (NO), on these responses. In both epithelium-intact and -denuded tertiary airway rings from rabbits, the adenosine receptor agonists 2-[p-(2-carboxyethyl)]phenylethylamino-5-N'-ethylcarboxamidoadenos ine (CGS-21680), 5'-(N-ethyl-carboxamido)adenosine (NECA), 2-chloroadenosine (CAD), and (-)-N6-(2-phenylisopropyl)adenosine (R-PIA) relaxed airway smooth muscle with a potency order of CGS-21680 > NECA > CAD > R-PIA. A 98.5, 89.7, 73.2, and 64.7% relaxation was observed at 10(-5) M by CGS-21680, NECA, CAD, and R-PIA in the epithelium-intact bronchial rings, respectively. The 50% maximum effective concentration (EC50; x 10(-7) M) values for CGS-21680, NECA, CAD, and R-PIA were 2, 4, 9, and 80, respectively. Denuded rings, however, showed much less relaxant responses to various adenosine agonists compared with epithelium-intact rings. The adenosine receptor antagonist 8-(sulfophenyl)theophylline significantly attenuated the relaxant responses to all the agonists in the epithelium-intact and -denuded rings. The epithelium-dependent relaxant effect of the agonists in airway rings was inhibited by NG-monomethyl-L-arginine (L-NMMA; 30 microM). The EC50 (x 10(-6) M) values for CGS-21680, NECA, CAD, and R-PIA in the presence of inhibitor were 5.5, 8, 30, and 200, respectively. The L-NMMA produced an insignificant inhibitory effect in the epithelium-denuded rings. L-Arginine but not D-arginine (100 microM) reversed the inhibitory effect of L-NMMA on adenosine agonist-induced relaxation. In primary epithelial cells in culture, CGS-21680 (10(-5) M) induced a fourfold increase in NO production over the control. The CGS-21680-induced NO production in epithelial cells was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME). Moreover, L-arginine reversed the inhibitory effect of L-NAME in the epithelial cells. The data suggest that adenosine relaxes rabbit airway smooth muscle through an A2 adenosine receptor and the epithelium serves as a source of NO.
Lipoprotein-stimulated surfactant secretion in alveolar type II cells: mediation by heterotrimeric G proteins
Authors: Pian M. S. , Dobbs L. G.
Published: December 2017

Low- and high-density lipoproteins (LDL and HDL, respectively) stimulate alveolar type II cells to secrete surfactant. Increases in phosphoinositide hydrolysis, cytosolic Ca2+, and membrane-associated protein kinase C activity precede LDL- and HDL-stimulated secretion. We report three lines of evidence supporting the hypothesis that Gi mediates LDL- and HDL-stimulated surfactant secretion and signal transduction in type II cells. First, pertussis toxin (PTX) inhibited secretion stimulated by the apolipoprotein ligands for either the LDL receptor or the HDL binding protein. Second, PTX inhibited protein kinase C activity in cell membranes stimulated by LDL or HDL. Third, treatment of cell membranes with LDL or HDL inhibited PTX-catalyzed labeling of substrates corresponding in molecular mass to Gi alpha. These observations suggest that receptor-mediated activation of Gi is required for LDL- and HDL-stimulated secretion and that LDL and HDL activate Gi. These studies in type II cells are the first to support the hypothesis that Gi mediates the effects of LDL or HDL on important phenotype-specific functions of differentiated cells.
Surfactant metabolic consequences of overexpression of GM-CSF in the epithelium of GM-CSF-deficient mice
Authors: Ikegami Machiko , Jobe Alan H. , Reed Jacquelyn A. Huffman , Whitsett Jeffrey A.
Published: December 2017

Granulocyte macrophage colony-stimulating factor (GM-CSF) is a regulator of surfactant metabolism because GM-CSF-deficient mice have abnormally slow clearance and catabolism of saturated phosphatidylcholine (Sat PC) and surfactant protein (SP)-A in airspaces and lung tissue. Overexpression of GM-CSF only in respiratory epithelial cells of mice deficient in GM-CSF using the SP-C promotor (GM−/−,SP-C-GM+/+) resulted in increased type II cell numbers and normalization of alveolar Sat PC pool sizes. Metabolic measurements demonstrated that incorporation of radiolabeled choline and palmitate was increased more than twofold, but the amount of radiolabeled Sat PC that accumulated in airspaces relative to the amount incorporated was decreased by 50% relative to normal GM+/+ mice. The clearance of dipalmitoylphosphatidylcholine and SP-B from the airspaces was more rapid for GM−/−,SP-C-GM+/+ mice than for GM+/+ mice. Loss of Sat PC and SP-B from the lungs (alveolar plus lung tissue) was similar in the two strains of mice. The normal surfactant pools in the GM−/−,SP-C-GM+/+ mice were achieved by the net effects of increases in type II cell numbers, increased incorporation, decreased accumulation, and increased reuptake rates for surfactant components, demonstrating the multiple effects of GM-CSF on surfactant metabolism.
Hyperoxia inhibits fetal rat lung fibroblast proliferation and expression of procollagens
Authors: Hussain Naveed , Wu Fengying , Christian Constance , Kresch Mitchell J.
Published: December 2017

The direct effects of hyperoxia on collagen production by fetal lung fibroblasts are unknown and would be important to the understanding of the molecular mechanisms involved in bronchopulmonary dysplasia in premature infants. We studied the effect of hyperoxia on 1) proliferation, 2) mRNA levels for type I and III procollagens, and 3) net collagen production in primary cultures of fetal rat lung fibroblasts. Fibroblasts from 19-day-old rat fetuses (term is 22 days) were obtained. Test plates were incubated in hyperoxia and controls in room air for varying time periods. Cell viability in both conditions was >97% as determined by trypan blue exclusion. Fibroblast proliferation in nonconfluent cultures was found to be significantly reduced with exposure to hyperoxia ( P< 0.001). Steady-state levels of type I and III procollagen mRNAs, analyzed on Northern blots hybridized to [32P]cDNA probes, were significantly decreased in hyperoxia ( P < 0.01). This effect was noted as early as 4 h of exposure to hyperoxia and persisted for 5 days. There was a significant inverse correlation between duration of exposure to O2and steady-state levels of mRNA for α1(I)-procollagen ( r = −0.904) and α1(III)-procollagen ( r = −0.971). There were no significant changes in steady-state levels of β-actin mRNA. We also found a significant decrease in collagen synthesis in hyperoxia-exposed fibroblasts ( P < 0.05). We conclude that hyperoxia directly effects a reduction in fetal lung fibroblast proliferation and net collagen production at a pretranslational level.
Oxidant stress regulates basal endothelin-1 production by cultured rat pulmonary endothelial cells
Authors: Michael John R. , Markewitz Boaz A. , Kohan Donald E.
Published: December 2017

Endothelin-1 (ET-1) is a pluripotent mediator that modulates vascular tone and influences the inflammatory response. Patients with inflammatory lung disorders frequently have elevated circulating ET-1 levels. Because these pathophysiological conditions generate reactive oxygen species that can regulate gene expression, we investigated whether the level of oxidant stress influences ET-1 production in cultured rat pulmonary arterial endothelial cells (RPAEC). Treatment with the antioxidant 1,3-dimethyl-2-thiourea (10 mM) or the iron chelator deferoxamine (1.8 μM) doubles basal ET-1 release. Conversely, exposing cells to H2O2generated by glucose and glucose oxidase (0.1–10 mU/ml) for 4 h causes a concentration-dependent decrease in ET-1 release. This effect occurs at concentrations of glucose oxidase that do not affect [3H]leucine incorporation or specific51Cr release from RPAEC. Catalase prevents the decrease in ET-1 synthesis caused by glucose and glucose oxidase. Glucose and glucose oxidase decrease not only ET-1 generation but also ET-1 mRNA as assessed by semiquantitative polymerase chain reaction. Our results indicate that changes in oxidative stress can either up- or downregulate basal ET-1 generation by cultured pulmonary endothelial cells.
Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury
Authors: Pitt Bruce R. , Schwarz Margaret , Woo Elizabeth S. , Yee Emily , Wasserloos Karla , Tran Sothi , Weng Weili , Mannix Robert J. , Watkins Simon A. , Tyurina Yulia Y. , Tyurin Vladimir A. , Kagan Valerian E. , Lazo John S.
Published: December 2017

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide ( t-BOOH), hyperoxia, or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.
Divergent regulation of 92-kDa gelatinase and TIMP-1 by HBECs in response to IL-1β and TNF-α
Authors: Yao P. M. , Maitre B. , Delacourt C. , Buhler J. M. , Harf A. , Lafuma C.
Published: December 2017

In this study, we addressed the question of whether human bronchial epithelial cells (HBECs) contribute to the regulation of 92-kDa gelatinase activity by secreting tissue inhibitor of metalloproteinase (TIMP)-1. We investigated expression of 92-kDa gelatinase and TIMP-1 in response to lipopolysaccharide (LPS) and to the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Confluent HBECs from explants were cultured in plastic dishes coated with type I and III collagen. We demonstrated that TIMP-1 was expressed at both the protein and mRNA levels by primary cultures of HBECs. Gelatin zymography of HBEC-conditioned media showed that exposure of HBECs to LPS, IL-1β, or TNF-α induced a twofold increase in the latent form of 92-kDa gelatinase production, as well as its activation. Also, quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) demonstrated a twofold increase in the 92-kDa mRNA level in response to both cytokines. In contrast, TIMP-1 production evaluated by immunoblotting was unchanged in the presence of LPS and IL-1β and was clearly decreased in the presence of TNF-α. Quantitative RT-PCR demonstrated that TIMP-1 mRNA levels remained unchanged in response to LPS or IL-1β but decreased by 70% in the presence of TNF-α. All of these results strongly suggest that the control mechanisms regulating the expression of 92-kDa gelatinase and TIMP-1 by HBECs in response to inflammatory stimuli are divergent and result in an imbalance between 92-kDa gelatinase and TIMP-1 in favor of the metalloproteinase. Such an imbalance may contribute significantly to acute airway inflammation.
Contribution of type I NOS to expired gas NO and bronchial responsiveness in mice
Authors: De Sanctis George T. , Mehta Sanjay , Kobzik Lester , Yandava Chandri , Jiao Aiping , Huang Paul L. , Drazen Jeffrey M.
Published: December 2017

Nitric oxide (NO) can be measured in the expired gas of humans and animals, but the source of expired NO (FENO) and the functional contribution of the various known isoforms of NO synthase (NOS) to the NO measured in the expired air is not known. FENO was measured in the expired air of mice during mechanical ventilation via a tracheal cannula. FENO was significantly higher in wild-type B6SV129J +/+ mice than in mice with a targeted deletion of type I (neural) NOS (nNOS, −/−) (6.3 ± 0.9 vs. 3.9 ± 0.4 parts/billion, P = 0.0345, for +/+ and −/− mice, respectively), indicating that ∼40% of the NO in expired air in B6SV129 mice is derived from nNOS. Airway responsiveness to methacholine (MCh), assessed by the log of the effective dose of MCh for a doubling of pulmonary resistance from baseline (ED200 R L), was significantly lower in the −/− nNOS mice than in the wild-type mice (logED200 R L, 2.24 ± 0.07 vs. 2.51 ± 0.06 μg/kg, respectively; P = 0.003). These findings indicate that nNOS significantly contributes to baseline FENO and promotes airway hyperresponsiveness in the mouse.
Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2
Authors: Jiménez Luis Albert , Zanella Christine , Fung Hua , Janssen Yvonne M. W. , Vacek Pam , Charland Colette , Goldberg Jonathan , Mossman Brooke T.
Published: December 2017

Stimulation of cell signaling cascades by oxidants may be important in the pathogenesis of pulmonary and pleural diseases. Here, we demonstrate in rat pleural mesothelial cells that apoptotic concentrations of crocidolite asbestos and H2O2induce phosphorylation and activation of extracellular signal-regulated protein kinases (ERK). Activation of c- jun-NH2-terminal protein kinases (JNK)/stress-activated protein kinases was also observed in response to H2O2. In contrast, asbestos caused more protracted activation of ERK without JNK activation. Both H2O2- and asbestos-induced activation of ERK was abolished by catalase. Moreover, chelation of surface iron from crocidolite fibers or addition of N-acetyl-l-cysteine prevented ERK activation and apoptosis by crocidolite, indicating an oxidative mechanism of cell signaling. The MEK1 inhibitor PD-98059 abrogated asbestos-induced apoptosis, confirming a causal relationship between ERK activation and apoptosis. These results suggest that distinct cell-signaling cascades may be important in phenotypic responses elicited by oxidant stresses.
Glucocorticoid regulation of surfactant components in immature lambs
Authors: Ballard Philip L. , Ning Yue , Polk Daniel , Ikegami Machiko , Jobe Alan H.
Published: December 2017

To assess effects of dose and duration of glucocorticoid exposure on maturation of the fetal lung, we administered single or multiple doses of betamethasone (0.5 mg/kg im) to pregnant sheep for 2 or 21 days before preterm delivery at 125 days of gestation. Lung function (compliance, lung volume at 40 cmH2O pressure, and ventilatory efficiency index) was increased after two to four weekly doses of glucocorticoid (2.5- to 4-fold increase) and after 48 h of exposure (1.4- to 2.3-fold). Total protein of lavage fluid decreased similarly with three doses, four doses, and 48 h of treatment. In lambs with long-term exposure to betamethasone, there was a similar, dose-dependent increase in concentrations of saturated phosphatidylcholine and surfactant proteins A (SP-A) and B (SP-B) (maximal 2- to 3-fold in tissue and 10- to 15-fold in lavage fluid). Levels of SP-A and SP-B were closely correlated in lavage fluid. In animals treated for 48 h, only tissue SP-B was increased (2.7-fold). We conclude that 48 h of glucocorticoid treatment improves lung function in the premature lamb without a detectable increase in lavage surfactant components and that longer exposure to antenatal glucocorticoid increases surfactant lipid and proteins in a coordinated fashion. The enhanced response with repetitive dosing indicates that the process of glucocorticoid-induced lung maturation is either reversible and/or gestational age dependent.
Rat alveolar macrophages express preprotachykinin gene-I mRNA-encoding tachykinins
Authors: Killingsworth Cheryl R. , Shore Stephanie A. , Alessandrini Francesca , Dey Richard D. , Paulauskis Joseph D.
Published: December 2017

Although the tachykinins substance P (SP) and neurokinin A have been largely localized to neurons, eosinophils have also been shown to express these peptides. Our aim was to determine whether rat alveolar macrophages (AM) express preprotachykinin gene-I (PPT-I) mRNA that encodes these tachykinins and to examine expression during inflammation. PPT-I mRNA was detected by reverse transcription (RT)-polymerase chain reaction (PCR) in AM and brain (control) but not in peritoneal macrophages. Northern analysis showed that PPT-I mRNA was induced two- to fourfold by in vivo treatment of rats with intratracheal lipopolysaccharide (LPS) and in vitro after 4 h of exposure to LPS. This increase was inhibited by dexamethasone. In situ RT-PCR and immunocytochemistry further confirmed that AM express PPT-I mRNA and SP-like immunoreactivity, respectively, which was enhanced by LPS treatment. A 1.3-kb transcript consistent with PPT-I mRNA was detected by Northern analysis of bronchoalveolar lavage neutrophils. Therefore, rat AM express PPT-I mRNA that is upregulated in AM by LPS and is attenuated by dexamethasone. PPT-I mRNA was also detected in lung neutrophils.
Induction of aquaporin 3 by corticosteroid in a human airway epithelial cell line
Authors: Tanaka Michiko , Inase Naohiko , Fushimi Kiyohide , Ishibashi Kenichi , Ichioka Masahiko , Sasaki Sei , Marumo Fumiaki
Published: December 2017

Although aquaporin 3 (AQP3) is expressed in many tissues in the kidney, gastrointestinal tract, lung, and other organs, its physiological significance in the body still remains to be clarified. To determine whether AQP3 expression is regulated by dexamethasone in human airway epithelium, we studied mRNA expression, protein expression, and water permeability of the cell membrane in a human airway epithelial cell line (A549 cells). Expression of AQP3 mRNA and protein was studied by Northern blot analysis and immunoblot analysis, and osmotic water permeability ( Pf) was measured by a stopped-flow light-scattering method. Expression of AQP3 mRNA and protein was detectable in A549 cells and was stimulated by dexamethasone. Pfin A549 cells after incubation with dexamethasone was ∼2.5-fold greater than that without dexamethasone. Moreover, this dexamethasone-induced increase in Pfwas inhibited after treatment with HgCl2. In conclusion, the present study shows that A549 cells express AQP3 and that dexamethasone upregulates the expression of AQP3 and increases the water permeability of the cell membrane. Dexamethasone-regulated AQP3 expression might be important in certain forms of pulmonary diseases accompanied by airway hypersecretion that are treated by corticosteroid administration.
Activated eosinophils elicit substance P release from cultured dorsal root ganglion neurons
Authors: Garland Allan , Necheles Jonathan , White Steven R. , Neeley Scott P. , Leff Alan R. , Carson Shannon S. , Alger Linda E. , McAllister Kyron , Solway Julian
Published: December 2017

This study was performed to test the hypothesis that activated eosinophils or their secretory products can directly stimulate sensory neurons to release their neuropeptides. Neurons derived from neonatal rat dorsal root ganglia (DRG), which synthesize and store sensory neuropeptides, were placed in primary cell culture and were exposed to eosinophils or their bioactive mediators. The resultant release of substance P (SP) was measured by enzyme-linked immunosorbent assay and was expressed as a percent (mean ± SE) of total neuronal SP content. Eosinophils were isolated from human volunteers with a history of allergic rhinitis and/or mild asthma and were activated by incubation with cytochalasin B (5 μg/ml) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 μM). Activated eosinophils [6 × 106/ml, suspended in Hanks’ buffered salt solution (HBSS)] applied to cultured DRG neurons for 30 min increased basal SP release 2.4-fold compared with HBSS-exposed neurons (activated eosinophils 11.10 ± 2.48% vs. HBSS 4.59 ± 0.99%; P = 0.002), whereas neither nonactivated eosinophils nor cytochalasin B and FMLP in HBSS influenced SP release. Additional cultured DRG neurons were exposed to soluble products made by eosinophils. Compared with SP release under control conditions (2.37 ± 0.34%), major basic protein (MBP) increased release in a concentration-related fashion (e.g., 3 μM MBP: 6.23 ± 0.67%, P = 0.006 vs. control), whereas neither eosinophil cationic protein (3 μM), eosinophil-derived neurotoxin (3 μM), leukotriene D4(500 nM), platelet-activating factor (100 nM), nor H2O2(100 μM) affected SP release. These studies demonstrate that activated eosinophils can stimulate cultured DRG neurons directly and suggest that MBP may be the responsible mediator.
Modulatory effects of PKC activity on increased 92-kDa gelatinase secretion by neonatal alveolar macrophages
Authors: Delacourt Christophe , Rouet-Benzineb Patricia , Delclaux Christophe , L’Hour Jeannique , Harf Alain , Lafuma Chantal
Published: December 2017

We previously demonstrated that alveolar macrophages (AMs) from neonatal rats can secrete more 92-kDa gelatinase than AMs from adult rats. In this study, we investigated the role of the protein kinase C (PKC) pathway in the transductional regulation of 92-kDa gelatinase secretion by rat AMs, and we also evaluated maturational changes in this role with increasing postnatal age. After AM stimulation by phorbol 12-myristate 13-acetate (PMA), we observed a dose-dependent increase in gelatinase secretion that was significantly more marked in AMs from 6-day-old rats than in AMs from adult rats and that was inhibited by the PKC inhibitor calphostin C. Adenosine 3′,5′-cyclic monophosphate mimetics or concanavalin A failed to induce an increase in gelatinase secretion by AMs. Time-dependent variations in PKC activity after PMA stimulation differed significantly between 6-day-old rats and adult rats; PKC activity decreased in adult AMs (50%) but remained stable in 6-day-old AMs. We therefore investigated age-related differences in the intracellular proteolytic degradation of PKC, which is thought to be mediated by calpains. Leupeptin, used as a calpain inhibitor, inhibited the decrease in PKC activity after exposure of adult AMs to PMA and induced a greater than threefold increase in PMA-induced gelatinase secretion. Calpain activity was significantly lower in AM extracts from 6-day-old than from adult rats. The physiological implication of these developmental changes in 92-kDa gelatinase regulation was demonstrated by investigation of AMs from 1-day-old rats that showed a high level of spontaneous PKC-dependent gelatinase secretion coexisting with very low calpain activity. We conclude that sustained PKC activity is a key factor in the increased gelatinase secretion by AMs seen during the postnatal period and is due, at least in part, to reduced PKC degradation.
Mechanism of capsaicin-induced relaxation in equine tracheal smooth muscle
Authors: Zhu Feng-Xia , Zhang Xiang-Yang , Olszewski Michal A. , Robinson N. Edward
Published: December 2017

The effects of capsaicin and neuropeptides were examined in equine tracheal smooth muscle (TSM). Neither capsaicin nor substance P (SP) contracted TSM. Capsaicin (100 μM) elicited relaxation in TSM contracted with methacholine. This relaxation was not mimicked by SP or calcitonin gene-related peptide (CGRP). Relaxation was not attenuated by removal of the epithelium or by pretreatment of tissue with meclofenamate or the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine. Previous exposure of TSM to capsaicin did not eliminate the relaxation responses to subsequent capsaicin. Although vasoactive intestinal peptide (VIP) elicited marked relaxation that was attenuated by α-chymotrypsin, α-chymotrypsin did not affect the capsaicin-induced relaxation. Capsaicin-induced relaxation was abolished by charybdotoxin, a blocker of large-conductance Ca2+-activated K+channels. These results indicate that capsaicin-induced equine TSM relaxation is not mediated either by neuropeptides such as SP or CGRP released from capsaicin-sensitive sensory nerves or by prostanoids, NO, or VIP. Relaxation is due to the effect of capsaicin on large-conductance Ca2+-activated K+channels. The peptidergic nerves play no important role in the regulation of TSM tone in horse airways.
Pneumocystis cariniiinduces ICAM-1 expression in lung epithelial cells through a TNF-α-mediated mechanism
Authors: Yu Mariette L. , Limper Andrew H.
Published: December 2017

Inflammatory cell recruitment contributes to respiratory impairment during Pneumocystis carinii pneumonia. We evaluated expression of intercellular adhesion molecule-1 (ICAM-1), a key participant in leukocyte accumulation, in rats with P. carinii pneumonia. Immunostaining for ICAM-1 was most marked on bronchiolar epithelium but was also evident on type II pneumocytes, endothelium, and macrophages. Lung from normal and dexamethasone-treated uninfected animals exhibited markedly less ICAM-1. We hypothesized that P. carinii promoted ICAM-1 expression in epithelium through tumor necrosis factor-α (TNF-α) release from macrophages or that P. carinii directly stimulated ICAM-1 expression. Alveolar macrophages were incubated with P. carinii, and the medium was added to A549 epithelial cells. Treatment of macrophages with P. carinii enhanced A549 ICAM-1, which was inhibited with antibody to TNF-α. To determine whether P. carinii alone also stimulated ICAM-1, A549 cells were cultured with P. carinii, also augmenting ICAM-1. Of note, A549 ICAM-1 expression from P. carinii alone was less than with P. carinii-exposed macrophages. Thus ICAM-1 is enhanced in lung epithelium during P. carinii infection, in part, through TNF-α-mediated mechanisms.
PGE2release by bradykinin in human airway smooth muscle cells: involvement of cyclooxygenase-2 induction
Authors: Pang Linhua , Knox Alan J.
Published: December 2017

Prostanoids may be involved in bradykinin (BK)-induced bronchoconstriction in asthma. We investigated whether cyclooxygenase (COX)-2 induction was involved in prostaglandin (PG) E2release by BK in cultured human airway smooth muscle (ASM) cells and analyzed the BK receptor subtypes responsible. BK stimulated PGE2release, COX activity, and COX-2 induction in a concentration- and time-dependent manner. It also time dependently enhanced arachidonic acid release. In short-term (15-min) experiments, BK stimulated PGE2generation but did not increase COX activity or induce COX-2. In long-term (4-h) experiments, BK enhanced PGE2release and COX activity and induced COX-2. The long-term responses were inhibited by the protein synthesis inhibitors cycloheximide and actinomycin D and the steroid dexamethasone. The effects of BK were mimicked by the B2-receptor agonist [Tyr(Me)8]BK, whereas the B1agonist des-Arg9-BK was weakly effective at high concentrations. The B2antagonist HOE-140 potently inhibited all the effects, but the B1antagonist des-Arg9,(Leu8)-BK was inactive. This study is the first to demonstrate that BK can induce COX-2. Conversion of increased arachidonic acid release to PGE2by COX-1 is mainly involved in the short-term effect, whereas B2receptor-related COX-2 induction is important in the long-term PGE2release.
Inhibition of nitric oxide synthase attenuates NNMU-induced alveolar injury in vivo
Authors: Cruz Wilhelm S. , Moxley Michael A. , Corbett John A. , Longmore William J.
Published: December 2017

The purpose of this study was to determine if the acute alveolar injury induced by subcutaneous injections of N-nitroso- N-methylurethane (NNMU) in rats is mediated by nitric oxide (NO ⋅). We show that intraperitoneal injections of the NO ⋅ synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) or aminoguanidine significantly attenuate the NNMU-induced alveolar injury as assessed by 1) normalization of the alveolar-arterial O2difference, 2) attenuation of the lowered phospholipid-to-protein ratio in the crude surfactant pellet (CSP), 3) attenuation of the elevated minimal surface tension of the CSP, and 4) attenuation of polymorphonuclear neutrophilic infiltration into the alveolar space. Injections of Nω-nitro-d-arginine methyl ester, the inactive stereoisoform ofl-NAME, did not affect the acute lung injury. Western blot analysis of whole lung homogenates demonstrate an elevated expression of transcriptionally inducible, Ca2+-independent NOS (iNOS) in NNMU-injected rats compared with control saline-injected rats. NOS inhibitors did not affect NNMU-induced iNOS expression. These investigations demonstrate that the inhibition of NOS attenuates NNMU-induced acute lung injury, suggesting a role for NO ⋅ in the progression of acute respiratory distress syndrome.
Biochemical detection of type I cell damage after nitrogen dioxide-induced lung injury in rats
Authors: McElroy Mary C. , Pittet Jean-François , Allen Lennell , Wiener-Kronish Jeanine P. , Dobbs Leland G.
Published: December 2017

We have previously shown that injury to lung epithelial type I cells can be detected biochemically by measuring the airway fluid content of a type I cell-specific protein, rTI40, in a model of severe acute lung injury [M. C. McElroy, J.-F. Pittet, S. Hashimoto, L. Allen, J. P. Wiener-Kronish, and L. G. Dobbs. Am. J. Physiol. 268 ( Lung Cell. Mol. Physiol. 12): L181–L186, 1995]. The first objective of the present study was to evaluate the utility of rTI40 in the assessment of alveolar injury in a model of milder acute lung injury. Rats were exposed to 18 parts/million NO2 for 12 h; control rats received filtered air for 12 h. In NO2-exposed rats, the total amount of rTI40 in bronchoalveolar fluid was elevated 2-fold compared with control values ( P < 0.001); protein concentration was 8.5-fold of control values ( P < 0.001). The increase in rTI40 was associated with morphological evidence of injury to type I cells limited to the proximal alveolar regions of the lung. The second objective was to correlate the severity of alveolar type I cell injury with functional measurements of lung epithelial barrier integrity. NO2 inhalation stimulated distal air space fluid clearance despite a significant increase in lung endothelial and epithelial permeability to protein. These data demonstrate that rTI40 is a useful biochemical marker for mild focal injury and that exposure to NO2 alters lung barrier function. Taken together with our earlier studies, these results suggest that the quantity of recoverable rTI40 can be used as an index of the severity of damage to the alveolar epithelium.
Surfactant modifies the lymphoproliferative activity of macrophages in hypersensitivity pneumonitis
Authors: Israël-Assayag Evelyne , Cormier Yvon
Published: December 2017

Alveolar macrophages (AM) from normal individuals suppress mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation, whereas cells from patients with hypersensitivity pneumonitis (HP) enhance PBMC. Because surfactant components can interfere with AM functions, we tested the effect of Survanta (a modified bovine surfactant) and surfactant fractions isolated from bronchoalveolar lavage of normal subjects and HP patients on AM-induced lymphoproliferation. Surfactant fractions were isolated from bronchoalveolar lavage fluids by differential centrifugation into total aggregates (TA) and large aggregates (LA). Surfactant preparations (200 μg/ml) from 10 normal subjects (N) or 12 HP patients or of Survanta were added to AM-PBMC cocultures stimulated with phytohemagglutinin (PHA) at 1:1 and 2:1 ratios. Results, expressed as percent of PHA-induced PBMC proliferation cocultures without surfactant, show that normal surfactant and Survanta decrease mitogen-induced proliferation of cells to a larger extent than surfactant from HP patients. For AM-to-PBMC ratios of 1:1, the results were as follows: N TA 10.58 ± 2.75% (mean ± SE), N LA 12.96 ± 2.78%, HP TA 43.09 ± 7.81%, HP LA 61.64 ± 7.77%, and Survanta 16.70 ± 2.95%. Similar data were obtained in 2:1 cocultures. Because surfactant components interact with AM, alterations of surfactant composition in lymphocytic diseases, mainly in the LA fraction, may account for the lack of suppressive activity in AM of HP patients and the observed alveolitis.