bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ca2+in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function
Authors: Usatyuk Peter V. , Fomin Victor P. , Shi Shu , Garcia Joe G. N. , Schaphorst Kane , Natarajan Viswanathan
Published: March 2015

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]iincludes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 μM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]iregulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.
p38 MAPK and NF-κB mediate COX-2 expression in human airway myocytes
Authors: Singer Cherie A. , Baker Kimberly J. , McCaffrey Alan , AuCoin David P. , Dechert Melissa A. , Gerthoffer William T.
Published: March 2015

We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-κB regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and interferon (IFN)-γ. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 μM) or the proteosome inhibitor MG-132 (1 μM). SB-203580 did not affect cytokine-stimulated IκBα degradation, NF-κB nuclear binding activity, or NF-κB-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-κB may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-κB, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-κB signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.
Elevated expression of hexokinase II protects human lung epithelial-like A549 cells against oxidative injury
Authors: Ahmad Aftab , Ahmad Shama , Schneider B. Kelly , Allen Corrie B. , Chang Ling-Yi , White Carl W.
Published: March 2015

Increased glucose utilization and hexokinase (HK)-II expression are adaptive features of lung cells exposed to hypoxia or hyperoxia. HK-II is the most regulated isoform of HK. Whether its overexpression could be protective against oxidative stress was explored in human lung epithelial-like (A549) cells. HK-II was overexpressed in A549 cells in a tetracycline-repressible retroviral vector system. Elevated expression of HK-II was confirmed by Western blot and activity measurements. Cell death caused by exposure to hyperoxia was decreased in HK-II-overexpressing cells. This effect was reversed when HK-II expression was suppressed with doxycycline. A similar protective effect was observed in HK-II-overexpressing cells after treatment with 1 mM hydrogen peroxide for 48 h. At baseline, fluorescence microscopy showed that overexpressed HK-II was localized to mitochondria. Electron microscopic studies showed that hyperoxia-exposed HK-II overexpressors had better-preserved and quantitatively smaller mitochondria than those in which the HK-II expression was suppressed or in the nontransduced A549 cells. Mitochondrial membrane potential was increased in HK-II-overexpressing cells exposed to hyperoxia compared with the nontransduced control cells under similar conditions. The present study demonstrates that HK-II protects human lung epithelial-like A549 cells against oxidative insults by protecting the mitochondria.
cAMP-induced changes of apical membrane potentials of confluent H441 monolayers
Authors: Lazrak Ahmed , Matalon Sadis
Published: March 2015

We recorded apical membrane potentials ( Va) of H441 cells [a human lung cell line exhibiting both epithelial Na+ (ENaC) and CFTR-type channels] grown as confluent monolayers, using the microelectrode technique in current-clamp mode before, during, and after perfusion of the apical membranes with 10 μM forskolin. When perfused with normal Ringer solution, the cells had a Va of -43 ± 10 mV (means ± SD; n = 31). Perfusion with forskolin resulted in sustained depolarization by 25.0 ± 3.5 mV (means ± SD; n = 23) and increased the number, open time, and the open probability of a 4.2-pS ENaC. In contrast to a previous report (Jiang J, Song C, Koller BH, Matthay MA, and Verkman AS. Am J Physiol Cell Physiol 275: C1610–C1620, 1998), no transient hyperpolarization was observed. The forskolin-induced depolarization of Va was almost totally prevented by pretreatment of monolayers with 10 μM amiloride or by substitution of Na+ ions in the bath solution with N-methyl-d-glucamine. These findings indicate that cAMP stimulation of Na+ influx across H441 confluent monolayers results from activation of an amiloride-sensitive apical Na+ conductance and not from Va hyperpolarization due to Cl- influx through CFTR-type channels.
Surfactant protein A regulates surfactant phospholipid clearance after LPS-induced injury in vivo
Authors: Quintero Omar A. , Korfhagen Thomas R. , Wright Jo Rae
Published: March 2015

Previous in vitro studies have suggested that surfactant protein A (SP-A) may play a role in pulmonary surfactant homeostasis by mediating surfactant secretion and clearance. However, mice made deficient in SP-A [SP-A (−/−) animals] have relatively normal levels of surfactant compared with wild-type SP-A (+/+) animals. We hypothesize that SP-A may play a role in surfactant homeostasis after acute lung injury. Bacterial lipopolysaccharide was instilled into the lungs of SP-A (−/−) mice and SP-A (+/+) mice to induce injury. Surfactant phospholipid levels were increased 1.6-fold in injured SP-A (−/−) animals, although injury did not alter [3H]choline or [14C]palmitate incorporation into dipalmitoylphosphatidylcholine (DPPC), suggesting no change in surfactant synthesis/secretion 12 h after injury. Clearance of [3H]DPPC from the lungs of injured SP-A (−/−) animals was decreased by ∼40%. Instillation of 50 μg of exogenous SP-A rescued both the clearance defect and the increased phospholipid defect in injured SP-A (−/−) animals, suggesting that SP-A may play a role in regulating clearance of surfactant phospholipids after acute lung injury.
Primary inflammation in human cystic fibrosis small airways
Authors: Tirouvanziam Rabindra , Khazaal Ibrahim , Péault Bruno
Published: March 2015

Most cystic fibrosis (CF) patients die of lung failure, due to the combined effects of bacterial infection, neutrophil-mediated inflammation, and airway obstruction by hyperviscous mucus. To this day, it remains unclear where and how this pathological vicious circle is initiated in vivo. In particular, it has proven difficult to investigate whether inflammatory pathways are dysregulated in CF airways independently of infection. Also, the relative involvement of large (tracheobronchial) vs. small (bronchiolar) airways in CF pathophysiology is still unclear. To help address these issues, we used an in vivo model based on the maturation of human fetal CF and non-CF small airways in severe combined immunodeficiency mice. We show that uninfected mature CF small airway grafts, but not matched non-CF controls, undergo time-dependent neutrophil-mediated inflammation, leading to progressive lung tissue destruction. This model of mature human small airways provides the first clear-cut evidence that, in CF, inflammation may arise at least partly from a primary defect in the regulation of neutrophil recruitment, independently of infection.
Neutrophils play a critical role in development of LPS-induced airway disease
Authors: Savov Jordan D. , Gavett Stephen H. , Brass David M. , Costa Daniel L. , Schwartz David A.
Published: March 2015

We investigated the role of neutrophils in the development of endotoxin-induced airway disease via systemic neutrophil depletion of C3H/HeBFeJ mice and coincident inhalation challenge with lipopolysaccharide (LPS) over a 4-wk period. Mice were made neutropenic with intraperitoneal injections of neutrophil antiserum before and throughout the exposure period. Experimental conditions included LPS-exposed, antiserum-treated; LPS-exposed, control serum-treated; air-exposed, antiserum-treated; and air-exposed, control serum-treated groups. Physiological, biological, and morphological assessments were performed after a 4-wk exposure and again after a 4-wk recovery period. After the 4-wk exposure, LPS-induced inflammation of the lower airways was significantly attenuated in the neutropenic mice, although airway responsiveness (AR) to methacholine (MCh) remained unchanged. After the recovery period, LPS-exposed neutrophil-replete mice had increased AR to MCh when compared with the LPS-exposed neutropenic animals. Morphometric data indicate that the 4-wk exposure to LPS leads to a substantial expansion of the subepithelial area of the medium-sized airways (90–129 μm diameter) in nonneutropenic mice but not neutropenic mice, and this difference persisted even after the recovery period. Expression of bronchial epithelial and subepithelial transforming growth factor-β1 (TGF-β1) was diminished in the challenged neutropenic mice compared with the neutrophil-sufficient mice. These studies demonstrate that neutrophils play a critical role in the development of chronic LPS-induced airway disease.
Serum SP-D is a marker of lung injury in rats
Authors: Pan Tianli , Nielsen Larry D. , Allen Martin J. , Shannon Kathleen M. , Shannon John M. , Selman Moises , Mason Robert. J.
Published: March 2015

Pulmonary surfactant protein D (SP-D) is expressed in alveolar type II and bronchiolar epithelial cells and is secreted into alveoli and conducting airways. However, SP-D has also been measured in serum and is increased in patients with acute respiratory distress syndrome, pulmonary fibrosis, and alveolar proteinosis. To demonstrate that SP-D can be measured in rat serum, we instilled rats with keratinocyte growth factor, which produces type II cell hyperplasia and an increase in SP-D in bronchoalveolar lavage fluid (BALF). To evaluate serum SP-D as a biomarker of lung injury, we examined several injury models. In rats treated with 1 unit of bleomycin, serum SP-D was elevated on days 3, 7, 14, and 28 after instillation, and SP-D mRNA was increased in focal areas as detected by in situ hybridization. However, there was no increase in whole lung SP-D mRNA when the expression was normalized to whole lung 18S rRNA. After instillation of 2 units of bleomycin, the serum levels of SP-D were higher, and SP-D was also increased in BALF and lung homogenates. In another model of subacute injury, serum SP-D was increased in rats treated with paraquat plus oxygen. Finally to evaluate acute lung injury, we instilled rats with HCl; SP-D was increased at 4 h after instillation. Our data indicate that serum SP-D may be a useful indicator of lung injury and type II cell hyperplasia in rats.
Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages
Authors: Araya Jun , Maruyama Muneharu , Inoue Akira , Fujita Tadashi , Kawahara Junko , Sassa Kazuhiko , Hayashi Ryuji , Kawagishi Yukio , Yamashita Naohiro , Sugiyama Eiji , Kobayashi Masashi
Published: March 2015

Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl2) and examined their apoptosis by DNA fragmentation assay, 4′,6-diamidino-2′-phenylindol dihydrochloride staining, and Western blot analysis. CoCl2 induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl2 inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl2 released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.
Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair
Authors: Ware Lorraine B. , Matthay Michael A.
Published: March 2015

A growing body of evidence indicates that the epithelial-specific growth factors keratinocyte growth factor (KGF), fibroblast growth factor (FGF)-10, and hepatocyte growth factor (HGF) play important roles in lung development, lung inflammation, and repair. The therapeutic potential of these growth factors in lung disease has yet to be fully explored. KGF has been best studied and has impressive protective effects against a wide variety of injurious stimuli when given as a pretreatment in animal models. Whether this protective effect could translate to a treatment effect in humans with acute lung injury needs to be investigated. FGF-10 and HGF may also have therapeutic potential, but more extensive studies in animal models are needed. Because HGF lacks true epithelial specificity, it may have less potential than KGF and FGF-10 as a targeted therapy to facilitate lung epithelial repair. Regardless of their therapeutic potential, studies of the unique roles played by these growth factors in the pathogenesis and the resolution of acute lung injury and other lung diseases will continue to enhance our understanding of the complex pathophysiology of inflammation and repair in the lung.
Surfactant protein A enhances apoptotic cell uptake and TGF-β1 release by inflammatory alveolar macrophages
Authors: Reidy Michael F. , Wright Jo Rae
Published: March 2015

The phagocytosis of apoptotic inflammatory cells by alveolar macrophages (AMs) is a key component of inflammation resolution within the air space. Surfactant protein A (SP-A) has been shown to stimulate the phagocytosis of apoptotic neutrophils (PMNs) by normal AMs. We hypothesized that SP-A promotes the resolution of alveolar inflammation by enhancing apoptotic PMN phagocytosis and anti-inflammatory cytokine release by inflammatory AMs. Using an LPS lung inflammation model, we determined that SP-A stimulates the phagocytosis of apoptotic PMNs threefold by normal AMs and AMs isolated after LPS injury. Furthermore, SP-A enhances transforming growth factor-β1 (TGF-β1) release from both AM populations. Inflammatory AMs release twofold more TGF-β1 in culture than do normal AMs. SP-A and apoptotic PMNs together stimulate TGF-β1 release equivalently from normal and inflammatory cultured AMs (330% of unstimulated release by normal AMs). In summary, SP-A enhances apoptotic PMN uptake, stimulates AM TGF-β1 release, and modulates the amount of TGF-β1 released when AMs phagocytose apoptotic PMNs. These findings support the hypothesis that SP-A promotes the resolution of alveolar inflammation.
Regulation of serum-induced fibronectin expression by protein kinases, cytoskeletal integrity, and CREB
Authors: Michaelson Jeffrey E. , Ritzenthaler Jeffrey D. , Roman Jesse
Published: March 2015

Lung injury, characterized by the flooding of interstitial and alveolar spaces with serum proteins, induces the expression of fibronectin (FN). This cell-adhesive extracellular matrix (ECM) glycoprotein is believed to modulate inflammation and wound repair. Murine NIH/3T3 fibroblasts transfected with a 1.2-kb human FN promoter-reporter gene were studied to gain insight into the mechanisms involved in the induction of FN by serum. Transcription of the FN gene, followed by FN protein production, was enhanced by 10% fetal bovine serum. This effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinases. ECMs typically found in injured tissues (i.e., type I collagen, fibrin, and FN) had no effect. Conversely, disruption of actin microfilaments inhibited, whereas disruption of microtubular assembly enhanced, the serum-induced FN response. The stimulatory effects of serum and microtubular disruption on FN gene transcription were related to increased DNA binding of the transcription factor cAMP response element binding protein. The data suggest that regulation of serum-induced FN expression in fibroblasts is dependent on protein kinases and on cytoskeletal integrity.
Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin
Authors: Samb Abdoulaye , Taillé Camille , Almolki Abdelhamid , Mégret Jérôme , Staddon James M. , Aubier Michel , Boczkowski Jorge
Published: March 2015

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3′,5′-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.
ATP- and UTP-activated P2Y receptors differently regulate proliferation of human lung epithelial tumor cells
Authors: Schäfer Rainer , Sedehizade Fariba , Welte Tobias , Reiser Georg
Published: March 2015

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP ≥ UTP > adenosine ≥ 2-methylthioadenosine-5′-diphosphate, with α,β-methylene adenosine 5′-triphosphate, 2′,3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, AMP, UMP, and ATPαS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-κB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1β and interleukin-6, but not tumor ncerosis factor-α. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.
Ionic mechanisms and Ca2+regulation in airway smooth muscle contraction: do the data contradict dogma?
Authors: Janssen Luke J.
Published: March 2015

In general, excitation-contraction coupling in muscle is dependent on membrane depolarization and hyperpolarization to regulate the opening of voltage-dependent Ca2+channels and, thereby, influence intracellular Ca2+concentration ([Ca2+]i). Thus Ca2+channel blockers and K+channel openers are important tools in the arsenals against hypertension, stroke, and myocardial infarction, etc. Airway smooth muscle (ASM) also exhibits robust Ca2+, K+, and Cl−currents, and there are elaborate signaling pathways that regulate them. It is easy, then, to presume that these also play a central role in contraction/relaxation of ASM. However, several lines of evidence speak to the contrary. Also, too many researchers in the ASM field view the sarcoplasmic reticulum as being centrally located and displacing its contents uniformly throughout the cell, and they have focused almost exclusively on the initial single [Ca2+] spike evoked by excitatory agonists. Several recent studies have revealed complex spatial and temporal heterogeneity in [Ca2+]i, the significance of which is only just beginning to be appreciated. In this review, we will compare what is known about ion channels in ASM with what is believed to be their roles in ASM physiology. Also, we will examine some novel ionic mechanisms in the context of Ca2+handling and excitation-contraction coupling in ASM.
Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction
Authors: Borbiev Talaibek , Verin Alexander D. , Birukova Anna , Liu Feng , Crow Michael T. , Garcia Joe G. N.
Published: March 2015

We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral α-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.
Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and in vivo
Authors: Pham Isabelle , Uchida Tokujiro , Planes Carole , Ware Lorraine B. , Kaner Robert , Matthay Michael A. , Clerici Christine
Published: March 2015

We investigated regulation of vascular endothelial growth factor (VEGF) expression by hypoxia in cultured and freshly isolated rat alveolar epithelial cells (AEC). In vitro, hypoxia increased VEGF mRNA and protein levels, with maximal stimulation at 0% O2for 18 h. A similar upregulation of VEGF expression was found in alveolar epithelial type II (ATII) cells freshly isolated from rats exposed to 8% O2for 24 h. In vitro , hypoxia-induced upregulation of VEGF mRNA was due to an increase in transcription, rather than an increase in RNA stability, inasmuch as the half-life of VEGF mRNA was unchanged. Upregulation of VEGF mRNA by hypoxia was mimicked by CoCl2and desferrioxamine in normoxic AEC and was not prevented by inhibitors of reactive oxygen species, suggesting that hypoxic VEGF regulation involved an O2-dependent protein that requires ferrous ions but is independent of reactive oxygen species generation. In polarized ATII cells, VEGF protein was secreted at the apical and basolateral sides. Similarly, in rats, VEGF was secreted in bronchoalveolar lavage fluid. Hypoxia induced a twofold increase in VEGF protein at the apical side of ATII cells in culture and in bronchoalveolar lavage fluid. These findings suggest that release of VEGF synthesized by AEC may target not only endothelial cells but also other alveolar cells, including macrophages and epithelial cells.
Alveolar macrophage activation after trauma-hemorrhage and sepsis is dependent on NF-κB and MAPK/ERK mechanisms
Authors: Jarrar Doraid , Kuebler Joachim F. , Rue Loring W. , Matalon Sadis , Wang Ping , Bland Kirby I. , Chaudry Irshad H.
Published: March 2015

The acute respiratory distress syndrome (ARDS) is a major cause of morbidity after injury. We hypothesized that alveolar macrophage (AMΦ) chemokine and cytokine release after hemorrhage and sepsis is regulated by NF-κB and MAPK. Adult male rats underwent soft tissue trauma and hemorrhagic shock (∼90 min) followed by crystalloid resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) 20 h after resuscitation. AMΦ were harvested, and TNF-α, IL-6, and macrophage inflammatory protein (MIP)-2 release and serum IL-6 and TNF-α levels were measured at 5 h after HCLP. Lung tissues were analyzed for activation of NF-κB, myeloperoxidase activity, and wet/dry weight ratio. In control animals, AMΦ were stimulated with LPS with or without inhibitors of NF-κB and MAPK. Serum TNF-α and IL-6 levels and spontaneous AMΦ TNF-α and MIP-2 release were elevated ( P < 0.05) after HCLP, concomitantly with the development of lung edema and leukocyte activation. Activation of NF-κB increased in lungs from the hemorrhage and CLP group compared with shams. Inhibition of NF-κB or the upstream MAPK significantly decreased LPS-stimulated AMΦ activation. Because enhanced release of inflammatory mediators by AMΦ may contribute to ARDS after severe trauma, inhibition of intracellular signaling pathways represents a target to attenuate organ injury under those conditions.
Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts
Authors: Carnevali Stefano , Petruzzelli Stefano , Longoni Biancamaria , Vanacore Renato , Barale Roberto , Cipollini Monica , Scatena Fabrizio , Paggiaro Pierluigi , Celi Alessandro , Giuntini Carlo
Published: March 2015

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 μM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.
Estradiol attenuates hypoxia-induced pulmonary endothelin-1 gene expression
Authors: Earley Scott , Resta Thomas C.
Published: March 2015

The ovarian hormone 17β-estradiol (E2β) attenuates chronic hypoxia-induced pulmonary hypertension. We hypothesized that E2β attenuates this response to hypoxia by decreasing pulmonary expression of the vasoactive and mitogenic peptide endothelin-1 (ET-1). To test this hypothesis, we measured preproET-1 mRNA and ET-1 peptide levels in the lungs of adult female normoxic and hypoxic (24 h or 4 wk at barometric pressure = 380 mmHg) rats with intact ovaries and in hypoxic ovariectomized (OVX) rats administered E2β or vehicle via subcutaneous osmotic pumps. Hypoxic exposure increased lung preproET-1 mRNA levels in OVX vehicle-treated rats, but not in rats with intact ovaries. In addition, E2β replacement prevented hypoxia-mediated increases in preproET-1 mRNA and ET-1 peptide expression. Considering that hypoxic induction of ET-1 gene expression is mediated by a hypoxia-inducible transcription factor(s) (HIF), we further hypothesized that E2β-induced attenuation of pulmonary ET-1 expression during hypoxia results from decreased HIF activity. We found that E2β abolished HIF-dependent increases in reporter gene activity. Further experiments demonstrated that overexpression of the transcriptional coactivator cAMP response element binding protein (CREB) binding protein (CBP)/p300, a factor common to both the estrogen receptor and HIF pathways, eliminated E2β-mediated attenuation of hypoxia-induced ET-1 promoter activity. We conclude that E2β inhibits hypoxic induction of ET-1 gene expression by interfering with HIF activity, possibly through competition for limiting quantities of CBP/p300.
Transfection of lung cells in vitro and in vivo: effect of antioxidants and intraliposomal bFGF
Authors: Luo Xiaoping , Belcastro Rosetta , Cabacungan Judy , Hannam Vicky , Negus Anna , Wen Yanxia , Plumb Jonathan , Hu Jim , Steer Brent , Koehler David R. , Downey Gregory P. , Tanswell A. Keith
Published: March 2015

We hypothesized that constitutive formation of reactive oxygen species by respiratory cells is a barrier to gene transfer when liposome-DNA complexes are used, by contributing to rapid degradation of plasmid DNA. When plasmid DNA is complexed to liposomes it is protected against H2O2-mediated degradation but not that mediated by the hydroxyl radical. Treatment of distal rat fetal lung epithelial cells (RFL19Ep) with the vitamin E analog Trolox (50 μM) reduced intracellular plasmid degradation. Both Trolox (50 μM) and an iron chelator, phenanthroline (0.1 μM), significantly increased transgene expression in RFL19Ep approximately twofold, consistent with a hydroxyl radical-mediated inhibition of transgene expression. When basic fibroblast growth factor (bFGF; 20 ng/ml), a growth factor with antioxidant properties, was included within liposomes, we observed a significantly greater enhancement of RFL19Ep transgene expression (∼2-fold) over that seen with Trolox or phenanthroline. Inclusion of bFGF within liposomes also significantly enhanced (∼4-fold) transgene expression in mice following intratracheal instillation.
Antioxidant responses to oxidant-mediated lung diseases
Authors: Comhair Suzy A. A. , Erzurum Serpil C.
Published: March 2015

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated throughout the human body. Enzymatic and nonenzymatic antioxidants detoxify ROS and RNS and minimize damage to biomolecules. An imbalance between the production of ROS and RNS and antioxidant capacity leads to a state of “oxidative stress” that contributes to the pathogenesis of a number of human diseases by damaging lipids, protein, and DNA. In general, lung diseases are related to inflammatory processes that generate increased ROS and RNS. The susceptibility of the lung to oxidative injury depends largely on its ability to upregulate protective ROS and RNS scavenging systems. Unfortunately, the primary intracellular antioxidants are expressed at low levels in the human lung and are not acutely induced when exposed to oxidative stresses such as cigarette smoke and hyperoxia. However, the response of extracellular antioxidant enzymes, the critical primary defense against exogenous oxidative stress, increases rapidly and in proportion to oxidative stress. In this paper, we review how antioxidants in the lung respond to oxidative stress in several lung diseases and focus on the mechanisms that upregulate extracellular glutathione peroxidase.
Molecular cloning of actin filament-associated protein: a putative adaptor in stretch-induced Src activation
Authors: Lodyga Monika , Bai Xiao-Hui , Mourgeon Eric , Han Bing , Keshavjee Shaf , Liu Mingyao
Published: March 2015

Mechanical stretch-induced activation of c-Src is an important step for signal transduction of stretch-induced fetal rat lung cell proliferation. This process appears to be mediated through actin filament-associated protein (AFAP), encoded by a gene originally cloned from the chicken. In the present study, we cloned the rat AFAP gene from fetal rat lungs. Its mRNA and protein are differentially expressed among various tissues. The protein is colocalized with actin filaments in fetal rat lung epithelial cells and fibroblasts. Mechanical stretch increased tyrosine phosphorylation of rat AFAP and its binding to c-Src within the initial several minutes. Src SH2 and SH3 binding motifs are highly conserved in the AFAP proteins (from chicken, rat to human). On the basis of the molecular structure of AFAP protein, we speculate that it is an adaptor in mechanical stretch-induced activation of c-Src. A novel model of mechanoreception is proposed.
Glucocorticoid-attenuated response genes induced in the lung during endotoxemia
Authors: Smith Jeffrey B. , Nguyen Tam T. , Hughes Heather J. , Herschman Harvey R. , Widney Daniel P. , Bui Kim C. , Rovai Leonor E.
Published: March 2015

Cytokines and other mediators whose induction in inflammatory lung disease is attenuated by glucocorticoids are potential targets for development of selective anti-inflammatory treatments. We refer to genes with these regulatory characteristics as glucocorticoid-attenuated response genes, or GARGs. Systematic identification of GARGs has not been attempted previously in vivo. Using an endotoxemia model in adrenalectomized mice, we constructed a subtracted lung library enriched in endotoxemia-induced genes and identified candidate GARGs by differential hybridization screening. Northern analysis confirmed induction in the lung during endotoxemia and attenuation by glucocorticoids of 36 genes of diverse types. The majority were genes of unknown function not previously implicated in the pulmonary response to inflammation, including a new member of a 2′–5′-oligoadenylate synthetase-like family and a novel lung inducible Neuralized-related C3HC4 RING protein. Our results suggest that a full understanding of glucocorticoid effects on lung inflammation will require elucidation of the roles of an extensive network of glucocorticoid-modulated genes.
Endogenous neurokinins facilitate synaptic transmission in guinea pig airway parasympathetic ganglia
Authors: Canning Brendan J. , Reynolds Sandra M. , Anukwu Linus U. , Kajekar Radhika , Myers Allen C.
Published: March 2015

Neurokinin-containing nerve fibers were localized to guinea pig airway parasympathetic ganglia in control tissues but not in tissues pretreated with capsaicin. The purpose of the present study was to determine whether neurokinins, released during axonal reflexes or after antidromic afferent nerve stimulation, modulate ganglionic synaptic neurotransmission. The neurokinin type 3 (NK3) receptor antagonists SB-223412 and SR-142801 inhibited vagally mediated cholinergic contractions of bronchi in vitro at stimulation voltages threshold for preganglionic nerve activation but had no effect on vagally mediated contractions evoked at optimal voltage or field stimulation-induced contractions. Intracellular recordings from the ganglia neurons revealed that capsaicin-sensitive nerve stimulation potentiated subsequent preganglionic nerve-evoked fast excitatory postsynaptic potentials. This effect was mimicked by the NK3 receptor agonist senktide analog and blocked by SB-223412. In situ, senktide analog markedly increased baseline tracheal cholinergic tone, an effect that was reversed by atropine and prevented by vagotomy or SB-223412. Comparable effects of intravenous senktide analog on pulmonary insufflation pressure were observed. These data highlight the important integrative role played by parasympathetic ganglia and indicate that activation of NK3 receptors in airway ganglia by endogenous neurokinins facilitates synaptic neurotransmission.
GLP-1 receptor signaling contributes to anorexigenic effect of centrally administered oxytocin in rats
Authors: Rinaman Linda , Rothe Elizabeth E.
Published: March 2015

The present study examined possible interactions between central glucagon-like peptide-1 (GLP-1) and oxytocin (OT) neural systems by determining whether blockade of GLP-1 receptors attenuates OT-induced anorexia and vice versa. Male rats were acclimated to daily 4-h food access. In the first experiment, rats were infused centrally with GLP-1 receptor antagonist or vehicle, followed by an anorexigenic dose of synthetic OT. Access to food began 20 min later. Cumulative food intake was measured every 30 min for 4 h. In the second experiment, rats were infused with OT receptor blocker or vehicle, followed by synthetic GLP-1 [(7–36) amide]. Subsequent food intake was monitored as before. The anorexigenic effect of OT was eliminated in rats pretreated with the GLP-1 receptor antagonist. Conversely, GLP-1-induced anorexia was not affected by blockade of OT receptors. In a separate immunocytochemical study, OT-positive terminals were found closely apposed to GLP-1-positive perikarya, and central infusion of OT activated c-Fos expression in GLP-1 neurons. These findings implicate endogenous GLP-1 receptor signaling as an important downstream mediator of anorexia in rats after activation of central OT neural pathways.
Molecular cloning, characterization, and distribution of the gerbil angiotensin II AT2receptor
Authors: Hoe Kwang-Lae , Armando Ines , Baiardi Gustavo , Sreenath Taduru , Kulkarni Ashok , Martiánez Alfredo , Saavedra Juan M.
Published: March 2015

We isolated a cDNA clone encoding the gerbil AT2receptor (gAT2) gene from a gerbil adrenal gland cDNA library. The full-length cDNA contains a 1,089-bp open reading frame encoding 363 amino acid residues with 90.9, 96.1, and 95.6% identity with the human (hAT2), rat (rAT2), and mouse AT2(mAT2) receptors, respectively. There are at least seven nonconserved amino acids in the NH2-terminal domain and in positions Val196, Val217, and Met293, important for angiotensin (ANG) II but not for CGP-42112 binding. Displacement studies in adrenal sections revealed that affinity of the gAT2receptor was 10-20 times lower for ANG II, ANG III, and PD-123319 than was affinity of the rAT2receptor. The affinity of each receptor remained the same for CGP-42112. When transfected into COS-7 cells, the gAT2receptor shows affinity for ANG II that is three times lower than that shown by the hAT2receptor, whereas affinities for ANG III and the AT2receptor ligands CGP-42112 and PD-123319 were similar. Autoradiography in sections of the gerbil head showed higher binding in muscles, retina, skin, and molars at embryonic day 19 than at 1 wk of age. In situ hybridization and emulsion autoradiography revealed that at embryonic day 19 the gAT2receptor mRNA was highly localized to the base of the dental papilla of maxillary and mandibular molars. Our results suggest selective growth-related functions in late gestation and early postnatal periods for the gAT2receptor and provide an essential basis for future mutagenesis studies to further define structural requirements for agonist binding.
Relationship between blood pressure, sleep K-complexes, and muscle sympathetic nerve activity in humans
Authors: Tank Jens , Diedrich Andre , Hale Nanette , Niaz Faiz E. , Furlan Raffaello , Robertson Rose Marie , Mosqueda-Garcia Rogelio
Published: March 2015

Stage 2 sleep is characterized by the EEG appearance of “K-complexes” and blood pressure oscillations. K-complexes may be directly related to blood pressure changes or they may reflect central sympathetic activation. We analyzed the temporal relationship among K-complexes, heart rate (HR), blood pressure (BP), and muscle sympathetic nerve activity (MSNA) during sleep in eight healthy volunteers (3 men and 5 women, age 22–41 yr). Most K-complexes presented as single large complexes (56 ± 20%), followed by single small complexes (15 ± 14%) and as couplets or triplets (13 ± 6%). Single large K-complexes were preceded by a baroreflex-mediated increase of MSNA in approximately one-half (55%) of the cases. Detailed analysis of HR, BP, and MSNA was possible in 63 (45%) large single K-complexes not disturbed by preceding baroreflex-related changes. Systolic and diastolic BP and MSNA increased significantly after single events (22.5 ± 13, 5.2 ± 2.1, and 6.5 ± 3.0%). Mean sympathetic baroreflex latency was similar after the single large K-complexes compared with the mean value during stage 2 sleep (1,290 ± 126 vs. 1,279 ± 61 ms). The area under the burst was significantly increased after single large K-complexes (median 3.9 vs. 9.0 arbitrary units, P < 0.03). The results support the hypothesis that K-complexes express cortical activation leading to temporary facilitation of sympathetic outflow in a graded fashion. Their functional effects appear to be independent of baroreflex modulation of MSNA in ∼50% of the cases.
Waterborne vs. dietary copper uptake in rainbow trout and the effects of previous waterborne copper exposure
Authors: Kamunde Collins , Clayton Cheryl , Wood Chris M.
Published: March 2015

Juvenile rainbow trout ( Oncorhynchus mykiss) were exposed to waterborne Cu (22 μg/l) in moderately hard water for up to 28 days. Relative to control fish kept at background Cu levels (2 μg/l), Cu-preexposed fish displayed decreased uptake rates of waterborne Cu via the gills but not of dietary Cu via the gut during 48-h exposures to64Cu-radiolabeled water and diet, respectively. At normal dietary and waterborne Cu levels, the uptake rates of dietary Cu into the whole body without the gut were 0.40–0.90 ng · g−1· h−1, >10-fold higher than uptake rates of waterborne Cu into the whole body without the gills, which were 0.02–0.07 ng · g−1· h−1. Previously Cu-exposed fish showed decreased new Cu accumulation in the gills, liver, and carcass during waterborne64Cu exposures and in the liver during dietary64Cu exposures. A 3-h gill Cu-binding assay showed downregulation of the putative high-affinity, low-capacity Cu transporters and upregulation of the low-affinity, high-capacity Cu transporters at the gills in Cu-preexposed fish. Exchangeable Cu pools in all the tissues were higher during dietary than during waterborne64Cu exposures, and previous Cu exposure reduced waterborne exchangeable Cu pools in gill, liver, and carcass. Overall, these results suggest a quantitatively greater role for the dietary than for the waterborne route of Cu uptake, a key role for the gill in Cu homeostasis, and important roles for the liver and gut in the normal metabolism of Cu in fish.
Comparison of the effects of ammonia on brain mitochondrial function in rats and gulf toadfish
Authors: Veauvy Clémence M. , Wang Yuxiang , Walsh Patrick J. , Pérez-Pinzón Miguel A.
Published: March 2015

We compared the effect of hyperammonemia on NADH levels in brain slices and on the rate of oxygen consumption from isolated nonsynaptic brain mitochondria in ammonia-sensitive Wistar rats with that in ammonia-tolerant gulf toadfish ( Opsanus beta). The NADH content was significantly decreased (12% less than control after 45 min with 1 mM NH4Cl) in rat brain slices, but it was not affected in brain slices from toadfish (with both 1 and 6 mM NH4Cl). The rates of oxygen consumption of different sets of enzymes of the electron transport chain (ETC; complexes I, II, III, and IV; II, III, and IV; and IV alone) were unaltered by hyperammonemic conditions in isolated nonsynaptic mitochondria from either rats or toadfish. These results lead us to conclude that the differing effects of ammonia on NADH levels in rat and toadfish brain slices must be due to aspects other than the direct effects of ammonia on enzymes of the ETC. Additionally, because these effects were seen in vitro, our studies enabled us to rule out the possibility that effects of ammonia on metabolism were via indirect systemic effects. These results are discussed in the context of current views on mechanisms of central nervous system damage in hyperammonemic states.
Endothelin-1 and -3 diminish neuronal NE release through an NO mechanism in rat anterior hypothalamus
Authors: Di Nunzio Andrea S. , Jaureguiberry María S. , Rodano Valeria , Bianciotti Liliana G. , Vatta Marcelo S.
Published: March 2015

The existence of endothelin binding sites on the catecholaminergic neurons of the hypothalamus suggests that endothelins (ETs) participate in the regulation of noradrenergic transmission modulating various hypothalamic-controlled processes such as blood pressure, cardiovascular activity, etc. The effects of ET-1 and ET-3 on the neuronal release of norepinephrine (NE) as well as the receptors and intracellular pathway involved were studied in the rat anterior hypothalamus. ET-1 (10 nM) and ET-3 (10 nM) diminished neuronal NE release and the effect blocked by the selective ET type B receptor antagonist BQ-788 (100 nM). Nω-nitro-l-arginine methyl ester (10 μM), methylene blue (10 μM), and KT5823 (2 μM), inhibitors of nitric oxide synthase activity, guanylate cyclase, and protein kinase G, respectively, prevented the inhibitory effects of both ETs on neuronal NE release. In addition, both ETs increased nitric oxide synthase activity. Furthermore, 100 μM picrotoxin, a GABAA-receptor antagonist, inhibited ET-1 and ET-3 response. Our results show that ET-1 as well as ET-3 has an inhibitory neuromodulatory effect on NE release in the anterior hypothalamus mediated by the ET type B receptor and the involvement of a nitric oxide-dependent pathway and GABAAreceptors. ET-1 and ET-3 may thus diminish available NE in the synaptic gap leading to decreased noradrenergic activity.