bluuebunny/crossref_metadata_2025 huggingface dataset:
Responsiveness of canine bronchial vasculature to excitatory stimuli and to cooling
Authors: Janssen Luke J. , Lu-Chao Hwa , Netherton Stuart
Published: December 2017

Changes in bronchial vascular tone, in part due to cooling during ventilation, may contribute to altered control of airflow during airway inflammation, asthma, and exercise-induced bronchoconstriction. We investigated the responses of canine bronchial vasculature to excitatory stimuli and cooling. Electrical stimulation evoked contractions in only some (8 of 88) tissues; these were phentolamine sensitive and augmented by Nω-nitro-l-arginine. However, sustained contractions were evoked in all tissues by phenylephrine [concentration evoking a half-maximal response (EC50) ≈2 μM] or the thromboxane A2mimetic U-46619 (EC50≈5 nM) and less so by β,γ-methylene-ATP or histamine. Cooling to room temperature markedly suppressed (≈75%) adrenergic responses but had no significant effect against U-46619 responses. Adrenergic responses, but not those to U-46619, were accompanied by an increase in intracellular Ca2+concentration. Chelerythrine (protein kinase C antagonist) markedly antagonized adrenergic responses (mean maxima reduced 39% in artery and 86% in vein) but had no significant effect against U-46619, whereas genistein (a nonspecific tyrosine kinase inhibitor) essentially abolished responses to both agonists. We conclude that cooling of the airway wall dramatically interferes with adrenergic control of bronchial perfusion but has little effect on thromboxane-mediated vasoconstriction.
Neutrophil defensins mediate acute inflammatory response and lung dysfunction in dose-related fashion
Authors: Zhang Haibo , Porro Giuliana , Orzech Neil , Mullen Brendan , Liu Mingyao , Slutsky Arthur S.
Published: December 2017

High concentrations of neutrophil defensins from airway and blood have been reported in patients with inflammatory lung diseases, but their exact role is unclear. We investigated the direct effect of defensins on the lungs of mice. Intratracheal instillation of purified defensins (5–30 mg/kg) induced a progressive reduction in peripheral arterial O2saturation, increased lung permeability, and enhanced the lung cytochrome c content. These indexes of acute lung dysfunction were associated with an increased total cell number and a significant neutrophil influx into the lung [5.1 ± 0.04% in control vs. 48.6 ± 12.7% in the defensin (30 mg/kg) group, P < 0.05]. Elastase concentrations in the bronchoalveolar lavage (BAL) fluids increased from 38 ± 11 ng/ml (control) to 80 ± 4 ng/ml (defensins, P < 0.05). Five hours after defensin instillation, concentrations of tumor necrosis factor- α and macrophage inflammatory protein-2 in BAL fluid were significantly increased. High levels of monocyte chemoattractant protein-1 in BAL fluid and plasma were also found after defensin stimulation. We conclude that intratracheal instillation of defensins causes acute lung inflammation and dysfunction, suggesting that high concentrations of defensins in the airways may play an important role in the pathogenesis of inflammatory lung diseases.
Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II
Authors: Borbiev Talaibek , Verin Alexander D. , Shi Shu , Liu Feng , Garcia Joe G. N.
Published: December 2017

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca2+-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca2+- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.
Potassium channels regulate tone in rat pulmonary veins
Authors: Michelakis Evangelos D. , Weir E. Kenneth , Wu Xichen , Nsair Ali , Waite Ross , Hashimoto Kyoko , Puttagunta Lakshmi , Knaus Hans Gunther , Archer Stephen L.
Published: December 2017

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K+ channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (KV) and inward rectifier (Kir) K+ channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba2+, and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several KV, Kir, and large-conductance Ca2+-sensitive K+channels are present in PVs. Immunohistochemistry showed that Kir channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K+ channels are present and functionally important in rat PVs. PVCMs form sphincters rich in Kir channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.
Cyclophosphamide stimulates lung fibroblasts to release neutrophil and monocyte chemoattractants
Authors: Koyama Sekiya , Takamizawa Akemi , Sato Etsuro , Masubuchi Takeshi , Nagai Sonoko , Izumi Takateru
Published: December 2017

Cyclophosphamide is an alkylating antineoplastic agent used in several conditions. However, little is known about the mechanism of its pulmonary toxicity. In the present study, we determined that human lung fibroblasts release activity for neutrophils and monocytes in response to cyclophosphamide in a dose- and time-dependent manner. Checkerboard analysis revealed that both neutrophil and monocyte activities were chemotactic. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular-sieve column chromatography revealed that both neutrophil (NCA) and monocyte (MCA) chemotactic activities had multiple peaks. NCA was inhibited by a leukotriene B4receptor antagonist and anti-interleukin-8 and anti-granulocyte colony-stimulating factor antibodies. MCA was attenuated by a leukotriene B4receptor antagonist and anti-monocyte chemoattractant protein-1 and anti-granulocyte-macrophage colony-stimulating factor antibodies. The concentrations of interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor significantly increased in response to cyclophosphamide. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to cyclophosphamide.
Lung uptake of antibodies to endothelial antigens: key determinants of vascular immunotargeting
Authors: Danilov Sergei M. , Gavrilyuk Vitaly D. , Franke Folker E. , Pauls Katarina , Harshaw David W. , McDonald Timothy D. , Miletich David J. , Muzykantov Vladimir R.
Published: December 2017

Vascular immunotargeting is a mean for a site-selective delivery of drugs and genes to endothelium. In this study, we compared recognition of pulmonary and systemic vessels in rats by candidate carrier monoclonal antibodies (MAbs) to endothelial antigens platelet endothelial cell adhesion molecule (PECAM)-1 (CD31), intercellular adhesion molecule (ICAM)-1 (CD54), Thy-1.1 (CD90.1), angiotensin-converting enzyme (ACE; CD143), and OX-43. Tissue immunostaining showed that endothelial cells were Thy-1.1 positive in capillaries but negative in large vessels. In the lung, anti-ACE MAb provided a positive staining in 100% capillaries vs. 5–20% capillaries in other organs. Other MAbs did not discriminate between pulmonary and systemic vessels. We determined tissue uptake after infusion of 1 μg of125I-labeled MAbs in isolated perfused lungs (IPL) or intravenously in intact rats. Uptake in IPL attained 46% of the injected dose (ID) of anti-Thy-1.1 and 20–25% ID of anti-ACE, anti-ICAM-1, and anti-OX-43 (vs. 0.5% ID of control IgG). However, after systemic injection at this dose, only anti-ACE MAb 9B9 displayed selective pulmonary uptake (16 vs. 1% ID/g in other organs). Anti-OX-43 displayed low pulmonary (0.5% ID/g) but significant splenic and cardiac uptake (7 and 2% ID/g). Anti-Thy-1.1 and anti-ICAM-1 displayed moderate pulmonary (4 and 6% ID/g, respectively) and high splenic and hepatic uptake (e.g., 18% ID/g of anti-Thy-1.1 in spleen). The lung-to-blood ratio was 5, 10, and 15 for anti-Thy-1.1, anti-ACE, and anti-ICAM-1, respectively. PECAM antibodies displayed low pulmonary uptake in perfusion (2% ID) and in vivo (3–4% ID/g). However, conjugation with streptavidin (SA) markedly augmented pulmonary uptake of anti-PECAM in perfusion (10–54% ID, depending on an antibody clone) and in vivo (up to 15% ID/g). Therefore, ACE-, Thy-1.1-, ICAM-1-, and SA-conjugated PECAM MAbs are candidate carriers for pulmonary targeting. ACE MAb offers a high selectivity of pulmonary targeting in vivo, likely because of a high content of ACE-positive capillaries in the lungs.
Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells
Authors: Ethier Michael F. , Yamaguchi Hiroshi , Madison J. Mark
Published: December 2017

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 μM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 μM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]ifrom 166 ± 11 to 671 ± 100 nM, and then [Ca2+]idecreased to a sustained level (CPA plateau; 236 ± 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]iwere observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]iduring stimulation with caffeine and, to a lesser extent, carbachol.
Tyrosine kinase receptor activation inhibits NPR-C in lung arterial smooth muscle cells
Authors: Sun Ju-Zhong , Oparil Suzanne , Lucchesi Pamela , Thompson John A. , Chen Yiu-Fai
Published: December 2017

We have previously demonstrated that expression of the atrial natriuretic peptide (ANP) clearance receptor (NPR-C) is reduced selectively in the lung of rats and mice exposed to hypoxia but not in pulmonary arterial smooth muscle cells (PASMCs) cultured under hypoxic conditions. The current study tested the hypothesis that hypoxia-responsive growth factors, fibroblast growth factors (FGF-1 and FGF-2) and platelet-derived growth factor-BB (PDGF-BB), that activate tyrosine kinase receptors can reduce expression of NPR-C in PASMCs independent of environmental oxygen tension. Growth-arrested rat PASMCs were incubated under hypoxic conditions (1% O2) for 24 h; with FGF-1, FGF-2, or PDGF-BB (0.1–20 ng/ml for 1–24 h); or with ANG II (1–100 nM), endothelin-1 (ET-1, 0.1 μM), ANP (0.1 μM), sodium nitroprusside (SNP, 0.1 μM), or 8-bromo-cGMP (0.1 mM) for 24 h under normoxic conditions. Steady-state NPR-C mRNA levels were assessed by Northern blot analysis. FGF-1, FGF-2, and PDGF-BB induced dose- and time-dependent reduction of NPR-C mRNA expression within 1 h at a threshold concentration of 1 ng/ml; hypoxia, ANG II, ET-1, ANP, SNP, or cGMP did not decrease NPR-C mRNA levels in PASMCs under the above conditions. Downregulation of NPR-C expression by FGF-1, FGF-2, and PDGF-BB was inhibited by the selective FGF-1 receptor tyrosine kinase inhibitor PD-166866 and mitogen-activated protein/extracellular signal-regulated kinase inhibitors U-0126 and PD-98059. These results indicate that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but neither hypoxia per se nor activation of G protein-coupled receptors, inhibits NPR-C gene expression in PASMCs. These results suggest that FGF-1, FGF-2, and PDGF-BB play a role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung.
Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia
Authors: Levy Mark A. , Tsai Yu-Hwai , Reaume Andrew , Bray Tammy M.
Published: December 2017

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.
Effect of chronic hypoxia on agonist-induced tone and calcium signaling in rat pulmonary artery
Authors: Bonnet Sébastien , Belus Alexandra , Hyvelin Jean-Marc , Roux Etienne , Marthan Roger , Savineau Jean-Pierre
Published: December 2017

The effect of chronic hypoxia (CH) for 14 days on Ca2+signaling and contraction induced by agonists in the rat main pulmonary artery (MPA) was investigated. In MPA myocytes obtained from control (normoxic) rats, endothelin (ET)-1, angiotensin II (ANG II), and ATP induced oscillations in intracellular Ca2+concentration ([Ca2+]i) in 85–90% of cells, whereas they disappeared in myocytes from chronically hypoxic rats together with a decrease in the percentage of responding cells. However, both the amount of mobilized Ca2+and the sources of Ca2+implicated in the agonist-induced response were not changed. Analysis of the transient caffeine-induced [Ca2+]iresponse revealed that recovery of the resting [Ca2+]ivalue was delayed in myocytes from chronically hypoxic rats. The maximal contraction induced by ET-1 or ANG II in MPA rings from chronically hypoxic rats was decreased by 30% compared with control values. Moreover, the D-600- and thapsigargin-resistant component of contraction was decreased by 40% in chronically hypoxic rats. These data indicate that CH alters pulmonary arterial reactivity as a consequence of an effect on both Ca2+signaling and Ca2+sensitivity of the contractile apparatus. A Ca2+reuptake mechanism appears as a CH-sensitive phenomenon that may account for the main effect of CH on Ca2+signaling.
Surfactant protein interactions with neutral and acidic phospholipid films
Authors: Rodriguez-Capote Karina , Nag Kaushik , Schürch Samuel , Possmayer Fred
Published: December 2017

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (POPG) at 50 μg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37°C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (γ), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in γminwas observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, γmaxwas lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low γminvalues with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low γ during film compression and lipid respreading during film expansion.
Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia
Authors: Gao Lin , Broughman James R. , Iwamoto Takeo , Tomich John M. , Venglarik Charles J. , Forman Henry Jay
Published: December 2017

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl−transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113–L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl−channel-forming peptide (N-K4-M2GlyR) and a K+channel activator (chlorzoxazone) increased Cl−secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 ± 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 ± 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.
Nitric oxide modulates capillary formation at the endothelial cell-tumor cell interface
Authors: Phillips Patricia G. , Birnby Linda M. , Narendran Amithi , Milonovich Wendy L.
Published: December 2017

Nitric oxide synthase expression has been documented in lung tumors, but a potential role for nitric oxide (NO) in induction of capillary formation remains to be elucidated. The purpose of this report was to characterize the direct effects of NO at the level of the tumor-endothelium interface with respect to angiogenesis. A Transwell two-compartment culture system, human endothelial cells (EC), and two human non-small cell lung cancer (CA) lines that constitutively produce NO were used to simulate the EC-tumor cell interface. Both histological types of lung CA, squamous and adenocarcinoma, induced baseline capillary formation by EC within 3 days. This process was inhibited by NO in the microenvironment because decreasing NO production with 100 μM aminoguanidine (AG) significantly increased capillary formation, whereas coincubation with 100 μM AG plus 400 μM l-arginine returned angiogenesis to baseline values. We demonstrate further that NO may exert its inhibitory effects by influencing matrix metalloproteinase expression/activity and tyrosine phoshorylation of proteins in the sprouting tips of nascent capillaries.
Partial restoration of defective chloride conductance in ΔF508 CF mice by trimethylamine oxide
Authors: Fischer Horst , Fukuda Norimasa , Barbry Pascal , Illek Beate , Sartori Claudio , Matthay Michael A.
Published: December 2017

This study was designed to test the in vivo efficacy of the chemical chaperone trimethylamine oxide (TMAO) in correcting the Cl−transport defect in a mouse model of cystic fibrosis (CF). Rectal potential difference (RPD) measurements were done in matched wild-type and ΔF508 CF mice. Mice were treated by subcutaneous injections of TMAO. Wild-type mice demonstrated a forskolin-stimulated, Cl−-dependent hyperpolarization of −6.4 ± 0.8 mV ( n = 11), which was significantly increased to −13.1 ± 1.4 mV after treatment with TMAO. ΔF508 CF mice showed no significant responses to forskolin. Treatment with TMAO recovered a forskolin-activated RPD in ΔF508 CF mice (−1.1 ± 0.2 mV; n = 17) but not in CFTR null mice. The effects of TMAO were dose dependent, resulting in a slope of −0.4 ± 0.1 mV · g−1· kg−1in ΔF508 CF mice. The forskolin-stimulated RPD in TMAO-treated ΔF508 CF mice was partially blocked by glibenclamide and further stimulated by apigenin. The total response to forskolin plus apigenin was −2.5 ± 0.45 mV ( n = 6 mice), corresponding to 39% of the response evoked by forskolin only in wild-type mice.
Augmented metalloproteinase activity and acute lung injury in copper-deficient rats
Authors: Lentsch Alex B. , Kato Atsushi , Saari Jack T. , Schuschke Dale A.
Published: December 2017

Dietary copper is required for normal function of >30 mammalian enzyme systems. Copper deficiency causes a number of cardiovascular defects as well as impaired immune cell function. Little is known regarding the effects of copper deficiency on acute inflammatory responses, but this topic is relevant because many members of the Western population receive less than the recommended dietary allowance of copper. In the current studies, we investigated the effects of dietary copper deficiency on acute lung injury induced by intrapulmonary deposition of IgG immune complexes. Weanling male Long-Evans rats were fed diets either adequate (5.6 μg/g) or deficient (0.3 μg/g) in copper. IgG immune complex lung injury was greatly increased in copper-deficient rats as determined by lung vascular leakage of albumin and histopathology. However, no change was observed in either the lung content of tumor necrosis factor-α or lung neutrophil accumulation. Lungs from copper-deficient rats had much higher levels of matrix metalloproteinase (MMP)-2 and MMP-9 than did copper-adequate control animals. This increased activity was not attributable to alveolar macrophages or neutrophils. These data suggest that the augmented lung injury caused by copper deficiency is due to increased pulmonary MMP-2 and MMP-9 activity and not a generalized amplification of the inflammatory response.
Interactions between CBP, NF-κB, and CREB in the lungs after hemorrhage and endotoxemia
Authors: Shenkar Robert , Yum Ho-Kee , Arcaroli John , Kupfner John , Abraham Edward
Published: December 2017

The transcriptional regulatory factor nuclear factor (NF)-κB has a central role in modulating expression of proinflammatory mediators that are important in acute lung injury. In vitro studies have shown that competition between NF-κB and cAMP response element binding protein (CREB) for binding to the coactivator CREB-binding protein (CBP) is important in regulating transcriptional activity of these factors. In the present study, we examined in vivo interactions between CBP, CREB, and NF-κB in hemorrhage- or endotoxemia-induced acute lung injury. Association of CBP with CREB or the p65 subunit of NF-κB increased in the lungs after hemorrhage or endotoxemia. Inhibition of xanthine oxidase before hemorrhage, but not before endotoxemia, decreased p65-CBP interactions while increasing those between CREB and CBP. These alterations in CREB-CBP and p65-CBP interactions were functionally significant because xanthine oxidase inhibition before hemorrhage resulted in increased expression of the CREB-dependent gene c-Fos and decreased expression of macrophage inflammatory protein-2, a NF-κB-dependent gene. The present results show that the coactivator CBP has an important role in modulating transcription in vivo under clinically relevant pathophysiological conditions.
Regulation of anion secretion by nitric oxide in human airway epithelial cells
Authors: Duszyk Marek
Published: December 2017

Nitric oxide (NO) is continuously produced and released in human airways, but the biological significance of this process is unknown. In this study, we have used Calu-3 cells to investigate the effects of NO on transepithelial anion secretion. An inhibitor of NO synthase, NG-nitro-l-arginine methyl ester, reduced short- circuit current ( Isc), whereas an NO donor, S-nitrosoglutathione (GSNO), increased Isc, with an EC50∼1.2 μM. The NO-activated current was inhibited by diphenylamine-2-carboxylate, clotrimazole, and charybdotoxin. Selective permeabilization of cell membranes indicated that NO activated both apical anion channels and basolateral potassium channels. An inhibitor of soluble guanylate cyclase, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one, prevented activation of Iscby NO but not by 8-bromo-cGMP, suggesting that NO acts via a cGMP-dependent pathway. Sequential treatment of cells with forskolin and GSNO or 1-ethyl-2-benzimidazolinone and GSNO showed additive effects of these chemicals on Isc. Interestingly, GSNO elevated intracellular Ca2+concentration ([Ca2+]i) but had no effect on Iscactivated by thapsigargin. These results show that NO activates transepithelial anion secretion via a cGMP-dependent pathway that involves cross talk between NO and [Ca2+]i.
Selective inhibition of COX-2 improves early survival in murine endotoxemia but not in bacterial peritonitis
Authors: Reddy Raju C. , Chen Gina H. , Tateda Kazuhiro , Tsai Wan C. , Phare Susan M. , Mancuso Peter , Peters-Golden Marc , Standiford Theodore J.
Published: December 2017

Prostaglandins of the E series are believed to act as important mediators of several pathophysiological events that occur in sepsis. Studies were performed to evaluate the effect of cyclooxygenase (COX)-2-specific inhibition on the outcome in murine endotoxemia and cecal ligation and puncture (CLP). We observed a significant time-dependent upregulation of PGE2production in both blood and lung homogenates of mice administered lipopolysaccharide intraperitoneally, which was nearly completely suppressed by the administration of the COX-2 inhibitor NS-398. Treatment with NS-398 significantly improved early but not late survival in lipopolysaccharide-challenged mice. On the contrary, elevated PGE2levels were found in bronchoalveolar lavage fluid but not in plasma of mice subjected to CLP (21 gauge). Pretreatment with NS-398 failed to significantly improve survival in CLP mice. No significant differences were noted in plasma or lung homogenate proinflammatory cytokine levels or lung neutrophil sequestration between the NS-398-treated and control groups. These results demonstrate that selective COX-2 inhibition confers early but not long-term benefits without affecting the expression of proinflammatory cytokines or the development of lung inflammation.
Alveolar fluid reabsorption is impaired by increased left atrial pressures in rats
Authors: Saldı́as F. J. , Azzam Z. S. , Ridge K. M. , Yeldandi A. , Rutschman D. H. , Schraufnagel D. , Sznajder J. I.
Published: December 2017

Cardiogenic pulmonary edema results from increased hydrostatic pressures across the pulmonary circulation. We studied active Na+ transport and alveolar fluid reabsorption in isolated perfused rat lungs exposed to increasing levels of left atrial pressure (LAP; 0–20 cmH2O) for 60 min. Active Na+ transport and fluid reabsorption did not change when LAP was increased to 5 and 10 cmH2O compared with that in the control group (0 cmH2O; 0.50 ± 0.02 ml/h). However, alveolar fluid reabsorption decreased by ∼50% in rat lungs in which the LAP was raised to 15 cmH2O (0.25 ± 0.03 ml/h). The passive movement of small solutes (22Na+ and [3H]mannitol) and large solutes (FITC-albumin) increased progressively in rats exposed to higher LAP. There was no significant edema in lungs with a LAP of 15 cmH2O when all active Na+ transport was inhibited by hypothermia or amiloride (10−4 M) and ouabain (5 × 10−4 M). However, when LAP was increased to 20 cmH2O, there was a significant influx of fluid (−0.69 ± 0.10 ml/h), precluding the ability to assess the rate of fluid reabsorption. In additional studies, LAP was decreased from 15 to 0 cmH2O in the second and third hours of the experimental protocol, which resulted in normalization of lung permeability to solutes and alveolar fluid reabsorption. These data suggest that in an increased LAP model, the changes in clearance and permeability are transient, reversible, and directly related to high pulmonary circulation pressures.
AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen
Authors: Andrew Angeline S. , Klei Linda R. , Barchowsky Aaron
Published: December 2017

Inhalation of nickel dust has been associated with an increased incidence of pulmonary fibrosis. Nickel may promote fibrosis by transcriptionally activating plasminogen activator inhibitor (PAI)-1 and inhibiting fibrinolysis. The current studies examined whether nickel stimulated the PAI-1 promoter though an oxidant-sensitive activator protein (AP)-1 signaling pathway. Addition of nickel to BEAS-2B human airway epithelial cells stimulated intracellular oxidation, induced c-Jun and c-Fos mRNA levels, increased phospho- and total c-Jun protein levels, and elevated PAI-1 mRNA levels over a 24-h time course. Pretreatment of the cells with antioxidants did not affect increased c-Jun protein or PAI-1 mRNA levels. Expression of the dominant negative inhibitor of AP-1, TAM67, prevented nickel-stimulated AP-1 DNA binding, AP-1-luciferase reporter construct activity, and PAI-1 mRNA levels. Overexpression of c-Jun, however, failed to induce the AP-1 luciferase reporter construct or PAI-1 mRNA levels. These data indicated that nickel activated AP-1 through an oxidant-independent pathway and that basal AP-1 is necessary for nickel-induced expression of PAI-1.
Norepinephrine induces alveolar epithelial apoptosis mediated by α-, β-, and angiotensin receptor activation
Authors: Dincer H. Erhan , Gangopadhyay Nupur , Wang Rongqi , Uhal Bruce D.
Published: December 2017

Norepinephrine (NE) induces apoptosis in cardiac myocytes, and autocrine production of angiotensin (ANG) II is required for apoptosis of alveolar epithelial cells (AECs) (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245–L1250, 1999; Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, and Uhal BD. J Cell Physiol 185: 253–259, 2000). On this basis, we hypothesized that NE might induce apoptosis of AECs in a manner inhibitable by ANG system antagonists. Purified NE induced apoptosis in the human A549 AEC-derived cell line or in primary cultures of rat AECs, with EC50 values of 200 and 20 nM, respectively. Neither the α-agonist phenylephrine nor the β-agonist isoproterenol could mimic NE when tested alone but when applied together could induce apoptosis with potency equal to NE. Apoptosis and net cell loss (47–59% in 40 h) in response to NE was completely abrogated by the ANG-converting enzyme inhibitor lisinopril or the ANG II receptor antagonist saralasin, each at concentrations capable of blocking Fas- or tumor necrosis factor-α-induced apoptosis. These data suggest that NE induces apoptosis of human and rat AECs through a mechanism involving the combination of α- and β-adrenoceptor activation followed by autocrine generation of ANG II.
Calcium sensitization produced by G protein activation in airway smooth muscle
Authors: Yoshimura Hayashi , Jones Keith A. , Perkins William J. , Kai Tetsuya , Warner David O.
Published: December 2017

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca2+concentration ([Ca2+]; i.e., the Ca2+sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in α-toxin-permeabilized canine tracheal smooth muscle were determined with Ca2+alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF[Formula: see text]; acute or 1-h exposure). Acute exposure to each compound increased Ca2+sensitivity without changing the response to high [Ca2+] (maximal force). However, chronic exposure to AlF[Formula: see text], but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF[Formula: see text] exposure required Ca2+during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.
H2O2signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells
Authors: Lee S. L. , Simon A. R. , Wang W. W. , Fanburg B. L.
Published: December 2017

Our previous studies have shown that 5-hydroxytryptamine (5-HT) induces cellular hyperplasia/hypertrophy through protein tyrosine phosphorylation, rapid formation of superoxide (O[Formula: see text]), and extracellular signal-regulated kinase (ERK)1/ERK2 mitogen-activated protein (MAP) kinase activation. Intracellularly released O[Formula: see text] is rapidly dismuted by superoxide dismutase (SOD) to H2O2, another possible cellular growth mediator. In the present study, we assessed whether H2O2participates in 5-HT-induced mitogenic signaling. Inactivation of cellular Cu/Zn SOD by copper-chelating agents inhibited 5-HT-induced DNA synthesis of bovine pulmonary artery smooth muscle cells (BPASMCs). Infection of BPASMCs with an adenovirus containing catalase inhibited both ERK1/ERK2 MAP kinase activation and DNA synthesis induced by 5-HT. Although we could not find evidence of p38 MAP kinase activation by 5-HT, SB-203580 and SB-202190, reported inhibitors of p38 MAP kinase, inhibited the 5-HT-induced growth of BPASMCs. However, these inhibitors also inhibited 5-HT-induced O[Formula: see text] release. Thus quenching of O[Formula: see text]may be their mechanism for inhibition of cellular growth unrelated to p38 MAP kinase inhibition. These data indicate that generation of O[Formula: see text] in BPASMCs in response to 5-HT is followed by an increase in intracellular H2O2that mediates 5-HT-induced mitogenesis through activation of ERK1/ERK2 but not of p38 MAP kinase.
Airway inflammation in nonasthmatic amateur runners
Authors: Bonsignore Maria R. , Morici Giuseppe , Riccobono Loredana , Insalaco Giuseppe , Bonanno Anna , Profita Mirella , Paternò Alessandra , Vassalle Cristina , Mirabella Angela , Vignola A. Maurizio
Published: December 2017

Elite athletes show a high prevalence of symptoms and signs of asthma, but no study has assessed the acute effects of endurance exercise on airway cells in nonasthmatic athletes. We measured exhaled nitric oxide (NO) and collected samples of induced sputum after 3% NaCl aerosol administration for 20 min in nonasthmatic middle-aged amateur runners after the Fourth Palermo International Marathon and 6–9 wk later (habitual training period) at baseline. After the marathon, exhaled NO ( n = 9 subjects) was higher [27 ± 9 parts/billion (ppb)] than at baseline (12 ± 4 ppb; P < 0.0005). Polymorphonuclear neutrophil (PMN) counts in induced sputum were much higher in runners (91.2 ± 3.6% of total cells postmarathon and 78.7 ± 9.1% at baseline) than in sedentary control subjects (9.9 ± 5.9%; P < 0.001). Expression of L-selectin and CD11b/CD18 in sputum PMNs was lower after the race than at baseline and inversely related to the amount of exhaled NO ( r = −0.66 and −0.69, respectively; P < 0.05). Our data indicate that sputum PMNs are increased in nonasthmatic runners both after a marathon and at baseline and suggest that NO may modulate exercise-associated inflammatory airway changes.
A low level of TNF-α mediates hemorrhage-induced acute lung injury via p55 TNF receptor
Authors: Song Yong , Ao Lihua , Raeburn Christopher D. , Calkins Casey M. , Abraham Edward , Harken Alden H. , Meng Xianzhong
Published: December 2017

Acute lung injury after hemorrhagic shock (HS) is associated with the expression of tumor necrosis factor (TNF)-α in the lung. However, the role of TNF-α and its receptors in this pulmonary disorder remains obscure. This study examined the temporal relationship of pulmonary TNF-α production to neutrophil accumulation during HS and determined the role of TNF-α in neutrophil accumulation and lung leak. HS was induced in mice by removal of 30% of total blood volume. Lung TNF-α was measured by ELISA. Neutrophil accumulation was detected by immunofluorescent staining, and microvascular permeability was assessed using Evans blue dye. Although HS induced a slight and transient increase in lung TNF-α, neutrophil accumulation preceded the increase in TNF-α. However, lung neutrophil accumulation and lung leak were abrogated in TNF-α knockout mice, and both were restored by administration of recombinant TNF-α to TNF-α knockout mice before HS. Neutrophil accumulation and lung leak were abrogated in mice lacking the p55 TNF-α receptor, but neither was influenced by p75 TNF-α receptor knockout. This study demonstrates that a low level of pulmonary TNF-α is sufficient to mediate HS-induced acute lung injury during HS and that the p55 TNF-α receptor plays a dominant role in regulating the pulmonary inflammatory response to HS.
SP-D and GM-CSF regulate surfactant homeostasis via distinct mechanisms
Authors: Ikegami Machiko , Hull William M. , Yoshida Mitsuhiro , Wert Susan E. , Whitsett Jeffrey A.
Published: December 2017

Both surfactant protein (SP) D and granulocyte-macrophage colony-stimulating factor (GM-CSF) influence pulmonary surfactant homeostasis, with the deficiency of either protein causing marked accumulation of surfactant phospholipids in lung tissues and in the alveoli. To assess whether the effects of each gene were mediated by distinct or shared mechanisms, surfactant homeostasis and lung morphology were assessed in 1) double-transgenic mice in which both SP-D and GM-CSF genes were ablated [SP-D(−/−),GM(−/−)] and 2) transgenic mice deficient in both SP-D and GM-CSF in which the expression of GM-CSF was increased in the lung. Saturated phosphatidylcholine (Sat PC) pool sizes were markedly increased in SP-D(−/−),GM(−/−) mice, with the effects of each gene deletion on surfactant Sat PC pool sizes being approximately additive. Expression of GM-CSF in lungs of SP-D(−/−),GM(−/−) mice corrected GM-CSF-dependent abnormalities in surfactant catabolism but did not correct lung pathology characteristic of SP-D deletion. In contrast to findings in GM(−/−) mice, degradation of [3H]dipalmitoylphosphatidylcholine by alveolar macrophages from the SP-D(−/−) mice was normal. The emphysema and foamy macrophage infiltrates characteristic of SP-D(−/−) mice were similar in the presence or absence of GM-CSF. Taken together, these findings demonstrate the distinct roles of SP-D and GM-CSF in the regulation of surfactant homeostasis and lung structure.
Expression of CFTR and Cl−conductances in cells of pulmonary neuroepithelial bodies
Authors: Yeger Herman , Pan Jie , Fu Xiao Wen , Bear Christine , Cutz Ernest
Published: December 2017

The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl−conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease.
VEGF induces airway epithelial cell proliferation in human fetal lung in vitro
Authors: Brown Karen R. S. , England Katherine M. , Goss Kelli L. , Snyder Jeanne M. , Acarregui Michael J.
Published: December 2017

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [3H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.
Timing of hyperoxic exposure during alveolarization influences damage mediated by leukotrienes
Authors: Manji Jacqueline S. , O'Kelly Cian J. , Leung Wynne I. , Olson David M.
Published: December 2017

Hyperoxic exposure of rat pups during alveolarization (postnatal days 4–14) severely retards alveolar development. Some aspects of this inhibition are mediated by leukotrienes (LTs) and may be time sensitive. We determined 1) the effects of exposure to hyperoxia (O2) during discrete periods before and during alveolarization on developing alveoli and 2) whether a relationship exists between O2and LTs in these periods. Pups were exposed to >95% O2from days 1 to 4, 4 to 9, 9 to 14, or 4 to 14 in the absence and presence of the LT synthesis inhibitor MK-0591. Both the level of in vitro lung tissue LT output on days 4, 9, and 14and the degree of alveolarization on day 14 were determined. Pups exposed to O2from days 4 to 9had a more profound inhibition of alveolarization on day 14compared with those exposed to O2from days 1 to 4 or 9 to 14. Peptido-LT levels were significantly higher in pups exposed to O2on days 9 and 14 compared with pups in air and returned to normal once normoxia was restored. LT inhibition from days 4to 14, 4 to 9, or 9 to 14 in pups exposed to O2from days 4to 14 prevented the O2-induced inhibition of alveolarization. These data suggest that developing alveoli are sensitive to LTs shortly before and after day 9, significantly retarding certain parameters of alveolarization on day 14. We conclude that some of the effects of O2are not uniform throughout different stages of alveolarization and that this is likely related to the timing of LT exposure.
Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA2
Authors: Sano Akiko , Zhu Xiangdong , Sano Hiroyuki , Muñoz Nilda M. , Boetticher Evan , Leff Alan R.
Published: December 2017

We examined the regulatory role of cytosolic phospholipase A2(cPLA2) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C4synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC4, which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-β2 and -γ2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca2+concentration was inhibited by ET-18-OCH3, a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA2blocker, had no inhibitory effect. Both TFMK and ET-18-OCH3attenuated stimulated arachidonate release and LTC4secretion, suggesting that activation of both PLC and cPLA2is essential for LTC4synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31–8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC4secretion. 1,2-bis(2-Aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid acetoxymethyl ester, an intracellular Ca2+chelator, suppressed both EPO release and LTC4secretion. We found that fMLP/B-induced LTC4secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA2. However, cPLA2does not regulate eosinophil degranulation.