bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental regulation of Na, K-ATPase in rat lung
Authors: Ingbar D. H. , Weeks C. B. , Gilmore-Hebert M. , Jacobsen E. , Duvick S. , Dowin R. , Savik S. K. , Jamieson J. D.
Published: December 2017

Late in gestation lung epithelium changes from net chloride and fluid secretion to sodium and fluid absorption. Fluid resorption is required for postnatal gas exchange and occurs by combined action of epithelial sodium channels and Na, K-ATPase. We hypothesized that alveolar epithelial Na, K-ATPase increases perinatally. Immunofluorescence (IF) and immunoelectron microscopy (IEM) with a monoclonal anti-alpha subunit antibody demonstrated that Na, K-ATPase was present on the basolateral surfaces of columnar epithelial cells at fetal day (FD) 17 and on type II cells throughout development. However, type I epithelial cells did not have detectable Na,K-ATPase. The steady-state levels of both the alpha 1 isoform and the beta-subunit mRNAs were maximal at FD20-neonatal day (ND) 1, with consistent increases from FD17 level. Na, K-ATPase alpha-subunit protein also increased from FD17 to FD20-22 and then decreased in the early postnatal period. The ouabain-inhibitable sodium pump activity per milligram membrane protein increased 2.6-fold from FD17 to FD22-ND1 (P < 0.05). The quantities of sodium pump mRNA, antigenic protein, and enzyme activity increase in late gestation in accord with a proposed role for Na, K-ATPase in resorption of alveolar sodium and fluid in preparation for birth.
Muscarinic receptor regulation of synaptic transmission in airway parasympathetic ganglia
Authors: Myers A. C. , Undem B. J.
Published: December 2017

Muscarinic receptor regulation of synaptic transmission in guinea pig bronchial parasympathetic ganglia was evaluated with the use of intracellular recording of the intrinsic ganglion neurons. Methacholine (1 microM) decreased the amplitude of vagus nerve-stimulated fast excitatory postsynaptic potentials (fEPSP) by 33 and 46% (at 0.8 and 8.0 Hz, respectively) but had no effect on the amplitude of the depolarizations evoked by a bath-applied nicotinic receptor agonist. Methoctramine (1 microM) inhibited methacholine's effect on fEPSP but alone did not influence the magnitude of the fEPSP evoked by vagus nerve stimulation. Methacholine (10 microM) depolarized a subpopulation of neurons (approximately 4 mV), which was blocked by pirenzepine (0.1 microM). In other neurons, either no effect or a small (1-5 mV) hyperpolarization was noted. Cholinergic contractions of bronchial smooth muscle elicited by electrical field stimulation were potentiated by methoctramine to the same extent as those evoked by vagus nerve (preganglionic) stimulation. The data indicate that M2 receptor activation can lead to inhibition of presynaptic acetylcholine release and consequently a significant inhibition of synaptic transmission in bronchial parasympathetic ganglia. The physiological role of this neuromodulatory effect appears limited, however, when studied in the in vitro setting.
Involvement of annexin II in exocytosis of lamellar bodies from alveolar epithelial type II cells
Authors: Liu L. , Wang M. , Fisher A. B. , Zimmerman U. J.
Published: December 2017

Annexins are a family of Ca(2+)- and phospholipid-binding proteins that have been implicated in exocytosis. In the present study, we investigated the participation of selected annexins in exocytosis of lamellar bodies by examining their liposome aggregation property and ability to reconstitute surfactant secretion from permeabilized rat lung alveolar type II cells. Annexins I, II, III, and VI were demonstrated in type II cells by immunoblot analysis, but annexin IV and V were not found. Annexins I-IV mediated liposome aggregation in the presence of 1 mM Ca2+. However, only annexin II tetramer had aggregation activity at 10 microM Ca2+. Annexins V and VI had negligible aggregation activity at any Ca2+ concentrations (up to 1 mM Ca2+). To study reconstitution of secretion by annexins, isolated type II cells were permeabilized with 40 microM beta-escin. Under these conditions, the permeabilized cells released approximately 30-40% lactic acid dehydrogenase into the medium. An underestimated fraction of cellular annexin content was lost during permeabilization. However, lamellar bodies in the permeabilized type II cells stained appropriately with the fluorescent dyes Nile red and quinacrine, indicating that they were intact. These permeabilized cells were secretion competent, since phosphatidylcholine (PC) secretion was stimulated by 0.2-1.0 microM Ca2+. Addition of an exogenous annexin mixture enhanced PC secretion from the permeabilized type II cells with maximal stimulation at 0.5 microM Ca2+. Of six purified annexins (I-VI) tested for their ability to reconstitute secretion from permeabilized cells, only annexin II was effective. Our results suggest that annexin II is not necessary for exocytosis of lamellar bodies.
Integrins inhibit LPS-induced DNA strand breakage in cultured lung endothelial cells
Authors: Hoyt D. G. , Mannix R. J. , Gerritsen M. E. , Watkins S. C. , Lazo J. S. , Pitt B. R.
Published: December 2017

Collagen inhibits acute DNA strand breakage and apoptosis in sheep pulmonary artery endothelial cells (SPAEC) treated with lipopolysaccharide (LPS). Here we tested the ability of major basement membrane components, type IV collagen, laminin and fibronectin, and integrin ligands and anti-integrin antibodies to inhibit DNA breakage caused by LPS in SPAEC and BALB/c murine lung endothelial cells (MLEC). In situ labeling of DNA strand breaks with terminal deoxynucleotidyl transferase revealed similar DNA breakage in attached SPAEC and MLEC within 2 h after incubation with 1 microgram LPS/ml. Acute DNA strand breakage was reduced in cells plated on gelatin, type IV collagen, laminin, cellular fibronectin, or plasma fibronectin. DNA breakage was also suppressed by plating cells on surfaces coated with the integrin ligand hexapeptide, GRGDSP (40 micrograms/cm2), but not with GRADSP. LPS-induced DNA strand breakage was inhibited in MLEC plated on surfaces coated with antibodies to murine alpha 5-, beta 1, or beta 3-integrin subunits. Addition of anti-integrin antibodies, but not GRGDSP, to the medium above cell monolayers inhibited strand breakage. Despite similar acute DNA breakage, MLEC exhibited less detachment and apoptosis than SPAEC, consistent with a difference in the sensing or processing systems for apoptosis in these two cell types. These results demonstrate that extracellular matrices and integrin activation can inhibit the genotoxicity of LPS.
Bradykinin regulation of airway submucosal gland secretion: role of bradykinin receptor subtype
Authors: Nagaki M. , Shimura S. , Irokawa T. , Sasaki T. , Oshiro T. , Nara M. , Kakuta Y. , Shirato K.
Published: December 2017

To clarify the role of bradykinin receptor subtypes, we examined the effect of bradykinin on feline tracheal and human airway submucosal gland secretion using an isolated gland preparation. Bradykinin induced a significant increase in [3H]glycoconjugate secretion in a dose-dependent manner from isolated glands, which was significantly inhibited by D-Arg-(Hyp3, Thi5,8, D-Phe7)-bradykinin (the B2-receptor antagonist), whereas Des-Arg9-(Leu8)-bradykinin (B1-receptor antagonist) or indomethacin did not significantly alter it. Nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) caused a significant inhibition of bradykinin-induced glycoconjugate secretion, which was reversed by the addition of L-arginine. Bradykinin evoked bidirectional current responses, and an initial inward current (Cl- current) was followed by an outward current (K+ current) of the acinar cells in a whole cell configuration by patch-clamp technique. Bradykinin induced an immediate increase in intracellular calcium concentration ([Ca2+]i) of the acinar cells followed by a prolonged plateau, and Ca2+ removal resulted in an initial increase alone. [Ca2+]i rise was significantly inhibited by the B2-receptor antagonist, whereas the B1-receptor antagonist did not significantly alter it. These findings suggest that B2-receptor stimulation and the resultant [Ca2+]i rise induced both mucus glycoprotein and electrolyte secretions, involving NO formation in airway submucosal gland cells.
Rate of nitric oxide release in the lung and factors influencing the concentration of exhaled nitric oxide
Authors: Sato K. , Sakamaki T. , Sumino H. , Sakamoto H. , Hoshino J. , Masuda H. , Sawada Y. , Mochida M. , Ohyama Y. , Kurashina T. , Nakamura T. , Ono Z.
Published: December 2017

The level of nitric oxide (NO) in exhaled air fluctuates in normal individuals depending on the physiological conditions. We evaluated the effects of duration of exhalation and breath-holding on the exhaled concentrations of NO in 16 normal human volunteers. Exhaled gas corresponding to vital capacity was collected in 6-liter Tedlar bags and analyzed by chemiluminescence. The NO concentration in exhaled gas increased significantly in proportion to the duration of exhalation [P = 0.009 +/- 0.011 (SD)] and was increased after breath-holding. There was no significant difference in the exhaled NO concentration among 10-s phases of a 30-s exhalation, as determined from multiple breath collections. The NO released from the airways is presumably unaffected by fluctuation of exhalation speed. The NO release rate, calculated from a single regression analysis between the NO concentration and the duration of exhalation, was 39 +/- 29 pmol/s, a value which was about fourfold greater in nine patients with bronchial asthma.
Attenuation of endotoxin-induced pulmonary vascular injury by antithrombin III
Authors: Uchiba M. , Okajima K. , Murakami K. , Okabe H. , Takatsuki K.
Published: December 2017

We evaluated the effects of antithrombin III (AT III) on the pulmonary vascular injury induced by injecting rats with lipopolysaccharide (LPS) to investigate the possible usefulness of AT III as a treatment for acute respiratory distress syndrome. The intravenous administration of AT III prevented the pulmonary accumulation of leukocytes (as evaluated by myeloperoxidase activity) and the increase in pulmonary vascular permeability to 125I-bovine serum albumin induced by LPS. The increase in pulmonary vascular permeability induced by LPS administration was unaffected by various anticoagulants but was inhibited by the leukocytopenia induced by nitrogen mustard or by the administration of a granulocyte elastase inhibitor, ONO-5046. AT III given alone, but not heparin plus AT III or Trp49-modified AT III, which lacks affinity for heparin, significantly increased the plasma concentration of 6-keto-prostaglandin F1alpha, suggesting that the interaction of AT III with heparin-like substances at the endothelial cell surface promotes the release of prostacyclin from endothelial cells in vivo. Trp49-modified AT III failed to prevent the LPS-induced accumulation of leukocytes and vascular injury. The pulmonary accumulation of leukocytes and vascular injury induced by LPS were not prevented by administering AT III to rats that were pretreated with indomethacin. The continuous intravenous infusion of prostacyclin prevented the LPS-induced pulmonary accumulation of leukocytes and vascular injury. Findings suggest that AT III depends on its ability to promote the release of prostacyclin, a potent inhibitor of leukocyte activation, from endothelial cells to prevent pulmonary vascular injury induced by LPS.
Peroxynitrite modulates receptor-activated Ca2+ signaling in vascular endothelial cells
Authors: Elliott S. J.
Published: December 2017

Peroxynitrite (ONOO-) is formed from superoxide (O2-) and .NO. We have previously reported that O2- does not alter endothelial cell Ca2+ signaling. To test whether .NO alters Ca2+ signaling, cells were incubated with the .NO donor, spermine NONOate. Neither spermine NONOate nor S-nitroso-N-acetyl penicillamine (SNAP) altered bradykinin-stimulated Ca2+ signaling. By contrast, 3-morpholinosydnonimine (SIN-1), which generates ONOO- by releasing O2- and .NO essentially in a simultaneous manner, significantly inhibited signaling. Initially, the inhibitory effect of 1 mM SIN-1 was selective toward agonist-stimulated influx of external Ca2+. At later time points, SIN-1 additionally depleted internal stores of releasable Ca2+. When cells were coincubated with SIN-1 plus superoxide dismutase, a technique designed to scavenge O2- and convert SIN-1 to purely an .NO-donor compound, Ca2+ signaling was identical to control. SIN-1C, the inactive metabolite of SIN-1, had no effect on [Ca2+]i. This study demonstrates that exogenously generated ONOO- modulates endothelial cell Ca2+ signaling, suggesting that ONOO- is of biological relevance to vasoregulation.
P2u-purinoceptor regulation of chloride secretion in cultured human tracheal submucosal glands
Authors: Yamaya M. , Sekizawa K. , Kakuta Y. , Ohrui T. , Sawai T. , Sasaki H.
Published: December 2017

We examined the purinergic regulation of chloride secretion and intracellular calcium ion concentration ([Ca2+]i) in the cultured epithelial cells passaged from human tracheal submucosal glands. Adenosine, AMP, ADP, ATP, and UTP induced transient short-circuit current (Isc) responses in a concentration-dependent fashion. The rank order of peak Isc responses at 10 microM concentrations was UTP = ATP > ADP > AMP = adenosine. Diphenylamine-2-carboxylic acid (1 mM) and 4,4'- diisothiocyano-2,2'-stilbene disulfonate (0.5 mM), and quinidine (1 mM) inhibited Isc responses induced by extracellular nucleotides. However, amiloride (10 microM) was without effect. Theophylline (1 mM) did not alter ATP (10 microM)-induced Isc response. Increases in Isc induced by ATP and UTP were significantly larger than those by alpha, beta-methylene ATP, beta, gamma-methylene ATP, or 2-methylthio ATP. Extracellular nucleotides caused increases in [Ca2+]i, and the rank order of [Ca2+]i responses was same as that of Isc responses. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (50 microM) inhibited increases in Isc and [Ca2+]i induced by extracellular nucleotides. These results suggest that extracellular nucleotides induce chloride secretion across cultured human tracheal glands via a P2u-purinoceptor, and this chloride secretion appears to be dependent on increases in [Ca2+]i and activation of Ca-dependent K channels.
Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells
Authors: Matalon S. , Benos D. J. , Jackson R. M.
Published: December 2017

The recent immunopurification and cloning of various lung Na+ channel proteins has provided the necessary tools to study Na+ transport at a fundamental level across a number of epithelial tissues. Various macroscopic measurements of Na+ transport have shown that Na+ ions enter the cytoplasm of alveolar cells mainly through amiloride-inhibitable Na+ channels. Molecular biology studies have shown the existence of three Na+ channel subunit mRNAs (alpha-, beta-, and gamma-rENaC) in mature fetal (FDLE) and adult alveolar type II (ATII) cells. Patch-clamp studies have demonstrated the existence of various types of amiloride-inhibitable Na+ channels, located in the apical membranes of FDLE and ATII cells. beta-Agonists and agents that enhance intracellular adenosine 3',5'-cyclic monophosphate levels increase the open probability of these channels, leading to increased Na+ transport across the alveolar epithelium in vivo. Immunopurification of a putative channel protein from adult ATII cells showed that it contains an amiloride-binding subunit with a molecular mass of 150 kDa. When this protein was reconstituted in planar lipid bilayers, it exhibited single channels with a conductance of 25 pS, which were moderately selective for Na+ over K+. The open probability of these channels was increased by the addition of protein kinase A (PKA) and ATP, and was decreased to the same extent by addition of [N-ethyl-N-isopropyl]-2'-4'-amiloride (EIPA) and amiloride (1 microM each) in the apical side of the bilayer, in agreement with the results of patch-clamp studies in ATII cells. Exposure of rats to sublethal hyperoxia increased alpha-rENaC mRNA and the functional expression of Na+ channels in alveolar epithelial cells and limited alveolar edema. These findings indicate that alveolar epithelial channels contain at least one family of amiloride-sensitive Na+ channel proteins, which displays a number of unique properties, including sensitivity to EIPA.
Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells
Authors: Wong H. R. , Finder J. D. , Wasserloos K. , Lowenstein C. J. , Geller D. A. , Billiar T. R. , Pitt B. R. , Davies P.
Published: December 2017

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L166-L171, 1996.-Interleukin-1 beta (IL-1 beta) is the critical cytokine affecting peripheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1 beta in stimulating iNOS transcription in cultured rat pulmonary artery smooth muscle cells (RPASMC). Treatment of RPASMC with IL-1 beta caused a concentration-dependent increase in iNOS gene expression by Northern and Western blotting. To demonstrate IL-1 beta-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promoter-luciferase constructs containing deletional mutations of the murine macrophage iNOS 5' flanking promoter region. IL-1 beta increased promoter activity approximately two- to threefold over baseline in fragments ranging from -1592 (full-length) to -242 bp. Activity was lost, however, when the promoter fragment was shorter than -242 bp. IL-1 beta-mediated increases in steady-state iNOS mRNA were sensitive to pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation. Nuclear proteins from IL-1 beta-stimulated cells demonstrated PDTC-sensitive binding to an oligonucleotide containing the sequence for the NF-kappa B binding element present in the region between -242 and -42 bp. These data document that IL-1 beta, by itself, increases iNOS expression in RPASMC by transcriptional activation, mediated in part by NF-kappa B.
Fibronectin modulates expression of interleukin-1 beta and its receptor antagonist in human mononuclear cells
Authors: Graves K. L. , Roman J.
Published: December 2017

Identification of factors that regulate production of proinflammatory cytokines may provide insight into mechanisms governing lung inflammation. One potential regulatory factor highly expressed in inflamed tissues is fibronectin (FN). To determine the potential effects of FN on interleukin (IL)-1 beta production, we exposed human peripheral blood mononuclear cells to soluble FN. This treatment resulted in the accumulation of IL-1 beta mRNA and enhancement of IL-1 beta protein synthesis and secretion. This effect was dose dependent and appeared to be mediated by the integrin alpha 5 beta 1. Treatment with FN also increased production of IL-1 receptor antagonist (IL-1ra), a naturally occurring inhibitor of IL-1 function. However, the stimulatory effect of FN on IL-1ra production was abolished by costimulation with type I collagen. We conclude that the increased deposition of FN in injured tissues may enhance the expression of IL-1 beta mRNA and augment the production and release of IL-1 beta protein by mononuclear cells. Differential expression of IL-1 beta and IL-1ra resulting in a high IL-1 beta-to-IL-1ra ratio in response to mixed matrices containing FN and type I collagen may be an important regulatory point in inflammation.
Higher lung antioxidant enzyme activity persists after single dose of corticosteroids in preterm lambs
Authors: Walther F. J. , David-Cu R. , Mehta E. I. , Polk D. H. , Jobe A. H. , Ikegami M.
Published: December 2017

Although administration of exogenous corticosteroids accelerates the late gestational rise in fetal rat and lamb lung antioxidant enzyme activity, the effect of dosing intervals on these responses remains uncertain. We studied the persistence and efficacy of the antioxidant response in fetal lamb lung to a single fetal dose of corticosteroids injected between 121 and 127 days gestational age. Fetal lambs received 0.5 mg/kg of betamethasone (n = 35) or saline (n = 26) by fetal intramuscular injection 24 h, 48 h, 4 days, or 7 days before preterm delivery at 128 days gestation (term = 150 days). After delivery, the lambs were ventilated for 40 min and killed. Total superoxide dismutase, catalase, glutathione peroxidase activities, and lipid hydroperoxide levels were measured, using homogenized lung. The saline-injected controls were similar at all time points. Lung antioxidant enzyme activity was consistently higher and lipid hydroperoxide presence was lower in the betamethasone-treated groups. We conclude that the positive effect of a single fetal dose of betamethasone on lung antioxidant enzyme activity occurs within 24 h after exposure, persists over a period of 7 days without a major change in the magnitude of the response, and leads to a reduction in lipid hydroperoxide formation during immediate postdelivery oxygen exposure.
Glibenclamide blockade of CFTR chloride channels
Authors: Schultz B. D. , DeRoos A. D. , Venglarik C. J. , Singh A. K. , Frizzell R. A. , Bridges R. J.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A- and ATP-regulated Cl- channel located in the apical membranes of epithelial cells. Previously Sheppard and Welsh (J. Gen. Physiol. 100: 573-591, 1992) showed that glibenclamide, a compound which binds to the sulfonylurea receptor and thus blocks nucleotide-dependent K+ channels, reduced CFTR whole cell current. The aim of this study was to identify the mechanism underlying this inhibition in cell-free membrane patches containing CFTR Cl- channels. Exposure to gliben-clamide caused a reversible reduction in current carried by CFTR which was paralleled by a decrease in channel open probability (Po). The decrease in Po was concentration dependent, and half-maximum inhibition (ki) occurred at 30 microM. Fluctuation analysis indicated a flickery-type block of open CFTR channels. Event duration analysis supported this notion by showing that the glibenclamide-induced decrease in Po was accompanied by interruptions of open bursts [i.e., an apparent reduction in the burst duration (Tburst)] with only a slight reduction in closed time (Tc). The plot of the corresponding open-to-closed (Tburst-1) and closed-to-open (Tc-1) rates as a function of glibenclamide concentration were consistent with a pseudo-first-order open-blocked mechanism and provided estimates of the on rate (kon = 1.17 microM-1S-1), the off rate (koff = 16 s-1), and the dissociation constant (Kd = 14 microM). The difference between the Ki (30 microM) and the Kd (14 microM) is the result expected for a closed-open-blocked model with an initial Po of 0.47. Since the initial Po was 0.50 +/- 0.02 (n = 12), we can conclude that glibenclamide blocks CFTR by a closed-open-blocked mechanism.
Leukotriene C4 inhibits ciliary activity in the presence of gamma-glutamyl transpeptidase
Authors: Ganbo T. , Hisamatsu K. , Inoue H.
Published: December 2017

We incubated human paranasal sinus mucosa in tissue culture with each of leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4). Ciliary beat frequency (CBF) was measured photoelectrically, and the concentrations of leukotrienes in the incubation medium were determined over time. LTC4 significantly decreased CBF with 2-h, 4-h, and 8-h exposures to 10(-6) M, 10(-8) M, and 10(-10) M, respectively. Moreover, LTC4 dose dependently reduced CBF to 81.4% of the initial value after 6-h exposure to 10(-6) M, to 82.5% after 8-h exposure to 10(-8) M, and to 89.7% after 12-h exposure to 10(-10) M. LTD4 also exhibited progressive ciliary inhibition, while LTE4 had a minimal effect on CBF. In the medium, LTC4 was changed to LTD4 and further to LTE4. gamma-Glutamyl transpeptidase (gamma-GTP), an enzyme that converts LTC4 to LTD4, was detected in the mucosa. Serine-borate complex, an inhibitor of gamma-GTP, blocked the inhibitory effect of LTC4 on CBF. These findings suggest that LTC4 may induce ciliary inhibition indirectly by conversion to LTD4.
Inhaled NO prevents IL-1-induced neutrophil accumulation and associated acute edema in isolated rat lungs
Authors: Guidot D. M. , Hybertson B. M. , Kitlowski R. P. , Repine J. E.
Published: December 2017

We determined previously that inhaled nitric oxide (NO) prevented oxidant-dependent capillary leak in isolated rat lungs perfused with human neutrophils and fMLP via a mechanism that was independent of vasodilatation. In the present investigation we determined that inhaled NO (50 ppm) prevented oxidant-dependent acute capillary leak (as reflected by weight gain and Ficoll retention) in isolated rat lungs given human recombinant interleukin-1 alpha (IL-1, 50 ng) intratracheally and perfused with human neutrophils. Inhaled NO also reduced neutrophil migration from the vascular to the airway compartment (as reflected by lung lavage fluid neutrophil numbers and levels of myeloperoxidase), in rats given IL-1 intratracheally and perfused with neutrophils. However, NO did not prevent IL-1-mediated increases in lung lavage levels of cytokine-induced neutrophil chemoattractant (CINC), a potent chemokine produced by alveolar macrophages and other resident cells that mediates IL-1-induced neutrophil infiltration in vivo. We conclude that inhaled NO prevented neutrophil migration and leak caused by intratracheal administration of IL-1 and neutrophil perfusion in isolated rat lungs. We speculate that NO directly inhibits neutrophil responsivity during lung inflammation, a premise that is consistent with the known effects of NO on neutrophil function in vitro. This study provides further evidence that inhaled NO may have important anti-inflammatory as well as vasodilator effects in acute lung injury.
Integrin mediation of type II cell adherence to provisional matrix proteins
Authors: Kim H. J. , Ingbar D. H. , Henke C. A.
Published: December 2017

Lung injury causes alveolar type I epithelial cell death, basement membrane denudation, and alveolar flooding with serum fibronectin and fibrinogen. For successful restoration of normal architecture, the epithelium must be regenerated from progenitor type II alveolar cells. Using adhesion assays, we examined whether type II alveolar cells adhere to the provisional matrix proteins fibronectin, fibrinogen, and fibrin, and whether integrins mediate this adherence. Rat type II cells adhered to fibronectin, vitronectin, fibrinogen, and fibrin. Synthetic RGD (arginine-glycine-aspartic acid) peptide blocked this adhesion. Flow cytometry and Western analysis indicated that type II cells expressed beta 1- and alpha v beta 3-integrins. Anti-beta 1-and anti-alpha v beta 3-integrin antibodies blocked type II cell adhesion to fibronectin and to fibronectin and fibrinogen, respectively. In summary, type II cells adhered to fibronectin, fibrinogen, and fibrin, and adhesion was partially mediated by integrins. This study provides the first evidence of type II cell adhesion to fibrin gels and vitronectin, beta 1- and alpha v beta 3-integrin mediation of type II cell adhesion, and the presence of the alpha v beta 3-integrin on type II epithelial cells.
Oxidant stress responses in influenza virus pneumonia: gene expression and transcription factor activation
Authors: Choi A. M. , Knobil K. , Otterbein S. L. , Eastman D. A. , Jacoby D. B.
Published: December 2017

The pathogenesis of influenza virus infections of the lungs is in part mediated by oxidative stress. Such infections might therefore be expected to induce expression of stress-response genes and genes encoding antioxidant enzymes and to activate transcriptional regulatory proteins. Mice (C57B1/6 and C3H/HeJ) were infected intranasally with influenza virus A/PR/8/34 (H1N1). Expression of the genes encoding the antioxidant enzymes manganese superoxide dismutase (Mn- SOD), indoleamine-2, 3-dioxygenase (IDO), heme oxygenase-1, and glutathione peroxidase were increased in the lungs of virus-infected animals. Cu/ZnSOD and catalase mRNA were not induced by viral infection. Activation of the transcriptional regulatory proteins AP-1, C/EBP, and NF-kappa B (which are known to be affected by oxidant stress) was demonstrated by electrophoretic mobility shift assay after viral infection. In the case of MnSOD, despite increased gene expression enzyme activity was not increased. In contrast, for heme oxygenase-1 both mRNA and activity were increased. C3H/ HeJ and C57B1/6 mice, which are known to have different responses to other types of oxidant stress, also differed in their responses to viral infection. Induction of heme oxygenase-1 expression was greater in C57B1/6 mice than in C3H/ HeJ mice, although inhibiting this enzyme did not alter virus-induced mortality. In contrast, IDO was more strongly induced in C3H/HeJ mice. Activation of NF-kappa B was much more marked in C57B1/6 mice than in C3H/HeJ mice. Although virus replication and inflammatory responses were equivalent in the two strains, lung injury (as measured by wet-to-dry wt ratios) and mortality were greater in C3H/HeJ mice than in C57B1/6 mice, a difference that may be related to differing oxidant stress responses. Thus influenza pneumonia causes an oxidant stress response in the lungs, the nature of which is determined in part by the genetic background of the host.
Evidence for a vitamin D paracrine system regulating maturation of developing rat lung epithelium
Authors: Nguyen T. M. , Guillozo H. , Marin L. , Tordet C. , Koite S. , Garabedian M.
Published: December 2017

Rat fetal lung is a target tissue for 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25 (OH)2 D3]. We have identified the cells that respond to the hormone and tested the hypothesis that the lung is also a source of 1 alpha,25(OH)2D3. We found that 1) at the end of pregnancy (days 20-21) alveolar type II cells (ATII) bore 1 alpha,25(OH)2D3 receptors and responded to the hormone. Incubating these cells with 10(-9) M 1 alpha,25(OH)2D3 for 48 h stimulated the synthesis (87.3 +/- 9.1%) and release (61.7 +/- 6.1%) of disaturated phosphatidylcholine; 2) EB-1213, a 1 alpha,25(OH)2D3 analogue with low calcemic activity, had similar effects on ATII; 3) neither fetal lung fibroblasts nor neonatal ATII (day 2 postpartum) expressed 1 alpha,25(OH)2D3 receptors; and 4) in contrast, fetal lung fibroblasts taken on days 19-22 of gestation converted [3H]25(OH)D3 to [3H]1 alpha,25(OH)2D3, whereas ATII and skin fibroblasts did not. These findings suggest that 1 alpha,25(OH)2D3 is a local mediator of epithelial-mesenchymal cell interactions in the developing rat lung and that 1 alpha,25(OH)2D3 or EB-1213 might be therapeutically useful in treating the respiratory distress syndrome of premature neonates.
Reduced in vivo plasma fibronectin content of lung matrix during postoperative sepsis
Authors: Rebres R. A. , Cho E. , Rotundo R. F. , Saba T. M.
Published: December 2017

Sepsis after surgery, trauma, or burn contributes to altered lung endothelial permeability and respiratory failure. Fibronectin (Fn), an opsonic and adhesive glycoprotein, exists in both a soluble form in plasma and an insoluble form in the extracellular matrix (ECM). Recent studies [E. M. Wheatley, P. J. McKeown-Longo, P. A. Vincent, and T. M. Saba, Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L148-L157, 1993] suggest that the ECM content of Fn may influence lung vascular permeability. We evaluated the incorporation of plasma-derived Fn (pFn) into the ECM of the lung during postoperative sepsis. Postoperative nonseptic and postoperative septic rats were compared, using a model of laparotomy followed by cecal ligation and puncture. To label the pFn pool, rats received intravenously 3 micrograms of purified rat 125I-labeled Fn/100 g body weight 6 h after surgery (laparotomy). 125I-Fn in the deoxycholate detergent-insoluble fraction of tissues was used to quantify matrix-incorporated Fn at 4 h after infusion with 125I-Fn. Septic rats exhibited a peripheral leukopenia as well as reduction in plasma volume, Fn halflife, and total pFn pool. Incorporation of pFn in the liver and spleen of postsurgical septic rats was not different (P > 0.05) from sham-operated (postsurgical nonseptic) rats, but incorporation was significantly decreased (P < 0.05) in vivo in the lung. However, under controlled in vitro conditions, lung tissue harvested from septic or sham-operated rats demonstrated a similar tissue incorporation of soluble 125I-pFn as well as similar rates of retention/turnover of ECM 125I-Fn, based on pulse-chase experiments. These data suggest that the in vivo inflammatory environment in the lung during postoperative sepsis, which cannot be reproduced in vitro, may alter the Fn content of the ECM of the lung. Such reduced levels of pFn in the lung ECM may be a factor influencing lung vascular integrity during postoperative sepsis.
Hypoxia-reoxygenation impairs NO-mediated vasodilation in rat lungs
Authors: Eddahibi S. , Raffestin B. , Tayarani Y. , Carville C. , Adnot S.
Published: December 2017

Isolated rat lungs subjected to hypoxia-reoxygenation (H/R) were used to study NO-mediated pulmonary vasodilation during oxidant-induced vascular injury. After ventilation with 3% O2, reoxygenation with 21% (H/R 21%) or 95% O2 (H/R 95%) caused lung edema and lipid peroxidation. Vasodilation to A23187 was attenuated after H/R 21% and abolished after H/R 95%. The vasodilator-response curve to NO was more shifted to the right after H/R 95% than after H/R 21%. Pretreatment with superoxide dismutase (SOD; 150 U/ml) and catalase (120 U/ml) prevented impairment of A23187- and NO-mediated vasodilation. SOD and catalase added after reoxygenation restored vasodilation to NO but not to A23187. In lungs obtained from chronically hypoxic rats but studied under conditions of normoxic ventilation, vasodilation to A23187 was abolished, but vasodilation to NO remained unchanged. The data suggest that generation of oxygen-derived reactive species after H/R produces impairment of NO formation as well as direct inactivation of NO. This does not explain the decreased endothelial NO-mediated pulmonary vasodilation in chronically hypoxic rats.
Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC
Authors: Papapetropoulos A. , Elmore L. A. , Catravas J. D.
Published: December 2017

It is unclear whether all or a fraction of the capillary plasma volume (Vcp) serves as the reaction volume (Vr) for pulmonary capillary endothelial ectoenzymes, in vivo. Cultured endothelial cell (EC) monolayers provide a convenient model for studying EC-bound enzyme-Vr relationships. Because the Michaelis-Menten parameter [maximum velocity of enzyme reaction (Vmax) = E x kcat/Vr, where E is enzyme mass and kcat is the constant of product formation] is inversely proportional to Vr, we hypothesized that increasing the volume of medium (Vm) bathing EC monolayers would proportionally reduce the calculated Vmax (or Vmax/K(m), where K(m) is the Michaelis constant) values of an ectoenzyme reacting with a substrate only if, and as long as, Vm = Vr. To test this hypothesis, studies were performed in bovine pulmonary arterial EC grown to confluence. Activities of angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT) were assayed in Earle's balanced salts solution utilizing [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) and 5'-[14C]AMP as substrates, respectively. Under first-order reaction conditions and at constant substrate concentrations ([BPAP] = 15 nM, [AMP] = 1 microM), Vmax/K(m) ratios of ACE and NCT declined to 20% of their original values, as Vm increased from 0.6 to 2 ml. ACE activity was also studied at constant substrate mass (BPAP = 7 pmol) under first-order reaction conditions. Again, enzyme activity (Vmax/K(m)) declined proportionally to increasing Vm. Under zero-order reaction conditions ([BPAP] = 250 microM), ACE activity (Vmax) was similarly related to Vm. Linear regression analyses revealed that ACE or NCT would recognize up to at least 3 ml Vm, a volume vastly exceeding that of Vcp in a section of the capillary bed composed of an equivalent number of ECs, thus suggesting that Vcp could serve as the reaction volume for pulmonary capillary EC ectoenzymes in vivo.
Lung epithelial cell-released nitric oxide protects against PMN-mediated cell injury
Authors: Su W. Y. , Day B. J. , Kang B. H. , Crapo J. D. , Huang Y. C. , Chang L. Y.
Published: December 2017

A calcium-independent type II nitric oxide (NO) synthase has been localized in lung epithelial cells; however, the function of NO. released by epithelial cells is unclear. We hypothesized that epithelial-derived NO may affect the interactions between polymorphonuclear neutrophils (PMN) and the alveolar epithelium and studied PMN adhesion and cytotoxicity to lung epithelial cells. A dose- and time-dependent production of NO. by L2 cells can be induced by a mixture of inflammatory mediators (cytomix) containing lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma. Increased NO. production by L2 cells was associated with decreased 51Cr release by the epithelial cells after they were incubated with activated PMN. Addition of NG-monomethyl-L-arginine or oxyhemoglobin reversed the protective effects of cytomix, suggesting that increased NO. production by L2 cells was responsible for the decreased 51Cr release. However, PMN adhesion and intercellular adhesion molecule-1, a major adhesion molecule involved in PMN adhesion to epithelium, were increased by cytomix. We conclude that NO. released by lung epithelial cells was involved in protecting epithelial cells from PMN-mediated cytotoxicity. NO.-mediated protection of lung epithelial cells occurred in spite of PMN adhesion being increased, suggesting that reduced adhesion is not required for NO.-mediated inhibition of PMN cytotoxicity.
Ion transport mechanisms in native rabbit nasal airway epithelium
Authors: Ropke M. , Carstens S. , Holm M. , Frederiksen O.
Published: December 2017

Net ion transports in rabbit nasal airway epithelium (RNAE) were estimated from unidirectional fluxes of 22Na+, 36Cl-, and 86Rb+ (K+ tracer), short-circuit current (Isc), and epithelial conductance (Gt) under short-circuit conditions in excised parallel RNAE from the two sides of the nasal septum mounted in Ussing chambers at 37 degrees C. Net Na+ absorption (JNa; 76 nmol.min-1.cm-2) was nearly equal to the net charge flux (Jnet), equivalent to the control Isc of 137 microA/cm2 (Jnet = Isc/zF = 85 neq.min-1.cm-2). Secretions of Cl- (9 nmol.min-1.cm-2) and K+ (Rb+) (1.2 nmol.min-1.cm-2) were small. Intra-animal variations between right- and left-side Isc values were small compared with large interanimal variations, suggesting long-term regulation of JNa. Serosal ouabain (10(-4) M) abolished Isc. Mucosal amiloride (10(-4) M) maximally inhibited Isc by 68% and JNa by 78%, abolished K+ (Rb+) secretion, increased Cl- secretion slightly (to 16 nmol.min-1.cm-2), and decreased control Gt (13.4 mS/cm2) by 26%. Amiloride-insensitive JNa was inhibited approximately 50% by mucosal hydrochlorothiazide (10(-4) M) but not by mucosal bumetanide, phloridzine, or ethoxzolamide. The Cl- secretion was abolished by serosal bumetanide (10(-4) M) and also by mucosal diphenylamine-2-carboxylate (2.5.10(-4) M) or bumetanide (10(-4) M). Serosal Ba2+ (2 mM) inhibited JNa and increased K+ (Rb+) secretion. The latter was blocked by mucosal Ba2+. Passive Cl- (but not Na+) fluxes varied proportionally with Gt and were approximately four times higher than passive Na+ fluxes, suggesting 1) significant anion selectivity of a low-resistance paracellular pathway and 2) separate routes for paracellular Cl- and Na+ fluxes. We conclude that RNAE is a suitable model organ for studies of regulation of JNa in native human airway epithelia.
Asbestos induces apoptosis in human alveolar macrophages
Authors: Hamilton R. F. , Iyer L. L. , Holian A.
Published: December 2017

Asbestos refers to a group of fibrous minerals implicated in the development of several lung diseases, including fibrosis (asbestosis), cancer, and malignant mesothelioma. Although major health risks exist in occupationally exposed individuals, low-level exposures of asbestos may still contribute to health problems. The mechanism by which asbestos causes lung disease is not clearly understood but has been proposed to involve the alveolar macrophage (AM). We propose that asbestos induces apoptosis of AM, resulting in the development of an inflammatory state. In this study, we examined two forms of asbestos, chrysotile (CHR) and crocidolite (CRO), along with a control fiber, wollastonite (WOL), to characterize their relative cytotoxicity and ability to stimulate apoptosis in vitro. AM were cultured for 24 h with these particulates and examined for cell viability (trypan blue exclusion) and apoptosis (morphology, levels of cytosolic oligonucleosomal DNA fragments, and DNA ladder). In the absence of a decrease in cell viability, both CHR and CRO produced changes in cell morphology consistent with apoptosis. In addition, levels of cytoplasmic oligonucleosomal DNA (Cell Death Detection enzyme-linked immunosorbent assay) were significantly enhanced for CHR (3-25 micrograms/ml) and CRO (25-75 micrograms/ml) in a dose-dependent manner (a process that was inhibitable by 10 microM Z-Val-Ala-Asp fluoromethyl ketone, an interleukin-converting enzyme inhibitor). In contrast, WOL (up to 400 micrograms/ml) produced no significant DNA fragmentation in a 24-h culture. Neither CHR nor CRO caused DNA ladder formation in 24-h cell cultures. However, in 48-h cell cultures, both CHR- and CRO-exposed cells, but not WOL, resulted in the formation of DNA ladders characteristic of apoptosis. In summary, these results suggest that, unlike nonfibrogenic particulates, low doses of asbestos fibers cause apoptosis in cultured human AM that may be an early step in the development of lung fibrosis.
Gestational and tissue-specific regulation of C1C-2 chloride channel expression
Authors: Murray C. B. , Chu S. , Zeitlin P. L.
Published: December 2017

Chloride channels supply critical functions in epithelial cells throughout the body. Although function of the volume- and voltage-gated C1C-2 is uncertain, its wide tissue distribution of mRNA suggests C1C-2 has important housekeeping functions. This study's objective was to identify the extent of not only C1C-2 mRNA expression but also protein expression as a measure of the capacity for C1C-2 chloride secretion in epithelial tissues. Using quantitative ribonuclease protection assay, we found that C1C-2 mRNA transcripts were abundant in fetal and postnatal brain, fetal kidney, liver, intestine, and lung. In contrast to brain, C1C-2 mRNA transcripts were downregulated during late gestation in lung, kidney, and intestine. The lung expressed the least C1C-2 mRNA. Immunoblotting demonstrated similar tissue- and gestation-dependent variations in C1C-2 protein expression. To determine if there is a correlation between the sites of C1C-2 protein expression and cystic fibrosis transmembrane conductance regulator (CFTR), another epithelial chloride channel, a polyclonal COOH-terminal C1C-2 antibody and an anti-R domain CFTR anti-body were used. C1C-2 and CFTR were expressed in different sites in lung and kidney.
Dimethyl sulfoxide decreases interleukin-8-mediated neutrophil recruitment in the airways
Authors: Massion P. P. , Linden A. , Inoue H. , Mathy M. , Grattan K. M. , Nadel J. A.
Published: December 2017

In this study, we investigated the role of dimethyl sulfoxide (DMSO) in inhibiting interleukin-8 (IL-8)-mediated neutrophil recruitment induced by Pseudomonas aeruginosa (PA) bacterial supernatant. First, we tested whether DMSO could inhibit IL-8 production induced by PA in human bronchial epithelial (16-HBE) cells in vitro. In these cells, exposure to PA or H2O2 induced IL-8 production dose dependently, an effect that was inhibited by 1% DMSO at both the protein and RNA level. Second, we tested whether DMSO could block the recruitment of neutrophils induced by PA in a bypassed segment of dog trachea in vivo. PA supernatant was placed in the tracheal segment for 6 h in four dogs, and neutrophil recruitment and IL-8 concentrations were measured in the superfusate. DMSO prevented the recruitment of neutrophils and IL-8 production induced by PA time dependently. The results suggest that DMSO may play an anti-inflammatory role in the airway by inhibiting IL-8 production in epithelial cells.
Soluble and insoluble fibronectin increases alveolar epithelial wound healing in vitro
Authors: Garat C. , Kheradmand F. , Albertine K. H. , Folkesson H. G. , Matthay M. A.
Published: December 2017

Adhesive interactions between cells and extracellular matrix proteins are important in cell attachment, migration, and proliferation. The present work defines the role of fibronectin (soluble and insoluble) compared with type I and type IV collagen on in vitro alveolar epithelial wound healing. Repeated video microscopy experiments demonstrated that the half-time of wound closure was decreased in the presence of soluble fibronectin (6.6 +/- 2.1 vs. 17.4 +/- 0.8 h in serum-free medium, P < 0.05). Video microscopy, electron microscopy, and vinculin distribution demonstrated the contribution of two main events during the repair process: the migration of epithelial cell sheets and the spreading of the cells. During the wound healing, the internuclear distance between two adjacent cells at the migrating edge of the wound was significantly increased 10 h after wounding in the presence of soluble fibronectin (67 +/- 3.0 vs. 45 +/- 1.5 microns in serum-free medium, P < 0.05), indicating that cell spreading is involved as part of the mechanism for wound closure. Compared with type I and type IV collagen, insoluble fibronectin was the most potent stimulus for alveolar type II cell motility and wound healing in the absence of other serum factors. These results demonstrate that alveolar epithelial wound healing can be modulated in vitro by the composition of the extracellular matrix, an effect that may be mediated by changes in cell shape.
Colchicine inhibits arachidonate release and 5-lipoxygenase action in alveolar macrophages
Authors: Peters-Golden M. , McNish R. W. , Davis J. A. , Blackwood R. A. , Brock T. G.
Published: December 2017

Although colchicine is known to inhibit leukotriene synthesis in neutrophils, its effect on other aspects of arachidonic acid (AA) metabolism as well as its mechanism of action are unknown. To address these questions, we investigated the effects of colchicine on resident rat alveolar macrophages (AM), cells that generate a variety of lipoxygenase and cyclooxygenase products after stimulation. Pretreatment of AM with 10 microM colchicine for 1 h dramatically inhibited the synthesis of all 5-lipoxygenase (5-LO) metabolites from endogenous AA in ionophore A-23187-stimulated cells. In addition, colchicine inhibited the total release of AA as well as prostanoids to a lesser extent. Similar effects were observed with the other microtubule-disruptive agents nocodazole and vinblastine, and 5-LO product formation stimulated by the particulate agonist zymosan was inhibited as well. A selective inhibitory effect of colchicine on the 5-LO pathway was demonstrated by monitoring the synthesis of 5-LO products from exogenously supplied AA. Cell-free enzyme assays showed that this effect was not through a direct inhibition of the 5-LO enzyme. Moreover, colchicine did not affect the translocation of 5-LO to the nuclear envelope. We next evaluated the effect of colchicine on the levels of the two 5-LO cofactors, ATP and Ca2+. Although colchicine did not affect ATP levels, it did abrogate the ionophore-induced increase in intracellular Ca2+ concentration; the inhibitory effect of colchicine on 5-LO metabolism in AM was partially overcome by stimulation with higher doses of A-23187. We conclude that microtubular disruption inhibits agonist-induced increase in intracellular Ca2+ concentration, with multiple consequences for AA metabolism. These include a reduction in the liberation of AA from membrane phospholipids as well as the selective inhibition of processing of AA by 5-LO.