bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydraulic conductivity of ischemic pulmonary venules
Authors: Qiao R. L. , Sadurski R. , Bhattacharya J.
Published: December 2017

We report the first determination of lung endothelial barrier properties in ischemic, nonreperfused microvessels. We quantified the endothelial barrier in terms of hydraulic conductivity (Lp) in single pulmonary venules (diameter 20–50 microns) of isolated blood perfused lungs (dog, rat), held at constant inflation pressure (5 cmH2O) with a gas mixture containing 21% oxygen. Lp were determined by our split-drop technique in which an oil drop is first microinjected into a venule and then split by microinjection of a protein solution. Lp was interpreted from measurements of the rate of oil drop movement. Baseline Lp recorded in the first 30 min of perfusion averaged 3.4 +/- 0.9 x 10(-7) ml/(cm2.s.cmH2O). Then, in two separate groups of venules in which we established 1.3 +/- 0.1 h and 3.4 +/- 0.8 h of ischemia, we determined Lp which were, respectively, 145 +/- 6.5 and 308 +/- 13% above baseline (P < 0.05). We conclude that ischemia alone, in the absence of reperfusion, significantly deteriorates the lung endothelial barrier.
Cell surface carbohydrates modulate neutrophil adherence to alveolar type II cells in vitro
Authors: Crestani B. , Rolland C. , Petiet A. , Colas-Linhart N. , Aubier M.
Published: December 2017

We characterized the influence of phosphorylated sugars and cell surface sialic acids on the adherence of human polymorphonuclear leukocyte (PMN) to rat alveolar type II cell (ATII cells) and human-derived A549 cell monolayers in vitro. Percent adherence of radiolabeled polymorphonuclear leukocytes was assessed after incubating cells with the carbohydrates, enzymes, or lectins to be tested. Lactose-1-phosphate (Lact1P) and maltose-1-phosphate (Malt1P) (10 mM) inhibited adherence of PMN to ATII cells and A549 cells. Maximal inhibition followed treatment of both PMN and rat ATII cells and amounted to 85 +/- 7% with Lact1P and 92 +/- 3% with Malt1P. Inhibition was concentration dependent. Incubation of PMN with mannose-6-phosphate reduced adherence to rat ATII cells and A549 cells by 36 +/- 11 and 39 +/- 8%, respectively. Maximal concentrations of sugars did not alter cellular viability. Neuraminidase-induced desialilation of ATII cells increased adherence of PMN by 36 +/- 7% to rat ATII cells and by 86 +/- 18% to A549 cells. Masking of terminal sialic acids on rat ATH cells with Limulus polyphemus agglutinin (100 micrograms/ml) increased adherence by 50 +/- 2%. These results indicate that cell surface carbohydrates are involved in the regulation of the adhesive interaction between PMN and ATII cells in vitro.
Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases
Authors: Richman-Eisenstat J. B. , Jorens P. G. , Hebert C. A. , Ueki I. , Nadel J. A.
Published: December 2017

Sputum from patients with cystic fibrosis, bronchiectasis, and chronic bronchitis contains neutrophils and neutrophil proteases, which have been implicated in the pathophysiology of mucus hypersecretion in airways. We asked whether interleukin-8 (IL-8), a potent neutrophil chemoattractant, might be involved in recruiting neutrophils into airways of patients with cystic fibrosis, bronchiectasis, and chronic bronchitis. We found significant neutrophil chemotactic activity in sputum obtained from these patients. The IL-8 concentrations that we measured in sputum of patients with cystic fibrosis (7.1 +/- 1.0 x 10(-9) M, mean +/- SE), bronchiectasis (9.6 +/- 2.9 x 10(-9) M), and chronic bronchitis (2.8 +/- 1.0 x 10(-9) M) have been reported to cause significant chemotaxis in vitro and in airways in vivo, whereas concentrations measured in induced sputum from healthy subjects (1.1 +/- 0.3 x 10(-10) M) do not. A monoclonal antibody to IL-8 significantly inhibited the chemotactic activity in patients' sputum by 75–98%, but not in induced sputum from healthy subjects (9%). We conclude that IL-8 is an important chemotactic factor in sputum of patients with cystic fibrosis, bronchiectasis, and chronic bronchitis, and we suggest that IL-8 accounts, at least in part, for neutrophil recruitment into airways of patients with these diseases.
ATP and adenosine 3',5'-cyclic monophosphate stimulate the synthesis of surfactant protein A in rat lung
Authors: Wali A. , Beers M. F. , Dodia C. , Feinstein S. I. , Fisher A. B.
Published: December 2017

Synthesis and secretion of surfactant protein A (SP-A) were studied in the isolated perfused rat lung using Trans35S-label (approximately 85% methionine, 15% cysteine) in the perfusate with or without 1 mM ATP or 0.1 mM 8-bromoadenosine 3',5',-cyclic monophosphate (8-BrcAMP) for up to 6 h of perfusion. By enzyme-linked immunosorbent assay, the SP-A content was 36 +/- 0.3% of total protein in extracellular surfactant and 10.8 +/- 1.9% of total protein in lamellar bodies of control lungs; these relativr proportions were maintained in the presence of ATP or 8-BrcAMP. Incorporation of [35S]methionine (cysteine) into the surfactant and lamellar body protein fraction could be detected at 4 h of perfusion. At 6 h, specific activity of total protein [disintegrations per minute (dpm)/micrograms)] was significantly increased in both the surfactant (54%) and lamellar body fractions (30%) under the influence of either secretagogue compared with control conditions. In the presence of ATP, there was a significant increase in the SP-A immunoprecipitable counts of 61 and 72% in extra- and intracellular compartments, respectively. However, no significant change was observed in the relative abundance of SP-A mRNA between control and secretagogue-treated lungs. This dissociation of SP-A mRNA abundance and label incorporation into protein indicates that alteration in translational efficiency or posttranslational factors may be involved in the secretagogue-induced stimulation of SP-A synthesis.
Transcellular eicosanoid synthesis in cocultures of alveolar epithelial cells and macrophages
Authors: Peters-Golden M. , Feyssa A.
Published: December 2017

We have examined the importance of the relative capacities for arachidonic acid (AA) liberation and oxygenation as determinants of the magnitude of transcellular eicosanoid synthesis in cocultures of [3H]AA-prelabeled alveolar macrophages (AM) and [14C]AA-prelabeled alveolar epithelial cells (AEC). Taking advantage of our previous observation that over time in culture, AEC exhibit increases in AA release (plateau at day 4) that precede increases in cyclooxygenase capacity (maximal at day 7), we studied cocultures of freshly harvested AM together with AEC at culture days 2, 4, and 7. In this model, ionophore stimulation resulted in transcellular eicosanoid synthesis via the bidirectional transfer of free AA, with AM synthesizing their exclusive 5-lipoxygenase products leukotriene B4 and 5-hydroxyeicosatetraenoic from AEC-derived AA, and AEC synthesizing their exclusive cyclooxygenase metabolites prostaglandin E2 and prostacyclin from AM-derived AA. 14C-labeled 5-lipoxygenase products were synthesized by cocultures in the rank order: day 4 > day 2 > day 7, paralleling the relative amounts of unmetabolized AEC-derived [14C]AA. Transcellular flow of [14C]AA to the AM 5-lipoxygenase pathway was also favored when the AEC cyclooxygenase pathway was inhibited with indomethacin. Inhibition of the AM 5-lipoxygenase pathway with the compound MK 886 similarly resulted in the accumulation of unmetabolized AM-derived [3H]AA and greater synthesis of 3H-labeled prostanoids. We conclude that 1) the quantity of unmetabolized AA made available by one cell is an important determinant of the extent to which the fatty acid is utilized for transcellular eicosanoid synthesis by the second cell; 2) the fate of AA derived from a donor cell is influenced by the expression of AA oxygenation pathways in both the donor cell and the recipient cell; and 3) intercellular exchange of AA favors its shunting from an inhibited oxygenation pathway to an uninhibited one.
Neutrophil-activating peptide-2 in patients with pulmonary edema from congestive heart failure or ARDS
Authors: Cohen A. B. , Stevens M. D. , Miller E. J. , Atkinson M. A. , Mullenbach G. , Maunder R. J. , Martin T. R. , Wiener-Kronish J. P. , Matthay M. A.
Published: December 2017

We carried out studies to determine whether the neutrophil-activation peptide-2 (NAP-2) plays a role in the recruitment and/or degranulation of neutrophils into the lungs of patients with the adult respiratory distress syndrome (ARDS) or congestive heart failure (CHF). NAP-2 precursors plus NAP-2 (beta-thromboglobulin-like antigen) were measured in lung fluids and plasmas with a radioimmunoassay, and NAP-2 was separated from its precursors by high-performance liquid chromatography. Pulmonary edema fluids (PEFs) from patients with CHF contained higher concentrations of the beta-thromboglobulin-like antigen than PEFs from patients with ARDS, and bronchoalveolar lavage fluids (BALs) from patients with ARDS contained higher concentrations of beta-thromboglobulin-like antigen than BALs from normal subjects. beta-Thromboglobulin-like antigen concentration was 4.1-fold greater in PEFs from patients with CHF than in their plasmas. Chemotactically active NAP-2 was also demonstrated in PEFs but not in plasmas from patients with CHF and ARDS. These data suggest that significant platelet degranulation occurred into the lungs of the patients with CHF and that NAP-2 and other platelet constituents may contribute to fluid formation in patients with CHF.
Fetal rat lung type II cell differentiation in serum-free isolated cell culture: modulation and inhibition
Authors: Fraslon C. , Lacaze-Masmonteil T. , Zupan V. , Chailley-Heu B. , Bourbon J. R.
Published: December 2017

Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal fibroblasts enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming growth factor-beta, a factor produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.
Heat stress regulates the human 70-kDa heat-shock gene through the 3'-untranslated region
Authors: Moseley P. L. , Wallen E. S. , McCafferty J. D. , Flanagan S. , Kern J. A.
Published: December 2017

Cells respond to a variety of stresses by synthesizing a family of proteins termed heat-shock proteins (HSP). Recently, the 3'-untranslated regions (UTRs) of some mRNAs have been shown to be important in the posttranscriptional regulation of protein production. Therefore, we hypothesized that heat could regulate HSP70 production through the HSP70 3'-UTR, in addition to its known effects on transcription. To test this hypothesis, cells were transfected with either a plasmid containing sequences encoding the human HSP70 or beta-globin 3'-untranslated region placed downstream of a chloramphenicol acetyltransferase (CAT) reporter gene. In both plasmids, the CAT gene was driven by an SV40 promoter. Following heat stress, cells transfected with the CAT construct containing the HSP70 3'-UTR showed increased CAT activity relative to the beta-globin 3'-UTR construct. This effect paralleled increases in HSP70 mRNA and levels of the inducible HSP70 protein by Western blot. These studies identify a heat-induced mechanism of posttranscriptional control of HSP70 synthesis utilizing the HSP70 3'-UTR, which may be important in the cells ability to regulate the heat-shock response.
Influence of hydroxyl radical on rabbit airway smooth muscle chronically exposed to H2O2 in vivo
Authors: Prasad K. , Gupta J. B.
Published: December 2017

The effects of .OH on the isolated tracheal smooth muscles (TSM), from control, polyethylene glycol (PEG)-glucose oxidase (GO)-, and GO+PEG catalase-treated rabbits were investigated. GO or GO+catalase were given intravenously each week for 4 mo. .OH produced relaxation of basal and ACh-precontracted tension in TSM of control rabbits. The relaxant effect was attenuated by removal of epithelium, whereas it was converted to contraction in the indomethacin-pretreated muscle. .OH produced contraction of TSM and ACh-precontracted muscle in GO-treated rabbits. The contractile response was abolished in preparations denuded of epithelium or pretreated with indomethacin. .OH produced relaxation in basal tension, but a small contraction in ACh-precontracted muscle of GO+catalase-treated rabbits. The contractile response to .OH was unaffected by indomethacin pretreatment; however, it was converted to relaxation in the preparations denuded of epithelium. Contractile response of TSM to ACh was augmented in deepithelialized, indomethacin-, or GO-treated preparations. H2O2 damaged the tracheal epithelium. These results suggest that 1) .OH-induced relaxation/contraction of TSM is partly epithelium dependent and is mediated by bronchodilator/bronchoconstrictor arachidonic acid metabolites, 2) the airway smooth muscle with healthy epithelium responds to .OH differently from those with dysfunctional or damaged epithelium, and 3) hyperresponsiveness of the airways to ACh may be related to the epithelial dysfunction.
Regulation of phosphatidylcholine synthesis in fetal type II cells by CTP:phosphocholine cytidylyltransferase
Authors: Zimmermann L. J. , Hogan M. , Carlson K. S. , Smith B. T. , Post M.
Published: December 2017

Phosphatidylcholine synthesis increases in fetal rat type II cells during late gestation, as demonstrated by an increased incorporation of radiolabeled palmitate, glycerol, acetate, and choline into phosphatidylcholine. However, the percentage of phosphatidylcholine present in the saturated form remains essentially constant. The developmental profile of the enzymes of the CDP-choline pathway suggests that CTP:choline-phosphate cytidylyltransferase catalyses a rate regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. When cytidylyltransferase activity is assayed in different subcellular fractions, the greatest increase, as a function of development, is found in microsomes. This developmental increase is accompanied by a shift in subcellular distribution of cytidylyltransferase activity from cytosol to microsomes in fetal type II cells during late gestation. This shift is evident even when cytidylyltransferase activity is assayed in the presence of 0.5 mM phosphatidylcholine/oleic acid (1/1 molar ratio) vesicles. We speculate that either a subcellular translocation of CTP:phosphocholine cytidylyltransferase from cytosol to microsomes or an increase in cytidylyltransferase gene expression are responsible for the developmental increase of de novo phosphatidylcholine synthesis by fetal type II cells.
Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury
Authors: Warner B. , Papes R. , Heile M. , Spitz D. , Wispe J.
Published: December 2017

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.
Antisense technology: biological utility and practical considerations
Authors: Neckers L. , Whitesell L.
Published: December 2017

Antisense RNA and DNA techniques have been developed as a relatively recent approach to the specific modulation of gene expression in vitro and in vivo. This review discusses general considerations for the application of antisense techniques. We shall examine the relative advantages and disadvantages of DNA versus RNA techniques, as well as the common pitfalls peculiar to each strategy.
Effect of immune sensitization on stimulated ACh release from trachealis muscle in vitro
Authors: Mitchell R. W. , Ndukuw I. M. , Ikeda K. , Arbetter K. , Leff A. R.
Published: December 2017

We assessed the effect of immune sensitization on acetylcholine (ACh) release from parasympathetic nerve terminals in tracheal smooth muscle (TSM) strips from ragweed-sensitized (RWS) and sham-sensitized, littermate control (LMC) dogs. Strips of TSM were tethered to force transducers at optimal length in perfusion chambers containing [3H]choline and a fixed volume of physiological perfusate. Tissues were equilibrated for 1 h by electrical field stimulation (EFS) every 5 min to facilitate uptake of label into parasympathetic nerves as ACh. Fresh perfusate (containing 3 x 10(-8) M physostigmine) was collected at 5-min intervals for 1 h, and a rate coefficient of [3H]ACh release was determined. Tissues were exposed to agonists in the seventh collection period, and the increase in label release (ratio change where < or = 1.00 = baseline) and force production were determined. Ragweed antigen challenge stimulated [3H]ACh release and contraction in RWS but not LMC tissues. [3H]ACh release was 1.93 +/- 0.22 x baseline in RWS vs. 0.92 +/- 0.02 in control tissues (P < 0.01); contraction was 31.2 +/- 9.5% of that elicited by EFS (% EFS) in RWS vs. 0% EFS in LMC tissues (P < 0.01). Strips of TSM from RWS but not LMC dogs demonstrated concentration-dependent, augmented release of ACh caused by histamine. After 10(-4) M histamine, [3H]ACh release in RWS was 1.94 +/- 0.37 x baseline vs. 1.05 +/- 0.06 for LMC tissues (P < 0.05); histamine also caused greater contraction in RWS (106.5 +/- 5.9% EFS) vs. LMC (86.5 +/- 5.6% EFS; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Halothane alters cytosolic calcium transient in tracheal smooth muscle
Authors: Jones K. A. , Housmans P. R. , Warner D. O. , Lorenz R. R. , Rehder K.
Published: December 2017

Halothane is a potent bronchodilator that attenuates vagally mediated constriction of canine airways, in part by a direct action on the smooth muscle cell. In tracheal smooth muscle, acetylcholine (ACh) produces a transient peak increase in cytosolic ionized calcium concentration ([Ca2+]i), which declines to a sustained plateau concentration that is higher than that of the unstimulated muscle, and a sustained increase in force. The transient peak [Ca2+]i is caused primarily by Ca2+ release from the sarcoplasmic reticulum (SR), whereas the plateau [Ca2+]i is caused primarily by influx of extracellular Ca2+ across the plasma membrane. This study was conducted to investigate the effects of halothane on the 1) transient peak [Ca2+]i during force development (i.e., developed force) and 2) plateau [Ca2+]i during force maintenance (i.e., sustained force) induced by ACh in isolated strips of canine tracheal smooth muscle. Changes in [Ca2+]i were measured with the photoprotein, aequorin. In muscle strips contracted after incubation with 1.1 (n = 5) or 1.5 (n = 5) minimum alveolar concentration (MAC) halothane, halothane significantly attenuated the transient peak aequorin luminescence (AL) and developed force and significantly prolonged the time necessary to reach peak AL and developed force; these effects were not dose dependent. In muscle strips (n = 3) contracted with ACh, addition of halothane caused a reduction in sustained force but no decrease in plateau AL.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyperoxic stress elevates p52(PAI-1) mRNA abundance in cultured cells and adult rat pulmonary tissue
Authors: White J. E. , Ryan M. P. , Tsan M. F. , Higgins P. J.
Published: December 2017

Hyperoxic stress alters expression of genes involved in extracellular matrix (ECM) remodeling. To identify novel ECM-associated gene products positively regulated by hyperoxia, rat kidney cells were exposed to 95% O2, and the complement of [35S]methionine-labeled, saponin-resistant, ECM-associated proteins was compared with normoxic controls. O2-stressed cells accumulated significantly greater ECM levels (approximately 3- to 4-fold that of control cells) of a 52-kDa glycoprotein (p52), recently identified as the matrix form of plasminogen activator inhibitor type 1 (PAI-1) (P.J. Higgins, P. Chaudhari, and M.P. Ryan. Biochem. J. 273: 651-658, 1991; P. J. Higgins, M. P. Ryan, R. Zeheb, T. D. Gelehrter, P. Chaudhari. J. Cell. Physiol. 143:321-329, 1990), which peaked at 48 h of exposure. Hyperoxia-associated increases in ECM p52(PAI-1) content reflected parallel elevations in p52(PAI-1) mRNA abundance. Similar results were obtained using secondary cultures of rat pulmonary fibroblasts. This 48-h period of maximal hyperoxia-induced p52(PAI-1) expression in vitro was used to design subsequent in vivo studies. Adult rats were exposed to 99% O2 for 24–50 h, and RNA was extracted from the pulmonary tissue of stressed and control animals. A 5- to 8-fold and 6- to 15-fold increase in lung p52(PAI-1) mRNA content was evident in hyperoxia-treated rats at 24 and 50 h, respectively. All of this increase occurred in the defined 3.2-kb species of rat p52(PAI-1) mRNA. Actin mRNA levels increased three- to sevenfold as a function of hyperoxic stress, whereas catalase and glyceraldehyde-3-phosphate dehydrogenase mRNA abundance was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of tachykinin receptors in ferret trachea by peptide agonists and nonpeptide antagonists
Authors: Geppetti P. , Bertrand C. , Bacci E. , Huber O. , Nadel J. A.
Published: December 2017

The tachykinin receptors mediating mucus secretion and smooth muscle contraction were studied in the ferret trachea in vitro. Substance P (SP) and the selective agonist for NK1 receptor ([Sar9,Met(O2)11]SP), but not selective agonists for NK2 ([Ala5,beta-Ala8]neurokinin A-(4–10)) and NK3 ([MePhe7]neurokinin B) receptors, induced secretion of macromolecules in a concentration-dependent fashion. The nonpeptide NK1 receptor antagonist, CP-96,345, but not the nonpeptide NK2 receptor antagonist, SR-48968, inhibited SP-induced secretion. Both neurokinin A (NKA) and [Ala5,beta-Ala8]NKA-(4-10), but not NK1 and NK3 selective agonists, evoked a concentration-dependent smooth muscle contraction. SR-48968, but not CP-96,345, inhibited in a concentration-dependent manner the response to NKA. CP-96,345 and SR-48968 did not affect the concentration-dependent increase in macromolecule secretion or smooth muscle contraction by carbachol. These findings indicate that NK1 receptors mediate secretion of macromolecules and NK2 receptors mediate smooth muscle contraction, in response to tachykinins in the ferret trachea in vitro.
Calcium-dependent chloride secretion across cultures of human tracheal surface epithelium and glands
Authors: Yamaya M. , Ohrui T. , Finkbeiner W. E. , Widdicombe J. H.
Published: December 2017

Surface epithelium and gland cells from human trachea were cultured on porous-bottom inserts and loaded with fura 2 to permit measurement of the intracellular calcium concentration ([Ca2+]i). Short-circuit current (Isc), an index of transepithelial active ion transport, was measured on cells from the same cultures. Surface epithelial [Ca2+]i of 82 +/- 15 nM was increased transiently by isoproterenol, histamine, and bradykinin with maximal increases of 88 +/- 17, 480 +/- 149, and 978 +/- 214 nM (n = 15), respectively. Baseline [Ca2+]i in cultured gland cells of 68 +/- 11 nM was increased transiently by isoproterenol, histamine, methacholine, and bradykinin with maximal increases of 105 +/- 19, 233 +/- 47, 327 +/- 121, and 634 +/- 151 nM (n = 17-21), respectively. In both cell types, mediators that increased [Ca2+]i also increased Isc with a time course identical to the increase in [Ca2+]i. Pretreatment with the calcium chelator, 1,2-bis-(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, acetoxymethyl ester (BAPTA-AM), had no effect on basal Isc or transepithelial resistance but markedly inhibited both the Isc and [Ca2+]i responses to agonists. Forskolin (10(-5) M), 3-isobutyl-1-methylxanthine (10(-3) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), and 8-(4-chlorophenylthio)-cAMP (10(-3) M) had no or only trivial effects on Isc and [Ca2+]i. We suggest that mediators increase Isc across human airway epithelium by activating Ca-dependent basolateral K channels, resulting in hyperpolarization and an increased driving force for Cl exit through apical membrane Cl channels.
Neutrophil-mediated endothelial angiotensin-converting enzyme dysfunction: role of oxygen-derived free radicals
Authors: Chen X. , Catravas J. D.
Published: December 2017

We examined the mechanisms whereby phorbol 12-myristate 13-acetate (PMA)-activated rabbit peritoneal neutrophils [polymorphonuclear leukocytes (PMN)] altered endothelial-bound angiotensin-converting enzyme (ACE) activity in cultured bovine pulmonary arterial endothelial cells (EC). PMA or PMN alone had no effect on ACE activity. When PMN were coincubated with PMA (10 ng/ml) for 4 h in Earle's salt solution, endothelial ACE activity was decreased by 87%. No EC cytotoxicity was observed at this time as determined by 51Cr release from prelabeled EC. Activated PMN-mediated decreased ACE activity was inhibited by catalase (2,000 U/ml) but not by superoxide dismutase (300 U/ml). The decrease in ACE activity was also inhibited by the hydroxyl radical scavenger dimethylthiourea (5 mM) but not mannitol (5 mM), which does not cross cell membranes. Pretreatment of EC with the iron chelator deferoxamine mesylate (1-10 mM) for 4 h attenuated the PMN-mediated decrease in ACE activity, as did the thiol reducing agent, 2-mercaptoethanol (0.1 mM), and the myeloperoxidase inhibitor, cyanide (5 mM), but not azide (1-50 mM). Treatment with the proteinase inhibitor phenylmethylsulfonyl fluoride, with human alpha-antitrypsin, or with the nitric oxide synthase inhibitor N omega-nitro-L-arginine had no effect on PMN-mediated ACE dysfunction. These results suggest that PMN-mediated ACE dysfunction may be due to the production of hydrogen peroxide by PMN and its subsequent conversion into hydroxyl radicals.
Neutrophil elastase increases secretory leukocyte protease inhibitor transcript levels in airway epithelial cells
Authors: Abbinante-Nissen J. M. , Simpson L. G. , Leikauf G. D.
Published: December 2017

Airway inflammation is often associated with the infiltration of activated neutrophils and subsequent protease release. Although aiding in the digestion and phagocytosis of foreign proteins and microorganisms, neutrophil proteases can indiscriminately damage healthy lung tissue. In the conducting airway, proteases, particularly neutrophil elastase, are counter-balanced by several antiproteases, including secretory leukocyte protease inhibitor (SLPI). SLPI can be produced locally by a number of cells including the airway epithelial cell. To examine the effects of neutrophil granule components on SLPI transcript levels, airway epithelial cells were treated (up to 96 h) with elastase, other proteases, or enzymes isolated from human sputum. We found that neutrophil elastase increased SLPI transcript levels in primary and transformed human airway epithelial cells in a time- and dose-dependent manner. Other neutrophil products, such as cathepsin G, myeloperoxidase, and lysozyme, had little or no effect on SLPI transcript levels. However, two nonneutrophil proteases, trypsin and pancreatic elastase, also increased SLPI transcript levels at higher doses than that required of neutrophil elastase. Two inflammatory cytokines, tumor necrosis factor-alpha and interleukin-8, produced little or no effect on SLPI transcript levels. This study demonstrates one way in which SLPI is regulated, via a protease that it inhibits, neutrophil elastase.
Effects of activated eosinophils cultured from human umbilical cord blood on guinea pig trachealis
Authors: Hamann K. J. , Strek M. E. , Baranowski S. L. , Munoz N. M. , Williams F. S. , White S. R. , Vita A. , Leff A. R.
Published: December 2017

We studied the biochemical indexes and corresponding induction of airway smooth muscle contraction and hyperresponsiveness in guinea pig trachealis in situ caused by cultured eosinophils derived from mononuclear cell fractions of human umbilical cord blood. A method was developed that permitted isolation of large numbers of cells (approximately 2.6 x 10(6)/ml cord blood) having morphological and immunohistological characteristics of human peripheral blood eosinophils. After activation with 10(-6) M formyl-Met-Leu-Phe + 5 micrograms/ml cytochalasin B (fMLP + B), in situ application to the epithelial surface of 6 x 10(6) cord-derived eosinophils (CDE)/surface area (cm2) caused 1.46 +/- 0.24 g/cm maximal active tracheal tension in guinea pig tracheal smooth muscle (P < 0.005 vs. zero baseline). Muscarinic responsiveness also was augmented in situ in trachealis preparations treated with activated 3-wk CDE. Contraction caused by 3 x 10(-7) mol/kg iv methacholine (MCh) was 0.94 +/- 0.18 g/cm at baseline vs. 1.80 +/- 0.24 g/cm after activated CDE (P = 0.02). Control (sham-activated) 3-wk CDE caused neither significant contraction [0.41 +/- 0.16 g/cm active tension (AT); P < 0.05 vs. fMLP+B] nor augmented muscarinic responsiveness. Cells cultured for 5 wk contained fewer granules than 3-wk CDE and also caused less direct contraction of trachealis (0.73 +/- 0.14 g/cm AT) after activation (P < 0.01 vs. 3-wk CDE). Both contraction and muscarinic augmentation were blocked in 3-wk CDE after blockade of leukotriene C4 (LTC4) synthesis by pretreatment with the 5-lipoxygenase inhibitor, A63162 (50 microM). Treatment with A63162 had no effect on the stimulated release of eosinophil peroxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Asbestos directly increases lung epithelial permeability
Authors: Peterson M. W. , Walter M. E. , Gross T. J.
Published: December 2017

Asbestos causes the fibrotic lung disease asbestosis, but the biologic basis for this is unknown. Lung epithelial dysfunction including increased permeability is hypothesized to contribute to lung scarring in other forms of pulmonary fibrosis. Lung epithelial permeability is increased in both animals and humans exposed to asbestos. It is not known whether the increased epithelial permeability results from direct effects of asbestos or occurs as a result of the inflammatory reaction to asbestos fibers. To address this question we used a cultured human lung epithelial model, and we measured the direct effect of asbestos on lung epithelial barrier integrity as measured by mannitol permeability. We exposed the monolayer to chryogenically ground, respirable-sized chrysotile asbestos particles. This chrysotile asbestos caused a dose- and time-dependent increase in mannitol permeability across the epithelial monolayer. Increased mannitol permeability occurred both in the presence and in the absence of serum, was not due to cytotoxicity as measured by lactate dehydrogenase release, and was not associated with altered actin cytoskeleton at the light microscopic level. Permeability to 70 kDa neutral dextran also increased after asbestos exposure; however, the absolute permeability to dextran was less than mannitol permeability. Neither latex beads nor tantalum caused any change in permeability, suggesting that our findings are not explained by nonspecific effects of particles. Increased permeability did not reverse in the continued presence of asbestos and persisted even after removing the asbestos. Finally, surface-bound iron did not appear to be necessary for this effect because neither chelating iron with deferoxamine nor iron-loading the asbestos altered the effect on mannitol permeability. These results show that asbestos has direct effects on lung epithelial permeability. Together with the recent observation that asbestos directly increases epithelial fibrinolytic activity, our results suggest a novel mechanism for asbestos-induced lung injury.
Postnatal lung growth: evidence that the gas-exchange region grows fastest at the periphery
Authors: Massaro G. D. , Massaro D.
Published: December 2017

The site(s) at which the gas-exchange region enlarges after alveoli are formed from the saccules of the immature lung is unclear; however, this information might be important to studies of the regulation of lung growth. Although aware of important assumptions on which it rests, we undertook this study to test the idea that the lung's gas-exchange region enlarges more rapidly in the immediate subpleural region than more centrally. To label the interstitium of the gas-exchange region, rats were provided silver-containing water from age 23 to 135 days (112 days). Some were killed at age 135 days, others 51 days after silver exposure ended (age 186 days). We considered silver grains that formed in the interstitium as a marker of lung present or formed during silver exposure; tissue added after exposure would diminish the numerical density of grains and the fastest growing sites would have the lowest grain density. Rats killed on the 112th day of silver exposure had a silver grain density in their immediate subpleural gas-exchange matrix that was 28% lower than in their more central gas-exchange matrix. Rats killed 51 days after silver exposure ended had a grain density in the immediate subpleural region that was 65% lower than in the central matrix of the gas-exchange region. The grain density in the rats killed 51 days after silver exposure ended was 33% lower in the central matrix and 67% lower in the peripheral matrix than in the respective regions of rats killed on day 112 of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Uptake of antioxidants in surfactant liposomes by cultured alveolar type II cells is enhanced by SP-A
Authors: Walther F. J. , David-Cu R. , Supnet M. C. , Longo M. L. , Fan B. R. , Bruni R.
Published: December 2017

Antioxidant delivery may be targeted toward the alveolar epithelium by encapsulating superoxide dismutase (SOD) and catalase in liposomes made from pulmonary surfactant. We studied whether antioxidant-surfactant liposomes increase cellular antioxidant activity in alveolar type II cells and whether this effect is influenced by the presence of surfactant protein A (SP-A). Cu,Zn SOD and catalase were encapsulated in liposomes made from synthetic phospholipids with or without 5% SP-A or from natural cow surfactant. Alveolar type II cells from adult rats were preincubated for 20 h, and liposome mixtures were added for 24 h, followed by measurement of cellular SOD and catalase activities (U/mg DNA). Antioxidant-surfactant liposomes increased alveolar type II cell antioxidant activity sharply. Uptake of SOD/catalase from liposomes with synthetic phospholipids and SP-A was twice that from liposomes without SP-A and did not further improve in the presence of SP-B and -C. Encapsulation of antioxidants diminished the surface activity of the surfactant liposomes, but this feature was absent in the presence of SP-A. These data suggest that: 1) antioxidant-surfactant liposomes augment alveolar type II cell antioxidant activity, 2) liposomal uptake is facilitated by the presence of SP-A, and 3) inhibition of surface activity of surfactant by encapsulated antioxidants can be reversed by SP-A.
Mitigation of oxidant injury to lung microvasculature by intratracheal instillation of antioxidant enzymes
Authors: Barnard M. L. , Baker R. R. , Matalon S.
Published: December 2017

We quantitated the ability of intratracheally administered liposome-encapsulated antioxidant enzymes to reduce reactive oxygen species injury to the pulmonary microvasculature. Cationic liposomes containing 3,500 U of Cu,Zn superoxide dismutase (Cu,Zn SOD) and 3,124 U of catalase were instilled into rabbits. The animals were killed 2-72 h later and their lungs were removed and perfused with Krebs Ringer with 5% wt/vol of fat-free bovine serum albumin. The pulmonary filtration co-efficient (Kf,c) was measured before and after adding 500 microM xanthine and 5 mU/ml xanthine oxidase (XO) into the lung perfusate. Two hours after a single intratracheal instillation of liposome-entrapped Cu,Zn SOD and catalase, lung antioxidant enzyme activities were 34 and 125% higher than the corresponding control values, remained virtually unchanged for up to 8 h post-instillation, and then decreased, reaching baseline values between 24 and 72 h. Addition of xanthine and XO into the lung perfusate of un-instilled rabbits, or rabbits that received liposomes with inactivated enzymes, caused a 100% increase in Kf,c (control value: 2 +/- 0.12 ml.min-1 x cmH2O-1 per 100 g dry lung weight). On the other hand, Kf,c values of rabbits lungs instilled with liposome-encapsulated active Cu,Zn SOD and catalase and challenged with xanthine and XO 8-24 h later remained at baseline levels. Instillation of liposomes containing either enzyme was equally effective in preventing the increase in Kf,c, indicating that both superoxide anions and hydrogen peroxide were necessary for the initiation of injury. We concluded that intratracheal instillation of liposome-encapsulated antioxidant enzymes caused a transient increase of lung antioxidant enzyme levels which protects the pulmonary microvasculature from free radical-initiated injury.
Pleural mesothelial cell response to inflammation: tumor necrosis factor-induced mitogenesis and collagen synthesis
Authors: Owens M. W. , Grimes S. R.
Published: December 2017

This study examined the effects of tumor necrosis factor-alpha on pleural mesothelial cell proliferation and collagen synthesis, functions which may be important in the response of the pleura to injury. Tumor necrosis factor-alpha caused a significant increase in proliferation and collagen production by rat pleural mesothelial cells in vitro. Proliferation increased in a time- and dose-dependent manner, resulting in an approximate twofold increase in the uptake of [3H]thymidine relative to control. The uptake of [3H]proline into collagenase-sensitive protein increased in a dose-dependent manner for concentrations of tumor necrosis factor-alpha > or = 1.0 ng/ml. The increase in collagen production were associated with increased steady-state levels of alpha 1(I)-procollagen mRNA. These results suggest that tumor necrosis factor-alpha may have a significant effect on pleural mesothelial cell function in vivo in the setting of inflammation. Increases in pleural mesothelial cell proliferation and collagen synthesis in response to inflammatory mediators, like tumor necrosis factor-alpha, may be important in healing the pleura after injury by a variety of disease processes.
Visualization of high- and low-affinity beta-adrenergic receptors in rat lung: upregulation by chronic hypoxia
Authors: Birnkrant D. J. , Davis P. B. , Ernsberger P.
Published: December 2017

Chronic hypoxia induces a hyperadrenergic state which down-regulates beta-adrenergic receptors (beta-AR) in the heart. We visualized lung beta-AR binding sites after adaptation to chronic hypoxia by autoradiography of whole lung sections labeled with 50 pM 125I-labeled pindolol. A low concentration of agonist (32 nM isoproterenol) selectively masked beta-ARs with high affinity for agonists. Total specific beta-AR binding increased twofold with hypoxia. In both the control and hypoxic lung sections, 60–70% of the beta-ARs were in a high-affinity state, which could be reversed by guanine nucleotide. Autoradiography revealed a high density of high- and low-affinity beta-AR sites in lung parenchyma, predominantly involving alveolar walls, but also the walls of airways and blood vessels. The distribution of high- and low-affinity beta-AR within the lung was qualitatively identical. Hypoxia increased beta-AR binding without affecting its distribution. The majority of the additional beta-ARs induced during adaptation to chronic hypoxia are in the high-affinity state, and are thus of probable functional significance.
Oxygen conformance of cellular respiration in hepatocytes
Authors: Schumacker P. T. , Chandel N. , Agusti A. G.
Published: December 2017

Cellular respiratory rates are normally determined by metabolic activity, but become rate limited by O2 availability if the cell O2 tension (PO2) falls below a critical value (typically 1–10 Torr). An ability to reduce metabolic activity and energy demand in response to a falling O2 availability might confer an increased resistance to a diminished O2 supply. Isolated rat hepatocytes were studied in primary culture under controlled O2 tensions. Cells were obtained by collagenase digestion and seeded into nutritive media in control and experimental spinner flasks at identical cell densities. Cells subjected to rapid reduction in PO2 (100⇢0 Torr over < 40 min) exhibited undiminished O2 uptake until PO2 fell below 10 Torr. By contrast, when cell PO2 was reduced over several hours, significant decreases in O2 uptake became evident at O2 tensions as high as 70 Torr. These decreases were associated with a reduction in ATP concentration and an increase in NAD(P)H, compared with rapidly deoxygenated cells at the same PO2. No loss in cell viability was detected after 24 h at reduced PO2. The decrease in respiratory rate was associated with an increased rate of lactic acid production relative to normoxic controls. Restoration of normoxia was associated with an immediate return of O2 uptake to control levels. These results demonstrate that hepatocytes are capable of reversibly decreasing metabolic activity and O2 demand during sustained moderate reductions in PO2, via a mechanism that appears to involve an inhibition of mitochondrial function other than O2 supply limitation. This response may alter cellular susceptibility to physiological stresses including hypoxia.
A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability
Authors: Partridge C. A. , Jeffrey J. J. , Malik A. B.
Published: December 2017

Tumor necrosis factor-alpha (TNF-alpha) may increase vascular endothelial permeability through alteration of the extracellular matrix (ECM). Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells grown to confluence on microporous filters with 10(4) U/ml TNF-alpha for 24 h increased monolayer permeability to 125I-labeled albumin two- to threefold. TNF-alpha treatment also induced expression of a 96-kDa gelatinolytic metalloproteinase that was present in the medium and bound to the ECM. The induced 96-kDa metalloproteinase was purified from conditioned medium and found to cleave fibronectin, laminin, types IV and V collagens, and gelatins from types I and III collagens, suggesting identity as a type IV collagenase-gelatinase. Incubation of BPMVE cells with the 96-kDa gelatinase increased monolayer permeability, an effect prevented by inclusion of either tissue inhibitor of metalloproteinase (TIMP) or 1,10-phenanthroline. When BPMVE cells were incubated with the 96-kDa gelatinase or 10(4) U/ml TNF-alpha and then stripped from the filters, the remaining ECM displayed increased permeability to 125I-albumin compared with matrix from untreated BPMVE. The ECM extracts from both TNF-alpha- and enzyme-treated cells were found to contain less fibronectin, whereas their total protein contents were similar to those of untreated controls. These results suggest that the 96-kDa metalloproteinase induced by TNF-alpha contributes to increased vascular endothelial permeability through the degradation of specific extracellular matrix components.
Release of ED1 fibronectin from matrix of perfused lungs after vascular injury is independent of protein synthesis
Authors: Vincent P. A. , Rebres R. A. , Lewis E. P. , Hurst V. , Saba T. M.
Published: December 2017

Fibronectin (Fn) is an adhesive protein found in the plasma and extracellular tissue matrix. Locally synthesized tissue or cellular Fn (cFn) has extra domains (ED1 and ED2) not present in liver synthesized plasma Fn (pFn). In the lung, Fn is found in the endothelial and epithelial basement membranes, as well as in the interstitial matrix. Utilizing murine monoclonal antibodies to ED1 of cFn, we studied the release of total Fn as well as ED1-Fn into the plasma-free perfusate of the isolated perfused rabbit lung in relation to changes in lung weight due to fluid accumulation after oxidant (H2O2) challenge. Both parameters were also studied after addition of cycloheximide (20 micrograms/ml perfusate) to the perfusion medium to inhibit lung protein synthesis. After continuous H2O2 challenge (11 nmol.ml buffer-1.min-1), there was a 2.25 +/- 0.62 g increase in lung weight over 60 min. Measurement of 125I-labeled albumin clearance at 20 min after the start of H2O2 infusion confirmed an increase in lung endothelial protein permeability after H2O2 treatment. Fn antigen was released into the perfusate as early as 15 min after oxidant challenge. By 60 min, total perfusate Fn increased in H2O2-treated lungs (n = 6) to 2.10 +/- 0.48 micrograms/ml compared with only 0.35 +/- 0.09 micrograms/ml in normal control lungs (n = 5). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of nonreduced samples revealed that the Fn released consisted of primarily intact (440 kDa) Fn as well as Fn fragments. A rapid release of ED1-Fn paralleled the increased release of total Fn.(ABSTRACT TRUNCATED AT 250 WORDS)
Postinsult treatment with N-acetyl-L-cysteine decreases IL-1-induced neutrophil influx and lung leak in rats
Authors: Leff J. A. , Wilke C. P. , Hybertson B. M. , Shanley P. F. , Beehler C. J. , Repine J. E.
Published: December 2017

We found that intratracheal administration of interleukin-1 alpha (IL-1) rapidly (5 h) increased leak of 125I-labeled albumin from the blood into the lung (lung leak), influx of neutrophils into lung lavages, lung oxidized glutathione (GSSG) levels, breath hydrogen peroxide (H2O2) concentrations, and lung histological abnormalities in intact rats. Since N-acetyl-L-cysteine (NAC) increases glutathione (GSH) levels in vivo and scavenges oxygen radicals in vitro, we tested the effect of NAC given intravenously on lung changes following intratracheal IL-1 administration. We found that administration of NAC immediately before or 2.5 h after intratracheal administration of IL-1 decreased lung leak, neutrophil influx into lung lavages, and defects in lung histology. NAC treatment also increased blood acid soluble sulfhydryl levels, reduced lung GSSG increases, and decreased breath H2O2 levels in rats given IL-1 intratracheally. The latter findings are consistent with the possibility that NAC is enhancing GSH or other sulfhydryls and, as a result, reducing oxidative stress due to H2O2 or H2O2-derived products. Since postinsult treatment with NAC is effective in this relevant intact animal model of acute lung injury, we speculate that NAC may have promise in the treatment of patients with the adult respiratory distress syndrome.