bluuebunny/crossref_metadata_2025 huggingface dataset:
Corticosteroids increase secretory leukocyte protease inhibitor transcript levels in airway epithelial cells
Authors: Abbinante-Nissen J. M. , Simpson L. G. , Leikauf G. D.
Published: December 2017

Secretory leukocyte protease inhibitor (SLPI) is the predominant antiprotease of the conducting airways and may play a role in reducing airway inflammation. In this study, the effect of corticosteroids used in the treatment of inflammatory airway disease on SLPI transcript levels was investigated. When human airway epithelial cells (9HTEo-) were treated continuously with 10 nM fluticasone propionate, SLPI transcript levels increased within 12 h, with maximal transcript accumulation occurring at 24–48 h. Several corticosteroids (0.1-1,000 nM) were compared, and the following potency in increasing SLPI transcript levels was observed: fluticasone > triamcinolone > or = dexamethasone > methylprednisolone > hydrocortisone. Fluticasone, the most potent corticosteroid, increased SLPI transcript levels at doses as low as 0.1 nM, whereas hydrocortisone, the least potent corticosteroid, was effective at 100 nM. Fluticasone-induced increases in SLPI transcript levels were inhibited by cycloheximide, suggesting protein synthesis may be required for this response. Because proteases are likely to be present when corticosteroids are administered therapeutically, we examined the interaction between elastase and fluticasone and found they act synergistically to increase SLPI transcript levels. Our findings suggest that corticosteroids may exert their antiinflammatory effects in part by increasing airway epithelial cell SLPI production.
Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury
Authors: Bui K. C. , Buckley S. , Wu F. , Uhal B. , Joshi I. , Liu J. , Hussain M. , Makhoul I. , Warburton D.
Published: December 2017

Hyperoxia causes a reproducible pattern of lung injury and repair in rodents, in which proliferation of alveolar epithelial cells (AEC) and fibroblasts is observed during recovery. We postulated that if quiescent cells are stimulated to reenter the cell cycle, then cyclin expression and cyclin-dependent protein kinase activity would be reactivated in AEC during the repair process after hyperoxic lung injury. To test this hypothesis, we exposed adult rats to short-term hyperoxia, followed by recovery for various times in room air. Cellular proliferation in vivo was confirmed by 1) flow cytometric analysis of DNA content (FACS) of freshly isolated AEC and 2) immunohistochemistry of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA on lung sections. The percentage of freshly isolated AEC in S phase and G2/M phase on FACS analysis increased twofold to a maximum of 16.5%, after 48 h in 100% oxygen and 48 h recovery in air. Cyclins A and D and p34cdc2 protein expression were also increased during the recovery period; while p33cdk2 and p34cdk4 increased only slightly. p34cdc2 histone H1 kinase activity, both in whole lung and in AEC, decreased initially after 48 h in oxygen. However, a marked increase in p34cdc2 kinase activity was observed at 48 h recovery in whole lung and returned to baseline by 72 h. In isolated and cultured AEC, p34cdc2 kinase activity was maximal at 24 h of recovery in air. We conclude that cyclins A and D and p34cdc2 protein expression and p34cdc2 kinase activity are increased in vivo during recovery from hyperoxic lung injury in both adult rat lungs and in AEC isolated from these lungs. We speculate that the induction of cyclin-dependent protein kinase activity is a key event in mediating the proliferative cellular repair response to lung injury.
Intracellular pH regulates voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells
Authors: Yamakage M. , Lindeman K. S. , Hirshman C. A. , Croxton T. L.
Published: December 2017

Changes in CO2 or in pH modify airway smooth muscle contractility. To investigate the mechanisms involved, we compared K(+)-induced contractions in porcine bronchial rings exposed to different CO2 concentrations and directly measured the effects of changes in intracellular (pHi) or extracellular pH (pHo) on Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDC) in porcine tracheal smooth muscle cells. Hypocapnia and hypercapnia caused leftward and rightward shifts, respectively, in the dose-response to K+ (P < 0.05) but did not change the maximum force obtained. Peak ICa (10 mM external Ca2+) elicited by depolarizing pulses from -80 mV was maximal [-265 +/- 12 pA (mean +/- SE), n = 19] at +10 mV. Intracellular acidification decreased the peak ICa at +10 mV from -261 +/- 20 pA to -177 +/- 12 pA (P < 0.05, n = 4), while intracellular alkalinization increased the peak ICa at +10 mV from -302 +/- 27 pA to -368 +/- 26 pA (P < 0.05, n = 4). Changes in pHo had little effect on ICa. There was no shift in the voltage-dependence of induced ICa with any change. We conclude that pHi, but not pHo, directly modulates the entry of Ca2+ into airway smooth muscle cells through VDC. This mechanism may contribute to regulation of airway tone by CO2.
Adenovirus-mediated gene transfer to human bronchial submucosal glands using xenografts
Authors: Pilewski J. M. , Engelhardt J. F. , Bavaria J. E. , Kaiser L. R. , Wilson J. M. , Albelda S. M.
Published: December 2017

The cystic fibrosis (CF) transmembrane conductance regulator has been localized to both submucosal glands and surface epithelium, suggesting that both glandular and surface epithelium may be important targets for gene therapy. To determine the distribution and efficiency of recombinant adenovirus-mediated gene transfer to human airway submucosal glands, an in vivo model was developed by heterotopically transplanting human bronchial segments from both normal and CF lung tissue into severe combined immunodeficient mice. A serotype 5 E1-deleted recombinant adenovirus containing a lacZ reporter gene driven by the cytomegalovirus promoter (H5.010CMVlacZ) was used to infect the xenografts. Transgene expression was correlated with three factors: 1) viral titer, 2) penetration of microspheres, and 3) dwell time of the viral instillate. At viral titers ranging from 10(8) to 10(11) plaque forming units/ml, expression of the lacZ gene was observed in a subpopulation of epithelial cells within approximately 40% of submucosal glands, with more efficient gene transfer to the ducts than the secretory tubules. Within individual glands, gene transfer varied from < 1% to approximately 20% of submucosal cells, including duct, mucous, and serous cells. Adenovirus-sized fluorescent microspheres were found to penetrate only some of the submucosal glands, suggesting that the gene transfer efficiency to submucosal tubules is due to limited viral particle penetration rather than tropism. Gene transfer to surface epithelial cells was inefficient. However, the percentage of transduced surface epithelial cells increased from < 1% to 5–10% as the dwell time of viral solution was increased from 5 min to 1 h, indicating that the time allowed for virus binding and entry is important for gene transfer efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)
The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide
Authors: Pryor W. A. , Squadrito G. L.
Published: December 2017

Nitric oxide and superoxide, which are produced by several cell types, rapidly combine to form peroxynitrite. This reaction can result in nitric oxide scavenging, and thus mitigation of the biological effects of superoxide. Also, superoxide can trap and hence modulate the effects of nitric oxide; superoxide dismutase, by controlling superoxide levels, therefore can influence the reaction pathways open to nitric oxide. The production of peroxynitrite, however, causes its own sequelae of events: Although neither .NO nor superoxide is a strong oxidant, peroxynitrite is a potent and versatile oxidant that can attack a wide range of biological targets. The peroxynitrite anion is relatively stable, but its acid, peroxynitrous acid (HOONO), rearranges to form nitrate with a half-life of approximately 1 s at pH 7, 37 degrees C. HOONO exists as a Boltzmann distribution of rotamers; at 5-37 degrees C HOONO has an apparent acidity constant, pKa,app, of 6.8. Oxidation reactions of HOONO can involve two-electron processes (such as an SN2 displacement) or a one-electron transfer (ET) reaction in which the substrate is oxidized by one electron and peroxynitrite is reduced. These oxidation reactions could involve one of two mechanisms. The first mechanism is homolysis of HOONO to give HO. and .NO2, which initially are held together in a solvent cage. This caged pair of radicals (the "geminate" pair) can either diffuse apart, giving free radicals that can perform oxidations, or react together either to form nitrate or to reform HOONO (a process called cage return). A large amount of cage return can explain the small entropy of activation (Arrhenius A-factor) observed for the decomposition of HOONO. A cage mechanism also can explain the residual yield of nitrate that appears to be formed even in the presence of high concentrations of all of the scavengers studied to date, since scavengers capture only free HO. and .NO2 and not caged radicals. If the cage mechanism is correct, the rate of disappearance of peroxynitrite be slower in solvents of higher viscosity, and we do not find this to be the case. The second mechanism is that an activated isomer of peroxynitrous acid, HOONO*, can be formed in a steady state. The HOONO* mechanism can explain the inability of hydroxyl radical scavengers to completely block either nitrate formation or the oxidation of substrates such as methionine, since HOONO* would be less reactive, and therefore more selective, than the hydroxyl radical itself.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of cigarette smoke extract on bovine bronchial epithelial cell attachment and migration
Authors: Cantral D. E. , Sisson J. H. , Veys T. , Rennard S. I. , Spurzem J. R.
Published: December 2017

The repair of injured epithelium involves a complex interaction between epithelial cells and the underlying extracellular matrix. We studied the effects of sublethal concentrations of cigarette smoke extract (CSE) and two volatile components of cigarette smoke, acetaldehyde and acrolein, on bovine bronchial epithelial cell (BBEC) attachment and migration in vitro. After short-term exposure (2 and 6 h) to CSE, BBEC attachment to fibronectin-coated dishes was decreased, and migration to fibronectin was unchanged. After a longer period of exposure, 24 h, attachment was increased and migration was unchanged. Exposure to a peptide containing the amino acid sequence arginine-glycine-aspartic acid (RGD) reduced attachment equally well for control and smoke-exposed cells. BBEC sheet migration was reduced over 72 h after exposure to CSE. Acrolein reduced BBEC migration to fibronectin but had no effect on attachment. Acetaldehyde had no effect on either attachment or migration. We conclude that exposure to CSE has important effects on bronchial epithelial cell migration and attachment, and that these effects change over time.
Neurokinin depletion attenuates pulmonary changes induced by antigen challenge in sensitized guinea pigs
Authors: Warth Mdo P. , Maldonado E. A. , Fernezlian S. M. , Leme A. S. , Perini A. , Saldiva P. H. , Martins M. A.
Published: December 2017

The role of neurokinins in the acute pulmonary response to antigen was studied in guinea pigs that received ovalbumin (50 mg/kg ip) on days 1 and 3 and capsaicin (50 mg/kg sc) on day 21 (OAC); ovalbumin on days 1 and 3 (OA1); capsaicin on day 1 and OA on days 8 and 10 (COA); and ovalbumin on days 8 and 10 (OA2). On day 28, guinea pigs were submitted to ovalbumin aerosol challenge. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) were significantly lower in OAC and COA groups compared with OA1 and OA2 groups (P < 0.001). There was no difference between maximal Edyn and RL values obtained in OAC and COA groups. Morphometric analysis of lungs showed significantly (P < 0.05) lower values of contraction index of airways, peribronchial edema, and alveoli over inflation in guinea pigs that received capsaicin compared with intact guinea pigs. Capsaicin treatment did not influence the formation of specific IgG1 anaphylactic antibodies. We conclude that neurokinin depletion results in a decrease in the pulmonary mechanical and inflammatory responses to antigen challenge in sensitized guinea pigs. These effects are observed when capsaicin is given either before or after sensitization.
Oxygen modulates surfactant protein mRNA expression and phospholipid production in human fetal lung in vitro
Authors: Acarregui M. J. , Brown J. J. , Mallampalli R. K.
Published: December 2017

We studied the effect of 20-95% O2 on mRNA levels for the surfactant-associated proteins (SP)-A, SP-B, and SP-C and [3H]choline incorporation into total phosphatidylcholine and type II cell-specific disaturated phosphatidylcholine (DPPC) in human fetal lung in culture. SP-A mRNA levels were increased by 25 and 39% in lung explants incubated in 70 and 95% O2, respectively, compared with levels in tissues incubated in 20% O2. SP-B mRNA levels were unaffected by O2, whereas SP-C mRNA levels were increased by 85, 102, and 115% in atmospheres of 35, 50, and 70% O2, respectively. [3H]choline incorporation into total phosphatidylcholine and DPPC were both increased in human fetal lung explants incubated in increased O2 concentrations compared with tissues incubated in 20% O2. Tissue levels of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) activity were not affected by O2 concentration, implying that the changes observed in SP mRNA levels and [3H]choline incorporation may not be mediated through alterations in PKA enzyme activity. These findings demonstrate that O2 regulates SP mRNA expression and phospholipid production in human fetal lung in vitro. We speculate that surfactant composition and possibly function may be regulated by O2 in human lung.
Stimulus and cell-specific expression of C-X-C and C-C chemokines by pulmonary stromal cell populations
Authors: Lukacs N. W. , Kunkel S. L. , Allen R. , Evanoff H. L. , Shaklee C. L. , Sherman J. S. , Burdick M. D. , Strieter R. M.
Published: December 2017

Chronic inflammatory responses in the lung rely on the continual recruitment of leukocytes to the site of inflammation. Recent data have demonstrated a possible role for stromal cell-derived chemokines in leukocyte recruitment. In the present study we examined the production of interleukin (IL)-8 and ENA-78, members of the C-X-C family of chemokines, and macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, members of the C-C chemokine family, from pulmonary smooth muscle and endothelial cells. The production of IL-8 and ENA-78 was induced by early response cytokines, IL-1 and tumor necrosis factor (TNF), but not by immune-associated cytokines, IL-4, IL-10, or interferon (IFN)-gamma. In contrast, the production of MIP-1 alpha and MIP-1 beta by pulmonary vascular smooth muscle cells increased when stimulated by immune-associated cytokines as well as with IL-1 beta and TNF. The level of MIP-1 alpha production induced in smooth muscle cells by the immune-associated cytokines, IL-4, IFN-gamma, and IL-10 ranged from 0 to 340 pg/ml. The production of MIP-1 beta in response to the immune-associated cytokines IL-4, IFN-gamma, and IL-10 in smooth muscle cells ranged from 260 to 940 pg/ml. Human pulmonary artery endothelial cells did not generate MIP-1 alpha or MIP-1 beta in response to graded doses of any of the cytokines. These data demonstrate differential induction of C-X-C and C-C chemokines from nonimmune stromal cell populations.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of CNP in human airways: cGMP-mediated stimulation of ciliary beat frequency
Authors: Geary C. A. , Davis C. W. , Paradiso A. M. , Boucher R. C.
Published: December 2017

Ciliated airway epithelial cells contribute to mucociliary transport systems via ciliary beating and electrolyte transport mechanisms. Both of these activities are regulated by agonists acting through intracellular calcium- and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent processes (5, 15, 18, 27). This study examines the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of both ciliary beat frequency (CBF) and electrolyte transport in human airway epithelia (HAE). In a previous report, cGMP production in HAE was observed after stimulation with either C-type natriuretic peptide (CNP) or sodium nitroprusside (SNP) (6). In this study, CNP was found to increase CBF by 30 +/- 6.9%, and this effect was mimicked by the cGMP analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), but not by sodium nitroprusside. CNP-induced increases in CBF do not appear to be mediated by changes in either intracellular calcium or cAMP levels. Using modified Ussing chambers, we also investigated CNP's potential modulation of sodium and chloride transport rates. Neither CNP, nor SNP, nor 8-BrcGMP altered active ion transport rates. We conclude that CNP regulates ciliary beat via cGMP-dependent mechanisms, whereas no effect of CNP or cGMP on ion transport was detected.
A1 adenosine receptor antagonists block ischemia-reperfusion injury of the lung
Authors: Neely C. F. , Keith I. M.
Published: December 2017

Ischemia-reperfusion (I-R) injury of the lung occurs after lung transplantation, pulmonary thromboembolectomy, or cardiopulmonary bypass. In the heart, adenosine, A1 adenosine receptor agonists, and a brief period of preconditioning ischemia attenuate I-R injury. Moreover, in the lung, thromboxane is released during ischemia and is an important mediator of I-R injury. We previously reported that adenosine produces vasoconstriction in the feline pulmonary vascular bed by acting on A1 receptors to induce the release of thromboxane and that these vasoconstrictor responses are desensitized by low doses of A1 receptor agonists. Because A1 receptor agonists mimic the effect of preconditioning ischemia, we hypothesized, in contrast to previously proposed mechanisms, that small amounts of adenosine released during preconditioning ischemia desensitize A1 receptors. Also, we hypothesized that greater amounts of adenosine are released after longer periods of ischemia, which activate A1 receptors. Thus if desensitization of A1 receptors is the mechanism by which preconditioning attenuates I-R injury of the heart and A1 receptor activation during ischemia plays an important role in I-R injury of the lung, A1 receptor antagonists should provide a protective effect in I-R injury of the lung. In this study, 2 h of ischemia and 2 h of reperfusion of the left lower lobe in intact-chest, spontaneously breathing cats caused lung injury characterized by the presence of neutrophils, macrophages, and RBCs in alveoli and caused alveolar edema, which was blocked in a highly significant manner by the A1 receptor antagonists xanthine amine congener (XAC) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). An intralobar arterial infusion of XAC (30 min before ischemia) reduced the %injured alveoli (defined as presence of 2 or more inflammatory cells or RBCs, or edematous fluid) from 60 +/- 10 to 7 +/- 2%, which was not significantly different from controls (5 +/- 1%; P < 0.0001). DPCPX (iv) reduced the %injured alveoli to 13 +/- 7% when administered 30 min before ischemia and to 6 +/- 2% when administered after 1 h of reperfusion, not significantly different from controls (P < 0.0001). Preconditioning ischemia (10-min ischemia +10-min reperfusion) also reduced the %injured alveoli after 2 h ischemia and 2 h reperfusion to 23 +/- 13%, almost identical to 2 h ischemia and 1 h reperfusion. These data support the hypothesis that A1 receptor antagonists block I-R injury of the lung. A1 receptor antagonists may be useful in preventing I-R injury after transplant surgery and during surgical procedures associated with I-R injury of the heart, brain, kidney, and spinal cord.
VIP inhibits basal and histamine-stimulated proliferation of human airway smooth muscle cells
Authors: Maruno K. , Absood A. , Said S. I.
Published: December 2017

Airway smooth muscle (ASM) cell proliferation contributes to increased airway resistance in bronchial asthma. We have examined the modulation of ASM proliferation by vasoactive intestinal peptide (VIP), a cotransmitter of airway relaxation. Human ASM cells were grown in culture as a monolayer. VIP (1.0 nM-1.0 microM) inhibited proliferation in a dose-dependent manner by up to 82% on day 2, but the related peptide glucagon had no effect. Histamine (100 nM-100 microM) increased cell counts by 66%, but in the presence of VIP, cell counts and [3H]thymidine incorporation were reduced by up to 55%. Adenosine 3',5'-cyclic monophosphate (cAMP)-promoting agents, including 3-isobutyl-1-methylxanthine, forskolin, and 8-bromo-adenosine 3',5'-cyclic monophosphate, alone and especially combined with VIP, reduced cell counts and [3H]thymidine incorporation, in correlation with cAMP levels. KT-5720 (1.0 nM-1.0 microM), a selective inhibitor of cAMP-dependent protein kinase A (PKA), abolished the inhibitory effect of VIP. The results show that VIP selectively and potently inhibits human ASM cell growth and multiplication, and nullifies the mitogenic effect of histamine, by a PKA-mediated mechanism. A deficiency of VIP may lead to ASM hyperplasia due to unopposed stimulation by endogenous mitogens.
Structure and function of the mouse surfactant protein B gene
Authors: Bruno Melanie A. , Bohinski Robert J. , Carter Jean E. , Foss Kathryn A. , Whitsett Jeffrey A.
Published: December 2017

Pages L381–L389: Melanie A. Bruno, Robert J. Bohinski, Jean E. Carter, Kathryn A. Foss, and Jeffrey A. Whitsett. “Structure and function of the mouse surfactant protein B gene.” On p. L383, Fig. 2 should appear as below (the nucleotide sequence from 4501 to 9882 was omitted).

(See PDF)
Muscarinic receptor subtypes are differentially distributed across brain stem respiratory nuclei
Authors: Mallios V. J. , Lydic R. , Baghdoyan H. A.
Published: December 2017

Cholinergic mechanisms are known to play a key role in the regulation of breathing, but the distribution of muscarinic receptor (mAChR) subtypes has not been localized within brain stem respiratory nuclei. This study examined the hypothesis that mAChR subtypes are heterogeneously distributed across brain stem nuclei that control breathing. With the use of in vitro receptor autoradiography, the results provide the first selective labeling and quantitative mapping of M1, M2, and M3 mAChR subtypes in cat brain stem regions known to regulate breathing. Among brain stem nuclei known to contain respiratory-related neurons, the greatest amount of mAChR binding was measured in the lateral and medial parabrachial nuclei and the lateral nucleus of the solitary tract. Fewer mAChRs were localized in nuclei comprising the ventral respiratory group (nucleus ambiguous, retrofacial nucleus) and ventral medulla (retrotrapezoid nucleus and ventrolateral medulla). The data provide an essential first step for future studies aiming to specify the regulatory role of mAChR subtypes within brain stem respiratory nuclei.
Proliferation of guinea pig tracheal epithelial cells in coculture with rat dorsal root ganglion neural cells
Authors: White S. R. , Garland A. , Gitter B. , Rodger I. , Alger L. E. , Necheles J. , Nawrocki A. R. , Solway J.
Published: December 2017

Neuropeptides secreted by sensory afferent nerves in airways may modulate growth of airway epithelial cells. To determine whether airway sensory C-fiber nerves secrete neuropeptides that stimulate airway epithelial cell proliferation, we measured S-phase traversal in guinea pig tracheal epithelial (GPTE) cells after coculture with rat dorsal root ganglion (DRG) cells. GPTE cells were grown in subconfluent culture on collagen-coated filters for 2 days. DRG cells were harvested from newborn rat pups and grown in primary culture for 7-10 days in separate wells. GPTE and DRG cells then were cocultured for 48 h, and 10 mM bromodeoxyuridine (BrdU), a thymidine analogue, was added in the final 24 h. Control GPTE cells were grown under similar conditions but without DRG cells. Coculture with DRG cells stimulated GPTE cell traversal of S phase. BrdU labeling in cocultured GPTE cells was 42.8 +/- 5.8 compared with 18.1 +/- 7.2% in control GPTE cells (P < 0.001, n = 6). Coculture in the presence of either the neurokinin (NK)1 receptor antagonists LY-297911 or CP-99,994, the NK2 receptor antagonist SR-48,968, or the calcitonin gene-related peptide (CGRP) receptor antagonist hCGRP-(8-37) (10(-7) M of each) during coculture attenuated proliferation of GPTE cells. Treatment with all three antagonists together during coculture decreased BrdU labeling to 2.4 +/- 0.9% of labeled cells vs. 8.5 +/- 0.5% of labeled cells during coculture without antagonists (n = 4, P < 0.02). DRG cells in coculture secreted substantial concentrations of CGRP [71.0 +/- 11.3 (+/- SE) pmol/ml], substance P (1.26 +/- 0.35 pmol/ml), and neurokinin A (0.45 +/- 0.10 pmol/ml) (n = 19 for each).(ABSTRACT TRUNCATED AT 250 WORDS)
Sodium-independent modulation of Na(+)-K(+)-ATPase activity by beta-adrenergic agonist in alveolar type II cells
Authors: Suzuki S. , Zuege D. , Berthiaume Y.
Published: December 2017

Although beta-adrenergic agonists are known to stimulate sodium transport in alveolar epithelial cells, the exact cellular mechanism involved in this process is unknown. We determined whether terbutaline, a beta-adrenergic agonist, modulated Na(+)-K(+)-ATPase in cultured rat alveolar type II cells by measuring the enzyme's activity via an adapted radiometric method. The assay conditions were optimized by evaluating permeabilization techniques and substrate concentrations for Na(+)-K(+)-ATPase measurement at maximum velocity enzyme reaction (Vmax). Terbutaline at 10(-2) M increased enzyme activity, with a maximal response at 15 min that was completely inhibited by 10(-2) M propranolol. This effect of terbutaline was dependent on the presence of serum as well as on the time the cells were in culture. The enhancement of Na(+)-K(+)-ATPase activity was reproduced by 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate and 5 x 10(-5) M forskolin. Neither 10(-4) M amiloride nor a sodium-free solution influenced the effect of terbutaline. Western blotting showed that terbutaline did not change the expression of the alpha 1-subunit of the enzyme, which is the predominant form in this cell type. We conclude that beta-adrenergic agonists can modulate Na(+)-K(+)-ATPase activity partially through adenosine 3',5'-cyclic monophosphate and this process is not secondary to an increase in intracellular sodium concentration.w
Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities
Authors: Verin A. D. , Patterson C. E. , Day M. A. , Garcia J. G.
Published: December 2017

Thrombin-induced cultured bovine endothelial cell (EC) gap formation and albumin permeability is initiated by contraction, which is dependent upon myosin light chain kinase-mediated myosin light chain (MLC) phosphorylation. MLC are then rapidly dephosphorylated (J. G. N. Garcia, H. W. Davis, and C. E. Patterson, J. Cell. Physiol. 163: 510–522, 1995), suggesting a role for MLC dephosphorylation in regulation of EC barrier function. Therefore, we studied the effect of semiselective protein phosphatase (PPase) inhibitors, calyculin A and okadaic acid, on MLC phosphorylation status, myosin-associated PPase activity, and EC monolayer permeability. Calyculin A (0.1–10 nM), but not okadaic acid (1–100 nM) produced significant dose-dependent enhancement of both MLC phosphorylation (three- to four-fold) and EC permeability (eightfold). EC homogenates were utilized to assess Ser/Thr PPase activities using either [32P]phosphorylase A or 32P-labeled skeletal MLC as substrates. Calyculin A at 5 nM (sufficient to inhibit type 1 and type 2A PPase) produced approximately 95% inhibition of all EC PPase activity against both substrates, whereas 2 nM okadaic acid (selective for PPase 2A) only partially inhibited EC PPase activity (40–60%). Fractionation of EC homogenates produced a supernatant fraction containing < 10% of total myosin and a pellet fraction with > 90% of total myosin. PPase activity in the myosin-enriched pellet was insensitive to 2 nM okadaic acid (0% inhibition) but sensitive to 5 nM calyculin (> 95% inhibition). Immunoreactive PPase 1 was present in both fractions, whereas PPase 2A was present only in the myosin-depleted fraction. We conclude that a type 1 myosin-associated PPase is involved in regulation of EC contractility and barrier function.
Effect of surfactant on basal and silica-induced eicosanoid production by the alveolar macrophage
Authors: Kuhn D. C. , Demers L. M.
Published: December 2017

Inflammation and fibrosis subsequent to the inhalation of certain mineral dust particles has been suggested to result from the activation of eicosanoid synthesis by the alveolar macrophage (AM). To determine if surfactant modifies dust-induced generation of eicosanoids by the AM, we evaluated the effect of the surfactant lipid dipalmitoyl lecithin (DPL) on the production of eicosanoids by rat AM exposed to respirable silica dust in vitro. During the first 24-h exposure period, DPL alone significantly increased basal production of eicosanoids but completely inhibited silica-induced thromboxane A2 synthesis. In contrast, leukotriene B4 (LTB4) production was only partially reduced by DPL. During a second 24-h exposure period, LTB4 production in response to the highest dose of silica remained significantly elevated in the presence of DPL. Similar results were obtained when the surfactant preparation Survanta was evaluated. These results suggest that 1) DPL and Survanta independently activate AM eicosanoid production, 2) DPL and Survanta modulate the response of the AM to silica dust, and 3) LTB4 may be the most important eicosanoid mediator of the long-term effects of silica dust exposure on lung pathophysiology.
Effects of cystic fibrosis airway secretions on rat lung: role of neutrophil elastase
Authors: Rees D. D. , Brain J. D.
Published: December 2017

Products of inflammatory cells present in pulmonary secretions may compromise lung structure and function. To investigate the pathogenic potential of cystic fibrosis (CF) airway secretions, we instilled CF sputum sol into the lungs of healthy rats and measured the resulting lung injury and inflammation. The ability of a neutrophil elastase (NE) inhibitor, L-680,833, to mitigate these responses was also investigated. CF sputum sol instilled into rat lungs induced hemorrhage, an increase in epithelial permeability, and neutrophil recruitment to the airspaces. However, when sputum sol was preincubated with NE inhibitor before instillation, hemorrhage was completely prevented, suggesting that NE within the CF airway secretions was responsible for the observed hemorrhagic injury. NE-inhibitor treatment had no effect on the observed increases in bronchoalveolar lavage albumin level or neutrophil numbers. Rats treated orally with NE inhibitor before instillation of sputum sol were also protected from hemorrhagic injury. These results demonstrate that NE within CF airway secretions causes lung tissue damage and that animals can be protected from such damage with an oral anti-elastase.
Surfactant treatment and ventilation effects on surfactant SP-A, SP-B, and SP-C mRNA levels in preterm lamb lungs
Authors: Woods E. , Ohashi T. , Polk D. , Ikegami M. , Ueda T. , Jobe A. H.
Published: December 2017

The effects of exogenous surfactant treatment combined with postnatal ventilation on surfactant protein (SP)-A, SP-B, and SP-C steady-state mRNA levels were evaluated in preterm sheep at 120, 132, and 139 days gestation. Three groups were studied at each gestation period: animals ventilated and treated with 100 mg/kg natural sheep surfactant, animals ventilated and not treated with surfactant, and a comparison group of lambs that were neither ventilated nor treated with surfactant. In unventilated lambs, SP-A and SP-C mRNA levels measured by densitometry from Northern blots increased between 120 and 132 days gestation (P < 0.05). At 120 days gestation, no differences in SP-A, SP-B, or SP-C mRNA levels were noted among the three groups. At 132 days gestation, SP-A mRNA levels increased in both ventilated groups (P < 0.01), but no additional surfactant effect on SP-A mRNA expression was detected. There were no changes in SP-B or SP-C mRNA levels among the groups at 132 days gestation. At 139 days gestation, mRNA levels for both SP-A and SP-B increased after ventilation, compared with the unventilated groups (P < 0.05). Furthermore, an additional effect of surfactant treatment to increase SP-A mRNA levels relative to animals undergoing ventilation alone was noted (P < 0.05). We conclude that postnatal changes in mRNA levels for the surfactant proteins are gestationally regulated and protein specific.
Keratinocyte growth factor increases mRNAs for SP-A and SP-B in adult rat alveolar type II cells in culture
Authors: Sugahara K. , Rubin J. S. , Mason R. J. , Aronsen E. L. , Shannon J. M.
Published: December 2017

The production of pulmonary surfactant, a complex of phospholipids and lung-specific surfactant proteins, is a primary function of alveolar type II cells. Although previous studies have demonstrated a role for cell-extracellular matrix interactions and normal cell shape in the maintenance of differentiated function in primary cultures of adult rat type II cells, a positive role for growth factors in surfactant protein gene expression in isolated normal adult type II cells has not been reported. In the present study, we have examined the effects of a panel of hormones, growth factors, and cytokines on the expression of mRNAs for surfactant proteins A, B, and C (SP-A, SP-B, and SP-C). Our results show that keratinocyte growth factor (KGF) induced a two- to threefold increase in steady-state levels of mRNAs for SP-A and SP-B, but had no effect on or decreased SP-C mRNA. The increase in SP-A mRNA was accompanied by an increase in SP-A protein. The effects of KGF were both dose and time dependent, and they could be neutralized by a monoclonal antibody against KGF. The effects of KGF were mimicked by acidic fibroblast growth factor, which will bind the KGF receptor. We conclude that KGF can support differentiation of alveolar type II cells as well as act as a mitogen, thus suggesting an important role for KGF in maintenance of the alveolar epithelium.
Expression of transforming growth factor-beta type II receptor in rat lung is regulated during development
Authors: Zhao Y. , Young S. L.
Published: December 2017

Transforming growth factor-beta (TGF-beta) is an autocrine/paracrine growth factor that regulates cell proliferation, differentiation, extracellular matrix production and various other cell functions in the lung. TGF-beta exerts its effects on cells by binding to transmembrane heteromeric serine-threonine kinase receptors. The expression and localization of specific TGF-beta receptors in the lung, however, have not yet been investigated. In the present studies, we isolated a 1,762-base pair cDNA containing the full-length coding sequence for TGF-beta type II receptor (T beta RII) from rat fetal lung with the use of polymerase chain reaction methods. The expression of T beta RII during lung development was examined by Northern analysis. A 5.1-kilobase T beta RII mRNA was detected in rat lung tissue. T beta RII mRNA was expressed in rat fetal lung tissue early in development, increased as development proceeded, reached maximal concentration postnatally, and then decreased to the adult level. The localization of T beta RII in fetal and postnatal rat lung tissue was investigated with the use of in situ hybridization performed with an antisense RNA probe. T beta RII gene was expressed in the mesenchymal tissue and in the epithelial lining of the developing airway at day 16 of gestation. The hybridization signal of T beta RII mRNA was also observed in the adventitial layer of small blood vessels. Expression of T beta RII gene in the developing airway epithelium occurred along a proximal-distal gradient. In postnatal lung, T beta RII mRNA was detected mainly in parenchymal tissues and blood vessels. Expression of T beta RII remained high in the interstitium of interalveolar septa.(ABSTRACT TRUNCATED AT 250 WORDS)
Cellular uptake of albumin from lungs of anesthetized rabbits
Authors: Hastings R. H. , Folkesson H. G. , Petersen V. , Ciriales R. , Matthay M. A.
Published: December 2017

Resolution of alveolar edema depends on clearance of serum protein, as well as liquid from the alveolar space. Protein clearance is slower than liquid clearance and may take days to weeks. Our earlier studies presented evidence for the importance of paracellular removal of soluble protein from the air spaces. However, long-term protein clearance may also depend on uptake by alveolar epithelial cells or macrophages. This study examined cellular uptake of soluble human albumin and insoluble colloidal gold-albumin from the lungs of anesthetized rabbits. Native albumin was endocytosed by both alveolar type I and type II cells and appeared in vesicles and endosomes. Neither cell type took up colloidal gold-albumin over periods as long as 8 h. Alveolar macrophages took up native albumin and colloidal gold-albumin to a greater extent and more rapidly than alveolar epithelial cells. The tracer proteins were found in vesicles, endosomes, and phagolysosomes. Similarly, cultured alveolar macrophages took up native albumin more rapidly than cultured type II cells. Thus macrophages may be important in clearing precipitated protein from the air spaces, and they may have a role in completing the clearance of soluble protein. The potential for transepithelial transport of soluble alveolar protein exists, but based on this work and our prior studies, it appears to be a low-capacity pathway.
Enhanced proliferation and IL-2 secretion by lung lymphocytes from HIV-infected subjects
Authors: Spain B. A. , Soliman D. M. , Sidner R. A. , Twigg H. L.
Published: December 2017

Human immunodeficiency virus (HIV)-positive patients frequently develop a CD3+/CD8+ cytotoxic T cell lymphocytic alveolitis. This could occur through in situ expansion of lung lymphocytes. We evaluated lung and blood lymphocyte proliferation in asymptomatic HIV-infected individuals by measuring spontaneous and cytokine-induced tritiated thymidine incorporation. Interleukin (IL)-2 and IL-4 secretion was determined with the use of enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques. Spontaneous proliferation by lung lymphocytes from HIV-positive patients was significantly greater than that of normal volunteers. Proliferation was confined to the CD8+ lymphocyte subset. Over time, spontaneous proliferation declined unless autologous alveolar macrophages (AM) were added, suggesting AM were providing additional stimulatory signals to lung lymphocytes. Lung and blood lymphocytes proliferated in response to IL-2 but not IL-4. Lymphocytes in HIV-infected lung spontaneously produced and secreted more IL-2 than either normal lung lymphocytes or autologous blood lymphocytes. IL-4 production was not detectable in either group. These findings support the hypothesis that lymphocytic alveolitis in asymptomatic HIV-positive patients results from IL-2-dependent in situ proliferation of CD3+/CD8+ cytotoxic T cells.
Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture
Authors: Johnson P. R. , Armour C. L. , Carey D. , Black J. L.
Published: December 2017

An increase in the bulk of the airway smooth muscle is a characteristic of asthma. Much of the research investigating the mechanisms of this increase in muscle has focused on mediators that are mitogenic for smooth muscle, while relatively few studies have focused on mediators inhibiting mitogenesis. In this study we have examined the effects of two mediators proposed as regulators of smooth muscle proliferation, namely heparin and prostaglandin (PG) E2, on human airway smooth muscle cells in culture stimulated with 1, 2.5, 5, and 10% fetal bovine serum (FBS) and platelet-derived growth factor AB (PDGF), 50 ng/ml. PGE2 had a biphasic effect on DNA synthesis in the presence of 1% FBS, with 10(-6) M causing inhibition and 10(-7) M causing an increase in DNA synthesis. PGE2 caused inhibition of DNA synthesis in the presence of 2.5, 5, and 10% FBS. Heparin (10 and 100 U/ml) caused an inhibition of DNA synthesis induced by 1% FBS, while 100 U/ml inhibited DNA synthesis induced by 5 and 10% FBS. PGE2 (10(-8), 10(-7), and 10(-6) M) inhibited the DNA synthesis induced by PDGF, while heparin (1, 10, and 100 U/ml) had no effect. These results indicate that both PGE2 and heparin may have a role in the control of human airway smooth muscle cell growth.
Role of CFTR in chloride secretion across human tracheal epithelium
Authors: Shen B. Q. , Mrsny R. J. , Finkbeiner W. E. , Widdicombe J. H.
Published: December 2017

We have tested two hypotheses: 1) the cystic fibrosis transmembrane conductance regulator (CFTR) represents the predominant Cl conductance in the apical membrane of human tracheal epithelium, and 2) CFTR in this tissue is close to maximally activated under baseline conditions. In support of the first hypothesis, we found 1) when the level of differentiation of cultures was varied by varying the culture conditions, there was a significant positive correlation between the levels of CFTR and the magnitude of mediator-induced Cl secretion. 2) Amiloride-insensitive baseline short-circuit current (Isc) and mediator-induced increases in Isc were inhibited by diphenylamine-2-carboxylic acid (DPAC) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a pharmacology consistent with passage of apical membrane Cl current through CFTR; Ca-activated Cl channels are inhibited by DIDS but not by DPAC. 3) Raising temperature from 22 degrees to 37 degrees C increased 125I efflux, and this increase was inhibited by DPAC and blockers of protein kinase A, but not by DIDS or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. In support of the second hypothesis, we have earlier shown [M. Yamaya, W.E. Finkbeiner, S.Y. Chun, and J.H. Widdicombe. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L713-L724, 1992] that adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents are essentially without effect on Isc across primary cultures of human tracheal epithelium. Here, we further show that these agents are also usually without effect on 125I efflux; the mean increase in efflux in response to elevating cAMP was approximately 20% that of raising temperature from 22 degrees to 37 degrees C.
Hypoxia inhibits L-arginine uptake by pulmonary artery endothelial cells
Authors: Block E. R. , Herrera H. , Couch M.
Published: December 2017

Under physiological conditions, L-arginine transport by porcine pulmonary artery endothelial cells (PAEC) is mediated by system y+, a sodium-independent transport system that accounts for 60 +/- 5% of L-arginine transport, and system Bo,+, a sodium-dependent system that accounts for 40 +/- 5% of transport. Because NO production is dependent on intracellular L-arginine content and intracellular L-arginine content depends on transport of extracellular L-arginine, we examined the effect of hypoxia on L-arginine transport and intracellular L-arginine content in PAEC. Exposure of passage 3-7 PAEC in monolayer culture to 0% O2 for 4 h decreased L-arginine transport via system y+ from 120 +/- 10 to 81 +/- 23 (in pmol.mg protein-1.30 s-1) (P < 0.001), whereas 20-h exposures decreased transport from 122 +/- 17 to 84 +/- 18 (P < 0.001) in system y+ and from 104 +/- 19 to 90 +/- 26 (P < 0.05) in system Bo,+. Exposure to 5% O2 for 3-5 wk decreased L-arginine transport via system y+ from 128 +/- 15 to 73 +/- 13 (P < 0.001) and via system Bo,+, from 105 +/- 25 to 65 +/- 13 (P < 0.001). Kinetic studies revealed that hypoxia decreased the maximal transport velocity but not the apparent Michaelis constant for both system y+ and system Bo,+, and the decreases in transport were not reversible after return to normoxia for up to 24 h. Long-term exposure, i.e., 3-5 wk, to 5% O2 also resulted in decreases in intracellular L-arginine content (0.75 +/- 0.10 vs. 0.49 +/- 0.09 nmol/10(6) cells, P < 0.05) which did not reverse after return to normoxia for 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells
Authors: Das K. C. , Lewis-Molock Y. , White C. W.
Published: December 2017

The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.
Dietary changes improve survival of CFTR S489X homozygous mutant mouse
Authors: Eckman E. A. , Cotton C. U. , Kube D. M. , Davis P. B.
Published: December 2017

Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Because the period of highest mortality is during the week after weaning, we hypothesized that providing a low-residue liquid diet would improve survival and growth. When 99 CF mice that survived to 10 days of age were fed Peptamen (Clintec Nutrition), an elemental liquid diet, and housed on corn-cob bedding, 88% of them survived to maturity (50 days). The diet causes only minor histologic and ion transport changes in the intestines of normal mice and does not reduce growth rate or size. CF mice raised on Peptamen continue to display severe pathological changes in the intestine and completely lack a adenosine 3',5'-cyclic monophosphate-inducible chloride current in the cecum. This combination of dietary and bedding changes provides a reliable method for keeping CF mice alive well into adulthood and will be useful for the evaluation of the effect and duration of potential therapies for CF.