bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in surfactant-associated protein mRNA profile in growth-restricted fetal sheep
Authors: Gagnon Robert , Langridge Johnathan , Inchley Kevin , Murotsuki Jun , Possmayer Fred
Published: December 2017

To test the hypothesis that chronic placental insufficiency resulting in fetal growth restriction causes an increase in fetal lung surfactant-associated protein (SP) gene expression, we embolized chronically catheterized fetal sheep ( n = 6) daily using nonradioactive microspheres in the abdominal aorta for 21 days (between 0.74 and 0.88 of gestation) until fetal arterial oxygen content was reduced by ∼40–50%. Control animals ( n = 7) received saline only. Basal fetal plasma cortisol concentration was monitored. At the end of the experiment, fetal lung tissues were collected, and ratios of tissue levels of SP-A, SP-B, and SP-C mRNA to 18S rRNA were determined by standard Northern blot analysis. Total DNA content of fetal lungs was reduced by 30% in the embolized group compared with control group ( P = 0.01). There was a 2.7-fold increase in fetal lung SP-A mRNA ( P< 0.05) and a 3.2-fold increase in SP-B mRNA ( P < 0.01) in the chronically embolized group compared with those in the control group. SP-A and SP-B mRNA tissue levels were highly correlated with the mean fetal plasma cortisol levels on days 20–21( r = 0.90, P < 0.01 for SP-A mRNA and r = 0.94, P < 0.01 for SP-B mRNA). SP-C mRNA tissue levels were not significantly affected by placental insufficiency. We conclude that fetal growth restriction due to placental insufficiency is associated with alterations in fetal lung SP, suggesting enhanced lung maturation that was highly dependent on the degree of increase in fetal plasma cortisol levels.
β2-Adrenoceptor agonists reduce the decline of rat diaphragm twitch force during severe hypoxia
Authors: van der Heijden H. F. M. , Heunks L. M. A. , Folgering H. , van Herwaarden C. L. A. , Dekhuijzen P. N. R.
Published: December 2017

The aim of the present study was to investigate the in vitro effects of the short-acting β2-adrenoceptor agonist salbutamol and the long-acting β2-adrenoceptor agonist salmeterol on hypoxia-induced rat diaphragm force reduction. In vitro diaphragm twitch force (Pt) and maximal tetanic force (Po) of isolated diaphragm muscle strips were measured for 90 min during hyperoxia (tissue bath [Formula: see text] 83.8 ± 0.9 kPa and [Formula: see text] 3.9 ± 0.1 kPa) or severe hypoxia ([Formula: see text] 7.1 ± 0.3 kPa and [Formula: see text] 3.9 ± 0.1 kPa) in the presence and absence of 1 μM salbutamol or 1 μM salmeterol. During hyperoxia, salbutamol and salmeterol did not significantly alter the time-related decreases in Ptand Po(to ∼50% of initial values). Salbutamol had no effects on Poor the Pt-to-Poratio. Salmeterol treatment significantly reduced Poand increased the Pt-to-Poratio during hyperoxia ( P < 0.05 compared with control value). Hypoxia resulted in a severe decrease in Pt(to ∼30% of initial value) and Poafter 90 min. Both salbutamol and salmeterol significantly reduced the decline in Ptduring hypoxia ( P < 0.05). The reduction in Powas not prevented. Salbutamol increased Ptrapidly but transiently. Salmeterol had a delayed onset of effect and a longer duration of action. Addition of 1 μM propranolol (a nonselective β-adrenoceptor antagonist) did not alter Pt, Po, or the Pt-to-Poratio during hypoxia but completely blocked the inotropic effects of salbutamol and salmeterol, indicating that these effects are dependent on β2-adrenoceptor agonist-related processes.
TNF-α increases ceramide without inducing apoptosis in alveolar type II epithelial cells
Authors: Mallampalli Rama K. , Peterson Erik J. , Carter Aaron Brent , Salome Ronald G. , Mathur Satya N. , Koretzky Gary A.
Published: December 2017

Ceramide is a bioactive lipid mediator that has been observed to induce apoptosis in vitro. The purpose of this study was to determine whether endogenous ceramide, generated in response to in vivo administration of tumor necrosis factor-α (TNF-α), increases apoptosis in primary rat alveolar type II epithelial cells. Intratracheal instillation of TNF-α (5 μg) produced a decrease in sphingomyelin and activation of a neutral sphingomyelinase. These changes were associated with a significant increase in lung ceramide content. TNF-α concomitantly activated the p42/44 extracellular signal-related kinases and induced nuclear factor-κB activation in the lung. Hypodiploid nuclei studies revealed that intratracheal TNF-α did not increase type II cell apoptosis compared with that in control cells after isolation. A novel observation from separate in vitro studies demonstrated that type II cells undergo a gradual increase in apoptosis after time in culture, a process that was accelerated by exposure of cells to ultraviolet light. However, culture of cells with a cell-permeable ceramide, TNF-α, or a related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-α activation of sphingomyelin hydrolysis might activate the mitogen-activated protein kinase and nuclear factor-κB pathways without increasing programmed cell death in type II cells.
Inducible NO synthase inhibition attenuates shear stress-induced pulmonary vasodilation in the ovine fetus
Authors: Rairigh Robyn L. , Storme Laurent , Parker Thomas A. , le Cras Timothy D. , Kinsella John P. , Jakkula Malathi , Abman Steven H.
Published: December 2017

Recent studies have suggested that type II (inducible) nitric oxide (NO) synthase (NOS II) is present in the fetal lung, but its physiological roles are uncertain. Whether NOS II activity contributes to the NO-mediated fall in pulmonary vascular resistance (PVR) during shear stress-induced pulmonary vasodilation is unknown. We studied the hemodynamic effects of two selective NOS II antagonists [aminoguanidine (AG) and S-ethylisothiourea (EIT)], a nonselective NOS antagonist [nitro-l-arginine (l-NNA)], and a nonselective vasoconstrictor (U-46619) on PVR during partial compression of the ductus arteriosus (DA) in 20 chronically prepared fetal lambs (mean age 132 ± 2 days, term 147 days). At surgery, catheters were placed in the left pulmonary artery (LPA) for selective drug infusion, an ultrasonic flow transducer was placed on the LPA to measure blood flow, and an inflatable vascular occluder was placed loosely around the DA for compression. On alternate days, a brief intrapulmonary infusion of normal saline (control), AG, EIT,l-NNA, or U-46619 was infused in random order into the LPA. The DA was compressed to increase mean pulmonary arterial pressure (MPAP) 12–15 mmHg above baseline values and held constant for 30 min. In control studies, DA compression reduced PVR by 42% from baseline values ( P < 0.01).l-NNA treatment completely blocked the fall in PVR during DA compression. AG and EIT attenuated the decrease in PVR by 30 and 19%, respectively ( P < 0.05). Nonspecific elevation in PVR by U-46619 did not affect the fall in PVR during DA compression. Immunostaining for NOS II identified this isoform in airway epithelium and vascular smooth muscle in the late-gestation ovine fetal lung. We conclude that selective NOS II antagonists attenuate but do not block shear stress-induced vasodilation in the fetal lung. We speculate that stimulation of NOS II activity, perhaps from smooth muscle cells, contributes in part to the NO-mediated fall in PVR during shear stress-induced pulmonary vasodilation.
Mediators of anaphylaxis but not activated neutrophils augment cholinergic responses of equine small airways
Authors: Olszewski Michal A. , Robinson N. Edward , Zhu Feng-Xia , Zhang Xiang-Yang , Tithof Patricia K.
Published: December 2017

Neutrophilic inflammation in small airways (SA) and bronchospasm mediated via muscarinic receptors are features of chronic obstructive pulmonary disease in horses (COPD). Histamine, serotonin, and leukotrienes (LTs) are reported to be involved in the exacerbation of COPD, and currently, histamine has been shown to increase tension response to electrical field simulation (EFS) in equine SA. We tested the effects of these mediators and the effects of activated neutrophils on the cholinergic responses in SA. Histamine, serotonin, and LTD4had a synergistic effect on EFS responses and only an additive effect on the tension response to exogenous ACh or methacholine. Atropine and TTX entirely eliminated the EFS-induced tension response in the presence of all three inflammatory mediators, indicating that augmentation of the EFS response applies only to the endogenous cholinergic response. Neutrophils isolated from control and COPD-affected horses were activated by zymosan, producing 18.1 ± 2.3 and 25.0 ± 2.3 nmol superoxide ⋅ 106cells−1⋅ 30 min−1, respectively. However, in contrast to the profound effect of mediators, incubation of SA for over 1 h in a suspension of up to 30 × 106zymosan-treated neutrophils/ml did not significantly affect EFS responses of SA isolated from either control or COPD-affected horses. We conclude that in equine SA 1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not affect cholinergic responses in SA; and 3) in acute bouts of equine COPD, histamine, LTD4, and serotonin (mediators primarily associated with type I allergic reaction) rather than mediators derived from neutrophils most likely contribute to increased cholinergic airway tone.
Formation of membrane lattice structures and their specific interactions with surfactant protein A
Authors: Palaniyar Nades , Ridsdale Ross A. , Hearn Stephen A. , Possmayer Fred , Harauz George
Published: December 2017

Biological membranes exist in many forms, one of which is known as tubular myelin (TM). This pulmonary surfactant membranous structure contains elongated tubes that form square lattices. To understand the interaction of surfactant protein (SP) A and various lipids commonly found in TM, we undertook a series of transmission-electron-microscopic studies using purified SP-A and lipid vesicles made in vitro and also native surfactant from bovine lung. Specimens from in vitro experiments were negatively stained with 2% uranyl acetate, whereas fixed native surfactant was delipidated, embedded, and sectioned. We found that dipalmitoylphosphatidylcholine-egg phosphatidylcholine (1:1 wt/wt) bilayers formed corrugations, folds, and predominantly 47-nm-square latticelike structures. SP-A specifically interacted with these lipid bilayers and folds. We visualized other proteolipid structures that could act as intermediates for reorganizing lipids and SP-As. Such a reorganization could lead to the localization of SP-A in the lattice corners and could explain, in part, the formation of TM-like structures in vivo.
Abrogation of cyclin D1 expression predisposes lung cancer cells to serum deprivation-induced apoptosis
Authors: Driscoll Barbara , Buckley Susan , Barsky Lora , Weinberg Kenneth , Anderson Kathryn D. , Warburton David
Published: December 2017

Cyclin D1 antisense (D1AS)-transfected lung epithelial cell lines were serum deprived and then analyzed for three hallmarks of apoptosis: appearance of single-strand DNA breaks, alteration of apoptosis-related protein expression, and induction of chromatin condensation. Single-strand DNA breaks appeared at significant levels 24 h after serum deprivation, whereas induction of chromatin condensation was observed after 72 h. The antioxidants dimethyl sulfoxide, ascorbate, and glutathione, as well as insulin-like growth factor-I, inhibited induction of DNA damage in this assay. Additionally, proliferating cell nuclear antigen expression is completely suppressed in the D1AS cells, indicating a mechanism to explain the reduced capacity for DNA repair. Increased expression of cyclin D1, which is a common lesion in lung cancer, may thus prevent induction of apoptosis in an oxidizing and growth factor-poor environment. Reducing cyclin D1 expression in lung cancer cells by expression of D1AS RNA disrupted these protective pathways.
Molecular embryology of the lung: then, now, and in the future
Authors: Warburton David , Zhao Jingsong , Berberich Mary Anne , Bernfield Merton
Published: December 2017

Complementary molecular and genetic approaches are yielding information about gain- versus loss-of-function phenotypes of specific genes and gene families in the embryonic, fetal, neonatal, and adult lungs. New insights are being derived from the conservation of function between genes regulating branching morphogenesis of the respiratory organs in Drosophila and in the mammalian lung. The function of specific morphogenetic genes in the lung are now placed in context with pattern-forming functions in other, better understood morphogenetic fields such as the limb bud. Initiation of lung morphogenesis from the floor of the primitive foregut requires coordinated transcriptional activation and repression involving hepatocyte nuclear factor-3β, Sonic hedgehog, patched, Gli2, and Gli3 as well as Nkx2.1. Subsequent inductive events require epithelial-mesenchymal interaction mediated by specific fibroblast growth factor ligand-receptor signaling as well as modulation by other peptide growth factors including epidermal growth factor, platelet-derived growth factor-A and transforming growth factor-β and by extracellular matrix components. A scientific rationale for developing new therapeutic approaches to urgent questions of human pulmonary health such as bronchopulmonary dysplasia is beginning to emerge from work in this field.
Cytokine gene expression after inhalation of corn dust
Authors: Wohlford-Lenane Christine L. , Deetz Daniel C. , Schwartz. David A.
Published: December 2017

To characterize the time course and localize the production of proinflammatory cytokines after inhalation of corn dust, we exposed mice (C3H/HeBFeJ) by inhalation challenge to sterile corn dust extract (CDE) and contrasted this response to inhalation of Escherichia coli 0111:B4 lipopolysaccharide (LPS) or pyrogen-free saline. After both CDE and LPS exposure, an increase in the concentration of bronchoalveolar lavage neutrophils was detected 1 h postinhalation and persisted for 48 h. Significant increases in the bronchoalveolar lavage concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and macrophage inflammatory protein (MIP)-2 resulted after inhalation of either CDE or LPS. Although the time courses of these cytokines were distinct, a similar pattern of release was observed after both CDE and LPS exposure. A single inhalation exposure of either CDE or LPS resulted in enhanced expression of mRNA for TNF-α, IL-1α, and MIP-2 that was evident and most pronounced within 1 h of the inhalation challenge. Although enhanced expression of mRNA for TNF-α was detectable 12 h after completion of the inhalation challenge, IL-1α and MIP-2 mRNA expression remained elevated through the 24-h time point. TNF-α, IL-1α, and MIP-2 expression was localized by in situ hybridization to inflammatory cells in the airways and alveoli from 1 to 24 h in both CDE- and LPS-exposed lungs. Interestingly, there was no convincing evidence that MIP-2 was substantially produced by airway epithelial cells. The pattern, timing, and location of expression of TNF-α, IL-1α, and MIP-2 mRNA after a single inhalation exposure of CDE in comparison with LPS is similar, supporting a common etiology and mechanism of inflammation in the lower respiratory tract. Moreover, our findings indicate that inhalation of corn dust or LPS results in an acute inflammatory process that is primarily mediated by inflammatory cells and appears to be self-limited.
Modulation of ACh release from airway cholinergic nerves in horses with recurrent airway obstruction
Authors: Zhang Xiang-Yang , Robinson N. Edward , Zhu Feng-Xia
Published: December 2017

To evaluate the functional status of neuronal α2-adrenoceptors (ARs) and β2-ARs on ACh release in horses with recurrent airway obstruction (RAO), we examined the effects of the physiological agonists epinephrine (Epi) and norepinephrine (NE) and the β2-agonists RR- and RR/ SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the β2-agonist, increases ACh release from airways of control horses only after the autoinhibitory muscarinic receptors are blocked by atropine, we also tested the hypothesis that if there is an M2-receptor dysfunction in equine RAO, SS-formoterol should increase ACh release even in the absence of atropine. ACh release was evoked by electrical field stimulation and measured by HPLC. Epi and NE caused less inhibition of ACh release in horses with RAO than in control horses. At the catecholamine concentration achieved during exercise (10−7 M), the inhibition induced by Epi and NE was 10.8 ± 13.2 and 3.4 ± 6.8%, respectively, in equine RAO versus 41.0 ± 6.4 and 27.1 ± 5.6%, respectively, in control horses. RR- and RR/ SS-formoterol (10−8 to 10−5 M) increased ACh release to a similar magnitude as that in control horses. These results indicate that neuronal β2-ARs are functioning; however, the α2-ARs are dysfunctional in the airways of horses with RAO in response to circulating catecholamines. SS-formoterol (10−8 to 10−5 M) facilitated ACh release in horses with RAO even in the absence of atropine. Addition of atropine did not cause significantly more augmentation of ACh release over the effect of SS-formoterol alone. The magnitude of augmentation in horses with RAO in the absence of atropine was similar to that in control horses in the presence of atropine. The latter observations could be explained by neuronal muscarinic-autoreceptor dysfunction in equine RAO.
Genetic disruption of atrial natriuretic peptide causes pulmonary hypertension in normoxic and hypoxic mice
Authors: Klinger James R. , Warburton Rod R. , Pietras Linda A. , Smithies Oliver , Swift Richard , Hill Nicholas S.
Published: December 2017

To determine whether atrial natriuretic peptide (ANP) plays a physiological role in modulating pulmonary hypertensive responses, we studied mice with gene-targeted disruption of the ANP gene under normoxic and chronically hypoxic conditions. Right ventricular peak pressure (RVPP), right ventricle weight- and left ventricle plus septum weight-to-body weight ratios [RV/BW and (LV+S)/BW, respectively], and muscularization of pulmonary vessels were measured in wild-type mice (+/+) and in mice heterozygous (+/−) and homozygous (−/−) for a disrupted proANP gene after 3 wk of normoxia or hypobaric hypoxia (0.5 atm). Under normoxic conditions, homozygous mutants had higher RVPP (22 ± 2 vs. 15 ± 1 mmHg; P < 0.05) than wild-type mice and greater RV/BW (1.22 ± 0.08 vs. 0.94 ± 0.07 and 0.76 ± 0.04 mg/g; P < 0.05) and (LV+S)/BW (4.74 ± 0.42 vs. 3.53 ± 0.14 and 3.18 ± 0.18 mg/g; P < 0.05) than heterozygous or wild-type mice, respectively. Three weeks of hypoxia increased RVPP in heterozygous and wild-type mice and increased RV/BW and RV/(LV+S) in all genotypes compared with their normoxic control animals but had no effect on (LV+S)/BW. After 3 wk of hypoxia, homozygous mutants had higher RVPP (29 ± 3 vs. 23 ± 1 and 22 ± 2 mmHg; P < 0.05), RV/BW (2.03 ± 0.14 vs. 1.46 ± 0.04 and 1.33 ± 0.08 mg/g; P < 0.05), and (LV+S)/BW (4.76 ± 0.23 vs. 3.82 ± 0.09 and 3.44 ± 0.14 mg/g; P < 0.05) than heterozygous or wild-type mice, respectively. The percent muscularization of peripheral pulmonary vessels was greater in homozygous mutants than that in heterozygous or wild-type mice under both normoxic and hypoxic conditions. We conclude that endogenous ANP plays a physiological role in modulating pulmonary arterial pressure, cardiac hypertrophy, and pulmonary vascular remodeling under normoxic and hypoxic conditions.
Postnatal maturation of carotid body and type I cell chemoreception in the rat
Authors: Bamford Owen S. , Sterni Laura M. , Wasicko Michael J. , Montrose Marshall H. , Carroll John L.
Published: December 2017

The site of postnatal maturation of carotid body chemoreception is unclear. To test the hypothesis that maturation occurs synchronously in type I cells and the whole carotid body, the development of changes in the intracellular Ca2+concentration responses to hypoxia, CO2, and combined challenges was studied with fluorescence microscopy in type I cells and compared with the development of carotid sinus nerve (CSN) responses recorded in vitro from term fetal to 3-wk animals. Type I cell responses to all challenges increased between 1 and 8 days and then remained constant, with no multiplicative O2-CO2interaction at any age. The CSN response to hypoxia also matured by 8 days, but CSN responses to CO2did not change significantly with age. Multiplicative O2-CO2interaction occurred in the CSN response at 2–3 wk but not in younger groups. We conclude that type I cell maturation underlies maturation of the CSN response to hypoxia. However, because development of responses to CO2and combined hypoxia-CO2challenges differed between type I cells and the CSN, responses to these stimuli must mature at other, unidentified sites within the developing carotid body.
Effects of a perfluorochemical emulsion on the fate of circulatingPseudomonas aeruginosa
Authors: Brain Joseph D. , Molina Ramon M. , DeCamp Malcolm M. , Warner Angeline E.
Published: December 2017

Because mononuclear phagocytes take up perfluorochemical emulsions (PFCE), we examined how prior treatment with PFCE affects the fate of circulating bacteria. Rats were preinjected with three daily intravenous injections of PFCE (2.0 ml/100 g) containing 12.5% (vol/vol) of a 4:1 mixture of F-dimethyl adamantane and F-trimethylbicyclo-nonane, 2.5% (wt/vol) Pluronic F-68 as the emulsifying agent, and 3% (wt/vol) hydroxyethyl starch as the oncotic agent. Pseudomonas aeruginosa or Staphylococcus aureus were injected 4 h after the third PFCE injection. PFCE pretreatment decreased the rate and extent of vascular clearance of P. aeruginosa, with decreased uptake by the liver. Importantly, there were significant decreases in killing of P. aeruginosa in the liver, lungs, spleen, and kidneys of PFCE animals. PFCE did not alter the clearance of S. aureus from the circulation. However, hepatic uptake was reduced, with concomitant increases in lung and kidney uptake. Ultrastructure of Kupffer cells revealed PFCE inclusions and extensive vacuolization. These experiments demonstrate that the clearance kinetics and organ distribution of circulating P. aeruginosa and their subsequent killing are altered by PFCE. Diminished hepatic phagocyte function leads to a decrease in vascular clearance of circulating bacteria, increased uptake in other reticuloendothelial organs, and decreased bactericidal activity versus P. aeruginosa.
Antisense oligonucleotides against the α-subunit of ENaC decrease lung epithelial cation-channel activity
Authors: Jain Lucky , Chen Xi-Juan , Malik Bela , Al-Khalili Otor , Eaton Douglas C.
Published: December 2017

Amiloride-sensitive Na+transport by lung epithelia plays a critical role in maintaining alveolar Na+and water balance. It has been generally assumed that Na+transport is mediated by the amiloride-sensitive epithelial Na+channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits α, β, and γ in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 ± 1.1 pS and an Na+-to-K+selectivity of 0.97 ± 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (α, β, and γ) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the α-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the β- and γ-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the α-subunit of ENaC alone or in combination with some protein other than the β- or γ-subunit protein is the major component of lung alveolar epithelial cation channels.
Effect of FK506 on ATP-induced intracellular calcium oscillations in cow tracheal epithelium
Authors: Kanoh Soichiro , Kondo Mitsuko , Tamaoki Jun , Shirakawa Hideki , Aoshiba Kazutetsu , Miyazaki Shunichi , Kobayashi Hideo , Nagata Naokazu , Nagai Atsushi
Published: December 2017

To elucidate the effect of FK506 on Ca2+oscillations in airway epithelium, we investigated cultured cow tracheal epithelial cells with a Ca2+image-analysis system. ATP (1 μM) induced long-lasting Ca2+oscillations, having nearly constant peak values (300–400 nM) and intervals (20–40 s) in subconfluent cells but not in confluent cells. These responses were gradually attenuated and abolished by the addition of FK506. Rapamycin, which binds the FK506-binding protein (FKBP), likewise inhibited Ca2+oscillations, whereas cyclosporin A, a calcineurin inhibitor, did not. Treatment of cells with FK506 decreased Ca2+content in thapsigargin-sensitive stores, suggesting that the partial depletion of the stores causes the inhibition of Ca2+oscillations. Immunocytochemistry revealed the existence of cytoplasmic FKBP-like immunoreactivities. The expression of a 12-kDa FKBP was greater in subconfluent cells than in confluent cells as determined by Western blotting, suggesting that the 12-kDa FKBP may be one of the factors that regulates Ca2+oscillations. Therefore, FK506 possesses an inhibitory action on the Ca2+response via intracellular FKBP but not via calcineurin, which may result in modification of airway epithelial functions.
Segmental microvascular permeability in ischemia-reperfusion injury in rat lung
Authors: Khimenko Pavel L. , Taylor Aubrey E.
Published: December 2017

Segmental microvascular permeabilities were measured using pre- and postalveolar vessel capillary filtration coefficient ( K fc) values (ml ⋅ min−1 ⋅ cmH2O−1 ⋅ 100 g−1) in isolated rat lungs subjected to ischemia-reperfusion (I/R). Total K fc values measured in flowing and nonflowing lungs were highly correlated ( r = 0.98, P < 0.0001). K fc values were then measured in another group of lungs under no-flow conditions when airway pressure was increased to 20 cmH2O and either the arterial or venous pressure was elevated to 7–8 cmH2O to measure the prealveolar and postalveolar K fc values. Control total and postalveolar K fc values were 0.0225 ± 0.001 and 0.0219 ± 0.001 ml ⋅ min−1 ⋅ cmH2O−1 ⋅ 100 g−1, respectively, and the prealveolar permeability was extremely small (0.00003 ± 0.00005 ml ⋅ min−1 ⋅ cmH2O−1 ⋅ 100 g−1). K fc values were again made in nonflowing lungs that had been subjected to 45 min of ischemia followed by 30 min of reperfusion. After I/R, the total membrane K fc increased 10-fold to 0.2597 ± 0.006 ml ⋅ min−1 ⋅ cmH2O−1 ⋅ 100 g−1, the prealveolar K fc increased to 0.0677 ± 0.003 ml ⋅ min−1 ⋅ cmH2O−1 ⋅ 100 g−1, and the postalveolar K fc increased to 0.1354 ± 0.008 ml ⋅ min−1 ⋅ cmH2O−1 ⋅ 100 g−1 ( P < 0.05 for all I/R values). These data indicate that normal solvent microvascular permeability was predominantly postalveolar, and after I/R damage, the postalveolar (venular) permeability comprised 52% of the total, whereas the prealveolar and alveolar vessels comprised only 27 and 23%, respectively, of the total K fc.
Abnormal glutathione transport in cystic fibrosis airway epithelia
Authors: Gao Lin , Kim Kwang Jin , Yankaskas James R. , Forman Henry Jay
Published: December 2017

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), ΔF508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or β-galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells ( P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. γ-Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.
Regulation of gelatinases in human airway smooth muscle cells: mechanism of progelatinase A activation
Authors: Foda Hussein D. , George Suni , Rollo Ellen , Drews Michelle , Conner Cathleen , Cao Jian , Panettieri Reynold A. , Zucker Stanley
Published: December 2017

Matrix metalloproteinases (MMPs) play an important role in tumor metastasis and invasion, inflammatory tissue destruction and remodeling, wound healing, and angiogenesis. The 72-kDa gelatinase A is the most widely distributed of all the MMPs, and along with the 92-kDa gelatinase B, both play an important role in the turnover of basement membrane. The role of MMPs in chronic airway inflammation and remodeling has received scant attention. In this study, we sought to examine the release and activation of gelatinases from human airway smooth muscle (ASM) cells and the effect of tumor necrosis factor-α and phorbol 12-myristate 13-acetate (PMA) on this release and activation. The role of membrane type 1 MMP (MT1-MMP) and tissue inhibitor of MMP (TIMP)-2 in activating progelatinase A has been explored. We have demonstrated, using human airway smooth muscle cells in culture, that 1) ASM releases gelatinase A constitutively and when stimulated with PMA and tumor necrosis factor-α releases gelatinase B, and the release of gelatinase B is protein kinase C dependent because it is blocked by H-7 and staurosporin; 2) treatment of ASM cells with PMA or concanavalin A failed to activate progelatinase A despite these agents increasing cell expression of MT1-MMP; and 3) the inability of ASM cell membranes to activate progelatinase A is most likely secondary to the high levels of TIMP-2 on the cell membrane. In conclusion, our results demonstrate that human ASM cells constitutively secrete progelatinase A and when stimulated with proinflammatory mediators secrete gelatinase B. The released gelatinases A and B may be important factors in the airway remodeling that occurs in asthma.
Differential expression of platelet-derived growth factor-α receptor by Thy-1−and Thy-1+lung fibroblasts
Authors: Hagood James S. , Miller Patrick J. , Lasky Joseph A. , Tousson Albert , Guo Benliu , Fuller Gerald M. , McIntosh J. Clarke
Published: December 2017

Fibroblasts are heterogeneous with respect to surface markers, morphology, and participation in fibrotic responses. This study was undertaken to determine whether Thy-1−and Thy-1+rat lung fibroblasts, which have distinct and relevant phenotypes, differ in their proliferative responses to platelet-derived growth factor (PDGF) isoforms. Homogeneous populations of Thy-1−and Thy-1+fibroblasts were found to proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated proliferation occurred only in Thy-1−cells. This differential activity correlated with significantly higher expression of PDGF-α receptor in Thy-1−fibroblasts as shown by immunoblotting, immunofluorescence, and Northern blotting. There was a rapid increase in c- myc mRNA in Thy-1−but not in Thy-1+fibroblasts on stimulation with PDGF-AA and PDGF-BB. The PDGF-α receptor, which mediates signaling by all PDGF isoforms, has been implicated in numerous clinical and experimental forms of fibrosis and regulates lung morphogenesis. Differential expression of the PDGF-α receptor supports distinct roles for Thy-1−and Thy-1+fibroblast populations in developmental and fibrotic processes in the lung.
Expression, pharmacological, and functional evidence for PACAP/VIP receptors in human lung
Authors: Busto Rebeca , Carrero Isabel , Guijarro Luis G. , Solano Rosa M. , Zapatero José , Noguerales Fernando , Prieto Juan C.
Published: December 2017

Pituitary adenylate cyclase-activating peptide (PACAP) type 1 (PAC1) and common PACAP/vasoactive intestinal peptide (VIP) type 1 and 2 (VPAC1and VPAC2, respectively) receptors were detected in the human lung by RT-PCR. The proteins were identified by immunoblotting at 72, 67, and 68 kDa, respectively. One class of PACAP receptors was defined from125I-labeled PACAP-27 binding experiments (dissociation constant = 5.2 nM; maximum binding capacity = 5.2 pmol/mg protein) with a specificity: PACAP-27 ≈ VIP > helodermin ≈ peptide histidine-methionine (PHM) ≫ secretin. Two classes of VIP receptors were established with125I-VIP (dissociation constants of 5.4 and 197 nM) with a specificity: VIP ≈ helodermin ≈ PACAP-27 ≫ PHM ≫ secretin. PACAP-27 and VIP were equipotent on adenylyl cyclase stimulation (EC50= 1.6 nM), whereas other peptides showed lower potency (helodermin > PHM ≫ secretin). PACAP/VIP antagonists supported that PACAP-27 acts in the human lung through either specific receptors or common PACAP/VIP receptors. The present results are the first demonstration of the presence of PAC1receptors and extend our knowledge of common PACAP/VIP receptors in the human lung.
Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells
Authors: Jilling Tamas , Haddad Imad Y. , Cheng Seng H. , Matalon Sadis
Published: December 2017

Nitric oxide (⋅ NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous ⋅ NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK1) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK1-WTCFTR). Exposure of these cells to 125 μM DETA NONOate at 37°C for 24 h (a chemical ⋅ NO donor) diminished Zn2+-induced and uninduced CFTR protein levels by 43.3 ± 5.1 and 34.4 ± 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of ⋅ NO, were added to the medium. Exposure to ⋅ NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK1-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible ⋅ NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM NG-monomethyl-l-arginine in the medium. Pretreatment of LLC-PK1-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of ⋅ NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that ⋅ NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.
Expression and activation of protein kinase C-ζ in eosinophils after allergen challenge
Authors: Evans David J. , Lindsay Mark A. , Webb Benjamin L. J. , Kankaanranta Hannu , Giembycz Mark A. , O’Connor Brian J. , Barnes Peter J.
Published: December 2017

Protein kinase (PK) C is an increasingly diverse family of enzymes that has been implicated in a range of cellular functions within the eosinophil. Using isoform-specific polyclonal antibodies, we have explored the expression of PKC isoforms in circulating eosinophils. Initial studies demonstrated the presence of the α, βI, βII, and ζ and the low-level expression of the δ, ε, ι, and μ isoforms but no detectable expression of the γ, η, and θ isoforms in both normal and asthmatic subjects. There was no difference in the total protein expression between these two groups. Subsequent studies examined the expression and activation of PKC isoforms in circulating eosinophils from asthmatic patients before and 24 h after a late asthmatic response to an inhaled allergen. Cellular fractionation showed PKC-α and PKC-βII to be mainly located in the cytosol, whereas PKC-βI was constitutively more expressed in the membrane. No changes in expression or subcellular localization of these isoforms were seen after allergen challenge. In contrast, PKC-ζ expression was increased after allergen challenge, and we demonstrated a significant PKC-ζ translocation to the membrane, in keeping with activation of the enzyme. Our results suggest that 24 h after allergen exposure of asthmatic patients, there is increased expression and activation of eosinophil PKC-ζ that correlates with late asthmatic responses recorded between 4 and 10 h postallergen challenge.
Surfactant secretagogue activation of protein kinase C isoforms in cultured rat type II cells
Authors: Gobran Laurice I. , Rooney Seamus A.
Published: December 2017

Several lung surfactant secretagogues are known to activate protein kinase C (PKC) in type II cells. Such agents include 12- O-tetradecanoylphorbol 13-acetate (TPA) and cell-permeable diacylglycerols that directly activate PKC. Other agents include ATP and UTP, which act at P2Y2receptors coupled to phosphoinositide-specific phospholipase C, activation of which leads to formation of diacylglycerols and consequent activation of PKC. Activation of PKC is associated with redistribution of enzyme from a cytosolic to a membrane fraction of the cell. We examined the PKC isomers that are translocated by ATP, UTP, TPA, and dioctanoylglycerol in cultured type II cells isolated from adult rats. PKC isoforms were identified by Western blotting using isoform-specific antibodies. Treatment of type II cells with ATP, UTP, TPA, and dioctanoylglycerol resulted in a significant redistribution of PKC-μ from cytosol to membrane. TPA and dioctanoylglycerol also activated PKC-α, -βI, -βII, -δ, and -η, but those isoforms were not activated by ATP or UTP. The effects of TPA and dioctanoylglycerol on PKC-μ were more pronounced than those of the P2Y2agonists, and the effect of TPA was also more rapid than that of ATP. The data show that direct activators and agents that generate endogenous diacylglycerols have different PKC activation patterns. Because it is activated by different types of secretagogues, PKC-μ may have an important role in the physiological regulation of surfactant secretion.
Urokinase-type plasminogen activator induces tyrosine phosphorylation of a 78-kDa protein in H-157 cells
Authors: Bhat G. Jayarama , Gunaje Jagadambika J. , Idell Steven
Published: December 2017

Studies from our laboratory have shown that exposure of human lung epithelial cells to urokinase plasminogen activator (uPA) induces their proliferation. This effect of uPA is likely to occur via activation of signal transduction pathways. To elucidate uPA-induced signal transduction mechanisms, we exposed H-157 cells to uPA and determined the induced tyrosine phosphorylation profile of proteins. We demonstrate that, in these cells, uPA prominently induced tyrosine phosphorylation of a 78-kDa protein. This effect was observed as early as 30 min and was sustained for at least 24 h. Treatment of cells with agents that abrogate uPA receptor (uPAR) function, including neutralizing anti-uPAR antibody, phosphatidylinositol-specific phospholipase C, or a selective antagonist that blocks the association of uPA with uPAR (Å5 compound), all failed to prevent uPA-induced tyrosine phosphorylation. B-428, an active site inhibitor of uPA activity, prevented the uPA effect. Treatment of cells with hepatocyte growth factor, vascular endothelial growth factor, or transforming growth factor-β, all of which are known to be activated by a uPA-dependent pathway, did not stimulate tyrosine phosphorylation of the 78-kDa protein. uPA induced an increase in [3H]thymidine incorporation into DNA, and cell numbers were unaffected in the presence of Å5. These results demonstrate that, in H-157 cells, uPA induces tyrosine phosphorylation of a 78-kDa protein via a proteolysis-dependent but uPAR-independent mechanism. This novel signaling pathway represents a putative mechanism by which uPA could influence epithelial cell proliferation.
P-selectin and ICAM-1 mediate endotoxin-induced neutrophil recruitment and injury to the lung and liver
Authors: Kamochi M. , Kamochi F. , Kim Y. B. , Sawh S. , Sanders J. M. , Sarembock I. , Green S. , Young J. S. , Ley K. , Fu S. M. , Rose C. E.
Published: December 2017

The role of leukocyte adhesion molecules in endotoxin-induced organ injury was evaluated by administering intraperitoneal Salmonella enteritidislipopolysaccharide (LPS) to wild-type (WT) mice, P-selectin-deficient mice, intercellular adhesion molecule (ICAM)-1-deficient mice, and P-selectin-ICAM-1 double-mutant mice. In WT mice, there was a sevenfold increase in the number of neutrophils present in the pulmonary vascular lavage fluid, and there were sevenfold more intracapillary neutrophils by electron-microscopic (EM) morphometry at 4 h after intraperitoneal LPS compared with that in control mice. Extravascular albumin accumulation increased approximately twofold in the lungs and liver of WT mice treated with LPS. In the double-mutant mice, although overall mortality after intraperitoneal LPS was not attenuated, there was a significant delay in mortality in the P-selectin-ICAM-1-deficient mutants compared with that in WT mice after intraperitoneal LPS ( P < 0.01). Moreover, compared with LPS-treated WT mice, lung and liver extravascular albumin accumulation was significantly lower in LPS-treated P-selectin-ICAM-1 double-mutant mice. Lung myeloperoxidase activity, normalized per 1,000 circulating neutrophils, increased after endotoxin in WT and P-selectin-deficient mice but not in P-selectin-ICAM-1 double-mutant mice. In addition, lung and liver myeloperoxidase activity per 1,000 circulating neutrophils in endotoxin-treated ICAM-1-deficient mice and P-selectin-ICAM-1 double mutants was significantly lower compared with that in endotoxin-treated WT mice. These data suggest that P-selectin and ICAM-1 significantly contribute to lung and liver injury after systemic endotoxemia.
Analysis of genomic regions involved in regulation of the rabbitsurfactant protein Agene in transgenic mice
Authors: Alcorn Joseph L. , Hammer Robert E. , Graves Katherine R. , Smith Margaret E. , Maika Shanna D. , Michael Laura F. , Gao Erwei , Wang Ying , Mendelson Carole R.
Published: December 2017

The gene encoding surfactant protein (SP) A, a developmentally regulated pulmonary surfactant-associated protein, is expressed in a lung-specific manner, primarily in pulmonary type II cells. SP-A gene transcription in the rabbit fetal lung is increased by cAMP. To delineate the genomic regions involved in regulation of SP-A gene expression, lines of transgenic mice carrying fusion genes composed of various amounts of 5′-flanking DNA from the rabbit SP-A gene linked to the human growth hormone structural gene as a reporter were established. We found that as little as 378 bp of 5′-flanking DNA was sufficient to direct appropriate lung cell-selective and developmental regulation of transgene expression. The same region was also sufficient to mediate cAMP induction of transgene expression. Mutagenesis or deletion of either of two DNA elements, proximal binding element and a cAMP response element-like sequence, previously found to be crucial for cAMP induction of SP-A promoter activity in transfected type II cells, did not affect lung-selective or temporal regulation of expression of the transgene; however, overall levels of fusion gene expression were reduced compared with those of wild-type transgenes.
Hormonal regulation and cellular localization of fatty acid synthase in human fetal lung
Authors: Wagle Sameer , Bui Anh , Ballard Philip L. , Shuman Henry , Gonzales John , Gonzales Linda W.
Published: December 2017

Fatty acid synthase (FAS; EC 2.3.1.85 ) supplies de novo fatty acids for pulmonary surfactant synthesis, and FAS gene expression is both developmentally and hormonally regulated in the fetal lung. To further examine hormonal regulation of FAS mRNA and to determine the cellular localization of FAS gene expression, we cultured human fetal lungs (18–22 wk gestation) as explants for 1–4 days in the absence (control) or presence of glucocorticoid [dexamethasone (Dex), 10 nM] and/or cAMP agents (8-bromo-cAMP, 0.1 mM and IBMX, 0.1 mM). FAS protein content and activity increased similarly in the presence of Dex (109 and 83%, respectively) or cAMP (87 and 111%, respectively), and responses were additive in the presence of both hormones (230 and 203%, respectively). With a rabbit anti-rat FAS antibody, FAS immunoreactivity was not detected in preculture lung specimens but appeared in epithelial cells lining the tubules with time in culture. Dex and/or cAMP markedly increased staining of epithelial cells, identified as type II cells, whereas staining of mesenchymal fibroblasts was very low under all conditions. With in situ hybridization, FAS mRNA was found to be enriched in epithelial cells lining the alveolar spaces, and the reaction product increased in these cells when the explants were cultured with the hormones. The increased FAS mRNA content in the presence of Dex and/or cAMP is primarily due to increased stabilization of mRNA, although Dex alone increased the transcription rate by ∼30%. We conclude that hormonal treatment of cultured human fetal lungs increases FAS gene expression primarily by increasing stability of the message. The induction of FAS during explant culture and by hormones occurs selectively in type II epithelial cells, consistent with the regulatory role of this enzyme in de novo synthesis of fatty acid substrate for surfactant synthesis in perinatal lungs.
Nifedipine inhibits pulmonary hypertension but does not prevent decreased lung eNOS in hypoxic newborn pigs
Authors: Fike Candice D. , Kaplowitz Mark R.
Published: December 2017

Therapies to prevent the onset or progression of pulmonary hypertension in newborns have received little study compared with those in adult models. We wanted to determine whether nifedipine treatment prevents the increased pulmonary vascular resistance, blunted pulmonary vascular responses to acetylcholine, and reduced lung endothelial nitric oxide synthase (eNOS) amounts that we have found in a newborn model of chronic hypoxia-induced pulmonary hypertension. Studies were performed with 1- to 3-day-old piglets raised in room air (control) or 10% O2 (hypoxia) for 10–12 days. Some piglets from each group were given nifedipine (3–5 mg/kg sublingually three times a day). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. Pulmonary vascular responses to acetylcholine and eNOS amounts were assessed in excised lungs. The calculated value of the pulmonary vascular resistance for nifedipine-treated hypoxic piglets (0.09 ± 0.01 cmH2O ⋅ ml−1 ⋅ min ⋅ kg) was almost one-half of the value for untreated hypoxic piglets (0.16 ± 0.01 cmH2O ⋅ ml−1 ⋅ min ⋅ kg) and did not differ from the value for untreated control piglets (0.05 ± 0.01 cmH2O ⋅ ml−1 ⋅ min ⋅ kg). Pulmonary arterial pressure responses to acetylcholine and whole lung homogenate eNOS amounts were less for both nifedipine-treated and untreated hypoxic piglets than for untreated control piglets. Nifedipine treatment attenuated pulmonary hypertension in chronically hypoxic newborn piglets despite the persistence of blunted responses to acetylcholine and reduced lung eNOS amounts.