bluuebunny/crossref_metadata_2025 huggingface dataset:
Constitutive endothelial nitric oxide synthase gene expression is regulated during lung development
Authors: Kawai N. , Bloch D. B. , Filippov G. , Rabkina D. , Suen H. C. , Losty P. D. , Janssens S. P. , Zapol W. M. , de la Monte S. , Bloch K. D.
Published: December 2017

Nitric oxide (NO), a potent vasodilator, is a free-radical gas synthesized from L-arginine by nitric oxide synthases (NOS). NO appears to have an important role in perinatal changes in pulmonary vascular resistance. We previously identified mRNA encoding the constitutive endothelial NOS (ceNOS) isoform in human pulmonary tissue. To begin investigating functions of this enzyme in perinatal pulmonary development, we measured ceNOS mRNA and immunoreactivity in the developing rat lung. With the use of RNA blot hybridization, abundant pulmonary ceNOS mRNA was detected during the late fetal and postnatal period. The highest levels were detected within 24 h after birth, and elevated mRNA levels persisted for 16 days. In contrast, much lower levels of ceNOS mRNA were found in adult rat lung. With the use of immunoblot techniques, ceNOS protein levels were found to be correlated with mRNA levels. To identify the pulmonary cell types expressing the ceNOS gene, in situ hybridization with a digoxigenin-labeled cRNA probe was performed on sections from lungs of 1-day-old and adult rats. In lungs from 1-day-old rats, ceNOS mRNA was detected in alveolar and serosal epithelial cells as well as in endothelial cells lining small and medium-sized blood vessels. In contrast, in adult lungs, ceNOS gene transcripts were detected in rare endothelial cells. These observations suggest that ceNOS gene expression is regulated during lung development and that ceNOS is available to participate in the postnatal reduction of pulmonary vascular resistance. ceNOS gene expression in nonendothelial cells in the neonatal rat lung suggests that NO may also contribute to nonvascular functions in the developing lung.
Functional activation of the cystic fibrosis trafficking mutant delta F508-CFTR by overexpression
Authors: Cheng S. H. , Fang S. L. , Zabner J. , Marshall J. , Piraino S. , Schiavi S. C. , Jefferson D. M. , Welsh M. J. , Smith A. E.
Published: December 2017

The most common mutation in the gene associated with cystic fibrosis (CF) causes deletion of phenylalanine at residue 508 (delta F508) of the gene product called CFTR. This mutation results in the synthesis of a variant CFTR protein that is defective in its ability to traffic to the plasma membrane. Because earlier studies showed delta F508-CFTR retains significant phosphorylation-regulated chloride (Cl-) channel activity, processes capable of restoring the mislocalized delta F508-CFTR to the correct cellular destination may have therapeutic benefit. Here we report one such process that involves overexpression of the mutant protein and appears to result in the escape of a small amount of delta F508-CFTR to the plasma membrane. In recombinant cells where expression of delta F508–CFTR is controlled by the metallothionein promoter, this effect can be brought about by treatment with sodium butyrate. Although cAMP-activated Cl- channel activity could also be detected in immortalized human airway epithelial cells homozygous for the delta F508 mutation at the single cell level, treatment with butyrate did not generate a measurable cAMP-stimulated Cl- current in polarized monolayers of primary CF airway epithelia. However, the observation that overexpression can effect the presence of recombinant delta F508-CFTR at the plasma membrane suggests that perhaps other butyrate-like compounds that are more potent and more specific for the promoter of the CF gene may be efficacious in alleviating the Cl- channel defect associated with CF.
Clearance of surfactant protein B from rabbit lungs
Authors: Ueda T. , Ikegami M. , Henry M. , Jobe A. H.
Published: December 2017

To characterize the metabolism of surfactant protein B (SP-B) in vivo, we measured the clearance of SP-B from adult rabbit lungs. Purified rabbit SP-B was radiolabeled with 125I by the Bolton-Hunter method. Trace amounts of 125I-labeled SP-B mixed with [14C]dipalmitoylphosphatidylcholine (DPPC) were given intratracheally via a bronchoscope to rabbits 0-16 h before collection of alveolar washes (AW). Macrophages were recovered from AW, and lamellar bodies (LB) were isolated from lung tissue by differential centrifugation. 125I-SP-B was cleared more rapidly from the airspaces and the total lung (half-life 7 h) than was DPPC (half-life 11 h in the total lung). There was an approximately threefold accumulation of SP-B relative to saturated phosphatidylcholine in macrophages at all times. The proportion of 125I and 14C radioactivities in lamellar bodies was similar at 2 and 4 h, but there was 14-fold less 125I-SP-B than [14C]DPPC in lamellar bodies by 16 h. This loss of SP-B from the lamellar body fraction is consistent with less recycling of SP-B. The results demonstrate different clearance kinetics of these two components of surfactant and indicate a significant role of macrophages in the clearance of SP-B.
Aberrant processing of surfactant protein C in hereditary SP-B deficiency
Authors: Vorbroker D. K. , Profitt S. A. , Nogee L. M. , Whitsett J. A.
Published: December 2017

Hereditary surfactant protein B (SP-B) deficiency causes lethal neonatal respiratory disease associated with abnormalities in pulmonary surfactant proteins and lipids. SP-C, a 4-kDa hydrophobic protein produced from a 21-kDa precursor, cooperates with SP-B to enhance the surface active properties of surfactant phospholipids. Anti-proSP-C polyclonal antisera were produced against fusion proteins containing 1) the amino terminus (amino acids 1–20), 2) the region carboxy-terminal to the mature SP-C peptide (amino acids 58–77), and 3) full-length 197-amino acid proSP-C and were characterized using immunoprecipitation, Western blot, and immunohistochemical techniques. Western blot analysis of bronchoalveolar lavage and amniotic fluid from hereditary SP-B-deficient patients allowed identification of a 12-kDa form of SP-C that contained epitopes consistent with the amino-terminal and active peptide regions of SP-C (amino acids 1–57). The 12-kDa SP-C peptide was not detected in bronchoalveolar lavage from healthy adults or adults with alveolar proteinosis or pneumonia. We conclude that SP-B deficiency is associated with the aberrant processing and secretion of an immature SP-C peptide, which may contribute to the respiratory failure associated with hereditary SP-B deficiency.
Role of endothelial cytoskeleton in high-permeability edema due to botulinum C2 toxin in perfused rabbit lungs
Authors: Ermert L. , Bruckner H. , Walmrath D. , Grimminger F. , Aktories K. , Suttorp N. , Duncker H. R. , Seeger W.
Published: December 2017

The cytoskeleton of the endothelial cell has been suggested to regulate endothelial barrier function. We investigated the role of actin in the maintenance of pulmonary capillary integrity in perfused rabbit lungs. As a tool for selective perturbation of actin, we employed Clostridium botulinum C2 toxin, which is composed of a membrane translocation component (C2II) and a component (C2I) effecting ADP-ribosylation of nonmuscle G-actin. ADP-ribosylated actin no longer capable of polymerization but acts as a barbed end-capping protein, thereby effecting selective loss of the nonmuscle F-actin content. In buffer-perfused rabbit lungs, combined application of both toxin components (range 50 pg/ml-5 ng/ml C2I) resulted in a time- and dose-dependent increase in the capillary filtration coefficient (Kfc) with concomitant edema formation. Only 300:600 pg/ml C2I:II sufficed to induce a > 10-fold rise of Kfc values within 110 min. This severe lung permeability increase occurred in the absence of vasomotor responses and potassium release or lactate dehydrogenase release. Application of each single toxin component displayed markedly reduced efficacy. Similar to the C2 toxin effect, severe permeability increase without concomitant hemodynamic changes was evoked by cytochalasin D, known to possess F-actin-disrupting properties. Preloading of lung cells with phallacidin, which in opposition to C2 toxin decreases F-actin depolymerization, significantly reduced the C2 toxin-induced increase in vascular permeability. Electron microscopic examination of C2 toxin-poisoned lungs showed early, extensive endothelial cell attenuations, followed by disruptions of the endothelial layer and marked interstitial edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Fetal lung cell-derived matrix alters distal lung epithelial ion transport
Authors: Pitkanen O. M. , Tanswell A. K. , O'Brodovich H. M.
Published: December 2017

Extracellular matrix (ECM) synthesized by the fetal mesenchymal cells provides a supporting structure for the growing airways and is important for airway branching and in the differentiation of the primitive epithelium. We studied whether ECM, in addition to its structural role in lung interstitium, influences the ion transport of rat fetal distal lung epithelial cells (FDLE). FDLE monolayers were cultured on two different fetal mixed lung cell (MLC)-derived matrix preparations and studied in Ussing chambers. FDLE on MLC matrix had an increased resting equivalent short-circuit current (Ieq). Amiloride (10(-4) M apically) decreased the Ieq significantly in all the FDLE monolayers. The residual Ieq was significantly larger in FDLE grown on MLC matrix (increased by 150 and 80% under baseline and beta 2-agonist-stimulated conditions, respectively) than on control filters and filters coated with type I collagen, and type IV collagen, laminin, or fibronectin. The matrix produced by MLC isolated at an earlier gestational stage decreased the FDLE's sensitivity to amiloride. The increased amiloride-insensitive Ieq was only modestly affected by the Na+/K+/Cl- cotransport inhibitor bumetanide (10(-4) M basally) but was abolished when the [Cl-] of the bathing solution was reduced to 10 mM. These observations demonstrated that MLC elaborated ECM is able to change the nature of the ion transport of FDLE. ECM may be an important factor governing the ion transporting phenotype of fetal type II alveolar epithelial cells.
Adenovirus-mediated gene transfer to human fetal lung ex vivo
Authors: Ballard P. L. , Zepeda M. L. , Schwartz M. , Lopez N. , Wilson J. M.
Published: December 2017

Gene therapy of the fetus or newborn infant is a potentially useful approach for prevention or treatment of specific lung diseases. To begin to address issues such as efficiency, duration, and cellular distribution of transgene expression, we studied transduction of human lung cells by recombinant, replication-deficient adenovirus containing the lacZ gene driven by the beta-actin promoter and cytomegalovirus enhancer (H5.010CBlacZ). Human fetal lungs of 20- to 24-wk gestation received approximately 10(11) viral particles by instillation into a major bronchus, and the tissue was cultured as explants in serum-free Waymouth's medium. beta-galactosidase staining (X-gal) was detected by 24 h in defined regions of treated tissue and localized to epithelial cells of airways and terminal saccules. beta-galactosidase activity in homogenate of treated tissue was maximal 3-5 days after exposure to virus, ranging from 0.2 to 1.5 A420.min-1.mg protein-1 in four experiments (control values were approximately 0.001). When virus was added directly to lung explants in culture, beta-galactosidase was expressed in most of the peripheral cells and rarely in interior cells, the level of activity was dose dependent between 10(8) and 10(11) viral particles/ml, and transgene expression was sustained for at least 28 days. Treatment of isolated cultured cells with virus resulted in equivalent staining of both epithelial cells and fibroblasts. We conclude that fetal lung cells are efficiently transduced by recombinant adenovirus, indicating the feasibility of gene therapy in the infant or fetus. Cultured fetal lung may be useful for testing gene constructs being considered for therapy.
TGF-beta regulates production of NO in pulmonary artery smooth muscle cells by inhibiting expression of NOS
Authors: Finder J. , Stark W. W. , Nakayama D. K. , Geller D. , Wasserloos K. , Pitt B. R. , Davies P.
Published: December 2017

We have previously reported that a mixture of lipopolysaccharide and cytokines stimulates cultured rat pulmonary artery smooth muscle cells (RPASM) to express elevated levels of mRNA for inducible nitric oxide synthase (iNOS), and to produce large amounts of nitric oxide (NO). The current study tests the hypothesis that transforming growth factor-beta (TGF-beta) modulates this process. Accordingly, RPASM were treated with a mixture of LPS (10 micrograms/ml) and the cytokines interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (500 U/ml), and interferon-gamma (100 U/ml). In the absence of TGF-beta 1, NO production (indicated by colorimetric assay of cumulative nitrite levels at 24 h) was greatly increased, as previously observed. Under identical conditions, TGF-beta 1 caused a concentration-dependent decrease in NO production. The addition of neither excess L-arginine nor sepiapterin reversed the inhibition, indicating that the effect of TGF-beta 1 was not due to limitation of enzyme substrate or cofactor tetrahydrobiopterin, respectively. Northern and Western analyses showed that TGF-beta 1 reduced levels of iNOS mRNA and protein to baseline at all time points examined up to 24 h. Complete suppression of iNOS protein expression was evident even when TGF-beta 1 was added at postinduction time points. One mechanism of action of TGF-beta 1 was demonstrated in experiments in which degradation of iNOS protein was greatly increased by the addition of TGF-beta 1. These results demonstrate that TGF-beta 1 regulates production of NO in RPASM by inhibiting iNOS expression in part by increasing degradation of existing iNOS protein.
Stimulatory effects of hepatocyte growth factor on normal and neoplastic human bronchial epithelial cells
Authors: Singh-Kaw P. , Zarnegar R. , Siegfried J. M.
Published: December 2017

We examined the mitogenic, chemoinvasive, and chemotactic effects of hepatocyte growth factor (HGF) toward normal and neoplastic human epithelial cells derived from the bronchial mucosa. Primary cultures of human bronchial epithelial cells (HBE cells), immortalized bronchial epithelial cells (IB3-1 cells), and cells derived from a squamous cell carcinoma of the lung (128-88T cells) were used as targets. HGF was mitogenic for all three cell types as measured by bromodeoxyuridine (BrdU) labeling and colony-forming efficiency (CFE). With the use of BrdU labeling, 9.8-16.8% of nuclei were labeled in controls vs. 56.9-65.6% labeled nuclei in cells treated with HGF. HGF stimulated colony formation 3.6-6.2-fold over untreated control. Analysis by reverse transcription-polymerase chain reaction demonstrated the presence of the c-met gene, the receptor for HGF, in all three cell types. Cell lysates from all three cell types contained proteins that were recognized by a c-met antibody as determined by Western blotting. The gene for HGF was not expressed in any of the cell types, although it was expressed in control MRC5 fibroblasts. No HGF protein could be detected by Western blotting in the conditioned medium from epithelial cells, although it was readily detectable in medium conditioned by lung fibroblasts. HGF proved to be a powerful chemotactic agent for all three cell types and also stimulated invasion into Matrigel, an artificial basement membrane. The results indicate HGF acts mainly as a paracrine growth factor for cells derived from the human bronchus, and may play a role in the growth and progression of lung tumors.
Dysfunction of cGMP-mediated pulmonary vasorelaxation in endotoxin-induced acute lung injury
Authors: Fullerton D. A. , McIntyre R. C. , Hahn A. R. , Agrafojo J. , Koike K. , Meng X. , Banerjee A. , Harken A. H.
Published: December 2017

Endothelial-dependent and -independent cGMP-mediated mechanisms of pulmonary vasorelaxation were studied in endotoxin-induced acute lung injury in the rat. Concentration-response curves were generated (10(-9) to 10(-6) M) for acetylcholine (ACh), A23187, and sodium nitroprusside (SNP) and for 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) (10(-9) to 10(-4) M) in isolated pulmonary arterial rings preconstricted with phenylephrine 6 h after endotoxin treatment (20 mg/kg ip). Endotoxin treatment produced significantly increased lung neutrophil accumulation (myeloperoxidase assay, 28 +/- 6 units/g lung tissue vs. 1.8 +/- 1 in controls) and lung leakage (lung/blood 125I-labeled albumin ratio, 0.06 +/- 0.01 vs. 0.028 +/- 0.01 in controls) as well as histological evidence of pulmonary vascular endothelial damage. The concentration-response curves demonstrated that pulmonary vasorelaxation by mechanisms that require generation of cGMP by either endothelial-dependent (both receptor-dependent, ACh, and receptor-independent, A23187) or endothelial-independent (SNP) pathways were significantly impaired after endotoxin treatment. Relaxation by stimulation with the cGMP analogue 8-BrcGMP was not different from control. Pulmonary vascular smooth muscle is able to relax in response to cGMP after endotoxin treatment, but relaxation by endothelial-dependent and -independent pathways that require generation of cGMP is significantly impaired.
PKG-I alpha phosphorylates the alpha-subunit and upregulates reconstituted GKCa channels from tracheal smooth muscle
Authors: Alioua A. , Huggins J. P. , Rousseau E.
Published: December 2017

Modulation of Ca(2+)-dependent K+ channel (GKCa) activities in airway smooth muscles (ASM) by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) is thought to play a central role in mediating the effect of some bronchodilator agents that elevate cytoplasmic basal cGMP concentrations. However, no direct evidence supports this hypothesis in ASM. In the present work, we provide evidence that PKG-I alpha upregulates GKCa channels derived from bovine tracheal smooth muscle cells and reconstituted into planar lipid bilayers. In two different experimental approaches, PKG increased the open probability as well as the mean open time of GKCa channels, without any effect on unitary current amplitudes and unit conductance. Our results indicate that the kinetics of GKCa channels are controlled by a phosphorylation step mediated by PKG, and thus might be modulated by intracellular cGMP. Biochemical assays demonstrated that PKG phosphorylates several protein bands in the membrane fraction. Two of those proteins co-migrate with the same relative molecular mass as the 62- and 30-kDa components of the purified channel complex, identified as GKCa-alpha and -beta subunits, respectively. Our results also indicate that PKG phosphorylates the GKCa-alpha subunit with an apparent stoichiometry of 0.89, which would be consistent with the presence of a single PKG-sensitive phosphorylating site within its amino acid sequence. Furthermore, these results demonstrate for the first time that PKG directly phosphorylates GKCa from airway smooth muscle cells and thereby activates the channels at negative voltage or at low free Ca2+ concentrations.
TNF-alpha and IL-1 beta upregulate nitric oxide-dependent ciliary motility in bovine airway epithelium
Authors: Jain B. , Rubinstein I. , Robbins R. A. , Sisson J. H.
Published: December 2017

Airway epithelial cells can be modulated by cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta that are released from inflammatory cells. Since ciliary motility is an important host defense function of airway epithelium, we hypothesized that cytokines, released from lung macrophages, upregulate ciliary motility. To test this hypothesis, ciliary beat frequency (CBF) was measured by video microscopy in cultured ciliated bovine bronchial epithelial cells (BBECs) incubated for 24 h with bovine alveolar macrophage-conditioned medium (AM-CM). Exposure to AM-CM resulted in a delayed (> or = 2 h) increase in CBF that was maximal after 24 h exposure (13.70 +/- 0.43 for AM-CM vs. 9.44 +/- 0.24 Hz for medium; P < 0.0001) and which was largely blocked by either anti-TNF-alpha or anti-IL-1 beta antibodies. rTNF-alpha or rIL-1 beta similarly increased CBF, which could be blocked by preincubation with either anti-rTNF-alpha or anti-rIL-1 beta antibodies. Preincubation of BBECs with actinomycin D or dexamethasone also blocked rTNF-alpha- and rIL-1 beta-induced cilia stimulation, suggesting that new protein synthesis is required for cytokine-induced upregulation of CBF. Since NO is known to upregulate ciliary motility and cytokines can induce NO synthase (NOS), we hypothesized that TNF-alpha and IL-1 beta increase CBF by inducing NOS in BBECs. The cilia stimulatory effects of TNF-alpha or IL-1 beta were inhibited by NG-monomethyl-L-arginine, a competitive NOS inhibitor, and restored by the addition of either L-arginine, an NOS substrate, or sodium nitroprusside, an NO donor.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of protein kinase C in nonsensitized and passively sensitized human isolated bronchial smooth muscle
Authors: Rossetti M. , Savineau J. P. , Crevel H. , Marthan R.
Published: December 2017

To examine the role of protein kinase C (PKC) activation in the control of the mechanical activity of human isolated bronchial smooth muscle obtained at thoracotomy, the effect of the phorbol ester phorbol 12,13-dibutyrate (PDB) was evaluated. PDB produced slowly developing and sustained contractions that were reduced 1) by the PKC inhibitor staurosporine and 2) after long-term (12 h) exposure to PDB, which downregulates PKC. Moreover, the inactive phorbol ester 4 alpha-phorbol 12,13 didecanoate had no contractile effect. Removal of external Ca2+ or addition of the Ca(2+)-channel antagonist verapamil reduced the PDB-induced contraction. Passive sensitization of human isolated bronchial rings, i.e., incubation overnight of tissues in serum from atopic asthmatic patients, decreased the maximal response to PDB to 28.9 +/- 8% of the maximal response to acetylcholine (ACh) when compared with that of paired nonsensitized rings, i.e., tissues incubated overnight in serum from normal subjects (46.7 +/- 9.4% of the maximal response to ACh, n = 5, P < 0.05). The decrease in the response to PDB induced by either long-term preexposure to PDB or passive sensitization was reversed when both types of tissues were allowed to recover unstimulated for 3 h before PDB application. These results show that 1) PKC activation induces maintained contractions in human isolated airway smooth muscle that are largely dependent on extracellular calcium; 2) passive sensitization alters the PKC-mediated response in a way similar to that induced by prolonged stimulation of PKC.
Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs
Authors: Guidot D. M. , Repine M. J. , Hybertson B. M. , Repine J. E.
Published: December 2017

We found that ventilation with nitric oxide (NO, 50 ppm) significantly (P < 0.05) reduced capillary leak (as reflected by weight gain and Ficoll retention) in isolated rat lungs perfused for 60 min with N-formyl-methionyl-leucyl-phenylalanine (fMLP; 10(-7) M) and human neutrophils (1,300/microliters). Perfusion with previously heated neutrophils (48 degrees C for 10 min, which inactivates NADPH oxidase) did not cause weight gain or Ficoll retention, indicating that neutrophil-derived oxidants mediated lung leak. Although perfusion with fMLP and neutrophils increased mean pulmonary artery pressures (PAP) from 7 to 11.7 +/- 0.5 mmHg at 10 min, lungs perfused with fMLP and neutrophils in which PAP was maintained at 7 mmHg by reducing perfusion flow rates also developed significant (P < 0.05) weight gain and Ficoll retention. Furthermore, inhaled NO did not reduce (P > 0.05) PAP at 10 min and only modestly reduced PAP at 30 and 60 min of perfusion. Our results suggest that oxidative endothelial damage, and not increased hydrostatic pressure, was the primary cause of the capillary leak, and that the protection provided by inhaled NO was not solely a consequence of vasodilation. We conclude that inhaled NO prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs, and speculate that inhaled NO has anti-inflammatory properties in addition to its ability to cause pulmonary vasodilation.
Hypoxia and reoxygenation stimulate biphasic changes in endothelial monolayer permeability
Authors: Partridge C. A.
Published: December 2017

Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells in low O2 content (95% N2-5% CO2) for 4 h increased monolayer permeability to dextran almost twofold and also increased the incidence of intercellular gaps and intracellular actin stress fibers. Hypoxic incubation decreased the extracellular matrix contents of fibronectin and vitronectin, proteins that serve as anchorage points for the endothelial cells. This state was reversed after 24 h of hypoxic incubation, and the BPMVE monolayer permeability to dextran was less than that of normoxic controls. The monolayer had fewer intercellular gaps and stress fibers, and the extracellular matrix contained increased amounts of fibronectin, vitronectin, and type I collagen. These alterations stimulated by 24 h of hypoxic incubation were resolved within 4 h of reoxygenation in room air supplemented with 5% CO2. These studies indicate that incubation of endothelial monolayers in hypoxic conditions first increases and then decreases monolayer permeability, through increased and decreased formation of intercellular gaps.
Leukotoxin, 9,10-epoxy-12-octadecenoate causes edematous lung injury via activation of vascular nitric oxide synthase
Authors: Ishizaki T. , Shigemori K. , Nakai T. , Miyabo S. , Ozawa T. , Chang S. W. , Voelkel N. F.
Published: December 2017

We examined the mechanism of leukotoxin, 9,10-epoxy-12-octadecenoate (Lx)-induced lung injury in blood-free, physiological salt solution-perfused rat lungs under constant flow conditions. Mean pulmonary arterial (Ppa) and pulmonary capillary pressure (Pcap, estimated by the double-occlusion method), wet lung weight (WLW), pulmonary capillary filtration coefficient (Kfc), lung perfusate lactate dehydrogenase (LDH) activity, and nitrite levels were assessed. Bolus injection of Lx (200 microM) caused insidious and significant lung weight gain, which was not associated with remarkable elevation of Ppa or Pcap but was associated with an increase of perfusate LDH activity and nitrite levels. Lx (20 microM) elevated Kfc, indicating that Lx had affected pulmonary vascular permeability. Because Lx causes endothelium dependent pulmonary vasodilation, we studied the effect of NG-monomethyl-L-arginine (L-NMMA), NG-monomethyl-D-arginine (D-NMMA), superoxide dismutase (SOD), human oxyhemoglobin (oxyHb), and methylene blue (MB) on Lx-induced lung injury. L-NMMA, SOD, and oxyHb, but not MB or D-NMMA, protected the lungs against Lx (200 microM)-induced injury. Lx increased pulmonary vascular permeability and caused lung injury. Because both nitric oxide synthase inhibitors and SOD inhibited the Lx-induced lung injury, it is possible that peroxynitrite is involved in the mechanism whereby Lx causes lung injury.
Reduction of thiazine dyes by bovine pulmonary arterial endothelial cells in culture
Authors: Bongard R. D. , Merker M. P. , Shundo R. , Okamoto Y. , Roerig D. L. , Linehan J. H. , Dawson C. A.
Published: December 2017

The uptake of methylene blue (MB), and toluidine blue O (TBO) by bovine pulmonary arterial endothelial cells grown on microcarrier beads was detected as a decrease in the concentration of dye in the medium after these thiazine dyes were added to the medium surrounding the cells. Because the reduced forms of these dyes are much more lipophilic than the oxidized forms, we considered the possibility that reduction of the dyes at the cell surface might have preceded the uptake by the cells. Therefore, we studied the ability of the cells to reduce a toluidine blue O-polyacrylamide polymer (TBOP), which was too large to enter the cells in either the oxidized or reduced form. The TBO moieties of the polymer were reduced by the cells, indicating that the dyes did not have to enter the cells to be reduced and that reduction can occur at, or near, the cell surface. The rate of TBOP reduction was about the same as the rate of uptake of the monomeric dyes, indicating that the cell surface reduction mechanism had a sufficient capacity to account for the monomer uptake by the cells. We also found that ferricyanide ion, which also did not permeate the cells, was reduced by the cells and that external ferricyanide inhibited the monomeric MB uptake. Thus the results with ferricyanide were also consistent with the concept that the monomeric thiazine dyes are reduced at the cell surface before the more lipophilic reduced forms are taken up by the endothelial cells.
Dipeptide transport characteristics of the apical membrane of rat lung type II pneumocytes
Authors: Meredith D. , Boyd C. A.
Published: December 2017

The transport of a hydrolysis-resistant dipeptide, D-phenylalanyl-L-alanine (D-Phe-L-Ala), has been studied by high-performance liquid chromatography in rat lung epithelial cells and apical membrane vesicles. Time-dependent uptake of D-Phe-L-Ala into isolated type II pneumocytes was shown. Uptake was saturable, and Michaelis-Menten kinetics were fitted to the data and gave an apparent Michaelis constant (Km) of 3.4 mM and a maximum velocity (Vmax) of 7.0 nmol.mg protein-1.min-1. However, known peptide transport inhibitors unexpectedly increased intracellular D-Phe-L-Ala concentration when initial rates of peptide uptake were studied. Apical (brush-border) membrane vesicles prepared from rat lung also showed time- and concentration-dependent influx of D-Phe-L-Ala (apparent Km 2.0 mM, Vmax 0.53 nmol.mg protein-1.min-1). Influx of this neutral dipeptide into the vesicles was shown to be both electrogenic and stimulated by an inwardly directed proton gradient. Influx was inhibitable by mercuric chloride and by the amino acid residue modifying compounds N-acetylimidazole and diethylpyrocarbonate. These findings strongly suggest the presence of a proton-coupled peptide transport protein in the apical surface of the type II cell. This transporter may play a role in lung homeostasis.
Leukotriene D4 facilitates airway smooth muscle cell proliferation via modulation of the IGF axis
Authors: Cohen P. , Noveral J. P. , Bhala A. , Nunn S. E. , Herrick D. J. , Grunstein M. M.
Published: December 2017

The insulin-like growth factor (IGF) axis is involved in regulating proliferation in a variety of cell types, including airway smooth muscle. Because airway hyperplasia is a characteristic feature of asthma and other lung diseases, we examined the interaction of the potent proinflammatory eicosanoid leukotriene D4 (LTD4) with the IGF axis in regulating airway smooth muscle cell mitogenesis. In cultured rabbit airway smooth muscle cells, IGF-I but not LTD4 was mitogenic at submaximal concentrations. The combination of the two agents exerted a significant synergistic effect on airway smooth muscle cell mitogenesis. Analysis of airway smooth muscle cell conditioned medium by Western ligand blotting demonstrated a marked LTD4-induced reduction in the levels of the predominant IGF binding protein IGFBP-2, which is elaborated into the conditioned medium. The latter effect on IGFBP-2 release was not associated with a reduction in IGFBP-2 mRNA levels; however, LTD4-treated airway smooth muscle conditioned medium demonstrated the presence of a lower molecular weight form of IGFBP-2 by cross-linking to IGFs and specific proteolysis of radiolabeled IGFBP-2. IGFBP-2 was also noted to be associated with airway smooth muscle cell membranes, where it was protected from LTD4-induced proteolysis. Finally, exogenous administration of IGFBP-2 was found to inhibit the promitogenic effect of IGF-I in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Reactive species in ischemic rat lung injury: contribution of peroxynitrite
Authors: Ischiropoulos H. , al-Mehdi A. B. , Fisher A. B.
Published: December 2017

Lung ischemia-reperfusion represents a potentially important mechanism for diverse forms of tissue injury associated with decreased pulmonary flow. Previous studies demonstrated oxidative injury in ischemic-reperfused lungs. The present study was designed to evaluate the contribution of nitric oxide and peroxynitrite in tissue injury. The levels of the stable decomposition products of nitric oxide and peroxynitrite, nitrite plus nitrate, were twofold greater than control during reperfusion after 60 min of ischemia. Inhibition of nitric oxide synthesis by endotracheal insufflation of 5 mM NG-nitro-L-arginine methyl ester, 30 min before the induction of ischemia, decreased the production of lung thiobarbituric acid reactive substances (TBARS) by 67% (P < 0.05, n = 5), TBARS released into the lung perfusate by 55% (P < 0.05, n = 5), lung-conjugated dienes by 61% (P < 0.05, n = 5), and dinitrophenylhydrazine-reactive protein carbonyl levels by 86% (P < 0.05, n = 5). Amino acid analysis of tissue homogenates from lungs exposed to 60 min of ischemia and 60 min of reperfusion revealed a 1.8-fold (P < 0.05, n = 5) increase in nitrotyrosine concentration compared with 2 h continuously perfused lungs. Inhibition of nitric oxide synthesis abolished the increase in nitrotyrosine levels. Furthermore, lungs exposed to 60 min of reperfusion after 60 min of ischemia showed specific binding of an anti-nitrotyrosine antibody. In reperfused tissues, antibody binding was observed throughout the lung. The binding was blocked with excess of nitrotyrosine, and minimal binding was observed in nonperfused blood-free control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of polycations on barrier and transport properties of alveolar epithelium in situ
Authors: Saumon G. , Soler P. , Martet G.
Published: December 2017

We examined the effect of polycations, classes of which are released by activated leukocytes, on the transport properties of the alveolar epithelium in isolated-perfused rat lungs. Protamine, polylysines, and ruthenium red produced rapid, dose-dependent increases in mannitol permeability (PAmann) when instilled into airspaces. The coupling between active transepithelial Na+ transport and alveolar fluid absorption was not altered, despite > 10-fold increases in PAmann. The increase in albumin permeability compared with that in mannitol suggested preservation of alveolar barrier-size selectivity. Tracheal instillation of protamine produced no cellular abnormality, whereas its addition to the perfusate resulted in damage to endothelial and type I cells. Protamine produced an even larger (P < 0.05) increase in PAmann in the presence of isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate + 3-isobutyl-1-methyl-xanthine. The stimulation of Na+ and fluid transport by these agents was unaffected by protamine. Mastoparan, a peptide that activates G proteins, produced effects comparable to those of the polycations. The protamine- and mastoparan-induced increase in PAmann was abolished by barium, a K+ channel blocker, but not by zinc, a membrane-protective cation. Other K+ channel blockers, tetraethylammonium and quinine, had no effect. Thus short-term apical application of polycations and mastoparan alter alveolar epithelium paracellular permeability by a noncytotoxic mechanism that is inhibited by barium. The resulting increase in paracellular permeability does not alter fluid absorption driven by active Na+ transport. Polycations have very different effects, depending on whether they are present on one side or the other side of the alveolar capillary barrier.
Time course of airway hyperresponsiveness and remodeling induced by hyperoxia in rats
Authors: Szarek J. L. , Ramsay H. L. , Andringa A. , Miller M. L.
Published: December 2017

The purpose of this study was to answer two questions concerning hyperoxia-induced airway hyperresponsiveness: 1) What is the time course of the development of airway hyperresponsiveness? 2) What is the relationship between the increase in responsiveness and smooth muscle area? Segments of intrapulmonary bronchi were isolated from male Sprague-Dawley rats that had been exposed to 80-85% O2 for a period of 1, 3, 5, or 7 days and from aged-matched control animals that breathed room air. Hyperoxia increased the sensitivity (log concentration or frequency that elicited a half-maximal response) and reactivity (maximum tension developed) of the airways to electrical field stimulation (EFS) after 3, 5, and 7 days; sensitivity to acetylcholine was not affected, but reactivity was increased after 7 days. Hyperoxia increased smooth muscle area beginning 5 days after commencing the exposure. After normalizing tension responses to smooth muscle area, reactivity of the airways to the stimuli was not different between the two groups, but sensitivity to EFS was still increased. The increase in reactivity observed after 5 and 7 days of exposure can be explained by an increase in smooth muscle area that occurred at these time points. The fact that the sensitivity of the airways to EFS remained increased after normalization, together with the fact that the increase in airway responsiveness after 3 days of exposure occurred at a time when smooth muscle area was not different from control, suggests that mechanisms other than increased smooth muscle area contribute to the development of hyperoxia-induced airway hyperresponsiveness.
Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5 beta 1, or TNF-alpha exposure
Authors: Curtis T. M. , McKeown-Longo P. J. , Vincent P. A. , Homan S. M. , Wheatley E. M. , Saba T. M.
Published: December 2017

Endothelial permeability can be altered by tumor necrosis factor-alpha (TNF-alpha), a cytokine released in association with inflammation-induced tissue injury. In the subendothelial matrix, fibronectin (Fn) influences endothelial cell adhesion by the interaction of integrins with RGD and non-RGD attachment sites in Fn. We compared the effect of TNF-alpha, RGD-containing peptides (GRGDSP), or antibody to alpha 5 beta 1-integrins on the protein permeability of bovine lung endothelial monolayers as assessed by transendothelial 125I-labeled albumin clearance. We also examined the influence of purified human plasma fibronectin (hFn) on this permeability response. TNF-alpha, RGD peptides, and antibodies to alpha 5 beta 1-integrins elicited a dose- and time-dependent increase in protein permeability as well as a reorganization and/or disruption of the endogenous Fn matrix. A control RGE peptide (GRGESP) as well as immunoglobulin G purified from nonimmune rabbit serum did not increase endothelial protein permeability or disrupt the endogenous fibrillar Fn pattern in the matrix. Likewise, a LDV peptide derived from the alternatively spliced type III connecting segment (IIICS) within bovine Fn (bFn) was unable to increase permeability of the bovine endothelial monolayer. Co-incubation of purified soluble hFn (300 or 600 micrograms/ml) with either TNF-alpha, the RGD peptide, or the antibody to alpha 5 beta 1-integrins prevented the increase in endothelial permeability. This protective effect was also observed when the purified hFn (600 micrograms/ml) was added after the TNF-alpha-induced increase in endothelial permeability had taken place. Immunofluorescent analysis confirmed the incorporation of the hFn into the subendothelial matrix and its co-localization with the endogenous bFn. The similar alteration of the subendothelial matrix after exposure to RGD peptides, anti-alpha 5 beta 1-antibodies, or TNF-alpha, coupled with the ability for hFn to attenuate the permeability increase typically elicited by all three agents, suggests that disruption of cell-matrix interactions may be the mechanism by which TNF-alpha alters endothelial permeability.
Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases
Authors: Bai T. R. , Zhou D. , Weir T. , Walker B. , Hegele R. , Hayashi S. , McKay K. , Bondy G. P. , Fong T.
Published: December 2017

The tachykinin neuropeptides substance P and neurokinin (NK) A have been postulated to participate in the inflammatory reaction in airways of smokers and asthmatics. We have examined the hypothesis that the expression of one or more of the three cloned tachykinin receptors (NK1, NK2, and NK3) is increased in inflammatory airway disorders, which could result in augmentation of the effect of released tachykinin neuropeptides. NK1 receptor and NK2 receptor but not NK3-receptor mRNA were detected by ribonuclease protection assay in RNA from both cartilaginous and membranous bronchi and subpleural lung. In lung samples containing membranous airways, NK2-receptor mRNA expression was increased fourfold in asthmatics compared with nonsmoking controls, whereas NK1-receptor mRNA levels were similar in the two groups. NK1- and NK2-receptor mRNA expression was increased twofold in smokers without airflow obstruction compared with nonsmokers, whereas NK1-receptor mRNA expression was significantly lower in patients with chronic obstructive pulmonary disease compared with smoking controls. In situ hybridization indicated NK1-receptor mRNA was expressed in submucosal glands and airway epithelial cells, whereas NK2-receptor and NK3-receptor mRNA were not detected. These observations have implications for the pathophysiology and treatment of both asthma and tobacco smoke-induced airway inflammation.
Bleomycin induces apoptosis in human alveolar macrophages
Authors: Hamilton R. F. , Li L. , Felder T. B. , Holian A.
Published: December 2017

Bleomycin (BLM) is an effective antineoplastic drug; however, cumulative dosage is often associated with inflammation that can progress to pulmonary fibrosis. The mechanisms by which this occurs are not understood, but they have been proposed to involve the alveolar macrophage (AM). In this study, we examined the in vitro effects of BLM on human AM cytotoxicity and the role of heat shock proteins (HSP or stress proteins) in this process. Although BLM did not cause marked necrosis, it caused significant DNA fragmentation detected by in situ DNA labeling and confirmed by BLM-induced DNA ladder formation after 24 h. The DNA fragmentation was significantly blocked by 10 and 50 microM ZnCl2, suggesting that BLM was inducing apoptosis. BLM did not alter intracellular protooncogene bcl-2 or glutathione levels. However, BLM significantly (50%) blocked HSP-72 expression by 4 h during a mild heat stress (39.8 degrees C). This inhibition occurs without affecting mRNA levels (in situ hybridization) for HSP-72 or overall protein synthesis ([35S]methionine incorporation), suggesting that BLM is blocking the stress response relatively specifically and post-transcriptionally. In summary, these results suggest that BLM causes apoptosis in human AM in vitro that is preceded by the inhibition of HSP-72 induction that appears to be caused by a posttranscriptional mechanism.
Leukotoxin, 9,10-epoxy-12-octadecenoate inhibits mitochondrial respiration of isolated perfused rat lung
Authors: Sakai T. , Ishizaki T. , Ohnishi T. , Sasaki F. , Ameshima S. , Nakai T. , Miyabo S. , Matsukawa S. , Hayakawa M. , Ozawa T.
Published: December 2017

To investigate how mitochondrial function was affected in leukotoxin (Lx)-,9,10-epoxy-12-octadecenoate-induced lung injury, lung mitochondria were extracted from isolated perfused rat lung with or without Lx-induced edematous injury. In the lung treated with 30 mumol of Lx, the mitochondrial respiration rate in states 3 and 4 significantly decreased (without mitochondrial uncoupling) concomitantly with increased release of lactate dehydrogenase (LDH), a parameter for cellular damage, into the perfusate and decreased ATP content in the lung tissue compared with those of untreated lung. Moreover, 30 mumol of Lx resulted in significant inhibition of cytochrome-c oxidase activity (vs. vehicle control). In contrast, lower doses of Lx (10 mumol) caused lung edema and cellular damage without evidence for mitochondrial dysfunction. We also examined cellular and mitochondrial damage in hydrostatic lung edema. Such edema showed neither suppressed mitochondrial respiration nor elevated LDH activity in perfusate, although lung wet weight increased as much as it did after 30 mumol Lx treatment. Our results suggest that the ex vivo mitochondrial dysfunction is one of the secondary (vs. initial augmented permeability) but specific manifestations of toxicity of Lx, and together with the previous reports, the ex vivo damaging effect of Lx against mitochondria may be ascribed not to its direct action on mitochondria but to Lx-derived cellular mechanism(s).
Electrophoretic analysis of contractile proteins of the diaphragm in chronically hypoxic rats
Authors: Mortola J. P. , Naso L.
Published: December 2017

We questioned whether the relative composition of the diaphragm proteins [myosin heavy chain (MHC), actin, tropomyosin (TM)-alpha and TM-beta, and MHC isoforms] was altered by chronic hypoxia. Rats were exposed to hypobaric hypoxia (inspired O2 pressure approximately 95 mmHg) from birth for 60 days or 9-11 mo. From the muscle homogenate, relative protein content was computed by densitometric analysis after protein separation with polyacrylamide gel electrophoresis and identification by immunoblotting. The relative concentrations of MHC, actin, TM-alpha and TM-beta did not differ from those of control rats for either duration of exposure. The main MHC isoforms (I, IIa, IIb, IIx) remained unaltered after 60 days but changed significantly after 9 mo of hypoxia, mostly because of a decrease in the slow (I) component and an increase of II complex. We conclude that chronic hypoxia of long duration modifies the expression of MHC isoforms in the rat diaphragm. Qualitative similar changes also occurred in a muscle of the thigh, the rectus femoris; hence, the changes in diaphragm MHC probably reflected the systemic effects of hypoxia rather than the increased mechanical load imposed by the chronic hyperventilation.
Retinoids, retinoic acid receptors, and cytoplasmic retinoid binding proteins in perinatal rat lung fibroblasts
Authors: McGowan S. E. , Harvey C. S. , Jackson S. K.
Published: December 2017

The early postnatal life of most mammals marks a period of extensive enlargement of the alveolar surface area and increase in the elastin content of the lung. The factors that regulate the onset and abatement of this burst of elastin synthesis have not been identified. Previous studies of lipid-laden rat pulmonary interstitial fibroblasts (LIF) have shown that their elastin synthesis is increased in vitro by retinoic acid (RA). We hypothesized that temporal changes in the endogenous RA content of LIF may correlate with changes in elastin synthesis by these cells. LIF were isolated from the lungs of rats at gestational day 19 and postnatal days 2, 4, 8, and 12 and their retinoid contents were quantitated. Retinyl esters were highest at gestational day 19 (2.9 +/- 0.6 pmol/10(6) cells, means +/- SE) and decreased to 1.6 +/- 0.2 pmol by postnatal day 2 (P < 0.05). This decrease in retinyl esters was accompanied by an increase in retinol from 0.4 +/- 0.1 to 2.0 +/- 0.6 pmol/10(6) cells (P < 0.05). RA increased in LIF from 0.07 +/- 0.04 pmol/10(6) cells at gestational day 19 to 0.29 +/- 0.05 pmol/10(6) cells at postnatal day 2 (P < 0.05) and increased in whole lung tissue from 0.07 +/- 0.04 to 0.29 +/- 0.05 nmol/g, over the same interval. The increase in RA content was accompanied by an increase in RA receptor (RAR)-beta and -gamma mRNAs. The steady-state mRNA level of cellular retinol binding protein (CRBP) was high in LIF, relative to whole lung tissue at day 2. Cellular RA binding protein (CRABP) mRNA rose fourfold from day 2 to day 8 and then fell by day 12. In summary, RA, RAR, and CRBP mRNA in LIF are highest before the period of maximal elastin synthesis, which occurs at postnatal days 8 and 12. These findings are consistent with the hypothesis that endogenous RA could contribute to the postnatal increase in elastin production by pulmonary fibroblasts.