bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization and activity of recombinant human CuZn superoxide dismutase after intratracheal administration
Authors: Sahgal N. , Davis J. M. , Robbins C. , Horowitz S. , Langenback E. G. , Perry R. H. , Colflesh D. , Tierney J. , Simon S. R.
Published: December 2017

Hyperoxia and mechanical ventilation cause acute lung injury which may be mitigated by prophylactic intratracheal (IT) administration of recombinant human CuZn superoxide dismutase (rhSOD). However, little is known about the localization, activity, and metabolism of rhSOD after IT administration by instillation or nebulization. Twenty-six newborn piglets were intubated, mechanically ventilated, and given either saline or fluorescently labeled rhSOD (5 mg/kg IT) by instillation or nebulization. Animals were killed 1, 6, or 12 h later. Intact rhSOD (% total fluorescence still associated with macromolecules) and total SOD activity in lung tissue were then determined. Results indicate that, after 1 and 6 h of administration, the majority of rhSOD present in the lung was still associated with the fluorescent label. By 12 h, most of the rhSOD was no longer fluorescently labeled. At 1 h, lung SOD activity increased by 100% compared with untreated control values, with activity remaining elevated at 6 and 12 h. Laser confocal microscopy of lung tissue showed that at 1 h, labeled rhSOD was found throughout the lung, inside a variety of cell types of airways, respiratory bronchioles, and alveoli. Deposition was more homogeneous after nebulization. Negative controls had minimal background fluorescence. These data indicate that after IT administration, rhSOD is rapidly incorporated into cells in the lung and significantly increases lung SOD activity. These observations have important implications for the clinical use of rhSOD in human trials.
Surfactant protein A is degraded by alveolar macrophages
Authors: Bates S. R. , Fisher A. B.
Published: December 2017

The metabolism of iodinated lung surfactant protein A (SP-A) by alveolar macrophages in primary culture was examined to determine the role these cells play in the degradation of this surfactant protein. SP-A was isolated from lung lavage obtained from normal bovines, patients with alveolar proteinosis, and silica-treated rats. SP-A (0.5 microgram/ml) was incubated for 3 h with rat alveolar macrophages obtained by lung lavage. Cell association and degradation of human and rat SP-A was three times greater than that of bovine SP-A. During the 3-h period, 50% of total macrophage-associated SP-A was degraded. Degradation was time-, temperature-, and concentration-dependent after a 1-h lag period. SP-A degradation was intracellular, since NH4Cl inhibited degradation > 50%, and macrophage-conditioned medium was ineffective. Tenfold more SP-A was degraded by macrophages than by type II cells isolated after elastase digestion of rat lungs. There was little degradation of SP-A by HeLa cells. We conclude that alveolar macrophages take up and degrade SP-A and thus could contribute to the catabolism of SP-A in the lung.
Effect of inspiratory training on mitochondrial DNA and cytochrome-c oxidase expression in the diaphragm
Authors: Akiyama Y. , Garcia R. E. , Bazzy A. R.
Published: December 2017

We have previously shown that respiratory training with inspiratory flow-resistive (IFR) loads improves diaphragm performance and is associated with an increase in cytochrome-c oxidase (COX) activity (1). The present study was conducted to define the level at which the increase in COX activity is controlled. Six sheep were trained with IFR loads for 3 h/day for 3 wk. The diaphragm was sampled from the six trained sheep and from six control sheep. Quantitative DNA and RNA slot-blot analyses with mitochondrially coded COX subunit III and nuclearly coded subunit IV probes and immunoblotting with anti-COX holoenzyme antibodies were performed. We found that in the diaphragm the amount of COX subunit proteins coded in either genetic system was greater in the trained than in the control sheep. Neither the amount of mitochondrial DNA nor mRNA for COX subunits was different between the two groups. We conclude that the increase in COX activity in the diaphragm after chronic respiratory training is determined by the amount of subunit proteins, possibly involving translation/degradation of these proteins.
Role of C fibers in the inflammatory response to intratracheal lipopolysaccharide
Authors: Long N. C. , Frevert C. W. , Shore S. A.
Published: December 2017

We proposed that C fibers play a role in mediating the inflammatory response to the intratracheal instillation of lipopolysaccharaide (LPS), a purified form of endotoxin. To test this hypothesis, we compared the inflammatory response to intratracheal LPS (0.1-2.5 mg/kg) in rats whose C fibers had been destroyed by neonatal capsaicin treatment to the response seen in animals that were treated with vehicle. Three hours after the instillation of LPS, we assessed pulmonary inflammation by performing bronchoalveolar lavage (BAL) on the animals. We measured the number of neutrophils, the concentration of protein as an index of vascular permeability, and the concentration of tumor necrosis factor (TNF). Our results indicate that capsaicin treatment resulted in more neutrophils and higher levels of protein and TNF in the BAL fluid in response to intratracheal LPS, compared with vehicle treatment. Using cells from both groups of rats, we also assessed the production of inflammatory mediators by alveolar macrophages incubated with LPS (0.3-30 ng/ml) in vitro. We found a modest increase in the concentration of TNF and nitrite in the supernatant of macrophages collected from capsaicin-treated rats, in comparison with vehicle-treated animals. These results are consistent with the hypothesis that intrinsic differences in the sensitivity of alveolar macrophages of capsaicin and vehicle-treated animals contribute to the greater inflammatory response of capsaicin-treated rat to intratracheal LPS.
SP-A-binding protein BP55 is involved in surfactant endocytosis by type II pneumocytes
Authors: Wissel H. , Looman A. C. , Fritzsche I. , Rustow B. , Stevens P. A.
Published: December 2017

The mechanism of surfactant protein (SP)-A-mediated lipid uptake by rat type II pneumocytes was investigated. In the absence of SP-A, freshly isolated type II pneumocytes actively take up very little if any liposomes. Most of the increase with time is independent of energy or temperature but is most likely due to spontaneous exchange of labeled lipids between liposomes and cell membranes. With 5 micrograms/ml SP-A, type II cells actively take up liposomes (244 pmol dipalmitoylphosphatidylcholine.h-1.10(6) cells-1). The effect of SP-A on uptake is temperature dependent and can be abolished by ATP depletion of the cells. Coincubation with an auto-anti-idiotypic antibody against the SP-A-binding protein BP55 on the cell membrane of type II pneumocytes inhibits SP-A-mediated lipid uptake by type II cells. With increasing amounts of extracellular SP-A present, increasing amounts of liposomes are taken up and directed toward a nondegrading compartment. We suggest that SP-A-mediated surfactant lipid uptake is a receptor-mediated endocytotic process involving BP55.
Effect of extracellular ATP on cytosolic Ca2+ concentration in rat pulmonary artery myocytes
Authors: Guibert C. , Pacaud P. , Loirand G. , Marthan R. , Savineau J. P.
Published: December 2017

Changes in cytosolic free Ca2+ concentration ([Ca2+]i) induced by ATP and other P2 purinoceptor agonists were investigated using indo 1 microspectrofluorimetry in single smooth muscle cells of the rat pulmonary artery. ATP (100 microM, 30 s) induced 3-4 cyclic rises in [Ca2+]i of decreasing amplitude. The first peak reached 743 +/- 24 nM from the resting value of 103 +/- 5 nM (n = 86). In approximately 50% of the cells, the ATP-induced [Ca2+]i oscillations were accompanied by a small but maintained rise in [Ca2+]i. In a series of 10 cells, the amplitude of this rise averaged 41 +/- 9 nM. The small rise 1) was also induced by 2-methylthio-ATP (2-MeS-ATP) and alpha,beta-methylene-ATP (alpha,beta-MeATP), 2) was insensitive to thapsigargin (TG, 1 microM), and 3) was abolished by the removal of external Ca2+. ATP-induced [Ca2+]i oscillations 1) were not abolished in the absence of external Ca2+, 2) were suppressed by treatment of the cells with TG (1 microM), and 3) were mimicked by UTP but not by 2-MeS-ATP or alpha,beta-MeATP. Both the number of cells that responded by [Ca2+]i oscillations and the maximal amplitude of the response depended on the agonist (ATP or UTP) concentration. The ATP-induced [Ca2+]i oscillations were not modified by tetracaine (500 microM) but were inhibited by forskolin (1 microM) and by phorbol 12,13-dibutyrate (PDB, 0.03 microM). The effect of PDB was reversed by the protein kinase C antagonist calphostin C (0.01 microM). These results suggest that the ATP-induced [Ca2+]i rise is mediated by the activation of P2x and P2u purinoceptors. Ca2+ entry through the P2x receptor channels produces a small and maintained [Ca2+]i rise. [Ca2+]i rise. Stimulation of P2u purinoceptor induces [Ca2+]i oscillations due to cyclic Ca2+ release from intracellular stores through inositol trisphosphate receptor channels.
Protective effects of BQ-123, an ETA receptor antagonist, against leukotoxin-induced injury in rat lungs
Authors: Ishizaki T. , Shigemori K. , Ameshima S. , Nakai T. , Miyabo S. , Hayakawa M. , Ozawa T. , Voelkel N. F.
Published: December 2017

We tested the hypothesis that BQ-123, a novel endothelin type A (ETA) receptor antagonist, protects the lung against leukotoxin 9,10-epoxy-12-octadecenoate (Lx), which causes acute lung injury in animals. In isolated rat lungs perfused with Earle's balanced salt solution, BQ-123 suppressed the Lx-induced increase in wet lung weight, wet lung weight/dry lung weight, the effluent perfusate lactic dehydrogenase activity, and effluent perfusate and lung tissue ET-1 levels. BQ-123 also significantly attenuated the Lx-induced increase of the pulmonary capillary filtration coefficient. Thus our experimental results indicate that the ETA receptor antagonist, BQ-123, protects against Lx-induced experimental lung vascular injury.
Capsaicin-induced release of VIP-like immunoreactivity: modulation by enzyme inhibitors
Authors: Leme A. S. , Arantes F. M. , Maldonado E. A. , Warth Mdo P. , Martins M. A.
Published: December 2017

Vasoactive intestinal peptide (VIP) is a potent bronchial smooth muscle relaxant. In the present study we measured the release of VIP-like immunoreactivity (VIP-LI) after tracheal infusion of capsaicin, histamine, and methacholine in isolated guinea pig lungs superfused through the trachea. We also studied if inhibition of VIP enzymatic cleavage using a combination of an inhibitor of neutral endopeptidase [thiorphan (Thio)] and an inhibitor of serine proteases [soybean trypsin inhibitor (STI)] influenced the airway effects of capsaicin. Infusion of capsaicin resulted in a significant increase in VIP-LI in the perfusate (12.32 +/- 4.80 to 33.52 +/- 8.46 fmol/5 min fraction; P < 0.001). There was no increase in VIP-LI after infusion of methacholine or histamine. Maximal changes in airway opening pressure (Pao) observed 0-10 min after tracheal infusion of capsaicin were significantly greater in the Thio group than the control group and the groups of lungs that received STI or STI + Thio (P < 0.005). In addition, recovery of VIP-LI in the superfusate after infusion of capsaicin was significantly greater in the group of lungs that was superfused with Thio + STI compared with STI, Thio, and control groups. Our results suggest that a bronchodilator peptide with the profile of enzymatic cleavage of VIP also modulates capsaicin effects, since the increase in Pao in the presence of Thio + STI was significantly lower than Thio alone.
Ability of adenovirus vectors containing different CFTR transcriptional cassettes to correct ion transport defects in CF cells
Authors: Jiang C. , O'Connor S. P. , Armentano D. , Berthelette P. B. , Schiavi S. C. , Jefferson D. M. , Smith A. E. , Wadsworth S. C. , Cheng S. H.
Published: December 2017

Cystic fibrosis (CF) airway epithelial cells exhibit defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated chloride (Cl) secretion, abnormal hyperabsorption of sodium (Na+), and aberrant fluid transport. Adenovirus-mediated transduction of cystic fibrosis transmembrane conductance regulator (CFTR) corrects these ion and fluid transport abnormalities in CF cells. However, several challenges remain pertaining to the use of adenovirus vectors for gene delivery, including the efficiency of gene transfer and the host response to the vector. To improve the efficacy of adenovirus-mediated gene transfer, we have constructed a series of recombinant adenoviruses containing different CFTR transcriptional units, and we have evaluated their relative ability to correct electrolyte and fluid transport in polarized CF airway epithelial cells. The ability of the vectors to correct the CF Cl- transport defects was greatest when the human cytomegalovirus promoter was used. The E1a and phosphoglycerate kinase promoters resulted in the greatest persistence of functional CFTR expression. Efficacy of gene expression by recombinant adenoviruses improved as the cells were treated with increasing multiplicities of infection, as the duration of viral contact with the target cells was lengthened, and when the virus concentration was increased. Transduction of functional CFTR Cl- channel activity reversed the abnormal Na+ hyperabsorption observed in CF cells in a dose-dependent manner, suggesting that Na+ channel activity is downregulated by CFTR. Although efficient correction of both cAMP-mediated Cl- transport and fluid secretion could be achieved readily with these vectors, normalization of the Na+ absorption required vector administration at high multiplicities of infection.
Role of the palmitoylation of surfactant-associated protein C in surfactant film formation and stability
Authors: Qanbar R. , Cheng S. , Possmayer F. , Schurch S.
Published: December 2017

The effect of palmitoylation of pulmonary surfactant-associated protein C (SP-C) on the surface activity of phospholipid mixtures of dipalmitoylphosphatidylcholine and phosphatidylglycerol was studied. Phospholipids reconstituted with palmitoylated or depalmitoylated bovine SP-C were examined at neutral and acidic pH using a captive bubble surfactometer. At low pH, effective lipid adsorption and near zero surface tensions upon compression were obtained even with protein-free samples. At physiological pH, only SP-C-containing samples achieved such properties. Lipid adsorption was decreased by prior SP-C depalmitoylation. Bubbles with palmitoylated SP-C were more mechanically stable and required less compression to reach low surface tensions. Subphase depletion experiments showed that dynamically cycled surface layers containing palmitoylated SP-C maintained their surface activity after subphase lipid depletion. In contrast, surface activity was rapidly lost where depalmitoylated SP-C or SP-B was included. Our results indicate that although SP-C palmitoylation has little effect on its ability to enhance lipid adsorption and surface tension reduction, it greatly enhances lipid respreading and film stability and is therefore important for surfactant function.
Culture and characterization of human nasal gland cells
Authors: Furukawa M. , Yamaya M. , Ikeda K. , Oshima T. , Suzuki T. , Sasaki H. , Takasaka T.
Published: December 2017

To study the regulation of ion transport by human nasal gland (HNG) cells, we developed a system for culturing HNG cells isolated by enzymatic digestion from human nasal polyps. When plated on flasks in media containing. Ultroser G serum substitute and a variety of growth factors, the HNG cells became confluent after 5-7 days. Cells were collected by trypsinization and replated at 10(6) cells/cm2 on porous filters. Confluent monolayers formed on days 3-5 after replating and were studied on days 5-7. Transepithelial resistance and short-circuit current (Isc) were 177 +/- 15 omega.cm2 and 12.2 +/- 1.3 microA/cm2 (means +/- SE, n = 35). Acetylcholine (ACh, 10(-5) M) induced a transient increase in Isc by 4.4 +/- 1.0 microA/cm2 when added to both apical and basolateral sides and returned to baseline levels within 5 min. Diphenylamine-2-carboxylate (10(-3) M) decreased baseline Isc by 30.2 +/- 1.7% (n = 6) and inhibited Isc responses induced by ACh. Amiloride also decreased baseline Isc by 12.3 +/- 3.4% (n = 6) but failed to inhibit ACh-induced Isc responses. ACh (10(-5) M) also induced an increase in intracellular Ca2+ concentration measured by fura 2-AM from 60.5 +/- 16.3 to 248.3 +/- 45.3 nM (n = 4). These findings suggest that gland cells from human nasal polyps can be grown in culture to produce epithelial cell sheets of high resistance, which secrete Cl- in response to cholinergic agent via an increase in intracellular Ca2+ concentration.
Effects of a short-chain phospholipid on ion transport pathways in rabbit nasal airway epithelium
Authors: Ropke M. , Hansen M. , Carstens S. , Christensen P. , Danielsen G. , Frederiksen O.
Published: December 2017

We investigated the mechanism of interference of mucosal application of the short-chain phospholipid didecanoyl-L-alpha-phosphatidylcholine (DDPC; 0.1-0.5%) with ion transport pathways in isolated rabbit nasal airway epithelium (RNAE). Transports of Na+ and Cl- were evaluated from tracer ion fluxes, short-circuit current (Isc), and epithelial conductance (Gt) under short-circuit conditions in Ussing chambers. DDPC rapidly and reversibly abolished net Na+ absorption, reduced control Isc (approximately 110 microA/cm2) by approximately 80%, and induced a small Cl secretion. Intracellular Ca2+ concentration ([Ca2+]i) increased dose dependently and transiently (measured by fura 2 in cultured rabbit airway epithelium), but ionomycin failed to mimic the decrease in Isc. The rise in [Ca2+]i may explain a Ba(2+)-sensitive transient activation of a basolateral K+ conductance. Indomethacin-sensitive prostaglandin E2 production in RNAE increased severalfold, but cyclooxygenase and lipoxygenase inhibitors did not prevent DDPC-induced changes in Isc. DDPC initially decreased control Gt (approximately 13 mS/cm2) by approximately 25% due to inhibition of amiloride-sensitive Na+ channels, and then reversibly increased Gt to approximately 45% above control values. Passive Na+ fluxes increased more than Cl fluxes, suggesting that the increase in Gt is due to formation of a paracellular shunt conductance in parallel with unaffected, anion-selective tight junction channels. The results suggest that DDPC inhibits apical membrane Na+ channels and causes structural changes in tight junctions after incorporation in apical cell membranes.
Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals
Authors: Stripp B. R. , Lund J. , Mango G. W. , Doyen K. C. , Johnston C. , Hultenby K. , Nord M. , Whitsett J. A.
Published: December 2017

Clara cell secretory protein (CCSP) is a product of nonciliated cells of the conducting airway epithelium. The normal physiological function of CCSP is unknown. However, the ability of CCSP to bind small lipophilic molecules, such as steroid hormones and certain pollutants, has led to speculation that this protein may mediate the biological accumulation of potentially harmful polychlorinated biphenyl (PCB) metabolites within the lung. To investigate the contribution of CCSP in the in vivo accumulation of methylsulfonyl-PCB, a line of mice was established that were homozygous for a null allele of the CCSP gene. CCSP-deficient mice were healthy and fertile, with no gross physiological or pathological abnormalities Parenteral challenge with the PCB metabolite 4-methylsulfonyl-2,2',4',5,5'-pentachlorobiphenyl (MeSO2-PCB) demonstrated that CCSP-deficient mice no longer accumulate this class of pollutants within lung and kidney tissues. These data demonstrate that CCSP is the determinant for MeSO2-PCB accumulation within mice and support the notion that bioconcentration of MeSO2-PCB pollutants occurs at sites of CCSP localization, such as the respiratory and reproductive tracts of humans.
Histamine activates phospholipase C in human airway epithelial cells via a phorbol ester-sensitive pathway
Authors: Rugolo M. , Barzanti F. , Gruenert D. C. , Hrelia S.
Published: December 2017

In human airway epithelial cell lines 9HTEo- and CFNPE9o, histamine causes a transient elevation of intracellular free calcium concentration ([Ca2+]i) detected by fura 2 fluorescence, which is due to both release from intracellular stores and extracellular Ca2+ entry. The effect of histamine is abolished by the Ca(2+)-ATPase inhibitor thapsigargin. Histamine also stimulates inositol phosphate accumulation. Changes in [Ca2+]i and inositol phosphate production exhibit a similar dose-response relationship for histamine (maximal effect at 10(-4) M), with both phenomena being blocked by the H1 antagonist mepyramine and being insensitive to pertussis toxin treatment. The effects of histamine on phosphoinositide metabolism and [Ca2+]i are abolished by a short-term preincubation with phorbol ester, and this effect is reversed by staurosporine and calphostin C, suggesting a feedback regulation by protein kinase C. The results indicate that human airway epithelial cells contain H1 receptors coupled to phospholipase C through a pertussis toxin-insensitive G protein.
Interactions of recombinant human pulmonary surfactant protein D and SP-D multimers with influenza A
Authors: Hartshorn K. , Chang D. , Rust K. , White M. , Heuser J. , Crouch E.
Published: December 2017

To further study the structure and function of surfactant protein D (SP-D), recombinant human SP-D (rhSP-D) was isolated from the culture medium of Chinese hamster ovary (CHO)-K1 cells stably transfected with a full-length hSP-D cDNA. Although a significant fraction of the secreted rhSP-D was recovered as dodecamers similar to recombinant rat SP-D (rrSP-D), a major fraction accumulated as multimers of dodecamers indistinguishable from human proteinosis SP-D. As previously shown for the rat protein, rhSP-D agglutinated specific strains of influenza A virus (IAV), inhibited viral hemagglutinin activity, and protected neutrophils (PMN) from deactivation by IAV. However, the potency of rhSP-D multimers was severalfold greater than for purified dodecamers, comparable to natural, proteinosis hSP-D. Although rhSP-D multimers were also more potent than the serum collectins in mediating viral aggregation and protection of PMN, they were less potent than conglutinin in inhibiting infectivity in vitro. These studies establish that the propensity of hSP-D to form multimers of dodecamers is determined by its primary structure and demonstrate carbohydrate recognition domain valency-dependent interactions of SP-D with IAV.
Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK
Authors: Devor D. C. , Singh A. K. , Bridges R. J. , Frizzell R. A.
Published: December 2017

We previously demonstrated that the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), stimulates a sustained Cl- secretory response across T84 monolayers by opening a Ca(2+)-dependent basolateral K+ channel. In the present work, we evaluated the effects on Cl-secretion of other benzimidazolones, NS-004 and NS-1619, which have been shown to open cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast to 1-EBIO, neither NS-004 nor NS-1619 stimulated a significant Cl- secretory current (Isc). Neither NS-004 nor NS-1619 increased Isc subsequent to forskolin stimulation. However, when added after 1-EBIO, NS-004 and NS-1619 stimulated large sustained increases in Isc. In addition, NS-004 and NS-1619 potentiated the effects of carbachol. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of NS-004 and 1-EBIO on the basolateral K+ (IK) and apical Cl- (ICl) currents. Both NS-004 and 1-EBIO increased ICl, and the stimulated currents were inhibited by glibenclamide. In contrast, NS-004 failed to significantly affect IK, but subsequent addition of 1-EBIO induced a large increase in IK. The effects of 1-EBIO, NS-004, and NS-1619 on the Ca(2+)-dependent K+ channel (KCa) in T84 cells was determined in excised inside-out patches. Neither NS-004 nor NS-1619 affected K+ channel activity, whereas the subsequent addition of 1-EBIO produced a marked channel activation. Results similar to those observed in T84 monolayers were obtained from murine airway cell primary cultures: NS-004 or NS-1619 had no effect on Isc, whereas 1-EBIO stimulated a sustained Cl- secretory response. The results demonstrate that activation of CFTR alone is insufficient to evoke transepithelial Cl- secretion. Activation of the basolateral membrane K+ channel is a necessary component of the secretory response. Thus the basolateral membrane KCa may be a novel pharmacological target in cystic fibrosis therapy.
Retinoic acid increases surfactant protein mRNA in fetal rat lung in culture
Authors: Bogue C. W. , Jacobs H. C. , Dynia D. W. , Wilson C. M. , Gross I.
Published: December 2017

Retinoic acid has both early or immediate (within hours) and late (after days) effects on gene expression. We studied the early effects of retinoic acid on the surfactant protein (SP) genes. Exposure of fetal rat lung explants to all trans-retinoic acid for 4 h resulted in a significant dose-dependent increase in SP-A, -B, and -C mRNA with markedly different dose-response characteristics. The maximal (2.5x) increase in SP-A mRNA was observed with 10(-10) M retinoic acid, whereas treatment with 10(-5) M resulted in a tendency to decreased levels. In contrast, maximal stimulation of SP-C (6x) was noted at 10(-5) M retinoic acid and that of SP-B (2x) at 10(-7) to 10(-5) M retinoic acid. Similar differences in the dose-response characteristics of SP-A and SP-C were observed with 9-cis-retinoic acid. A retinoic acid response element consensus sequence was identified in the rat SP-A gene; we hypothesize that retinoic acid-receptor complexes act directly on the SP-A gene via this response element.
Leukotriene D4 induces MMP-1, which functions as an IGFBP protease in human airway smooth muscle cells
Authors: Rajah R. , Nunn S. E. , Herrick D. J. , Grunstein M. M. , Cohen P.
Published: December 2017

We have previously demonstrated that the asthma-associated proinflammatory eicosanoid leukotriene D4 (LTD4) is comitogenic with insulin-like growth factors (IGF) in airway smooth muscle (ASM) cells. This synergistic effect of LTD4 and IGF on ASM cell growth involves proteolysis of ASM-produced inhibitory IGF-binding proteins (IGFBP). In this report, we analyzed the conditioned media (CM) from LTD4-treated human ASM cells (ASM-LTD4-CM) by Western ligand blotting and demonstrated a marked LTD4-induced reduction in the levels of the intact IGFBP (predominantly IGFBP-2) secreted by these cells. The IGFBP-2 in the ASM-LTD4-CM was identified as lower-molecular-weight fragments by Western immunoblotting. Incubation with 125I-labeled IGFBP demonstrated that an IGFBP protease was induced in the ASM cells in response to LTD4 treatment. Immunodepletion of ASM-LTD4-CM with anti-matrix metalloproteinase (MMP)-1 antibodies demonstrated a dose-dependent reduction of IGFBP proteolysis. Tissue inhibitor of MMP-1 and Batimastat (synthetic) inhibited proteolysis of IGFBP. Immunoblotting the ASM-LTD4-CM with anti-MMP-1 demonstrated a dose-dependent increase in MMP-1 protein. Similar results were also obtained by immunocytochemistry. Collectively, these observations demonstrate that MMP-1 is an IGFBP protease induced by leukotrienes that plays a significant role in modulating IGF action in ASM cells. A similar mechanism may be applicable in vivo in the airways of patients with asthma.
Basic fibroblast growth factor induces expression of the PDGF receptor-alpha on human bronchial smooth muscle cells
Authors: Bonner J. C. , Badgett A. , Lindroos P. M. , Coin P. G.
Published: December 2017

Bronchial smooth muscle cell (SMC) hyperplasia is a key feature in the pathology of asthma. Platelet-derived growth factor (PDGF) isoforms are SMC mitogens. We investigated the effect of basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-beta 1), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) on the PDGF receptor system on human bronchial SMC from three different donors. bFGF induced gene expression of the PDGF alpha-receptor (PDGF-R alpha) approximately threefold without altering the PDGF beta-receptor (PDGF-R beta). IL-1 beta and TNF-alpha did not affect the PDGF receptor system. TGF-beta 1 downregulated PDGF-R alpha mRNA approximately 60% without changing PDGF-R beta mRNA levels. Receptor assays showed that bFGF increased the [125I]PDGF-AA binding site approximately twofold, whereas TGF-beta 1 reduced [125I]PDGF-AA binding approximately 60%. TGF-beta 1, but not latent TGF-beta 1, counteracted the bFGF-induced increase in [125I]PDGF-AA binding. PDGF-AA-stimulated tyrosine phosphorylation on the PDGF-R alpha was enhanced after treatment with bFGF, bFGF pretreatment enhanced the mitogenic response of SMC to PDGF-AA and PDGF-AB. These findings suggest that upregulation of the PDGF-R alpha by bFGF could contribute to SMC hyperplasia during chronic airway inflammation in asthma.
Effect of okadaic acid on elastin gene expression in interstitial lung fibroblasts
Authors: Berk J. L. , Massoomi N. , Krupsky M. , Goldstein R. H.
Published: December 2017

Okadaic acid (OA), a specific serine/threonine protein phosphatase inhibitor, downregulated tropoelastin formation and elastin mRNA levels in a dose-related and cycloheximide-sensitive fashion in cultured lung fibroblasts. Treatment with a tyrosine phosphatase inhibitor at high concentrations did not alter elastin mRNA levels, however. Nuclear run-on analysis indicated that OA primarily suppressed elastin gene expression through a transcriptional mechanism. In contrast to its effects on elastin expression, OA downregulated alpha 1(I) mRNA to significantly lesser degrees. The mechanism by which OA decreased elastin mRNA levels did not appear to involve protein kinase C or share the signaling pathway of IL-1 beta. Prolonged treatment with phorbol ester promoted the inhibitory effects of OA on elastin, as did shorter treatment with IL-1 beta. Moreover, transient transfection studies indicated that OA and IL-1 beta do not act through the same cis-acting element in the elastin promoter. Finally, unlike the transient effects of IL-1 beta, OA induced persistent inhibition of elastin expression by a transcriptional mechanism. Taken together, these data indicate that serine/threonine protein phosphorylation can regulate the amount and composition of extracellular matrix secreted by fibroblasts into the interstitium of the lung.
Effects of hypoxia on expression of superoxide dismutases in cultured ATII cells and lung fibroblasts
Authors: Jackson R. M. , Parish G. , Ho Y. S.
Published: December 2017

This study investigated whether hypoxia affected the expression of mitochondrial manganese-containing superoxide dismutase (Mn-SOD) and the cytosolic copper and zinc-containing superoxide dismutase (Cu,Zn-SOD) in alveolar type II epithelial (ATII) cells and lung fibroblasts. Cells were exposed in vitro to air (controls) or to 2.5% oxygen (hypoxia) for 24 h. Mn-SOD and Cu,Zn-SOD mRNA expression was measured by quantitative reverse transcriptase-polymerase chain reaction. Both Mn-SOD and Cu,Zn-SOD mRNA expression in ATII cells decreased significantly after 1 day in hypoxic conditions. The decrease in Mn-SOD mRNA (-69%) was greater than that in Cu,Zn-SOD mRNA (-48%). ATII cell surfactant protein A transcript expression remained constant. Mn-SOD (-52%) and Cu,Zn-SOD (-54%) mRNA expression decreased similarly in lung fibroblasts cultured during hypoxia. The half-life of the Mn-SOD mRNA measured in lung fibroblasts exposed to air or hypoxia for 24 h decreased significantly from 5.8 +/- 0.1 to 3.8 +/- 0.7 h (-34%). The half-life for the Cu,Zn-SOD decreased significantly from 4.0 +/- 0.3 to 2.4 +/- 0.1 h (-40%). Neither Mn-SOD nor Cu,Zn-SOD protein expression in ATII cells changed significantly during hypoxia. Hypoxia decreases expression of Mn-SOD and Cu,Zn-SOD mRNA in ATII cells and lung fibroblasts in part by decreasing stability of the mRNA transcripts.
Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication
Authors: Fiedler M. A. , Wernke-Dollries K. , Stark J. M.
Published: December 2017

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.
Effect of lowering serum cholesterol on the composition of surfactant in adult rat lung
Authors: Davidson K. G. , Acton S. M. , Barr H. A. , Nicholas T. E.
Published: December 2017

Treatment of rats with 10 mg.kg body wt-1 day-1 4-aminopyrazolo[3,4-d]pyrimidine (APP) for 2 days markedly reduced serum cholesterol and phospholipids. This was associated with large decreases in the principal component of alveolar surfactant, the disaturated phospholipids (DSP), in the lamellar body and in the tubular myelin-rich and -poor alveolar fractions, but with no concomitant change in cholesterol or surfactant protein A. These decreases in DSP were associated with a decrease in the synthesis of surfactant phospholipids. Despite these large changes in composition of alveolar surfactant, we could detect no change in either static or dynamic lung compliance. However, the treatment markedly increased both the minimum and maximum surface tension of the lipid extract of the tubular myelin-rich fraction, as measured by bubble surfactometry. Whereas these changes appeared unimportant in the isolated perfused lung at resting tidal volume, they were associated with edema after an increase in tidal volume. The ability of APP to inhibit phospholipid synthesis selectively makes it a useful tool in investigating the surfactant system.
Phosphorylation of calponin in airway smooth muscle
Authors: Pohl J. , Winder S. J. , Allen B. G. , Walsh M. P. , Sellers J. R. , Gerthoffer W. T.
Published: December 2017

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.
Endothelial gaps: time course of formation and closure in inflamed venules of rats
Authors: Baluk P. , Hirata A. , Thurston G. , Fujiwara T. , Neal C. R. , Michel C. C. , McDonald D. M.
Published: December 2017

In the rat trachea, substance P causes rapid but transient plasma leakage. We sought to determine how closely the number, morphology, and size of endothelial gaps correspond to the time course of this leakage. Endothelial gaps were examined by scanning electron microscopy (EM), by transmission EM, or by light microscopy after silver nitrate staining. Substance P-induced leakage of the particulate tracer Monastral blue peaked at 1 min but decreased with a half-life of 0.3 min. The number of silver-stained gaps also peaked at 1 min then decreased significantly more slowly (half-life 1.9 min) than the leakage. Scanning EM revealed two types of endothelial gaps, designated vertical gaps and oblique slits. Vertical gaps predominated at peak leakage, whereas oblique slits became more common as the leakage diminished. Measurements of the mean diameter of vertical gaps made by light microscopy, scanning EM, and transmission EM were all in the range of 0.36-0.47 micron. Fingerlike endothelial cell processes that appeared during gap formation became shorter as the leakage diminished (mean length: 1.44 microns at 1 min compared with 1.06 microns at 3 min after substance P), suggesting a role in gap closure. We conclude that the plasma leakage occurring immediately after an inflammatory stimulus results from the rapid formation of endothelial gaps. Multiple factors, including alterations in gap morphology, gap closure, and changes in driving force, are likely to participate in the rapid decrease in the leakage.
Strain increases airway smooth muscle contractile and cytoskeletal proteins in vitro
Authors: Smith P. G. , Moreno R. , Ikebe M.
Published: December 2017

Mechanical stress contributes to lung development and the progression of some lung diseases, although its effects on individual lung cells are unknown. Because increased airway smooth muscle (ASM) is found in lung diseases where abnormal stress is present, we determined if strain (change in resting length) causes ASM hypertrophy independently of other in vivo influences. Cultured canine ASM cells were subjected to two levels of cyclic deformational strain for 14 days and compared with nonstrained cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Cells subjected to 16-30% strain demonstrated increases in total cellular protein, myosin, myosin light chain kinase (MLCK), and desmin, whereas the cellular contents of actin, vimentin, and tubulin were similar. Changes in myosin appeared mostly due to the smooth muscle isoform, whereas nonmuscle myosin was unchanged. The increases in myosin and MLCK were disproportionate to increases in total protein, suggesting selective changes in contractile proteins. These relative increases in content of proteins were not as pronounced with 0-16% strain, suggesting a graded response. These data suggest that strain per se can increase the contractile proteins of ASM cells independently of other in vivo factors and modulates cultured cell phenotype to a more differentiated state, since it increases smooth muscle-specific proteins, such as smooth muscle myosin isoforms and desmin.
Cytosolic Ca2+ and adenylyl cyclase responses in phenotypically distinct pulmonary endothelial cells
Authors: Stevens T. , Fouty B. , Hepler L. , Richardson D. , Brough G. , McMurtry I. F. , Rodman D. M.
Published: December 2017

Pulmonary microvascular endothelium forms a tighter barrier than does pulmonary artery endothelium; the mechanism of this important phenotypic difference is uncertain. We examined two regulators of endothelial permeability, cytosolic Ca2+ concentration ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP), in microvascular (PMVEC) and pulmonary conduit artery (PAEC) endothelium. Both resting and stimulated [Ca2+]i were lower in PMVEC compared with PAEC (resting [Ca2+]i, 94 +/- 7 vs. 123 +/- 8 nM; ATP-stimulated peak, 1.04 +/- 0.14 vs. 1.98 +/- 0.13 microM). Sustained Ca2+ transients in response to either ATP or thapsigargin were reduced in PMVEC compared with PAEC (ATP, 199 +/- 22 vs. 411 +/- 43 nM; thapsigargin, 195 +/- 13 vs. 527 +/- 65 nM), suggesting reduced Ca2+ influx in PMVEC. Reduced Ca2+ influx in PMVEC was confirmed by Mn2+ quenching and patch-clamp experiments. mRNA for Ca(2+)-inhibitable and protein kinase C-stimulated adenylyl cyclases was detected in both cell types. Whereas ATP caused a [Ca2+]i-mediated decrease in cAMP in PAEC, ATP caused a protein kinase C-mediated increase in cAMP in PMVEC. We conclude that PMVEC express a unique phenotype that favors enhanced barrier function through attenuated Ca2+ influx and preservation of cAMP content.
Site of methacholine reactivity in the peripheral airways: analysis using lung explants
Authors: Wright J. L. , Sun J. P. , Churg A.
Published: December 2017

We used a recently developed lung explant technique to investigate the partitioning of airway contractility in the bronchioles of normoresponsive and hyperresponsive rats. Specifically, we addressed the questions 1) whether airway response to methacholine varied with airway size and 2) whether airways from rats known to be innately hyperresponsive to methacholine (Fisher) would have responses different from normoresponsive rats (Sprague-Dawley). We found that, in both strains of rats, contraction to methacholine occurred primarily in the medium- and larger-sized bronchioles (airways of diameter > 0.32 mm) and that, at the higher methacholine concentrations, the Fisher rats had greater degrees of contraction than did the Sprague-Dawley rats. These results suggest that the increased airway responsiveness seen in Fisher rats is due to an intrinsic increase in responsiveness (increased contractility) of their airways, which may be related to amount of smooth muscle, rather than an increase in airway sensitivity to methacholine. They do not, however, completely rule out the possibility of in vivo species-dependent differences in airway-parenchymal interactions.
Surfactant phospholipid secretion from rat alveolar type II cells: possible role of PKC isozymes
Authors: Linke M. J. , Burton F. M. , Fiedeldey D. T. , Rice W. R.
Published: December 2017

Protein kinase C (PKC) plays an integral role in control of many type II cell functions, including regulation of surfactant phospholipid secretion. To determine which isozymes of PKC may regulate type II cell functions, we identified those PKC isozymes activated in type II cells in association with surfactant phospholipid secretion after phorbol ester treatment. Transcripts encoding PKC-alpha, -beta, -delta, -epsilon, -eta, and -zeta were detected in type II cells by reverse transcriptase-polymerase chain reaction, whereas PKC-alpha, -beta, -delta, -eta, and -zeta were detected in type II cells by immunoblotting. PKC-alpha and -beta were only present in the cytosol in unstimulated type II cells, whereas PKC isozymes delta, eta, and zeta were found in cytosol and membrane fractions in unstimulated type II cells. 12-O-tetradecanoylphorbol-13-acetate stimulated surfactant secretion and activated PKC-alpha, -beta, -delta, and -eta isozymes in a dose-dependent manner. The inactive analogue 4alpha-phorbol 12,13-didecanoate neither activated PKC isozymes nor stimulated surfactant phospholipid secretion. PKC-zeta was not activated by any of the phorbol esters. PKC isozymes alpha, beta, delta, and eta are present in purified type II epithelial cells and are activated in a dose-dependent manner in alveolar type II cells in association with surfactant phospholipid secretion after phorbol ester treatment.
Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1
Authors: Chin J. E. , Hatfield C. A. , Winterrowd G. E. , Brashler J. R. , Vonderfecht S. L. , Fidler S. F. , Griffin R. L. , Kolbasa K. P. , Krzesicki R. F. , Sly L. M. , Staite N. D. , Richards I. M.
Published: December 2017

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.
Human airway epithelial cells express interleukin-2 in vitro
Authors: Aoki Y. , Qiu D. , Uyei A. , Kao P. N.
Published: December 2017

Human airway epithelial cells (AEC) produce the T cell growth factor interleukin (IL)-2 that likely modulates the T cell lung inflammatory response. IL-2 mRNA from cultured AEC and from Jurkat T cells was analyzed by reverse transcription-polymerase chain reaction and Northern hybridization. IL-2 mRNA is present constitutively in AEC and is enhanced twofold after stimulation with phorbol 12-myristate 13-acetate (PMA; 20 ng/ml) + histamine (2 mM). Normal human AEC secrete IL-2 at rest (7 pg/ml), and IL-2 secretion is increased threefold after stimulation with PMA + histamine; this increase is inhibited by dexamethasone and diphenhydramine. Transcriptional regulation of IL-2 was investigated with a transgenic human AEC line, 16HBE/IL-2 luciferase; there is constitutive IL-2 transcription at rest, and IL-2 transcription is enhanced 8-fold by PMA and 25-fold by PMA + histamine. IL-2 regulation differs fundamentally between AEC and Jurkat T cells. AEC IL-2 likely promotes local proliferation of T cells and may contribute to pathological airway inflammation in asthma.