bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of vascular endothelial barrier function
Authors: Lum H. , Malik A. B.
Published: December 2017

The increase in endothelial permeability in response to inflammatory mediators such as alpha-thrombin and histamine is accompanied by cell rounding and interendothelial gap formation, implicating that the predominant transport pathway is a diffusive one [i.e., via cellular junctions (paracellular transport)]. However, the possible contribution by vesicle-mediated transport (i.e., via albumin binding protein gp60) to the overall permeability increase needs investigation. Regulation of paracellular transport in endothelial cells is associated with modulation of actin-based systems which anchor the cell to its neighbor or extracellular matrix, thus maintaining endothelial integrity. At the cell-cell junctions, actin is linked indirectly to the plasma membrane by linking proteins (e.g., vinculin, catenins, alpha-actinin) to cadherins, which function in homophilic intercellular adhesion. Cadherins may also play a role in regulating the formation of tight junctions, which also may be associated with actin. At endothelial focal contacts, the transmembrane receptors (integrins) for matrix proteins are linked to actin via linking proteins (i.e., vinculin, talin, alpha-actinin). In response to inflammatory mediators, second messengers signal two regulatory pathways which modulate the actin-based systems, which may lead to impairment of the endothelial barrier integrity. One pathway is based on protein kinase C (PKC) isozyme-specific phosphorylation of linking proteins at the cell-cell and cell-matrix junctions. The increased phosphorylation is associated with actin reorganization, cell rounding, and increased paracellular transport. The other is the activation of myosin light-chain kinase, (MLCK), which causes an actin-myosin-based contraction that may lead to a centripetal retraction of endothelial cells. Current research is in the identification of protein substrates of PKC isozymes, the specific role of their phosphorylation in barrier function, and determining the precise role of MLCK in modulation of endothelial barrier function.
Cadmium selectively inhibits fibroblast procollagen production and proliferation
Authors: Chambers R. C. , McAnulty R. J. , Shock A. , Campa J. S. , Newman Taylor A. J. , Laurent G. J.
Published: December 2017

Chronic inhalation of cadmium fumes has been associated with the development of emphysema, a disease characterized by extensive disruption of lung connective tissue. Cadmium is also an important contaminant of tobacco and may play a role in cigarette smoking-related emphysema. In this paper we investigated the effect of nontoxic doses of cadmium chloride (CdCl2) on fibroblast procollagen production and proliferation, key features of connective tissue repair following injury. CdCl2 inhibited fibroblast procollagen production in a dose-dependent manner in two different cell lines. For fetal rat fibroblasts, maximal effects were observed at 10 microM CdCl2, with values reduced by 82 +/- 6% (mean +/- SE, n = 6, P < 0.01) relative to control cells. In contrast, noncollagen protein synthesis by these cells was enhanced in the presence of CdCl2. In human fetal lung fibroblasts (HFL1), maximal inhibition of procollagen production (83 +/- 2%, P < 0.01) was observed at 40 microM CdCl2, whereas noncollagen protein synthesis was unaffected. In both cell lines the inhibition of procollagen production was shown to be due to decreased procollagen synthesis and an increase in the proportion of newly synthesized procollagen degraded. Cadmium also affected fibroblast proliferation in response to 2% serum, with values for fetal rat cells depressed by 17 +/- 4, 72 +/- 2, and 86 +/- 4% (all P < 0.01) compared with controls at 1, 5, and 10 microM CdCl2, respectively. These data show that cadmium selectively inhibits fibroblast procollagen production and also attenuates their mitogenic response to serum.(ABSTRACT TRUNCATED AT 250 WORDS)
Immortalization of subpopulations of respiratory epithelial cells from transgenic mice bearing SV40 large T antigen
Authors: Ikeda K. , Clark J. C. , Bachurski C. J. , Wikenheiser K. A. , Cuppoletti J. , Mohanti S. , Morris R. E. , Whitsett J. A.
Published: December 2017

Murine lung epithelial (MLE) cell lines were produced from lung tumors derived from transgenic mice bearing the viral oncogene, SV40 large T antigen, under transcriptional control of the promoter-enhancer region of the human surfactant protein C (SP-C) gene. Cells were selected on the basis of increased murine cystic fibrosis transmembrane conductance regulator (mCFTR) mRNA content and were dilution cloned to produce distinct immortalized epithelial cell lines. MLE-13a3 cell lines expressing high levels of mCFTR mRNA also expressed apolipoprotein J (apoJ) mRNA, a developmentally regulated glycoprotein expressed preferentially in fetal lung. SP-A, -B, and -C were not detected or were present at low levels in the MLE cells that contained abundant CFTR and apoJ mRNA. In contrast, MLE cells, cloned on the basis of abundant surfactant protein mRNAs, expressed apoJ and mCFTR mRNAs at low levels. Forskolin-stimulated short-circuit current, typical of CFTR-mediated chloride transport activity, was generated by monolayers of subclones of the MLE-13a3 cell lines. Tumor necrosis factor-alpha stimulated mCFTR mRNA, whereas dexamethasone, retinoic acid, and phorbol ester had no effect on the levels of mCFTR mRNA in MLE-13a3 cells.
Isolation and maintenance of human pulmonary artery endothelial cells in culture isolated from transplant donors
Authors: Visner G. A. , Staples E. D. , Chesrown S. E. , Block E. R. , Zander D. S. , Nick H. S.
Published: December 2017

Even though endothelial cells from different locations have similarities, there are potential morphological and functional differences between cells from different vascular regions, as well as between species. Our laboratory is interested in studying the molecular regulation of vasoactive substances in pulmonary vasculature. Therefore, we have developed reproducible methodology to isolate and maintain cultures of human pulmonary artery endothelial cells. The major innovation has been the employment of sections of pulmonary artery from heart transplant donors, from which endothelial cells are isolated. Cell monolayers were identified as endothelial cells by phase-contrast microscopy. Representative dishes of cells were further characterized by indirect immunofluorescent staining for factor VIII antigen, uptake of acetylated low-density lipoprotein, and electron microscopy. These cells were also evaluated for the expression of endothelin-1 (ET-1), a vasoactive 21-amino acid peptide derived from endothelial cells. The cells expressed ET-1 peptide and mRNA as determined by radioimmunoassay and Northern analysis, respectively. We also demonstrated that these cells are useful in transient transfection experiments for potential evaluation of promoter elements. The availability and relevance of these cells provide an important investigative tool for studies on human pulmonary vascular disease.
Regulation of type II alveolar epithelial cell proliferation by TGF-beta during bleomycin-induced lung injury in rats
Authors: Khalil N. , O'Connor R. N. , Flanders K. C. , Shing W. , Whitman C. I.
Published: December 2017

Three isoforms of transforming growth factor-beta (TGF-beta) are found in mammalian cells and are potent regulators of inflammation, connective tissue synthesis, cellular proliferation, and differentiation. To determine the distribution and regulation of TGF-beta isoforms during pulmonary injury, a rat model of bleomycin-induced lung inflammation and repair was used. Using immunohistochemistry, we demonstrate that TGF-beta 2 and TGF-beta 3 were localized to alveolar macrophages as well as epithelial and smooth muscle cells of both normal rat lungs and rat lungs obtained at all time intervals after bleomycin administration. Early in bleomycin-induced lung injury, when there is active proliferation of type II alveolar epithelial cells, there was an increase in the number of type II alveolar epithelial cells isolated per lung and an increase in DNA synthesis by explanted type II alveolar epithelial cells. At this time, the secretion of biologically active TGF-beta 1–3, which are potent inhibitors of epithelial cell proliferation, was decreased. However, the secretion of TGF-beta 1–3 activity was markedly increased later in the injury response and coincided with a reduction in the number of type II alveolar epithelial cells isolated per lung and DNA synthesis in vitro. Furthermore, the addition of TGF-beta 1, 2, and 3 to cultures of actively proliferating type II alveolar epithelial cells resulted in inhibition of [3H]thymidine incorporation, whereas, in the presence of anti-TGF-beta 1-3 antibody, there was an increase in [3H]thymidine incorporation. Our findings suggest that altered secretion of TGF-beta 1-3 activity by type II alveolar epithelial cells during bleomycin-induced lung injury may regulate pulmonary alveolar epithelial cell proliferation during injury and repair phases.
Endotoxin responsiveness and grain dust-induced inflammation in the lower respiratory tract
Authors: Schwartz D. A. , Thorne P. S. , Jagielo P. J. , White G. E. , Bleuer S. A. , Frees K. L.
Published: December 2017

To identify the role of endotoxin responsiveness in grain dust-induced airway disease, we used two models of extotoxin hyporesponsiveness to perform inhalation exposure studies in mice. In the first model, we investigated whether genetic resistance to endotoxin would alter the inflammatory response to inhaled grain dust by comparing the inflammatory response in the lower respiratory tract of endotoxin-sensitive and -resistant male mice after inhalation of pyrogen-free saline, corn dust extract (CDE), sterile CDE (SCDE), or lipopolysaccharide (LPS). Endotoxin-sensitive and -resistant mice were exposed for 4 h to nebulized solutions of LPS, SCDE, or CDE. Another group of endotoxin-sensitive and -resistant mice was sham exposed for 4 h to nebulized sterile saline. Dose-response relationships for endotoxin were explored for LPS, SCDE, and CDE. Bronchoalveolar lavage (BAL) 5 h after the start of exposure demonstrated a higher concentration of total cells, neutrophils (PMNs), and tumor necrosis factor-alpha (TNF-alpha) in BAL fluid after inhalation of CDE, SCDE, or LPS in endotoxin-sensitive than in endotoxin-resistant mice. Whereas endotoxin-sensitive mice demonstrated a dose-response relationship between the endotoxin concentration in each of the solutions and the concentration of cells, PMNs, and TNF-alpha in BAL fluid, concentrations of TNF-alpha were significantly higher only in BAL fluid of endotoxin-resistant mice exposed to higher concentrations of SCDE or CDE. In the second model, we investigated whether acquired endotoxin tolerance would alter the inflammatory response to SCDE.(ABSTRACT TRUNCATED AT 250 WORDS)
Protein kinase C in ATP regulation of lung surfactant secretion in type II cells
Authors: Chander A. , Sen N. , Wu A. M. , Spitzer A. R.
Published: December 2017

Previous studies provided indirect evidence for a role for protein kinase C (PKC) in ATP stimulation of surfactant secretion. The present study demonstrates that ATP increases PKC activity in the membrane fraction and decreases PKC activity in the cytosol fraction of alveolar type II cells, indicating translocation of PKC to the membranes. The kinetics of ATP concentration dependence of increases in phosphatidylcholine secretion and diacylglycerol content were similar, suggesting direct correlation between these two parameters. ATP also increased membrane PKC activity in a concentration-dependent manner. Almost one-half of the PKC activity in the cytosol and membrane fractions was Ca2+ independent. The ATP-induced increase was greater in membrane-associated Ca(2+)-dependent enzyme (233%) than in Ca(2+)-independent enzyme (121%). Desensitization of PKC by exposure of cells to phorbol esters decreased PKC activity in the membrane and cytosol fractions. In cells pretreated for 3 h with phorbol esters, PKC activity was near minimum, and ATP-stimulated secretion was lowest (; 40% of that observed in untreated cells). These results indicate that a major part of ATP-stimulated surfactant secretion in type II cells is mediated via activation of PKC.
Effects of neuropeptides on human lung fibroblast proliferation and chemotaxis
Authors: Harrison N. K. , Dawes K. E. , Kwon O. J. , Barnes P. J. , Laurent G. J. , Chung K. F.
Published: December 2017

An increase in subepithelial mesenchymal cells and associated connective tissue is a feature of bronchial asthma. We determined whether neuropeptides could modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. Human lung fibroblasts were cultured with neurokinin A (NKA), substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin-gene-related peptide (CGRP). After 48 h, fibroblast proliferation was measured by a colorimetric assay based on the uptake and subsequent release of methylene blue. The chemotactic response to neuropeptides was determined with the use of a modified Boyden chamber. Both NKA and SP (10(-7)-10(-4) M) stimulated human lung fibroblast proliferation in HFL1 and IMR-90 fibroblasts. VIP and CGRP had no effect on fibroblast proliferation. NKA alone stimulated fibroblast chemotaxis maximally at 10(-10) M. Neutral endopeptidase (NEP) activity of 0.52 and 5.2 pmol/10(6) cells was assayed in IMR-90 and Hs68 fibroblasts, respectively. Phosphoramidon (5 x 10(-6)-10(-5) M), an NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, and neuropeptide release may contribute to the structural abnormalities observed in asthmatic airways.
Turnover of extracellular matrix by type II pulmonary epithelial cells
Authors: Dunsmore S. E. , Rannels D. E.
Published: December 2017

Rat type II pulmonary epithelial cells synthesize and assemble a multicomponent extracellular matrix (ECM) which can modulate cellular differentiation in primary culture. This study defines turnover of the type II cell matrix. Turnover kinetics were analyzed in two types of pulse-chase protocols based on loss of radioactive ECM components. To estimate turnover of previously synthesized ECM, type II cells were plated on extracted matrix that was radiolabeled 2, 3, or 6 days; alternatively, ECM was radiolabeled in pulse-chase experiments to measure turnover by the same cells that synthesized the matrix. Rapid initial rates of ECM turnover were evident in both cases. While overall matrix stability appeared to change with culture time, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a similar spectrum of proteins in the ECM over the course of kinetic studies. The results reveal rapid turnover of ECM by type II cells and suggest that matrix stability may be regulated. These observations provide a basis for future investigations of the physiological significance of turnover of individual ECM components by the alveolar epithelium.
Catecholamine affects acetylcholine release in trachea: alpha 2-mediated inhibition and beta 2-mediated augmentation
Authors: Zhang X. Y. , Robinson N. E. , Wang Z. W. , Lu M. C.
Published: December 2017

We investigated the effects of catecholamines on acetylcholine (ACh) release from equine airway parasympathetic nerves. Trachealis strips were suspended in 2-ml tissue baths with Krebs-Henseleit solution containing atropine (10(-7) M), neostigmine (10(-6) M), and guanethidine (10(-5) M). Electrical field stimulation (20 V, 0.5 ms, 0.5 Hz, for 15 min) was applied, and ACh was measured by high-performance liquid chromatography with electrochemical detection. Epinephrine (Epi) and norepinephrine (NE) inhibited ACh release in a concentration-dependent manner. Inhibition was attenuated by the alpha 2-adrenoceptor antagonist idazoxan (10(-6) M) but not by the alpha 1-antagonist prazosin (10(-6) M). After alpha 2-blockade with idazoxan (10(-5) to 10(-4) M), Epi but not NE augmented ACh release. Isoproterenol (10(-7) to 10(-5) M) increased ACh release, an effect that was reversed by the beta 2-adrenoceptor antagonist ICI-118,551 (10(-5) M) but not by the beta 1-adrenoceptor antagonist atenolol (10(-5) M). Our results indicate that horse airway cholinergic nerves are modulated by both alpha 2-inhibitory and beta 2-excitatory adrenoceptors, with the former being predominant.
Oxygen metabolites modulate sodium transport in gerbil middle ear epithelium: involvement of PGE2
Authors: Herman P. , Tu T. Y. , Loiseau A. , Clerici C. , Cassingena R. , Grodet A. , Friedlander G. , Amiel C. , Tran Ba Huy P.
Published: December 2017

The middle ear epithelium and respiratory epithelia share basic properties such as homeostasis of air-filled cavities and mucociliary clearance toward the pharynx. With the middle ear SV40-transformed (MESV) cell line, we used the short-circuit current (Isc) technique to investigate changes in ion transport induced by oxidants. Xanthine and xanthine oxidase on the basal side of the monolayers dramatically increased Isc up to 50%. This effect was not affected by superoxide dismutase or mannitol, but could be blunted by catalase or 1,3-dimethyl-2-thiourea. Increasing concentrations of H2O2 from 10(-5) to 5 x 10(-4) M produced a dose-dependent increase in Isc from 0.26 +/- 0.16 up to 4.21 +/- 0.43 microA/cm2 (P < 0.05, n = 5). Concentration of half-maximal stimulation (EC50) was 4.68 x 10(-5) M. This effect was inhibited by indomethacin and was related to a sodium transport, since the H2O2-induced increase in Isc could be prevented or abolished by 1) apical addition of benzamil (10(-6)M) and 2) substitution of sodium with N-methyl-glucamine. H2O2 exposure also induced indomethacin-sensitive increase in released prostaglandin (PG) E2 (EC50 = 5.62 x 10(-5) M) and in cAMP content (EC50 = 3.95 x 10(-5) M) with similar kinetics. These results suggest that exposure of MESV cells to oxidants stimulates the production of PGE2, which in turn increases the transepithelial sodium transport rate.
Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles
Authors: Jiang H. , Rao K. , Liu X. , Liu G. , Stephens N. L.
Published: December 2017

The increased shortening velocity and capacity of airway smooth muscle (ASM) from ragweed pollen-sensitized dogs, which may be responsible for its in vivo airway hyperresponsiveness, have been shown to be associated with higher actomyosin adenosinetriphosphatase activity and greater level of phosphorylation of the 20-kDa myosin light-chain (MLC20) at rest and during contraction. Current studies show that the elevated level of phosphorylation may be the result of an increased myosin light-chain kinase (MLCK) activity due to excessive quantity of MLCK. There were no significant changes in total activity of calmodulin, a protein that binds and activates MLCK, in sensitized dog ASM (SASM) compared with control ASM (CASM). When normalized to the relative calmodulin content in the tissues, the specific calmodulin activities (means +/- SE) in sensitized tracheal smooth muscle (STSM) and sensitized bronchial smooth muscle (SBSM) and in their controls were not different (STSM 0.359 +/- 0.117, CTSM 0.339 +/- 0.136. SBSM 0.201 +/- 0.098, and control bronchial smooth muscle 0.213 +/- 0.056 nmol Pi.calmodulin content-1.min-1, respectively). Intracellular Ca2+ levels indicated by fura 2 fluorescent dye remained unaltered in SASM. We conclude that airway hyperresponsiveness may result from higher MLCK content in SASM rather than from changes in Ca(2+)-calmodulin activities, which is an example of alteration in Ca2- sensitivity of ASM.
In vivo effects of endothelin A- and B-receptor antagonists in guinea pigs
Authors: Nagase T. , Fukuchi Y. , Matsui H. , Aoki T. , Matsuse T. , Orimo H.
Published: December 2017

Endothelin (ET)-1, a novel 21-amino acid constrictor peptide, has been recently reported to have a potential pathophysiological role in asthma. We hypothesized that ET-1 might affect guinea pig lung via different ET receptor subtypes, i.e., ETA and ETB, in vivo. To test this hypothesis, we investigated the effects of ET-1 on airways in anesthetized, open-chest, mechanically ventilated [frequency (f) = 1 Hz; tidal volume (VT) = 9 ml/kg; positive end-expiratory pressure (PEEP) = 4 cmH2O] guinea pigs in the absence or the presence of ETA and ETB selective antagonists, i.e., BQ-123 and BQ-788, respectively. We affixed alveolar capsules to the lungs to measure alveolar pressure and calculated the elastance of lung (EL) and the resistance of lung (RL), tissue (Rti), and airway (R(aw)) under control conditions and after intravenous administration of ET-1 (10(-8) mol/kg). ET-1 induced a concentration-dependent increase in RL, Rti, R(aw), and EL.BQ-123 (2 mg/kg) partially blocks delta RL and delta R(aw) during ET-1 induced constriction, while delta Rti and delta EL were not significantly affected. BQ-788 (2 mg/kg) significantly inhibited delta RL, delta Rti, delta R(aw), and delta EL during ET-1-induced constriction. The combination of BQ-123 and BQ-788 completely ablated the response to ET-1. These data suggest that both ET receptor subtypes, i.e., ETA and ETB, may have physiological roles in guinea pig airways in response to ET-1, a potential mediator of asthma.
Tannin inhibits adenylate cyclase in airway epithelial cells
Authors: Cloutier M. M. , Guernsey L.
Published: December 2017

Tannin, isolated from cotton bracts extract and implicated in the pathogenesis of byssinosis, inhibits adenosine 3',5'-cyclic monophosphate (cAMP) production and Cl- secretion in bovine airway epithelial cells in part by inhibiting adrenergic receptor binding. The purpose of this study was to determine whether tannin affected other parts of the adrenergic-cAMP signal transduction pathway by examining the effect of tannin on guanosine 5'-triphosphate (GTP)-regulatory pathways (G proteins) and on adenylate cyclase activity. cAMP production in confluent airway epithelial cells was measured in the presence of cholera toxin (100 micrograms/ml), an activator of GS proteins, and forskolin (0.1-1,000 microM), a direct activator of adenylate cyclase. Cholera toxin stimulated cAMP production; this response, however, was inhibited in cells pretreated with 50 micrograms/ml tannin. Forskolin (100 microM) stimulated cAMP production 13-fold above baseline values. Tannin pretreatment inhibited the stimulatory effect of forskolin on cAMP release in a dose-dependent manner with a tannin concentration causing 50% inhibition of 7.5 micrograms/ml. The stimulatory effect of forskolin on cAMP release was completely inhibited in cells pretreated with 50 micrograms/ml tannin. The inhibition was reversible 3 h after removal of tannin from the solution. Tannin also inhibited forskolin-stimulated adenylate cyclase activity in a dose-dependent, noncompetitive manner. We conclude that forskolin and cholera toxin stimulate cAMP production in airway epithelial cells and that tannin inhibits the production of cAMP in airway epithelial cells by a direct effect on adenylate cyclase activity.
Lung-specific direct in vivo gene transfer with recombinant plasmid DNA
Authors: Tsan M. F. , White J. E. , Shepard B.
Published: December 2017

A number of gene delivery methods have been developed to facilitate gene transfer into mammalian somatic cells in vivo. In this study, we demonstrated that tracheal insufflation of two recombinant plasmids containing a bacterial gene, chloramphenicol acetyltransferase (CAT), driven by the cytomegalovirus (CMV) immediate early promoter/enhancer, either alone or complexed to cationic liposomes, resulted in efficient and selective transfection of the lungs in rats. When the simian virus 40 (SV40) promoter/enhancer was used, there was no detectable transfection. Insufflation of plasmid DNA was as efficient as plasmid-liposome complexes in transfecting the lungs. Expression of the CAT gene in the lungs was noted as early as 1 day after insufflation of plasmid DNA alone or plasmid-liposome complexes, and lasted for > 21 days. In contrast, intravenous injection of plasmid alone or plasmid-liposome complexes did not result in transfection of the lungs. Because of its simplicity, without the potential adverse effect of any gene delivery systems, intratracheal delivery of recombinant plasmid DNA may have potential implication for lung-specific gene therapy.
Adrenal hormone regulation of extracellular matrix synthesis by type II cells
Authors: Dunsmore S. E. , Rannels S. R. , Grove R. N. , Rannels D. E.
Published: December 2017

Previous studies suggested an association between changes in extracellular matrix (ECM) and pulmonary epithelial cell growth and differentiation in lungs of adrenalectomized rats. The role of adrenal hormones to modulate synthesis of cell and ECM proteins by type II epithelial cells was thus investigated. In cells isolated from lungs of adrenalectomized animals, synthesis of ECM proteins was stimulated to a greater extent than was synthesis of cell proteins. This resulted in increased relative synthesis of matrix components. Conversely, dexamethasone (200 nM) treatment in vitro reduced the effects of adrenalectomy and, in control cells, decreased relative ECM synthesis. The magnitude of these effects depended on time in culture and serum conditions. For example, culture of normal cells in serum-free medium mimicked the effects of adrenalectomy, whereas there were no effects of dexamethasone in the presence of serum. These results indicate a role for adrenal steroid hormones to modulate ECM metabolism by type II pulmonary epithelial cells and suggest that consequent effects on ECM biological activity may modify epithelial cell differentiation or growth.
Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis
Authors: Kelleher M. D. , Abe M. K. , Chao T. S. , Jain M. , Green J. M. , Solway J. , Rosner M. R. , Hershenson M. B.
Published: December 2017

Abnormal growth of airway smooth muscle may play an important role in the pathogenesis of human airway diseases. Little is known about the proliferative responses of cultured airway smooth muscle cells, nor of the precise pathways responsible for mitogenesis in these cells. We assessed DNA synthesis, cell proliferation, and mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes after exposure to four potential mitogens: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and 5-hydroxytryptamine (5-HT). Stimulation with either PDGF or IGF-1 induced substantial increases in DNA synthesis and cell number, as reflected by [3H]thymidine incorporation, flow cytometry, and methylene blue staining. Treatment with EGF or 5-HT, on the other hand, induced only modest DNA synthesis and no increase in cell number. Immunoblots and kinase renaturation assays of cell extracts demonstrated activation of both the 42- and 44-kDa MAP kinases within minutes of either PDGF, IGF-1, EGF, or 5-HT exposure. However, relative to EGF and 5-HT stimulation, late-phase MAP kinase activation was significantly greater after treatment with the mitogens PDGF and IGF-1. We conclude that in cultured bovine tracheal myocytes 1) PDGF and IGF-1 are potent mitogens; 2) MAP kinase may be activated subsequent to stimulation of either receptor tyrosine kinases (PDGF, EGF, IGF-1) or G protein-linked receptors lacking in known tyrosine kinase activity (5-HT); and 3) unsustained MAP kinase activation is insufficient for mitogenesis. Finally, the finding that mitogenicity correlates with the late phase of MAP kinase activation is consistent with the notion that sustained MAP kinase activation is important for bovine tracheal myocyte proliferation.
Inhibition of cytochrome-c oxidase activity during prolonged hypoxia
Authors: Chandel N. , Budinger G. R. , Kemp R. A. , Schumacker P. T.
Published: December 2017

During acute (< 30 min) hypoxia, cellular respiration is independent of the O2 concentration as long as PO2 remains above a critical value (5–10 Torr). Similarly, state 3 respiration by isolated mitochondria is independent of PO2 above a critical tension of 2–4 Torr. However, rat hepatocytes demonstrate a reversible suppression of respiration and an increase in NAD(P)H concentration during prolonged (2–24 h), but not acute hypoxia [P. T. Schumacker, N. Chandel, and A. G. N. Augusti. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L395–L402, 1993]. This study tested whether respiration is similarly inhibited in isolated mitochondria exposed to low PO2 for prolonged periods and whether cytochrome-c oxidase participates in this response. Coupled rat liver mitochondria were incubated under low oxygen conditions (PO2 < 2 Torr) for 2 h. State 3 respiration after reoxygenation to PO2 = 20 Torr was then compared with the value obtained subsequently at 100 Torr. Using succinate and ADP as substrates, we determined that state 3 respiration at 20 Torr was 61.0 +/- 8.4% of the subsequent value at 100 Torr (P < 0.05). By contrast, control mitochondria reoxygenated to 100 Torr first and 20 Torr subsequently showed no significant difference at the two O2 tensions (P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)
Basenji-greyhound dog: increased m2 muscarinic receptor expression in trachealis muscle
Authors: Emala C. W. , Aryana A. , Levine M. A. , Yasuda R. P. , Satkus S. A. , Wolfe B. B. , Hirshman C. A.
Published: December 2017

Airway smooth muscle from asthmatic humans and from the Basenji-greyhound dog (BG) dog is hyporesponsive to beta-adrenergic agonist stimulation. Because adenylyl cyclase is under dual regulation in airway smooth muscle, we compared muscarinic receptor-coupled inhibition of adenylyl cyclase in airway smooth muscle from BG and mongrel dogs. Inhibition of forskolin-stimulated adenylyl cyclase activity by the muscarinic M2 agonist oxotremorine was greater in airway smooth muscle membranes from BG compared with mongrel controls. Quantitative immunoprecipitation studies showed increased numbers of m2 but not m3 muscarinic receptors in the BG airway smooth muscle. The enhanced ability of muscarinic agonists to inhibit adenylyl cyclase in BG airway smooth muscle may be due to the greater numbers of muscarinic m2 receptors, which may account in part for impaired airway smooth muscle relaxation in the BG model of airway hyperresponsiveness.
Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles
Authors: Jiang He , Rao Kang , Liu Xueliang , Liu Gang , Stephans Newman L.
Published: December 2017

Page L739: He Jiang, Kang Rao, Xueliang Liu, Gang Liu, and Newman L. Stephans. “Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles.” This title is incorrect and conveys the opposite meaning of the article. The correct title should read: “Intracellular Ca2+, myosin light chain phosphorylation, and calmodulin activity in ragweed pollen-sensitized airway smooth muscles.”
Regulation of interleukin-1 receptor antagonist by Th1 and Th2 cytokines
Authors: Kline J. N. , Fisher P. A. , Monick M. M. , Hunninghake G. W.
Published: December 2017

Airway inflammation is an important aspect of asthma. Recent studies of airway inflammation in asthma have focused attention on cytokines released by T helper lymphocyte type 1 (Th1)- and Th2-like T cells. Interleukin (IL)-1 is also increased in the airways of asthmatics, and it is most likely derived from airway and alveolar macrophages. The effects of Th1 or Th2 cytokines on the release of IL-1 or its specific antagonist, IL-1ra, have not been well studied. We examined the response of THP-1 cells, a myelomonocytic cell line, to stimulation with various Th1 and Th2 cytokines and found that IL-4, IL-10, and IFN-gamma increased IL-1ra mRNA and protein release. The increase in mRNA was not due to an increase in IL-1ra mRNA stability. IL-4 (10 ng/ml) increased IL-1ra release from 9,641 +/- 322 [from cells stimulated with lipopolysaccharide (LPS) alone] to 50,796 +/- 1,917 pg/ml (from cells stimulated with LPS and IL-4). IL-10 (10 ng/ml) caused a similar upregulation of IL-1ra from LPS-stimulated cells: 87,478 +/- 7,808 compared with 8,004 +/- 1,166 pg/ml released from the cells stimulated with LPS alone. Cells stimulated with IFN-gamma (100 U/ml) and LPS released 27,854 +/- 3,626 pg/ml of IL-1ra, compared with 9,069 +/- 236 pg/ml in the presence of LPS alone. In addition, the Th1 cytokine, IFN-gamma, but not the Th2 cytokines, IL-4 and IL-10, upregulated IL-1 beta mRNA and increased the release of IL-1 beta protein. Similar studies were performed using freshly isolated monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypoxia decreases endothelin-1 synthesis by rat lung endothelial cells
Authors: Markewitz B. A. , Kohan D. E. , Michael J. R.
Published: December 2017

Endothelin-1 (ET-1) is a 21-amino acid peptide synthesized by several cell types in the lung. Locally, ET-1 regulates vascular and airway tone and is mitogenic for vascular and airway smooth muscle cells. Little, however, is known about the regulation of ET-1 in pulmonary endothelial cells. Cultured rat lung endothelial cells (RLECs) release significant amounts of ET-1 into the supernatant, and isolation of RNA followed by reverse transcription and polymerase chain reaction amplification confirms the presence of ET-1 mRNA. Exposure of RLECs to a hypoxic environment for 24 h decreases ET-1 production by approximately 50% compared with normoxic controls. The effect of hypoxia is reversible upon restoration of a normoxic environment. RNase protection studies reveal decreased ET-1 mRNA in hypoxic cells. Inhibition of nitric oxide (NO) synthase increases ET-1 synthesis during normoxia and hypoxia without altering the inhibitory effect of hypoxia. The addition of 10% carbon monoxide (CO) to the hypoxic environment does not erase the effect of hypoxia on ET-1 production, suggesting that the transduction process does not involve a heme sensor. In summary, we conclude that 1) RLECs synthesize ET-1; 2) hypoxia reversibly decreases ET-1 production; 3) constitutive NO production decreases ET-1 release during normoxia and hypoxia; 4) inhibiting constitutive NO synthesis does not prevent the decrease in ET-1 release caused by hypoxia; and 5) this effect of hypoxia appears to be transduced without the involvement of a heme sensor.
Serotonin-evoked calcium transients in airway smooth muscle cells
Authors: Tolloczko B. , Jia Y. L. , Martin J. G.
Published: December 2017

Serotonin (5-HT) is an important mediator of allergic airway narrowing in several animal species. The present study was designed to characterize the receptor subtypes that mediate 5-HT-induced airway responses in the rat. To do this, we measured Ca2+ transients and adenosine 3',5'-cyclic monophosphate (cAMP) production evoked by 5-HT in cultured rat tracheal smooth muscle cells as well as 5-HT-induced contractions of isolated tracheal rings. 5-HT (10(-6) to 10(-4) M) triggered a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a second phase of sustained, elevated, and sometimes oscillating levels of [Ca2+]i. Sustained but not peak [Ca2+]i levels were dependent on Ca2+ influx but were not attenuated by nifedipine (10(-5) M). Oscillations were observed in cells in Ca2+ -free medium, suggesting Ca2+ -induced Ca2+ release independent of Ca2+ influx. The effects of 5-HT were inhibited by thapsigargin (10(-6) M) and ketanserin (10(-7) M). In cells incubated with LY-278,584 (5-HT3 antagonist) and (-)pindolol (5-HT1 antagonist), 5-HT-evoked responses were not significantly different from the control values. 5-methyltryptamine (5-MT), a ligand with higher affinity for 5-HT2C receptors than "classical" 5-HT2 receptors, elicited higher responses than dipropyl-5-carboxamidotryptamine (DP-5-CT), which possesses lower affinity for 5-HT2C receptors than 5-MT, but an affinity for the classical 5-HT2 receptor similar to 5-MT, but an affinity for the classical 5-HT2 receptor similar to 5-MT.(ABSTRACT TRUNCATED AT 250 WORDS)
Influence of PLC and MLCK inhibitors and the role of L-calcium channels in the cat pulmonary vascular bed
Authors: Kaye A. D. , Nossaman B. D. , Ibrahim I. N. , Kadowitz P. J.
Published: December 2017

The effects of U-73122, a phospholipase C (PLC) inhibitor, on pressor responses to angiotensin II (ANG II), norepinephrine (NE), serotonin (5-HT), BAY K 8644, and the thromboxane A2 (TxA2) mimic, U-46619, were studied in the pulmonary vascular bed of the intact-chest cat. Under conditions of constant lobar blood flow, injections of ANG II, NE, 5-HT, U-46619, and the calcium channel opener, BAY K 8644, into the lobar arterial perfusion circuit caused dose-related increases in lobar arterial pressure, which were reproducible with respect to time. Infusion of U-73122, a PLC inhibitor, into the perfused lobar artery at 10–100 micrograms/kg for 10 min significantly reduced responses to ANG II, serotonin, and NE; however, U-73122 did not alter responses to BAY K 8644 or to U-46619. In a separate series of animals, the effects of the myosin light chain kinase inhibitor, KT-5926, were investigated, and after infusion of KT-5926 into the perfused lobar artery at 1–2 micrograms/kg for 10 min, responses to ANG II, NE, 5-HT, BAY K 8644, and U-46619 were reduced significantly. In a final series of experiments, the effects of the L-type calcium channel blocker, nicardipine, were investigated, and infusion of the L-type calcium channel blocker into the perfused lobar artery at 0.5-1 microgram/kg for 10 min reduced responses to ANG II, BAY K 8644, and NE. However, nicardipine did not alter pressor responses to 5-HT or the TxA2 mimic, U-46619.(ABSTRACT TRUNCATED AT 250 WORDS)
Estrogen modulates the dimensions of the lung's gas-exchange surface area and alveoli in female rats
Authors: Massaro G. D. , Mortola J. P. , Massaro D.
Published: December 2017

Sexually mature virgin female rats and mice have a higher mass-specific gas-exchange surface area (Sa) and smaller alveoli than same-aged males even though mass-specific oxygen consumption (VO2) is the same, within species, in both sexes (G. D. Massaro, J. P. Mortola, and D. Massaro. Proc. Natl. Acad. Sci. USA 92: 1105-1107, 1995). We now report that rats subjected to ovariectomy at age 21 days had, at age 59 days, a smaller mass-specific Sa and larger alveoli than sham-ovariectomized rats; these differences were not due to differences in VO2 and were prevented by estrogen therapy. Furthermore, sham-ovariectomized rats treated with estrogen had smaller and more alveoli than sham-operated females not given estrogen. Androgenization of newborn female rats did not alter mass-specific Sa or alveolar size. Male mice genetically deficient in androgen receptors had the same mass-specific Sa as normal male littermates. We conclude that estrogen is responsible for the sexual dimorphism of the lung's gas-exchange region and induces the formation of smaller, more numerous alveoli in otherwise untreated female rats.
Endothelin receptors and activity differ in human, dog, and rabbit lung
Authors: McKay K. O. , Armour C. L. , Black J. L.
Published: December 2017

In this study, we have examined dog and rabbit airways as potential models for human airways in regard to the activity of endothelin. The receptors involved in the response to endothelin-1 (ET-1) in airway tissue from human, rabbit, and dog lung were investigated, as was the mechanism responsible for the contraction to ET-1 in tissue from the three species. By using specific endothelin receptor agonists and antagonists, we have demonstrated that ETB receptors predominate in rabbit and human airways and ETA receptors in dog airways. The contraction to ET-1 is not dependent on cyclooxygenase products of arachidonic acid, as indomethacin had no effect on the response to ET-1. Extracellular calcium influx via voltage-dependent channels is necessary for contraction to ET-1 in rabbit and dog airways. These results are in contrast to our previously reported results in human airways, in which neither removal of extracellular calcium nor verapamil affected the ET-1 response. The sustained phase of the contraction to ET-1 in all three species may be mediated in part by activation of protein kinase C (PKC), as the inhibitor staurosporine significantly altered the time course of the response to endothelin. We therefore conclude that in rabbit airways ET-1 activates ETB receptors, triggers the influx of extracellular calcium through voltage-dependent channels, and induces a contractile response that is, in part, dependent upon stimulation of PKC. The same mechanism is triggered in dog bronchus; however, the receptors involved in this species are of the ETA type. Finally, in human airways, the contractile response to ET-1, while independent of extracellular calcium influx, is dependent upon PKC activation after binding of the peptide to ETB receptors.
Extracellular glutathione peroxidase in human lung epithelial lining fluid and in lung cells
Authors: Avissar N. , Finkelstein J. N. , Horowitz S. , Willey J. C. , Coy E. , Frampton M. W. , Watkins R. H. , Khullar P. , Xu Y. L. , Cohen H. J.
Published: December 2017

The epithelial cells of the lower respiratory tract are exposed to high levels of inhaled oxygen and other oxidants. We hypothesized that lung cells would secrete the antioxidant enzyme, extracellular glutathione peroxidase (eGPx), into epithelial lining fluid (ELF). To investigate this hypothesis, we used specific immunoprecipitations of GPx enzymes from ELF, specific immunoprecipitations of 75Se metabolically labeled proteins from lung cells in culture, and in situ hybridization, Northern blot, and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Fifty-seven percent of ELF GPx activity was due to eGPx and 40% was due to cellular GPx (cGPx). Primary bronchial epithelial cells (BEC), primary alveolar macrophages (AM), and two human bronchial epithelial cell lines, BEP2D and A549, synthesized both eGPx and cGPx and secreted eGPx into the medium. Freshly isolated human AM and BEC expressed eGPx mRNA, while freshly isolated rabbit type 2 pneumocytes did not. In lung tissue, eGPx mRNA was found mainly in interstitial cells of tissue surrounding airways. It is concluded that more than half of GPx activity in BAL is due to eGPx, and that BEC, AM, and interstitial cells are potential sources of pulmonary eGPx.
Platelet-activating factor stimulates lung pericyte growth in vitro
Authors: Khoury J. , Langleben D.
Published: December 2017

Platelet-activating factor (PAF) is released from activated leukocytes and endothelial cells in sepsis, lung injury, and the adult respiratory distress syndrome. With these disorders, pulmonary hypertension develops, partly due to muscularization of the microvasculature by proliferation of pericytes. PAF may be a mediator of this process. Therefore, we examined the effects of PAF on in vitro growth of rat lung pericytes. Compared with control growth, semisynthetic PAF (10(-9) M) stimulated the 7-day mean growth of proliferating pericytes by 31% in medium with serum and 29% without serum and of previously growth-arrested pericytes by 12% with serum and 23% without serum. These effects were blocked by the PAF-receptor blocker CV-3988. PAF also increased [3H]thymidine incorporation into pericytes by 79%. Synthetic 16:0 PAF stimulated pericyte growth, but 18:0 PAF did not. PAF exposure did not induce apoptosis in pericytes. Thus PAF compounds, similar to those found in vivo, stimulate lung pericyte growth in vitro. PAF may act as a direct cytokine on cells involved in muscularization of the pulmonary vessel walls.
Compartmentalization of glucose utilization after intravenous vs. intratracheal challenge with LPS
Authors: De Boisblanc B. P. , Dobrescu C. , Skrepnik N. , Nelson S. , Spitzer J. J. , Bagby G. J.
Published: December 2017

The rate of glucose utilization (Rg) of various tissues including lung and bronchoalveolar lavage (BAL) fluids cells was measured, using the 2-deoxyglucose technique in Sprague-Dawley rats 4 h after challenge with either 1 mg/kg intravenous or 0.3 mg/kg intratracheal lipopolysaccharide (LPS). After intravenous LPS, Rg increased in whole lung and nonrespiratory tissues, but was unaltered in BAL cells. After intratracheal LPS, the Rg of nonrespiratory tissues was unchanged, but the Rg of BAL cells increased from 3.7 +/- 0.3 to 71.5 +/- 16.0 nmol/min. This increase in the Rg of BAL cells was explained by a doubling of the macrophage specific Rg, by a 100-fold increase in polymorphonuclear leukocytes (PMN) number, and by a higher Rg in PMN than in macrophages. These results demonstrate that increased glucose utilization after intratracheal LPS is confined to the respiratory system and that intra-alveolar phagocytes participate in this increase.
Identification of receptors binding fibronectin and laminin on fetal rat lung cells
Authors: Caniggia I. , Liu J. , Han R. , Wang J. , Tanswell A. K. , Laurie G. , Post M.
Published: December 2017

Fibronectin and laminin have been implicated in regulating lung morphogenesis. In the present study, the cell surface receptors involved in fetal lung cell binding to laminin and fibronectin were identified. Messages for alpha5- and beta1-integrin subunits were detected in both fetal lung epithelial cells and fibroblasts. The presence of alpha5 beta1 -integrin on both cell types was demonstrated by immunocytochemistry and confirmed by cell adhesion experiments with fibronectin and RGD-containing peptides. Epithelial cells adhered more readily to laminin than fibroblasts. The alpha4 beta1-integrin, and RGD-independent fibronectin receptor, was weakly expressed on either cell type. Both cell types expressed alpha6-integrin subunit mRNA and stained immunopositive for the alpha6-subunit. Although either cell type expressed nonintegrin 67-kDa laminin-elastin receptor mRNA, no positive immunoreactivity for this laminin-elastin binding protein was detected. None of these findings explain the enhanced attachment of distal fetal lung epithelial cells to laminin compared with fibroblasts. Previously, we have reported that epithelial cells were enriched in alpha3-integrin subunit mRNA and protein expression. Herein, we found that epithelial cell attachment to laminin was nearly completely inhibited by alpha3- but only partially by alpha6 -monoclonal antibodies. A peptide near the globular region at the long arm of the laminin A-chain, which contained the IKVAV sequence, and the laminin A-chain amino acid sequence representing the alpha3 beta1 -integrin binding site, inhibited the adherence of epithelial cells to laminin. Fetal lung epithelial cells attached to substrata coated with the alpha3 beta1-integrin binding site peptide and the peptide containing the IKVAV sequence. These data suggest that both fetal lung cell types bind to fibronectin via the fibronectin receptor, alpha5 beta1, and fetal lung epithelial cells interact with laminin via alpha3 beta1 and proteins that recognize the IKVAV-containing sequence on the laminin A-chain.
Regulation of xanthine dehydrogenase and xanthine oxidase activity by hypoxia
Authors: Poss W. B. , Huecksteadt T. P. , Panus P. C. , Freeman B. A. , Hoidal J. R.
Published: December 2017

The present study determined the effect of hypoxia on xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activity and gene and protein expression in cultured bovine aortic endothelial cells (BAEC). BAEC were exposed to hypoxia (3% O2) or anoxia (0% O2) for 24 or 48 h and to 24 h of hypoxia followed by 24 h of reoxygenation. Hypoxia- and anoxia-exposed BAEC demonstrated a greater than twofold increase in XDH/XO activity at 24 and 48 h compared with timed controls. Hypoxic cells that were subsequently reoxygenated in 21% O2 also demonstrated a similar increase in XDH/XO activity vs. timed controls. No differences were seen in mRNA levels at any time point. Similarly, no difference was noted in XDH/XO protein expression after hypoxic exposure, as determined by Western blot analysis. The increase in XDH/XO activity was not prevented by cyclohexamide, indicating that protein synthesis was not required. Thus the increased XDH/XO activity observed in response to hypoxia in the present study was due to posttranslational modulation of the enzyme.