bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression of Tie-2 receptors and angiopoietins in response to in vivo hypoxia in rats
Authors: Abdulmalek Kefeya , Ashur Fathia , Ezer Nadine , Ye Fengchun , Magder Sheldon , Hussain Sabah N. A.
Published: December 2017

In this study, we assessed the effects of in vivo hypoxia on the expression of Tie-2 receptors and angiopoietins in various organs of conscious rats and correlated these effects with the expression of hypoxia-inducible factor-1 (HIF-1). RT-PCR and Southern blotting were used to amplify mRNA expression of angiopoietin-1, -2, and -3, Tie-2, and HIF-1α in tissues of normoxic and hypoxic (fraction of inspired oxygen of 9–10% for either 12 or 48 h) rats. Hypoxia provoked a decline in angiopoietin-1 mRNA and Tie-2 mRNA, protein, and phosphorylation levels in the lung, liver, cerebellum, and heart but not in the kidney and diaphragm. In comparison, hypoxia raised the levels of angiopoietin-2 mRNA in the cerebellum and angiopoietin-3 mRNA in the lung, kidney, and diaphragm. HIF-1α mRNA was abundant in most organs of normoxic rats but was significantly induced in the kidney and diaphragm of hypoxic rats. We conclude that in vivo hypoxia exerts inhibitory effects on the activity of the angiopoietin-1/Tie-2 receptor pathway through reduction of angiopoietin-1 and upregulation of angiopoietin-2 and -3. Induction of angiopoietin-3 in the kidney and diaphragm of hypoxic rats could be mediated through the HIF-1 transcription factor.
Respiratory distress after intratracheal bleomycin: selective deficiency of surfactant proteins B and C
Authors: Savani Rashmin C. , Godinez Rodolfo I. , Godinez Marye H. , Wentz Erica , Zaman Aisha , Cui Zheng , Pooler Patricia M. , Guttentag Susan H. , Beers Michael F. , Gonzales Linda W. , Ballard Philip L.
Published: December 2017

Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4–7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.
G551D CF mice display an abnormal host response and have impaired clearance ofPseudomonaslung disease
Authors: McMorran B. J. , Palmer J. S. , Lunn D. P. , Oceandy D. , Costelloe E. O. , Thomas G. R. , Hume D. A. , Wainwright B. J.
Published: December 2017

Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-α, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.
Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia
Authors: Allen Corrie B. , Schneider B. Kelly , White Carl W.
Published: December 2017

Exposure of cultured cells to changing gaseous environments is used as a model for understanding both the immediate and long-term effects of such exposures on lung cells in vivo. We conducted experiments with polystyrene tissue culture flasks and plates to determine the time course of changes in oxygen concentration occurring under in vitro conditions. Only a few minutes were required for the concentration of oxygen in the environmental chamber to reach equilibrium with that of the flushing gas. However, >3 h were required for the oxygen content in the medium in the tissue culture flasks and plates to achieve equilibrium. The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium are the major barriers to rapid oxygen transport into the culture medium. The delay in achieving the desired Po 2 within the culture medium limits the temporal precision of any assessment of the correlation of cellular events with the concentration of oxygen to which those cells are exposed.
Ion transport by sheep distal airways in a miniature chamber
Authors: Al-Bazzaz Faiq J. , Gailey Cynthia
Published: December 2017

Ion transport and the electric profile of distal airways of sheep lungs were studied in a miniature polypropylene chamber with a 1-mm aperture. Small airways with an inner diameter < 1 mm were isolated, opened longitudinally, and then mounted as a flat sheet onto the 1-mm aperture where it was glued and secured with an O-ring. Both sides of the tissue were bathed with identical physiological solutions at 37°C and oxygenated. Pooled data from 27 distal airways showed an inner airway diameter of 854 ± 22 (SE) μm and a transepithelial potential difference (PD) of 1.86 ± 0.29 mV, lumen negative. Short-circuit current ( I sc) was 25 ± 3.5 μA/cm2, tissue resistance was 96 ± 14 Ω, and conductance was 15.2 ± 1.7 mS/cm2. At baseline, amiloride-sensitive Na transport accounted for 51% of I sc (change in I sc = 9.7 ± 2.6 μA/cm2; n = 8 airways), corresponding to 0.36 μeq · cm−2 · h−1. Treatment with 0.1 mM bumetanide did not reduce the I sc( n = 5 airways). Exposure to 1 μM Ca ionophore A-23187 raised the I sc by 9 μA/cm2(47%; P < 0.03; n = 6 airways). The latter effect was blunted by bumetanide. Carbachol at 1 μM provoked a biphasic response, an initial rapid rise in I scfollowed by a decline ( n = 3 airways). There was no significant increase in PD or I sc in response to isoproterenol or dibutyryl cAMP. The data suggest that Na absorption constitutes at least 50% of baseline transport activity. Cl or other anion secretion such as HCO3 appears to be present and could be stimulated by raising intracellular Ca.
Regulation of apoptosis by vasoactive peptides
Authors: Filippatos Gerasimos S. , Gangopadhyay Nupur , Lalude Omosalewa , Parameswaran Narayanan , Said Sami I. , Spielman William , Uhal Bruce D.
Published: December 2017

Although originally discovered because of their ability to affect hemodynamics, vasoactive peptides have been found to function in a variety of capacities including neurotransmission, endocrine functions, and the regulation of cell proliferation. A growing body of evidence describes the ability of vasoactive peptides to regulate cell death by apoptosis in either a positive or negative fashion depending on the peptide and the type of target cell. The available evidence to date is strongest for the peptides endothelin, angiotensin II, vasoactive intestinal peptide, atrial natriuretic peptide, and adrenomedullin. Each of these peptides is discussed, with specific regard to apoptosis, in terms of regulatory activity, target cell specificity, and potential role in pulmonary physiology.
Functional components of basic fibroblast growth factor signaling that inhibit lung elastin gene expression
Authors: Carreras Isabel , Rich Celeste B. , Jaworski Julie A. , Dicamillo Sandra J. , Panchenko Mikhail P. , Goldstein Ronald , Foster Judith Ann
Published: December 2017

Previously, we have demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in confluent rat lung fibroblasts via the binding of a Fra-1-c-Jun heterodimer to an activator protein-1-cAMP response element in the distal region of the elastin promoter. In the present study, we show that bFGF activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, resulting in the translocation of phosphorylated extracellular signal-regulated kinase 1/2 into the nucleus followed by increased binding of Elk-1 to the serum response element of the c-Fos promoter, transient induction of c-Fos mRNA, and sustained induction of Fra-1 mRNA. The addition of PD-98059, an inhibitor of mitogen-activated protein kinase kinase, abrogates the bFGF-dependent repression of elastin mRNA expression. Comparative analyses of confluent and subconfluent fibroblast cultures reveal significant differences in elastin mRNA levels and activator protein-1 protein factors involved in the regulation of elastin transcription. These findings suggest that bFGF modulates specific cellular events that are dependent on the state of the cell and provide a rationale for the differential responses that can be expected in development and injury or repair situations.
Interaction of bacterial lipopolysaccharide with mouse surfactant protein C inserted into lipid vesicles
Authors: Augusto Luis , Le Blay Karine , Auger Genevieve , Blanot Didier , Chaby Richard
Published: December 2017

Infection of the respiratory tract is a frequent cause of lung pathologies, morbidity, and death. When bacterial endotoxin [lipopolysaccharide (LPS)] reaches the alveolar spaces, it encounters the lipid-rich surfactant that covers the epithelium. Although binding of hydrophilic surfactant protein (SP) A and SP-D with LPS has been established, nothing has been reported to date on possible cross talks between LPS and hydrophobic SP-B and SP-C. We designed a new binding technique based on the incorporation of surfactant components to lipid vesicles and the separation of unbound from vesicle-bound LPS on a density gradient. We found that among the different hydrophobic components of mouse surfactant separated by gel filtration or reverse-phase HPLC, only SP-C exhibited the capacity to bind to a tritium-labeled LPS. The binding of LPS to vesicles containing SP-C was saturable, temperature dependent, related to the concentrations of SP-C and LPS, and inhibitable by distinct unlabeled LPSs. Unlike SP-A and SP-D, the binding of SP-C to LPS did not require calcium ions. This LPS binding capacity of SP-C may represent another antibacterial defense mechanism of the lung.
Signal transduction pathways of IL-1β-mediated iNOS in pulmonary vascular smooth muscle cells
Authors: Finder Jonathan D. , Petrus Jennifer L. , Hamilton Andrew , Villavicencio Raphael T. , Pitt Bruce R. , Sebti Saïd M.
Published: December 2017

Interleukin (IL)-1β is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1β-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1β induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1β activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1β-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1β in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.
Identification of a region of strong discrimination in the pore of CFTR
Authors: McCarty Nael A. , Zhang Zhi-Ren
Published: December 2017

The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl−channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl−and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl−and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl−and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.
Redox regulation of endothelial barrier integrity
Authors: Zhao Xiangyi , Alexander J. Steven , Zhang Shu , Zhu Yanan , Sieber Nola J. , Aw Tak Yee , Carden Donna L.
Published: December 2017

Intestinal ischemia-reperfusion is associated with the generation of reactive oxygen metabolites as well as remote, oxidant-mediated lung injury. Oxidants elicit endothelial redox imbalance and loss of vascular integrity by disorganizing several junctional proteins that contribute to the maintenance and regulation of the endothelial barrier. To determine the specific effect of redox imbalance on pulmonary vascular barrier integrity, microvascular permeability was determined in lungs of animals subjected to chemically induced redox imbalance. The effect of redox imbalance on microvascular permeability and endothelial junctional integrity in cultured lung microvascular cells was also determined. Whole lung and cultured pulmonary endothelial cell permeability both increased significantly in response to chemical redox imbalance. Thiol depletion also resulted in decreased endothelial cadherin content and disruption of the endothelial barrier. These deleterious effects of intracellular redox imbalance were blocked by pretreatment with exogenous glutathione. The results of this study suggest that redox imbalance contributes to pulmonary microvascular dysfunction by altering the content and/or spatial distribution of endothelial junctional proteins.
Targeted transgenic expression of β2-adrenergic receptors to type II cells increases alveolar fluid clearance
Authors: McGraw Dennis W. , Fukuda Norimasa , James Paul F. , Forbes Susan L. , Woo Alison L. , Lingrel Jerry B. , Witte David P. , Matthay Michael A. , Liggett Stephen B.
Published: December 2017

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous β-agonists. To selectively delineate the effects of alveolar type II (ATII) cell β2-adrenergic receptors (β2-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human β2-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater β2-ARs than nontransgenic (NTG) mice (939 ± 113 vs. 194 ± 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was ∼40% greater than that in untreated NTG mice (15 ± 1.4 vs. 10.9 ± 0.6%; P < 0.005) and approached that of NTG mice treated with the β-agonist formoterol (19.8 ± 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 ± 0.5% but had no effect on NTG mice (11.5 ± 1.0%). Na+-K+-ATPase α1-isoform expression was unchanged, whereas α2-isoform expression was ∼80% greater in the TG mice. These findings show that β2-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of β2-ARs specifically expressed on ATII cells.
Neuroepithelial bodies in mammalian lung express functional serotonin type 3 receptor
Authors: Fu X. W. , Wang D. , Pan J. , Farragher S. M. , Wong V. , Cutz E.
Published: December 2017

Serotonin (5-HT) type 3 receptor (5-HT3-R) is a ligand-gated ion channel found primarily in the central and peripheral nervous system. We report expression and functional characterization of 5-HT3-R in pulmonary neuroepithelial body (NEB) cells. Using nonisotopic in situ hybridization, we demonstrate expression of 5-HT3-R mRNA in NEB cells in the lungs of different mammals (hamster, rabbit, mouse, and human). Dual immunocytochemistry (for 5-HT and 5-HT3-R) and confocal microscopy localized 5-HT3-R on NEB cell plasma membrane from rabbit. The electrophysiological characteristics of 5-HT3-R in NEB cells were studied in fresh slices of neonatal hamster lung using the whole cell patch-clamp technique. Application of the 5-HT (5–150 μM) and 5-HT3-R agonist 2-methyl-5-HT (5–150 μM) induced inward currents in a concentration-dependent manner. The 5-HT-induced current was blocked (76.5 ± 5.9%) by the specific 5-HT3-R antagonist ICS-205–930 (50 μM), whereas katanserin and p-4-iodo- N-{2-[4-(methoxyphenyl)-1-piperazinyl]ethyl}- N-2-pyridinylbenzamide had minimal effects. Forskolin had no effect on desensitization and amplitude of the 5-HT-induced current. The reduction of Ca2+ and Mg2+ in the extracellular solution enhanced the amplitude of the 5-HT-induced current because of slower desensitization. Our studies suggest that 5-HT3-R in NEB cells may function as an autoreceptor and may potentially be involved in modulation of hypoxia signaling.
Oxygen dependency of monoamine oxidase activity in the intact lung
Authors: Audi Said H. , Dawson Christopher A. , Ahlf Susan B. , Roerig David L.
Published: December 2017

Hydrogen peroxide generated by monoamine oxidase (MAO)-mediated deamination of biogenic amines has been implicated in cell signaling and oxidative injury. Because the pulmonary endothelium is a site of metabolism of monoamines present in the venous return, this brings into question a role for MAO in hyperoxic lung injury. The objective of this study was to evaluate the O2dependency of the MAO reaction in the lung. To this end, we measured the pulmonary venous effluent concentrations of the MAO substrate [14C]phenylethylamine and its metabolite [14C]phenylacetic acid after the bolus injection of either phenylethylamine or phenylacetic acid into the pulmonary artery of perfused rabbit lungs over a range of Po2values from 16 to 518 Torr. The apparent Michaelis constant for O2was ∼18 μM, which is more than an order of magnitude less that measured for purified MAO. The results suggest a minimal influence of high O2on MAO activity in the normal lung and demonstrate the importance of measuring reaction kinetics in the intact organ.
Genes other than TLR4 are involved in the response to inhaled LPS
Authors: Lorenz Eva , Jones Michael , Wohlford-Lenane Christine , Meyer Nicole , Frees Kathy L. , Arbour Nancy C. , Schwartz David A.
Published: December 2017

For several decades, the mouse strains C3H/HeJ and C57BL/10ScNCr have been known to be hyporesponsive to endotoxin or lipopolysaccharide (LPS). Recently, mutations in Toll-like receptor (TLR) 4 have been shown to underlie this aberrant response to LPS. To further determine the relationship between TLR4 and responsiveness to LPS, we genotyped 18 strains of mice for TLR4 and evaluated the physiological and biological responses of these strains to inhaled LPS. Of the 18 strains tested, 6 were wild type for TLR4 and 12 had mutations in TLR4. Of those strains with TLR4 mutations, nine had mutations in highly conserved residues. Among the strains wild type for TLR4, the inflammatory response in the airway induced by inhalation of LPS showed a phenotype ranging from very sensitive (DBA/2) to hyporesponsive (C57BL/6). A broad spectrum of airway hyperreactivity after inhalation of LPS was also observed among strains wild type for TLR4. Although the TLR4 mutant strains C3H/HeJ and C57BL/10ScNCr were phenotypically distinct from the other strains with mutations in the TLR4 gene, the other strains with mutations for TLR4 demonstrated a broad distribution in their physiological and biological responses to inhaled LPS. The results of our study indicate that although certain TLR4 mutations can be linked to a change in the LPS response phenotype, additional genes are clearly involved in determining the physiological and biological responses to inhaled LPS in mammals.
Autocrine signaling by IL-10 mediates altered responsiveness of atopic sensitized airway smooth muscle
Authors: Grunstein M. M. , Hakonarson H. , Leiter J. , Chen M. , Whelan R. , Grunstein J. S. , Chuang S.
Published: December 2017

To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.
Induction of c-junand TGF-β1 in Fischer 344 rats during amiodarone-induced pulmonary fibrosis
Authors: Chung William H. , Bennett Brian M. , Racz William J. , Brien James F. , Massey Thomas E.
Published: December 2017

Amiodarone (AM) is an antidysrhythmic agent with a propensity to cause pulmonary toxicity, including potentially fatal fibrosis. In the present study, the potential roles of c-Jun and transforming growth factor (TGF)-β1 in AM-induced inflammation and fibrogenesis were examined after intratracheal administration of AM (1.83 μmol/day on days 0 and 2) or an equivalent volume (0.4 ml) of distilled water to male Fischer 344 rats. Northern and immunoblot analyses demonstrated that lung TGF-β1 (mRNA and protein) expression was increased 1.5- to 1.8-fold relative to control during the early inflammation period and 1 day, 1 wk, and 2 wk post-AM treatment. Lung c-Jun protein expression was increased concomitantly with evidence of AM-induced fibrosis; at 5 wk post-AM treatment, c-Jun protein was increased 3.3-fold relative to control. The results indicate a role for induction of c- jun and TGF-β1 expression in the development of AM-induced pulmonary fibrosis in the Fischer 344 rat and provide potential targets for therapeutic intervention.
Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice
Authors: Wesselkamper Scott C. , Chen Lung Chi , Kleeberger Steven R. , Gordon Terry
Published: December 2017

After repeated exposures, many individuals develop tolerance to the adverse health effects of inhaled pollutants. Pulmonary tolerance can be characterized as the ability of the lung to withstand the adverse actions of a toxic compound after repeated exposures. To determine whether genetic background is important to the development of pulmonary tolerance to inhaled pollutants, 11 inbred strains of mice were exposed once (1×) or for 5 consecutive days (5×) to 1.0 mg/m3of zinc oxide (ZnO). Development of pulmonary tolerance was assessed by measuring polymorphonuclear leukocyte and protein levels in bronchoalveolar lavage fluid and comparing the responses of the 1× and 5× groups. Significant interstrain variation in polymorphonuclear leukocyte and protein responses was observed between the groups with 1× and 5× exposures, which indicates that genetic background has an important role in the development of pulmonary tolerance. The BALB/cByJ strain and the DBA/2J strain were the most tolerant and nontolerant, respectively. The CByD2F1/J offspring were uniformly nontolerant. The development of tolerance was also investigated in BALB/cByJ and DBA/2J mice after 1× and 5× exposure to ozone and aerosolized endotoxin. Discordance in the phenotypic pattern of pulmonary tolerance among strains after exposure to ZnO, ozone, and endotoxin suggested that different mechanisms may be responsible for the development of pulmonary tolerance to these agents.
Human mast cell and airway smooth muscle cell interactions: implications for asthma
Authors: Page Severine , Ammit Alaina J. , Black Judith L. , Armour Carol L.
Published: December 2017

Asthma is characterized by inflammation, hyperresponsiveness, and remodeling of the airway. Human mast cells (HMCs) play a central role in all of these changes by releasing mediators that cause exaggerated bronchoconstriction, induce human airway smooth muscle (HASM) cell proliferation, and recruit and activate inflammatory cells. Moreover, the number of HMCs present on asthmatic HASM is increased compared with that on nonasthmatic HASM. HASM cells also have the potential to actively participate in the inflammatory process by synthesizing cytokines and chemokines and expressing surface molecules, which have the capacity to perpetuate the inflammatory mechanisms present in asthma. This review specifically examines how the mediators of HMCs have the capacity to modulate many functions of HASM; how the synthetic function of HASM, particularly through the release and expression of stem cell factor, has the potential to influence HMC number and activation in an extraordinarily potent and proinflammatory manner; and how these interactions between HMCs and HASM have potential consequences for airway structure and inflammation relevant to the disease process of asthma.
Oxidative stress in lavage fluid of preterm infants at risk of chronic lung disease
Authors: Schock Bettina C. , Sweet David G. , Halliday Henry L. , Young Ian S. , Ennis Madeleine
Published: December 2017

There is evidence that oxidative stress plays a role in the development of chronic lung disease (CLD), with immature lungs being particularly sensitive to the injurious effect of oxygen and mechanical ventilation. We analyzed total ascorbate, urate, and protein carbonyls in 102 bronchoalveolar lavage fluid samples from 38 babies (33 preterm, 24–36 wk gestation; 5 term, 37–39 wk gestation). Preterm babies had significantly decreasing concentrations of ascorbate, urate, and protein carbonyls during the first 9 days of life ( days 1–3, 4–6, and 7–9, Kruskal-Wallis ANOVA: P = 0.016, P < 0.0001, and P = 0.010, respectively). Preterm babies had significantly higher protein carbonyl concentrations at days 1–3 and 4–6( P = 0.005 and P = 0.044) compared with term babies. Very preterm babies (24–28 wk gestation) had increased concentrations of protein carbonyls at days 4–6 ( P = 0.056) and significantly decreased ascorbate concentrations at days 4–6 ( P= 0.004) compared with preterm babies (29–36 wk gestation). Urate concentrations were significantly elevated at days 1–3( P = 0.023) in preterm babies who subsequently developed CLD. This study has shown the presence of oxidative stress in the lungs of preterm babies during ventilation, especially in those who subsequently developed CLD.
RT-PCR reveals muscarinic acetylcholine receptor mRNA in the pre-Bötzinger complex
Authors: Lai Jiunu , Shao Xuesi M. , Pan Richard W. , Dy Edward , Huang Cindy H. , Feldman Jack L.
Published: December 2017

Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized site for respiratory rhythm generation. Because pharmacological tools for identifying the subtypes of the muscarinic receptors that underlie these effects are limited, we probed for mRNA for these receptors in the pre-BötC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from rat medullary slices. Cholinergic receptor subtype M2and M3mRNAs were observed in the first round of PCR amplification. All five subtypes, M1–M5, were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BötC are M2and M3, with minor expression of M1, M4, and M5.
Albumin uptake and transcytosis in endothelial cells in vivo induced by albumin-binding protein
Authors: Vogel Stephen M. , Minshall Richard D. , Pilipović Milena , Tiruppathi Chinnaswamy , Malik Asrar B.
Published: December 2017

The 60-kDa endothelial cell surface albumin-binding glycoprotein (gp60) is postulated to be a docking site for albumin that mediates the uptake of albumin and its transport in cultured microvessel endothelial cells. In the present study, we used an isolated Krebs-perfused rat lung preparation to address the in vivo role of gp60 in mediating albumin uptake and transport. Addition of primary anti-gp60 antibody to the perfusate followed by the secondary antibody to cross-link gp60 increased the vessel wall125I-albumin permeability-surface area ( PS) product 2.5-fold without affecting the capillary filtration coefficient ( Kf,c;a measure of liquid permeability). In contrast, EDTA (5 mM), which induces interendothelial gap formation, produced parallel increases in both Kf,cand125I-albumin PS product. Increasing perfusate albumin concentration to >1 g/100 ml (EC501.2 g/100 ml) was sufficient to block125I-albumin PS product, indicating that the perfusate albumin competed with tracer albumin for transendothelial albumin transport. Cross-linking of gp60 in lungs perfused with saturating concentration of albumin resulted in a greater increase in125I-albumin PS product, indicating that gp60 function was capable of being modulated. These results show that activation of gp60 in pulmonary microvessels induces albumin uptake and its transport through a nonhydraulic pathway that fits with a model of albumin permeability via the transcellular pathway.
TGF-β isoform release and activation during in vitro bronchial epithelial wound repair
Authors: Howat William J. , Holgate Stephen T. , Lackie Peter M.
Published: December 2017

Restitution of an epithelial layer after environmental or biological damage is important to maintain the normal function of the respiratory tract. We have investigated the role of transforming growth factor (TGF)-β isoforms in the repair of layers of 16HBE 14o−bronchial epithelial-derived cells after damage by multiple scoring. ELISA showed that both latent TGF-β1 and TGF-β2 were converted to their active forms 2 h after wounding. Time-lapse microscopy showed that the addition of TGF-β1, but not TGF-β2, progressively increased the rate of migration of damaged monolayers at concentrations down to 250 pg/ml. This increase was blocked by addition of a neutralizing TGF-β1 antibody. Phase-contrast microscopy and inhibition of proliferation with mitomycin C showed that proliferation was not required for migration. These results demonstrate that conversion of latent to active TGF-β1 and TGF-β2 during in vitro epithelial wound repair occurs quickly and that TGF-β1 speeds epithelial repair. A faster repair may be advantageous in preventing access of environmental agents to the internal milieu of the lung although the production of active TGF-β molecules may augment subepithelial fibrosis.
A2 adenosine receptors regulate CFTR through PKA and PLA2
Authors: Cobb B. R. , Ruiz F. , King C. M. , Fortenberry J. , Greer H. , Kovacs T. , Sorscher E. J. , Clancy J. P.
Published: December 2017

We investigated adenosine (Ado) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo. A2B Ado receptors were identified in Calu-3, IB-3-1, COS-7, and primary human airway cells. Ado elevated cAMP in Calu-3, IB-3-1, and COS-7 cells and activated protein kinase A-dependent halide efflux in Calu-3 cells. Ado promoted arachidonic acid release from Calu-3 cells, and phospholipase A2(PLA2) inhibition blocked Ado-activated halide efflux in Calu-3 and COS-7 cells expressing CFTR. Forskolin- and β2-adrenergic receptor-stimulated efflux were not affected by the same treatment. Cytoplasmic PLA2(cPLA2) was identified in Calu-3, IB-3-1, and COS-7 cells, but cPLA2 inhibition did not affect Ado-stimulated cAMP concentrations. In cftr(+) and cftr(−/−) mice, Ado stimulated nasal Cl− secretion that was CFTR dependent and sensitive to A2 receptor and PLA2 blockade. In COS-7 cells transiently expressing ΔF508 CFTR, Ado activated halide efflux. Ado also activated G551D CFTR-dependent halide efflux when combined with arachidonic acid and phosphodiesterase inhibition. In conclusion, PLA2 and protein kinase A both contribute to A2 receptor activation of CFTR, and components of this signaling pathway can augment wild-type and mutant CFTR activity.
Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR
Authors: Zhang Zhi-Ren , Zeltwanger Shawn , Smith Stephen S. , Dawson David C. , McCarty Nael A.
Published: December 2017

A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current ( i)-voltage ( V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current ( I)- V relationship ( I = N × Po× i, where N is the number of active channels and Pois open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the Poof V317E CFTR is voltage dependent. The decrease in Poat negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the Po- V relationship did not depend on the direction of Cl−movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.
p38 MAPK activation by TGF-β1 increases MLC phosphorylation and endothelial monolayer permeability
Authors: Goldberg Peter L. , MacNaughton Darren E. , Clements Richard T. , Minnear Fred L. , Vincent Peter A.
Published: December 2017

Transforming growth factor (TGF)-β1 increases endothelial monolayer permeability and myosin light chain phosphorylation (MLC-P) beginning 1–2 h posttreatment, suggesting that changes in gene expression may be required for these responses. The role of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38 MAPK) was investigated because both kinases have been implicated in regulating gene expression after TGF-β1. ERK1/2 phosphorylation increased threefold above the control level, and the increase was temporally associated with the increase in MLC-P. Inhibition of ERK1/2 phosphorylation with the MAPK kinase inhibitor U-0126 did not prevent the increase in either monolayer permeability or MLC-P. p38 MAPK phosphorylation increased fourfold above the control level, but unlike ERK1/2, this increase peaked 30 min and 1 h post-TGF-β1 treatment. Inhibition of p38 MAPK activity with SB-203580 prevented the increases in both monolayer permeability and MLC-P. Treatment of the monolayers with cycloheximide in conjunction with TGF-β1-inhibited MLC-P, showing a requirement for protein synthesis. These studies demonstrate that p38 MAPK activation and subsequent protein synthesis are part of the signal transduction pathway leading to the TGF-β1-induced increases in monolayer permeability and MLC-P.
Tryptase's potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions
Authors: Brown James K. , Jones Cary A. , Rooney Leeann A. , Caughey George H. , Hall Ian P.
Published: December 2017

We reported previously that mast cell tryptase is a growth factor for dog tracheal smooth muscle cells. The goals of our current experiments were to determine if tryptase also is mitogenic in cultured human airway smooth muscle cells, to compare its strength as a growth factor with that of other mitogenic serine proteases, and to determine whether its proteolytic actions are required for mitogenesis. Highly purified preparations of human lung β-tryptase (1–30 nM) caused dose-dependent increases in DNA synthesis in human airway smooth muscle cells. Maximum tryptase-induced increases in DNA synthesis far exceeded those occurring in response to coagulation cascade proteases, such as thrombin, factor Xa, or factor XII, or to other mast cell proteases, such as chymase or mastin. Irreversibly abolishing tryptase's catalytic activity did not alter its effects on increases in DNA synthesis. We conclude that β-tryptase is a potent mitogenic serine protease in cultured human airway smooth muscle cells. However, its growth stimulatory effects in these cells occur predominantly via nonproteolytic actions.
Maintenance of renal autoregulation during infusion of aminophylline or adenosine
Authors: Premen A. J. , Hall J. E. , Mizelle H. L. , Cornell J. E.
Published: December 2017

Adenosine has been postulated to link control of glomerular filtration rate (GFR) and renal blood flow (RBF) with changes in renal metabolism. In the present study, we examined the role of adenosine in renal autoregulation by comparing the responses of normal anesthetized dogs to step decreases in renal artery pressure (RAP) to the response obtained after receptor blockade of adenosine with aminophylline or by flooding the kidney with exogenous adenosine. In six dogs at normal RAP, intrarenal infusion of aminophylline (10 mumol/min) did not alter renal hemodynamics. GFR and RBF were well autoregulated (greater than 90% of control) at RAP values equal to or greater than 85 mmHg before and after aminophylline. At RAP equal to 75 mmHg, GFR and RBF decreased by 27 +/- 10 and 20 +/- 8%, respectively, before aminophylline and by 25 +/- 7 and 13 +/- 6% after aminophylline. In a different group of six dogs, intrarenal infusion of adenosine (6 mumol/min) significantly increased RBF (32 +/- 9%) and decreased GFR (38 +/- 10%) at normal RAP. However, GFR and RBF were both well autoregulated (greater than 90% of control) at RAP values equal to or greater than 85 mmHg before and after adenosine. At RAP equal to 75 mmHg, GFR and RBF decreased by 10 +/- 5 and 7 +/- 3%, respectively, before adenosine and by 12 +/- 6 and 17 +/- 5% after adenosine. Neither aminophylline nor adenosine attenuated the elevations in plasma renin activity associated with reductions in RAP. These data fail to provide evidence that adenosine is an important factor in autoregulation of GFR and RBF during acute reductions in RAP within the autoregulatory range.
Defined human renal tubular epithelia in culture: growth, characterization, and hormonal response
Authors: Wilson P. D. , Dillingham M. A. , Breckon R. , Anderson R. J.
Published: December 2017

Individually microdissected nephron segments of defined epithelial origin from human kidneys were cultured in vitro in the present studies. Nephron segments of proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb of Henle, and cortical collecting tubule were grown in defined media. Each cell type retained differentiated characteristics as assessed by ultrastructural morphology, marker enzyme profiles, and adenylate cyclase response to selected hormones. These studies demonstrate the feasibility of using primary cultures of well-defined segments of the human nephron to study human renal tubular epithelia in vitro.
Interactions of vasopressin, cAMP, and prostaglandins in toad urinary bladder
Authors: Schlondorff D. , Satriano J. A.
Published: December 2017

Prostaglandin (PG) inhibits the hydroosmotic effect of vasopressin. We therefore reexamined the interaction of vasopressin (VP), cAMP, and prostaglandins in toad bladder epithelial cells. Vasopressin slightly, but reproducibly, stimulated PGE2 and thromboxane B2 (TXB2) synthesis in cells prepared by the use of collagenase. When cells were prepared in the presence of a readily reversible cyclooxygenase inhibitor, ibuprofen, subsequent PGE2 synthesis was enhanced sevenfold but that of TXB2 was not. Increasing cAMP by either phosphodiesterase inhibition or 8-bromo-cAMP significantly inhibited both basal and VP-stimulated PGE2 synthesis. This inhibition was overcome by addition of arachidonic acid. Future studies employing these agents will have to consider these effects. VP enhanced 32P labeling of phosphatidylinositol (PI) and phosphatidic acid. This effect was prevented by the phosphodiesterase inhibitor, which also decreased phosphatidylcholine labeling. The results indicate that the phosphodiesterase inhibitor for cAMP may decrease PG formation by interfering with phospholipase activation. Furthermore, VP, similar to its effect in the liver, also increases PI turnover in toad bladder. This may initiate PG synthesis and provide a link among VP, cAMP, and calcium. A double-reciprocal feedback is proposed, whereby VP stimulates PG synthesis in a cAMP-independent manner and also inhibits PG synthesis in a cAMP-dependent manner.
Localization of diuretic action in microperfused rat distal tubules: Ca and Na transport
Authors: Costanzo L. S.
Published: December 2017

Experiments were performed in rats to examine the distal site of action of thiazide diuretics and the additive hypocalciuric properties of thiazides and amiloride. In clearance experiments, the maximal natriuretic and hypocalciuric dose of chlorothiazide was established. When amiloride was added, there was further augmentation of Ca reabsorption (P less than 0.025) but no additional natriuresis. Amiloride blunted thiazide-induced kaliuresis (P less than 0.001). Localization of the thiazide effect was studied in early and late distal tubules microperfused in vivo with control and thiazide-containing solutions. The maximally effective luminal drug concentration, 5 X 10(-4) M, inhibited Na transport (P less than 0.001) and enhanced Ca transport (P less than 0.01) in the early distal segments; late segments were on the average unaffected. It is suggested that thiazides interact with the distal convoluted tubule cell, whose predominant location is the early distal tubule. In two long distal tubules, with early and late segments, a maximal concentration of chlorothiazide increased Ca transport and decreased Na transport. Addition of 10(-5) M amiloride caused an additional increment in Ca reabsorption. As amiloride's action is located in the late distal tubule, it is suggested from these experiments that a basis for additive hypocalciuric actions of thiazides and amiloride is separate sites of action in the distal tubule.
Anion transport inhibitors: effects on water and sodium transport in the toad urinary bladder
Authors: Brem A. S. , Eich E. , Pearl M. , Taylor A.
Published: December 2017

Acidification of the medium bathing the serosal surface of the toad urinary bladder results in impairment of the water permeability response to vasopressin. The magnitude of the hydrosmotic response to a maximal concentration of either vasopressin or the cyclic nucleotide analogue 8-(p-chlorophenylthio)-cyclic 3',5'-adenosine monophosphate (C1PhS-cAMP) was progressively reduced when serosal bath pH was decreased from 8.5 to 6.5. The disulfonic stilbenes SITS and DIDS and the diuretic furosemide, agents known to interfere with anion transport and with the regulation of intracellular pH in other tissues, inhibited the water flow response to vasopressin and C1PhS-cAMP in a pH-dependent manner when added to the serosal bathing medium. Inhibition of the hydrosmotic response to 10(-5) M C1PhS-cAMP was estimated to be half-maximal at 1.5 X 10(-4) M SITS, 2 X 10(-5) M DIDS, and 1 X 10(-5) M furosemide. The degree of inhibition induced by the anion transport inhibitors varied inversely with the concentration of exogenous cyclic nucleotide. SITS, DIDS, and furosemide had no effect on either basal or vasopressin-stimulated short-circuit current at serosal pH 8.5; all three agents inhibited basal short-circuit current at pH 7.1 but had no effect on the natriferic response to vasopressin. These results are consistent with the view that changes in intracellular hydrogen ion and/or anion concentration can selectively inhibit the increase in water permeability elicited by vasopressin at a step(s) distal to the generation of cAMP.
Rapid correction of severe hyponatremia in the rat: histopathological changes in the brain
Authors: Ayus J. C. , Krothapalli R. K. , Armstrong D. L.
Published: December 2017

The purpose of the present studies was to examine the effects of rapid correction of severe hyponatremia (serum sodium less than 120 meq/liter) either to mildly hyponatremic levels (serum sodium = 130 meq/liter) or to normonatremic levels (serum sodium = 150 meq/liter) on the brain histology of rats. In group I, 13% of the rats revealed brain lesions following correction to mildly hyponatremic levels by the administration of 855 mM NaCl. All the rats (100%) in group II had brain lesions following correction to normonatremic levels by 24 h of water restriction. Similarly, all the rats in group III showed brain lesions following correction to normonatremic levels by the administration of 855 mM NaCl. Severe hyponatremia by itself did not cause any brain lesions in another group. We conclude that rapid correction of severe hyponatremia to mildly hyponatremic levels by the administration of 855 mM NaCl does not cause significant brain lesions. On the other hand, rapid correction to normonatremic levels either by water restriction or by the administration of 855 mM NaCl results in significant brain lesions.