bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Na(+)-H+ exchange in human normal and cystic fibrotic ciliated airway epithelium
Authors: Paradiso A. M.
Published: December 2017

The pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein was used to measure intracellular pH (pHi) and test for Na(+)-H+ exchange in single ciliated human nasal epithelial (HNE) cells from normal and cystic fibrosis (CF) subjects. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered NaCl Ringer solution, average pHi for normal and CF ciliated HNE cells was 7.15 +/- 0.02 and 7.18 +/- 0.03, respectively. Utilizing the NH+4-loading technique to acid load cells, we found that pHi decreased from approximately 7.2 to approximately 6.6; subsequent alkalinization of normal and CF ciliated cells required Na+ and was independent of Cl- but was blocked by amiloride (500 microM). In the presence of extracellular Na+, initial rates (delta pHi/min) of recovery from an acid load in normal and CF cells were 0.09 +/- 0.03 and 0.12 +/- 0.05, respectively. Pretreatment of cells with phorbol 12-myristate 13-acetate (PMA, 100 nM) caused initial rates of recovery to increase over control values by approximately twofold in both cell preparations. Other studies showed that the native H+ conductance of the plasma membrane was negligible in both normal and CF cell preparations. The results provide evidence in support of an amiloride-sensitive PMA-activated Na(+)-H+ exchanger in ciliated HNE cells from normal and CF donors.
Differential extraction for the rapid purification of bovine surfactant protein B
Authors: Beers M. F. , Bates S. R. , Fisher A. B.
Published: December 2017

Surfactant protein B (SP-B), a peptide found in organic solvent extracts of mammalian surfactant, has been isolated from surfactant previously by column chromatography and/or preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). We have developed a method for isolation of SP-B from bovine surfactant utilizing differential organic extraction. Dried surfactant, isolated from lavage of excised cow lungs, was delipidated by extraction with diisopropyl ether-butanol (3:2). The aqueous layer, containing surfactant proteins, was dried and then was sequentially extracted with diethyl ether-ethanol (3:1) and CHCl3:MeOH:HCl (3:2:0.005 N). SP-B partitioned into chloroform-methanol, which was evaporated under N2. Purified SP-B, quantitated by Coomassie dye binding, represented 1% (wt/wt) of the original surfactant with a final phospholipid-to-protein ratio less than 1. Silver-stained SDS/PAGE of the SP-B extract revealed a single band at 9 kDa (reduced) and 18 kDa (nonreduced), which by immunoblotting reacted strongly with monospecific anti-SP-B antibody. Amino acid sequence analysis confirmed the presence of NH2 and N-1 terminal sequences of bovine SP-B. This procedure offers a rapid, reliable method for isolation of purified SP-B from whole surfactant.
Cloning and expression of an airway epithelial 12-lipoxygenase
Authors: Marzo Nicoletta De , Sloane David L. , Dicharry Sherry , Highland Ella , Sigal Elliott
Published: December 2017

Pages L198–L207: Nicoletta De Marzo, David L. Sloane, Sherry Dicharry, Ella Highland, and Elliott Sigal. “Cloning and expression of an airway epithelial 12-lipoxygenase.” Page L202, Fig. 3, nucleotide sequence 1321–1380 should read:

TCCTTCTGTCCCCCTGATGACCTGGCTGAC

CGGGGGCTCCTGGGAGTCAAGTCTTCTTTC

The deduced amino acid sequence for this region in Figs. 3 and 7 is correct. Also, the nucleotide sequence submitted to GenBank (accession number M62516) is correct.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia
Authors: Anderson M. P. , Sheppard D. N. , Berger H. A. , Welsh M. J.
Published: December 2017

Cl- channels located in the apical membrane of secretory epithelia play a key role in epithelial fluid and electrolyte transport. Dysfunction of one of these channels, cystic fibrosis transmembrane conductance regulator (CFTR), causes the genetic disease cystic fibrosis (CF). We review here the properties and regulation of the different types of Cl- channels that have been reported in airway and intestinal epithelia. We begin by describing the properties of the CFTR Cl- channel and then use those properties as a point of reference. We focused particularly on the evidence that localizes specific types of Cl- channel to the apical membrane. With that background, we assess the biological function of various Cl- channels in airway and intestinal epithelia.
Intracellular Ca2+ and regulation of ion transport across rabbit Clara cells
Authors: Van Scott M. R. , Paradiso A. M.
Published: December 2017

We investigated whether Ca2+ was involved in regulation of ion transport across rabbit distal airway epithelial cells by studying the effects that elevation of intracellular Ca2+ (Cai) had on the bioelectric properties of nonciliated bronchiolar (Clara) cell epithelia in culture. Exposure of Clara cells to 5 x 10(-7) M ionomycin increased Cai concentration and transepithelial short-circuit current (Isc). Changing extracellular Ca2+ concentration in the presence of ionomycin demonstrated that changes in Isc paralleled changes in Cai. Another ionophore, 4-bromo-A23187, also increased Cai and Isc. Ionomycin-induced changes in Isc were insensitive to amiloride and were inhibited greater than 50% by pretreating the cells with bumetanide or substituting gluconate for Cl- in the bathing solution. Bradykinin and carbachol, which increased Cai and caused an increase in Isc across tracheal cell cultures, had no effect on Cai or Isc in Clara cell preparations. These results support the hypothesis that changes in Cai are linked to regulation of Cl- secretion across bronchiolar epithelial cells, but physiological regulators of Cai in Clara cells remain to be defined.
Chronic hypoxia selectively augments rat pulmonary artery Ca2+ and K+ channel-mediated relaxation
Authors: Rodman D. M.
Published: December 2017

The initiating event in hypoxic pulmonary hypertension is felt to be sustained hypoxic vasoconstriction, ultimately leading to vascular remodeling and fixed pulmonary hypertension. During the initial vasospastic phase endogenous vasodilatory pathways may serve to ameliorate the development of pulmonary hypertension. However, various studies in the systemic and pulmonary circulations have shown that chronic hemodynamic stress alters both endothelial and smooth muscle cell function. The effect of chronic hypoxia in rats was therefore tested on three major vasodilatory pathways: 1) endothelium-dependent relaxation (using endothelium-derived relaxing factor agonists and antagonists); 2) smooth muscle cell cyclic nucleotide-mediated relaxation [using guanosine and adenosine 3',5'-cyclic monophosphate (cGMP and cAMP) agonists]; and 3) ion channel-dependent relaxation (using K+ channel agonists and Ca2+ channel antagonists). It was found that short-term exposure (72 h) to hypoxia caused augmentation of K+ and Ca2+ channel-dependent relaxation with no effect on endothelium-dependent or cyclic nucleotide-mediated relaxation. More prolonged exposure (4–5 wk) was additionally associated with inhibition of endothelium-dependent relaxation and smooth muscle cell cGMP-mediated relaxation. There was no effect on either basal modulation of tone by the endothelium, cAMP-mediated relaxation, or systemic vessel relaxation. It is concluded that an early response to hemodynamic stress in the pulmonary circulation is alteration in smooth muscle cell ion channel function and/or Ca2+ homeostasis.
Structural and functional impairment of surfactant protein A after exposure to nitrogen dioxide in rats
Authors: Muller B. , Barth P. , von Wichert P.
Published: December 2017

The major surfactant protein A (SP-A) determines the dynamics of surfactant metabolism and function. We now for the first time studied the in vivo exposure of the SP-A to nitrogen dioxide (NO2) and compared it to in vitro exposure effects. Several properties that are believed to possess physiological functions were investigated. Exposure of rat SP-A to NO2 showed 1) a higher alveolar pool size after in vivo exposure, 2) a slightly decreased mannose binding capacity for the in vivo exposed form but a highly decreased binding after in vitro exposure, 3) an affected protein-lipid aggregation that was impaired most by the in vitro exposed protein, 4) a low inhibition for the secretion of phosphatidylcholine in isolated type II pneumocytes, and 5) no difference in apparent molecular weight and immunological reactivity of the differently exposed SP-A. The NO2-induced alterations of the SP-A may contribute to the pulmonary toxicity of this oxidant. It is clear from this study that the extend of the effects from in vivo and in vitro exposure are different. Therefore both forms of exposure are necessary to assess the biological significance of NO2 exposure.
Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+i and inositol polyphosphates
Authors: Lum H. , Aschner J. L. , Phillips P. G. , Fletcher P. W. , Malik A. B.
Published: December 2017

The temporal relationship between the alpha-thrombin-induced increase in transendothelial permeability and the alpha-thrombin-mediated changes in several key transmembrane signaling events was examined in confluent monolayers of bovine pulmonary artery endothelial cells (BPAEC). The time courses of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] generation, changes in cytosolic [Ca2+] ([Ca2+]i), and reorganization of cytoskeletal F-actin were determined to assess the relationship between these events and the onset of the alpha-thrombin-induced increase in endothelial permeability. alpha-Thrombin (10(-7) M) increased the transendothelial 125I-albumin clearance rate half-maximally by approximately 1 min and maximally by approximately 2 min (160% over control level). The increase in permeability occurred concomitantly with reorganization of F-actin cytoskeleton (i.e., loss of peripheral band and increased stress fiber density) and increased actin polymerization. Stimulation of fura-2-loaded BPAEC with 10(-7) M alpha-thrombin produced a typical biphasic rise in [Ca2+]i. The initial rapid increase in [Ca2+]i peaked by approximately 16 s after thrombin challenge and the [Ca2+]i response showed a slow decrease to half-maximal within 50 s. The alpha-thrombin-induced increase in permeability as well as the increase in [Ca2+]i were consistently preceded by increased Ins(1,4,5)P3 generation detectable within 10 s after thrombin challenge. These results indicate that alpha-thrombin triggers a cascade of events (i.e., Ins(1,4,5)P3 generation and the ensuing rise in [Ca2+]i), which may comprise the second messengers that mediate F-actin reorganization and the increase in endothelial permeability.
Bronchial epithelial cells release neutrophil chemotactic activity in response to tachykinins
Authors: Von Essen S. G. , Rennard S. I. , O'Neill D. , Ertl R. F. , Robbins R. A. , Koyama S. , Rubinstein I.
Published: December 2017

The purpose of this study was to determine whether substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) induce the release of neutrophil chemotactic activity (NCA) from bovine bronchial epithelial cells (BBEC) and whether neutral endopeptidase (NEP), a membrane-bound metalloenzyme that hydrolyzes tachykinins, modulates these effects. BBEC monolayers were exposed to SP, NKA, and NKB in the absence or presence of phosphoramidon (10(-6) M), a selective NEP inhibitor, for 72 h. Using a modified blind-well in vitro neutrophil chemotaxis assay, we found that tachykinin-exposed BBEC culture supernatant fluids induced significant neutrophil chemotaxis compared with supernatants obtained from unstimulated BBEC. Maximal effect was observed after 48 h of incubation and at SP concentration of 10(-13) M [92 +/- 3 (SP) vs. 64 +/- 2 (media) cells/high-power field (HPF), mean +/- SE, n = 7, P less than 0.05]. Release of NCA was mediated by the COOH-terminal of the SP molecule. The rank order of potency of tachykinins in inducing release of NCA was SP greater than NKA = NKB. SP-induced response was significantly potentiated by phosphoramidon (109 +/- 3 vs. 92 +/- 3 cells/HPF, n = 7, P less than 0.05), whereas other proteinase inhibitors had no effect. The released NCA was composed of protein and lipid-soluble components. These data indicate that mammalian tachykinins induce the release of NCA from BBEC and that NEP modulates these effects. We suggest that tachykinins regulate neutrophil recruitment into the lower respiratory tract, in part, by inducing the release of NCA from airway epithelial cells.
Generation of the neutrophil-activating peptide-2 by cathepsin G and cathepsin G-treated human platelets
Authors: Cohen A. B. , Stevens M. D. , Miller E. J. , Atkinson M. A. , Mullenbach G.
Published: December 2017

The neutrophil-activating peptide-2 (NAP-2) is a cytokine that is generated by the proteolytic cleavage of a precursor protein and that causes neutrophil degranulation and chemotaxis. NAP-2 precursors are produced in platelets and are normally found in the circulation. We showed that NAP-2 is generated by the action of neutrophil cathepsin G on two of the precursors, the connective tissue-activating peptide-III (CTAP-III) and beta-thromboglobulin (beta-TG). However, neutrophil elastase degraded the precursors to inactive peptides. The specific binding of cathepsin G to platelets caused the platelets to secrete NAP-2, and cathepsin G bound to the platelets could still generate NAP-2 from its precursor proteins. In addition, activated neutrophils in the presence of platelets generated NAP-2 from its precursors and caused platelets to secrete NAP-2. These studies demonstrate a unique mechanism for the activation of neutrophils through the interaction of neutrophils, platelets, and NAP-2 precursors that are released either by activated platelets or are present in circulation. It is therefore possible that NAP-2 may be generated at sites where aggregations of neutrophils and platelets occur in vessels such as pulmonary capillaries in patients with the adult respiratory distress syndrome and coronary arteries in patients with evolving myocardial infarctions.
Surfactant proteins and lipids are regulated independently during hyperoxia
Authors: Minoo P. , King R. J. , Coalson J. J.
Published: December 2017

Adult hamsters were exposed to 100% oxygen for up to 8 days. At time of death lung tissue was analyzed for the expression of surfactant protein (SP) genes, and surfactant was isolated from alveolar lavage fluid. Surfactant was analyzed for the composition of proteins and phospholipids and for its surface properties. We found, over the 8 days of exposure, that an alveolitis composed of polymorphonuclear leukocytes (PMNs) and alveolar macrophages, accompanied by exudation of edema fluid, appeared in the alveolar spaces. The steady-state levels of SP mRNAs declined after 8 days of exposure to 100% oxygen, but the patterns indicated individual genetic control. SP-A was elevated early in the course of the hyperoxic exposure but decreased significantly by day 8; SP-B decreased continuously; SP-C was unchanged (or slightly elevated) through day 2 and then declined. The amounts of recoverable lavage surfactant increased by greater than threefold, and the phospholipid composition showed increasing percentages of disaturated phosphatidylcholine. All surfactants lowered surface tension to less than 10 dyn/cm, but the adsorption rates decreased as exposure progressed. The results indicate that lung injury induced by 100% oxygen is accompanied by altered patterns of surfactant metabolism, possibly because of a changing type II cell phenotype or alterations in Clara cell-derived surfactant. These changes may result in perturbed physiological function contributing to decreased lung compliance.
CFU-rAM, the origin of lung macrophages, and the macrophage lineage
Authors: Sorokin S. P. , McNelly N. A. , Hoyt R. F.
Published: December 2017

Macrophage precursors and their progeny have been identified in early rat embryos with the use of a peroxidase-coupled marker, isolectin B4 of Griffonia simplicifolia (GSA I-B4). The macrophage lineage can be traced back to actively dividing GSA-positive angular cells present in the mesenchyme of late neurulas. These increase in number and establish residence successively in rudiments of the central nervous system, liver, and lungs. In organ-cultured fetal lungs they transform directly into a self-replicating population of macrophages responsive to colony-stimulating factors. The angular cell therefore can be seen as the source of lung macrophages during prenatal life. The extent to which this manner of production continues in postnatal life is unclear, but it appears that central hematopoietic tissues (bone marrow, spleen) may generate macrophages by a direct pathway from early-committed progenitors as well as indirectly through a series of intermediate stem cells. Considering the wealth of new information available from diverse studies in specialized culture environments and to a lesser extent from studies in vivo, it is time to integrate these findings into a more comprehensive theory of macrophage origin and fate than we have at present.
Kinetics of pulmonary superoxide dismutase in interleukin-1-induced oxygen-tolerant rats
Authors: Tsan M. F. , White J. E.
Published: December 2017

We have previously demonstrated that tracheal insufflation of interleukin-1 alpha (IL-1) enhances pulmonary Mn superoxide dismutase (Mn SOD) activity and protects rats against O2 toxicity (M. F. Tsan, C. Y. Lee, and J. E. White. J. Appl. Physiol. 71: 688–697, 1991). In this study, we investigated the kinetics of mRNA, specific (immunoreactive) proteins, and enzyme activities of pulmonary Mn SOD and Cu,Zn SOD in IL-1-induced O2-tolerant rats. At 1 day after IL-1 (5 micrograms) insufflation and O2 exposure, levels of Mn SOD mRNA and specific protein, but not enzyme activity, were markedly elevated. At 2.3 and 7 days after O2 exposure, levels of Mn SOD mRNA, specific protein, and enzyme activity were all increased in IL-1-treated animals. In contrast, in control rats at 2.3 days after O2 exposure, level of Mn SOD mRNA was markedly elevated, whereas levels of specific protein and enzyme activity were decreased. Levels of pulmonary Cu,Zn SOD mRNA, specific protein, and enzyme activity were unchanged in control and IL-1-treated rats, except that in IL-1-induced long-term O2-tolerant rats (7 days after O2 exposure), they were all increased. Since at 7 days after IL-1 insufflation, normoxia-exposed rats did not show increased levels of pulmonary Mn SOD or Cu,Zn SOD mRNA, the increased levels of pulmonary SOD seen in IL-1-induced long-term O2-tolerant rats are, at least in part, due to the effect of O2 exposure
Effect of food restriction on hyperoxia-induced lung injury in preterm guinea pig
Authors: Langley S. C. , Kelly F. J.
Published: December 2017

Undernutrition may exacerbate hyperoxia-induced lung injury, a finding that may be of significance in the early clinical management of the premature human infant. Addressing this specific problem, we found that 72 h of food restriction in guinea pig pups delivered 3 days preterm increased mortality rates among pups exposed to 95% oxygen (8/18) and yet had no effect on 21% oxygen (air)-exposed pups (0/10). Reduced tolerance of hyperoxic conditions was not, however, associated with increased lung injury, assessed as pulmonary microvascular leakage. Pulmonary antioxidant enzyme activities [Cu,Zn superoxide dismutase (SOD), Mn SOD, glutathione peroxidase, and catalase] were unaltered by starvation or hyperoxia. Lung glutathione concentration was slightly decreased after food restriction, whereas hyperoxic exposure did not change either lung or bronchoalveolar lavage fluid glutathione concentrations or lung antioxidant enzyme activities. Increased susceptibility to the lethal effects of oxygen in the starved preterm guinea pig pup could not be attributed to a deficiency of pulmonary antioxidant defenses.
Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells
Authors: Madden J. A. , Vadula M. S. , Kurup V. P.
Published: December 2017

Smooth muscle cells (SMC) were isolated from cat cerebral arteries and three sizes of pulmonary artery (less than 200-, 200- to 600-, and greater than 800-microns diameter) and used within 72–96 h. Change in cell length in response to hypoxia and other vasoactive agents was measured in a specially constructed cell chamber on an inverted microscope. Pulmonary artery SMC responded to hypoxia differently according to artery size. SMC from less than 200- and 200- to 600-microns-diameter pulmonary arteries shortened 18.6 +/- 4.85 and 24.2 +/- 2.70%, respectively. However, SMC from greater than 800-microns-diameter pulmonary arteries shortened 0.81 +/- 0.44%. Cerebral artery SMC plated on a flexible polydimethyl siloxane membrane showed loss of tension during exposure to hypoxia. The shortening of SMC from the 200- to 600-microns pulmonary arteries was accompanied by myosin phosphorylation. SMC from greater than 800-microns-diameter pulmonary arteries and cerebral arteries contained myosin that did not phosphorylate during hypoxia. The SMC from both artery types responded to norepinephrine, serotonin, prostaglandin F2 alpha and indomethacin and exhibited alpha-adrenergic receptor population patterns similar to those of intact arteries. The pattern of hypoxic responses exhibited by these nondedifferentiated pulmonary and cerebral artery SMC supports the idea that, at least in the cat, the hypoxic sensor is located within the SMC.
Intact lung cytochrome P-450 is not required for hypoxic pulmonary vasoconstriction
Authors: Chang S. W. , Dutton D. , Wang H. L. , He L. S. , Stearns R. , Hui A. , Giacomini K. M. , Ortiz de Montellano P. , Voelkel N. F.
Published: December 2017

Lung cytochrome P-450 has been suggested to play a role in hypoxic pulmonary vasoconstriction. We reexamined this hypothesis using specific suicide substrate inhibitors of cytochrome P-450, 1-aminobenzotriazole (1-ABT), and chloramphenicol. In isolated, blood-perfused rat lungs, 1-ABT (0.5 mg/ml) and chloramphenicol (1 mg/ml) inhibited lung microsomal cytochrome P-450 (ethoxycoumarin O-deethylase) activity to 24 and 44% of control, respectively, and blunted hypoxia and angiotensin II-induced vasoconstriction. The depression of vascular contraction by 1-ABT was not due to an effect on calcium channels, since similar concentrations of 1-ABT had no inhibitory activity on electrical field-stimulated contractile response in rabbit papillary muscle strips. However, when 1-ABT was washed out of the lung after preincubation, the vascular reactivity to hypoxia and angiotensin II was restored despite persistent depression of lung cytochrome P-450 activity to 26% of control values. In isolated rat aortic and pulmonary arterial rings, addition of 1-ABT or metyrapone to the organ bath acutely reversed norepinephrine-induced contraction but preincubation with 1-ABT, metyrapone, or chloramphenicol had no effect on subsequent norepinephrine contractions. We conclude that 1-ABT inhibited lung vascular reactivity by a mechanism independent of cytochrome P-450 inhibition or calcium channel blockade and that an intact lung cytochrome P-450 system is not required for hypoxic pulmonary vasoconstriction in rat lungs.
Differential effect of platelet-derived growth factor on glycosaminoglycan synthesis by fetal rat lung cells
Authors: Caniggia I. , Post M.
Published: December 2017

Lung morphogenesis is in part regulated by extracellular matrix (ECM), and cytokines may indirectly control lung development via modulation of ECM. In the present study, we investigated the effect of different platelet-derived growth factor (PDGF) isoforms AA, AB, and BB on the synthesis of glycosaminoglycans (GAG) by fetal rat lung cells. Independent of gestational age, PDGF-BB, but not PDGF-AA or -AB, stimulated GAG synthesis of fetal lung fibroblasts. In contrast, GAG synthesis by epithelial cells was not affected by any of the PDGF molecules. The stimulatory effect of PDGF-BB on fibroblast GAG biosynthesis was dose (> 10 ng/ml) and time (> 8 h) dependent. The relative proportion of the individual GAG molecules was not altered by PDGF-BB exposure. Blockage of tyrosine kinase activity with staurosporine did abolish the effect of PDGF-BB on fibroblast GAG formation. Actinomycin D and cycloheximide did not abrogate the PDGF-BB effect, suggesting that no new RNA or protein synthesis is required. The proteoglycan synthesis blocker, beta-D-xyloside, also did not inhibit the PDGF-BB action on fibroblast GAG synthesis. These data suggest that the effect of PDGF on GAG synthesis is cell type and isoform specific and is most likely a direct effect on the GAG chain elongation enzymes.
Maturation of inositol 1,4,5-trisphosphate receptor binding in rabbit tracheal smooth muscle
Authors: Schramm C. M. , Chuang S. T. , Grunstein M. M.
Published: December 2017

The mechanisms underlying maturational changes in agonist-mediated airway contractility remain to be identified. Since the signal transduction process coupled to airway contraction involves the Ca(2+)-mobilizing action of the second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], the present study examined 1) whether Ins(1,4,5)P3 binding to its intracellular receptor varies with age in rabbit tracheal smooth muscle (TSM), and 2) whether Ca2+ exerts a modulatory effect on Ins(1,4,5)P3 receptor binding that is age dependent. [3H]Ins(1,4,5)P3 binding was assayed in crude TSM membrane preparations isolated from 2-week-old and adult rabbits. Monophasic Scatchard plots were obtained, reflecting a single binding site, with Hill coefficients of 0.988–0.996. The mean +/- SE values for receptor density (Bmax) and binding affinity [i.e., the dissociation constant (Kd)] were similar in the adult and immature tissues, wherein Bmax = 211 +/- 6 238 +/- 60 fmol/mg protein, respectively; and Kd = 14.1 +/- 0.2 and 11.6 +/- 1.2 nM, respectively. Addition of Ca2+ (10(-8)-10(-3) M) significantly modulated Ins(1,4,5)P3 binding, with opposing maturational effects. In adult tissues, Ca2+ produced dose-dependent inhibition of Ins(1,4,5)P3 binding to 59.5% control, whereas Ins(1,4,5)P3 binding in response to Ca2+ was significantly enhanced in the 2-week-old tissues to 183% control. Collectively, these observations demonstrate that the inherent basal binding characteristics of the Ins(1,4,5)P3 receptor are similar in maturing TSM, but that Ca2+ exerts opposite modulatory actions on Ins(1,4,5)P3 receptor binding in immature and adult tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Sodium- and chloride-conductive pathways in cultured mouse tracheal epithelium
Authors: Clarke L. L. , Burns K. A. , Bayle J. Y. , Boucher R. C. , Van Scott M. R.
Published: December 2017

The utility of a transgenic murine model of cystic fibrosis (CF) lung disease will likely depend on whether the mouse's proximal airway epithelium is characterized by Na(+)- and Cl(-)-conductive pathways comparable to those found in human airways. Therefore, the electrophysiological properties of primary cultures of mouse tracheal epithelium (MTE) were investigated using double-barreled, Cl(-)-selective microelectrodes. Epithelial cells isolated from freshly excised mouse tracheae formed confluent polarized monolayers on permeable collagen supports and developed significant transepithelial potential differences (approximately -10 mV) within 5-6 days postseeding. Under basal conditions, the MTE monolayers had an equivalent short-circuit current (Ieq) of -21.1 +/- 2.1 microA/cm2 and a transepithelial resistance of 424 +/- 49 omega.cm2. Intracellular measurements indicated that the apical (Va) and basolateral (Vb) membrane potential differences were -16.9 +/- 1.5 and -25.4 +/- 1.5 mV, respectively; apical membrane fractional resistance was 0.36 +/- 0.03; and intracellular Cl- activity was 56.1 +/- 2.3 mM. The presence of an apical Na+ conductance was demonstrated by luminal amiloride application (10(-4)M), which decreased Ieq, hyperpolarized Va, and increased the fractional resistance of the apical membrane. The presence of an apical Cl- conductance was demonstrated by substitution of Cl- with gluconate in the luminal bath, which decreased intracellular Cl- activity and increased the fractional resistance of the apical membrane. Luminal application of ATP (10(-4) M was also found to increase the rate of Cl- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential systemic and intrapulmonary TNF-alpha production in Candida sepsis during immunosuppression
Authors: Lechner A. J. , Tredway T. L. , Brink D. S. , Klein C. A. , Matuschak G. M.
Published: December 2017

Candida albicans (CA) increasingly causes septic shock, acute lung injury, and multiple organ damage during immunosuppression-related neutropenia. However, the effects of neutrophil (PMN) depletion on induction of tumor necrosis factor-alpha (TNF) by CA and its potential mediation of Candida septic shock are unknown. We hypothesized that reduced CA uptake by circulating PMNs during cyclophosphamide (CY)-related neutropenia sensitizes to TNF-mediated shock from enhanced cytokine production after phagocytosis by tissue macrophages. Absolute or relative neutropenia (PMNs < or = 500/microliters or 2,500/microliters) was modeled in rats by intraperitoneal CY 4-8 days before 10(9) yeast-phase CA (acute studies < or = 24 h, n = 81 animals) or 10(6) CA (subacute studies < or = 72 h, n = 25). Compared with neutrophil-sufficient rats, absolute neutropenia accelerated hemodynamic collapse and respiratory distress after 10(9) CA, and pulmonary microvascular permeability was amplified. These changes evolved without increased candidemia or elevations in bioactive or antigenic serum TNF, which remained low even at death (42.3 +/- 14.8 U/ml vs. 12.6 +/- 2.9 U/ml for CY + saline, means +/- SE, P = NS). In contrast, significant TNF in lung tissue and bronchoalveolar lavage fluid (BALF) was evident within 6 h in CY + 10(9) CA rats. Electron microscopy confirmed hyphal proliferation into alveoli from yeast within mononuclear cells in lung capillaries. Subacute disseminated candidiasis after 10(6) CA was not associated with elevated serum, lung, or BALF TNF. We conclude that differential systemic and intrapulmonary TNF production occur in CA septic shock during preexisting neutropenia, with compartmentalized TNF production in the lower respiratory tract accompanying yeast-mycelial transformation. Thus TNF is not an obligate mediator of acute candidemic shock or subacute disseminated candidiasis during CY-induced immunosuppression but may initiate pulmonary injury accompanying high-grade candidemia.
Murine pulmonary surfactant SP-A gene: cloning, sequence, and transcriptional activity
Authors: Korfhagen T. R. , Bruno M. D. , Glasser S. W. , Ciraolo P. J. , Whitsett J. A. , Lattier D. L. , Wikenheiser K. A. , Clark J. C.
Published: December 2017

SP-A is an abundant pulmonary surfactant-associated protein whose expression is controlled in a cell- and developmental-specific manner. To analyze regulation of SP-A gene expression, the murine SP-A gene was cloned and sequenced. The murine DBA/2J gene was approximately 4.6 kb in length comprised of six exons and five introns. Three mRNAs of 3.0, 1.7, and 0.9 kb were detected by Northern blot analysis of murine lung mRNA. Expression of the SP-A mRNAs was first detected at day 15 of gestation and increased dramatically before birth. A single SP-A gene was detected in the DBA/2J mouse genome. SP-A mRNA was detected in lung but not in the gastrointestinal tract, kidney, brain, liver, or heart and was detected by in situ hybridization in bronchial and alveolar cells of the murine lung. Primer extension analysis with a primer to exon three revealed two extension products differing by 9 bp in length, suggesting two closely juxtaposed transcription initiation sites. Chimeric gene(s) containing 1.8 kb of 5' SP-A sequences and the bacterial chloramphenicol acetyltransferase gene were expressed in pulmonary adenocarcinoma cells and in HeLa cells. Expression of the murine SP-A gene is partially controlled by non-cell-selective transcriptionally active sequences.
Characteristics of magnetically separated rat tracheal epithelial cell populations
Authors: Ford J. R. , Terzaghi-Howe M.
Published: December 2017

A simple magnetic separation technique has been developed using lectins specific for two of the cell types found in the tracheal mucosa. The resulting populations of basal and secretory cells were examined for proliferative capacity in culture and in vivo. The basal cell fraction contains the cells that proliferate in culture and respond to 12-O-tetradecanoylphorbol-13-acetate. In addition, the basal cell fraction exhibited the highest proliferative capacity in vivo during the first few days after transplantation. Repopulation of inverted intestinal segments showed that only with suspensions containing a significant proportion of basal cells could a mucociliary lining be established. Segments receiving the same number of unsorted or predominantly mucous secreting cells did not repopulate in vivo. These data support the hypothesis that the basal cell is most likely the stem cell of the tracheal epithelium.
Methylene blue inhibits neurogenic cholinergic vasodilator responses in the pulmonary vascular bed of the cat
Authors: McMahon T. J. , Kadowitz P. J.
Published: December 2017

The effects of methylene blue, an inhibitor of soluble guanylate cyclase, on pulmonary vasodilator responses to efferent vagal stimulation were investigated in the intact-chest cat under conditions of controlled blood flow and constant left atrial pressure. In animals pretreated with reserpine or phenoxybenzamine, under elevated tone conditions, efferent vagal stimulation at frequencies of 2-16 Hz caused stimulus-frequency-dependent decreases in lobar arterial pressure and pulmonary lobar vascular resistance. The vasodilator response to vagal stimulation was reproducible, blocked by atropine, and reduced by methylene blue. Intralobar infusion of methylene blue increased lobar arterial pressure without significantly altering systemic arterial or left atrial pressure. Methylene blue had no significant effect on vasodilator responses to isoproterenol, albuterol, atriopeptin III, lemakalim, adenosine, ATP, and pituitary adenylate cyclase-activating polypeptide-27 but significantly decreased vasodilator responses to acetylcholine, nitric oxide (NO), sodium nitroprusside, and the S-nitrosothiol, S-nitroso-N-acetyl-penicillamine. The effects of methylene blue on responses to vagal stimulation were reversible and were similar with the addition of a NO synthase inhibitor. The present data suggest that vasodilator responses to cholinergic nerve stimulation involve an increase in the production of guanosine 3',5'-cyclic monophosphate in the pulmonary vascular bed. These results provide additional evidence to support the hypothesis that neurogenically released acetylcholine induces endothelium-dependent, muscarinic, guanylate cyclase-mediated vasodilation.
Kinetics of urea exchange in air-filled and fluid-filled rat lungs
Authors: Effros R. M. , Murphy C. , Ozker K. , Hacker A.
Published: December 2017

Urea has been used as an indicator for estimating 1) the dilution of epithelial lining fluid (ELF) that occurs during bronchoalveolar lavage (BAL) and 2) the permeability-surface area product (PS) of the pulmonary endothelium to this solute. Because relatively little is known about how urea equilibrates with fluid in the lung tissues and airspaces, we have undertaken a study of the kinetics of movement from the vasculature into the tissues of isolated, perfused rat lungs. Although instillation of 5 ml of 154 mM saline into the airspaces of this preparation increased the calculated extravascular volume of 3HOH from 0.64 +/- 0.23 to 2.10 +/- 0.58 ml (SE, n = 6) during a single transit through the pulmonary circulation, it did not have a detectable effect on the distribution of [14C]urea in the lung tissues. However, leakage of [14C]urea into saline within the airspaces was detected during constant infusions: concentrations in the airspace fluid reached 1.1 +/- 0.2% of those in the perfusate by 90 s and 1.90 +/- 0.2% at 120 s, levels that would significantly reduce estimates of the dilution of ELF by BAL. In contrast, concentrations of 99mTc-diethylenetriaminepentaacetic acetic acid (DTPA) in the airspaces remained < 0.2% of those in the perfusate, suggesting that 99mTc-DTPA may be a superior indicator for estimating dilution of ELF by BAL.(ABSTRACT TRUNCATED AT 250 WORDS)
Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs
Authors: Boggaram V. , Margana R. K.
Published: December 2017

Surfactant protein C (SP-C), a hydrophobic protein of pulmonary surfactant is essential for surfactant function. Toward elucidating molecular mechanisms that mediate regulation of SP-C gene expression in rabbit lung, we isolated and characterized cDNAs encoding rabbit SP-C and studied the regulation of SP-C gene expression during fetal lung development and by adenosine 3',5'-cyclic monophosphate (cAMP) and dexamethasone in fetal lung tissues in vitro. We found that rabbit SP-C is highly homologous to SP-C of other species and is encoded by two mRNAs that differ by an insertion of 31 nucleotides in the 3' untranslated regions. SP-C mRNAs were classified into two types based on the nucleotide sequence; type I represents RNA without the 31 nucleotide insert and comprises approximately 80–90% of total SP-C mRNA content, whereas type II represents RNA containing the insert and comprises approximately 10–20% of total SP-C mRNA content. SP-C mRNAs were induced in a coordinate manner during fetal lung development and by cAMP and dexamethasone in fetal lung tissues in vitro. Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase [corrected] chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene.
Stimulation of bovine pulmonary artery endothelial cell ACE by dexamethasone: involvement of steroid receptors
Authors: Dasarathy Y. , Lanzillo J. J. , Fanburg B. L.
Published: December 2017

After exposure of bovine pulmonary artery endothelial cells in culture to 1 microM dexamethasone for 24–48 h, angiotensin-converting enzyme (ACE) activity of these cells was elevated severalfold. The increase in ACE activity was preceded by an increase in ACE mRNA, which could be detected after treatment of cells with dexamethasone for 4 h. When the increase in ACE mRNA produced by dexamethasone at 4 h was blocked by alpha-amanitin, an RNA polymerase II inhibitor, the increase in ACE activity detected at 48 h was inhibited. RU 38486, a steroid receptor antagonist, inhibited the elevation of both ACE activity and mRNA produced by dexamethasone. Among other steroids tested, only hydrocortisone, aldosterone and corticosterone-21-acetate had a stimulatory effect on ACE activity. RU 38486 effectively blocked the elevation in ACE activity produced by both aldosterone and dexamethasone, but had no effect on the elevation of ACE activity produced by other agents (3-isobutyl-1-methylxanthine, A23187, and dibutyryl adenosine 3',5' cyclic monophosphate). From these data we conclude that dexamethasone and certain other steroids with an hydroxyl group in the 11th carbon position regulate ACE gene expression of bovine endothelial cells at the transcriptional level via a steroid receptor-mediated mechanism.
PMA-activated neutrophils decrease pulmonary endothelial ectoenzyme activities in perfused rabbit lungs
Authors: Chen X. , Catravas J. D.
Published: December 2017

We have studied the effects of phorbol 12-myristate 13-acetate (PMA, 15 micrograms) on pulmonary endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] function in isolated rabbit lungs perfused in situ with platelet-poor (PPP) or platelet-rich (PRP) plasma in the presence or absence of neutrophils. Enzyme activities were estimated from the hydrolysis of the substrates [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) by ACE and 14C-labeled AMP by NCT during a single transpulmonary passage, using indicator-dilution techniques. In all treatment groups PMA produced a delayed increase in pulmonary vascular resistance to about three times the control value. PMA alone [in lungs perfused with PPP (n = 5 animals) or PRP (n = 6)] or neutrophils alone (in PPP-perfused lungs, n = 5) had no effect on enzyme activity. However, PMA-activated neutrophils (n = 5) decreased percent metabolism (%M) of [3H]BPAP from 87 +/- 3 to 77 +/- 4% (30 min after PMA), and the apparent first-order parameter [ratio of maximum activity to Michaelis constant (Amax/Km)] for ACE from 821 +/- 114 to 613 +/- 61 ml/min (30 min after PMA). At the same time, Km values of BPAP for ACE and AMP for NCT were elevated from 9.2 +/- 2.2 to 19.3 +/- 3 microM and 6.7 +/- 1.2 to 15.1 +/- 3.6 microM, respectively, whereas Amax (product of enzyme mass and rate of product formation, thus an index of perfused microvascular surface area) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)
PMA-activated neutrophils decrease ectoenzyme activities in rabbit aortic endothelial cells in culture
Authors: Chen X. , Tzanela M. , Baumgartner M. K. , McCormick J. R. , Catravas J. D.
Published: December 2017

We have studied the effects of phorbol 12-myristate 13-acetate (PMA)-activated neutrophils [polymorphonuclear leukocytes (PMN)] on endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] activities in cultured rabbit aortic endothelial cells (EC) with the use of [3H]benzoyl-Phe-Ala-Pro and 14C-labeled AMP as substrates, respectively, under first-order reaction conditions. PMA (1–1,000 ng/ml) or PMN alone had no effect on ACE activity. When PMA was incubated together with PMN (PMN/EC = 1.25:1 or 2.5 x 10(5) neutrophils/ml) for 4 h in Earle's salts, a PMA dose-dependent decrease in ACE activity was observed. Threshold PMA concentration was 2 ng/ml. At 8 ng PMA/ml, ACE activity was totally abolished, without any evidence of cytotoxicity, as inferred from release of 51Cr from prelabeled EC. The decrease in ACE activity was also dependent on PMN concentration and was detectable at PMN/EC values as low as 1.25:10 (0.25 x 10(5) PMN/ml). Inhibition of ACE occurred as early as 1 h after incubation (PMA 10 ng/ml, PMN/EC = 1.25:1). PMA alone caused a small but significant increase in NCT activity, whereas PMA coincubation with PMN produced a significant decrease in NCT activity (20–29%), which was PMA and PMN concentration independent. PMA increased PMN adherence to endothelial monolayers in a concentration-dependent manner. Pretreating PMN with monoclonal antibody 60.3 (raised against the adhesion glycoprotein CD18) or placing a 2-microns filter between PMN and EC, protected the decrease in ACE activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Platelets and a platelet-released factor enhance endothelial barrier
Authors: Haselton F. R. , Alexander J. S.
Published: December 2017

The role of platelets in the maintenance of endothelial barrier is examined in an in vitro model of the microvasculature. Human platelets (6,000/microliters) perfused through a cell column of endothelial-covered microcarriers decrease paracellular permeability of sodium fluorescein (mol wt 342) to 63% of baseline values. This effect is reversible and a second application and removal of platelets produces a similar response. This effect occurs within 5 min and reverses within 10 min after platelet removal. The reduction in permeability is not due to mechanical obstruction of endothelial junctions, since the number of recirculating platelets is not reduced and releasate from unstimulated 2-h platelet incubations also decreases permeability. Releasate from platelets stimulated with 0.1 U/ml of thrombin for 15 min have the same permeability reducing effect. In this system, the platelet factors serotonin (10(-3) M) and ADP (10(-4) M) have no effect on permeability. However, the platelet factors adenosine (10(-4) M), ATP (10(-5) M), and beta-agonists decrease permeability. None of these appear to account for platelet permeability activity, since activity is not blocked by agents directed against these mediators (adenosine deaminase, apyrase, 8-phenyltheophylline, or propranolol). The active factor(s) is stable at -20 degrees C, heat stable, sensitive to trypsin, and has an apparent molecular weight > 100. We conclude that unstimulated platelets release a factor(s) that enhances endothelial barrier in vitro and may be important in maintenance of the normal in vivo barrier.
Effect of in vivo TNF administration on superoxide production and PKC activity of rat alveolar macrophages
Authors: Mayer A. M. , Pittner R. A. , Lipscomb G. E. , Spitzer J. A.
Published: December 2017

After the intravenous injection of recombinant human tumor necrosis factor (TNF)-alpha (6.0 x 10(5) U) into rats, phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide anion (O2-) secretion was enhanced in suspensions of alveolar macrophages (AM phi) compared with saline-treated controls. No enhancement of spontaneous, A23187-stimulated, or opsonized zymosan (OPZ)-stimulated O2- release was observed. Intratracheal injection of TNF-alpha (6.0 x 10(5) U) did not result in enhancement of spontaneous or A23187-, OPZ-, or PMA-stimulated O2- release. Although no TNF-alpha was detected in the bronchoalveolar lavage fluid, small quantities of TNF-alpha and/or other mediators secreted by polymorphonuclear leukocytes present in the lung capillaries, veins, and arteries may have leaked into the alveolar compartment and primed AM phi for enhanced PMA-stimulated O2- release. The respiratory burst in macrophages and neutrophils appears to be dependent on the translocation of protein kinase C. We have demonstrated protein kinase C translocation in both TNF-alpha- and saline-treated AM phi on PMA stimulation, although no differences were observed due to TNF-alpha treatment.
ED1-containing cellular fibronectin release into lung lymph during lung vascular injury with postoperative bacteremia
Authors: Rizk T. , Rebres R. , Vincent P. , Lewis E. , McKeown-Longo P. , Saba T. M.
Published: December 2017

Fibronectin (Fn) exists in both a soluble and insoluble form. Soluble Fn in plasma and lymph is an opsonic molecule that enhances phagocytic host defense. Insoluble Fn in the subendothelial and extracellular matrix is an adhesive molecule that mediates cell adhesion to substratum. The extracellular matrix of tissues such as the lung contains a mixture of both plasma-derived fibronectin (pFn) as well as locally synthesized cellular fibronectin (cFn). cFn is antigenically related to pFn, but cFn has extra domains (ED1 and ED2) that do not exist in liver synthesized pFn. The purpose of this study was to determine whether ED1-Fn was released into lung lymph before an increase in lung vascular permeability following postoperative bacteremia. Male sheep (n = 8) with surgically prepared lung lymph fistulae were infused intravenously with a sublethal dose (5 x 10(8)) of Pseudomonas aeruginosa 2 days following surgery. Lymph flow (QL), lymph-to-plasma (L/P) total protein ratio, lung protein clearance (QL x L/P), and hemodynamics were measured over 48 h following bacterial challenge. The lymph and plasma ED1-Fn concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using a murine monoclonal antibody specific to the ED1 region of human cFn. There was a rapid rise of ED1-Fn flux in lung lymph which was evident 60 min after the start of bacterial infusion, resulting in a maximum three- to fourfold increase (P < 0.05) in this parameter. In contrast, the ED1-Fn concentration in plasma before bacterial infusion was less than lung lymph and it did not increase over the initial 6 h following bacterial infusion.(ABSTRACT TRUNCATED AT 250 WORDS)