bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogen peroxide-induced pulmonary vasodilation: role of guanosine 3',5'-cyclic monophosphate
Authors: Burke-Wolin T. , Abate C. J. , Wolin M. S. , Gurtner G. H.
Published: December 2017

Hydrogen peroxide (H2O2), but not tertbutyl hydroperoxide, produces a concentration-dependent vasodilation of the pulmonary circulation in isolated saline perfused rabbit lungs when pulmonary arterial pressures (PAP) are raised with the thromboxane analogue U-46619. This vasodilation was enhanced in the presence of indomethacin, suggesting that H2O2 possesses both a prostaglandin-mediated constrictor and an additional dilator mechanism. In isolated rabbit intrapulmonary arteries the endothelium did not alter the dose-dependent relaxation of arterial rings to H2O2, and indomethacin enhanced the relaxant response of the peroxide. The decrease in PAP and relaxation of isolated pulmonary arteries observed with H2O2 was attenuated with 10 microM methylene blue, an inhibitor of soluble guanylate cyclase activation. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP)-selective phosphodiesterase inhibitor, enhanced the vasodilation or relaxation to the peroxide in both preparations. These changes were not endothelium dependent. Inhibition of the cGMP-associated endothelium-derived relaxant factor (EDRF) with nitro-L-arginine, did not alter relaxation of arterial rings to peroxide. Thus H2O2 appears to produce pulmonary vasodilation through the activation of guanylate cyclase and accumulation of cGMP. Both H2O2 and EDRF may function as tonic stimulators of guanylate cyclase in the pulmonary circulation and contribute to the maintenance of low basal pressures.
Comparison of response of bovine and porcine pulmonary arterial endothelial cells to monocrotaline pyrrole
Authors: Reindel J. F. , Hoorn C. M. , Wagner J. G. , Roth R. A.
Published: December 2017

Monocrotaline (MCT)-induced vascular injury in liver and lung may be caused by interaction of MCT metabolites such as monocrotaline pyrrole (MCTP) with vascular cells. Responses of bovine and porcine pulmonary artery endothelial cells (BECs and PECs, respectively) to a single administration of MCTP were compared. MCTP caused a delayed and progressive release of lactate dehydrogenase (LDH) activity from BECs and a gradual decrease in monolayer cellularity. Surviving cells became markedly hypertrophic. PECs were less sensitive to the cytolytic effects of MCTP, showing minimal cell detachment and little release of LDH activity. However, monolayer cellularity, as assessed by PEC enumeration, decreased in a dose-dependent manner. Hypertrophy of surviving PECs was less pronounced than in BECs. MCTP caused enhanced release of prostacyclin from monolayers of BECs and PECs exposed to 10 micrograms MCTP/ml, and concentrations of 0.5 microgram/ml or greater caused equivalent reduction in colony-forming efficiency in both cell types. In summary, whereas BECs were more susceptible to the cytolytic and hypertrophic effects of MCTP, BECs and PECs responded similarly with regard to prostacyclin release and were equally sensitive to the cytostatic effects of this compound.
Surfactant lipid uptake and secretion in type II cells in response to lectins and secretagogues
Authors: Griese M. , Gobran L. I. , Rooney S. A.
Published: December 2017

Secretion of surfactant phospholipids can be inhibited by the surfactant-associated protein SP-A. SP-A was reported to stimulate lipid uptake by type II cells, whereas surfactant secretagogues were reported to have the same effect in isolated perfused lungs. We examined the effect of such secretagogues on uptake of liposomes containing L-alpha-[2-palmitoyl-9,103H(N)]-dipalmitoyl phosphatidylcholine in primary cultures of type II cells. As SP-A contains a lectinlike domain and other lectins were reported to inhibit surfactant secretion, we also examined the effect of such lectins on lipid uptake. At concentrations at which they maximally stimulate phosphatidylcholine secretion in type II cells, several secretagogues had no effect on liposome uptake. Maclura pomifera agglutinin (MPA) stimulated uptake approximately 10-fold with a concentration eliciting 50% maximum stimulation (EC50) of 17 micrograms/ml. The effect of MPA on uptake was considerably greater than that of SP-A. However, although the stimulatory effect of ATP on phosphatidylcholine secretion was almost completely antagonized by SP-A, it was maximally inhibited only 75% by MPA. The concentration eliciting 50% maximum inhibition (IC50) for MPA inhibition of secretion was 0.5 micrograms/ml. Concanavalin A, another lectin, had no effect on lipid uptake but completely inhibited secretion. These data show that a lectin other than SP-A can stimulate phospholipid uptake by type II cells cultured on plastic and suggest that surfactant secretion and reuptake are independently regulated processes.
Basal activation of protein kinase C in rat alveolar macrophages: implications for arachidonate metabolism
Authors: Peters-Golden M. , McNish R. W. , Sporn P. H. , Balazovich K.
Published: December 2017

Alveolar macrophages (AM) exhibit numerous functional differences from other mononuclear phagocyte populations, even though they are derived from a common circulating monocytic precursor. Yet no differences in fundamental signaling mechanisms uniquely expressed by AM have been elucidated to date. Protein kinase C (PKC) is one signal transduction mechanism thought to have an important role in regulating macrophage function and about which little information exists for AM. This study was undertaken to assess the state of activation of PKC in cultured resident rat AM compared with resident rat peritoneal macrophages (PM) and the means by which active PKC regulates arachidonic acid (AA) metabolism in the two cell types. As assessed by a histone phosphorylation assay, resting AM, in contrast to PM, exhibited constitutive activation of PKC as evidenced by localization of a majority of PKC activity to the membrane fraction. Ionophore A23187-stimulated release and metabolism of AA were attenuated by depletion of or inhibition of cellular PKC activity in AM but not in PM. In contrast, A23187-stimulated AA metabolism was augmented by activation of PKC to a greater extent in PM than in AM. Results from both cell types indicated that the 5-lipoxygenase pathway was particularly upregulated by PKC activation. We conclude that activation of PKC occurs uniquely during macrophage residence in the alveolar space and that this property as well as the downregulation of PKC which results have profound consequences for the regulation of at least one important macrophage function, the synthesis of bioactive eicosanoids.
Endothelin increases [Ca2+]i, protein phosphorylation, and O2-. production in human alveolar macrophages
Authors: Haller H. , Schaberg T. , Lindschau C. , Lode H. , Distler A.
Published: December 2017

The goal of the present study was to investigate whether endothelin (ET) increases O2-.production in alveolar macrophages and to establish which second messengers are activated by ET. We measured the effects of ET on cytosolic free calcium concentration [Ca2+]i, protein phosphorylation, and O2-.production in human alveolarmacrophages (HAM). Human macrophages were obtained by bronchoalveolar lavage. [Ca2+]i was measured by the fura-2 method both in cell suspensions and in isolated single macrophages. Protein phosphorylation was assessed by gel electrophoresis and autoradiography after labeling the cells with 32P. ET increased [Ca2+]i in a dose-dependent manner (50% effective concentration = 1 x 10(-7)M). At a concentration of 10(-6)M ET, [Ca2+]i rose from basal values of 121 +/- 23 to 456 +/- 41 nM. This increase was comparable with the increase of [Ca2+]i induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine and platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine). The initial rise in [Ca2+]i and the sustained phase were significantly reduced in calcium-free medium (1 mM ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and after preincubation of HAM with the calcium channel blocker nitrendipine (10(-7)M). Exposure of 32P-labeled HAM to ET for 1 min induced the phosphorylation of a group of 48-kDa proteins and of a 35-kDa protein. The ET-induced 32P incorporation into the 48-kDa proteins was less pronounced than the 12-O-tetra-decanoylphorbol-13-acetate or PAF-induced response (168 +/- 24 vs. 356 +/- 68 and 278 +/- 61% of control).(ABSTRACT TRUNCATED AT 250 WORDS)
Inositol 1,4,5-trisphosphate, inositide flux rates and pool sizes during smooth muscle relaxation
Authors: Baron C. B. , Pompeo J. N. , Coburn R. F.
Published: December 2017

Decreases in D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content and changes in inositol phospholipid contents occurred during the time of atropine-induced relaxation of swine tracheal smooth muscle contracted with 55 microM carbachol. Decrease in Ins(1,4,5)P3 occurred in a pool which makes up 40% of the total content of this inositol phosphate and which has access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. A 50% decrease in this pool occurred at 16 s after addition of atropine and within 6-10 s after inhibition of phospholipase C (PLC). The maximal fall in Ins(1,4,5)P3 occurred at a time when force had only decreased 30% of the maximal response. A phosphatidylinositol 4-phosphate (PIP) pool linked to muscarinic receptor-activation increased 160% after addition of atropine, the maximal response occurring at a time when relaxation was 80% complete. The mechanisms for this increase were the maintained formation of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) even though PIP2 hydrolysis was inhibited and the apparent chemical equilibrium between PIP and PIP2.
Distinct effects of oxygen on surfactant protein B expression in bronchiolar and alveolar epithelium
Authors: Wikenheiser K. A. , Wert S. E. , Wispe J. R. , Stahlman M. , D'Amore-Bruno M. , Singh G. , Katyal S. L. , Whitsett J. A.
Published: December 2017

Hyperoxia causes severe lung injury in association with altered expression of surfactant proteins and lipids. To test whether oxygen induces surfactant protein B (SP-B) expression in specific respiratory epithelial cells, adult B6C3F1 and FVB/N mice were exposed to room air or 95% oxygen for 1–5 days. Northern blot analysis demonstrated an 8- to 10-fold increase in SP-B mRNA after 3 days that was maintained thereafter. In situ hybridization localized SP-B mRNA to bronchial, bronchiolar, and alveolar epithelial cells. Hyperoxia was associated with increased SP-B mRNA, noted primarily in the bronchiolar epithelium and decreased SP-B mRNA in the alveolar epithelium. After 5 days, central regions of lung parenchyma were nearly devoid of SP-B mRNA, while SP-B mRNA was maintained in alveolar cell populations close to vascular structures. To determine whether increased bronchiolar expression of SP-B mRNA during hyperoxia was a specific response, the abundance of CC10 mRNA (a Clara cell protein) was assessed. CC10 mRNA was detected in tracheal, bronchial, and bronchiolar, but not alveolar epithelium and was decreased upon exposure to hyperoxia. Immunocytochemistry demonstrated that SP-B proprotein was detected in bronchial, bronchiolar, and alveolar epithelial cells with staining increased in the bronchial and bronchiolar epithelium upon exposure to hyperoxia. SP-B gene expression in the respiratory epithelium is regulated at a pretranslational level and occurs in a cell specific manner during hyperoxic injury in the mouse.
Bombesin stimulates human nasal mucous and serous cell secretion in vivo
Authors: Baraniuk J. N. , Silver P. B. , Lundgren J. D. , Cole P. , Kaliner M. A. , Barnes P. J.
Published: December 2017

Bombesin, gastrin-related peptide (GRP), and related peptides sharing the common carboxyterminal sequence stimulate lactoferrin (serous cell marker) and glycoconjugate (mucous cell and goblet cell marker) release from human nasal mucosal explants in vitro. In vivo, GRP released from trigeminal sensory nerves may act upon GRP-bombesin binding sites on respiratory epithelial cells and submucosal glands. To determine whether GRP-bombesin can stimulate nasal secretion in vivo, bombesin was administered to eight normal subjects by unilateral, topical administration. Secretions from both nostrils were collected for measurement of total protein, lysozyme, hexose-containing glycoconjugates, and albumin (marker of vascular permeability). Baseline secretions contained 72.0 +/- 17.3 micrograms/ml of total protein, 14 +/- 2 micrograms/ml of lysozyme, 113 +/- 44 micrograms/ml of hexose-containing glycoconjugates, and 7.8 +/- 3.4 micrograms/ml of albumin. Hexose-containing glycoconjugate secretion was significantly increased after 1 nmol (385 +/- 63 micrograms/ml, P less than 0.001 by analysis of variance), 10, 100, and 1,000 nmol of bombesin, but the secretion was not dose dependent. Significant lysozyme (24 +/- 3 micrograms/ml, P less than 0.05) and total protein (155 +/- 23 micrograms/ml, P less than 0.01) secretion occurred after 1,000 nmol. No statistically significant changes in albumin secretion occurred at any dose. Saline had no significant effects on secretion. Therefore, bombesin stimulated secretion from submucosal glands and possibly epithelial cells in the human nose without affecting vascular permeability.
Lipopolysaccharide stimulates de novo synthesis of PGH synthase in human alveolar macrophages
Authors: Pueringer R. J. , Hunninghake G. W.
Published: December 2017

Lipopolysaccharide (LPS)-stimulated human alveolar macrophages (HAMs) produce large amounts of prostaglandin (PG) E2 for up to 72 h. The mechanism of this enhanced and prolonged metabolism of arachidonic acid to PGE2 is unknown. To determine whether LPS-stimulated HAM PGE2 production is due in part to an increase in the new synthesis of the first committed enzyme, PGH synthase, we measured PGE2 formation, PGH synthase activity, and newly synthesized PGH synthase at 2, 6, and 24 h after LPS-stimulation. PGE2, measured by radioimmunoassay, was not increased at 2 h but was increased at 6 and 24 h after stimulating HAMs with LPS. Unstimulated HAMs did not produce PGE2. Likewise, the activity of PGH synthase extracted from HAMs was not increased at 2 h but was increased at 6 and 24 h after LPS stimulation. Cycloheximide and actinomycin D markedly inhibited PGE2 production in LPS-stimulated HAMs. Newly synthesized PGH synthase measured by immunoprecipitating 35S-labeled PGH synthase was not detected at 2 h but was detected at 6 and 24 h after stimulation. The parallel increases in PGE2 production, PGH synthase activity, and newly synthesized PGH synthase coupled with the dependence of PGE2 on the ability of the alveolar macrophage to synthesize protein suggest that LPS-stimulated HAM PGE2 production is in part regulated by the de novo synthesis of PGH synthase.
Expression of peroxidase activity in rat tracheal epithelial cells associated with Mycoplasma pulmonis
Authors: Kinbara M. , Ueda T. , Hirai K.
Published: December 2017

Endogenous peroxidase activity has not been localized in the tracheal mucosal epithelial cells of specific pathogen-free (SPF) rats. After natural infection with Mycoplasma pulmonis in SPF rats, peroxidase activity became localized to the endoplasmic reticulum, nuclear envelope, and Golgi apparatus of tracheal ciliated or mucous secretory cells. Some secretory cells occasionally had peroxidase-positive secretory granules. At 1 wk M. pulmonis was found to attach to these epithelial cells, which then showed positive peroxidase activity at 2 wk. Serum antibody titers against M. pulmonis were positive at 5 wk. These results suggest that virulent mycoplasma infection and interaction with the tracheal epithelial cells trigger the de novo expression of peroxidase activity, which seems to play a role in mucosal anti-microbial defense mechanisms.
Platelet-derived growth factor and growth-related genes in rat lung. II. Effect of exposure to 85% O2
Authors: Han R. N. , Buch S. , Freeman B. A. , Post M. , Tanswell A. K.
Published: December 2017

The expression of platelet-derived growth factor (PDGF), its receptor, and related genes was studied in the lung tissue of rats exposed to air or 85% O2. PDGF-B chain mRNA was increased by 6 days and PDGF B-type receptor mRNA was increased by 4 and 6 days of exposure to 85% O2. Despite a continued increase of cell division, both PDGF-B chain and B-type receptor mRNAs had returned to control values by 14 days of exposure to 85% O2. PDGF-A chain mRNA was unaffected by exposure to 85% O2. Nuclear runoff transcription analysis confirmed increased transcription of PDGF-B chain mRNA, whereas Western blot analysis of lung homogenates suggested consequent increased translation of PDGF-B chain mRNA to PDGF-BB at 7 days of exposure to 85% O2. Combined immunocytochemistry and autoradiography localized PDGF-BB to the major site of cell division, the pulmonary interstitium. We speculate that the early pulmonary fibroblast hyperplasia observed following exposure to 85% O2 is mediated by increased PDGF-B chain gene expression and may also be mediated by changes in PDGF B-type receptor gene expression.
Effects of indomethacin on PMA-induced pulmonary endothelial enzyme dysfunction in vivo
Authors: Chen X. , Orfanos S. E. , Catravas J. D.
Published: December 2017

We investigated the effects of phorbol myristate acetate (PMA) on metabolic pulmonary endothelial ectoenzyme dysfunction. Anesthetized rabbits were placed on total heart bypass, and the single-pass transpulmonary metabolism of [3H]benzoyl-Phe-Ala-Pro (BPAP) by endothelial-bound angiotensin-converting enzyme (ACE) and [14C]adenosine 5'-monophosphate (AMP) by 5'-nucleotidase (NCT) was calculated before and after PMA (10 micrograms/kg iv), a dose that does not produce histologically evident endothelial damage. Under conditions of partial microvascular recruitment (blood flow = 400 ml/min through the entire lung), PMA, but not the vehicle, significantly reduced substrate utilization of both BPAP and adenosine 5'-monophosphate (AMP) and increased the apparent Michaelis constant (Km) values of ACE for BPAP, indicative of metabolic dysfunction. These changes were completely prevented by pretreatment with indomethacin. Under conditions of near full microvascular recruitment (blood flow = 640 ml/min through the left lung only), PMA similarly reduced substrate utilization and increased the apparent Km of ACE for BPAP. In this case, however, indomethacin failed to prevent the observed PMA-induced metabolic dysfunction. We conclude that PMA alters endothelial ectoenzyme substrate metabolism independently from changes in pulmonary blood flow; indomethacin appears to antagonize the effects of PMA under conditions of partial microvascular recruitment only, perhaps by diverting flow to previously unperfused, unexposed to PMA, and hence metabolically healthy vessels.
Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism
Authors: Liedtke C. M.
Published: December 2017

A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)
Cloning and expression of an airway epithelial 12-lipoxygenase
Authors: De Marzo N. , Sloane D. L. , Dicharry S. , Highland E. , Sigal E.
Published: December 2017

Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region. On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA. Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase. Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase. This is the first sequence of an epithelial lipoxygenase from any species. A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase. We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme.
Changes in lung eicosanoid content during normal and abnormal transition in perinatal lambs
Authors: Abman S. H. , Stenmark K. R.
Published: December 2017

To study the potential contribution of eicosanoids in maintaining high vascular tone in utero or in modulating resistance during the normal or abnormal transition of the pulmonary circulation, we performed serial measurements of hemodynamic parameters and lung eicosanoid content in perinatal sheep with and without pulmonary hypertension. Prostacyclin (6-keto-PGF1 alpha), thromboxane (TxB2), and leukotriene contents were measured in fetal lung liquid (FLL), bronchoalveolar lavage fluid (BALF), and lung tissue samples. Leukotriene content was barely detectable above background in FLL samples from 11 late-gestation fetuses, and lung leukotriene content in fetal lung was one-third of that measured in maternal lung (P less than 0.01). Tissue samples from serial lung biopsies obtained before and after cesarean-section delivery of late-gestation lambs demonstrated increased lung prostacyclin content after delivery (P less than 0.04), but no changes in total leukotriene or thromboxane contents were found. In an experimental model of perinatal pulmonary hypertension, prostanoid and leukotriene content of FLL obtained immediately before delivery were not different from an age-matched nonhypertensive control group. Leukotriene content in BALF and lung tissue obtained 2 h after delivery was not increased in the hypertensive group. TxB2, but not 6-keto-PGF1 alpha, content was higher in lung tissue from the hypertensive group (P less than 0.02). Thus lung leukotriene content did not decrease from fetal values after cesarean-section delivery, and the lipoxygenase pathway was not significantly activated with delivery after chronic intrauterine pulmonary hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)
Permeability: theory vs. practice in lung research
Authors: Peterson B. T.
Published: December 2017

An increase in permeability of the pulmonary endothelium or epithelium to proteins and other macromolecules is an important part of many types of lung injury. Assessment of lung alveolar-capillary membrane integrity has been the focus of many investigations, but no single method is best for all conditions. Methods using in vitro preparations, such as cell monolayers, provide the most accurate estimates of biophysical parameters defined from membrane theory, but these models may not accurately represent the in vivo condition. Methods using in vivo preparations, such as indicator dilution techniques and the sheep lung-lymph preparation, are more accurate representations of the clinical condition, but considerable assumptions must be made to estimate the parameters of permeability. The most productive studies of lung permeability make use of at least two methods carefully selected according to their strengths and weaknesses. The challenge is to select the right tools for the job and to interpret the results in light of the weaknesses of the methods used. This will lead to a better understanding of the role of altered permeability in the pathophysiology of lung injury.
Differential sensitivity to fibrinogen inhibition of SP-C- vs. SP-B-based surfactants
Authors: Seeger W. , Gunther A. , Thede C.
Published: December 2017

Inhibition of surfactant function by plasma-derived proteins is assumed to occur under conditions of alveolar protein leakage. We investigated surface properties and sensitivity to the inhibitory capacity of fibrinogen (Fbg) of artificial surfactants in a pulsating-bubble surfactometer. Phospholipid-fatty acid mixtures (PLM) with or without hydrophobic apoproteins [natural bovine surfactant proteins (SP) B and C, recombinant human SP-C] were used. Without Fbg, all apoprotein-based surfactants exhibited rapid absorption facilities and reduced surface tension to near zero values under dynamic compression. Fbg caused a dose-dependent reduction of adsorption kinetics and dynamic surface tension-lowering properties of all surfactant preparations, but there was a marked rank order of “Fbg sensitivity”: PLM without apoprotein being the most sensitive and SP-B-based PLM the least. Fbg sensitivity of recombinant SP-C-based PLM could be lowered dose dependently by adding small amounts of SP-B. For further support of a putative role of SP-B in resistance to Fbg inhibition, calf lung surfactant extract was incubated with monoclonal antibody against SP-B. Without Fbg, anti-SP-B had little influence. However, sensitivity to the inhibitory effect of Fbg was markedly and dose dependently increased by anti-SP-B but not by control immunoglobulin. We conclude that SP-B-based surfactant preparations display markedly lower susceptibility to Fbg inhibition than SP-C combinants.
Surfactant phospholipids: synthesis and storage
Authors: Batenburg J. J.
Published: December 2017

Pulmonary surfactant, a complex consisting of 90% lipids and 10% specific proteins, lines the alveoli of the lung and prevents alveolar collapse and transudation by lowering the surface tension at the air-liquid interface. Dipalmitoylphosphatidylcholine constitutes approximately 50% of the surfactant lipids and is primarily responsible for the surface tension-lowering property of the surfactant mixture. This phospholipid, together with the other surfactant phospholipids, is produced at the endoplasmic reticulum of the alveolar type II epithelial cells. The characteristic lamellar bodies in these cells serve as storage depot for the surfactant before this is secreted onto the alveolar surface. This article reviews the pathways via which the surfactant lipids are synthesized, our current knowledge of the regulation of these pathways, and what is known about intracellular traffic of phospholipids from their site of synthesis to the lamellar bodies.
Characterization of L-glutamine transport by type II alveolar cells
Authors: Hautamaki R. D. , Greene B. , Souba W. W.
Published: December 2017

This study characterized the sodium-dependent transport of L-glutamine (L-Gln) by rat type II alveolar cells. Uptake of 50 microM glutamine (Gln) was determined and found to be linear for at least 15 min. The sodium-dependent velocity represented greater than 80% of the total uptake at all time points. Kinetic studies of sodium-dependent Gln uptake at concentrations between 0.005 and 8 mM showed uptake to occur via two saturable transport systems, a high-affinity carrier [Michaelis constant (Km) = 259 +/- 19 microM, maximum velocity (Vmax) = 0.942 +/- 0.08 nmol.mg protein-1.30 s-1] and a low-affinity transporter (Km = 4.96 +/- 1.2 mM, Vmax = 2.98 +/- 0.19 nmol.mg protein-1.30 s-1). Uptake of Gln via the low-affinity system was nearly completely blocked by 10 mM 2-methylaminoisobutyric acid (MeAIB), and increasing concentrations of Gln almost totally inhibited transport of MeAIB, indicating the presence of system A. Further inhibition studies of the high-affinity transporter showed marked inhibition by serine, cysteine, and nonradioactive Gln. Lithium did not substitute for sodium, strongly suggesting that L-Gln was not transported by system N. Furthermore, transport of Gln by the high-affinity carrier was not affected by hormones or by changes in external pH. We conclude that sodium-dependent L-Gln transport by rat type II alveolar cells occurs predominantly via system A and system ASC.
Surfactant protein A expression is delayed in fetuses of streptozotocin-treated rats
Authors: Guttentag S. H. , Phelps D. S. , Stenzel W. , Warshaw J. B. , Floros J.
Published: December 2017

The content and distribution of the 26-to 38-kDa surfactant protein (SP-A) and its mRNA were determined in fetuses of control and streptozotocin (STZ)-treated Sprague-Dawley rats using immunohistochemistry, RNA blotting, and in situ hybridization. Female rats were treated with 50 mg/kg STZ before mating, and the fetuses were killed at fetal days 18-21 or on neonatal days 1 and 2 (day of birth = end of day 22). SP-A was barely detectable on fetal day 18 in controls and easily detected by fetal day 21. In the STZ group, SP-A was decreased compared with controls at fetal days 18-21. However, by neonatal days 1–2, there were no significant differences in SP-A levels between groups. SP-A mRNA was detectable at fetal day 18 in controls, but it was decreased in the STZ group at day 18-21 (P less than 0.02) and differences were no longer detected by neonatal days 1–2. SP-A and SP-A mRNA accumulated with advancing gestational age in both groups until neonatal days 1–2. The differences in SP-A and SP-A mRNA levels in the two groups diminished with advancing age but remained significant at fetal day 21. These data suggest that STZ-induced diabetes interferes with normal expression of SP-A in the developing fetal lung.
Human tracheobronchial epithelial cells direct migration of lung fibroblasts in three-dimensional collagen gels
Authors: Infeld M. D. , Brennan J. A. , Davis P. B.
Published: December 2017

Normal airway morphogenesis and repair after injury depend in part on the interaction between the mesenchymal and epithelial cells in the tracheobronchial tree. We cultured human lung fibroblasts between layers of type I collagen gel and examined sections through these three-dimensional matrices to assess fibroblast migration. The migration assay used in these experiments allowed simultaneous assessment of directed and random fibroblast migration as well as cell number. We tested the hypothesis that human tracheobronchial epithelial (HTBE) cells direct the migration of fibroblasts. When fibroblasts were cultured alone, migration was nearly equivalent in the upper and lower collagen layers. When HTBE cells were plated on the upper collagen lattice, there was a net migration of fibroblasts toward the HTBE cells. The differential migration was evident early in culture but became maximal after 1 wk. Differences increased at higher HTBE cell inoculation densities. No epithelial chemokinetic or mitogenic influence was evident: total cell migration and total fibroblast number were not significantly different between the control and coculture sections. HTBE fibronectin production may contribute to directed migration because fibronectin, added to the upper lattice, reproduced a portion of the directed migration seen in coculture. Our data support the hypothesis that epithelial cells direct fibroblast migration.
Bovine tracheal serous cell secretion: role of cAMP and cAMP-dependent protein kinase
Authors: Finkbeiner W. E. , Widdicombe J. H. , Hu L. , Basbaum C. B.
Published: December 2017

The role of adenosine 3',5'-cyclic monophosphate (cAMP) and protein phosphorylation during beta-adrenergic receptor stimulation of bovine tracheal gland serous cells was investigated in vitro. Isoproterenol, a beta-adrenergic agonist, increased the secretion of 35S-labeled molecules. Intracellular cAMP levels were increased within 1 min after stimulation of bovine tracheal gland serous cells with isoproterenol. The dose-response relationship for isoproterenol-stimulated generation of cAMP correlated with the dose-response relationship for isoproterenol-stimulated secretion of 35S-labeled molecules. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated both isoproterenol-evoked secretion of 35S-labeled molecules and the production of intracellular cAMP, and the beta-adrenergic receptor antagonist propranolol completely blocked both effects. The secretory response of the cells to isoproterenol could be mimicked by the cAMP analogues 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate. Activity of cAMP-dependent kinase was measured in soluble and particulate cell extracts. cAMP effected the state of phosphorylation of proteins associated with the soluble but not the particulate fraction. These studies are consistent with the hypothesis that beta-adrenergic stimulation of secretion from bovine tracheal gland serous cells occurs via a cAMP-mediated pathway and that one of the molecular events in this pathway is cAMP-dependent protein phosphorylation.
Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension
Authors: Stelzner T. J. , O'Brien R. F. , Yanagisawa M. , Sakurai T. , Sato K. , Webb S. , Zamora M. , McMurtry I. F. , Fisher J. H.
Published: December 2017

The role of endogenous circulating or locally produced endothelin-1 (ET-1) in pulmonary hypertensive states is unknown. To investigate this we measured ET-1 levels and preproendothelin-1 (prepro-ET-1) mRNA expression at various ages in control Sprague-Dawley (SDR) rats and in fawn-hooded rats (FHR), a strain which develops idiopathic pulmonary hypertension. Although serum ET-1 levels were similar in SDR and FHR, we found twofold increases in FHR whole lung homogenate ET-1 levels by radioimmunoassay. Coexisting threefold increases in preproET-1 mRNA expression were found in FHR lungs by densitometric analysis of Northern blots and by filter hybridization, suggesting the increase in lung ET-1 was due to enhanced intrapulmonary production of the peptide. To test whether the increase in lung preproET-1 mRNA was primary or secondary to established pulmonary hypertension, we compared preproET-1 mRNA expression prior to development of pulmonary hypertension in fetal (19 day gestation) and neonatal (5 day old) FHR and SDR. Despite similar right ventricular size in SDR and FHR, preproET-1 mRNA was already elevated in neonatal FHR lungs. Furthermore, we found no increase in lung preproET-1 mRNA or ET-1 levels in adult SDR with an equivalent degree of pulmonary hypertension due to chronic hypoxia, implying that the increases in ET-1 production in FHR were not a common consequence of all pulmonary hypertensive states. The functional significance of these observations remains unclear but raises the possibility of a role for ET-1 in the pathophysiology of pulmonary hypertension in the FHR.
Effects of TNF-alpha and phorbol ester on human surfactant protein and MnSOD gene transcription in vitro
Authors: Whitsett J. A. , Clark J. C. , Wispe J. R. , Pryhuber G. S.
Published: December 2017

Tumor necrosis factor-alpha (TNF-alpha) and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) decrease the synthesis of surfactant proteins association with decreased SP-A and SP-B mRNA. Nuclear run-on assays were utilized to test whether the inhibitory effects of TNF-alpha and TPA were associated with changes in surfactant protein gene transcription. SP-A gene transcription was inhibited by both TNF-alpha and TPA as assessed by nuclear run-on assays. Inhibitory effects of both agents on SP-A gene transcription were readily detected within 6 h after exposure and persisted for 24 h. While TNF-alpha and TPA decreased cellular SP-B mRNA content, transcription of the SP-B gene was not influenced by these agents. In contrast to the inhibitory effects of TPA and TNF-alpha on SP-A and SP-B mRNAs, steady-state mRNA and rate of transcription of human manganese superoxide dismutase (MnSOD) were increased by both agents. The time course and extent of increased MnSOD gene transcription by TNF-alpha and TPA were distinct. Transcription of the human beta-actin gene was not altered by either agent. The inhibitory effects of TPA and TNF-alpha on SP-A expression in pulmonary adenocarcinoma cells are associated with the inhibition of SP-A gene transcription. Loss of SP-B mRNA was not accompanied by decreased SP-B gene transcription. Actinomycin D blocked the inhibitory effects of TNF-alpha and TPA on SP-A and SP-B mRNA, supporting a role for posttranscriptional events in the modulation of the expression of the surfactant proteins SP-A and SP-B.
Binding, uptake, and localization of surfactant protein B in isolated rat alveolar type II cells
Authors: Breslin J. S. , Weaver T. E.
Published: December 2017

This study reports the ability of rat alveolar type II cells to internalize mature bovine surfactant protein B (SP-B) in vitro. Isolated type II cells were incubated with labeled SP-B, and binding and internalization were studied biochemically and morphologically. Biochemical analyses demonstrated a time-dependent association of 125I-labeled SP-B with type II cells; binding steadily increased through 4 h and then remained constant through 20 h of incubation. The association of [3H]SP-B with type II cells was characterized via light and electron microscopic autoradiography. Significant quantities of [3H]SP-B were found at the plasma membrane, in the endocytic pathway, and in lamellar bodies. The pathway of SP-B internalization was not altered by the presence of whole rat surfactant; however, the quantity of SP-B internalized into lamellar bodies was increased. 3[H]SP-B was not associated with coated pits and colocalized with horseradish peroxidase (HRP), consistent with receptor-independent internalization. Cell-associated SP-B was not degraded and was detected in lamellar bodies undergoing exocytosis. These results suggest that SP-B may follow a recycling pathway similar to that previously reported for surfactant phospholipids.
Staphylococcus aureus alpha-toxin permeabilizes the basolateral membrane of a Cl(-)-secreting epithelium
Authors: Ostedgaard L. S. , Shasby D. M. , Welsh M. J.
Published: December 2017

Apical membrane ion channels control the rate of transepithelial electrolyte transport in many epithelia. One way to study such channels in their native location, the apical membrane, is to eliminate the resistance of the basolateral membrane to ion flow. Then the opening and closing of apical channels can be measured as a transepithelial current, free from the influence of basolateral membrane transport processes. To develop a method that would permeabilize an epithelial basolateral membrane to ions and nucleotides, we examined the effect of Staphylococcus aureus alpha-toxin on the Cl(-)-secreting T84 epithelial cell line. alpha-Toxin permeabilized the basolateral, but not the apical membrane to Cl-, adenosine 3',5'-cyclic monophosphate (cAMP), and GTP. However, the integrity of signal-transduction pathways, the regulation of apical membrane Cl- channels, and the transepithelial resistance remained intact. In the course of examining the effect of ATP, we found that the basolateral membrane contained purinergic receptors that both stimulated Cl- secretion on their own and, at high concentrations, inhibited cAMP-induced Cl- secretion. These effects of extracellular ATP were eliminated after prolonged exposure to ATP, suggesting receptor downregulation. In addition, depletion of intracellular ATP following permeabilization prevented cAMP-dependent regulation of apical Cl- channels. We conclude that alpha-toxin may prove to be a useful tool for studying the regulation and properties of apical membrane ion channels.
In vitro responses of ovine intrapulmonary arteries and veins to endothelin-1
Authors: Toga H. , Ibe B. O. , Raj J. U.
Published: December 2017

We determined responses of third-generation intrapulmonary arteries and veins of fetal, newborn, and adult sheep to endothelin-1 (ET) and the role of endothelium and cyclooxygenase metabolites in ET effects in adult sheep lung vessels. Presence of endothelium in vessel rings was confirmed by response to endothelium-dependent vasodilators, acetylcholine or bradykinin. Vessel tension induced by ET was expressed as a percentage of tension induced by 100 mM KCl. We found that arteries and veins contracted to 10(-9) to 10(-6) M ET in a dose-dependent manner. Veins exhibited greater sensitivity to ET than arteries in all age groups. Arteries and veins of adult sheep lungs were more sensitive to ET than those of fetal and newborn lambs. In adult sheep lung vessels, pretreatment with indomethacin (5 x 10(-6) M) and SQ 29548, a thromboxane A2-prostaglandin H2 receptor antagonist (10(-5) M), significantly attenuated venous contraction to ET; arterial contraction was unaffected. Denuding vessels of endothelium did not affect responses to ET. We conclude that, in ovine lungs, veins are more sensitive to ET than arteries and that developmental differences in pulmonary vascular responses to ET exist.
Prostaglandin D2 production and identification of prostaglandin H synthase within canine mast cell granule
Authors: Thomas P. S. , Wilson A. N. , Schreck R. E. , Lazarus S. C.
Published: December 2017

We have identified the presence of functional prostaglandin H synthase (PGH synthase, E.C. 1.14.99.1, or cyclooxygenase) within canine mast cell granules by demonstrating the generation of prostaglandin (PG) D2 from isolated and purified granules incubated with substrate as arachidonic acid or stimulated with calcium ionophore, A23187. This confirms the presence of both enzyme and substrate within the granule. Localization of PGH synthase to the granule was confirmed by immunoblotting of the pure granule preparation and by immunocytochemistry using the whole cell. In functional studies, colchicine, a microtubule polymerization inhibitor, caused a fall of up to 70%, both in the amount of histamine released and in the amount of PGD2 generated. This suggests either that functional PGH synthase is closely associated and coactivated with granules or that there is an independent association of this enzyme with the microtubule system. Release of the preformed and newly formed mediators of the mast cell appear to be closely linked, and prevention of degranulation may therefore attenuate the effects of both classes of mediators.
Localization of alveolar surfactant clearance in rabbit lung cells
Authors: Rider E. D. , Ikegami M. , Jobe A. H.
Published: December 2017

Localization of surfactant phospholipid clearance in lung cells was investigated in vivo in rabbits using radiolabeled dipalmitoylphosphatidylcholine (DPPC) and 1,2-dihexa-decyl-sn-glycero-3-phosphocholine (DPPC-ether), a phospholipase A1- and A2-resistant analogue of DPPC. After intratracheal injection of liposomes of the labeled lipids associated with unlabeled surfactant, adult rabbits were killed in groups of three to five at 0, 4, 12, and 24 h with recovery of bronchoalveolar lavages for alveolar macrophages and surfactant. Type II cells and tissue-associated macrophages were isolated on Percoll gradients following elastase and trypsin digestion of the lungs. Radiolabel recoveries as saturated phosphatidylcholine were measured in alveolar wash, alveolar macrophages, lung tissue, and the type II cell and mixed cell bands from the Percoll gradients. Cost accounting of label demonstrated similar recoveries at 0 h, but significantly more DPPC-ether compared with DPPC in cells at later times, indicating ineffective degradation of the DPPC-ether. Internalization of the lung tissue-associated labels into cells was time dependent. At all times, greater than 65% of the cell-associated labels were recovered in type II cells, indicating the primary role for these cells in clearing alveolar surfactant phospholipid in vivo. The total contribution of alveolar macrophages to the overall clearance was approximately 20%.
Control of the beat cycle of respiratory tract cilia by Ca2+ and cAMP
Authors: Lansley A. B. , Sanderson M. J. , Dirksen E. R.
Published: December 2017

Beat frequency and the duration of the constituent recovery, effective, and rest phases of the beat cycle of respiratory tract cilia were measured photoelectronically before and after manipulation with ionomycin or isoproterenol. Both ionomycin, acting by increasing intracellular Ca2+, and isoproterenol, acting by elevating intracellular adenosine 3',5'-cyclic monophosphate (cAMP), increased beat frequency by reducing the duration of the three phases of the ciliary beat cycle in a similar manner. The addition of increasing concentrations of ATP to ciliated cells permeabilized by exposure to saponin caused a pattern of phase reduction indistinguishable from that observed in whole cells. The beat frequency of permeabilized cells was slower than that of whole cells and insensitive to changes in Ca2+ and cAMP. Ca2+ and cAMP may regulate ciliary beat frequency by acting at a common site within intact cells, possibly regulating the rate at which the axoneme can use ATP or the availability of ATP to the axoneme.
Influences of endogenous and exogenous TGF-beta on elastin in rat lung fibroblasts and aortic smooth muscle cells
Authors: McGowan S. E.
Published: December 2017

The factors that regulate elastin production during neonatal lung development have not been elucidated. Previous investigations suggested that transforming growth factor-beta (TGF-beta) increases elastin production by neonatal rat lung fibroblasts (LF). We examined whether this effect of TGF-beta was unique to these cells or was evident in other neonatal cells, which constitutively produce elastin, or in cells from adults, whose constitutive elastin production is low. We have quantitated soluble elastin, elastin mRNA, and TGF-beta production in primary cultures of smooth muscle cells (SMC) and LF from neonatal and adult rats and have examined the alterations in soluble elastin and elastin mRNA that result from adding 100 pM exogenous TGF-beta 1 to these cultures. Unsupplemented cultures of LF and SMC obtained from neonatal rats exhibited higher steady-state levels of elastin mRNA and contained more soluble elastin in their culture medium than did cells from adult animals. When neonatal LF were supplemented with 100 pM TGF-beta 1, they showed a significant increase in the soluble elastin content of their culture medium and their steady-state elastin mRNA. Neither LF obtained from adults nor SMC obtained from neonatal or adult rats significantly increased their soluble elastin or steady-state elastin mRNA after the addition of exogenous TGF-beta. When neonatal LF were supplemented with an anti-TGF-beta neutralizing antibody, the soluble elastin content of the culture medium decreased significantly. These data suggest that the responsiveness of elastin expression to TGF-beta is limited to neonatal LF and that endogenous TGF-beta influences elastin production by neonatal LF.