bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of hypoxia on phospholipid metabolism in porcine pulmonary artery endothelial cells
Authors: Bhat G. B. , Block E. R.
Published: December 2017

The effect of exposure of porcine pulmonary artery endothelial cells to hypoxic (0% O2) and normoxic (20% O2) conditions for 24 and 48 h on phospholipid metabolism was studied. Sonicates prepared from endothelial cells that were exposed to 24 h of hypoxia showed significant increases in phospholipase A1 (91%), phospholipase C (75%), and diacylglycerol lipase (57%) activities. Hypoxic exposure of cells for 48 h caused an increase in diacylglycerol lipase activity (54%) only. Hypoxia also caused significant decreases in ATP levels and ATP-dependent arachidonyl coenzyme A (CoA) synthetase activity. Phospholipase A2, lysophosphatidylcholine acyltransferase, and diacylglycerol acyltransferase activities were not influenced by 24 or 48 h of hypoxia. When endothelial cells were prelabeled with [3H]arachidonic acid and then exposed to hypoxia, increased counts were recovered from the free fatty acid fraction of medium and from the cell fatty acid esters, lysophospholipids, diacylglycerols, and triacylglycerols. There was a concomitant decreased recovery of counts from cell phospholipids. These results indicate that hypoxic exposure of endothelial cells altered phospholipid metabolism by activating deacylation pathways and inhibiting reacylation via ATP-dependent arachidonyl CoA synthetase.
Exogenous glutathione protects endothelial cells from menadione toxicity
Authors: Chang M. , Shi M. , Forman H. J.
Published: December 2017

Administration of menadione (vitamin K3; 2-methyl-1,4-naphthoquinone) to cultured bovine pulmonary artery endothelial cells caused a dose- and time-dependent depletion of cellular ATP and depletion of intracellular glutathione (GSH). The toxicity of menadione was correlated with an increase of menadione-glutathione conjugate in the medium and with an increase in H2O2 generation. Recent studies have suggested that GSH may be useful as a pharmacological agent to prevent oxidant injury. In the present study, treatment with exogenous GSH prevented the loss of cellular GSH and ATP caused by menadione. Protection by extracellular GSH involved two mechanisms. In one mechanism, extracellular GSH was degraded by gamma-glutamyl transpeptidase, producing substrates for subsequent intracellular de novo GSH synthesis. We found that the protective effect of extracellular GSH was decreased by inhibiting gamma-glutamyl transpeptidase. In the other mechanism, GSH reacted in the medium with menadione to form a conjugate. Although formation of the menadione-glutathione conjugate within cells may contribute to menadione toxicity, addition of menadione-glutathione conjugate to the medium was found to be nontoxic to endothelial cells. Thus exogenous GSH protected endothelial cells by two mechanisms: maintenance of intracellular GSH and prevention of menadione entrance into cells.
Structure, function, and regulation of cellular tight junctions
Authors: Schneeberger E. E. , Lynch R. D.
Published: December 2017

The tight junction (TJ) is a dynamic structure that is controlled, in part, by the activity of the cytoskeleton. It has become abundantly clear that, in the presence of Ca2+, assembly of the TJ is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation of an organized network of TJ elements, the composition of which remains unknown. The TJ functions both as a barrier between two fluid compartments and, to a lesser extent, as a fence between apical and basolateral membrane domains. To meet the many physiological and pathological challenges to which epithelia and endothelia are subjected, the TJ must be capable of a rapid and coordinated response, which depends on complex regulatory mechanisms. The precise characterization of the mechanisms involved in the assembly and regulation of the TJ is an area of current active investigation. However, until the biochemical composition of this structure has been defined and its gene identified, the TJ will continue to be an elusive yet tantalizing challenge to the cell biologist.
Interleukin-1 alpha and -beta augment pulmonary artery transendothelial albumin flux in vitro
Authors: Campbell W. N. , Ding X. , Goldblum S. E.
Published: December 2017

Human recombinant interleukin-1 alpha (rIL-1 alpha) and -beta were studied to determine whether either could alter the permeability of bovine pulmonary artery endothelial cell monolayers. Endothelial cells were grown to confluence on filters mounted in chemotaxis chambers placed in wells. Barrier function of the monolayers was assessed by placing 14C-labeled bovine serum albumin ([14C]BSA) in the upper chamber and sampling the lower well for [14C]BSA. rIL-1 alpha induced a significant (P less than 0.01) dose- and time-dependent increase in transendothelial [14C]BSA flux. rIL-1 alpha exposures as brief as 30 min increased permeability, but the increased albumin transfer could not be demonstrated before 4 h after exposure. Exposures up to 6 h were reversible at 24 h. rIL-1 alpha induced significantly (P less than 0.01) greater increments in [14C]BSA flux than did equivalent exposures to rIL-1 beta. No important differences between bovine and human rIL-1 beta were demonstrated. Increased transendothelial flux could not be ascribed to either endothelial cytotoxicity or growth inhibition. There was no additive or synergistic relationship between rIL-1 alpha and human recombinant tumor necrosis factor-alpha. Our studies suggest that IL-1 alpha and -beta may play a role in the pathogenesis of pulmonary vascular leak.
Restricted diffusion of macromolecules by endothelial monolayers and small-pore filters
Authors: Schaeffer R. C. , Gong F. , Bitrick M. S.
Published: December 2017

We studied the size-selective permeability and restricted diffusion (Rd) properties of macromolecules across bovine pulmonary artery endothelial cell (BPAEC) or epithelial (LLC-PK1) monolayers grown on polycarbonate (PC) filter supports using fluorescein isothiocyanate-hydroxyethyl starch (FITC-HES, 16 A less than ae less than 180 A). Most BPAEC seeded at subconfluent density and grown for 3–6 days produced barriers with marked Rd. This characteristic was similar to that measured across PC filters with pore radii (rp) of 150 and 250 A without cells. Rd of LLC-PK1 monolayers was consistent with a transport pathway rp of much less than 75A. BPAEC monolayers prepared by supraconfluent seeding showed convective solute transport due to a significant number of incompletely formed intracellular junctions. Most monolayers grown to confluence, or a thin layer of collagen type I prevented this effect, Rd was enhanced when BPAEC monolalyers were grown on this collagen network. These data suggest that the subendothelial layers, which includes basal lamina, pericyte, and interstitial collagen, may act as series resistors with the endothelium to provide the Rd observed in the microvascular wall in vivo. This may explain why BPAEC monolayers grown to confluence without subendothelial layers in vitro showed Rd consistent with large (150–250 A) rp that was significantly greater than those modeled as the small (approximately 50 A) “pore” or 6-A fiber matrix of the in vivo capillary wall.
Superoxide responses of endothelial cells to C5a and TNF-alpha: divergent signal transduction pathways
Authors: Murphy H. S. , Shayman J. A. , Till G. O. , Mahrougui M. , Owens C. B. , Ryan U. S. , Ward P. A.
Published: December 2017

There is increasing evidence that endothelial cells respond to a variety of mediators. In the current studies rat pulmonary artery endothelial cells (RPAEC) responded to human recombinant C5a and tumor necrosis factor-alpha (TNF-alpha) with the generation of superoxide (O2-). RPAEC responsiveness was dependent on whether cells had been obtained from confluent or subconfluent cell monolayers. RPAEC responded to C5a and TNF-alpha in a dose-dependent manner, with increases in intracellular Ca2+ (Cai2+), formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], and generation of O2-. Optimal O2- responses occurred in cells that had been pretreated with the inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate, and O2- responses were allopurinol insensitive. Pertussis toxin pretreatment abolished the ability of C5a to cause increases in Ins(1,4,5)P3 and Cai2+ and formation of O2- but did not inhibit the changes in Cai2+ and formation of O2- after addition of TNF-alpha. The O2- response to C5a but not to TNF-alpha was abolished by pretreatment with the inhibitor of protein kinase C, staurosporine. These data indicate that signal transduction events in response to C5a and TNF-alpha were fundamentally different.
Tobacco smoke releases performed mediators from canine mast cells and modulates prostaglandin production
Authors: Thomas P. S. , Schreck R. E. , Lazarus S. C.
Published: December 2017

The role of an extract of tobacco smoke in activating mast cells was studied. With the use of isolated, canine mast cells as a model, we found that cigarette smoke solution (CSS) induced the release of the performed mediators histamine and tryptase from these cells in an energy- and temperature-dependent, non-cytotoxic manner. There was no requirement for extracellular calcium. Nicotine tartrate did not reproduce the effect of CSS. Interestingly, mast cells produced little prostaglandin D2 (PGD2) in response to the CSS, and there was a concentration-related inhibition of calcium ionophore A23187-induced PGD2 synthesis. This suggests at least two mechanisms acting on the mast cell: tobacco smoke can directly activate mast cells to release performed mediators and can simultaneously inhibit prostaglandin production. These observations suggest a mechanism by which mast cells may participate in the bronchospastic and proinflammatory changes seen in the lungs and airways of smokers.
Selective induction of intercellular adhesion molecule-1 by interferon-gamma in human airway epithelial cells
Authors: Look D. C. , Rapp S. R. , Keller B. T. , Holtzman M. J.
Published: December 2017

To evaluate the factors controlling migration of leukocytes into pulmonary airway epithelium, we determined the biochemical mechanisms responsible for the regulation of intercellular adhesion molecule-1 (ICAM-1) expression on cultured monolayers of human tracheal epithelial cells (HTECs) or SV40 virus-transformed human bronchial epithelial cells (BEAS-2B). Validation experiments with human umbilical vein endothelial cells (HUVECs) demonstrated little detectable ICAM-1 expression on unstimulated cells or on cells incubated with interferon-gamma (IFN-gamma), but HUVEC monolayers responded to interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) with significant increases in ICAM-1 and ICAM-1-dependent adherence of polymorphonuclear leukocytes (PMNs). HTEC monolayers also exhibited no significant basal ICAM-1 expression but, in contrast to HUVEC monolayers, had marked increases in ICAM-1 expression and ICAM-1-dependent PMN adherence only after incubation with IFN-gamma (and not after IL-1 beta or TNF-alpha) treatment. BEAS-2B cells also exhibited relatively selective IFN-gamma stimulation of ICAM-1 expression and ICAM-1-dependent PMN adherence but (like late passage HTEC) showed significant basal ICAM-1 expression. Differences in IFN-gamma effect on ICAM-1 levels between HUVEC and HTEC monolayers were not due to differences in number or responsiveness of IFN-gamma receptors, because both cell types exhibited a similar number of receptors and other IFN-gamma-dependent responses of HUVECs remained active. In all analyses, ICAM-1 mRNA levels correlated closely with detection of ICAM-1 on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
L-arginine restores endothelium-dependent relaxation in pulmonary circulation of chronically hypoxic rats
Authors: Eddahibi S. , Adnot S. , Carville C. , Blouquit Y. , Raffestin B.
Published: December 2017

We investigated whether loss of endothelial-derived relaxing factor (EDRF) activity in the pulmonary vessels of chronically hypoxic rats could be restored by pretreatment with L-arginine. We measured vasodilation to acetylcholine (ACh), calcium ionophore A23187, or linsidomine (Sin-1) under conditions of increased vascular tone induced by U-46619 (50 pmol/min), as well as vasoconstriction to endothelin-1 (ET) in isolated lungs pretreated with meclofenamate (3 microM). In lungs from normoxic (N) rats, in vitro L- or D-arginine (10(-3) M) did not alter vasodilation to the endothelium-dependent agents ACh (10(-9)-10(-6) M) and A23187 (10(-9)-10(-7) M), but NG-monomethyl-L-arginine (10(-3) M) completely abolished it. In lungs from rats exposed to 3 wk of hypoxia (H), vasodilation to ACh or A23187 was fully restored after in vitro L-arginine (10(-3) M) or N alpha-benzoyl-L-arginine (5 x 10(-5) M) but remained abolished after D-arginine, L-citrulline, L-ornithine, or L-argininosuccinic acid. In vivo pretreatment of H rats with L-arginine (300 mg/kg iv) 30 min before isolating the lung also restored vasodilation to A23187. Vasodilation to the endothelium-independent agent Sin-1 was similar in both groups of lungs and was not altered by in vitro L-arginine. L-arginine attenuated the increased pressor response to ET (300 pmol) of H rat lungs but had no effect in N rats. Our results demonstrate that loss of EDRF activity associated with hypoxic pulmonary hypertension may be reversed by supplying L-arginine.
Pulmonary SP-A enhances adsorption and appears to induce surface sorting of lipid extract surfactant
Authors: Schurch S. , Possmayer F. , Cheng S. , Cockshutt A. M.
Published: December 2017

The effect of surfactant concentration and supplementation with surfactant-associated protein A (SP-A) on the surface activity of lipid extract surfactant (LES) was examined using a captive bubble technique. Adsorption of LES is strongly concentration dependent over the range of 50-1,000 micrograms/ml. Addition of SP-A to LES at low concentrations in the presence of calcium dramatically increases the rate of adsorption. In quasistatic cycling experiments, samples containing SP-A require less compression to achieve low surface tensions even during the first compression cycle. Calculated film compressibilities at 15 mN/m indicate that SP-A alters the surfactant monolayer behavior such that in a small number of cycles the compressibility is indistinguishable from that of pure DPPC. Furthermore, SP-A reduces the incidence of bubble "clicking," suggesting a stabilization of the monolayer at low surface tensions. In dynamic cycling experiments, SP-A reduces compression of the film area required to achieve a low surface tension of approximately 1 mN/m. SP-A eliminated the plateau just below 25 mN/m normally observed during the compression phase with low concentrations of LES and the shoulder observed at approximately 35 mN/m during expansion. In the presence of SP-A and, to a lesser extent with high concentrations of LES, there is a marked lag in the increase in surface tension during the initial part of the dynamic expansion loop, with surface tensions remaining near 1 mN/m for approximately 10% of the increase in bubble area. The results indicate that SP-A enhances phospholipid adsorption during dynamic cycling and may enhance elimination of non-DPPC lipids during cycling.(ABSTRACT TRUNCATED AT 250 WORDS)
Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid
Authors: Joris L. , Quinton P. M.
Published: December 2017

We describe techniques which we developed to study the composition and regulation of airway surface fluid (ASF). ASF from isolated equine tracheal mucosa was absorbed onto or equilibrated with small strips of ashless filter paper after timed incubations in a water-saturated, 37 degrees C environment. After expression of the fluid from the paper, constant-volume aliquots of the samples were pipetted with a microvolumetric pipette on a filmed grid, together with standards containing known concentrations for Na, Cl, K, Ca, S, and P. Analysis of the microdroplets by dispersive X-ray microanalysis on a scanning electron microscope demonstrated that ASF from "unstimulated" tracheal tissue is significantly altered compared with the Ringer solution which was used to rinse the mucosal surface before incubation, in that Na is significantly decreased (approximately 130 mM), whereas K is significantly increased (approximately 16 mM). Furthermore, when a beta-adrenergic agonist (isoproterenol 10(-4) M) was added to the serosal surface, the Na concentration significantly increased to values which approached Ringer solution (146 +/- 5.3 mM) and were significantly different compared with the Na values obtained in unstimulated tissues. In contrast, Na remained unchanged after addition of a cholinergic agonist (metacholine, 10(-4) M) to the serosal side of the tissue; however, as with beta-adrenergic stimulation, most other minor elements and especially K significantly decreased compared with their values in unstimulated tissue. These results clearly suggest that ASF composition is clearly controlled by active transport of the airway epithelium and may be influenced by neurohumoral stimulation.
IGF-I regulation of elastogenesis: comparison of aortic and lung cells
Authors: Rich C. B. , Ewton D. Z. , Martin B. M. , Florini J. R. , Bashir M. , Rosenbloom J. , Foster J. A.
Published: December 2017

Rat neonatal aortic smooth muscle and pulmonary fibroblast cell cultures were exposed to different amounts of insulin-like growth factor-I (IGF-I, 1-100 ng/ml of medium) for 24 h. Aortic smooth muscle cells exhibited an increase in both steady-state levels of tropoelastin mRNA and soluble elastin with increasing amounts of IGF-I, suggesting that the growth factor is acting by increasing transcription or transcript stability. In contrast, pulmonary fibroblast cultures did not exhibit an elastogenic response to IGF-I because neither the steady-state levels of tropoelastin mRNA nor soluble elastin were affected. Transient transfection of the two cell cultures with a chimeric construct containing 500 bp of the elastin gene 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene showed that reporter activity was increased threefold in smooth muscle cells treated with IGF-I, whereas activity remains essentially the same in control and growth factor-treated pulmonary fibroblast cells. Receptor binding analyses revealed that both cell types possess the type I IGF-I receptor. Therefore, the lack of an elastogenic response in the lung cells cannot be attributed to lack of the appropriate receptor. These data, obtained in vitro with cell types that are principal producers of lung and aortic elastin, agree with results obtained in vivo. This agreement suggests that the regulation of elastin gene expression varies among cells derived from different tissues and furthermore provides model systems to investigate differential regulation of the elastin gene.
Binding and uptake of surfactant protein B by alveolar type II cells
Authors: Bates S. R. , Beers M. F. , Fisher A. B.
Published: December 2017

Surfactant protein B (SP-B, mol wt 9,000, reduced) is a low-molecular-weight hydrophobic protein found in organic extracts of lung surfactant. The interaction of iodinated bovine SP-B (125I-SP-B) and isolated rat alveolar type II cells was examined. The association of SP-B with the lung cells was time and temperature dependent; type II cells exhibited time-dependent binding (at 4 degrees C) and uptake (at 37 degrees C) of SP-B. Binding of phospholipid-poor 125I-SP-B was linearly related to the external SP-B concentration from 0.25 to 60 microgram/ml and was not inhibited by a 60-fold excess of unlabeled SP-B. However, the binding of 125I-SP-B reconstituted with bovine surfactant or with phospholipid-containing liposomes occurred through a high-affinity, saturable process and could be inhibited with unlabeled SP-B. By Scatchard analysis, half-maximum binding in the presence of surfactant occurred at 3.1 +/- 0.7 micrograms SP-B/ml. Saturable binding of SP-B reconstituted with surfactant also occurred with other cell types. The results indicate that SP-B was bound and internalized by type II cells. The apparent lack of specificity in the absence of phospholipid may have been due to the self-association of SP-B. The reconstitution of SP-B with phospholipid altered the binding of phospholipid-poor SP-B from a nonspecific process to a high-affinity process consistent with a cell surface binding site.
Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch
Authors: Liu M. , Skinner S. J. , Xu J. , Han R. N. , Tanswell A. K. , Post M.
Published: December 2017

On the basis of in vivo animal studies and on experiments of nature, it has been suggested that fetal breathing movements are essential for normal lung growth in utero. To study this hypothesis in vitro, we examined the effect of mechanical stretch on proliferation of fetal rat lung cells maintained in organotypic culture to provide a three-dimensional matrix. Initial studies demonstrated that stretch-mediated effects on cell division and DNA synthesis in such cultures were influenced by cell inoculation density, fetal calf serum concentration, and by the amplitude, frequency, periodicity, and duration of the applied stretch. After a 48-h exposure to an intermittent stretch pattern (5% elongation, 60 stretches/min for 15 min of each hour), cell number increased 10% (P less than 0.05), cell doubling time was reduced from 71 to 55 h (P less than 0.05), [3H]thymidine incorporation into DNA increased 61% (P less than 0.01), and the [3H]thymidine-labeling index increased 2.8-fold (P less than 0.001) compared with nonstretched controls. This effect did not appear to be mediated by prostaglandins or leukotrienes because the prostaglandin synthase inhibitors ibuprofen (2.5–50 microM) or BW 755C (5 microM), leukotriene biosynthesis inhibitors BW 755C (5 microM) or MK-886 (0.3 microM), and leukotriene D4 receptor antagonist MK-571 (0.3 microM) did not block stretch-mediated effects. We conclude that mechanical forces act directly to stimulate fetal rat lung cell growth and that these results are compatible with a significant role for fetal breathing in normal fetal lung growth.(ABSTRACT TRUNCATED AT 250 WORDS)
Mucins: structure, function, and role in pulmonary diseases
Authors: Rose M. C.
Published: December 2017

Mucins, major components of the extracellular mucus blanket that protect and lubricate mammalian epithelia, are high-molecular-mass glycoconjugates (154 to > or = 7,000 kDa) with hundreds of oligosaccharide chains in O-glycosidic linkages to a protein backbone. The apparent expression of more than one type of oligosaccharide core structure in mucins isolated from pathological material may reflect either inherent limitations in analysis, disease-related alterations in parameters affecting glycosylation and post-translational modifications (e.g., nucleotide-sugar concentrations, expression of specific glycosyltransferases, rates of transport through the endoplasmic reticulum and Golgi) or the activation of mucin protein genes that are more highly expressed in disease states with different glycosylation patterns. Recent studies have revealed the existence of a family of at least four human mucin proteins; MUC1, MUC2, MUC3, MUC4, each of which contains a variable number of tandem repeats that differ in sequence and size. Full-length sequences of cDNA clones encoding human mucin proteins are currently available only for MUC1 which, in contrast to most airway and intestinal mucins, is membrane associated and not secreted. Current information on mucin oligosaccharides and proteins is reviewed herein. More detailed knowledge of the protein and oligosaccharide structures of mucins will be important in identifying specific role(s) in health and disease, i.e., in the physiological functions of mucus.
Expression of aminopeptidase N in fetal rat lung during development
Authors: Jiang X. , Tangada S. , Peterson R. D. , Funkhouser J. D.
Published: December 2017

The ectopeptidase, aminopeptidase N, serves as a cell surface marker of the apical surface of the alveolar type II epithelial cell in adult lung. It is also present in fetal lung before differentiation of morphologically mature type II alveolar epithelial cells, suggesting that it is expressed by precursors of the type II cells. We have examined the mRNA coding for the aminopeptidase in adult and fetal lung and in mature type II cells and determined levels of mRNA and immunoreactive protein during fetal lung development. Comparison of the temporal patterns of steady-state levels of aminopeptidase mRNA and immunoreactive protein during development show that the expression of the protein is developmentally regulated and that expression is regulated, at least in part, at a pretranslational level. Both mRNA and immunoreactive protein levels increase severalfold on the final gestational day, suggesting that the function of the aminopeptidase may be associated with air breathing.
Cigarette smoking decreases bioactive interleukin-6 secretion by alveolar macrophages
Authors: Soliman D. M. , Twigg H. L.
Published: December 2017

Studies suggest smokers have decreased alveolar macrophage (AM) accessory cell (AC) function and a reduced incidence of immune-mediated lung diseases such as sarcoidosis. Impaired AM secretion of cytokines important in T-cell immune responses could explain this observation. We investigated production and secretion of interleukin-1 (IL-1) and interleukin-6 (IL-6) in smokers and nonsmokers. Lipopolysaccharide-induced AM IL-1 secretion in smokers was significantly reduced compared with nonsmoker AM. However, intracellular IL-1 in smoker AM was higher than in nonsmokers, suggesting that reduced IL-1 secretion was due to impaired release rather than reduced production. Smoker AM secreted significantly less bioactive IL-6 measured in a bioassay compared with nonsmoker AM. Intracellular IL-6 was virtually undetectable in both groups. In some smokers IL-6 production determined by immunoprecipitation was reduced. However, as a group antigenic IL-6 secretion determined by enzyme-linked immunoabsorbent assay was similar in smokers and nonsmokers, suggesting that smoker AM may cosecrete an inhibitor of IL-6 bioactivity. Indeed, AM supernatants from smokers inhibited B9 proliferation in response to maximal recombinant IL-6 stimulation, whereas supernatants from nonsmokers did not. We conclude that AM from smokers secrete less cytokines important in T-cell proliferation than AM from nonsmokers and suggest that for IL-6 this impairment is related to both decreased production of antigenic protein as well as cosecretion of an IL-6 inhibitor.
Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment
Authors: Voorhout W. F. , Veenendaal T. , Haagsman H. P. , Weaver T. E. , Whitsett J. A. , van Golde L. M. , Geuze H. J.
Published: December 2017

The hydrophobic surfactant protein B (SP-B) is synthesized in alveolar type II cells as a 40-kDa precursor protein that is processed via 39- and 23-kDa intermediates to the mature 8-kDa size. To determine the site of SP-B processing, the subcellular distribution of the precursor and mature forms of SP-B was investigated on ultrathin cryosections of human lung using two polyclonal antibodies that discriminate between precursor forms and the mature form of SP-B. An antibody against the human lysosomal membrane glycoprotein CD63 was used to identify organelles in the endosomal/lysosomal pathway. Precursor SP-B was present in the endoplasmic reticulum, the Golgi complex, and multivesicular bodies but was absent from lamellar bodies and plasma membrane. Mature SP-B was present in multivesicular bodies and lamellar bodies but almost completely absent in the endoplasmic reticulum and the Golgi complex. Semiquantitative evaluation of the immunogold labeling by counting the gold particles representing precursor or mature SP-B in the different compartments showed that the mature-to-precursor ratio was low in the endoplasmic reticulum and the Golgi complex (0.13 and 0.11, respectively), increased in the multivesicular bodies to 3.4, and was very high (65) in lamellar bodies. Multivesicular bodies and lamellar bodies contain the lysosomal membrane marker CD63 and are therefore part of the lysosomal pathway. These data strongly suggest that precursor SP-B is proteolytically processed to its mature 8-kDa form intracellularly in an endosomal/lysosomal compartment, most probably in multivesicular bodies.
Insulin inhibits beta-adrenergic responses in fetal rabbit lung in explant culture
Authors: Davis D. J. , Hickman J. M. , Lefebvre C. A. , Lyon M. E.
Published: December 2017

Infants of diabetic mothers are at increased risk of a number of problems at birth. Among these problems are increased risks of respiratory distress syndrome and transient tachypnea of the newborn. Because surfactant synthesis, surfactant secretion, and lung fluid resorption are all mediated in part by beta-adrenergic responses, we asked if excess insulin interferes with the beta-adrenergic response cascade in fetal lung. Lungs from fetal rabbits (26 day) were grown in explant culture in hormone-supplemented culture medium. The explants were harvested after 48 h exposure to hormones and processed for determination of beta-adrenergic receptor concentration, guanine nucleotide regulatory proteins (Gs, Gi), beta-agonist stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation, cAMP-dependent phosphodiesterase activity, and choline incorporation into phosphatidylcholine. Although insulin did not change the concentration of beta-adrenergic receptors, it decreased the ability of isoproterenol to stimulate cAMP generation. Increase in stimulation over basal was similar in explants treated with dexamethasone and dexamethasone plus insulin, but absolute levels of isoproterenol-stimulated cAMP were less in the explants treated with dexamethasone plus insulin. We speculate that insulin inhibition of cAMP generation may be important in the pathogenesis of the respiratory problems of infants of diabetic mothers.
Surfactant protein C is recycled from the alveoli to the lamellar bodies
Authors: Baritussio A. , Pettenazzo A. , Benevento M. , Alberti A. , Gamba P.
Published: December 2017

To clarify the life cycle of surfactant protein C (SP-C), we obtained alveolar surfactant from 3-day-old rabbits killed 15 h after the tracheal administration of 32P and L-[35S]methionine. Small amounts of this surfactant were administered into the tracheas of 3-day-old rabbits, which were killed after 1 min and 2 and 4 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine (DPPC) in lung lavage fluid and in a lamellar body-rich fraction isolated from the lung homogenate. We found that SP-C and DPPC are both taken up from the alveolar spaces and incorporated into lamellar bodies, but the time courses of reuptake are different. Our results indicate that in the alveoli SP-C is turned over faster than DPPC.
Formation of alveoli in rats: postnatal effect of prenatal dexamethasone
Authors: Massaro Gloria D. , Massaro Donald
Published: December 2017

Pages L37–L41: Gloria D. Massaro and Donald Massaro. “Formation of alveoli in rats: postnatal effect of prenatal dexamethasone.” Page L39: In Table 7 the values for the number of saccules or alveoli per rat should be x 10-6, not x 106. The corrected table appears below.

(See PDF)
Influence of extracellular matrix in tumor necrosis factor-induced increase in endothelial permeability
Authors: Partridge C. A. , Horvath C. J. , Del Vecchio P. J. , Phillips P. G. , Malik A. B.
Published: December 2017

We examined the possibility that alterations of the extracellular matrix (ECM) contribute to the tumor necrosis factor-alpha (TNF-alpha)-induced increase in endothelial monolayer permeability. Endothelial permeability to 125I-labeled albumin was determined using bovine pulmonary microvessel endothelial cell (BPMVE) monolayers grown to confluence on microporous (0.8 microns diam) gelatin- and fibronectin-coated polycarbonate filters. Treatment of BPMVE with TNF-alpha (10(2) to 10(4) U/ml for 4–24 h) produced concentration- and time-dependent increases in endothelial permeability that paralleled the changes in morphology from cobblestone to elongated cells and the formation of prominent intercellular gaps and actin stress fibers. We examined the role of ECM in these changes using filters coated with ECM made by the BPMVE. Fresh BPMVE seeded onto filters coated with ECM produced by TNF-alpha-treated BPMVE had two- to threefold higher 125I-albumin permeability values than BPMVE monolayers seeded onto filters coated with ECM from control cells (P < 0.05). BPMVE seeded onto ECM from TNF-alpha-treated BPMVE also developed intercellular gaps and centralized actin filaments characteristic of the TNF-alpha-treated BPMVE. This effect was not attributable to TNF-alpha adsorbed to ECM. Polyacrylamide gel electrophoresis of ECM extracted from BPMVE treated with TNF-alpha showed decreased fibronectin. These findings suggest that the TNF-alpha-induced increase in endothelial permeability involves the loss of fibronectin and remodeling of the ECM. The increase in endothelial permeability may be secondary to decreased endothelial cell-ECM contacts resulting in elongation of cells and formation of intercellular gaps.
Immunolocalization of antioxidant enzymes and isozymes of glutathione S-transferase in normal rat lung
Authors: Coursin D. B. , Cihla H. P. , Oberley T. D. , Oberley L. W.
Published: December 2017

Polyclonal antisera to manganese and copper-zinc superoxide dismutases, catalase, glutathione peroxidase (GPx), and isozymes of glutathione S-transferase (liver and placental isolates, GST-L and GST-P, respectively) were used to localize these enzymes in normal rat lung by immunostaining. Light-microscopic results, using an immunoperoxidase technique, were expanded on by electron-microscopic immunogold localization. The findings were consistent with previous biochemical work. However, both GPx and GST-P were predominantly localized to extracellular connective tissue of the lung. These findings demonstrate the basal antioxidant enzyme phenotypes for parenchymal lung tissue at light- and electron-microscopic levels. Significant components of enzymatic defense to oxidant stress are heterogeneously distributed throughout rat lung tissue including both epithelial cell surfaces and the extracellular matrix.
Interleukin-8 induces neutrophil accumulation but not protease secretion in the canine trachea
Authors: Jorens P. G. , Richman-Eisenstat J. B. , Housset B. P. , Graf P. D. , Ueki I. F. , Olesch J. , Nadel J. A.
Published: December 2017

The neutrophil enzyme elastase is a potent secretagogue of airway secretory cells, and elastase is present in high concentrations in sputum of patients with hypersecretion (e.g., cystic fibrosis, bronchiectasis). Interleukin-8 (IL-8), a recently discovered cytokine with potent neutrophil chemotactic properties in vitro, is also found in the sputum of these patients. We used an isolated tracheal segment in dogs in vivo to study the effect of IL-8 in causing neutrophil accumulation, elastase release, and secretion (by measuring lysozyme concentrations) in the luminal superfusate. IL-8 caused a potent time-dependent neutrophil accumulation at between 3 and 6 h. The effect was significant at 10(-9) and maximum at 10(-8) M. No increase in free elastase, cathepsin G, or lysozyme was detected in the superfusate. Thus, in contrast to previous studies showing that ragweed antigen causes the accumulation of neutrophil elastase which in turn causes lysozyme secretion, IL-8 causes neutrophil accumulation without granule secretion (or subsequent secretagogue activity). The findings were confirmed with dog and human neutrophils in vitro.
Effect of an anti-Mo1 MAb on ozone-induced airway inflammation and airway hyperresponsiveness in dogs
Authors: Li Z. , Daniel E. E. , Lane C. G. , Arnaout M. A. , O'Byrne P. M.
Published: December 2017

Ozone inhalation causes neutrophil migration into the airway and airway hyperresponsiveness in dogs. The leukocyte adhesion molecule Mo1 (CD11b/CD18) is a heterodimeric glycoprotein the expression of which is necessary for neutrophil adhesion to endothelium. To evaluate the contribution of Mo1 to ozone-induced neutrophil influx and airway hyperresponsiveness, six dogs were treated intravenously with an Anti-Mo1 monoclonal antibody (3.75 mg/kg in normal saline) that binds to both human and canine Mo1, or the diluent alone, 1.5 h before inhaling ozone (3 ppm for 30 min), or dry air. Airway responses to doubling doses of inhaled acetylcholine (ACh) were measured before and after inhalation of ozone. Neutrophil influx was assessed by bronchoalveolar lavage (BAL) performed after the second ACh inhalation. Treatment with anti-Mo1 prevented the ozone-induced influx of neutrophils into BAL. After diluent and inhaled dry air, the neutrophil count in BAL was 1.49 +/- 1.26 (SE) x 10(4) (5.0% of total cells). After diluent and inhaled ozone, the neutrophil count increased to 7.27 +/- 3.22 (SE) x 10(4) (22.6% of total cells) (P < 0.05). After anti-Mo1 and inhaled ozone, the neutrophil count was 1.48 +/- 0.62 (SE) x 10(4) (8.5% of total cells). Treatment with anti-Mo1 also significantly reduced the number of eosinophils in BAL after ozone. Ozone-induced ACh airway hyperresponsiveness was not prevented by treatment with anti-Mo1. These results indicate that expression of Mo1 is necessary for ozone-induced neutrophil migration into the airway lumen.
Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs
Authors: Boggaram Vijayakumar , Margana Ramgopal K.
Published: December 2017

Pages L634–L644: Vijayakumar Boggaram and Ramgopal K. Margana. “Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.” Page L634, left-hand column, in the abstract, the sentence beginning on line 22 should read: Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase 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. Page L634, left-hand column, paragraph 2, line 4 should read: SP-A [relative molecular weight ( Mr)≈35,000] and SP-D ( Mr ≈43,000) are hydrophilic proteins that are similar in having collagenous amino-terminus domain and noncollagenous lectinlike carboxy-terminus domain. SP-B ( Mr ≈8,000) and SP-C ( Mr ≈5,000) are hydrophobic proteins derived from larger precursor proteins by amino- and carboxy-terminus proteolytic processing.
Smooth muscle guanine nucleotides and receptor-effector coupling following inhibition of oxidative energy production
Authors: Coburn R. F. , Azim S. , Fillers W. S. , Baron C. B.
Published: December 2017

We measured total concentrations of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) in rabbit aortic smooth muscle under several different conditions. We computed free [GDP] and free GTP/GDP (using a Keq of 1.0 for the nucleoside diphosphate kinase reduction) under these conditions. At a time when muscle was contracted by 15 microM norepinephrine (NE) for 5 min under normoxia, [GTP], free [GDP], and free GTP/GDP were 0.29 +/- 0.03 mM, 3.5 microM, and 82, respectively. Following rapid inhibition of oxidative energy production during NE-evoked maintained force, which is associated with slow decreases in mean tissue [PCr], and [ATP] and force relaxation, [GTP] and free GTP/GDP were decreased at relaxation threshold to 0.22 +/- 0.02 (SE) mM and 43, respectively, and progressively fell further, paralleling decreases in force and [ATP] and [PCr]. There were marked decreases in the sum of GTP + GDP contents under conditions where muscle energy stores were decreased (i.e., low [PCr] + [ATP]). Similar data were obtained during a 50 mM KCl-evoked contracture. Free [GDP] increased from normoxic values of 3.5 microM to values as great as 6.0 microM at low energy store states. Free GDP was equivalent to 6% of total GDP under normoxia and increased to 16–21% of total GDP under conditions of low energy stores. Evidence was obtained that decreases in [GTP] or free GTP/GDP seen under conditions of low total energy stores were not sufficient to inhibit heterotrimeric G protein function and uncouple receptor-effector coupling.
Expression and secretion of transforming growth factor-beta by bleomycin-stimulated rat alveolar macrophages
Authors: Denholm E. M. , Rollins S. M.
Published: December 2017

Bleomycin-induced fibrosis in rodents has been used extensively as a model of human pulmonary fibrosis. The influx of monocytes observed during the early stages of fibrosis is at least partially regulated by the elaboration of chemotactic factors in the lung. Exposure of alveolar macrophages (AM phi) to bleomycin either in vivo or in vitro stimulated secretion of monocyte chemotactic activity (MCA). This MCA has been previously characterized as being primarily due to fibronectin fragments. The present experiments revealed that bleomycin also induced AM phi to secrete a second chemotactic factor, transforming growth factor-beta (TGF-beta). However, the TGF-beta secreted by macrophages was in latent form, since no TGF-beta activity was detected unless AM phi conditioned medium (CM) was acid-activated. After acidification, chemotactic activity in CM from AM phi stimulated with bleomycin in vitro was increased by 3.6, whereas activity in AM phi CM from fibrotic rats increased by 2 and that of a bleomycin-stimulated AM phi cell line increased by 1.6. This acid-activatable chemotactic activity was inhibited by antibody to TGF-beta. Bleomycin-stimulated AM phi s secreted significantly more TGF-beta than did unstimulated controls. Further, in vitro exposure of AM phi to bleomycin induced TGF-beta mRNA expression in a time- and concentration-dependent manner, with maximal mRNA being detected following a 16-h incubation with 1 microgram/ml bleomycin.
Formation of alveoli in rats: postnatal effect of prenatal dexamethasone
Authors: Massaro Gloria D. , Massaro Donald
Published: December 2017

Pages L37–L41: Gloria D. Massaro and Donald Massaro. “Formation of alveoli in rats: postnatal effect of prenatal dexamethasone.” Page L39: Tables 4–7 show incorrect sex. The 14-day-old pups were male. The sex of the pups is correct in Tables 1–3.
Ionic currents in rat pulmonary and mesenteric arterial myocytes in primary culture and subculture
Authors: Yuan X. J. , Goldman W. F. , Tod M. L. , Rubin L. J. , Blaustein M. P.
Published: December 2017

The electrophysiological properties of cultured single vascular smooth muscle (VSM) cells from rat pulmonary (PA) and mesenteric (MA) arteries were studied using the whole cell patch-clamp technique. Cells were studied at 3–7 days as primary cultures, or were replated after 10–20 days and subcultured for 2–5 days. In the standard physiological bath solution (containing 1.8 mM Ca2+), and with 125 mM K+ + 10 mM ethylene glycol-bis(beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA)-filled pipettes, both PA and MA primary cultured cells had high input resistances (mean = 2-3 G omega) and resting membrane potentials of about -40 mV. The cells were clamped at a holding potential of -70 mV. Depolarization to -20 mV or more evoked a transient inward current (Iin) that was eliminated in Ca(2+)-free bath solution; this indicates that Iin was carried by Ca2+. Iin was substantially smaller in subcultured cells from both PA and MA. Depolarization also activated three components of outward current (Iout) in primary cultured PA and MA cells: a rapidly inactivating transient component (Irt), a slowly inactivating transient component (Ist), and a steady-state (noninactivating) component (Iss). All three components of Iout were inhibited to varying degrees by 5 mM 4-aminopyridine and were eliminated by replacing intracellular K+ with Cs+, but were only minimally affected by removal of extracellular Ca2+. These results suggest that this Iout was carried by K+ and was voltage gated. Little external Ca(2+)-dependent Iout was observed under these conditions, but a substantial Ca(2+)-dependent component was seen when the EGTA concentration in the pipettes was reduced to 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine-induced inhibition of vagal motoneuron excitability: receptor subtype and mechanisms
Authors: Marks J. D. , Donnelly D. F. , Haddad G. G.
Published: December 2017

With the use of intracellular techniques and a medullary slice preparation, we examined the changes in cellular and membrane properties of single adult rat vagal motoneurons during exposure to potent and specific agonists and antagonists of adenosine A1 and A2 receptors. A1 receptor stimulation increased input resistance, but markedly reduced spontaneous neuronal firing and increased rheobase. A1 agonists also increased the amplitude of the action potential afterhyperpolarization (AHP) in a dose-dependent manner. Prior treatment with A1 antagonists blocked these electrophysiological effects. Blockade of Ca2+ entry with Co2+ abolished the AHP increase. Synaptic blockade with both tetrodotoxin and high Mg2+, low Ca2+ solutions prevented the increase in input resistance. A2 receptor stimulation was without effect. Perfusion with adenosine in the presence of dipyridamole caused effects similar to A1 agonist stimulation, but no effect in the absence of dipyridamole. These results show that adenosine decreases vagal motoneuron excitability by stimulating A1 receptors. Our data also support the idea that the adenosine-induced decrease in excitability is presynaptic in nature. In addition, the AHP increase may be mediated by enhanced Ca2+ entry into the postsynaptic neuron.
Activation of phospholipase D in rat type II pneumocytes by ATP and other surfactant secretagogues
Authors: Rooney S. A. , Gobran L. I.
Published: December 2017

Surfactant phospholipid secretion can be stimulated by a variety of agonists acting via a number of signal-transduction mechanisms. To determine whether phospholipase D has a role in surfactant secretion, we examined phosphatidylethanol formation in response to surfactant secretagogues in primary cultures of rat type II cells. Phosphatidylethanol formation was stimulated by ATP, 12-O-tetradecanoylphorbol-13 acetate (TPA), and dioctanoylglycerol, surfactant secretagogues that also activate protein kinase C. Surfactant secretagogues that act via other signaling mechanisms had no effect on phosphatidylethanol formation. The effect of ATP on phosphatidylethanol formation was dependent on time, with the maximum stimulation being achieved in approximately 10 min. It was also dependent on ATP concentration. The ATP concentration eliciting 50% of the maximum effect (EC50) was 2.45 x 10(-6) M. This was similar to the EC50 reported for ATP stimulation of surfactant secretion. ATP analogues also stimulated phosphatidylethanol formation with a potency order generally similar to that reported for surfactant secretion. The effects of ATP, TPA, and dioctanoylglycerol were antagonized by protein kinase C inhibitors. We speculate that activation of protein kinase C either directly by TPA and dioctanoylglycerol or indirectly subsequent to phosphoinositide-specific phospholipase C activation by ATP leads to initial stimulation of surfactant secretion as well as activation of phospholipase D. The action of phospholipase D on cellular phospholipids then leads to further generation of diacylglycerols, continued activation of protein kinase C, and sustained surfactant secretion.