bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of alveoli in rats: postnatal effect of prenatal dexamethasone
Authors: Massaro G. D. , Massaro D.
Published: December 2017

We administered a glucocorticosteroid (dexamethasone) or its diluent to pregnant rats on gestation days 17, 18, and 19. In male offspring we determined the lung's gas exchange surface area (S(a)), the average volume (v) of gas exchange saccules at age 2 days and alveoli at age 14 days, and their number (N) on these days. S(a), v, and N at 2 days and v at 14 days were not affected by the prenatal administration of dexamethasone. However, S(a) and N were lower in 14-day-old pups from dexamethasone-treated dams than in pups from diluent-treated dams. In separate experiments we found the responsiveness to prenatal dexamethasone, as a depressor of the postnatal increase in S(a), appeared earlier in female than male fetuses; it was present in female but not in male fetuses on days 16–18 and was found in male fetuses on days 17–19. We conclude 1) prenatal administration of dexamethasone diminishes the postnatal increase in S(a), 2) responsiveness to this action of dexamethasone occurs earlier in gestation in female than in male fetuses, and 3) prenatal dexamethasone does not effect the postnatal volume of an average alveolus but diminishes their number in male pups.
Stimulation of phosphatidylcholine hydrolysis in type II alveolar epithelial cells
Authors: Dubrovin L. C. , Brown L. A.
Published: December 2017

The effects of phorbol 12-myristate 13-acetate (TPA) or ATP on phosphatidylcholine (PC) hydrolysis were investigated in cultured type II pneumocytes prelabeled with [3H]choline or 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine ([3H]lyso-PAF). In cells prelabeled with [3H]choline, TPA or ATP stimulated an increase in [3H]choline, [3H]phosphocholine, and [3H]glycerophosphocholine. The formation of these choline metabolites was associated with a concomitant loss of [3H]PC but not from disaturated PC or phosphatidylinositol. In cells prelabeled with [3H]lyso-PAF, the formation of [3H]phosphatidic acid (PA) and then [3H]1,2-DG was stimulated by TPA or ATP and was associated with a loss of 3H from PC but not from disaturated PC or phosphatidylinositol. There was a concentration-dependent formation of [3H]1,2-DG and [3H]PA in response to ATP. Downregulation of protein kinase C with TPA abolished the stimulation of PC hydrolysis. In addition to the generation of metabolites indicative of phospholipase C and/or D activity, [3H]lyso-PC, a product of phospholipase A2, was also generated in response to TPA. These findings suggest an important role for PC breakdown in signal transduction in type II pneumocytes.
Surfactant protein D: subcellular localization in nonciliated bronchiolar epithelial cells
Authors: Crouch E. , Parghi D. , Kuan S. F. , Persson A.
Published: December 2017

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated carbohydrate binding protein that was initially characterized as a biosynthetic product of type II pneumocytes. Immunoperoxidase studies of formaldehyde solution-fixed and paraffin-embedded rat lung demonstrated staining for SP-D in the cytoplasm of a subpopulation of bronchiolar epithelial cells as well as type II cells. Accordingly, immunogold-labeling techniques were used to further examine the cellular distribution and subcellular localization of SP-D in the small airways. Lung tissues were fixed with 0.5% glutaraldehyde-3% paraformaldehyde and embedded in LR White resin. Sections were reacted with affinity purified polyclonal antibodies to SP-D, and sites of antibody binding were demonstrated using a biotinylated secondary antibody-streptavidin-gold detection system. Anti-SP-D selectively decorated secretory compartments of nonciliated bronchiolar cells (Clara cells) with strong and specific labeling of apical electron-dense secretory granules. Almost all of the granules in nonciliated columnar cells were labeled; however, labeling was typically nonuniform, with preferential decoration of the periphery of the granule. The largest numbers of immunoreactive epithelial cells were observed in the distal membranous bronchioles, with progressively smaller numbers of cells in more proximal bronchioles. There was no detectable labeling of cells lining the large cartilagenous airways or trachea. These studies provide evidence that SP-D is a secretory product of nonciliated bronchiolar cells. We suggest that Clara cell-derived SP-D is a component of bronchiolar lining material, consistent with our hypothesis that SP-D contributes to surfactant metabolism and/or host defense within small airways.
Surfactant protein C: a review of its unique properties and metabolism
Authors: Beers M. F. , Fisher A. B.
Published: December 2017

Traditionally, our thinking about surfactant proteins has centered around their effects on the biophysical properties of surfactant phospholipids. It is now apparent that the three major surfactant proteins (SP-A, SP-B, and SP-C) are a biochemically and functionally diverse group of mammalian peptides. Accumulated data suggest that they have roles beyond modulation of alveolar surface tension. SP-C is a 33-35 amino acid peptide found in organic extracts of pulmonary surfactant. In part, because of its extreme hydrophobicity, a full understanding of SP-C is presently incomplete. Progress to date has included evaluation of the biophysical properties and investigations of the SP-C gene, including studies of the SP-C promoter. This review describes the unique structural and functional properties of the SP-C molecule and summarizes available data on its molecular biology and metabolism. Studies from literature show that SP-C represents a physiologically important peptide with novel structural properties; namely, extreme hydrophobicity, an alpha-helical membrane spanning region, and a unique posttranslational modification: palmitoylation. From data on similarly modified proteins, we propose that the properties of SP-C, including the covalent addition of palmitic acid, render it capable of being targeted to and interacting with specific cell membranes. A complete understanding of SP-C, especially with regard to its metabolism and function, may require a reorientation of our thinking to consider SP-C as a membrane peptide and not just as a "surfactant protein."
Spontaneous production of PDGF A-chain homodimer by rat lung fibroblasts in vitro
Authors: Fabisiak J. P. , Absher M. , Evans J. N. , Kelley J.
Published: December 2017

Platelet-derived growth factor (PDGF) is considered a decisive mediator of fibroblast growth and phenotype within the lung. The cellular sources of PDGF within the lung remain undefined. The ability of lung fibroblasts themselves to produce PDGF in vitro was therefore investigated. Northern and Western blot analyses revealed the expression of PDGF-A mRNA and secretion of A-chain containing proteins by fibroblasts derived from adult and fetal rat lung. PDGF-A gene or protein expression were below the limits of detection in two human lung fibroblast lines examined in a similar manner. PDGF-B transcripts or proteins were not detected in any lung fibroblast line examined. Conditioned medium (CM) was collected from these same lung fibroblast lines and tested for its ability to promote cell growth using human fetal lung fibroblasts as targets. Both adult and fetal rat lung fibroblasts were found to produce a potent and efficacious stimulus for cell growth. Growth-promoting activity in rat fibroblast-derived CM functioned as a "competence" factor and was partially inhibited by anti-PDGF antibody. Thus rat lung fibroblasts in vitro produce potent growth factors of which at least one appears to be PDGF-AA. Differences in the expression of PDGF-AA between rat and human lung fibroblasts exist. Growth factor-producing fibroblasts may play a role in lung repair and remodeling through production PDGF-AA in vivo.
Cat tracheal gland cells in primary culture
Authors: Culp D. J. , Lee D. K. , Penney D. P. , Marin M. G.
Published: December 2017

Conditions for the primary culture of isolated cat tracheal gland cells were established. Cells plated onto glutaraldehyde-fixed gels of rat tail collagen grew to confluency after 8 days of culture forming a monolayer of cuboidal cells with ultrastructural characteristics of epithelial cells and immunoreactivity to antikeratins. Cultured cells synthesized and released radiolabeled high-molecular-weight glycoconjugates. Glycoconjugate secretion was increased approximately 10% in response to the cholinergic agonist, carbachol. Secretion of glycoconjugates was unrelated to regulated exocrine secretion, since these cells were devoid of secretion granules as assessed by light and electron microscopy. Confluent cultures also generated a spontaneous potential difference and short-circuit current, which were both inhibited by ouabain and increased by carbachol. This suggested gland cells contribute to fluid secretion by active ion-transport mechanisms. We also plated cells onto unfixed collagen gels that were released from the culture dish at confluency. Cells were columnar with apically oriented secretion granules that stained with alcian blue and for blood group A immunoreactivity. Secretion of radiolabeled high-molecular-weight glycoconjugates was increased 27% by carbachol. These cell culture systems may serve as models to investigate glandular secretory mechanisms and their regulation.
Differential collagen and fibronectin production by Thy 1+ and Thy 1- lung fibroblast subpopulations
Authors: Derdak S. , Penney D. P. , Keng P. , Felch M. E. , Brown D. , Phipps R. P.
Published: December 2017

Pulmonary fibrosis resulting from diverse etiologies is characterized by proliferation of fibroblasts and excessive accumulation of interstitial collagen. Whether fibrosis is associated with selective expansion of fibroblast subpopulations differing in amounts or types of collagens synthesized is unknown. We have previously isolated lines and clones of normal murine lung fibroblasts based on the presence of the Thy 1 surface antigen. These subpopulations differ in morphology, growth characteristics, and display of class II major histocompatibility complex antigens (R.P. Phipps, D.P. Penney, P. Keng, H. Quill, A. Paxhia, S. Derdak, and M. E. Felch. Am. J. Respir. Cell Mol. Biol. 1: 65-74, 1989). We evaluated the amounts and types of collagen and fibronectin synthesized by Thy 1+ (Fib2-T-3+) and Thy 1- (Fib2-T-4-) lung fibroblast lines and clones. Thy 1+ fibroblast line synthesized two- to threefold more collagen and noncollagen protein than the Thy 1- line. In contrast, both the Thy 1+ and Thy 1- lines synthesized similar amounts of fibronectin. Thy 1+ and Thy 1- lines and clones expressed mRNA for alpha 1(I)-and alpha 1(III)-procollagen and synthesized both types (predominantly type I and lesser amounts of type III) of collagen, protein, and mRNA. The fibroblast clones varied significantly in total collagen and fibronectin production, with one Thy 1- clone (D3) synthesizing the largest amount of collagen but relatively little fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)
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)
Oxygen affects human endothelial cell proliferation by inactivation of fibroblast growth factors
Authors: Grant M. M. , Koo H. C. , Rosenfeld W.
Published: December 2017

The fibroblast growth factors (FGF), including endothelial cell growth factor (ECGF)/acidic FGF and basic FGF, are important modulators of endothelial cell replication in vitro and in vivo. Premature infants and adults with lung injuries are often treated with high levels of inspired O2, which can be necessary for survival but potentially injurious to developing lungs and in tissue repair following injury. Human umbilical artery and vein endothelial cells were grown in ECGF- or FGF-supplemented Medium 199 and exposed to ambient levels of O2 from 10 to 95%. Endothelial cell growth, measured by [3H]thymidine incorporation, was inhibited by increasing levels of O2 and ceased above 50% O2. Vein endothelial cells could recover from up to 24 h of hyperoxic exposure when given fresh medium, but not after 48 h. Artery-derived cells were more sensitive to O2 than were vein-derived cells. Complete medium without endothelial cells, preincubated 24 h in 95% O2, lost its ability to support cell growth under normoxic conditions. Exposing individual medium components to high O2 demonstrated that purified natural ECGF and recombinant acidic or basic FGF were all inactivated by O2. Human recombinant superoxide dismutase prevented FGF inactivation. O2 inactivation of essential growth factors could thus have major consequences for lung development or repair of injured capillaries in infants or adults inspiring high levels of O2
Circulating xanthine oxidase mediates lung neutrophil sequestration after intestinal ischemia-reperfusion
Authors: Terada L. S. , Dormish J. J. , Shanley P. F. , Leff J. A. , Anderson B. O. , Repine J. E.
Published: December 2017

Injury to nonpulmonary organ systems often initiates systemic processes that cause recruitment of neutrophils to the lung. We found that rats subjected to intestinal ischemia-reperfusion (I/R) had increased transvascular leak of 125I-labeled albumin into lungs and decreased lung ATP levels (P less than 0.05). In addition, rats subjected to intestinal I/R had increased plasma xanthine oxidase (XO) activity, plasma leukotactic activity for neutrophils, and lung neutrophil retention (assessed by morphometry and myeloperoxidase activity) compared with sham-treated rats (P less than 0.05). By comparison, after intestinal I/R, rats fed an allopurinol- or tungsten-enriched diet had decreased plasma and intestinal XO activities, decreased plasma leukotacic and lung myeloperoxidase (MPO) activities, decreased lung leak, and increased lung ATP levels compared with rats fed control diets (P less than 0.05). Further studies suggested a more specific role for circulating rather than tissue XO in mediating lung neutrophil accumulation but not lung leak. Plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, increased in rats administered purified XO intravenously. In addition, plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, decreased in rats administered antisera against XO and then subjected to intestinal I/R. We conclude that circulating XO increases acutely and may contribute to pulmonary retention of neutrophils after an ischemic intestinal insult.
Effects of hydrogen peroxide on the responsiveness of isolated canine bronchi: role of prostaglandin E2 and I2
Authors: Gao Y. , Vanhoutte P. M.
Published: December 2017

The present study was design to determine the role of prostaglandin E2 and I2 in the responses of isolated canine airways to H2O2. Rings of canine third-order bronchi, some of which had undergone mechanical denudation of the epithelium, were suspended in organ chambers; isometric tension was recorded. During contractions to acetylcholine, H2O2 induced concentration-dependent relaxations. The relaxations were attenuated significantly by indomethacin, acetylsalicylic acid, and methylene blue. H2O2 increased the release of prostaglandin E2 and 6-keto-prostaglandin F1 alpha and the content of adenosine 3',5'-cyclic monophosphate (cAMP). These effects were abolished by indomethacin or methylene blue. H2O2 did not affect the content of guanosine 3',5'-cyclic monophosphate significantly. These observations suggest that 1) H2O2 relaxes canine bronchial smooth muscle and 2) elevation of tissue content of cAMP induced by prostaglandin E2 and I2 may be involved. These phenomena did not appear to be modulated by the respiratory epithelium, since H2O2-induced relaxations and increases in the release of PGE2 and 6-ketoprostaglandin F1 alpha were similar in preparations with and without epithelium. However, after treatment with methylene blue, H2O2 induced contractions only in preparations with epithelium. These epithelium-dependent contractions were not affected by inhibitors of cyclooxygenase and lipoxygenase.
Mechanisms of fibrin formation and lysis by human lung fibroblasts: influence of TGF-beta and TNF-alpha
Authors: Idell S. , Zwieb C. , Boggaram J. , Holiday D. , Johnson A. R. , Raghu G.
Published: December 2017

Fibrin gels form within the alveolar and interstitial compartments of the injured lung, and fibroblasts invade and facilitate organization of these transitional gels. We studied the effects of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) on fibrinolytic and procoagulant activities of human lung fibroblasts (HLF) to determine their capacity to regulate pulmonary fibrin deposition. Fibrinolytic activity of cell lysates and media (n = 6 HLF cultures) were uniformly depressed by TGF-beta or TNF-alpha. In dose and time-course studies, HLF plasminogen activator inhibitor-1 (PAI-1) was increased by TGF-beta, whereas TNF-alpha induced release of PAI-1 into the media. HLF and media urokinase concentrations were depressed by TGF-beta, whereas urokinase was unchanged or increased by TNF-alpha. Tissue plasminogen activator was mainly cell associated and unchanged by TGF-beta or TNF-alpha. HLF antiplasmin activity was not detected. Plasma recalcification times of HLF media were decreased by TNF-alpha but unchanged by TGF-beta. These studies suggest that TGF-beta and TNF-alpha impair the ability of HLF to degrade fibrin by disturbing the balance of HLF plasminogen activators and PAI and that these cytokines concurrently leave unchanged or increase the capacity of HLF to initiate fibrin formation. Cytokines likely to occur in the injured lung induce abnormalities of fibrinolysis in HLF from adults; such abnormalities favor extravascular fibrin deposition, a characteristic feature of alveolitis.
Quantitation of alveolar distribution of liposome-entrapped antioxidant enzymes
Authors: Baker R. R. , Czopf L. , Jilling T. , Freeman B. A. , Kirk K. L. , Matalon S.
Published: December 2017

Liposome-encapsulated Cu,Zn superoxide dismutase (Cu,Zn SOD) and catalase (CAT) were instilled intratracheally in rabbits, and the temporal and spatial distribution of Cu,Zn SOD and CAT within the lung was assessed at the organ and cellular levels. Specific activities of Cu,Zn SOD and CAT were increased in both lung homogenates and isolated alveolar type II pneumocytes. Peak Cu,Zn SOD activities in lung homogenates and alveolar type II cells were observed 4 h after liposome instillation and returned to control levels by 24 h, whereas CAT activities remained significantly above controls. There were no significant differences in liposome distribution or antioxidant enzyme uptake among lung lobes. The distribution of fluorescently labeled Cu,Zn SOD and CAT was assessed with the use of epifluorescence microscopy and digital image processing to determine patterns of cellular incorporation of liposome-entrapped Cu,Zn SOD and CAT within the lung. Although the mean fluorescence intensity of alveoli from rabbits instilled with liposomes containing labeled Cu,Zn SOD and CAT was greater than autofluorescence observed with either no liposome or empty liposome instillation, fluorescence intensity varied between adjacent alveoli. Both fluorescently labeled Cu,Zn SOD and CAT were located cytosolically, and uptake was not limited to alveolar type II pneumocytes. These results demonstrate that a single intratracheal instillation of liposomes can effect increases in Cu,Zn SOD and CAT activities in distal lung cells, including alveolar type I and type II cells and macrophages.
Stretching increases calcium influx and efflux in cultured pulmonary arterial smooth muscle cells
Authors: Bialecki R. A. , Kulik T. J. , Colucci W. S.
Published: December 2017

To determine the effect of a single static stretch on calcium fluxes in cultured pulmonary arterial smooth muscle cells (PASMC), calcium influx and efflux were evaluated in PASMC on a collagen-coated silicone membrane using 45Ca2+ as a tracer. A single 20% linear stretch of the silicone membrane of 1 min in duration increased calcium uptake by 71%. This effect was partially inhibited by verapamil or gadolinium, but was not altered by staurosporine, pertussis toxin, or removal of extracellular sodium. Stretch-stimulated calcium uptake attenuated over time, such that uptake during the last minute of a 5-min sustained stretch was 46% of that during the first minute of stretch. A single 20% stretch sustained for 6 min caused a 47% increase in calcium efflux, the magnitude of which was linearly related to the degree of cell stretch. Gadolinium and removal of extracellular calcium each partially inhibited stretch-induced calcium efflux. We conclude that a single static stretch of PASMC causes increases in both calcium influx and efflux. Stretch-stimulated calcium influx does not require sodium influx and is mediated in part by a pathway sensitive to both gadolinium and verapamil. Stretch-stimulated calcium efflux is due to both calcium influx via a gadolinium-sensitive pathway and mobilization of intracellular stores. Because calcium is a key cellular second messenger, these effects of stretch on cellular calcium handling may play a role in the regulation of vascular smooth muscle cell phenotype and function.
Mechanism of H2O2-induced modulation of airway smooth muscle
Authors: Gupta J. B. , Prasad K.
Published: December 2017

We investigated the effects of H2O2 generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. H2O2 generated by G+GO was measured with luminol-dependent chemiluminescence. G+GO in the concentrations of 1x (1.80 microM G, 0.075 U/ml GO) and 2, 4, and 8x generated 1.35, 3.2, 6.10, and 6.00 microM of H2O2, respectively. H2O2 produced relaxation of rabbit tracheal smooth muscle, relaxed acetylcholine (ACh)-precontracted muscle, and reduced muscle responsiveness to ACh. These effects were concentration dependent. H2O2, however, produced contraction of guinea pig tracheal smooth muscle. Catalase completely inhibited the H2O2-induced relaxation of ACh-precontracted tracheal smooth muscle. H2O2-induced relaxation was greater in preparations with intact epithelium (65%) than in those denuded of epithelium (40%). The relaxant effects of H2O2 in the presence of an inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine), an inhibitor of guanylate cyclase (methylene blue), an inhibitor of cyclooxygenase (indomethacin), and an ATP-sensitive K+ channel blocker (glipizide) were 44, 44, 39, and 48%, respectively. H2O2-induced relaxation in the presence of indomethacin in preparations with denuded epithelium was 29%. These results suggest that H2O2-induced relaxation of tracheal smooth muscle is partly epithelium dependent and is mediated by inhibitory arachidonic acid metabolites, epithelium-derived relaxing factor (nitric oxide), ATP-sensitive K+ channels, and the synthesis and release of prostaglandins from epithelium and the underlying smooth muscle.
Contractile effect of bombesin on guinea pig lung in vitro: involvement of gastrin-releasing peptide-preferring receptors
Authors: Lach E. , Haddad E. B. , Gies J. P.
Published: December 2017

Bombesin (Bn) and related agonists produce a potent contractile response in guinea pig peripheral airways in vitro, with the following relative potencies: bombesin > gastrin-releasing peptide (GRP) > neuromedin C ≫ neuromedin B. Specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6–13)methyl ester and [D-Phe6,Cpa14,psi 13–14]Bn(6–14)-NH2, inhibited bombesin-induced lung contraction with high potencies [negative logarithm of the molar concentration of antagonist that produces a twofold shift to the right in the agonist dose-response curve (pA2) of 7.1 and 7.2, respectively], whereas the less-specific antagonist [Leu14,psi 13–14]Bn has a lower one (pA2 of 5.6). In binding studies, the high affinities of GRP, [D-Phe6]Bn(6–13)methyl ester and [D-Phe6,Cpa14,psi 13–14]Bn(6–14)NH2 in contrast with the low affinity of neuromedin B agree with the hypothesis that GRP-preferring receptors are involved in bombesin-induced bronchoconstriction. Bombesin-induced bronchoconstriction is unaffected by atropine, hexamethonium, propranolol, triprolidine, methysergide, Ro 19–3704, and indomethacin or AA-861, suggesting that the Bn response does not occur via a mechanism involving the corresponding endogeneous agents or via the release of arachidonic acid metabolites. Moreover, the effect of Bn is insensitive to capsaicin pretreatment, excluding the involvement of endogeneous neuropeptides. Present results provide evidence that Bn-induced bronchoconstriction results from a direct effect of Bn on bronchial smooth muscle GRP-preferring receptors.
O2-dependent modulation of calf pulmonary artery tone by lactate: potential role of H2O2 and cGMP
Authors: Omar H. A. , Mohazzab K. M. , Mortelliti M. P. , Wolin M. S.
Published: December 2017

Lactate was found to produce a relaxation of isolated endothelium-removed calf pulmonary arteries precontracted with 20–30 mM K+. Examination of the mechanism of this response indicates that it appears to be O2 dependent and mediated via guanosine 3',5'-cyclic monophosphate (cGMP), since it is reduced by hypoxia (N2 atmosphere, PO2 = 8-10 Torr) and because the relaxation was both eliminated by inhibition of soluble guanylate cyclase activation with methylene blue and enhanced by an antagonist of cGMP-selective phosphodiesterases (M & B 22948). Relaxation to lactate is not mediated via prostaglandin formation or arginine-derived nitric oxide, since indomethacin or nitro-L-arginine, respectively, did not alter the response. Lucigenin-elicited chemiluminescence, a potential detector of superoxide anion, was significantly increased by lactate only after inhibition of Cu-Zn-superoxide dismutase (via pretreatment with diethyldithiocarbamate). Pyruvate (5 mM) produced only minimal relaxation and did not significantly increase chemiluminescence. In the homogenate fraction of the arterial smooth muscle, NAD plus lactate or NADH was required to observe increased chemiluminescence. The calf pulmonary arterial smooth muscle contraction to hypoxia and relaxation to posthypoxic reoxygenation was observed to be increased by lactate, associated with a reduced level tone generation under O2 but not N2 atmosphere. Thus lactate, but not pyruvate, appears to cause a cGMP-mediated relaxation in the calf pulmonary artery through an increased intracellular H2O2 generation via the NADH-dependent production of superoxide anion, and activation of this relaxing mechanism modulates O2-elicited tone responses.
Adenine nucleotide degradation in ischemic rabbit lung tissue
Authors: De Leyn P. , Lerut T. , Schreinemakers H. , van Belle H. , Lauwerijns J. , van Lommel F. , Verbeken E. , Flameng W.
Published: December 2017

The aim of the study was to determine the pathways and site of adenosine triphosphate (ATP) catabolism during lung ischemia, which thus far are largely unknown. For this purpose we used the isolated rabbit lung. Rabbit lungs were flushed in situ with a modified Krebs-Henseleit solution (60 ml/kg), the deflated heart lung blocks were isolated, immersed in saline solution, and stored at 37 degrees C. In group I (normothermic ischemia; n = 6) tissue content of ATP decreased progressively from 9.42 +/- 0.58 mumol/g dry wt to 3.42 +/- 0.24 mumol/g dry wt after 30 min of ischemia and further to 0.51 mumol/g dry weight after 4 h. Hypoxanthine was the major catabolite (92% of the nucleoside and purine base fraction at 4 h ischemia). Adenosine did not accumulate (preischemic 0.08 +/- 0.02 mumol/g dry weight vs. 0.13 +/- 0.01 mumol/g dry weight; P > 0.05). AMP accumulated, but also inosine monophosphate (IMP), which was undetectable before ischemia, increased significantly during ischemia. To determine the breakdown pathway of AMP, 400 microM of the adenosine deaminase inhibitor EHNA was added to the flush solution in group II (n = 6). During ischemia, ATP breakdown was unaltered but adenosine became the major catabolite (2.8 times the concentration of hypoxanthine at 4 h ischemia). By pretreatment of the rabbits with the nucleoside transport inhibitor R 75231 (group III; n = 6) no effect was observed on the concentrations during ischemia of inosine and hypoxanthine and only a minor increase of adenosine was found. Cytochemical localization of nucleoside phosphorylase revealed activity predominantly in the endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mediation of endothelial injury following neutrophil adherence to extracellular matrix
Authors: Kaslovsky R. A. , Lai L. , Parker K. , Malik A. B.
Published: December 2017

Since polymorphonuclear leukocytes (PMN) rapidly migrate across the endothelial barrier and attach to extracellular matrix components, we tested the hypothesis that adhesion of PMN to matrix proteins can mediate endothelial injury following PMN activation. Studies were made using gelatin- and fibronectin-coated polycarbonate microporous filters (10 microns thick) on which confluent monolayers of bovine pulmonary microvessel endothelial cells were grown. PMN were layered either directly onto endothelial cells (at a ratio of 10:1) (“upright system”) or onto gelatin- and fibronectin-coated filters with the endothelial monolayer grown on the underside of the filter without contact between PMN and endothelial cells (“inverted system”). PMN were activated with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) in both systems. PMN activation increased endothelial permeability to 125I-labeled albumin in upright as well as inverted systems. Pretreatment of PMN with anti-CD18 monoclonal antibodies IB4 or R15.7, which inhibited PMN adherence to matrix constituents as well as to endothelial cells, prevented the permeability increase in both configurations. This effect of anti-CD18 monoclonal antibodies (mAbs) was not ascribed to a reduction in PMN activation, since PMA-induced superoxide generation was unaffected. We conclude that activation of PMN adherent to extracellular matrix proteins increases endothelial permeability to albumin and that this response is dependent on PMN adhesion to the matrix. The results support the concept that PMN-mediated increase in endothelial permeability is the result of “targeted” release of PMN products independent of whether the PMN are adherent to the extracellular matrix or the endothelium.
Hydrogen peroxide-induced increase in endothelial adhesiveness is dependent on ICAM-1 activation
Authors: Lo S. K. , Janakidevi K. , Lai L. , Malik A. B.
Published: December 2017

Reactive oxygen radicals (ROS) generated by phagocytes promote human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein endothelial cells (EC). We determined the effects of hydrogen peroxide (H2O2), a phagocyte-derived ROS, on EC adhesiveness by determining steady-state intracellular adhesion molecule 1 (ICAM-1) mRNA and ICAM-1 protein expression. The adhesion of PMN to H2O2-treated EC was concentration dependent with maximal adhesion achieved at 0.1 mM H2O2. PMN adhesion occurred rapidly, reaching its maximum value within a 1-h treatment time. The PMN adhesion was dependent on the interaction between CD11/CD18 integrins on PMN and ICAM-1 on EC, since either anti-CD18 or anti-ICAM-1 monoclonal antibodies (MAbs) inhibited (by > 90%) the adhesive interaction between PMN and EC. In parallel with the increases in EC adhesivity, we detected a two- to threefold increase in EC surface expression of ICAM-1 between 0.5 and 1 h after H2O2 challenge. Northern analysis revealed an increase in the steady-state ICAM-1 mRNA signal within 0.5 h after H2O2 exposure, and the response was sustained up to 2 h. Inhibition of intracellular catalase in H2O2-treated EC by 3-amino-1,2,3-triazole augmented the PMN adhesion by 20%, whereas enhancement of EC H2O2-scavenging activity by addition of catalase abrogated the H2O2-induced PMN adhesion, indicating that oxidant-antioxidant balance at the EC interface is a critical factor modulating PMN-EC adhesive interactions. The results suggest that H2O2-induced PMN adhesion is dependent on the rapid induction of the ICAM-1 mRNA signal and the surface expression of ICAM-1 on EC.(ABSTRACT TRUNCATED AT 250 WORDS)
Oxygen modulates the differentiation of human fetal lung in vitro and its responsiveness to cAMP
Authors: Acarregui M. J. , Snyder J. M. , Mendelson C. R.
Published: December 2017

Previously, it was found that lung explants from mid-trimester human abortuses differentiate spontaneously in organ culture in serum-free defined medium in an atmosphere of 95% air-5% CO2. Dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) treatment of human fetal lung in culture increases the rate of morphological differentiation and enhances expression of the surfactant protein A (SP-A) gene. To begin to define the factors responsible for this accelerated in vitro differentiation, we analyzed the effects of atmospheric oxygen on the morphological and biochemical development of human fetal lung in culture and on responsiveness of the cultured tissue to DBcAMP. We found that when lung explants were maintained in an atmosphere containing 1% oxygen they failed to differentiate spontaneously and no induction of SP-A gene expression was apparent. Furthermore, at 1% oxygen, DBcAMP had no effect to stimulate morphological differentiation or SP-A gene expression. When lung tissues that had been maintained for 5 days in 1% oxygen were transferred to an environment containing 20% oxygen, there was rapid morphological development and induction of SP-A gene expression. The effects on morphological development were manifest within 24 h of transfer to the 20% oxygen environment; within 72 h, a marked stimulatory effect of DBcAMP on SP-A gene expression also was observed. Our findings further suggest that the effects of oxygen on the levels of SP-A and SP-A mRNA are concentration dependent. Interestingly, the inductive effects of DBcAMP on SP-A gene expression were apparent only at oxygen concentrations > or = 10%. Morphological differentiation of the cultured human fetal lung tissue also was influenced by oxygen in a concentration-dependent manner. These findings suggest that oxygen plays an important permissive role in the spontaneous differentiation of human fetal lung in vitro.
Variable glutathione levels and expression of antioxidant enzymes in human endothelial cells
Authors: Jornot L. , Junod A. F.
Published: December 2017

We have investigated the relationship between intracellular glutathione levels and the inducibility of the mRNAs encoding the major antioxidant enzymes Cu,Zn superoxide dismutase (Cu,Zn SOD), catalase (CAT), glutathione peroxidase (GP), and the stress protein heme oxygenase (HO) following exposure of human umbilical vein endothelial cells (HUVEC) to either hypoxanthine-xanthine oxidase or 95% O2. Treatment of HUVEC with 2 and 200 microM buthionine sulfoximine (BSO) for 16 h reduced total glutathione (GSH) levels by 51 and 95%, respectively, whereas treatment with 100 microM diethylmaleate (DEM) for 24 h increased the cellular GSH content by 58%. None of these treatments affected the responsiveness of HUVEC to a subsequent oxidant challenge, in terms of antioxidant enzymes activities and mRNA levels. On the contrary, HO mRNA was significantly induced by both BSO and DEM, as well as by hyperoxia, albeit to a different extent. We conclude that intracellular redox changes do not appear to regulate the expression of the mRNAs encoding Cu,Zn SOD, CAT, and GP. Furthermore, factors other than endogenous thiols may play a role in the control of HO mRNA expression.
Calcium mobilization by muscarinic cholinergic stimulation in bovine single airway smooth muscle
Authors: Kajita J. , Yamaguchi H.
Published: December 2017

Cellular mechanisms for Ca2+ mobilization by muscarinic cholinergic stimulation were investigated in dispersed bovine airway smooth muscle cells by the fura-2 method. Because the dissociation constant (Kd) of the intracellular fura-2 was not known, we first determined the Kd as 386 +/- 6 nM, which ranged from 368 to 394 nM. By using the Kd, resting intracellular Ca2+ concentration ([Ca2+]i) was calculated to be 177 +/- 11 nM (n = 22). Carbachol (CCh) at 10(-7) - 10(-5) M caused two distinct increases in [Ca2+]i; an initial transient which reached 1.3-2.7 microM followed by a sustained increase in [Ca2+]i of 250-400 nM. The sustained Ca2+ elevation was not observed in the absence of extracellular Ca2+. D 600 (10(-5) - 3 x 10(-5) M) also eliminated this response. An analogue of diacylglycerol (DAG), 1,2-dioctanoyl-sn-glycerol, caused a gradual increase in [Ca2+]i to 303 +/- 24 nM (n = 13), which was equivalent to the sustained response to 10(-7) and 10(-6) M CCh and was again blocked by D 600. These findings are consistent with the idea that inositol 1,4,5-trisphosphate releases stored Ca2+ as the main cause for the initial transient and that activation of DAG by CCh may result in the sustained increase in [Ca2+]i through voltage-dependent Ca2+ channels.
Effects of sodium and proton pump activity on respiratory burst and pH regulation of rat alveolar macrophages
Authors: Murphy J. K. , Forman H. J.
Published: December 2017

At pH 7.4, extracellular Na+ removal inhibited the rat alveolar macrophage respiratory burst (RB) stimulated by phorbol 12-myristate 13-acetate (PMA) or zymosan-activated serum (ZAS). At pH 6.8, the RB was lower and decreased the Na+ effect. Amiloride inhibited the ZAS RB independently of effects on Na(+)-H+ exchange, but did not affect PMA stimulation. NBD-Cl, an H(+)-ATPase inhibitor, significantly inhibited the PMA or ZAS RB. Na+ removal caused sustained elevation of intracellular free [Ca2+], which previous studies suggested inhibits the RB. Intracellular pH (pHi) was lower at pHo 6.8 compared with pHo 7.4, but not altered by Na+ removal. PMA stimulation resulted in acidification corresponding with onset of superoxide production. At pHo 7.4, recovery to baseline pHi occurred that was not inhibited by amiloride or Na+ removal. In contrast, amiloride slowed pHi recovery after an exogenous acid load. Addition of H(+)-ATPase inhibitors, NBD-Cl or bafilomycin, following PMA stimulation or acid loading, inhibited pHi restoration. These studies suggest that pHi regulation following stimulation was mainly through a proton pump, whereas Na(+)-H+ exchange occurred only after greater acid loading. Nevertheless, Na+ and pH interacted to modulate the RB independent of Na(+)-H+ exchange.
Enhanced Na+ transport in an air-liquid interface culture system
Authors: Johnson L. G. , Dickman K. G. , Moore K. L. , Mandel L. J. , Boucher R. C.
Published: December 2017

Use of the air-liquid interface culture technique has produced improved morphological differentiation of rodent, canine, and human tracheal epithelia. We have investigated the effect of this culture technique on ion transport activities of cultured canine bronchial epithelia. These cells were isolated from excised airways by enzymatic digestion and plated on permeable collagen membrane substrates. All cultures were maintained utilizing standard culture techniques, by bathing both apical and basolateral sides with hormone supplemented, serum-free media until confluent (days 4–6). Half of the cultures were converted to air-liquid interface cultures (ALIC) by gentle aspiration of the apical medium and half were continued under standard technique culture (STC) conditions. After three additional days, preparations cultured under both conditions were mounted in modified Ussing chambers where bioelectric properties were measured under short-circuit conditions. Mean short-circuit current (Isc) was significantly greater in ALIC (-91.3 +/- 7.84 microA/cm2) than in STC (-54.8 +/- 5.03 microA/cm2). The sodium channel blocker, amiloride, reduced Isc by 68.4 +/- 5.0% in STC and by 84.8 +/- 3.0% in ALIC. 22Na and 36Cl fluxes confirmed the presence of enhanced sodium absorption in ALIC when compared with STC. The depth of the apical fluid, measured by microelectrodes during ALIC, was approximately 15 microns. Studies of cellular metabolism demonstrated a shift in metabolism from an anaerobic to an oxidative pattern in ALIC. This change in the pattern of metabolism suggests that the ALIC technique enhanced sodium transport in canine bronchial epithelia by increasing oxygen delivery to the epithelium.
Protein kinase C activity during the process of lung liquid clearance
Authors: Sapijaszko M. , Mackenzie J. , Walsh M. P. , Berthiaume Y.
Published: December 2017

Although active transport of sodium plays an important role in the resolution of pulmonary edema, the biochemical regulation of this process is still under investigation. The purpose of this study was to evaluate the activity of protein kinase C during the process of lung liquid clearance. Alveolar flooding was induced by instilling 5% bovine serum albumin solution, saline, or heterologous serum in the air spaces of rats. The activity of protein kinase C was measured in both the instilled and control lungs at 10 min and 1 and 4 h after fluid instillation. Four hours after instillation of 5% bovine serum albumin, the ratio of protein kinase C activity in the instilled lung compared with the control lung was 2.2 +/- 0.3. Similar results were obtained following instillation with heterologous serum or saline. Since we measured a clearance rate of 0.8 ml/h in anesthetized rats, we can postulate that the activation of protein kinase C occurred when > 40% of the liquid had been cleared from the lung. This increased activity of protein kinase C was not due to an increase in kinase activity in the inflammatory cells or an increase in enzyme quantity but due to a decrease of protein kinase C inhibitory activity in the lung. These results suggest that protein kinase C second messenger system may play a regulatory role in lung liquid clearance.
Effects of beta-adrenergic receptor activation on alveolar macrophage cytoplasmic motility
Authors: Fukushima T. , Sekizawa K. , Jin Y. , Yamaya M. , Sasaki H. , Takishima T.
Published: December 2017

We studied the effects of fenoterol, a beta-adrenoceptor agonist, on the cytoplasmic motility of alveolar macrophages (AM) from dog lungs in vitro. Four days after the instillation of Fe3O4 particles (3 mg/kg) into the lower lobe bronchus, AM were harvested by bronchoalveolar lavage. Remanent field strength (RFS) from the AM containing Fe3O4 particles (5 x 10(6) cells) was measured immediately after magnetization. RFS decreased with time due to particle rotation (relaxation), which is related to cytoplasmic motility of AM. Fenoterol (10(-9) M to 10(-5) M) decreased the relaxation rate (lambda 0; min-1) in a concentration-dependent fashion with the maximum effect at 10(-6) M. Both forskolin (10(-6) M to 10(-4) M) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (10(-3) M) mimicked fenoterol-induced inhibitory effects on lambda 0. Fenoterol and forskolin concentration-dependently increased intracellular levels of cAMP, which were parallel to decreases in lambda 0 induced by these drugs. KT 5720 (10(-5) M), a specific inhibitor of protein kinase A, significantly inhibited fenoterol (10(-6) M)-induced inhibitory effects on lambda 0 (P < 0.01). These results imply that beta-adrenergic receptor activation inhibits cytoplasmic motility of AM via increases in intracellular levels of cAMP, which may be coupled with activation of a cAMP-dependent protein kinase.
Glucocorticoid elevation of mRNA encoding epinephrine-forming enzyme in lung
Authors: Kennedy B. , Elayan H. , Ziegler M. G.
Published: December 2017

The epinephrine-forming enzyme phenylethanolamine N-methyltransferase (PNMT) is present in lung and its activity is increased by the glucocorticoid dexamethasone. Chronic administration of dexamethasone (0.5 mg/kg twice daily) doubled levels of mRNA coding for PNMT in rat lung. Administration of the glucocorticoid antagonist RU 486 after 7 days of dexamethasone treated reduced PNMT mRNA by about two-thirds within 24 h. Lung epinephrine (E) levels correlated with lung PNMT activity in these dexamethasone-treated and control rats. In a separate experiment, lung PNMT mRNA levels were nearly tripled 6 h after dexamethasone (1 mg/kg sc). In a third experiment chronic administration of the PNMT inhibitor SKF 64139 (50 mg/kg twice daily) reduced in vitro lung PNMT activity in chronically dexamethasone-treated adrenalectomized rats by approximately 96% and reduced lung E levels by approximately 75%. We conclude that glucocorticoids increase lung PNMT activity by increasing levels of mRNA coding for this enzyme. The data also suggest that a substantial fraction of lung E is locally synthesized. We speculate that enhanced lung E synthesis may participate in glucocorticoid-induced dilation of the bronchioles.
Prostaglandin secretion by guinea pig tracheal epithelial cells caused by eosinophil major basic protein
Authors: White S. R. , Sigrist K. S. , Spaethe S. M.
Published: December 2017

We examined the effect of eosinophil major basic protein (MBP) on prostaglandin (PG) secretion from guinea pig tracheal epithelial (GPTE) cells. Primary cultures of GPTE cells were incubated with 10(-6) M MBP for up to 6 h and then stimulated with 10(-6) M bradykinin (BK). PGE2, 6-ketoprostaglandin F1 alpha (PGF1 alpha), PGF2 alpha, and thromboxane B2 (TxB2) concentrations in media were determined by enzyme-linked immunoabsorbent assay (EIA). Incubation with MBP for 6 h caused secretion of both PGE2 (17,614 +/- 4,416 vs. 1,426 +/- 555 pg/10(6) cells at baseline, P < 0.001, n = 7) and PGF2 alpha (20,303 +/- 5,724 vs. 3,790 +/- 1.075 pg/10(6) cells at baseline, P < 0.002, n = 7). Secretion of PGE2 and PGF2 alpha stimulated by MBP required at least 2 h. Incubation with MBP for 6 h also augmented the subsequent response to BK: PGE2 secretion was 29,215 +/- 6,853 vs. 3,445 +/- 1,041 pg/10(6) cells for BK alone (P < 0.0001), and PGF2 alpha secretion was 25,407 +/- 6,237 vs. 5,213 +/- 1,535 pg/10(6) cells for BK alone (P < 0.0001). MBP did not change 6-keto-PGF1 alpha and TxB2 secretion. Incubation of GPTE cells from seven animals with polylysine, a protein with mass and ion charge similar to MBP, for 2 h, both caused secretion of PGE2 (8,579 +/- 3,244 vs. 788 +/- 419 pg/10(6) cells at baseline, P < 0.01) and augmented the response to BK (12,732 +/- 4,788 vs. 1,653 +/- 680 pg/10(6) cells after BK alone, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of tunicamycin on maturation of fetal mouse lung
Authors: Webster E. H. , Hilfer S. R. , Searls R. L. , Kornilow J.
Published: December 2017

The mesodermal capsule of the fetal lung plays a role in differentiation of the respiratory region. It has been proposed for other epithelial organs that the mesodermal capsule influences development by modifying the basal lamina or the extended extracellular matrix. The effect could be on deposition or turnover of collagens, proteoglycans, and/or glycoproteins. This study tests the role of glycoproteins in differentiation of respiratory endings by inhibiting their synthesis with the antibiotic tunicamycin (TM). Lungs at 16 and 18 days gestation and 3 days after birth were cultured with TM and examined for morphological and biochemical differences from normal controls. With TM, alveolar regions did not expand properly and formed fewer type I pneumocytes, although type II pneumocytes were unaffected. The epithelium of untreated respiratory regions showed greater incorporation of radioactive mannose than the airways region or mesenchyme. This incorporation was diminished in TM, but the pattern persisted. Comparison with the results obtained with beta-xyloside suggested that differentiation of type I and type II pneumocytes is under separate control.