bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of amiloride on alveolar epithelial PD and fluid composition in rabbits
Authors: Nielson D. W. , Lewis M. B.
Published: December 2017

To look for evidence of active absorption of Na+ in the alveolus in vivo in air-filled lungs, we measured [K+] and [Cl-] in the alveolar lining fluid and the potential difference (PD) across the alveolar epithelium by puncturing alveoli in lungs of anesthetized rabbits with nonselective and ion-selective microelectrodes. After intravenous doses of amiloride, the PD and [K+] decreased (-1.0 +/- 0.3 to -0.3 +/- 0.1 mV, 7.4 +/- 1.1 to 4.2 +/- 0.4 meq/l, P less than 0.001), but [Cl-] did not change (96 +/- 9, 94 +/- 4 meq/l). In another set of experiments, the PD was measured with microelectrodes filled with an electrolyte solution, and midway through each measurement some of the solution was injected into the alveolar lumen. Injecting the solution without amiloride did not alter the alveolar PD (-1.0 +/- 0.4 before and -1.1 +/- 0.5 after injection). The alveolar PD decreased to -0.1 +/- 0.2 mV after injecting the solution with 10(-5) M amiloride into the alveolar interior. These results support the hypothesis that alveolar epithelium in air-filled lungs actively absorbs sodium in vivo, which accounts for the majority of the transepithelial PD.
Mechanisms of decrease in cytoplasmic motility of alveolar macrophages during immediate asthmatic response in dogs
Authors: Yamaya M. , Zayasu K. , Sekizawa K. , Yamauchi K. , Fukushima T. , Sasaki H. , Takishima T.
Published: December 2017

To study the cytoplasmic motility (CM) of alveolar macrophages (AM) during immediate asthmatic response, we measured remanent filed strength (RFS) in dogs in vivo after Ascaris suum-antigen (AA) inhalation. Four days after instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the right lower lobe bronchus, RFS was measured at the body surface immediately after magnetization of the Fe3O4 particles by an externally applied magnetic field. RFS decreased with time because of particle rotation (relaxation). Relaxation is thought to be correlated to CM of AM (J. Appl. Physiol. 55: 1196-1202, 1983). AA inhalation increased respiratory resistance (Rrs) in 10 dogs (responders; P less than 0.01), and did not change Rrs in five dogs (nonresponders; P greater than 0.50). The relaxation rate (lambda 0) significantly decreased because of AA inhalation in responders (P less than 0.01), whereas lambda 0 did not change in nonresponders (P greater than 0.50). Intravenous injection of indomethacin or diclofenac sodium (5 mg/kg) 30 min before AA inhalation completely inhibited the decrease in lambda 0 induced by AA inhalation, although both drugs did not inhibit the increase in Rrs (P greater than 0.10). In in vitro experiments, AM (5 x 10(6) cells) cells) harvested by bronchoalveolar lavage showed a significant dose-dependent decrease in lambda 0 with prostaglandins (PG) E1 and E2. However, histamine and platelet-activating factor increased lambda 0 significantly (P less than 0.01), whereas PGD2, PGF2 alpha, PGI2, thromboxane A2, leukotrienes B4, C4, and D4, and AA itself were without effect (P greater than 0.50). Furthermore, PGE2 aerosols mimicked AA-induced effects on AM.(ABSTRACT TRUNCATED AT 250 WORDS)
Epithelial modulation of airway smooth muscle
Authors: Morrison K. J. , Gao Y. , Vanhoutte P. M.
Published: December 2017

The responsiveness of airway smooth muscle is influenced by the functional integrity of the respiratory epithelium. The nature of this regulatory action by the epithelium remains largely unresolved. Several explanations may account for the epithelium-dependent responses induced by numerous stimuli. This review will present and discuss the evidence suggesting that the epithelium generates an inhibitory signal or signals that function to modulate the responsiveness of the underlying smooth muscle. In addition, the possible candidates for the identity of this epithelium-derived relaxing factor or factors will be assessed. Finally, the mechanisms by which the epithelium-derived relaxing factors may act to modulate bronchomotor tone will be discussed.
Movement of receptors and ligands through the endocytic apparatus in alveolar macrophages
Authors: Kaplan J. , Ward D. M.
Published: December 2017

The ability of alveolar macrophages to remove potentially injurious agents is essential for normal lung function. One mechanism by which macrophages affect this function is by the internalization and clearance of noxious agents from lung fluid. Extracellular macromolecules may be removed as a result of nonspecific uptake via fluid-phase pinocytosis or as a result of binding to surface receptors followed by a highly efficient internalization of the receptor-ligand complex. The ability of macrophages to bind, internalize, and thus sequester extracellular macromolecules is facilitated by the fact that membrane components involved in this process may be reutilized. That is, once internalized, membrane components are not invariably degraded but can be recycled for further use. In this commentary we examine some of the features known about the recycling process in both macrophages and other cell types. We demonstrate that although the rate of internalization of a membrane component is unique to that component, the rate of movement of internalized components within cells is a function of the endocytic apparatus and is independent of the ligands or receptors contained in the internalized vesicle. We also demonstrate how alterations in the rates of recycling may result in changes in the distribution of membrane proteins expressed on the cell surface and affect the ability of a cell to respond to the environment.
Depletion of tissue glutathione with diethyl maleate enhances hyperbaric oxygen toxicity
Authors: Weber C. A. , Duncan C. A. , Lyons M. J. , Jenkinson S. G.
Published: December 2017

Rats exposed to hyperbaric hyperoxia experience severe central nervous system and lung toxicity. Exogenous glutathione administration has been shown to protect rats from the effects of hyperbaric hyperoxia. To explore the hypothesis that decreases in tissue glutathione (GSH) could increase the susceptibility of rats to hyperbaric hyperoxia, we administered diethyl maleate (DEM) (a compound that conjugates with GSH and rapidly lowers tissue levels) and measured tissue GSH levels. DEM administration decreased plasma GSH by 86%, liver GSH by 82%, and brain GSH by 45% between 2 and 4 h after injection with values returning to normal by 24 h. We then treated rats with DEM or saline and began exposure at 2 h after treatment to 100% oxygen at 4 ATA. Time-to-convulsion and time-to-death were recorded. Rats that received DEM 2 h before exposure seized earlier and died earlier than controls. Intraperitoneal administration of GSH to DEM-treated rats abolished the enhanced toxicity occurring during a hyperbaric hyperoxic exposure. DEM appears to increase the toxicity of rats exposed to hyperbaric hyperoxia by lowering tissue GSH levels, and replenishment of lung and brain GSH by exogenous administration reverses these effects.
A genetic model for evaluation of susceptibility to ozone-induced inflammation
Authors: Kleeberger S. R. , Bassett D. J. , Jakab G. J. , Levitt R. C.
Published: December 2017

We examined ozone-induced airway inflammatory responses in inbred mice, and progeny of crosses between them, to investigate genetic susceptibility to ozone. Nine strains of male mice (18–23 g, 5–7 wk) were exposed for 3 h to 2 ppm ozone (O3) or filtered air (control), and pulmonary inflammation was assessed 2, 6, and 24 h after exposure by inflammatory cell counts and total protein content in bronchoalveolar lavage (BAL). The time course of the response to O3 was consistent between the strains. The maximum change in polymorphonuclear leukocytes (PMNs) was detected 6 h after O3, and the maximum increase in BAL protein occurred 24 h postexposure. Air controls exhibited no detectable changes in the parameters of inflammation at any time. The phenotypes of the C57BL/6J (B6, termed susceptible) and C3H/HeJ (C3, termed resistant) strains were easily distinguished by the magnitude of their inflammatory responses to O3. A 22-fold difference in PMNs was detected between the two strains 2 h after O3 (P less than 0.001), and a sixfold difference was found 6 h after O3 (P less than 0.001). Total BAL proteins were also significantly different between the B6 and C3 strains 6 h (P less than 0.01) and 24 h after O3 (P less than 0.001). To further evaluate the potential genetic contribution to the inflammatory response, the F1, F2, and backcross progeny from crosses between B6 and C3 strains were examined. The phenotypes of these progeny were consistent with the hypothesis that a single autosomal recessive gene at the Inf locus confers susceptibility to acute O3-induced influx of PMNs, but the genetic control of altered permeability is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)
Basolateral K+ channels in airway epithelia. II. Role in Cl- secretion and evidence for two types of K+ channel
Authors: McCann J. D. , Welsh M. J.
Published: December 2017

We previously described a Ca2(+)-activated K+ channel (KCLIC) in airway epithelial cells [J. D. McCann, J. Matsuda, M. Garcia, G. Kaczorowski, and M. J. Welsh. Am. J. Physiol 258 (Lung Cell. Mol. Physiol. 2): L334-L342, 1990]. To determine whether the KCLIC channel is a basolateral membrane channel and to understand its role in Cl- secretion, we studied airway epithelial cells grown on permeable supports. When cells were stimulated with A23187, charybdotoxin (ChTX) inhibited Cl- secretion and 86Rb efflux at the same concentrations, indicating that the KCLIC channel is required for Ca2(+)-stimulated Cl- secretion. We also investigated the function of K+ channels in adenosine 3',5'-cyclic monophosphate-stimulated secretion. Addition of isoproterenol caused a biphasic increase in Cl- secretion; the time course of the transient component correlated with the time course of the isoproterenol-induced increase in Ca2+ concentration [( Ca2+]c). ChTX inhibited the transient component, but not the prolonged component of secretion; Ba2+ inhibited the sustained component. These results suggest that when cells are grown on permeable supports isoproterenol-induced secretion depends on activation of two types of K+ channel: the KCLIC channel that is stimulated initially and a ChTX-insensitive K+ channel that is stimulated during sustained secretion. This conclusion was supported by measurement of 86Rb efflux from cell monolayers
Lung epithelial ion transport and fluid movement during the perinatal period
Authors: Bland R. D.
Published: December 2017

During fetal life, the mammalian lung is a secretory organ that serves no respiratory function. Its potential airspaces are filled with liquid that flows from the pulmonary circulation across the epithelium in response to the osmotic force generated by Cl- secretion of airway and distal lung epithelial cells. As birth approaches, net Cl- secretion across the respiratory tract epithelium decreases, and this is associated with a reduction in the flow of liquid into the lung lumen. The cause for this change is unknown, but several recent studies indicate that it may be related to alterations in the hormonal milieu to which the lung epithelium is exposed late in gestation. The switch from placental to pulmonary gas exchange at birth requires rapid removal of liquid from the lung lumen. During labor and the immediate postnatal period, the pulmonary epithelium changes from a predominantly Cl- secreting membrane to a predominantly Na(+)-absorbing membrane, with resultant reversal of the direction of flow of lung liquid. There is considerable evidence that this change reflects an active metabolic process involving increased Na(+)-K(+)-ATPase activity in lung epithelial cells, which drives liquid from the lung lumen into the interstitium, with subsequent absorption into the pulmonary circulation.
Metabolic changes in alveolar type II cells after exposure to hydrogen peroxide
Authors: LaCagnin L. B. , Bowman L. , Ma J. Y. , Miles P. R.
Published: December 2017

Since oxygen metabolites may play an important role in pulmonary oxidant injury, the effects of hydrogen peroxide (H2O2) on energy metabolism in alveolar type II cells isolated from rats were studied. The major effect of H2O2 is a rapid and dramatic reduction in the steady-state level of cellular ATP; e.g., ATP levels are reduced by 77 +/- 3% after only 5 min of exposure to H2O2 (0.5 mM). Cellular oxygen consumption is affected in a similar manner, suggesting that ATP synthesis is impaired. Experiments with isolated lung mitochondria demonstrate that exposure to 0.5 mM H2O2 for 5 min inhibits the rate of mitochondrial ATP synthesis by 51 +/- 3%. The site of mitochondrial ATP synthesis inhibition by H2O2 appears to be the adenosinetriphosphatase-synthase enzyme complex which phosphorylates ADP to ATP. Mitochondrial electron transport is unaffected. The association of 3-O-methylglucose with type II cells and glycolytic metabolism, measured as lactate production, are reduced by 25-35% by H2O2. The data also show that the cells are capable of recovery following exposure to H2O2, at least at lower exposure levels. These results indicate that exposure of type II cells to H2O2 alters the energy state of the cells by decreasing ATP synthesis. In turn, other important cellular functions may be impaired.
Arachidonate metabolism increases as rat alveolar type II cells differentiate in vitro
Authors: Lipchik R. J. , Chauncey J. B. , Paine R. , Simon R. H. , Peters-Golden M.
Published: December 2017

Rat type II alveolar epithelial cells are known to undergo morphological and functional changes when maintained in culture for several days. Having previously demonstrated that these cells can deacylate free arachidonic acid (AA) and metabolize it to products of the cyclooxygenase pathway, the present study was undertaken to determine whether in vitro differentiation was accompanied by alterations in the availability and metabolism of AA. We assessed the constitutive and ionophore A23187-induced deacylation and metabolism of endogenous AA, as well as the metabolism of exogenously supplied AA, in primary cultures of rat type II cells at days 2, 4, and 7 after isolation. Levels of free endogenous AA were increased at day 4, whereas eicosanoid synthesis, predominantly prostaglandin E2 and prostacyclin, increased markedly only at day 7. A similar time course of augmentation of prostanoid release was seen in response to exogenous AA. Type II cells cultured on fibronectin, intended to hasten cell flattening and spreading, demonstrated accelerated increases in available free AA in response to A23187; cells cultured on basement membrane derived from Engelbreth-Holm-Swarm mouse sarcoma, known to maintain the type II phenotype, exhibited diminished levels of available free AA. From these findings, we conclude that alterations in arachidonate metabolism are linked to alterations in cellular phenotype. The potentiation of eicosanoid synthesis accompanying in vitro differentiation suggests a possible role for the alveolar epithelium in the modulation of inflammation and fibrosis in the distal lung.
Bronchial epithelial cells exposed to isocyanates potentiate activation and proliferation of T-cells
Authors: Mattoli S. , Miante S. , Calabro F. , Mezzetti M. , Fasoli A. , Allegra L.
Published: December 2017

Bronchial epithelial cells release chemotactic factors for lymphocytes and express HLA-DR antigens. Thus they may contribute to the T-cell-mediated inflammatory responses involved in a number of pulmonary diseases such as asthma. In this study, the in vitro exposure of human bronchial epithelial cells to toluene 2,4-diisocyanate (TDI), an inflammatory and asthmogenic stimulus presumed to act at least in part through immunological mechanisms, provoked cell damage followed by proliferation of the cells that survived the injury. At the time of the proliferative response, epithelial cells released factors that upregulated the activation and proliferation of T lymphocytes presensitized by antigen receptor triggering. The T-cell activating factors were interleukin (IL) 1- and 6-like substances, as demonstrated by the ability of specific antisera to inhibit most of the biological effect, and by the ability of recombinant IL-1 and IL-6 to reproduce it. Appreciable amounts of immunoreactive IL-1 and IL-6 were indeed recovered in the supernatants of TDI-exposed epithelial cells. The release of these cytokines may represent an important mechanism by which epithelial cells respond to some environmental stimuli and contribute to the persistence of inflammatory responses in the airways.
Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression
Authors: Panettieri R. A. , Yadvish P. A. , Kelly A. M. , Rubinstein N. A. , Kotlikoff M. I.
Published: December 2017

Although chronic severe asthma is characterized by increased smooth muscle mass in the airways, the physiological stimuli that promote airway smooth muscle (ASM) proliferation (hyperplasia) or increase ASM protein expression (hypertrophy) are unknown. We examined the effects of histamine, an autocoid associated with airway hyperresponsiveness, on protein synthesis, myosin heavy chain expression, and cell proliferation in cultured canine ASM cells. In confluent ASM cells, histamine significantly increased incorporation of [35S]-methionine in protein. Maintenance of the proportion of smooth muscle-specific myosin heavy chain to total myosin heavy chain suggested a nonspecific increase in contractile protein expression. DNA synthesis, as measured by [3H]thymidine incorporation, was significantly increased by histamine in a concentration-dependent manner. Cell proliferation paralleled [3H]thymidine incorporation; histamine significantly increased cell numbers at 24 and 48 h of stimulation. Because growth of mesenchymal-derived cells is associated with transcription of c-fos mRNA, we examined whether histamine altered expression of this proto-oncogene. Histamine-treated cells showed marked increases in expressions of steady-state c-fos mRNA, with a time course of mRNA induction similar to cells exposed to platelet-derived growth factor or serum, known smooth muscle and fibroblast cell mitogens. Therefore, histamine is an ASM mitogen with an action similar to other mesenchymal cell growth factors and may play a role in the hyperplasia of ASM in asthma.
Mechanical removal of airway epithelium disrupts mast cells and releases granules
Authors: Franconi G. M. , Rubinstein I. , Levine E. H. , Ikeda S. , Nadel J. A.
Published: December 2017

Many previous investigators have utilized mechanical rubbing as a method for examining effects of epithelial removal. In the present study, we examined whether this procedure also affects mast cell integrity in the underlying lamina propria. We isolated bronchial rings from six ferrets, and we found that removal of epithelium by rubbing decreased the total number of intact mast cells from 10.0 +/- 1.9 to 2.2 +/- 0.6 (SE) mast cells/mm luminal perimeter (P less than 0.01). In addition, we found a very large number of metachromatic particles that appeared to be mast cell granules unassociated with identifiable mast cells. Their identity was confirmed the presence of free mast cell granules and showed that they contained intact membranes. These effects were not observed when the epithelium was removed by enzymatic digestion, but they were observed after mechanical deformation of the bronchi alone. We suggest that mast cell components released by removing or damaging the epithelium may affect the function of various cells in the airway.
Potassium currents in canine airway smooth muscle cells
Authors: Kotlikoff M. I.
Published: December 2017

The electrical properties of dissociated canine tracheal smooth muscle cells were examined using the whole cell patch-clamp technique. In current clamp mode, current clamp steps did not initiate action potentials but showed clear outward rectification, which was abolished when cells were loaded with Cs+ ions and when tetraethylammonium (TEA+) ions replaced Na+ in the bath solution. In voltage-clamp experiments, depolarizations positive to -45 mV evoked brief voltage-dependent inward Ca2+ currents [Am. J. Physiol. 254 (Cell Physiol. 23): C793-C801, 1988], followed by sustained outward currents, which did not completely inactivate. Outward currents were identified as K+ currents on the basis of the reversal potential of the current and by ion-substitution experiments. The currents were further defined as Ca2(+)-insensitive delayed rectifier currents, since they were unaltered under conditions in which 1) the Ca2+ current was completely blocked by Mn2+ or nifedipine (10 microM); 2) Ba2+ ions were substituted for Ca2+ as the inward current charge carrier; or 3) charybdotoxin (40 nM) or TEA+ (up to 10 mM) were added to the bath. A Ca2(+)-activated potassium [K(Ca)] current was activated by application of methacholine (100 microM), or A23187 (1 microM), under conditions of low Ca2+ buffering capacity in the internal solution [0.3 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N-,N--tetraacetic acid (EGTA)]. The K(Ca) current was blocked by 10 mM TEA+ and was not observed under conditions of high intracellular Ca2+ buffering (11 mM EGTA). These data indicate that canine airway smooth muscle cells contain voltage-dependent delayed rectifier channels that underlie membrane rectification and K(Ca) channels that are activated by agents which release intracellular Ca2+ stores.
Calcium ionophores injure alveolar epithelial cells: relation to phospholipase activity
Authors: Rice K. L. , Duane P. G. , Mielke G. , Sinha A. A. , Niewoehner D. E.
Published: December 2017

Phospholipases and certain of their hydrolytic products are toxic to alveolar epithelial cells. Since many intracellular phospholipases are Ca2+ dependent, we postulated that elevating cytosolic Ca2+ with ionophores might cause epithelial injury via phospholipase activation. Isolated perfused hamster lungs exposed to an Ca2+ ionophore A23187 develop functional evidence of severe epithelial injury. Ultrastructural studies show widespread lysis of type I epithelial cells, with only minimal abnormalities in other lung cells, including the microvascular endothelium. Analysis of whole lung lipid extracts reveals a modest elevation in free arachidonic acid but no changes in other putative products of phospholipase activity. Parallel studies were performed in cultured cells of pulmonary origin. As measured by 51Cr release, A23187 causes substantial cytotoxicity in 3-day-old cultures of rat type II alveolar epithelial cells (RAEC) but not in cultured bovine pulmonary artery endothelial cells (BPAEC). RAEC prelabeled with [14C]stearic acid [( 14C]SA) and [3H]arachidonic acid [( 3H]AA) release radiolabeled free fatty acids (FFA) in response to A23187 in a dose- and time-dependent manner that parallels the cytotoxicity index. Analyses of putative phospholipase products in cells radiolabeled with [14C]SA and [3H]AA, with [14C]choline, or with [14C]ethanolamine suggest that liberation of radiolabeled FFA may be due to several phospholipases but with principal activity being exhibited by a phospholipase C having specificity toward phosphatidylcholine and phosphatidylethanolamine. Prelabeled BPAEC release only minimal quantities of FFA in response to A23187 under the same conditions. These studies demonstrate that elevations of intracytoplasmic Ca2+ are capable of severely and selectively damaging alveolar epithelial cells and that the injury is associated with activation of intracellular phospholipases. These findings may have implications in regard to the pathogenesis of acute lung injury in humans.
Regulation of surfactant protein A mRNA by hormones and butyrate in cultured fetal rat lung
Authors: Nichols K. V. , Floros J. , Dynia D. W. , Veletza S. V. , Wilson C. M. , Gross I.
Published: December 2017

We have previously shown that dexamethasone, triiodothyronine (T3) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulate phosphatidylcholine (PC) synthesis in fetal rat lung explants in culture. There are also additive interactions between these agents with regard to PC synthesis. In this study we examined the regulation of surfactant protein A (SP-A) mRNA in fetal rat lung in culture. Dexamethasone increased SP-A mRNA in the explants in a dose-dependent fashion (1–200 nM), but T3 did not. Whereas 8-bromo-cAMP increased SP-A mRNA, a decrease was observed with dibutyryl cAMP. These findings support the view that at least some of the genes involved in the synthesis of the various components of surfactant are independently regulated. Since we observed differences in the effects of a cAMP analogue which contained butyrate and one that did not, explants were then cultured with Na butyrate, a known regulator of gene expression. A significant decrease in SP-A mRNA was observed at mM concentrations. Exposure of the explants to alpha-aminobutyric acid, a butyric acid analogue which is elevated in the blood of infants of diabetic mothers, resulted in a significant decrease in SP-A mRNA at a concentration 1/25 of that required for Na butyrate. This observation raises the question of whether the decreased SP-A levels reported in fetuses of diabetic mothers may, at least in part, be related to this metabolite.
Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia
Authors: Fleming R. E. , Whitman I. P. , Gitlin J. D.
Published: December 2017

To determine the effect of inflammation on extrahepatic ceruloplasmin gene expression we examined the ceruloplasmin mRNA content of adult rat tissues after endotoxin injection. Within 8 h of a dose of endotoxin ceruloplasmin mRNA content increased in the liver as expected and was also detectable in the lung. The effect of endotoxin was tissue specific because ceruloplasmin mRNA was not consistently detected in other extrahepatic tissues. The kinetics of ceruloplasmin mRNA accumulation in lung and liver tissue were similar with a maximum seven- to ninefold increase in ceruloplasmin mRNA content in each tissue within 24 h. The relative rate of ceruloplasmin gene transcription was increased in both tissues within 3 h of endotoxin, suggesting similar mechanisms of regulation of ceruloplasmin gene expression during inflammation. One cellular site of ceruloplasmin production in the inflamed lung was found to be the alveolar macrophage, which expressed the ceruloplasmin gene and synthesized ceruloplasmin protein in response to endotoxin in vitro. Because of these findings we also examined the effects of hyperoxia on ceruloplasmin gene expression. Exposure of adult rats to 95% O2 resulted in a five- to sixfold induction of ceruloplasmin mRNA in lung tissue within 46 h, and this response was time dependent, reaching maximum values at 86 h. Hyperoxic induction of ceruloplasmin mRNA was specific to the lung and not the result of systemic inflammation because hepatic ceruloplasmin mRNA content remained constant. These data indicate that the lung is a prominent site of ceruloplasmin gene expression during inflammation and hyperoxia and suggest that this protein may play a previously unappreciated role in pulmonary injury or repair.
cAMP increases synthesis of surfactant-associated protein A by perfused rat lung
Authors: Fisher A. B. , Arad I. , Dodia C. , Chander A. , Feinstein S. I.
Published: December 2017

Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8-BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27–36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant-associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A.
Complement activation by cigarette smoke
Authors: Robbins R. A. , Nelson K. J. , Gossman G. L. , Koyama S. , Rennard S. I.
Published: December 2017

Lung disease secondary to cigarette smoking is associated with an influx of neutrophils and monocytes into the lower respiratory tract. To determine whether cigarette smoke can generate chemotactic activity, human serum was exposed to cigarette smoke and evaluated for neutrophil and monocyte chemotactic activity. Serum exposed to cigarette smoke attracted significantly greater numbers of neutrophils and monocytes compared with normal human serum exposed to air (P less than 0.01, both comparisons). The increase in chemotactic activity was partially attenuated by EDTA but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and MgCl2 (P greater than 0.05, both comparisons), suggesting activation of the alternate complement pathway. To evaluate the capacity of cigarette smoke to activate the complement system, smoke-exposed serum was evaluated for cleavage of properdin factor B and C3 using immunoelectrophoresis and for C5a using a radioimmunoassay. Cleavage of properdin factor B and C3 was observed in the smoke-exposed serum and C5a was detected in the smoke-exposed serum (112 +/- 31 ng/ml). These data suggest that complement activation may play a role in directing the influx of neutrophils and monocytes into the lungs of cigarette smokers.
p146 type II alveolar epithelial cell antigen is identical to aminopeptidase N
Authors: Funkhouser J. D. , Tangada S. D. , Jones M. , O S. J. , Peterson R. D.
Published: December 2017

A prominent membrane protein of rat type II alveolar cells, p146, was originally identified by one of many mouse monoclonal antibodies that were produced to rat lung cells in the course of a search for differentiation antigens that might prove useful in studying lung differentiation. We report here results from analysis of the primary structure of this molecule and, based on this knowledge, the elucidation of the function of the protein. Amino acid sequencing of the NH2-terminal portion of the p146 protein, plus partial sequencing of several peptides obtained by limited proteolysis, indicates it is identical to aminopeptidase N. Further, the immunoaffinity purified p146 protein has aminopeptidase N activity. The discussion includes references to other molecules such as CD 13 and CD 10 (CALLA) that were recognized as differentiation antigens and subsequently found to be peptidases. The possible biological implications of such a peptidase on the luminal surface of type II alveolar cells are also considered.
Tumor necrosis factor-alpha increases Mn-SOD expression: protection against oxidant injury
Authors: Warner B. B. , Burhans M. S. , Clark J. C. , Wispe J. R.
Published: December 2017

Antioxidant enzymes, including superoxide dismutase, are important for protecting the lung against O2 injury. Manganese superoxide dismutase (Mn-SOD) is a superoxide anion (O2-.) scavenger located in the mitochondria, a primary site of O2-. production during hyperoxia. We studied the effects of tumor necrosis factor (TNF-alpha), a macrophage-derived cytokine, on Mn-SOD expression in human pulmonary adenocarcinoma cells. TNF-alpha significantly increased Mn-SOD activity and mRNA in a dose-and time-dependent manner. Mn-SOD activity was increased 3-fold and mRNA 20-fold after a 48-h incubation with TNF-alpha (25 ng/ml). To examine the mechanism of this increase, cells were incubated for 48 h with TNF-alpha (25 ng/ml) with or without cycloheximide (10 microns) or actinomycin D (10 micrograms/ml). Actinomycin D blocked the induction of Mn-SOD mRNA by TNF-alpha, but cycloheximide did not. These findings suggest that the effect of TNF-alpha requires gene transcription but not synthesis of new protein intermediates. To test the hypothesis that increased Mn-SOD protects against oxidative injury, pulmonary adenocarcinoma cells were incubated in TNF-alpha (25 ng/ml) for 48 h and then exposed to paraquat (PQ+), an intracellular O2-. generator. Cells pretreated with TNF-alpha had significantly improved survival in PQ+ compared with controls. At the LD50 (6 microns) for control cells, 95% of TNF-alpha-treated cells survived, 85% at the LD75 (10 microns), and 77% at the LD90 (14 microns). Our results suggest that the induction of Mn-SOD by TNF-alpha in pulmonary adenocarcinoma cells is pretranslationally mediated and that increasing Mn-SOD activity with TNF-alpha confers protection against O2 radicals.
Injury of rat pulmonary alveolar epithelial cells by H2O2: dependence on phenotype and catalase
Authors: Simon R. H. , Edwards J. A. , Reza M. M. , Kunkel R. G.
Published: December 2017

In a variety of inflammatory lung diseases, type I alveolar epithelial cells are more likely to be injured than are type II cells. Because oxidants have been implicated as a cause of injury in various inflammatory lung diseases, we evaluated the effects of differentiation on alveolar epithelial cell susceptibility to H2O2-induced injury. With the use of isolated rat type II cells in culture, we found that the cytotoxic effect of H2O2 increased between days 2 and 7, when type II cells are known to lose their distinctive type II properties and assume a more type I-like appearance. We previously reported that type II cells utilized both intracellular catalase and glutathione-dependent reactions to protect against H2O2. We therefore examined whether alterations in either of these protective mechanisms were responsible for the differentiation-dependent changes in sensitivity to H2O2. We found that catalase activity within alveolar epithelial cells decreased between 2 and 7 days in culture, whereas no changes were detected in glutathione-dependent systems. We then used a histochemical technique that detects catalase activity and found that type II cells within rat lungs possessed numerous catalase-containing peroxisomes, whereas very few were detected in type I cells. These findings demonstrate that as type II cells assume a type I-like phenotype, they become more susceptible to H2O2-induced injury. This increased susceptibility is associated with reductions in intracellular catalase activity, both in vitro and in vivo.
Fetal lung development in rats with a glycogen storage disorder
Authors: Rannels S. R. , Rannels S. L. , Sneyd J. G. , Loten E. G.
Published: December 2017

A New Zealand strain of rats (NZR/Mh) is unable to mobilize liver glycogen due to a deficiency of phosphorylase b kinase. Affected homozygous rats (gsd/gsd) were used to assess the developmental relationship between lung glycogen loss and surfactant phospholipid and protein biosynthesis. Phosphorylase a and phosphorylase b kinase activities were negligible in gsd/gsd fetal lungs compared with controls from gestational day (D18) until postnatal day 1 (D + 1). At D20, tissue glycogen content was 158 +/- 5 and 181 +/- 6 mumol/g lung for control and gsd/gsd, respectively. Control rats mobilized 84% of their lung glycogen by D + 1, whereas the gsd/gsd strain retained 70–80% of D19–20 levels. This apparent fall in gsd/gsd glycogen per gram lung was due to an increase in cellular protein and size. Thus, in controls, total glycogen per lung decreased 65% from D20 to D + 1, whereas DNA doubled. In contrast, gsd/gsd lung growth resulted in a doubling of total lung glycogen, whereas the glycogen-to-DNA ratio remained constant. A lack of cellular glycogenolysis was confirmed by electron microscopy where gsd/gsd type II cells remained large and glycogen-rich over the entire perinatal interval. The potential for glycogen breakdown by a lysosomal alpha-amyloglucosidase in gsd/gsd lungs was estimated in tissue homogenates, whereas rates of hydrolysis of glycogen or p-nitrophenylglucoside were significant and equal to controls at all ages tested. Incorporation of [14C]choline into phosphatidylcholine (PC) of incubated lung slices increased 1.7-fold in control lungs from D20–D21. Over the same interval, PC synthesis in gsd/gsd lungs was 40% lower and did not change.(ABSTRACT TRUNCATED AT 250 WORDS)
Perinatal rat lung catalase gene expression: influence of corticosteroid and hyperoxia
Authors: Clerch L. B. , Iqbal J. , Massaro D.
Published: December 2017

Dexamethasone accelerates the late gestational rise in rat lung catalase activity; neonatal hyperoxia elevates rat lung catalase activity. We studied the regulation of catalase gene expression in these instances. Catalase mRNA/mg DNA increased to gestation day 22 and then fell to the concentration in adult lungs. The rate of transcription of catalase mRNA was higher on gestation day 22 than gestation day 19, whereas the half-life of catalase mRNA (approximately 7 h) was the same on both days. Dexamethasone given 48 and 24 h before expected birth (gestation 22 days) increased catalase mRNA concentration at days 20 and 22 without a change in catalase mRNA stability. Early postnatal hyperoxia (greater than 95% O2, 72 h) elevated catalase mRNA/mg DNA and doubled its half-life without changing its rate of transcription. We conclude the normal late gestational elevation of catalase activity and the increase of activity during prenatal dexamethasone treatment are regulated at the level of gene transcription. By contrast, the elevation of catalase activity during neonatal hyperoxia is mediated posttranscriptionally by increased catalase mRNA stability.
Sulfation by human lung fibroblasts: SO4(2-) and sulfur-containing amino acids as sources for macromolecular sulfation
Authors: Elgavish A. , Meezan E.
Published: December 2017

Studies were carried out in human lung fibroblasts (IMR-90) to investigate 1) the relative contribution of two extracellular pools, inorganic sulfate and sulfur-containing amino acids, to the intracellular fraction precipitable by trichloroacetic acid and 2) the possibility that the transport of these sulfur-containing substrates at the plasma membrane may be a limiting step for macromolecular sulfation. Our studies indicate that the ability to use SO4(2-) released by intracellular catabolism of the sulfur-containing amino acid L-cysteine differs from one cell system to another. In contrast to smooth muscle cells, in the human lung fibroblast, L-cysteine contributes significantly to the intercellular pool of SO4(2-) used for sulfation at extracellular [SO4(2-)] less than 100 microM. However, under physiological conditions with respect to SO4(2-) ([SO4(2-)]0 = 300 microM), L-cysteine does not contribute greater than 30% of the sulfate incorporated into the cellular fraction. Taurine (2-aminoethanesulfonic acid) inhibits SO4(2-) incorporation into the cell-associated macromolecular fraction. However, results suggest that the effect is not due to either SO4(2-) released by its catabolism or to an effect on SO4(2-) transport into the cell. The fact that the transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate incorporation indicates that carrier-mediated sulfate transport at the cellular plasma membrane may be a limiting step for sulfate incorporation. In conclusion, under physiological conditions with respect to SO4(2-), inorganic sulfate is a major source of sulfate for sulfation in human lung fibroblasts and macromolecular sulfation may be limited by its transport into the cells.
Intracellular pH: effect on pulmonary arterial smooth muscle
Authors: Krampetz I. K. , Rhoades R. A.
Published: December 2017

To investigate the effect of changes in intracellular H+ concentration [( H+]i) on pulmonary arterial tone, isolated canine intrapulmonary arteries were exposed to NH4Cl (4-120 mM). Above 4 mM, NH4Cl caused contraction. When induced by less than or equal to 30 mM, contraction was not endothelium dependent and was unaffected by calcium-free medium or by nifedipine (10 microM). Exposure to ryanodine or norepinephrine in calcium-free medium reduced subsequent NH4Cl-induced contraction. Measurements of [H+]i and tension indicated that contraction induced by 30 mM NH4Cl occurred simultaneously with development of peak intracellular alkalosis (delta [H+]i = -47 +/- 9 nM, n = 7). Withdrawal of NH4Cl produced a rapid cytosolic acidification (delta [H+]i = + 131 +/- 29 nM, n = 7), which coincided with a transient nifedipine-sensitive contraction. In the isolated perfused rat lung, addition and removal of NH4Cl similarly increased pulmonary arterial pressure. These data suggest that intracellular alkalosis stimulates pulmonary arterial smooth muscle contraction directly and through release of calcium from intracellular sites. Intracellular acidification appears to stimulate transient contraction by allowing calcium entry from extracellular sources through voltage-activated channels. We conclude that modifications in intracellular pH effect pulmonary arterial smooth muscle tone and consequently can alter blood flow through the lung.
Normalization of force generated by canine airway smooth muscles
Authors: Jiang H. , Halayko A. J. , Rao K. , Cunningham P. , Stephens N. L.
Published: December 2017

A variety of normalizations have been employed to compare maximal isometric force (Po) produced by smooth muscles at different locations and stages of maturation. Because these procedures have not always been based on rigorous principles, confusion has resulted. To obtain a less ambiguous index of force production, we measured in vitro Po from mongrel canine tracheal (TSM) and bronchial (BSM) smooth muscle with an electromagnetic lever and normalized it to force per unit cross-sectional area of whole tissue (tissue stress), to force per unit cross-sectional area of muscle in the cross section of total tissue (muscle stress), and to force per fractional unit of myosin in the tissue cross section (myosin stress). Proportion of myosin in cross-sectional area of tissue was deduced from data obtained by sodium dodecyl sulfate gel electrophoresis of crude muscle extracts. For TSM, tissue stress was 1.499 X 10(5) N/m2 +/- 0.1 (SE), whereas it was only 0.351 X 10(5) N/m2 +/- 0.05 (SE) for BSM, representing a 4.27-fold difference (P less than 0.01). There was a 1.60-fold difference (P less than 0.05) in muscle stress, which was correlated to the morphometric finding that 79 +/- 1.4% (SE) of the tracheal strip cross section was muscle, whereas only 30 +/- 1.0% (SE) of bronchial tissue was occupied by muscle. Average myosin content was the same in smooth muscle cells of TSM and BSM, indicating that total amount of myosin in tissue cross sections was essentially a function of proportional area of muscle cells in total tissue cross sections.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP-stimulated inositol phospholipid metabolism and surfactant secretion in rat type II pneumocytes
Authors: Griese M. , Gobran L. I. , Rooney S. A.
Published: December 2017

Extracellular ATP (10(-3) M) stimulated [3H]phosphatidylcholine secretion approximately 3.4-fold in rat type II pneumocytes prelabeled overnight with [3H]choline. The same concentration of ATP caused a rapid increase in [3H]inositol trisphosphate (IP3) and a decrease in [3H]phosphatidylinositol bisphosphate (PIP2) in [3H]inositol-prelabeled cells. ATP also caused a biphasic increase in 1,2-[3H]diacylglycerol in cells prelabeled with [3H]arachidonic acid: a rapid increase that peaked at 10 s followed by a larger increase that peaked at 5-10 min. The first peak in diacylglycerol and the increase in IP3 are consistent with phospholipase C action on PIP2 and generation of second messengers that promote mobilization of intracellular Ca2+ and activation of protein kinase C. However, at the level of phosphatidylcholine secretion the stimulatory effects of ATP and of direct activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol, were at least additive, suggesting that activation of protein kinase C may not be the major signal transduction mechanism in ATP action or alternatively that ATP activates a different isoform of protein kinase C. Pretreatment of type II cells with TPA for 30 min led to a subsequent 40% diminution in the stimulatory effects of ATP on both phosphatidylcholine secretion and IP3 generation.
Leukotriene receptor on U-937 cells: discriminatory responses to leukotrienes C4 and D4
Authors: Wetmore L. A. , Gerard N. P. , Herron D. K. , Bollinger N. G. , Baker S. R. , Feldman H. A. , Drazen J. M.
Published: December 2017

Dimethyl sulfoxide (DMSO)-differentiated U-937 cells develop cell surface receptors for leukotrienes that, when stimulated, initiate a transient increase in intracellular calcium concentration [( Ca2+]i). We investigated the calcium transient that occurs after addition of leukotriene C4 (LTC4) to determine whether it occurs due to 1) the bioconversion of LTC4 to leukotriene D4 (LTD4), which then acts at the LTD4 receptor; 2) the direct action of LTC4 at the LTD4 receptor; or 3) the action of LTC4 at a receptor selective for LTC4. Bioconversion of [3H]LTC4 to [3H]LTD4 was inhibited by 98% when DMSO-differentiated U-937 cells were incubated with 10 mM AT-125 compared with control cells. The dose-response curve for LTC4, with [Ca2+]i as the index of response, was parallel to that for LTD4 but was significantly (P less than 0.0001) shifted 1.6 +/- 0.11 log units to the right. AT-125 did not change the response to LTD4 but the LTC4 dose-response curve was shifted on additional 1.7 logo units to the right. The antagonists SKF 104353 (1 microM) and LY 171883 (10 microM) shifted the dose-response curve for LTD4 3.0 +/- 0.23 and 2.5 +/- 0.23 log units, respectively, to the right and completely inhibited the change in [Ca2+]i due to LTC4 in the presence of 10 mM AT-125. Molecular modeling studies demonstrated a striking difference in the spatial configuration of LTC4 and LTD4, likely accounting for the ability of cell surface receptors to discriminate between the effects of these two molecules.(ABSTRACT TRUNCATED AT 250 WORDS)