bluuebunny/crossref_metadata_2025 huggingface dataset:
Cigarette smoking induces an elastolytic cysteine proteinase in macrophages distinct from cathepsin L
Authors: Reilly J. J. , Chen P. , Sailor L. Z. , Wilcox D. , Mason R. W. , Chapman H. A.
Published: December 2017

Degradation of the interstitium of the lung by elastolytic enzymes is thought to be a critical component of the pathogenesis of emphysema. Alveolar macrophages are increased in numbers in cigarette smokers and contain the elastolytic cysteine proteinase cathepsin L. We sought to determine if cigarette smoking induces a change in cathepsin L levels in alveolar macrophages which would, in turn, alter the expression of elastolytic activity. Lysates of smokers' macrophages, assayed at pH 5.50, degraded more than seven times as much [3H]elastin as did lysates from nonsmokers' macrophages (44 +/- 20.8 vs. 6 +/- 1.6 micrograms.10(6) cells-1.24 h-1). Little or no activity was demonstrable at neutral pH. Immunoblots of macrophage lysates demonstrated that smokers' cells contain 3.7 +/- 1.1 times as much 25-kDa cathepsin L antigen as nonsmokers' cells. However, as judged by active site labeling, levels of active cathepsin L in smokers and nonsmokers are indistinguishable, suggesting that most of the 25-kDa antigen found in smokers' macrophages is inactive. Inhibitors of cathepsin L had little effect on lysate elastolytic activity, confirming that an enzyme other than cathepsin L is responsible for the increased elastolytic activity seen in smokers' macrophages. Further experiments demonstrated that this second enzyme(s) has a profile of inhibition indicating that it is a cysteine proteinase with optimal activity at pH 5.50. It is this second elastolytic cysteine proteinase(s) that is induced by exposure to cigarette smoke and is responsible for the sevenfold increase in elastolytic activity found in smokers' macrophage lysates.
Expression of SV40 T antigen under control of rabbit uteroglobin promoter in transgenic mice
Authors: DeMayo F. J. , Finegold M. J. , Hansen T. N. , Stanley L. A. , Smith B. , Bullock D. W.
Published: December 2017

The rabbit uteroglobin gene is expressed in the lungs and reproductive tracts of male and female rabbits. To examine whether the promoter region of the uteroglobin gene could be used to target a heterologous gene to the lungs of transgenic mice, a fusion gene consisting of 3.3 kb of the 5'-flanking region of the rabbit uteroglobin gene and the large T antigen gene of the SV40 virus was constructed and microinjected into the pronuclei of one-cell mouse embryos. Eleven founder transgenic mice (5 female and 6 male) were generated. Seven of these mice developed bronchioalveolar neoplasms. Four of the founder males also developed primitive undifferentiated urogenital tract tumors. One founder female and one female offspring of a founder male developed glandular paraovarian tumors. Northern analysis revealed that the predominant site of expression of the transgene was the lung. Immunohistochemical staining showed T antigen predominantly in epithelial cells lining the bronchioles, the submucosal glands of the trachea, and the neoplasms. There appeared to be a high level of mosaicism for the transgene in the founder mice, with poor transmission of the transgene to subsequent generations. This suggests that, under the control of the uteroglobin promoter, the T antigen gene may be lethal to the fetus.
Composition changes in human tracheal cartilage in growth and aging, including changes in proteoglycan structure
Authors: Roberts C. R. , Pare P. D.
Published: December 2017

High-buoyant-density proteoglycans were extracted and purified from tracheal cartilage obtained from nine individuals aged 1-58 yr. Cartilage from young individuals contained one major species of extractable aggregating proteoglycan and very little link protein. Link protein concentration relative to proteoglycan was observed to increase during the first 14 years of life, consistent with the increased formation of proteoglycan aggregates that are stabilized by link protein, as the process of tracheal cartilage growth and airway lumen widening ends. With increasing age after maturity, two further populations of proteoglycans became more abundant; these were characterized by higher mobility in composite agarose-polyacrylamide gel electrophoresis. The ability of the proteoglycans to associate with hyaluronan decreased with increasing age, although members of each of the three proteoglycan species contained functional hyaluronan-binding domains. Link proteins showed evidence of increasing proteolysis with age. Hydroxyproline content of the cartilage decreased with age; total tissue glycosaminoglycan and water contents showed no significant changes. Altered proteoglycan charge density, proteoglycan size, and aggregation properties, as well as changes in distribution of proteoglycans, may contribute to the changes in cartilage biomechanics that are associated with age-dependent changes in human lung function.
Ca2+-ATPase and function of sarcoplasmic reticulum during cardiac hypertrophy
Authors: Levitsky Dmitri , Bastie Diane De La , Schwartz Ketty , Lompré Anne-Marie
Published: December 2017

The properties of the calcium pump system of sarcoplasmic reticulum (SR) were studied in a series of 34 rats subjected to cardiac overload and 19 sham-operated animals. Total homogenates of left ventricle were analyzed by measuring the oxalate-supported Ca2+ uptake rate, the steady-state level of the phosphorylated intermediate of Ca2+-adenosine triphosphatase (Ca2+-ATPase) (E-P), and the amount of Ca2+-ATPase mRNA. All three parameters decreased gradually as a function of the relative left ventricular weight increase. The calcium-sensitivity curves showed that the velocity of Ca2+ transport in SR from the hypertrophied heart is diminished at low as well as optimal Ca2+2 concentrations, with the dissociation constant (Kd) value for Ca2+ unchanged from that of the control preparation. Taken together with the results presented in our recent publication (De la Bastie, Levitsky, Mercadier, Marotte, Wisnewsky, Brovkovivh, Schwartz, and Lompré, Circ. Res. 66: 554–564, 1990), these data strongly indicate that differences in the Ca2+ pump activities of SR from normal and hypertrophied rat hearts are due to quantitative rather than qualitative changes of the Ca2+-ATPase protein.

calcium ion uptake; calcium ion adenosine triphosphatase messenger ribonucleic acid; cardiac hypertrophy; monoclonal antibody
TGF-beta 1 produces a "prooxidant" effect on bovine pulmonary artery endothelial cells in culture
Authors: Das S. K. , Fanburg B. L.
Published: December 2017

The pulmonary endothelium is known to be sensitive to oxidant injury, including that of hyperoxia. Similar to effects of exposure to 80–95% O2, porcine platelet transforming growth factor (TGF)-beta 1 at concentrations of greater than or equal to 0.3 ng/ml inhibited proliferation and caused enlargement of bovine pulmonary artery endothelial cells after 24 h of incubation in room air. Uptake of [3H]thymidine, but not of [3H]deoxycytidine, was suppressed by both hyperoxia and TGF-beta 1. The cellular enlargement produced by TGF-beta 1 in room air was attenuated in the presence of anoxia, indicating a need for O2 for TGF-beta 1 to have an effect on cell size. In the presence of 20 microM FeCl3, both TGF-beta 1 and 80% O2 produced marked cellular desquamation from culture dishes. The antioxidants dimethyl sulfoxide and vitamin E partially counteracted the growth inhibitory effect of TGF-beta 1 on endothelial cells. In contrast to its effect on endothelial cells, TGF-beta 1 only moderately altered size and proliferation of smooth muscle cells from the same pulmonary vessels. Uptake of [3H]thymidine by smooth muscle cells was uninfluenced in 48 h by TGF-beta 1, and little, if any, desquamation of these cells occurred with TGF-beta 1 in the presence of 20 microM FeCl3. We propose from these experiments that TGF-beta 1 may produce an oxidant effect on vascular endothelium that is capable of causing injury to this tissue.
Effect of work on intracellular calcium of the intact heart
Authors: Fralix Teresa A. , Heineman Frederick W. , Balaban Robert S.
Published: December 2017

Intracellular calcium has been proposed to play a key role in the orchestration of metabolic rate with contractile activity in the mammalian heart. Calcium is believed to accomplish this task by modulating the contractile apparatus as well as the metabolic process directly, and perhaps simultaneously, during alterations in cardiac work. The purpose of this study was to evaluate whether appropriate changes in intracellular calcium accompany alterations in cardiac work in the intact working rabbit heart. A range of myocardial oxygen consumption was obtained from 0.94 to 6.51 μmol·g LV wt-1·min-1 by changing afterload or beta-agonist addition. With the increase in work and associated increase in respiration, an increase in intracellular calcium was observed, on the basis of indo-1 fluorescence. These results indicate that intracellular calcium is a valid candidate as a cytosolic transducer contributing to the orchestration of myofibril adenosinetriphosphatase activity and oxidative phosphorylation in the intact heart.

oxygen consumption; indo-1; fluorescence; rabbit; afterload; isoproterenol
Alveolar size, number, and surface area: developmentally dependent response to 13% O2
Authors: Blanco L. N. , Massaro D. , Massaro G. D.
Published: December 2017

Nonpregnant female rats were kept in 13% O2 for greater than 3 wk before being bred, throughout pregna ncy, and, with their pups, after birth; control rats were only in air. The average volume (v), number (N) and surface area (Sa) of gas-exchange structures (saccules or alveoli) were estimated by stereological means. Saccule conversion t o alveoli by septation between age 2 and 14 days was impaired in 13% O2 rats; there was less decrease in v (signifying less septation) and less increase in N (indicating the formation of fewer alveoli) in 13% O2 pups than in air pups. Be tween age 14 and 40 days, v rose 2-fold in air pups and 1.3-fold in 13% O2 pups; N increased 1.7-fold in air rats and 2.8-fold in 13% O2 rats. In other experiments, 23-day-old rats, previously only in air, were continued in air or were placed in 13% O2 until 44-days-old. At age 44 days, Sa was 25% and v 27% greater in 13% O2 rats than in air rats, but N was the same in both groups. We conclude there are multiple mechanisms for forming alveoli and increasing Sa and the se mechanisms exhibit a developmentally dependent response to 13% O2.
Phagocytosis of surfactant by alveolar macrophages in vitro
Authors: Grabner R. , Meerbach W.
Published: December 2017

Because of their localization and function as phagocytes, alveolar macrophages could take part in the catabolism of surfactant, including surfactants used for treatment. Conditions of the ingestion of the surfactant preparation AWD 56-02 by alveolar macrophages in vitro are described in this paper. The surfactant was labeled with rhodaminyl phosphatidylethanolamine and incubated with alveolar macrophages lavaged from rat lungs. Membrane binding and phagocytosis were evaluated by fluorescence microscopy and quantified fluorimetrically after extraction of the dye. The surfactant was phagocytosed only in the presence of rat serum. The opsonins in the serum are related to complement and Fc receptors as demonstrated by the heat lability of the factor and the inhibition by aggregated gamma-globulins. Furthermore, the phagocytosis depends on calcium and on a factor in the surfactant preparation. Experiments with inhibitors and competition with unlabeled surfactant show that the phagocytosis is a specific and energy-dependent process. Catabolism by alveolar macrophages might be an important step in the metabolism of surfactant, especially when administered in pathological conditions characterized by the presence of serum in the airspaces.
Regulation of intracellular pH in alveolar epithelial cells
Authors: Lubman R. L. , Crandall E. D.
Published: December 2017

Alveolar type II epithelial cells in adult mammalian lungs actively transport salt and water, secrete surfactant, and differentiate into type I cells under normal conditions and following lung injury. It has become increasingly apparent that, like all epithelial cells, alveolar pneumocytes have evolved specialized ion transport mechanisms by which they regulate their intracellular pH (pHi). pHi is an important biological parameter in all living cells whose regulation is necessary for normal cellular homeostasis. pHi, and the ion transport mechanisms by which it is regulated, may contribute to many cellular processes, including transcellular transport, cell volume and osmolarity regulation, and intracellular transport, cell volume and osmolarity regulation, and intracellular electrolyte composition. Moreover, changes in pHi may serve as intracellular signals for biological processes such as cell growth, proliferation, and differentiation. We review herein the general principles of pHi regulation in epithelia and describe the mechanisms and effects of pHi regulation in alveolar pneumocytes. Many of the critical issues in current pulmonary research involve processes that pHi is most likely to affect, including maintenance of alveolar epithelial barrier integrity, development and maintenance of epithelial polarity, epithelial proliferation and differentiation, and regulation of transepithelial transport with respect to alveolar fluid balance in normal individuals and in those with excess alveolar fluid (i.e., pulmonary edema). Investigations into the regulation of pHi in alveolar pneumocytes and the regulatory effects of pHi in turn on other cellular processes are likely to yield information important to the understanding of lung biology and pulmonary disease.
Long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions
Authors: Siminski J. T. , Kavanagh T. J. , Chi E. , Raghu G.
Published: December 2017

Current concepts of the pathogenesis of lung injury and repair are derived from in vitro cellular and in vivo investigations. Studies with viable ex vivo models may offer additional insights into disease processes, since essential cellular interactions would be maintained. However, a major limiting factor has been the availability of a model that maintains normal parenchymal structure, viability, and homeostasis beyond 4 wk in serum-free conditions. We have succeeded in establishing an ex vivo lung culture system which reproducibly maintains parenchymal architecture for up to 9 wk. Our method is a simple, modified version of previously utilized techniques. Thin slices of mature murine lung were inflated with agar-defined medium and cultured on Gelfoam saturated with serum-free medium. Normal pulmonary parenchyma, with the exception of endothelial cells, was maintained for up to 60 days as assessed chronologically by light and electron microscopy. The integrity of the microvasculature and endothelial cells was lost beyond 7 days. The adult lung ex vivo culture system maintained necessary epithelial and interstitial cellular interactions in the alveolar wall without systemic circulatory influences. Future studies with this model may provide important insights in assessing the pathogenesis of many acute and chronic lung diseases and clarify existing controversies raised from in vitro and in vivo studies.
Sequence, ontogeny, and cellular localization of murine surfactant protein B mRNA
Authors: D'Amore-Bruno M. A. , Wikenheiser K. A. , Carter J. E. , Clark J. C. , Whitsett J. A.
Published: December 2017

We have isolated and characterized murine surfactant protein (SP-B) cDNAs and determined both the cellular distribution and developmental expression of SP-B mRNA. The nucleotide sequence of the composite SP-B cDNAs and cloned genomic SP-B DNA predicted a primary translation product of 377 amino acids (molecular mass = 41.7 kDa) that contained a single asparagine linked glycosylation site. The 410 base 3' untranslated region contained a novel sequence consisting of a (CCA)21 repeat. Tissue specificity and developmental expression of SP-B mRNA was assessed by Northern blot analysis. A single 1.6-kb mRNA species was detected specifically in lung tissue. In fetal mouse lung SP-B mRNA was detected at low levels on day 15 of gestation and increased markedly between days 16 and 17, reaching adult levels by birth. In situ hybridization analysis of SP-B mRNA demonstrated SP-B expression primarily in bronchiolar and alveolar epithelium, although discrete bronchial cells also contained the SP-B mRNA. The murine SP-B mRNA was expressed in proximal and distal epithelial cells within the respiratory tract and shares a close structural relationship to SP-B from other species.
Tracheal epithelial cell fatty acid composition modulates prostaglandin E2 and cAMP production
Authors: Alpert S. E. , Walenga R. W. , Kramer C. M. , Silski C. L. , Davis P. B.
Published: December 2017

Tracheal epithelial (TE) cells from both rabbits and humans, when cultured in defined serum-free media without lipid supplements, develop fatty acid profiles significantly different from freshly isolated epithelium, including a markedly decreased cellular content of arachidonic acid (AA). In rabbit TE cells, supplementation of media with a phospholipid-rich lipoprotein extract (Excyte III) plus 1 microM bovine serum albumin-complexed AA (Excyte/AA) restored the fatty acid composition of the cultured cells more similar to that of native airway epithelium than did supplementation of media with 5% fetal bovine serum (FBS). In human TE cells, Excyte/AA or 5% FBS increased AA content, but neither lipid supplement completely “normalized” the fatty acid profiles. Compared with lipid-unsupplemented cultures, basal production of prostaglandin E2 (PGE2) was increased by approximately four- to eightfold in rabbit and human TE cells supplemented with 5% FBS or Excyte/AA. In Excyte/AA-supplemented human TE cells, PGE2 production induced by 5 microM calcium ionophore A23187 was more than threefold greater than that of companion ionophore-stimulated unsupplemented monolayers, but PGE2 production was similar in both culture conditions in response to 10 microM exogenous AA. Thus increased cellular content and availability of AA, rather than changes in cyclooxygenase activity, appear to be responsible for the elevated PGE2 production in Excyte/AA-supplemented human TE cells. Secondary effects of lipid supplementation were also observed; Excyte/AA-supplemented human TE cells produced significantly less adenosine 3',5'-cyclic monophosphate (cAMP) in response to exogenous PGE2 and isoproterenol than did lipid-unsupplemented cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelin regulation of mucus glycoprotein secretion from feline tracheal submucosal glands
Authors: Shimura S. , Ishihara H. , Satoh M. , Masuda T. , Nagaki N. , Sasaki H. , Takishima T.
Published: December 2017

We examined the effects of endothelin on both the trichloroacetic acid precipitable 3H-labeled glycoconjugate release and intracellular Ca2+ concentration ([Ca2+]i]) measured by the usage of fura-2 in submucosal glands isolated from feline trachea. Endothelin-1 produced a significant increase in glycoconjugate release from the isolated glands in a dose-dependent fashion, reaching a response of 161% of the control at 10(-6) M. Atropine, propranolol, phentolamine, or indomethacin did not produce any significant alterations in the ET-1-evoked glycoconjugate secretion from the isolated glands. In contrast, in tracheal explants which contained epithelium, ET-1 produced a significant reduction in the glycoconjugate secretion in a dose-dependent fashion, reaching a response of 59% of the control at 10(-6) M. In the presence of cultured epithelial cells, ET-1 also produced a significant reduction in the glycoconjugate secretion from isolated glands. In isolated glands, ET-1 produced a sustained increase in the [Ca2+]i which was abolished by the removal of Ca2+ from the medium or by the presence of cultured epithelial cells. Pretreatment with indomethacin failed to alter the epithelial inhibitory action evoked by ET-1 in both the glycoconjugate secretion and the [Ca2+]i in isolated glands. ET-2 and ET-3 failed to produce significant alterations in the glycoconjugate secretion or [Ca2+]i. These findings indicate 1) that ET-1 induces mucus glycoprotein secretion via a Ca2+ influx and 2) that it possibly augments the an epithelial action inhibitory to the mucus glycoprotein secretion from airway submucosal glands.
Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats
Authors: Hershenson M. B. , Aghili S. , Punjabi N. , Hernandez C. , Ray D. W. , Garland A. , Glagov S. , Solway J.
Published: December 2017

We exposed 21-day-old rats to either normoxia or hyperoxia (greater than 95% O2) for 8 days and assessed in vivo airway responsiveness to aerosolized and intravenous methacholine (MCh) and airway architecture. Airway responsiveness was determined using a plethysmographic method. Hyperoxia increased airway cholinergic responsiveness, as reflected in a decreased mean ED200 (concentration of MCh required to increase respiratory system resistance by 100%) for both aerosolized MCh [air exposed, 5.94 +/- 2.50 vs. O2 exposed, 0.29 +/- 3.34 (SD) mg/ml, P = 0.0013, unpaired t test] and intravenous MCh (air, 1.40 x 10(-8) vs. O2, 2.45 x 10(-10) mol/kg, P = 0.0002). Airway morphometry was studied in a separate cohort of animals. After fixation by distension with Formalin at 25 cmH2O pressure, each airway cross section was photographed, and airway circumference, epithelial area, and smooth muscle layer area were determined by means of contour tracing using a digitizing pad and microcomputer. For the small airways (circumference less than 1,000 microns), hyperoxia increased both mean epithelial thickness (air, 4.88 +/- 0.53; O2, 8.64 +/- 0.90 microns) and mean smooth muscle layer thickness (air, 2.69 +/- 0.11; O2, 4.79 +/- 0.56 microns; P less than 0.0001 for each). O2 had similar effects on the larger (1,000-3,000 microns) central airways (P less than 0.0001 for both layers). We conclude that chronic hyperoxic exposure induces both airway hyperresponsiveness and airway wall thickening in immature rats.
Developing bronchopulmonary epithelium of the human fetus secretes fluid
Authors: McCray P. B. , Bettencourt J. D. , Bastacky J.
Published: December 2017

We studied human fetal lung tissue in submersion organ culture to determine whether the bronchopulmonary epithelium secretes fluid during development. In this system the acinar tubules continued to grow, secrete fluid, and become progressively dilated. Baseline transepithelial potential differences (psi t) of -0.5 to -11 mV (mean, -3.8 mV, lumen negative, n = 27) were measured with microelectrodes after 3-8 days in culture, suggesting active electrolyte transport. Bumetanide (500 microM), an inhibitor of chloride secretion in other systems, decreased the basal psi t from -5 +/- 1.5 to -3.2 +/- 1.6 (SE) mV (P less than 0.05, n = 6), suggesting that chloride transport contributed to the voltage. Isoproterenol (5 microM) increased the baseline psi t from -5.6 +/- 2.1 to -9.2 +/- 2.5 (SE) mV (P less than 0.05, n = 4). Subsequent addition of bumetanide inhibited the isoproterenol-induced stimulation of the psi t by 20% (P less than 0.05). 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. (CPT-cAMP, 50 microM) and 3-isobutyl 1-methylxanthine (IBMX, 100 microM) had similar effects, causing an increase in the psi t from -2.2 +/- 0.5 to -8 +/- 1.6 (SE) mV, an effect that was inhibited by the addition of bumetanide (P less than 0.005, n = 6). Both isoproterenol and CPT-cAMP/IBMX produced significant increases in the percentage luminal area of the explants at 12 and 24 h after exposure compared with control. We conclude that 1) the developing bronchopulmonary epithelium (acinar tubules) contributes to lung fluid production in the human fetus, 2) fetal lung fluid secretion is chloride dependent, and 3) chloride secretion and fluid secretion may be stimulated by a beta-agonist and cAMP.
Different desipramine-sensitive pulmonary removals of plasma epinephrine and norepinephrine in dogs
Authors: Eisenhofer G. , Smolich J. J. , Esler M. D.
Published: December 2017

The lungs are an important site for the clearance of norepinephrine from the circulation, but the mechanism of removal and its importance for clearance of epinephrine require clarification. In the present study, 3H-labeled epinephrine and norepinephrine were infused intravenously into anesthetized dogs, and blood samples were taken from the pulmonary artery and aorta to compare pulmonary extractions of both catecholamines before and after administration of the neuronal uptake blocking drug, desipramine. Circulating [3H]norepinephrine and [3H]-epinephrine were both extracted by the lungs, but fractional extraction of [3H]epinephrine (8.1 +/- 1.1%) was half that of [3H]norepinephrine (16.9 +/- 1.9%). Pulmonary extractions of both catecholamines were completely blocked by desipramine. Desipramine-sensitive removal accounted for a tenth of the total body extraction of [3H]epinephrine and a third that of [3H]norepinephrine. Pulmonary removal of [3H]epinephrine was 14% that of the total body clearance, whereas pulmonary removal of [3H]norepinephrine was 25% that of the total body clearance. The study showed the following: removal of catecholamines by the canine lung in vivo is by desipramine-sensitive uptake; uptake is half as efficient for epinephrine as norepinephrine: and, pulmonary removal contributes importantly to the desipramine-sensitive component of total body catecholamine clearance.
Requirements for extracellular metabolism of pulmonary surfactant: tentative identification of serine protease
Authors: Gross N. J. , Schultz R. M.
Published: December 2017

Pulmonary alveolar surfactant is secreted by the alveolar epithelium in the form of lamellar bodylike structures that evolve sequentially into tubular myelin and vesicular forms that can be separated by centrifugation. Using an in vitro procedure by which the extracellular metabolism of pulmonary surfactant can be mimicked, namely cyclic variation in surface area, we previously reported that serine protease activity, which we called “convertase,” was required for the conversion of tubular myelin to the vesicular form. In the present studies we explored the biochemical requirements of this activity and sought the enzyme in alveolar products. Convertase activity has unusual requirements; in addition to being dependent on repetitive variations in surface area (cycling), it requires the presence of a high g fraction of lung secretions that is heat stable and not inhibitable by diisopropyl fluorophosphate (DFP) or alpha 1-antitrypsin, both typical serine protease inhibitors. The enzyme does not require calcium ions and has a pH optimum of 7.4. Convertase appears to be a component of surfactant itself because the ability of purified surfactant to convert to the vesicular form on cycling is impaired by pretreating it with DFP. A protein of Mr 75,000 that reacts with DFP and is heat sensitive was found in alveolar lavage, lamellar body preparations, and lung homogenate. It copurifies with lung surfactant in sucrose gradients. A similar DFP-reactive protein was observed in stable human neoplastic peripheral airway cell lines that express type II properties, suggesting that it may be a product of type II cells. We tentatively conclude that surfactant convertase is a 75,000 serine protease that is closely associated with surfactant phospholipid and that may be a product of alveolar type II cells.
Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity
Authors: Koyama S. , Rennard S. I. , Robbins R. A.
Published: December 2017

Bronchial asthma is accompanied by inflammatory cell infiltration in the airway. Increased bronchial reactivity to cholinergic stimulation is well recognized in patients with bronchial asthma. Thus, we postulated that acetylcholine (ACh) stimulates bronchial epithelial cells (BEC) to release neutrophil and monocyte chemotactic activity (NCA and MCA). To test this hypothesis, bovine BEC monolayers were tested for NCA and MCA by a blind-well chemotactic chamber technique. BEC released NCA and MCA in response to ACh in a dose-dependent and time-dependent manner. Molecular sieve column chromatography revealed that ACh induced a single low-molecular-weight peak (near 400) for NCA and two low-molecular-weight peaks (near 12,000 and 400) for MCA. The release of NCA and MCA was inhibited by the lipoxygenase inhibitors, nordihydroguaiaretic acid and diethylcarbamazine. Cigarette smoke is a well-recognized stimulus for airway inflammation. To determine whether smoke might activate BEC to release NCA by stimulating nicotinic ACh receptors, we further characterized the ACh receptors, using nicotine and nicotinic and muscarinic receptor antagonists. Nicotine, the nicotinic receptor antagonist d-tubocurarine, and the M2 receptor antagonist gallamine did not modulate the release of NCA in response to ACh. In contrast, atropine and the M1 receptor antagonist, pirenzepine, inhibited the release of NCA. These data demonstrate that ACh stimulates BEC to release lipoxygenase-derived NCA and MCA through the muscarinic receptor.
Expression of CFTR and presence of cAMP-mediated fluid secretion in human fetal lung
Authors: McCray P. B. , Reenstra W. W. , Louie E. , Johnson J. , Bettencourt J. D. , Bastacky J.
Published: December 2017

We studied the developmental expression of the cystic fibrosis (CF) gene in human lung tissue from normal and CF-affected fetuses. Two unrelated CF fetuses, both homozygous for the delta F508 deletion, were examined. Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was present in second-trimester CF lung and in first- and second-trimester normal lung as assessed by amplification of reverse transcribed total RNA with the use of the polymerase chain reaction. CFTR protein was identified by immunoprecipitation in normal second-trimester fetal lung explants. To evaluate possible functional consequences of CF in the fetus, lung tissue explants were grown in submersion organ culture. By light and electron microscopy, the CF fetal lung explants appeared normal. When explants from normal fetal lung were exposed to 8-(4-chlorophenylthio) adenosine 3',-5'cyclic monophosphate (CPT-cAMP), and 3-isobutyl-1-methylxanthine (IBMX) for 24 h, the intraluminal fluid content increased, as assessed by a 40 +/- 4% increase in cross-sectional diameter. In contrast, identically treated CF explants showed no significant change in explant diameter (3 +/- 1.6%). The transepithelial potential (psi t) across fetal lung explants was measured with microelectrodes. In normal second-trimester explants, CPT-cAMP and IBMX caused hyperpolarization of psi t (-0.93 +/- 14 mV to -4.3 +/- 1.2 mV); in contrast, CF fetal lung explants showed no significant change in psi t with CPT-cAMP and IBMX (-0.84 +/- 0.07 mV to -1.21 +/- 0.26 mV). This study confirms the presence of CFTR mRNA and protein in human fetal lung and suggests that although the CF fetal lung appears normal morphologically, there is a defect in cAMP-mediated fluid secretion in the lung of the CF fetus.
Role of eicosanoids in staphylococcal alpha-toxin-induced lung injury in the rat
Authors: Chang S. W. , Czartolomna J. , Voelkel N. F.
Published: December 2017

We investigated the role of arachidonic acid-derived eicosanoids in staphylococcal alpha-toxin (alpha-T)-induced lung injury. Bolus injection of 200 and 500 micrograms alpha-T into isolated perfused rat lungs resulted in increased pulmonary perfusion pressure followed by lung weight gain. Inhibition of pressure change with papaverine (10(-4) M) failed to abolish lung edema. Furthermore, alpha-T increased the permeability-surface area product in papaverine-treated lungs and caused marked endothelial cell injury and interstitial edema as documented by electron microscopy. alpha-T dose dependently increased lung tissue thromboxane B2 (TxB2) levels and leukotriene C4 levels. In lungs given 0, 200, and 500 micrograms of alpha-T, TxB2 (in micrograms/g wet lung) values were 16.3 +/- 2.8, 25.0 +/- 3.0, and 54.2 +/- 6.2; and leukotriene C4 values were 4.6 +/- 1.1, 6.7 +/- 1.2, and 22.1 +/- 3.8, respectively. Inhibition of cyclooxygenase enzyme with indomethacin (10(-5) M) or lipoxygenase enzyme with 2(12-hydroxydodeca-5,10-dinyl)-3,5,6-trimethyl-1,4-benzoq uin one (AA861, 10(-5) M) attenuated the vasoconstriction and prevented lung edema due to low dose (200 micrograms) but not high dose (500 micrograms) alpha-T. The protective effect of these inhibitors on lung edema is in part due to decreases in alpha-T-stimulated venoconstriction because alpha-T-induced increase in lung microvascular pressure was attenuated by indomethacin and AA861 pretreatment. We conclude that both eicosanoid-dependent and eicosanoid-independent mechanisms contribute to alpha-T-induced lung edema in the rat.
The lungs and vitamin A
Authors: Chytil F.
Published: December 2017

Evidence is reviewed supporting the view that vitamin A (retinol) and its metabolite, retinoic acid, called natural retinoids, are major factors involved in differentiation and in maturation of the lungs. This conclusion is based on morphological observation that lack of this dietary micronutrient causes keratinizing squamous metaplasia of the bronchopulmonary tree that can be reversed by refeeding the animal with retinol. In addition to these observations suggesting an indirect participation of retinol and/or retinoic acid in the differentiation of this organ, more direct evidence is presented that this vitamin is involved in pulmonary gene expression. Adult as well as fetal lungs accumulate retinyl esters, the storage form of vitamin A, contain specific retinol- and retinoic acid-binding proteins and, more importantly, express several isoforms of nuclear retinoic acid receptors. These proteins are involved in activation and repression of specific genes regulated by retinoic acid. That retinol and/or retinoic acid may be involved in lung maturation is suggested by experimental results showing depletion of lung retinyl esters at birth, by significant alterations in the levels of the cellular retinoic acid-binding protein during lung maturation and, more importantly, by reduction in the morbidity of prematurely born human neonates who are given vitamin A because they are susceptible to bronchopulmonary dysplasia.
An allelic variant of the 6A gene for human surfactant protein A
Authors: Rishi A. , Hatzis D. , McAlmon K. , Floros J.
Published: December 2017

Two human surfactant protein A (SP-A) cDNA clones (1A and 6A) encoding human SP-A genes have previously been identified and characterized by our laboratory. The present study was undertaken to assess potential heterogeneity in the 6A locus among specimens from white adults and infants with respiratory distress syndrome (RDS) as well as black adults. The entire and/or a portion of the published SP-A gene was amplified from different genomic DNAs using the polymerase chain reaction (PCR), cloned, and sequenced. The data revealed the presence of a variant (6A1) of the 6A gene with characteristics of the 1A gene. The most notable changes were a one-base change in exon 1 and an 11-base insertion in the 3' untranslated region (3'UT). Both changes are found in 1A gene. Analysis of the reverse-transcribed (RT) PCR products from lung mRNAs using 6A specific oligos showed that the 6A1 variant is transcribed. The prevalence of each SP-A allele was then assessed in a group of 20 Nigerian Blacks and 43 Whites (26 infants with RDS and 17 adults). Although the data did not reach statistical significance, a trend was shown where the 6A/6A genotype appeared with higher frequency in Blacks.
Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells
Authors: Sisson Joseph H. , Tuma Dean J. , Rennard Stephen I.
Published: December 2017

Pages L29–L36: Joseph H. Sisson, Dean J. Tuma, and Stephen I. Rennard. “Acetaldehyde-mediated cilia dysfunction in bovine bronchial epithelial cells.” In Figs. 1, 2, 4, and 5, the concentration of acetaldehyde in the figures and their legends should read mM (millimolar) instead of μM (micromolar). Page L34: right-hand column, the first sentence of the paragraph should read: The threshold concentrations of acetaldehyde-induced impairment in these studies was 30 mM for ATPase inhibition and ciliostasis
Arachidonic acid increases cholinergic secretory responsiveness of ferret tracheal glands
Authors: McBride R. K. , Stone K. K. , Marin M. G.
Published: December 2017

The purpose of this study was to determine if arachidonic acid could alter ferret tracheal gland secretory responsiveness to a cholinergic agonist. We prepared glandular explants and incubated the explants in medium containing [3H]glucosamine. Secretory responsiveness was expressed as the percent change in basal secretion of acid-precipitable [3H]glucosamine-labeled glycoconjugates induced by the addition of agonist with and without arachidonic acid [mean +/- SE (n)]. Addition of 10(-3) M arachidonic acid caused a significant increase in secretion [28 +/- 6% (n = 6)] compared with untreated control tissues [-10 +/- 4% (n = 7), P less than or equal to 0.05]. Carbachol (10(-7) M) increased secretion 39 +/- 9% (n = 7). The combination of 10(-3) M arachidonic acid and 10(-7) M carbachol elicited a significantly greater change in secretion compared with either agent alone [173 +/- 50% (n = 5)]. The addition of nordihydroguaiaretic acid (10(-6) M) or indomethacin (10(-6) M) partially attenuated the arachidonic acid-enhanced secretory responsiveness to carbachol. Treatment with both blockers completely inhibited the arachidonic acid-enhanced secretory responsiveness to carbachol. The effect of arachidonic acid on cholinergic stimulation was also abolished by treating the explant cultures with tetrodotoxin (10(-7) M). This hypersecretory state is most likely mediated by eicosanoid-induced release of neurotransmitters from nerve terminals.
Monocrotaline pyrrole alters DNA, RNA and protein synthesis in pulmonary artery endothelial cells
Authors: Hoorn C. M. , Roth R. A.
Published: December 2017

Administration of monocrotaline pyrrole (MCTP) to animals results in pulmonary vascular injury. Pulmonary vascular endothelium is a likely target for this pneumotoxicant. Cultured porcine pulmonary artery endothelial cells (PECs) treated with MCTP remain viable but are unable to divide and exhibit an altered morphology. Such responses raise a question about the extent to which affected cells carry out normal functions such as RNA and protein synthesis. Accordingly, the cellular activity of MCTP-treated PECs was examined in this study. PECs were treated with a single administration of MCTP or vehicle, and determinations of cell number, protein, and DNA content were made at times up to 7 days posttreatment. DNA, RNA, and protein synthesis were quantified by incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine, respectively. Increases in cell number that occurred with time in the control cells were reduced in MCTP-treated cells. At 7 days posttreatment, both protein and DNA content increased above control levels. Synthesis of DNA, RNA, and protein continued in all treatment groups throughout the posttreatment period, but cells treated with high concentrations of MCTP showed less synthetic activity than controls during the initial 48 h posttreatment. By 7 days, MCTP-treated cells were producing significantly more DNA, RNA, and protein. These results indicate that cells treated with MCTP continue to synthesize DNA, resulting in an increased DNA content. In addition, treated cells continue to synthesize RNA and translate RNA into protein. Thus, cellular activity is maintained but altered substantially by MCTP exposure.
Surfactant protein A metabolism in preterm ventilated lambs
Authors: Ikegami M. , Lewis J. F. , Tabor B. , Rider E. D. , Jobe A. H.
Published: December 2017

Surfactant protein A (SP-A) metabolism was studied in vivo in 33 preterm ventilated lambs at 138 +/- 1 days gestational age by measuring recoveries of exogenously administered surfactant containing both radiolabeled SP-A and labeled saturated phosphatidylcholine (Sat PC) given via the trachea at birth. Endogenously secreted SP-A was also labeled with [35S]methionine and followed over 24 h. The exogenously labeled SP-A left the alveolar pool more rapidly than did Sat PC over the first 5 h of life (P less than 0.05), and both exogenously labeled SP-A and Sat PC were detected within lamellar bodies by 2 h, indicating uptake from the airspaces. The quantity of SP-A in alveolar washes increased about twofold from birth to 5 h of age, whereas alveolar Sat PC pools were constant over 24 h. The SP-A endogenously labeled with [35S]methionine was recovered at highest specific activities in the alveolar washes at 10 and 45 min after birth with no labeled SP-A detectable in lamellar body fractions until 2 h. The curve for endogenous SP-A labeling of lamellar bodies was similar to that for exogenous labeling, indicating that SP-A was initially secreted by a pathway independent of lamellar bodies with subsequent SP-A labeling of lamellar bodies. The kinetics of SP-A metabolism were very different than for Sat PC in preterm lambs.
Cloning and sequence analysis of rat cystic fibrosis transmembrane conductance regulator
Authors: Fiedler M. A. , Nemecz Z. K. , Shull G. E.
Published: December 2017

A complementary DNA (cDNA) encoding the rat cystic fibrosis transmembrane conductance regulator (CFTR) has been isolated and the tissue distribution of the rat CFTR mRNA has been determined. Northern blot analysis revealed that the highest levels of the 6.3 kilobase (kb) CFTR mRNA were expressed in the colon, with expression also noted in uterus, lung, stomach, and small intestine. A 7.5-kb mRNA was expressed in skeletal muscle, and in testes both the 7.5-kb mRNA and a 6.0-kb mRNA were expressed. Five cDNAs were isolated from a rat colon library, the longest corresponding to codons 684 through the poly A tail. Three other clones, corresponding to codons 213 through 245, 372 through 574, and 656 through 886 were also isolated. Polymerase chain reaction amplification of cDNA prepared from rat colon mRNA was utilized to clone the remainder of the cDNA. The predicted amino acid sequence of the rat CFTR is 79% identical to the human CFTR, with 73% identity noted in the R domain, and 81 and 83% identities noted in nucleotide binding folds 1 and 2, respectively. Thirty-two of the 38 potential phosphorylation sites identified in the human CFTR were also present in the rat CFTR.
Bioassay of a tracheal smooth muscle-constricting factor released by respiratory epithelial cells
Authors: Wilkens J. H. , Becker A. , Wilkens H. , Emura M. , Riebe-Imre M. , Plein K. , Schober S. , Tsikas D. , Gutzki F. M. , Frolich J. C.
Published: December 2017

Epithelium-derived factors of unknown identity have been proposed to modulate airway smooth muscle tone. We developed a novel sensitive bioassay system that allows serial perfusion of cultured respiratory epithelial cells and guinea pig trachea (GPT). GPT responses were assessed as diameter changes by computerized video microscopy (resolution, 15 microns). A permanent hamster lung epithelial cell line was grown on microcarrier beads and perfused in a cell column. When the outflow tubing from the epithelial cell column was connected to the inflow cannula, the detector GPT contracted, reaching 28 +/- 6% of the maximum methacholine (100 microM)-induced contraction (n = 12, P less than 0.001). Perfusion of the cell column with diclofenac (10 microM) or lysin-mono-acetylsalicylic acid (100 microM) abolished the GPT contraction, whereas selective perfusion of the detector GPT with either agent did not block the contraction. Analysis of the effluent of the epithelial cell column demonstrated a significant basal release of prostaglandins F2 alpha and E2 (PGF2 alpha and PGE2) and 6-ketoprostaglandin F1 alpha, whereas only marginal amounts of thromboxane B2 were detected. When given exogenously, PGF2 alpha, PGE2, PGI2, and U-46619 all contracted the GPT. It is concluded that lung epithelial cells can contract GPT by releasing a transferable factor. This factor is likely to be a constrictor cyclooxygenase product that is not produced in epithelium-denuded GPT.
Direct measurement of acetylcholine release in guinea pig trachea
Authors: Baker D. G. , Don H. F. , Brown J. K.
Published: December 2017

In this study, we applied high-performance liquid chromatography with electrochemical detection (HPLC-EC) to the measurement of acetylcholine (ACh) release from nerve endings in guinea pig tracheal smooth muscle. We also tested for muscarinic inhibitory regulation of ACh release in this species, which is widely used for studies of airway neural control. Clip-connected segments of the posterior membrane of the guinea pig trachea were mounted in organ baths between stimulating electrodes and incubated in Krebs-Henseleit buffer containing (in microM) 10 indomethacin, 1 neostigmine, 1 phentolamine, and 1 propranolol. To measure ACh, the bath was emptied and aliquots of buffer were injected directly into the HPLC-EC; the lower limit of detection was 1 pmol/200 microliters sample. Electrical field stimulation (EFS) at 5 Hz for 10 or 30 min increased ACh release from 1.8 +/- 1.4 (SE) to 6.2 +/- 1.3 pmol.mg protein-1.min-1 (n = 15). The effect of atropine was examined by comparing amounts of ACh released by EFS before and after exposure to either atropine (0.3 microM) or vehicle. Before atropine treatment, EFS-evoked ACh release was 4.9 +/- 0.6 pmol.mg protein-1.min-1; after atropine exposure, EFS-evoked release of ACh increased significantly to 15.0 +/- 2.2 pmol.mg protein-1.min-1 (n = 11; P less than 0.05). Corresponding values before and during exposure to vehicle were 9.3 +/- 4.4 and 10.7 +/- 4.7 pmol.mg protein-1.min-1, respectively. The ratios of the changes in EFS-evoked ACh release were 3.1 +/- 0.3 and 1.3 +/- 0.1 in atropine-treated and vehicle-treated groups, respectively (P less than 0.05). We conclude that HPLC-EC is a reliable and sensitive technique for the detection of EFS-evoked release of ACh from clip-connected segments of guinea pig tracheal smooth muscle.
IL-6 enhances TNF-alpha- and IL-1-induced increase of Mn superoxide dismutase mRNA and O2 tolerance
Authors: Tsan M. F. , White J. E. , Del Vecchio P. J. , Shaffer J. B.
Published: December 2017

Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1), and interleukin-6 (IL-6) are multifunctional cytokines produced by a number of cells in response to endotoxin. We have previously demonstrated (M.-F. Tsan, J. E. White, T. A. Santana, and C. Y. Lee. J. Appl. Physiol. 68: 1211–1219, 1990, and M.-F. Tsan, C. Y. Lee, and J. E. White. J. Appl. Physiol. 71: 688–697, 1991) that tracheal insufflation of 5 micrograms of TNF-alpha or 1 microgram of IL-1 markedly protects rats against O2 toxicity and enhances pulmonary Mn superoxide dismutase (Mn SOD) activity. We now report that TNF-alpha and IL-1 at subprotective doses, e.g., 1 and 0.2 micrograms, respectively, act synergistically in protecting rats against O2 toxicity. Likewise, TNF-alpha and IL-1 at 0.005 microgram/ml each act synergistically in enhancing endothelial cell Mn SOD, but not Cu,Zn SOD mRNA levels. IL-6 at 5 or 10 micrograms provides no protective effect in rats against O2 toxicity and at up to 0.5 microgram/ml has no apparent effect on endothelial cell Mn or Cu,Zn SOD mRNA levels. However, IL-6 markedly enhances TNF-alpha- and IL-1-induced increases in Mn SOD mRNA levels and O2 tolerance. These results support an important role of Mn SOD in the protection against O2 toxicity.