bluuebunny/crossref_metadata_2025 huggingface dataset:
Carl J. Wiggers and the pulmonary circulation: a young man in search of excellence
Authors: Reeves John T.
Published: December 2017

Oddly, Carl Wiggers (1883–1962), who is remembered for his work on the systemic circulation, may be considered the “American father of the pulmonary circulation.” In nearly 20 papers published in the American Journal of Physiology between 1909 and 1925, he reported the first reliable pressure contours in the pulmonary artery, inquired into the relationship between respiration and pulmonary arterial pressure, examined right atrial and right ventricular function, and demonstrated how right and left heart dynamics relate to heart sounds. He also stimulated direct visualization of the lung microcirculation. Method and concept are inextricably linked in the progress of science. His contributions to the pulmonary circulation were based on his high-fidelity pressure and sound recording instruments, which he ultimately applied in the left heart. Wiggers’ search for excellence in method brought him well-deserved fame in the systemic circulation, but the search began in the lung.
Neonatal Clara cell toxicity by 4-ipomeanol alters bronchiolar organization in adult rabbits
Authors: Smiley-Jewell Suzette M. , Nishio Susan J. , Weir Alison J. , Plopper Charles G.
Published: December 2017

Nonciliated bronchiolar (Clara) cells metabolize environmental toxicants, are progenitor cells during development, and differentiate postnatally. Because differentiating Clara cells of neonatal rabbits are injured at lower doses by the cytochrome P-450-activated cytotoxicant 4-ipomeanol than are those of adults, the impact of early injury on the bronchiolar epithelial organization of adults was defined by treating neonates (3–21 days) and examining them at 4–6 wk. Bronchiolar epithelium of 6-wk-old animals treated on day 7 was most altered from that of control animals. Almost 100% of the bronchioles were lined by zones of squamous epithelial cells. Compared with control animals, the distal bronchiolar epithelium of 4-ipomeanol-treated animals had more squamous cells (70–90 vs. 0%) with a reduced overall epithelial thickness (25% of control value), fewer ciliated cells (0 vs. 10–20%), a reduced expression of Clara cell markers of differentiation (cytochrome P-4502B, NADPH reductase, and 10-kDa protein), and undifferentiated nonciliated cuboidal cell ultrastructure. We conclude that early injury to differentiating rabbit Clara cells by a cytochrome P-450-mediated toxicant inhibits bronchiolar epithelial differentiation and greatly affects repair.
Mechanism of alltrans-retinoic acid and glucocorticoid regulation of surfactant protein mRNA
Authors: George Thomas N. , Miakotina Olga L. , Goss Kelli L. , Snyder Jeanne M.
Published: December 2017

The surfactant proteins (SPs) are required for the normal function of pulmonary surfactant, a lipoprotein substance that prevents alveolar collapse at end expiration. We characterized the effects of cortisol and all trans-retinoic acid (RA) on SP-A and SP-B gene expression in H441 cells, a human pulmonary adenocarcinoma cell line. Cortisol, at 10−6M, caused a significant inhibition of SP-A mRNA to levels that were 60–70% of controls and a five- to sixfold increase in the levels of SP-B mRNA. RA alone (10−6M) had no effect on SP-A mRNA levels and modestly reduced the inhibitory effect of cortisol. RA alone and the combination of cortisol and RA both significantly increased SP-B mRNA levels. RA had no effect on the rate of SP-A gene transcription or on SP-A mRNA stability. Cortisol alone and the combination of cortisol and RA significantly inhibited the rate of SP-A gene transcription but had no effect on SP-A mRNA half-life. RA at 10−6M had no effect on the rate of SP-B gene transcription but prolonged SP-B mRNA half-life. Cortisol alone and the combination of cortisol and RA caused a significant increase in the rate of SP-B gene transcription and also caused a significant increase in SP-B mRNA stability. We conclude that RA has no effect on SP-A gene expression and increases SP-B mRNA levels by an effect on SP-B mRNA stability and not on the rate of SP-B gene transcription. In addition, the effects of the combination of RA and cortisol were generally similar to those of cortisol alone.
G protein-coupled prostaglandin receptor modulates conductive Na+uptake in lung apical membrane vesicles
Authors: Mukhopadhyay Somnath , Dutta-Roy Asim K. , Fyfe Gregor K. , Olver Richard E. , Kemp Paul J.
Published: December 2017

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2receptor controls perinatal lung alveolar Na+transport. [3H]PGE2binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2= PGE1> PGD2> PGF2α. Kinetic analysis demonstrated both high-affinity [dissociation constant ( KD) = 2.1 ± 0.7 nM; maximal binding (Bmax) = 27 ± 7 fmol/mg protein] and low-affinity ( KD= 28 ± 2 nM; Bmax= 265 ± 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50= 11 mM for PGF2αvs. 10–50 μM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 μM PGE2(≈IC25) increased conductive22Na+uptake into membrane vesicles only in the presence of 100 μM intravesicular GTP. The KDvalue for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3subtype, whereas Na+uptake studies suggest that it helps maintain perinatal lung Na+homeostasis.
Acrolein-induced MUC5ac expression in rat airways
Authors: Borchers Michael T. , Wert Susan E. , Leikauf George D.
Published: December 2017

Acrolein, a low-molecular-weight aldehyde found in photochemical smog and tobacco smoke, can induce mucus hypersecretion, inflammation, and airway hyperreactivity. To determine whether changes in steady-state mucin gene expression (MUC2 and MUC5ac) are associated with histological signs of mucus hypersecretion, rats were exposed to acrolein (3.0 parts/million, 6 h/day, 5 days/wk, 2 wk), and the trachea with the main stem bronchi was separated from the intrapulmonary airways (lung). The temporal expression of MUC2 and MUC5ac mRNA was determined by RT-PCR, and acidic mucin glycoproteins were detected by Alcian blue histochemical analysis. MUC5ac protein content in the airways was determined by immunohistochemical analysis. Tracheal MUC5ac mRNA increased within 2 days and was accompanied by an increase in MUC5ac immunostaining on the surface of the airways and in submucosal gland epithelium. By comparison, increases in lung MUC5ac mRNA and mucin glycoproteins were delayed and were elevated after exposures on days 5 and 9, respectively. Increased MUC5ac immunostaining was detected within the lumen and airway epithelium of the lung on day 12. In contrast, MUC2 mRNA levels were not significantly changed in the trachea or lung. These findings indicate that acrolein-induced mucus hypersecretion is due, in part, to increases in MUC5ac rather than to MUC2 gene expression. These findings suggest that aldehyde-induced increases in MUC5ac may play a role in chronic mucus hypersecretion, a pathognomonic feature of chronic obstructive pulmonary disease.
Molecular basis and function of voltage-gated K+channels in pulmonary arterial smooth muscle cells
Authors: Yuan Xiao-Jian , Wang Jian , Juhaszova Magdalena , Golovina Vera A. , Rubin Lewis J.
Published: December 2017

K+-channel activity-mediated alteration of the membrane potential and cytoplasmic free Ca2+concentration ([Ca2+]cyt) is a pivotal mechanism in controlling pulmonary vasomotor tone. By using combined approaches of patch clamp, imaging fluorescent microscopy, and molecular biology, we examined the electrophysiological properties of K+channels and the role of different K+currents in regulating [Ca2+]cytand explored the molecular identification of voltage-gated K+(KV)- and Ca2+-activated K+(KCa)-channel genes expressed in pulmonary arterial smooth muscle cells (PASMC). Two kinetically distinct KVcurrents [ IK(V)], a rapidly inactivating (A-type) and a noninactivating delayed rectifier, as well as a slowly activated KCacurrent [ IK(Ca)] were identified. IK(V)was reversibly inhibited by 4-aminopyridine (5 mM), whereas IK(Ca)was significantly inhibited by charybdotoxin (10–20 nM). K+channels are composed of pore-forming α-subunits and auxiliary β-subunits. Five KV-channel α-subunit genes from the Shaker subfamily (KV1.1, KV1.2, KV1.4, KV1.5, and KV1.6), a KV-channel α-subunit gene from the Shab subfamily (KV2.1), a KV-channel modulatory α-subunit (KV9.3), and a KCa-channel α-subunit gene ( rSlo), as well as three KV-channel β-subunit genes (KVβ1.1, KVβ2, and KVβ3) are expressed in PASMC. The data suggest that 1) native K+channels in PASMC are encoded by multiple genes; 2) the delayed rectifier IK(V)may be generated by the KV1.1, KV1.2, KV1.5, KV1.6, KV2.1, and/or KV2.1/KV9.3 channels; 3) the A-type IK(V)may be generated by the KV1.4 channel and/or the delayed rectifier KVchannels (KV1 subfamily) associated with β-subunits; and 4) the IK(Ca)may be generated by the rSlo gene product. The function of the KVchannels plays an important role in the regulation of membrane potential and [Ca2+]cytin PASMC.
Inhibition of vascular and epithelial differentiation in murine nitrofen-induced diaphragmatic hernia
Authors: Coleman C. , Zhao J. , Gupta M. , Buckley S. , Tefft J. D. , Wuenschell C. W. , Minoo P. , Anderson K. D. , Warburton D.
Published: December 2017

Neonates with congenital diaphragmatic hernia (DH) die of pulmonary hypoplasia and persistent pulmonary hypertension. We used immunohistochemical localization of α-smooth muscle actin (α-SMA), platelet endothelial cell adhesion molecule (PECAM)-1, thyroid transcription factor (TTF)-1, surfactant protein (SP) A, SP-C, and competitive RT-PCR quantitation of TTF-1, SP-A, SP-C, and α-SMA mRNA expression to characterize the epithelial and vascular phenotype of lungs from ICR fetal mice with a nitrofen-induced DH. Nitrofen (25 mg) was gavage fed to pregnant mice on day 8 of gestation. Fetal mice were delivered on day 17. The diaphragm was examined for a defect, and the lungs were either fixed, sectioned, and immunostained or processed for mRNA isolation. In comparison with control lungs, DH lungs showed increased expression of α-SMA mRNA, fewer and more muscular arterioles (α-SMA), less well-developed capillary networks (PECAM-1), delayed epithelial development marked by a persistence of TTF-1 in the periphery, and decreased SP-A mRNA and SP-A expression. These data suggest that in the murine nitrofen-induced DH, as in human congenital DH, pulmonary insufficiency is due to an inhibition of peripheral pulmonary development including terminal airway and vascular morphogenesis.
Hypoxic pulmonary endothelial cells release a diffusible contractile factor distinct from endothelin
Authors: Gaine Sean P. , Hales Mariesa A. , Flavahan Nicholas A.
Published: December 2017

Hypoxia (0% O2) evokes a late-phase, endothelium-dependent contractile response in porcine isolated pulmonary arteries that may be caused by a cyclooxygenase-independent, endothelium-derived contractile factor. The aim of this study was to further analyze the mechanism underlying this hypoxic response. Proximal porcine pulmonary arterial rings were suspended for isometric tension recording in organ chambers. Hypoxia (0% O2) caused a late-phase, endothelium-dependent contractile response that was not inhibited by the endothelin (ET)A-receptor antagonist BQ-123 (10−6M), by the ETB-receptor antagonist BQ-788 (10−7M), or by their combination. In contrast, ET-1 caused a concentration-dependent contraction of arterial rings that was inhibited by BQ-123 (10−6M) and a relaxation that was abolished by BQ-788 (10−7M) or by endothelial cell removal. Therefore, the endothelium-dependent contraction to hypoxia is not mediated by ET. Hypoxia caused only relaxation in endothelium-denuded rings. However, when a pulmonary valve leaflet, a rich source of pulmonary endothelial cells, was placed into the lumen of endothelium-denuded rings, hypoxia caused a late-phase contractile response that was similar to that observed in arterial rings with native endothelium. This hypoxic contraction persisted in the presence of indomethacin (10−5M) and N-nitro-l-arginine methyl ester (3 × 10−5M) to block cyclooxygenase and nitric oxide synthase, respectively. These results suggest that hypoxic contraction of pulmonary arteries is mediated by a diffusible, contractile factor released from hypoxic endothelial cells. This contractile mediator is distinct from ET.
Intracellular uptake of recombinant superoxide dismutase after intratracheal administration
Authors: Das Santanu , Horowitz Stuart , Robbins Carolyn G. , El-Sabban Marwan Eid , Sahgal Namita , Davis Jonathan M.
Published: December 2017

We have previously demonstrated that recombinant human copper-zinc superoxide dismutase (rhCu,ZnSOD) is rapidly incorporated into cells of airways, respiratory bronchioles, and alveoli after intratracheal administration. The present study examines whether this cellular uptake is specific for rhCu,ZnSOD or whether other proteins are similarly incorporated into lung cells. Twenty-two newborn piglets (2–3 days old, 1.2–2.0 kg) were intubated and mechanically ventilated. Eight piglets received fluorescently labeled recombinant human manganese superoxide dismutase (rhMnSOD), six received fluorescently labeled albumin, two received free (unbound) fluorescent label intratracheally, and two piglets served as untreated controls. To determine whether endogenous surfactant was important in the process of intracellular uptake, four additional piglets were made surfactant deficient by repeated bronchoalveolar lavage and then given rhCu,ZnSOD intratracheally. All animals were killed after 30–60 min. Lung sections were examined blindly by laser confocal microscopy. Similar to our previous observations with rhCu,ZnSOD, intracellular uptake of rhMnSOD and albumin was noted throughout the lung. The free label did not localize intracellularly. The uptake of proteins did not appear to be affected by surfactant deficiency. rhMnSOD administration was associated with a greater than twofold increase in lung MnSOD activity. Data suggest that the cellular uptake of antioxidants and other proteins in the lung may reflect a nonspecific host defense system for clearing proteins from the lumen of airways and alveoli.
Paraquat-induced phosphatidylserine oxidation and apoptosis are independent of activation of PLA2
Authors: Fabisiak James P. , Kagan Valerian E. , Tyurina Yulia Y. , Tyurin Vladimir A. , Lazo John S.
Published: December 2017

Paraquat is a pneumotoxin that causes lung injury by enhancing oxidative stress; however, the cellular responses to these redox events are undefined. We previously showed that paraquat produced selective peroxidation of phosphatidylserine that preceded apoptosis in 32D cells. We now report that the phospholipase A2(PLA2) inhibitor quinacrine can attenuate phosphatidylserine oxidation and also block paraquat-induced apoptosis. Therefore, we investigated the potential for PLA2to mediate apoptosis after paraquat. We found that, in contrast to quinacrine, the PLA2inhibitors manoalide, aristolochic acid, and arachidonyl trifluoromethylketone failed to prevent paraquat-induced apoptosis. Moreover, no evidence of PLA2activation was observed within 7 h after paraquat exposure. Finally, quinacrine failed to inhibit basal and 4-bromo-A-23187-induced release of [3H]arachidonic acid at concentrations that protected paraquat-induced apoptosis. We conclude that paraquat-induced phosphatidylserine oxidation and apoptosis occurred in the absence of PLA2activation and that quinacrine protected phosphatidylserine and cell viability after paraquat in a PLA2-independent manner.
Inhibition of voltage-gated K+current in rat intrapulmonary arterial myocytes by endothelin-1
Authors: Shimoda Larissa A. , Sylvester J. T. , Sham James S. K.
Published: December 2017

Although endothelin (ET)-1 is an important regulator of pulmonary vascular tone, little is known about the mechanisms by which ET-1 causes contraction in this tissue. Using the whole cell patch-clamp technique in rat intrapulmonary arterial smooth muscle cells, we found that ET-1 and the voltage-dependent K+(KV)-channel antagonist 4-aminopyridine, but not the Ca2+-activated K+-channel antagonist charybdotoxin (ChTX), caused membrane depolarization. In the presence of 100 nM ChTX, ET-1 (10−10to 10−7M) caused a concentration-dependent inhibition of K+current (56.2 ± 3.8% at 10−7M) and increased the rate of current inactivation. These effects of ET-1 on K+current were markedly reduced by inhibitors of protein kinase C (staurosporine and GF 109203X) and phospholipase C (U-73122) or under Ca2+-free conditions and were mimicked by activators of protein kinase C (phorbol 12-myristate 13-actetate and 1,2-dioctanoyl- sn-glycerol). These data suggest that ET-1 modulated pulmonary vascular reactivity by depolarizing pulmonary arterial smooth muscle, due in part to the inhibition of KVcurrent that occurred via activation of the phospholipase C-protein kinase C signal transduction pathway.
Leukotriene B4mediates histamine induction of NF-κB and IL-8 in human bronchial epithelial cells
Authors: Aoki Yosuke , Qiu Daoming , Zhao Guo Hua , Kao Peter N.
Published: December 2017

In 16HBE transformed human bronchial epithelial cells, histamine stimulated interleukin (IL)-8 mRNA and protein secretion, and this histamine stimulation was inhibited by the H1-receptor antagonist diphenhydramine (DPH), by the inhibitor of 5-lipoxygenase-activating protein (FLAP) MK-886, by the 5-lipoxygenase inhibitor Zileuton, and by dexamethasone. Histamine stimulated bronchial epithelial cell production of leukotriene B4(LTB4), and this production was inhibited by FLAP inhibitors MK-886 and L-655,238 and Zileuton. Histamine stimulated IL-8 luciferase reporter gene activity that was inhibited with DPH, dexamethasone, MK-886 and L-655,238, and Zileuton. The inhibition of IL-8 transcription and protein secretion by FLAP inhibitors and Zileuton was reversed with exogenous LTB4. There was increased IL-8 nuclear factor-κB (NF-κB) DNA-binding activity after histamine stimulation, and this was inhibited by DPH and MK-886. Cytoplasmic phospholipase A2mRNA levels were also potently induced by histamine. Thus histamine stimulation of bronchial epithelial cells involves binding at H1receptors, production of LTB4, activation of NF-κB and increased expression of IL-8.
Modulation of aquaporin 4 and the amiloride-inhibitable sodium channel in perinatal rat lung epithelial cells
Authors: Ruddy M. K. , Drazen J. M. , Pitkanen O. M. , Rafii B. , O’Brodovich H. M. , Harris H. W.
Published: December 2017

During the perinatal period, a dramatic reversal of lung transepithelial ion and water transport occurs that involves the amiloride-inhibitable Na+channel (ENaC). Aquaporin (AQP) water channel proteins facilitate cell membrane water transport. We now report that AQP-4, localized to basolateral membranes of airway epithelial cells, increases its mRNA expression in developing lung eightfold during the 2 days before birth to reach a peak on the first postnatal day in the lungs but not in brains or kidneys of neonatal rats. AQP-4 and the α-, β-, and γ-subunits of ENaC are both expressed by cultured rat fetal distal lung epithelial (FDLE) cells. AQP-4 and ENaC expression increase in FDLE cells cultured on uncoated permeant filters compared with matched control cells cultured on filters containing extracellular matrix derived from fetal lung epithelial cells. Similarly, AQP-4 expression increases in FDLE cells exposed to 21% O2compared with cells exposed to 3% O2. These data demonstrate that AQP-4 expression is highest on the first day after birth in neonatal rat lungs. Exposure to ambient 21% O2may contribute to increases in AQP-4 and ENaC expression to facilitate water transport across neonatal airway epithelia in the immediate postnatal period.
Pseudomonaspyocyanine alters calcium signaling in human airway epithelial cells
Authors: Denning Gerene M. , Railsback Michelle A. , Rasmussen George T. , Cox Charles D. , Britigan Bradley E.
Published: December 2017

Pseudomonas aeruginosa, an opportunistic human pathogen, causes both acute and chronic lung disease. P. aeruginosa exerts many of its pathophysiological effects by secreting virulence factors, including pyocyanine, a redox-active compound that increases intracellular oxidant stress. Because oxidant stress has been shown to affect cytosolic Ca2+concentration ([Ca2+]c) in other cell types, we studied the effect of pyocyanine on [Ca2+]cin human airway epithelial cells (A549 and HBE). At lower concentrations, pyocyanine inhibits inositol 1,4,5-trisphosphate formation and [Ca2+]cincreases in response to G protein-coupled receptor agonists. Conversely, at higher concentrations, pyocyanine itself increases [Ca2+]c. The pyocyanine-dependent [Ca2+]cincrease appears to be oxidant dependent and to result from increased inositol trisphosphate and release of Ca2+from intracellular stores. Ca2+plays a central role in epithelial cell function, including regulation of ion transport, mucus secretion, and ciliary beat frequency. By disrupting Ca2+homeostasis, pyocyanine could interfere with these critical functions and contribute to the pathophysiological effects observed in Pseudomonas-associated lung disease.
PKC isoforms and other signaling proteins involved in surfactant secretion in developing rat type II cells
Authors: Gobran Laurice I. , Xu Zhi-Xin , Rooney Seamus A.
Published: December 2017

We previously reported that there is a developmental increase in surfactant secretion in response to P2Y2 purinoceptor agonists. UTP does not stimulate secretion in type II cells from 1- or 2-day-old rats; there is a small response to UTP in cells from 4-day-old animals, and the response increases with increasing age thereafter. Second messenger formation in response to P2Y2 agonists has a similar developmental pattern. We have investigated whether the failure to respond to P2Y2 agonists is due to a deficiency in the P2Y2 receptor or in downstream signaling factors. We compared type II cells from adult and 1- to 2-day-old rats with respect to expression of the P2Y2 receptor gene and the levels of phospholipase C-β (PLC-β) and protein kinase C (PKC) isomers and of the α-subunit of the GTP-binding protein Gq. We measured gene expression by reverse transcriptase-polymerase chain reaction and protein levels by immunoblotting. We identified PKC-α, -βI, -βII, -δ, -η, -ζ, -θ, and -μ, PLC-β3, and Gqα in adult and newborn type II cells. PKC-ε, -γ, and -λ and PLC-β1, -β2, and -β4 were not present in adult or newborn type II cells. Expression of the P2Y2 receptor gene was essentially the same in newborn and adult cells. However, the levels of PKC-α, -βI, -βII, and -ζ in newborn type II cells were only 43–57% those of adult cells. The level of PKC-θ also tended to be lower in the newborn cells. There was little difference between newborn and adult type II cells in the levels of PKC-δ, -η, and -μ, PLC-β3, and Gqα. These data suggest that the lack of response of early newborn type II cells to P2Y2 agonists is not due to a lack of expression of the receptor gene but possibly to insufficient amounts of one or more of the α, βI, βII, or ζ PKC isoforms.
Regulation of myosin heavy chain gene expression after short-term diaphragm inactivation
Authors: Yang Liying , Bourdon Johanne , Gottfried Stewart B. , Zin Walter A. , Petrof Basil J.
Published: December 2017

Although prolonged diaphragm denervation (DNV) produces myofiber atrophy and a loss of type I myosin heavy chain (MHC) expression, short-term DNV leads to significant diaphragm hypertrophy. The purpose of this study was to explore the regulation of MHC isoform expression and muscle remodeling during DNV hypertrophy of the diaphragm. Both unilateral and bilateral DNV led to similar changes, with a significant increase in total RNA content and muscle mass but no change in dry-to-wet weight ratio. Sarcomere number was also increased in diaphragm myofibers after DNV (∼20%), suggesting an adaptive response to muscle stretch. There was hypertrophy of type I myofibers and increased coexpression of type I and type II MHCs within single myofibers by immunocytochemistry as well as increased type I MHC (25–46%) and decreased type IIb MHC (14–39%) by SDS-PAGE. Contractility parameters were also consistent with a type II-to-type I MHC phenotype transformation. Importantly, DNV-induced modulation of MHC isoform mRNA transcript levels did not correspond to changes in their cognate proteins, suggesting a major degree of posttranscriptional control. We conclude that DNV hypertrophy of the diaphragm is associated with reciprocal changes in type I and type II MHC isoforms that are directly opposed to the type I-to-type II MHC phenotype transformation reported in the diaphragm DNV atrophy model. Furthermore, in contradistinction to most hypertrophy models, control of MHC gene expression and myofibrillar remodeling after short-term DNV appears to entail major involvement of posttranscriptional regulatory mechanisms.
Interaction of ratPneumocystis cariniiand rat alveolar epithelial cells in vitro
Authors: Beck James M. , Preston Angela M. , Wagner John G. , Wilcoxen Steven E. , Hossler Paul , Meshnick Steven R. , Paine Robert
Published: December 2017

During Pneumocystis carinii pneumonia, P. carinii trophic forms adhere tightly to type I alveolar epithelial cells (AECs). However, the manner in which the interaction between P. cariniiorganisms and AECs results in clinical pneumonia has not been explored. To investigate this interaction in vitro, we established a culture system using rat P. carinii and primary cultures of rat AECs. We hypothesized that binding of P. carinii to AECs would alter the metabolic, structural, and barrier functions of confluent AECs. Using fluorescently labeled P. carinii, we demonstrated that P. carinii bound to AECs in a dose-dependent manner. During P. carinii-AEC interaction, both the AECs and the P. carinii organisms remained metabolically active. Immunofluorescent staining demonstrated that AEC expression of the junctional proteins E-cadherin and occludin and the structural protein cytokeratin 8 were unaffected by P. carinii binding. To evaluate the effect of P. carinii on AEC barrier function, transepithelial resistance across AEC monolayers was measured during interaction with organisms. Culture with P. carinii did not result in loss of AEC barrier function but in fact increased AEC transepithelial resistance in a dose- and time-dependent manner. We conclude that the direct interaction of P. carinii with AECs does not disrupt AEC metabolic, structural, or barrier function. Therefore, we speculate that additional inflammatory cells and/or their signals are required to induce the epithelial derangements characteristic of P. carinii pneumonia.
Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane
Authors: Zen K. , Notarfrancesco K. , Oorschot V. , Slot J. W. , Fisher A. B. , Shuman H.
Published: December 2017

Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic βHC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.
Mechanism for secretagogue-induced surfactant protein A binding to lung epithelial cells
Authors: Chen Qiping , Fisher Aron B. , Strayer David S. , Bates Sandra R.
Published: December 2017

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of125I-A2R and125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of ∼32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was ∼210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.
Potassium channels modulate hypoxic pulmonary vasoconstriction
Authors: Barman Scott A.
Published: December 2017

The role of Ca2+-activated K+-channel, ATP-sensitive K+-channel, and delayed rectifier K+-channel modulation in the canine pulmonary vascular response to hypoxia was determined in the isolated blood-perfused dog lung. Pulmonary vascular resistances and compliances were measured with vascular occlusion techniques. Under normoxia, the Ca2+-activated K+-channel blocker tetraethylammonium (1 mM), the ATP-sensitive K+-channel inhibitor glibenclamide (10−5 M), and the delayed rectifier K+-channel blocker 4-aminopyridine (10−4 M) elicited a small but significant increase in pulmonary arterial pressure. Hypoxia significantly increased pulmonary arterial and venous resistances and pulmonary capillary pressure and decreased total vascular compliance by decreasing both microvascular and large-vessel compliances. Tetraethylammonium, glibenclamide, and 4-aminopyridine potentiated the response to hypoxia on the arterial segments but not on the venous segments and also further decreased pulmonary vascular compliance. In contrast, the ATP-sensitive K+-channel opener cromakalim and the L-type voltage-dependent Ca2+-channel blocker verapamil (10−5 M) inhibited the vasoconstrictor effect of hypoxia on both the arterial and venous vessels. These results indicate that closure of the Ca2+-activated K+ channels, ATP-sensitive K+ channels, and delayed rectifier K+ channels potentiate the canine pulmonary arterial response under hypoxic conditions and that L-type voltage-dependent Ca2+ channels modulate hypoxic vasoconstriction. Therefore, the possibility exists that K+-channel inhibition is a key event that links hypoxia to pulmonary vasoconstriction by eliciting membrane depolarization and subsequent Ca2+-channel activation, leading to Ca2+ influx.
Differential regulation of glutathione by oxidants and dexamethasone in alveolar epithelial cells
Authors: Rahman Irfan , Bel Agnes , Mulier Brigitte , Donaldson Kenneth , MacNee William
Published: December 2017

We studied the regulation of GSH and the enzymes involved in GSH regulation, γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyl transpeptidase (γ-GT), in response to the oxidants menadione, xanthine/xanthine oxidase, hyperoxia, and cigarette smoke condensate in human alveolar epithelial cells (A549). Menadione (100 μM), xanthine/xanthine oxidase (50 μM/10 mU), and cigarette smoke condensate (10%) exposure produced increased GSH levels (240 ± 6, 202 ± 12, and 191 ± 2 nmol/mg protein, respectively; P < 0.001) compared with the control level (132 ± 8 nmol/mg protein), which were associated with a significant increase in γ-GCS activity (0.18 ± 0.006, 0.16 ± 0.01, and 0.17 ± 0.008 U/mg protein, respectively; P < 0.01) compared with the control level (0.08 ± 0.001 U/mg protein) at 24 h. Exposure to hyperoxia (95% O2) resulted in a time-dependent increase in GSH levels. γ-GCS activity increased significantly at 4 h ( P < 0.001), returning to control values after 12 h of exposure. Dexamethasone (3 μM) exposure produced a significant time-dependent decrease in the levels of GSH and γ-GCS activity at 24–96 h. The activity of γ-GT did not change after oxidant treatment; however, it was decreased significantly by dexamethasone at 24–96 h. Thus oxidants and dexamethasone modulate GSH levels and activities of γ-GT and γ-GCS by different mechanisms. We suggest that the increase in γ-GCS activity but not in γ-GT activity may be required for the increase in intracellular GSH under oxidative stress in alveolar epithelial cells.
Human lung fibroblasts release chemokinetic activity for monocytes constitutively
Authors: Koyama Sekiya , Sato Etsuro , Masubuchi Tsuyoshi , Takamizawa Akemi , Nomura Hiroshi , Kubo Keishi , Nagai Sonoko , Izumi Takateru
Published: December 2017

We determined whether human lung fibroblasts (HLFs) might release mediators that are responsible for monocyte chemokinetic activity (MCA) constitutively. HLF supernatant fluids showed MCA in a time-dependent manner ( P < 0.001). Checkerboard analysis of 24- and 72-h supernatant fluids showed that the activity was chemokinetic. Partial characterization of 24- and 72-h supernatant fluids revealed that the mediators released after 24 h were predominantly composed of lipid-soluble activity, and MCA was blocked by lipoxygenase inhibitors. The mediators released after 72 h were predominantly trypsin sensitive and blocked by cycloheximide. Molecular-sieve column chromatography identified four peaks of MCA. A polyclonal antibody to monocyte chemoattractant protein-1 (MCP-1) inhibited MCA by 20% after 24 h and by 40% after 72 h. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-β (TGF-β) antibodies attenuated MCA released after 72 h by 30 and 10%, respectively. These antibodies inhibited corresponding molecular-weight peaks separated by molecular-sieve column. The concentrations of MCP-1, GM-CSF, and TGF-β were 4,698 ± 242, 26.8 ± 3.8, and 550 ± 15 pg/ml, respectively. A leukotriene B4(LTB4)-receptor antagonist attenuated the total MCA and the lowest molecular weight peak of MCA. The concentrations of LTB4were 153.4 ± 12.4 (24 h) and 212 ± 16.6 (72 h) pg/ml. These findings suggest that HLFs may modulate the recruitment of monocytes into the lung by releasing MCP-1, GM-CSF, TGF-β, and LTB4constitutively.
Neuropeptides mediate the ozone-induced increase in the permeability of the tracheal mucosa in guinea pigs
Authors: Nishiyama Harumi , Ikeda Hirotada , Kaneko Takeshi , Fu Li , Kudo Makoto , Ito Takaaki , Okubo Takao
Published: December 2017

We examined the effects of acute exposure to ozone on the permeability of the tracheal mucosa and the contribution of neural pathways to the effects of ozone using horseradish peroxidase (HRP; mol wt 40,000) as a marker of lumen-to-blood transfer of a macromolecule in guinea pigs in vivo. Each guinea pig was anesthetized and exposed for 30 min to either ozone [0.5 or 3 parts/million (ppm)] or air. Immediately after exposure, a tracheal segment was isolated between two polyethylene cannulas in vivo and filled with HRP solution (50 mg/ml). Blood samples were drawn before and 10, 20, 30, and 40 min after the intratracheal instillation of HRP. The plasma levels of HRP in guinea pigs exposed for 30 min to 3 ppm of ozone, but not to 0.5 ppm of ozone, were significantly greater than those in guinea pigs exposed to air. Although the increased plasma HRP levels after exposure to 3 ppm of ozone were unaffected by propranolol or atropine, they were completely inhibited by pretreatment with capsaicin (50 mg/kg sc, injected in two doses). These results suggest that endogenous neuropeptides mediate the ozone-induced increase in the permeability of the tracheal mucosa in guinea pigs in vivo, but neither an adrenergic nor a cholinergic pathway appears to be involved.
Tumor necrosis factor induces neuroendocrine differentiation in small cell lung cancer cell lines
Authors: Haley Kathleen J. , Patidar Kirit , Zhang Fan , Emanuel Rodica L. , Sunday Mary E.
Published: December 2017

We studied tumor necrosis factor (TNF)-α as a candidate cytokine to promote neuroendocrine cell differentiation in a nitrosamine-hyperoxia hamster lung injury model. Differential screening identified expression of the genes modulated by TNF-α preceding neuroendocrine cell differentiation. Undifferentiated small cell lung carcinoma (SCLC) cell lines NCI-H82 and NCI-H526 were treated with TNF-α for up to 2 wk. Both cell lines demonstrated rapid induction of gastrin-releasing peptide (GRP) mRNA; H82 cells also expressed aromatic-l-amino acid decarboxylase mRNA within 5 min after TNF-α was added. Nuclear translocation of nuclear factor-κB immunostaining occurred with TNF-α treatment, suggesting nuclear factor-κB involvement in the induction of GRP and/or aromatic-l-amino acid decarboxylase gene expression. We also demonstrated dense core neurosecretory granules and immunostaining for proGRP and neural cell adhesion molecule in H82 cells after 7–14 days of TNF-α treatment. We conclude that TNF-α can induce phenotypic features of neuroendocrine cell differentiation in SCLC cell lines. Similar effects of TNF-α in vivo may contribute to the neuroendocrine cell differentiation/hyperplasia associated with many chronic inflammatory pulmonary diseases.
Enhancement of fibronectin expression in rat lung by ozone and an inflammatory stimulus
Authors: Gupta S. K. , Reinhart P. G. , Bhalla D. K.
Published: December 2017

This study investigated the relationship of fibronectin expression and induction of pulmonary inflammation by ozone (O3). Rats were exposed to 0.8 parts/million O3to induce lung inflammation. A second inflammatory stimulus, rabbit serum, was applied intratracheally to augment O3-induced inflammation. Bronchoalveolar lavage fluid (BALF) and lung tissues were analyzed for fibronectin protein and mRNA expression. Blood plasma was analyzed to investigate the potential of a minimally invasive procedure in predicting lung inflammation and fibronectin levels. Significant increases in the levels of fibronectin protein in the BALF and lung tissue after O3exposure were further enhanced by pretreatment with normal serum. An increase in fibronectin mRNA following O3exposure was also enhanced by serum pretreatment, which by itself had no effect on lung fibronectin mRNA expression. Plasma fibronectin levels were comparable in air-PBS and O3-PBS groups but increased in the O3-serum group. The results suggest leakage of fibronectin from blood plasma into the lung following intratracheal application of rabbit serum and upregulation of local synthesis following O3exposure.
Delayed rectifier K+ current of dog bronchial myocytes: effect of pollen sensitization and PKC activation
Authors: Waldron Gareth J. , Sigurdsson Stefan B. , Aiello Ernesto A. , Halayko Andrew J. , Stephens Newman L. , Cole William C.
Published: December 2017

The properties of delayed rectifier K+ current [ I K(dr)] of canine airway smooth muscle cells isolated from small bronchi and its modulation by protein kinase C (PKC) were studied by whole cell patch clamp. I K(dr) activated positive to −40 mV, with half-maximal activation at −16 ± 1.2 mV ( n = 15) and average current density of 31 ± 2.6 pA/pF ( n = 15) at +30 mV. The capacitive surface area, current density, and voltage dependence of activation of I K(dr) of myocytes of ragweed pollen-sensitized dogs were not different from age-matched control dogs. However, the sensitization reduced the availability of I K(dr) between −40 and −20 mV due to a hyperpolarizing shift in the voltage dependence of steady-state inactivation (−29.9 ± 1.2 in sensitized versus −26.0 ± 0.7 mV in control dogs, n = 9 and 11, respectively; P < 0.05). PKC activation with diacylglycerol analog or phorbol ester depressed I K(dr) amplitude, whereas an inactive diacylglycerol analog had no effect. The hyperpolarizing shift in voltage dependence of inactivation and/or modulation of I K(dr) by PKC may be two mechanisms that contribute to the enhanced reactivity of bronchial tissues from ragweed pollen-sensitized dogs.
Clara cell secretory protein deficiency increases oxidant stress response in conducting airways
Authors: Mango Gregory W. , Johnston Carl J. , Reynolds Susan D. , Finkelstein Jacob N. , Plopper Charles G. , Stripp Barry R.
Published: December 2017

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP −/−) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP −/− mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP −/− mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.
Ca2+ and K+ currents regulate accommodation and firing frequency in guinea pig bronchial ganglion neurons
Authors: Myers Allen C.
Published: December 2017

Intracellular microelectrode recordings were obtained from neurons located in adult guinea pig bronchial parasympathetic ganglia in situ to determine the calcium and potassium currents regulating repetitive action potential activity and firing rates by these neurons. Neurons in these ganglia respond to prolonged suprathreshold depolarizing current steps with either a burst of action potentials at the onset of the stimulus (accommodating or phasic neurons) or repetitive action potentials throughout the stimulus (nonaccommodating or tonic neurons). Instantaneous and adapted firing rates during prolonged threshold and suprathreshold stimuli were lower in tonic than in phasic neurons, indicating a longer interspike interval between repetitive action potentials in tonic neurons. In tonic neurons, blockade of A-type current with 4-aminopyridine increased accommodation; 4-aminopyridine or apamin decreased the interspike interval in tonic neurons. Calcium-free buffer, cadmium ions, or ω-conotoxin GVIA also increased accommodation in tonic neurons but did not affect the interspike interval; nifedipine or verapamil did not affect the tonic firing pattern. Accommodation in phasic neurons could be decreased by a conditioning hyperpolarization step of the resting potential, which could be subsequently blocked by 4-aminopyridine or calcium-free buffer. Accommodation in phasic neurons could also be decreased by apamin or barium ions: the repetitive action potentials observed during these treatments could be reversed by cadmium ions or calcium-free buffer. These results indicate that tonic and phasic neurons in guinea pig bronchial parasympathetic ganglia have similar types of calcium currents, but potassium channels may ultimately regulate the accommodation pattern, the firing rate, and, consequently, the output from these neurons.
Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin
Authors: Folkesson Hans G. , Nitenberg Gerard , Oliver Bonnie L. , Jayr Christian , Albertine Kurt H. , Matthay Michael A.
Published: December 2017

Alveolar epithelial fluid transport was studied 10 days after subacute lung injury had been induced with intratracheal bleomycin (0.75 U). An isosmolar Ringer lactate solution with 5% bovine serum albumin and125I-labeled albumin as the alveolar protein tracer was instilled into the right lung; the rats were then studied for either 1 or 4 h. Alveolar fluid clearance was increased in bleomycin-injured rats by 110% over 1 h and by 75% over 4 h compared with control rats ( P < 0.05). The increase in alveolar fluid clearance was partially inhibited by amiloride (10−3 M). Alveolar fluid clearance decreased toward normal levels in rats that were studied 60 days after bleomycin instillation. Remarkably, the measured increase in net alveolar fluid clearance occurred in the presence of a significant increase in alveolar epithelial permeability to protein. Moreover, the increase in alveolar epithelial fluid clearance occurred even though the mRNA for the α-subunit of the epithelial sodium channel was decreased in alveolar epithelial type II cells isolated from these rats. In addition,22Na uptake by isolated alveolar epithelial type II cells from rats treated with bleomycin demonstrated a 52% decrease in uptake compared with type II cells from control rats. Morphological results demonstrated a significant hyperplasia of alveolar type II epithelial cells 10 days after bleomycin injury. Thus, these results provide evidence that proliferation of alveolar epithelial type II cells after acute lung injury may upregulate the transport capacity of the alveolar epithelium, even though the expression of epithelial sodium channels is reduced and the uptake of22Na per cell is also reduced. These results may have clinical relevance for the resolution of alveolar edema in the subacute phase of lung injury.