bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged isoproterenol infusion impairs the ability of β2-agonists to increase alveolar liquid clearance
Authors: Morgan Eric E. , Hodnichak Cheryl M. , Stader Sonya M. , Maender Kay C. , Boja John W. , Folkesson Hans G. , Maron Michael B.
Published: March 2015

We determined if prolonged isoproterenol (Iso) infusion in rats impaired the ability of the β2-adrenergic agonist terbutaline to increase alveolar liquid clearance (ALC). We infused rats with Iso (at rates of 4, 40, or 400 μg · kg−1· h−1) or vehicle (0.001 N HCl) for 48 h using subcutaneously implanted miniosmotic pumps. After this time, the rats were anesthetized, and ALC was determined (by mass-balance after instillation of Ringer lactate containing albumin into the lungs) under baseline conditions and after terbutaline administration. Baseline and terbutaline-stimulated ALC in vehicle-infused rats averaged, respectively, 19.6 ± 1.2% (SE) and 44.7 ± 1.5%/h. The ability of terbutaline to increase ALC was eliminated at 400 μg · kg−1· h−1Iso, inhibited by 26% at 40 μg · kg−1· h−1Iso, and was not affected by 4 μg · kg−1· h−1Iso. β-adrenergic receptor (βAR) density of freshly isolated alveolar epithelial type II (ATII) cells from Iso-infused rats was reduced by the 40 and 400 μg · kg−1· h−1infusion rates. These data demonstrate that prolonged exposure to β-agonists can impair the ability of β2-agonists to stimulate ALC and produce ATII cell βAR downregulation.
Mechanical ventilation-induced pneumoprotein CC-16 vascular transfer in rats: effect of KGF pretreatment
Authors: Lesur Olivier , Hermans Cédric , Chalifour Jean-François , Picotte Johanne , Lévy Bruno , Bernard Alfred , Lane Denis
Published: March 2015

After air-blood barrier injury, “pneumoproteins” specific to lung epithelial distal airspaces reaching the bloodstream are putative markers of lung hyperpermeability. The contribution of mechanical ventilation (MV) to this leakage is unknown. To explore this issue, 16-kDa Clara cell protein (CC-16) concentration was quantified in bronchoalveolar lavages (BALFs) and/or sera of rats first exposed either to ambient air or to 48 h of hyperoxia-induced acute lung injury and then ventilated for 2 h according to one of the following strategies: 1) spontaneous ventilation (SV), 2) very-low-volume high PEEP (VLVHP, where PEEP is positive end-expiratory pressure), 3) low-volume zero PEEP, 4) moderate-volume low PEEP, and 5) high-volume zero PEEP (HVZP). Results show that total proteins in BALFs increased with time and MV, with little impact from hyperoxia preexposure. CC-16 content decreased in BALFs but increased in the bloodstream during MV, suggesting intravascular leakage. Lung overdistension may result either from high-volume (HVZP) or high-PEEP (VLVHP) MV, and it was the most potent inducer of CC-16 leakage ( P < 0.05 vs. SV). In the VLVHP group, pretreatment with keratinocyte growth factor was efficient in reducing blood CC-16 transfer.
Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension
Authors: Mata-Greenwood Eugenia , Meyrick Barbara , Soifer Scott J. , Fineman Jeffrey R. , Black Stephen M.
Published: March 2015

Utilizing in utero aortopulmonary vascular graft placement, we developed a lamb model of congenital heart disease and increased pulmonary blood flow. We showed previously that these lambs have increased pulmonary vessel number at 4 wk of age. To determine whether this was associated with alterations in VEGF signaling, we investigated vascular changes in expression of VEGF and its receptors, Flt-1 and KDR/Flk-1, in the lungs of shunted and age-matched control lambs during the first 8 wk of life. Western blot analysis demonstrated that VEGF, Flt-1, and KDR/Flk-1 expression was higher in shunted lambs. VEGF and Flt-1 expression was increased at 4 and 8 wk of age ( P <0.05). However, KDR/Flk-1 expression was higher in shunted lambs only at 1 and 4 wk of age ( P <0.05). Immunohistochemical analysis demonstrated that, in control and shunted lambs, VEGF localized to the smooth muscle layer of vessels and airways and to the pulmonary epithelium while increased VEGF expression was localized to the smooth muscle layer of thickened media in remodeled vessels in shunted lambs. VEGF receptors were localized exclusively in the endothelium of pulmonary vessels. Flt-1 was increased in the endothelium of small pulmonary arteries in shunted animals at 4 and 8 wk of age, whereas KDR/Flk-1 was increased in small pulmonary arteries at 1 and 4 wk of age. Our data suggest that increased pulmonary blood flow upregulates expression of VEGF and its receptors, and this may be important in development of the vascular remodeling in shunted lambs.
Changes in biophysical and biochemical properties of single bronchial smooth muscle cells from asthmatic subjects
Authors: Ma Xuefei , Cheng Zhaoqin , Kong Hong , Wang Ying , Unruh Helmut , Stephens Newman L. , Laviolette Michel
Published: March 2015

Whether contractility of bronchial smooth muscle cells (BSMC) from asthmatic subjects is significantly altered has never been validated. We tested the hypothesis that such BSMC show increased contractility. Cells were isolated from endobronchial biopsies. BSMC shortening was measured under an inverted microscope. Statistically significant increases in maximum shortening capacity (Δ Lmax) and velocity ( Vo) were found in asthmatic BSMC compared with normal cells. Mean Δ Lmaxin asthmatic BSMC was 39.05 ± 1.99% (SE) of resting cell length compared with 28.6 ± 1.1% in normal cells; mean Vowas 7.2 ± 0.8% of resting cell length/s in asthmatic cells and 5.23 ± 0.46% in normal cells. To investigate the mechanism of the increased contractility, we measured mRNA abundance of smooth muscle types of myosin light chain kinase (smMLCK) and myosin heavy chain. RT-PCR data revealed that smMLCK mRNA was higher in asthmatic BSMC (0.106 ± 0.021 arbitrary densitometric units, n = 7) than in control cells (0.04 ± 0.008, n = 11; P < 0.05). Messages for myosin heavy chain isoforms showed no difference. Increased kinase message content is an index of the mechanism for the increased velocity and capacity of shortening we report.
Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts
Authors: Sanchez-Esteban Juan , Wang Yulian , Cicchiello Lawrence A. , Rubin Lewis P.
Published: March 2015

Development of the pulmonary air sacs is crucial for extrauterine survival. Late fetal lung development is characterized by a thinning of the mesenchyme, which brings pneumocytes and endothelial cells into apposition. We hypothesized that mechanical stretch, simulating fetal breathing movements, plays an important role in this remodeling process. Using a Flexercell Strain Unit, we analyzed the effects of intermittent stretch on cell proliferation and apoptosis activation in fibroblasts isolated from fetal rat lungs during late development. On day 19, intermittent stretch increased cells in G0/G1by 22% ( P = 0.001) and decreased in S phase by 50% ( P = 0.003) compared with unstretched controls. Cell proliferation analyzed by 5-bromo-2′-deoxyuridine incorporation showed a similar magnitude of cell cycle arrest ( P = 0.04). At this same gestational age, stretch induced apoptosis by two- to threefold over controls, assayed by DNA flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick-end labeling, and caspase-3 activation. These results indicate that mechanical stretch of fibroblasts isolated during the canalicular stage inhibits cell cycle progression and activates apoptosis. These findings are cotemporal with the mesenchymal thinning that normally occurs in situ.
What leads to different mediators of alkalosis-induced vasodilation in isolated and in situ pulmonary vessels?
Authors: Gordon John B. , VanderHeyden Michele A. , Halla Ted R. , Cortez Edmundo P. , Hernandez Guillermo , Haworth Steven T. , Dawson Christopher A. , Madden Jane A.
Published: March 2015

We previously found that nitric oxide synthase (NOS) inhibition fully blocked alkalosis-induced relaxation of piglet pulmonary artery and vein rings. In contrast, NOS inhibition alone had no effect on alkalosis-induced pulmonary vasodilation in isolated piglet lungs. This study sought to identify factors contributing to the discordance between isolated and in situ pulmonary vessels. The roles of pressor stimulus (hypoxia vs. the thromboxane mimetic U-46619), perfusate composition (blood vs. physiological salt solution), and flow were assessed. Effects of NOS inhibition on alkalosis-induced dilation were also directly compared in 150–350-μm-diameter cannulated arteries and 150–900-μm-diameter, angiographically visualized, in situ arteries. Finally, effects of NOS inhibition on alkalosis-induced vasodilation were measured in intact piglets. NOS inhibition with N ω-nitro-l-arginine fully abolished alkalosis-induced vasodilation in all cannulated arteries but failed to alter alkalosis-induced vasodilation in intact lungs. The results indicate that investigation of other factors, such as perivascular tissue (e.g., adventitia and parenchyma) and remote signaling pathways, will need to be carried out to reconcile this discordance between isolated and in situ arteries.
Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development
Authors: Luyet Cédric , Burri Peter H. , Schittny Johannes C.
Published: March 2015

Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1–0.01 μg/g, days 1–4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19–21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa ( day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.
Zinc chelators inhibit eotaxin, RANTES, and MCP-1 production in stimulated human airway epithelium and fibroblasts
Authors: Richter Martin , Cantin André M. , Beaulieu Claudia , Cloutier Alexandre , Larivée Pierre
Published: March 2015

Asthma is characterized by an increased production of eosinophil-active C-C chemokines by the airway epithelium. Recent studies have identified the presence of important quantities of labile zinc in the conducting airways. We hypothesized that modulation of this labile zinc could influence the production of proinflammatory chemokines in respiratory epithelial cells. The zinc chelator N,N,N′ ,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the heavy metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS) were used to reduce the labile zinc content of A549, BEAS-2B, and HFL-1 cells. Northern blot analysis and RNase protection assay were used to study the effects of the zinc chelators on mRNA expression. DMPS and TPEN specifically inhibited the production of eotaxin, regulated on activation, normal T-cell expressed, and presumably secreted, and monocyte chemotactic protein-1 in TNF-α-stimulated respiratory epithelial cells and fibroblasts through labile zinc chelation. The inhibitory effects of DMPS and TPEN were associated with the decreased binding of the zinc-finger transcription factor GATA-1, whereas no change in NF-κB activation was observed. Together these results demonstrate that modulation of the labile pool of zinc can regulate gene expression and protein synthesis of C-C chemokines in lung epithelial cells and fibroblasts.
Injury, inflammation, and remodeling in fetal sheep lung after intra-amniotic endotoxin
Authors: Kramer Boris W. , Kramer Susanne , Ikegami Machiko , Jobe Alan H.
Published: March 2015

Chorioamnionitis is frequent in preterm labor and increases the risk of bronchopulmonary dysplasia. We hypothesized that intra-amniotic endotoxin injures the lung in utero, causing a sequence of inflammation and tissue injury similar to that which occurs in the injured adult lung. Preterm lamb lungs at 125 days gestational age were evaluated for indicators of inflammation, injury, and repair 5 h, 24 h, 72 h, and 7 days after 4 mg of intra-amniotic endotoxin injection. At 5 h, the epithelial cells in large airways expressed heat shock protein 70, and alveolar interleukin-8 was increased. Surfactant protein B (SP-B) decreased in alveolar type II cells at 5 h, and SP-B in lung tissue and alveolar lavage fluid increased by 72 h. By 24 h, neutrophils were recruited into the large airways, and cell death was the highest. Alveolar type II cells decreased by 25% at 24 h, and proliferation was highest at 72 h, consistent with tissue remodeling. Intra-amniotic endotoxin caused surfactant secretion, inflammation, cell death, and remodeling as indications of lung injury. The recovery phase was accompanied by maturational changes in the fetal lung.
Regulation of mucin secretion from human bronchial epithelial cells grown in murine hosted xenografts
Authors: Conway Jason D. , Bartolotta Tracy , Abdullah Lubna H. , Davis C. William
Published: March 2015

Studies of regulated mucin secretion from goblet cells in primary cultures of human bronchial epithelial (HBE) cells have suffered, generally, from poor signal-to-noise ratios, with reported secretory responses of <100% (less than onefold) relative to baseline. Using, instead, HBE cells grown as xenografts in the backs of nude mice, we found that UTP (100 μM) stimulated strong mucin secretory responses from isolated, luminally perfused preparations. The peak response (10 min) for 11 control experiments (37 xenografts) was 3.3 ± 0.05-fold relative to baseline, and the time-integrated response (60 min) was 23.4 ± 0.5-fold. Because responses to ATP and UTP were approximately equal, an apical membrane P2Y2-receptor (R) is suggested. Additionally, ADP activated mucin release from HBE xenografts, whereas UDP and 2-methlythio-ADP did not, a pattern of response inconsistent with known purinoceptors. Hence, either a novel receptor to ADP is suggested or there is significant conversion of ADP to ATP by ecto-adenylate kinase activity. Adenosine and a nitric oxide donor were without effect. Consistent with P2Y2-R coupling to phospholipase C, HBE xenografts responded to ionomycin and PMA; however, they were recalcitrant to forskolin and chlorophenylthio-cAMP, and to 8-bromo-cGMP. Hence, human airway goblet cells, like those of other species, appear to be regulated primarily via phospholipase C pathways, activated particularly by apical membrane P2Y2-R agonists.
Iron regulates xanthine oxidase activity in the lung
Authors: Ghio Andrew J. , Kennedy Thomas P. , Stonehuerner Jacqueline , Carter Jacqueline D. , Skinner Kelly A. , Parks Dale A. , Hoidal John R.
Published: March 2015

The iron chelator deferoxamine has been reported to inhibit both xanthine oxidase (XO) and xanthine dehydrogenase activity, but the relationship of this effect to the availability of iron in the cellular and tissue environment remains unexplored. XO and total xanthine oxidoreductase activity in cultured V79 cells was increased with exposure to ferric ammonium sulfate and inhibited by deferoxamine. Lung XO and total xanthine oxidoreductase activities were reduced in rats fed an iron-depleted diet and increased in rats supplemented with iron, without change in the ratio of XO to total oxidoreductase. Intratracheal injection of an iron salt or silica-iron, but not aluminum salts or silica-zinc, significantly increased rat lung XO and total xanthine oxidoreductase activities, immunoreactive xanthine oxidoreductase, and the concentration of urate in bronchoalveolar fluid. These results suggest the possibility that the production of uric acid, a major chelator of iron in extracellular fluid, is directly influenced by iron-mediated regulation of the expression and/or activity of its enzymatic source, xanthine oxidase.
Nerve growth factor and nerve growth factor receptors in respiratory syncytial virus-infected lungs
Authors: Hu Chengping , Wedde-Beer Katrin , Auais Alexander , Rodriguez Maria M. , Piedimonte Giovanni
Published: March 2015

Nerve growth factor (NGF) controls sensorineural development and responsiveness and modulates immunoinflammatory reactions. Respiratory syncytial virus (RSV) potentiates the proinflammatory effects of sensory nerves in rat airways by upregulating the substance P receptor, neurokinin 1 (NK1). We investigated whether the expression of NGF and its trkA and p75 receptors in the lungs is age dependent, whether it is upregulated during RSV infection, and whether it affects neurogenic inflammation. Pathogen-free rats were killed at 2 (weanling) to 12 (adult) wk of age; in addition, subgroups of rats were inoculated with RSV or virus-free medium. In pathogen-free rats, expression of NGF and its receptors in the lungs declined with age, but RSV doubled expression of NGF, trkA, and p75 in weanling and adult rats. Exogenous NGF upregulated NK1receptor expression in the lungs. Anti-NGF antibody inhibited NK1receptor upregulation and neurogenic inflammation in RSV-infected lungs. These data indicate that expression of NGF and its receptors in the lungs declines physiologically with age but is upregulated by RSV and is a major determinant of neurogenic inflammation.
Stem cell antigen-1 expression in the pulmonary vascular endothelium
Authors: Kotton Darrell N. , Summer Ross S. , Sun Xi , Ma Bei Yang , Fine Alan
Published: March 2015

Although the function of the cell surface protein stem cell antigen-1 (Sca-1) has not been identified, expression of this molecule is a characteristic of bone marrow-derived hematopoietic stem cell populations. Expression of Sca-1, however, is not restricted to hematopoietic tissue. By RT-PCR and Western analysis, we found that Sca-1 is expressed in the adult mouse lung. Sca-1 immunohistochemistry revealed a linear staining pattern on the endothelial surface of large and small pulmonary arteries and veins and alveolar capillaries. Expression of Sca-1 in the pulmonary endothelium was confirmed by dual fluorescent microscopy on lung sections and by fluorescence-activated cell sorting analysis of digested lung tissue; each of these methods showed colocalization with the endothelial marker platelet/endothelial cell adhesion molecule-1. In the kidney, Sca-1 expression was also noted in large vessels, but, in contrast to the lung, was not observed in capillaries. Overall, our data indicate that Sca-1 expression helps define the surface phenotype of endothelial cells throughout the pulmonary vasculature.
Ca2+-dependent stimulation of alveolar fluid clearance in near-term fetal guinea pigs
Authors: Norlin Andreas , Folkesson Hans G.
Published: March 2015

We investigated the importance of changes in intracellular Ca2+concentration ([Ca2+]i) for amiloride-sensitive alveolar fluid clearance (AFC) in late-gestational guinea pigs. Fetal guinea pigs of 61, 68, and 69 days (term) gestation were investigated under normal conditions and after oxytocin-induced preterm labor. AFC or alveolar fluid secretion was measured using an impermeable tracer technique. At 61 days gestation there was net secretion of fluid into the lungs, and at birth the lungs cleared 49 ± 7% of the instilled fluid volume over 1 h. Induction of preterm labor with oxytocin induced AFC at 61 days gestation. When present, AFC was inhibited or reversed to net fluid secretion by amiloride (10−3M). Inhibition of membrane Ca2+channels by verapamil (10−4M) or depletion of intracellular Ca2+by thapsigargin (10−5M) reduced AFC when net AFC was evident. Amiloride lacked an inhibitory effect on AFC when instilled with verapamil or thapsigargin. The results indicate that AFC via amiloride-sensitive pathways develops during late gestation, and that inducing preterm labor precociously may activate such pathways. Our results suggest that Ca2+may act as a second messenger in mediating catecholamine-stimulated AFC.
Contractile activity and smooth muscle α-actin organization in thrombin-induced human lung myofibroblasts
Authors: Bogatkevich Galina S. , Tourkina Elena , Abrams Charles S. , Harley Russell A. , Silver Richard M. , Ludwicka-Bradley Anna
Published: March 2015

Activated fibroblasts, or myofibroblasts, are crucial players in tissue remodeling, wound healing, and various fibrotic disorders, including interstitial lung fibrosis associated with scleroderma. Here we characterize the signaling pathways in normal lung fibroblasts exposed to thrombin as they acquire two of the main features of myofibroblasts: smooth muscle (SM) α-actin organization and collagen gel contraction. Our results show that the small G protein Rho is involved in lung myofibroblast differentiation. Thrombin induces Rho-35S-labeled guanosine 5′- O-(3-thiotriphosphate) binding in a dose-dependent manner. It potently stimulates Rho activity in vivo and initiates protein kinase C (PKC)-ϵ-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM α-actin organization, collagen gel contraction, and PKC-ϵ-SM α-actin and PKC-ϵ-RhoA coimmunoprecipitation. However, it has no effect on PKC-ϵ activation or translocation of PKC-ϵ to the membrane. Overexpression of constitutively active PKC-ϵ and constitutively active RhoA induces collagen gel contraction or SM α-actin organization, whereas, individually, they do not perform these functions. We therefore conclude that the contractile activity of myofibroblasts induced by thrombin is mediated via PKC-ϵ- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-ϵ-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-ϵ-SM α-actin coimmunoprecipitation, which leads to the PKC-ϵ-RhoA-SM α-actin ternary complex formation.
PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction
Authors: Qiao Jing , Huang Fei , Lum Hazel
Published: March 2015

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 μM forskolin plus 1 μM IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of32P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of ∼23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.
ICAM-1 facilitates alveolar macrophage phagocytic activity through effects on migration over the AEC surface
Authors: Paine Robert , Morris Susan B. , Jin Hong , Baleeiro Carlos E. O. , Wilcoxen Steven E.
Published: March 2015

We postulate that intercellular adhesion molecule-1 (ICAM-1) on type I alveolar epithelial cells (AEC) facilitates phagocytic activity of alveolar macrophages (AM) in the alveolus. When wild-type and ICAM-1-deficient mice were inoculated intratracheally with FITC-labeled microspheres, AM phagocytosis of beads (after 1 and 4 h) was significantly reduced in ICAM-1−/− mice compared with controls. To focus on ICAM-1-mediated interactions specifically involving AM and AEC, rat AM were placed in culture with rat AEC treated with neutralizing anti-ICAM-1 F(ab′)2fragments. Blocking ICAM-1 significantly decreased the AM phagocytosis of beads. Planar chemotaxis of AM over the surface of AEC was also significantly impaired by neutralization of AEC ICAM-1. ICAM-1 in rat AEC is associated with the actin cytoskeleton. Planar chemotaxis of AM was also significantly reduced by pretreatment of the AEC monolayer with cytochalasin B to disrupt the actin cytoskeleton. These studies indicate that ICAM-1 on the AEC surface promotes mobility of AM in the alveolus and is critically important for the efficient phagocytosis of particulates by AM.
Prostacyclin analogs inhibit fibroblast migration
Authors: Kohyama Tadashi , Liu Xiangde , Kim Hui Jung , Kobayashi Tetsu , Ertl Ronald F. , Wen Fu-Qiang , Takizawa Hajime , Rennard Stephen I.
Published: March 2015

The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PGI2) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI2on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PGI2analog carbaprostacyclin (10−6M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 μg/ml) 58.0 ± 13.2% ( P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 ± 4.6% ( P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI2analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both 10−6M), significantly inhibited fibroblast migration to fibronectin. In summary, PGI2appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.
Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema
Authors: Palestini Paola , Calvi Chiara , Conforti Elena , Botto Laura , Fenoglio Carla , Miserocchi Giuseppe
Published: March 2015

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml · kg−1· min−1saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (∼4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.
Heterogeneity of bronchial endothelial cell permeability
Authors: Moldobaeva Aigul , Wagner Elizabeth M.
Published: March 2015

In vivo models of airway inflammation suggest that most protein transudation occurs from bronchial microcirculation. However, due to technical limitations in the isolation and culture of bronchial endothelial cells, most studies of lung vascular permeability have focused on pulmonary endothelium. Thus conditions for culture of sheep bronchial artery endothelial cells (BAEC) and bronchial microvascular endothelial cells (BMVEC) were established. The bronchial artery and the mainstem bronchi, stripped of epithelium, were dissected, and endothelial cells were isolated by enzymatic treatment. BAEC and BMVEC demonstrated positive staining for factor VIII-related antigen, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein, and PECAM-1. Radioligand binding studies confirmed equivalent numbers of bradykinin B2 receptors on BAEC and BMVEC. Permeability of BAEC and BMVEC was determined after treatment with bradykinin and thrombin by comparing the translocation of FITC-dextran (mol wt 9,500) across confluent monolayers ( n = 10–12). Bradykinin caused a maximal increase in permeability in BAEC (165% increase) and BMVEC (144% increase) by 15 min compared with vehicle controls. Thrombin treatment altered BMVEC permeability only, reaching a maximal response at 60 min (109% increase). These results demonstrate bronchial endothelial cell heterogeneity and establish methods to determine intracellular mechanisms contributing to airway disease in relevant cell systems.
Nitric oxide modulates branching morphogenesis in fetal rat lung explants
Authors: Young Stephen L. , Evans Katherine , Eu Jerry P.
Published: March 2015

Cells of the developing lung express the constitutive nitric oxide synthases (NOSs) I and III. The developmental importance of these enzymes is largely unknown, although a role for nitric oxide (NO) in the regulation of pulmonary blood flow at birth is established. Known effects of NO on transcription factors, apoptosis, and cellular proliferation, plus the time and spatial limits of pulmonary NOS expression, suggest that NO might influence lung development. We tested the potential of NO to modulate lung branching morphogenesis by exposing lung explants from gestational day 13 rat fetuses to varying doses of several NO donors (NONO-ate). We counted the number of airway branches that were added between the first and 72nd h of culture. NO released only from a NONO-ate with a long half-life {( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl)amino]-diazen-1-ium-1,2-diolate-NO}, increased branching in ambient O2by twofold. The NO effect was not mimicked with a cyclic guanine monophosphate analog; nonspecific NOS inhibitors in millimolar concentrations inhibited branching. We conclude that endogenous and exogenous NO can modulate branching morphogenesis in the rat lung.
Physiological properties and functions of Ca2+sparks in rat intrapulmonary arterial smooth muscle cells
Authors: Remillard Carmelle V. , Zhang Wei-Min , Shimoda Larissa A. , Sham James S. K.
Published: March 2015

Ca+spark has been implicated as a pivotal feedback mechanism for regulating membrane potential and vasomotor tone in systemic arterial smooth muscle cells (SASMCs), but little is known about its properties in pulmonary arterial smooth muscle cells (PASMCs). Using confocal microscopy, we identified spontaneous Ca2+ sparks in rat intralobar PASMCs and characterized their spatiotemporal properties and physiological functions. Ca2+ sparks of PASMCs had a lower frequency and smaller amplitude than cardiac sparks. They were abolished by inhibition of ryanodine receptors but not by inhibition of inositol trisphosphate receptors and L-type Ca2+ channels. Enhanced Ca2+ influx by BAY K8644, K+, or high Ca2+ caused a significant increase in spark frequency. Functionally, enhancing Ca2+ sparks with caffeine (0.5 mM) caused membrane depolarization in PASMCs, in contrast to hyperpolarization in SASMCs. Norepinephrine and endothelin-1 both caused global elevations in cytosolic Ca2+ concentration ([Ca2+]), but only endothelin-1 increased spark frequency. These results suggest that Ca2+ sparks of PASMCs are similar to those of SASMCs, originate from ryanodine receptors, and are enhanced by Ca2+ influx. However, they play a different modulatory role on membrane potential and are under agonist-specific regulation independent of global [Ca2+].
Evidence for the role of p38 MAP kinase in hypoxia-induced pulmonary vasoconstriction
Authors: Karamsetty M. R. , Klinger J. R. , Hill N. S.
Published: March 2015

Mitogen-activated protein (MAP) kinases regulate smooth muscle cell contraction. Hypoxia contracts pulmonary arteries by mechanisms that are incompletely understood. We hypothesized that hypoxic contraction of pulmonary arteries involves activation of the MAP kinases. To test this hypothesis, we studied the effects of SB-202190, a p38 MAP kinase inhibitor, PD-98059 and UO-126, two structurally different MEKK inhibitors, and anisomycin, a stimulator of p38 MAP kinase on acute hypoxia-induced contraction in rat conduit pulmonary artery rings precontracted with phenylephrine or KCl. Hypoxia induced a transient contraction, followed by a relaxation, and then a slowly developing sustained contraction. Hypoxia also significantly increased phosphorylation of p38 MAP kinase. SB-202190 did not affect the transient phase but abrogated the sustained phase of hypoxic contraction, whereas anisomycin enhanced both phases of contraction. SB-202190 also attenuated and anisomycin enhanced the phenylephrine-induced contraction. In contrast, PD-98059 and UO-126 had minimal effects on either hypoxic or phenylephrine-induced contraction. None of the treatments modified KCl-induced contraction. We conclude that p38, but not the ERK1/ERK2 MAP kinase pathway, mediates the sustained phase of hypoxic contraction in isolated rat pulmonary arteries.
Potential switch from eupnea to fictive gasping after blockade of glycine transmission and potassium channels
Authors: St.-John Walter M. , Rybak Ilya A. , Paton Julian F. R.
Published: March 2015

This study evaluated possible neuronal mechanisms responsible for the transition from normal breathing (eupnea) to gasping. We hypothesized that a blockade of both inhibitory glycinergic synaptic transmission and potassium channels, combined with an increase in extracellular concentration of potassium, would induce a switch from an eupneic respiratory pattern to gasping. Efferent activities of the phrenic, vagal, and hypoglossal nerves were recorded during eupnea and ischemia-induced gasping in a perfused in situ preparation of the juvenile rat (4–6 wk of age). To block potassium channels, 4-aminopyridine (4-AP, 1–10 μM) was administered. Strychnine (0.2–0.6 μM) was used to block glycinergic neurotransmission. After administrations of 4-AP, excess extracellular potassium (10.25–17.25 mM), and strychnine, the incrementing pattern of eupneic phrenic activity was altered to a decrementing discharge. Hypoglossal and vagal activities became concentrated to the period of the phrenic burst with expiratory activity being reduced or eliminated. These changes in neural activities were similar to those in ischemia-induced gasping. Results are consistent with the concept that the elicitation of gasping represents a switch from a network-based rhythmogenesis for eupnea to a pacemaker-driven mechanism.
Synergistic interactions between airway afferent nerve subtypes mediating reflex bronchospasm in guinea pigs
Authors: Mazzone Stuart B. , Canning Brendan J.
Published: March 2015

The hypothesis that airway afferent nerve subtypes act synergistically to initiate reflex bronchospasm in guinea pigs was addressed. Laryngeal mucosal application of capsaicin or bradykinin or the epithelial lipoxygenase metabolite 15( S)-hydroxyeicosatetraenoic acid evoked slowly developing but pronounced and sustained increases in tracheal cholinergic tone in situ. These reflexes were reversed by atropine and prevented by vagotomy, trimethaphan, or laryngeal denervation. Central nervous system-acting neurokinin receptor antagonists also abolished the reflexes without altering baseline cholinergic tone. Baseline tone was, however, reversed by disrupting pulmonary afferent innervation while preserving the innervation of the trachea and larynx. Surprisingly, selective pulmonary denervation also prevented the laryngeal capsaicin-induced tracheal reflexes, suggesting that laryngeal C-fibers act synergistically with continuously active intrapulmonary mechanoreceptors to initiate reflex bronchospasm. Indeed, reflex bronchospasm evoked by histamine was markedly potentiated by bradykinin, an effect mimicked by intracerebroventricular, but not intravenous, substance P. These data, as well as anatomic evidence for afferent nerve subtype convergence in the commissural nucleus of the solitary tract, suggest that airway nociceptors and mechanoreceptors may act synergistically to regulate airway tone.
Wide-band spectral tuning of heat receptors in the pit organ of the copperhead snake (Crotalinae)
Authors: Moiseenkova Vera , Bell Brent , Motamedi Massoud , Wozniak Edward , Christensen Burgess
Published: March 2015

Receptors located in the facial pit organ of certain species of snake signal the presence of prey. Infrared radiation is an effective stimulus suggesting that these receptors may be low-threshold temperature receptors. We recorded from the nerve innervating the pit organ of snakes belonging to the family of Crotalinae while stimulating the receptive area with well-defined optical stimuli. The objective was to determine the sensitivity of these receptors to a wide range (0.400–10.6 μm) of optical stimuli to determine if a temperature-sensitive or photosensitive protein initiated signal transduction. We found that receptors in the pit organ exhibited a unique broad response to a wide range of electromagnetic radiation ranging from the near UV to the infrared. The spectral tuning of these receptors parallels closely the absorption spectra of water and oxyhemoglobin, the predominant chromophore in tissue. Our results support the hypothesis that these are receptors activated by minute temperature changes induced by direct absorption of optical radiation in the thin pit organ membrane.
Differential regulation of adrenal corticosteroids after restriction-induced drinking in rats
Authors: Wotus Cheryl , Engeland William C.
Published: March 2015

Water-restricted rats exhibit a rapid decrease in plasma corticosterone after drinking. The present study examined the effect of restriction-induced drinking on plasma aldosterone and plasma clearance of corticosterone. Rats were water restricted for 6–7 days and then killed before or 15 min after water administration; plasma and adrenal hormones were assayed. Plasma and adrenal corticosterone decreased after drinking without a change in plasma corticosteroid-binding globulin; plasma ACTH decreased or did not change. In contrast, plasma aldosterone did not change or increased after drinking; plasma renin activity was elevated by water restriction and increased further after drinking. In another experiment, rats were adrenalectomized, and corticosterone and aldosterone were replaced with pellets and osmotic minipumps, respectively. Rats were water restricted and killed. There was a small decrease in plasma corticosterone but no change in aldosterone after drinking in adrenalectomized animals. These data suggest that changes in plasma steroids after restriction-induced drinking result from zone-specific responses of the adrenal to known secretagogues, with minimal contribution from increased plasma clearance.
Chronic hypoxia abolishes posthypoxia frequency decline in the anesthetized rat
Authors: Ilyinsky Oleg , Tolstykh Gleb , Mifflin Steve
Published: March 2015

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 ± 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats ( P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats ( P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.
Role of hypoxemia for the cardiovascular responses to apnea during exercise
Authors: Lindholm Peter , Nordh Jessica , Linnarsson Dag
Published: March 2015

We sought to define the role of hypoxemia in eliciting the cardiovascular responses to apnea during exercise. Eleven men performed repeated apneas during 100-W steady-state exercise, either with normoxic gas (air) or 95% oxygen (oxygen). Beat-by-beat arterial blood pressure, arterial oxygen saturation, and heart rate (HR) were determined, and stroke volume (SV) was estimated from impedance cardiography calibrated with soluble gas rebreathing. There were large interindividual variabilities of HR, mean arterial pressure (MAP), and total peripheral resistance (TPR) at end-apnea (ea). However, for each individual, HRea, MAPea, and TPRea were highly correlated between air and oxygen ( R = 0.94, 0.78, and 0.93). HR decreased and MAP increased faster during apnea with air than with oxygen (ANOVA, P < 0.05), but MAPea was not different between conditions. Cardiac output was reduced by 33% with air and by 11% with oxygen ( P < 0.001 for air vs. oxygen). We conclude that the hypoxemia component cannot account for the wide interindividual differences of HR and TPR responses to apnea. However, hypoxemia augments the HR and TPR responses and may limit the MAP response to apnea by preventing a bradycardia-associated increase of SV.
Development of β-cell mass in fetuses of rats deprived of protein and/or energy in last trimester of pregnancy
Authors: Bertin Eric , Gangnerau Marie-Noëlle , Bellon Georges , Bailbé Danièle , Arbelot De Vacqueur Annick , Portha Bernard
Published: March 2015

Fetal malnutrition is now proposed as a risk factor of later obesity and type II diabetes. We previously analyzed the long-term impact of reduced protein and/or energy intake strictly limited to the last week of pregnancy in Wistar rats. Three protocols of gestational malnutrition were used: 1) low-protein isocaloric diet (5 instead of 15%) with pair feeding to the mothers receiving the control diet, 2) restricted diet (50% of control diet), and 3) low protein-restricted diet (50% of low-protein diet). Only isolated protein restriction induced a long-term β-cell mass decrease. In the present study, we used the same protocols of food restriction to analyze their short-term impact (on day 21.5 of pregnancy) on β-cell mass development. A 50% β-cell mass decrease was present in the three restricted groups, but low-protein diet, either associated or not to energy restriction, increased fetal β-cell insulin content. Among all the parameters analyzed to further explain our results, we found that the fetal plasma level of taurine was lowered by low-protein diet and was the main predictor of the fetal plasma insulin level ( r = 0.63, P < 0.01). In conclusion, rat fetuses exposed to protein and/or energy restriction during the third part of pregnancy have a similar dramatic decrease in β-cell mass, and their ability to recover β-cell mass development retardation depends on the type of malnutrition used. Moreover, our results support the hypothesis that taurine might play an important role in fetal β-cell mass function.
Relationship between renal sympathetic nerve activity and arterial pressure during REM sleep in rats
Authors: Miki Kenju , Kato Makiko , Kajii Suzuko
Published: March 2015

The relationship between renal sympathetic nerve activity (RSNA) and systemic arterial pressure obtained during rapid eye movement (REM) sleep was compared with that obtained in other sleep and awake states. Electrodes for the measurements of RSNA, electrocardiogram, electromyogram, and electroencephalogram and a catheter for the measurement of systemic arterial pressure were implanted while the animals were under aseptic conditions at least 5 days before the experiment. During the transition from non-REM (NREM) to REM sleep, RSNA and heart rate (HR) decreased immediately by 46 ± 2% ( P < 0.05) and 22 ± 3 beats/min ( P < 0.05), respectively, over 3 s after the onset of REM sleep. Meanwhile, systemic arterial pressure increased gradually after the onset of REM sleep, which was apparently independent of the changes in RSNA. During REM sleep, the relationships between RSNA/HR and systemic arterial pressure were dissociated compared with that obtained during the other behavioral states. These data indicate that the interdependency between systemic arterial pressure and RSNA during REM sleep is likely to be modified compared with other behavioral states.