bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor necrosis factor-alpha activates pulmonary artery endothelial protein kinase C
Authors: Ferro T. J. , Parker D. M. , Commins L. M. , Phillips P. G. , Johnson A.
Published: December 2017

We investigated the hypothesis that tumor necrosis factor-alpha (TNF) activates pulmonary endothelial protein kinase C (PKC). Confluent bovine pulmonary artery endothelial monolayers were exposed to recombinant human TNF, and the translocation of PKC, an indicator of enzyme activation, was studied using both slot immunoblotting and immunofluorescence. For slot immunoblot analysis, membrane and cytosol lysate fractions were prepared, and PKC antigen was assessed using MC5 monoclonal anti-PKC antibody. TNF (1,000 U/ml for 15 min) induced translocation of PKC into the membrane. Immunofluorescence analysis with the MC5 antibody was also used. Monolayers treated with culture medium showed diffuse cytoplasmic fluorescence. In contrast, treatment with either TNF (1,000 U/ml for 15 min) or 1,2-dioctanoylglycerol (4 x 10(-5) M for 5 min), a diacylglycerol that activates PKC, resulted in translocation of fluorescence to the cell periphery; fine, punctate PKC-associated fluorescence was localized to the margins of cells. The TNF-induced translocation of PKC was inhibited using either IP-300 polyclonal anti-TNF antibody (indicating that the TNF effect was not due to the vehicle or contaminating endotoxin) or calphostin C (10(-6) M for 15 min), which inhibits PKC activation by interacting with the regulatory diacylglycerol-binding domain. TNF treatment had no effect on either the content of PKC, or of total protein, in the membrane + cytosol, and cycloheximide (40 microM for 5 min) did not alter the translocation of PKC induced by TNF; these results indicate that the effect of TNF on PKC translocation was related to neither de novo membrane synthesis of PKC (as opposed to translocation per se) nor nonspecific augmentation of protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Increased expression of PDGF A- and B-chain genes in rat lungs with hypoxic pulmonary hypertension
Authors: Katayose D. , Ohe M. , Yamauchi K. , Ogata M. , Shirato K. , Fujita H. , Shibahara S. , Takishima T.
Published: December 2017

To study the molecular basis of vascular remodeling in pulmonary hypertension, we developed an experimental system in which male Sprague-Dawley rats were exposed to hypoxia for up to 3 wk. Both the right ventricular systolic pressure and gravimetric index for right ventricular hypertrophy were higher in rats exposed to hypoxia for 3 wk than those of age-matched control rats (P < 0.01), indicating that pulmonary hypertension was established under conditions used. To examine the possible involvement of platelet-derived growth factor (PDGF) in the pulmonary vascular remodeling caused by hypoxia, we cloned rat PDGF A- and B-chain cDNA and prepared specific cRNA probes. Northern blot analysis revealed that PDGF B-chain mRNA levels in the lungs were increased, reached a maximum of day 1, and were sustained at day 3, whereas PDGF A-chain mRNA levels reached a maximum on day 3. Thus the increase in the PDGF B-chain mRNA level precedes that in the PDGF A-chain mRNA level. These results suggest that the PDGF A- and B-chain products may be coordinately and sequentially involved in hypoxic pulmonary vascular remodeling.
Type II pneumocyte hypertrophy without activation of surfactant biosynthesis after partial pneumonectomy
Authors: Uhal B. D. , Etter M. D.
Published: December 2017

Hypertrophic and normotrophic type II pneumocytes were isolated from pneumonectomized adult rats by unit gravity (1 g) sedimentation or by fluorescence-activated cell sorting (FACS). In vivo or in vitro, hypertrophic cells incorporated significantly more 5-bromo-2'-deoxyuridine or tritiated thymidine into acid-insoluble material than did normotrophic cells. By FACS analysis of cell subpopulations isolated by 1 g, > 97% of normotrophic cells had G0-phase DNA content. In contrast, the cell cycle distribution of hypertrophic cells was 75% G1, 5% S, and 20% G2/M phases. Rates of incorporation of tritiated choline into total cellular phosphatidylcholine (PC) were identical in type II cells isolated from normal or pneumonectomized rats. The intracellular contents of disaturated phosphatidylcholine (DSPC) and total PC, as well as the ratio of these two lipids, were the same in hypertrophic and normotrophic cells from pneumonectomized rats and in cells isolated from normal rats. No significant difference was observed in the rate at which hypertrophic or normotrophic cells incorporated choline into DSPC. These results demonstrate that type II pneumocyte hypertrophy after pneumonectomy reflects balanced cell growth secondary to cell cycle progression in vivo. The data also indicate that epithelial cell hypertrophy after pneumonectomy, in contrast to that which develops after more acute lung injury, occurs without activation of surfactant biosynthesis or storage.
cAMP inhibition of interleukin-1-induced interleukin-6 production by human lung fibroblasts
Authors: Zitnik R. J. , Zheng T. , Elias J. A.
Published: December 2017

We characterized the effects of agents that alter intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on the interleukin (IL)-6 production of human lung fibroblasts. Unstimulated fibroblasts did not produce significant amounts of IL-6. Recombinant (r) tumor necrosis factor (TNF) weakly stimulated, recombinant interleukin-1-alpha (rIL-1 alpha) strongly stimulated, and rIL-1 alpha and rTNF in combination synergistically augmented fibroblast IL-6 production. Prostaglandin (PG)E1, forskolin, dibutyryl cAMP (DBcAMP), 3-isobutyl-1-methylxanthine (IBMX), and cholera toxin did not cause a detectable alteration in the IL-6 production of unstimulated fibroblasts. However, these agents inhibited the IL-6 production of rIL-1 and rIL-1 plus rTNF-stimulated cells. These effects were dose dependent with a concentration of 2 x 10(-9) M PGE1, 5 x 10(-6) M forskolin, 5 x 10(-4) M DBcAMP, and 1 x 10(-3) M IBMX decreasing rIL-1 alpha (2.5 ng/ml)-induced IL-6 production by approximately 50%. The inhibitory effects of these agents, correlated with their ability to induce fibroblast cAMP accumulation, could not be explained by alterations in cell number or viability and were appreciable even when cAMP modifiers were added to fibroblast culture, 1 h after rIL-1. They were also at least partly specific for rIL-1, since these agents increased the IL-6 production of rTNF-stimulated cells. These cAMP-induced alterations in IL-6 production were associated with corresponding alterations in IL-6 mRNA accumulation. Nuclear run-on analysis demonstrated that the inhibitory effects of PGE1 were associated with a comparable decrease in IL-6 transcription. Agents that increase the levels of intracellular cAMP inhibit rIL-1-induced IL-6 by human lung fibroblasts.
Asymmetric effects of H2O2 on alveolar epithelial barrier properties
Authors: Kim K. J. , Suh D. J.
Published: December 2017

The effects of H2O2 on active ion transport and resistance to passive solute flow were studied utilizing rat alveolar epithelial cell monolayers cultured on permeable supports. Type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer solution. These monolayers have a high transepithelial resistance (> 2,000 omega.cm2) and actively transport Na+ from apical fluid. H2O2 (0-100 mM) was then delivered to either apical or basolateral fluid. The changes in short-circuit current (Isc) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against H2O2 injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ; an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results indicated that H2O2 decreased Isc and R gradually in a dose-dependent manner. The effective concentration of apical H2O2 at which Isc (or R) was decreased by 50% at 1 h (ED50) was approximately 4 mM. However, basolateral H2O2 exposure led to ED50 for Isc (and R) of approximately 0.04 mM. Inhibition of cellular catalase yielded ED50 for Isc (and R) of approximately 0.4 mM when H2O2 was given apically, while ED50 for basolateral exposure to H2O2 did not change in the presence of ATAZ. The rate of H2O2 consumption in apical and basolateral bathing fluids was the same, while cellular catalase activity rose gradually with time in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes
Authors: Salvaterra C. G. , Goldman W. F.
Published: December 2017

The effects of hypoxia on the cytosolic Ca2+ concentration, [Ca2+]i, were characterized in cultured pulmonary arterial smooth muscle (PASM) cells. Reducing O2 tension (PO2) from 150 to < 25 Torr induced a reversible 100-200% increase in [Ca2+]i that was characterized by two components: an early rise in [Ca2+]i that was dependent on the rate, as well as the magnitude, of decline in PO2 and a later, steady-state increase that was independent of the rate at which PO2 changed. Caffeine lowered [Ca2+]i during normoxia and blocked the early component of the response to hypoxia, whereas the steady-state hypoxic response was only partially inhibited. Like hypoxia, thapsigargin (TG) elevated [Ca2+]i, and there was no additional hypoxia-induced elevation in [Ca2+]i at any time after exposure to TG. At steady state, the hypoxic responses were completely reversed by removal of extracellular Ca2+, whereas, on average, verapamil and nifedipine attenuated the hypoxia-induced increases in [Ca2+]i by only 44 and 35%, respectively. These results suggest that hypoxia-induced elevation of [Ca2+]i in PASM cells consists of an early release of Ca2+ from the sarcoplasmic reticulum and a later influx of extracellular Ca2+, in part, through nifedipine- and verapamil-insensitive Ca2+ channels. The results are consistent with the idea that hypoxia and thapsigargin may share common mechanisms for tonically increasing [Ca2+]i.
Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage
Authors: Wright J. R. , Youmans D. C.
Published: December 2017

Pulmonary surfactant modulates several functions of alveolar macrophages including phagocytosis, killing, and chemotaxis. We hypothesized that the reported stimulatory effect of surfactant on macrophage migration was mediated by one of the surfactant proteins, SP-A. We found that macrophage migration was stimulated by SP-A in a concentration-dependent manner. A concentration of 105 micrograms SP-A/ml enhanced migration approximately 10-fold. Heat treatment or reduction and alkylation of SP-A reduced its stimulatory effect. A checker-board analysis showed that SP-A stimulated migration primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). The interaction of SP-A with macrophages may be mediated at least partly by the collagen-like domain of SP-A. We speculate that SP-A may play a multifunctional role in regulating pulmonary immune response by stimulating multiple macrophage functions.
Characterization of L-arginine transport by pulmonary artery endothelial cells
Authors: Greene B. , Pacitti A. J. , Souba W. W.
Published: December 2017

The transport of L-arginine by porcine pulmonary artery endothelial cells (PAECs) was characterized. Uptake of 50 microM L-arginine was time dependent and linear in the presence and absence of sodium, with approximately 70% of uptake occurring via a carrier-mediated Na(+)-independent process. Kinetic studies of saturable Na(+)-independent transport revealed two transport components: a high-affinity transporter [Michaelis constant (Km) = 304 +/- 23 microM, maximal transport velocity (Vmax) = 679 +/- 34 pmol.mg protein-1.30 s-1], and a low-affinity carrier (Km = 3.9 +/- 1.0 mM, Vmax = 2.8 +/- 0.7 nmol/mg protein-1.30 s-1). Saturable Na(+)-independent uptake of 50 microM L-arginine transport showed no significant variation in uptake between pH 6.0 and 8.0 and was blocked by the system y+ substrates L-arginine, L-homoarginine, L-lysine, and L-ornithine. Na(+)-dependent L-arginine transport occurred via a single high-affinity system (Km = 62 +/- 3 microM, Vmax = 211 +/- 24 pmol.mg protein-1.30 s-1) which was significantly inhibited by L-arginine, L-lysine, L-ornithine, L-leucine, L-alanine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. Na(+)-dependent arginine uptake was pH and hormone insensitive, and lithium did not substitute effectively for sodium. These data are consistent with mediation of high-affinity arginine transport by PAECs via Na(+)-independent system y+ and Na(+)-dependent system BO,+.
Secretory product expression during Clara cell differentiation in the rabbit and rat
Authors: Cardoso W. V. , Stewart L. G. , Pinkerton K. E. , Ji C. , Hook G. E. , Singh G. , Katyal S. L. , Thurlbeck W. M. , Plopper C. G.
Published: December 2017

One function of the nonciliated (Clara) cells of bronchiolar epithelium is to synthesize, store, and release small-molecular-mass (6–12 kDa) secretory proteins or Clara cell secretory protein (CCSP). This study compares the emergence of this secretory function during Clara cell differentiation in rabbits and rats. Lungs of fetal and postnatal animals were evaluated by ultrastructural morphometry and immunohistochemistry. Secretory granules were rarely seen in perinatal animals and increased to adult levels of abundance earlier in rats (1 wk postnatal) than in rabbits (3–4 wk). In contrast, rough endoplasmic reticulum was abundant in perinatal animals and decreased with age. Antibodies raised against CCSP revealed little CCSP in fetal animals; however, after birth CCSP increased to adult levels earlier in rats (1 wk postnatal) than in rabbits (3 wk). We conclude that the maturation of Clara cell secretory function 1) occurs postnatally, 2) involves a decrease in biosynthetic organelles, 3) shows close association between CCSP expression and secretory granule abundance, and 4) varies by species in timing and cellular abundance of biosynthetic machinery.
Effects of substrate and inhibition of oxidative metabolism on contraction and myosin phosphorylation in ASM
Authors: Hai C. M. , Watson C. , Wallach S. J. , Reyes V. , Kim E. , Xu J.
Published: December 2017

Steady-state active stress in smooth muscle is maintained by cross bridges which undergo continuous cycling and myosin phosphorylation, and the two processes both consume ATP. In this study, we investigated whether energy utilization by cross-bridge cycling and myosin phosphorylation is compartmentalized and examined their relative affinities for ATP in airway smooth muscle. We measured active stress, myosin phosphorylation, O2 consumption, and tissue ATP content in bovine tracheal smooth muscle activated by K+ depolarization when glucose was replaced by pyruvate and when oxidative metabolism was inhibited by hypoxia or uncoupled by 2,4-dinitrophenol. The results indicate that ATP produced from both glycolysis and oxidative metabolism is available to both cross-bridge cycling and myosin phosphorylation. However, steady-state myosin phosphorylation was insensitive to the inhibition of oxidative metabolism by hypoxia and mitochondrial uncoupling when steady-state isometric stress and tissue ATP content were significantly reduced. These results suggest that, relative to actomyosin adenosine 5'-triphosphatase, myosin light chain kinase has a higher affinity for ATP in intact airway smooth muscle. However, peak myosin phosphorylation associated with the initial rapid stress development was sensitive to inhibition of oxidative metabolism, probably reflecting a lower content of intracellular calcium store as a result of metabolic inhibition.
Electrophysiological properties of guinea pig tracheal epithelium determined by cable analysis
Authors: Croxton T. L.
Published: December 2017

Electrophysiological characteristics of guinea pig tracheae were measured in vitro using an adaptation of cable analysis. This method allowed the repeated measurement of luminal diameter and epithelial electrical potential, resistance, and short-circuit current (Isc) during treatments known to affect smooth muscle contraction and epithelial ion transport. Stable values taken 3 h after mounting were as follows: diameter, 2.27 +/- 0.10 mm; potential, -28.3 +/- 2.3 mV; resistance, 327 +/- 30 omega.cm2; and Isc, 91.2 +/- 6.8 microA/cm2. These electrophysiological results are comparable to reported values for other species. However, the resistance and potential obtained in this study were larger than those previously reported for the guinea pig. Tracheal diameter was decreased 15% by methacholine and was increased 43% by subsequent isoproterenol treatment. Isoproterenol caused a small but significant increase in Isc when this quantity was normalized to tracheal length rather than to the apparent surface area. In contrast, apical amiloride decreased Isc by 51% and did not change diameter. These data validate this implementation of cable analysis, demonstrate that sodium absorption is the predominant mechanism of active ion transport by guinea pig tracheal epithelium, and indicate that this tissue has little capacity for stimulated chloride secretion.
Maturational changes induced by 1 alpha,25-dihydroxyvitamin D3 in type II cells from fetal rat lung explants
Authors: Marin L. , Dufour M. E. , Nguyen T. M. , Tordet C. , Garabedian M.
Published: December 2017

Specific binding sites for 1 alpha,25 dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] localized to type II pneumocytes have been evidenced in fetal rat lung at the end of gestation, suggesting a role for vitamin D3 in the control of lung maturation. In this study, we describe the morphological changes that occur in lung explants from 18-day-old rat fetuses grown for 1 and 2 days in control conditions and in the presence of 1 alpha,25(OH)2D3 (10(-9) M) or dexamethasone (10(-7) M). Point counting and planimetric measurements on light and electron micrographs show that 1 alpha,25-(OH)2D3 1) dramatically decreases the mean glycogen content of type II cell profiles between days 1 and 2 of the culture, suggesting an acceleration of the glycogenolytic processes normally occurring at that stage and 2) does not change the intracellular osmiophilic lamellar body (OLB) content of cell profiles, but increases the amount of intraluminal surfactant by 126% when expressed as surfactant clusters surface area/section surface area and by 129% when expressed on a per cell basis, suggesting a stimulation of surfactant synthesis and secretion. By contrast, dexamethasone increases the mean intracellular OLB content of type II cell profiles by 306% and decreases the relative surface area of secreted material by 53 and 73%. In conclusion, 1 alpha,25(OH)2D3 accelerates the physiological maturation of fetal rat type II pneumocytes and could represent a key factor for the onset of normal lung function at birth.
Fluoroaluminate- and GTP gamma S-induced stress, shortening, and myosin phosphorylation in airway smooth muscle
Authors: Hai C. M. , Ma C. B.
Published: December 2017

GTP-binding proteins in bovine tracheal smooth muscle were activated by fluoroaluminate and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and the sensitivities of fluoroaluminate- and GTP gamma S-induced active stress and myosin phosphorylation to muscle shortening were compared. Relative to the value of myosin phosphorylation at L0, unloaded shortening induced a 63% decrease in fluoroaluminate-activated steady-state myosin phosphorylation, but had no significant effect on GTP gamma S-activated myosin phosphorylation. These results were consistent with the hypothesis that shortening-sensitive and shortening-insensitive signal-transduction pathways coexist in airway smooth muscle. However, unlike myosin phosphorylation, active stress induced by fluoroaluminate was actually less sensitive to shortening. The amount of shortening necessary to reduce active stress to half of that at Lo was 65% in fluoroaluminate-activated tissues, but was only 34% in GTP gamma S-activated tissues. The observation of different sensitivities of fluoroaluminate-activated myosin phosphorylation and active stress suggests that GTP-binding proteins modulate the dependence of active stress on muscle length in smooth muscle.
Pulmonary vein contracts in response to hypoxia
Authors: Zhao Y. , Packer C. S. , Rhoades R. A.
Published: December 2017

Hypoxic pulmonary vasoconstriction (HPV) is an important regulatory mechanism in matching regional blood flow and ventilation. The HPV response has been well documented on the arterial side, but the response of pulmonary veins to hypoxia has received little attention. The purpose of the present study was to determine whether isolated rat pulmonary veins contract in response to decreased PO2 and, if so, to compare the venous response with that of the pulmonary artery. Rat pulmonary venous and arterial rings were attached to force transducers and precontracted with either a submaximal dose of KCl or norepinephrine under normoxic conditions and then made hypoxic. The pulmonary venous hypoxic response consisted of a single sustained contraction, whereas the arterial response to hypoxia was biphasic, consisting of an initial rapid contraction and then a slowly developed but sustained contraction. The venous hypoxic contraction was significantly greater in magnitude than either phase 1 or phase 2 of the arterial response. Endothelium denudation did not affect the venous hypoxic response. However, the venous hypoxic response was dependent on the level of precontractile tone and also appeared to be dependent on the specific contractile agonist. Unlike the isolated arterial phase 1 hypoxic response (but similar to the arterial phase 2 response) the pulmonary venous hypoxic contraction was inhibited in Ca(2+)-free media or by Ca2+ channel blockers. In summary, pulmonary venous smooth muscle contracts to a relatively greater degree in response to severe hypoxia than does pulmonary arterial smooth muscle. The venous hypoxic response is endothelium independent, as is phase 2 of the arterial response.(ABSTRACT TRUNCATED AT 250 WORDS)
Nitric oxide synthesis by rat pleural mesothelial cells: induction by cytokines and lipopolysaccharide
Authors: Owens M. W. , Grisham M. B.
Published: December 2017

The close proximity of pleural mesothelial cells (PMC) and mononuclear cells during pleural inflammation suggests that leukocyte-derived products (e.g., cytokines) may play an important role in modulating PMC function. The purpose of this study was to determine whether certain cytokines and bacterial products induce PMC to produce nitric oxide (NO). Confluent monolayers of rat PMC were exposed to tumor necrosis factor (TNF), interleukin-1 beta (IL-1), gamma-interferon (IFN), or lipopolysaccharide (LPS) individually and in various double and triple combinations for 6–72 h. Concentrations of nitrite and nitrate were quantified and used as indirect indices of NO production. Nitrite/nitrate accumulation was maximal at 72 h, with most of the increase occurring from 48 to 72 h. Maximal nitrite/nitrate production was observed with triple combinations with the combination of LPS, IL-1, and TNF giving the highest concentration (137.4 +/- 2.8 microM). Nitrite/nitrate production was significantly inhibited by NG-nitro-L-arginine methyl ester, suggesting that nitrite and nitrate were derived from the L-arginine-dependent formation of NO. These data indicate that PMC can be induced to produce large amounts of NO in response to specific combinations of proinflammatory cytokines and LPS.
Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells
Authors: Babij P. , Zhao J. , White S. , Woodcock-Mitchell J. , Mitchell J. , Absher M. , Baldor L. , Periasamy M. , Low R. B.
Published: December 2017

RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.
Apoprotein-based synthetic surfactants inhibit plasmic cleavage of fibrinogen in vitro
Authors: Gunther A. , Bleyl H. , Seeger W.
Published: December 2017

Fibrinogen (Fbg) leakage and intra-alveolar fibrin accumulation are commonly noticed in adult respiratory distress syndrome and interstitial lung diseases. Activation of the extrinsic coagulation pathway and elevation of antiplasmin- and plasminogen-activator inhibitor levels are assumed to favor alveolar clot formation and to inhibit fibrinolysis under these conditions. We investigated the influence of synthetic surfactants on the plasmic cleavage of fibrinogen in vitro. Fibrinogenolysis was quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with densitometric evaluation and fragment E enzyme-linked immunosorbent assay. A synthetic phospholipid mixture (PLM) (dipalmitoyl-DL-alpha-phosphatidylcholine:L-alpha-phosphatidyl-DL-gly cer ol: palmitic acid 68.5:22.5:9) caused a dose-dependent inhibition of fibrinogenolysis in a concentration range between 0.1 and 2 mg/ml. This inhibitory capacity was markedly amplified upon reconstitution of PLM with natural and recombinant surfactant protein (SP)-C as well as natural SP-B. Natural SP-A and recombinant SP-A were far less effective in this respect. In the absence of phospholipids, the hydrophobic apoproteins revealed only moderate plasmin inhibitory capacity (recombinant SP-C > natural SP-C and SP-B). Natural calf lung surfactant extract displayed comparable inhibitory capacity on plasmic Fbg cleavage as PLM. We conclude that hydrophobic surfactant material may suppress plasmin activity and thus may contribute to the finding of delayed alveolar fibrin clearance in inflammatory lung diseases with Fbg leakage.
Neurotransmitter-induced exocytosis in goblet and acinar cells of rat nasal mucosa studied by video microscopy
Authors: Kamijo A. , Terakawa S. , Hisamatsu K.
Published: December 2017

To investigate the mechanism and neural control of the nasal secretion, we observed the isolated rat nasal mucosa by video-enhanced differential interference contrast microscopy. This technique allowed us to visualize abrupt changes of the individual granules leading to degranulation in the acinar cells and in epithelial goblet cells during secretory stimulation. This image provided evidence that exocytosis is the major mode for regulated secretion in the nasal acinar cells and goblet cells. Acetylcholine (ACh, 0.1–100 microM), substance P (SP, 0.1–10 microM), and vasoactive intestinal peptide (VIP, 0.1–1 microM) induced exocytotic responses and shrinkage of the acinus in a concentration-dependent manner. The effects of ACh (10 microM) on the acinus were clearly inhibited by atropine (5 microM), but the effects of SP (1 microM) and VIP (1 microM) were not. The acinar shrinkage always started before exocytosis, suggesting that the fluid secretion precedes the mucus release. In goblet cells, SP (1 microM) and ACh (10 microM) increased the frequency of exocytotic responses significantly, suggesting that these substances truly play the role of a neurotransmitter for nasal secretion. Histamine (HIST) induced no visible response. The effect of HIST on secretory cells may be neuronally mediated in vivo.
Neuromodulation mediated by neurokinin-1 subtype receptors in adult rabbit airways
Authors: John C. , Brunner S. , Tanaka D. T.
Published: December 2017

The neuromodulatory actions of Septide, a selective neurokinin-1 (NK-1) agonist, and substance P (SP) were investigated in isolated rabbit tracheal smooth muscle (TSM) segments. The tissues were placed in organ baths containing modified Krebs-Ringer solution and contracted with either the agonists or electrical field stimulation (ES) with frequencies ranging from 1 to 50 Hz. The neutral endopeptidase (NEP) inhibitor, phosphoramidon (10(-6) M), had no significant effect on Septide-mediated contractions. Septide-mediated contractions were augmented in the presence of neostigmine, eliminated in the presence of atropine, and diminished in the presence of tetrodotoxin. Both SP and Septide increased ES-induced contractions in a dose-dependent manner. On the other hand, the presence of both Septide and SP did not further augment this neuromodulatory action. After NK-1 desensitization, Septide-mediated contractions were also virtually eliminated; however, the peptide's neuromodulatory action was unaffected. In contrast, the presence of GR-82334, a selective NK-1 antagonist, eliminated Septide's neuromodulatory activity. These findings provide evidence that 1) NK-1 receptors facilitate both the direct release of ACh as well as augment the release of ACh by ES and 2) the cholinergic pathways involved with these two processes may represent different mechanisms.
Endotoxin enhances hypoxic constriction of rat aorta and pulmonary artery through induction of EDRF/NO synthase
Authors: Zelenkov P. , McLoughlin T. , Johns R. A.
Published: December 2017

The vascular response to hypoxia in endotoxin (lipopolysaccharide; LPS)-exposed rat pulmonary artery (PA) and thoracic aorta (AO) was investigated and the mechanism of the observed hypoxic responses defined. In isometric tension studies, LPS-treated AO and PA rings, with and without endothelium, demonstrated decreased (P < 0.05) contractile response to phenylephrine (PE EC50), and the dose response was shifted to the right (P < 0.01) compared with non-LPS treated rings. Both vessel types responded to hypoxia with a markedly increased (P < 0.01) and sustained (P < 0.01) constriction when preexposed to LPS. Control non-LPS rings with endothelium intact had a transient vasoconstriction in early hypoxia, which was abolished with removal of the endothelium. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, increased the PE EC50 tension in LPS-treated rings, markedly reduced the duration and magnitude of the hypoxic vasoconstriction in LPS-treated rings, and attenuated the transient vasoconstriction seen in endothelium-intact, non-LPS rings (all P < 0.05). L-Arginine reversed the L-NAME effects. Hypoxia decreased guanosine 3',5'-cyclic monophosphate (cGMP) content 54 +/- 4% in all LPS and 33 +/- 4% in the non-LPS intact rings (P < 0.05). L-NAME reduced cGMP content 90 +/- 5% in all LPS rings. Indomethacin inhibited formation of a constriction factor in aortic LPS-treated rings (P < 0.01) that was endothelium dependent and unaffected by the presence of L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)
Paraquat-induced lung injury: prevention by vasoactive intestinal peptide and related peptide helodermin
Authors: Pakbaz H. , Foda H. D. , Berisha H. I. , Trotz M. , Said S. I.
Published: December 2017

We earlier showed that the neuropeptide vasoactive intestinal peptide (VIP) reduces or prevents acute injury produced in rat lungs by xanthine and xanthine oxidase. We have now examined whether VIP can protect against lung injury induced by paraquat, a prooxidant pesticide. Isolated guinea pig lungs were perfused for 60 min with Krebs-4% albumin and mechanically ventilated with 95% O2-5% CO2. Infusion of paraquat (100 mg/kg) into the pulmonary artery (n = 9 observations) increased peak airway pressure from 10.1 +/- 0.6 to 54.7 +/- 6.5 cmH2O, perfusion pressure from 8.0 +/- 0.5 to 14.9 +/- 3.0 cmH2O, wet-to-dry lung weight ratio to 7.17 +/- 0.37, and bronchoalveolar lavage protein content to 2.70 +/- 0.83 mg/ml (P < 0.01). Pretreatment with 1–3 micrograms.kg-1 x min-1 VIP markedly attenuated or prevented all abnormalities. Of the related peptides tested, helodermin was as effective as VIP, but secretin and glucagon were ineffective. The results demonstrate that VIP and helodermin protect perfused guinea pig lungs against paraquat-induced injury and support the view that VIP has antioxidant activity.
Retinoic acid increases elastin in neonatal rat lung fibroblast cultures
Authors: Liu B. , Harvey C. S. , McGowan S. E.
Published: December 2017

The factors that regulate elastin synthesis during pulmonary alveolar septal formation have not been identified. Because maximal alveolar elastin synthesis occurs over a relatively brief period (postnatal days 4–14 in the rat), we hypothesized that changes in the local concentrations of factors that regulate elastin synthesis may precede or accompany this period. Because pulmonary retinoid stores decline just before the fourth postnatal day, we also hypothesized that this decline could be accompanied by the utilization of retinoic acid, one of the most biologically active retinoids, in a regulatory process that increases elastin synthesis. If these hypotheses are correct, then retinoic acid should increase elastin synthesis by pulmonary cells. Therefore, cultures of neonatal rat lung fibroblasts were exposed to retinoic acid, and elastin production was quantitated. Retinoic acid produced a two- to threefold increase in the steady-state level of elastin mRNA, in soluble elastin, and in insoluble elastin. The transcriptional initiation rate of the elastin gene was 1.8-fold higher in nuclei that were isolated from retinoic acid-treated cells than in nuclei that were isolated from control cells. This indicates that the increase in steady-state elastin mRNA results, at least partially, from an increase in elastin transcription. Lung fibroblasts that were isolated from 8-day-old rats, but not cultured, contained retinoic acid. These findings suggest that retinoic acid is a potential regulator of elastin synthesis in developing pulmonary alveoli.
Lung morphometry: a new generation of tools and experiments for organ, tissue, cell, and molecular biology
Authors: Bolender R. P. , Hyde D. M. , Dehoff R. T.
Published: December 2017

Today all structural information of the lung can be quantified and interpreted in the three-dimensional space of real-world biology. Remarkable achievements in the theory and practice of biological stereology are creating a new generation of data suitable for constructing structural hierarchies. Such hierarchies serve to organize and link biological data, thereby providing a framework on which to build new information systems. In this review, we describe the new tools of quantitative morphology and show how they can be used to design new experiments for lung research.
Alveolar macrophage secretion of a 92-kDa gelatinase in response to bleomycin
Authors: Denholm E. M. , Rollins S. M.
Published: December 2017

These experiments were conducted to study the possible involvement of macrophage-derived gelatinases in the bleomycin-induced model of pulmonary fibrosis. Normal rat alveolar macrophages and the rat alveolar macrophage cell line NR8383 were stimulated in vitro with 0-1.0 microgram/ml bleomycin for 18 h. Gelatinase activity in the medium was assayed on zymograms in which gelatin or collagen were used as substrates. Macrophages stimulated with 0.01-1.0 microgram/ml of bleomycin secreted significantly more of a 92-kDa gelatinase than did unstimulated controls. Addition of cycloheximide during stimulation decreased gelatinase activity by 86 +/- 4%, and activity was completely inhibited by the addition of EDTA to zymograms. This gelatinase degraded denatured type I collagen and native type IV collagen. Western blot analysis using a monoclonal mouse anti-rat antibody demonstrated that this enzyme was the same as a metalloproteinase secreted by rat mammary carcinoma cells. Gelatinase secreted by macrophages in fibrotic lungs may enhance macrophage migration through the lung and may also be active in the remodeling process.
Effect of hypoxia and norepinephrine on cytoplasmic free Ca2+ in pulmonary and cerebral arterial myocytes
Authors: Vadula M. S. , Kleinman J. G. , Madden J. A.
Published: December 2017

The fluorescent calcium indicator, fura 2, was used to test whether contraction of primary cultured smooth muscle cells (SMC) from small pulmonary arteries in response to hypoxia and the relaxation by large pulmonary and cerebral artery SMC were mediated by changes in cytoplasmic free Ca2+ (Ca2+c). Because SMC from large pulmonary and cerebral arteries contract to norepinephrine (NE), Ca2+c levels during NE exposure were measured to determine if they differed from those seen with hypoxia. Under hypoxic conditions, Ca2+c increased 64.1 +/- 11.1% above the normoxic baseline in small pulmonary artery SMC. In SMC from large pulmonary and cerebral arteries, Ca2+c decreased 25.2 +/- 9.20 and 28.1 +/- 5.80%. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and ryanodine attenuated the Ca2+c increase in the small pulmonary artery SMC. On exposure to NE, Ca2+c increased markedly in all three SMC types. EGTA and ryanodine treatment also attenuated NE-induced Ca2+c increases in all three SMC types. These results show that the three SMC types mobilize their available Ca2+ stores differently when exposed to hypoxia but similarly when exposed to NE. The data also suggest that a change in the Ca2+c concentration, rather than a change in the Ca2+ sensitivity of the contractile apparatus, is involved in the response to hypoxia.
Synthesis and vectorial export of cGMP in airway epithelium: expression of soluble and CNP-specific guanylate cyclases
Authors: Geary C. A. , Goy M. F. , Boucher R. C.
Published: December 2017

Guanosine 5'-cyclic monophosphate (cGMP) is an important modulator of fluid balance in many epithelia. We examined its metabolism in primary cultures of human airway epithelia. Sodium nitroprusside increased cGMP levels 30-fold, suggesting that the respiratory epithelium expresses a soluble guanylate cyclase; however, endogenous nitric oxide production was not detected. cGMP levels could also be increased by C-type natriuretic peptide (CNP), but not by atrial natriuretic peptide, brain natriuretic peptide, or Escherichia coli heat-stable enterotoxin, indicating expression of a CNP-specific membrane-bound guanylate cyclase. The one-half effective concentration for CNP was 40 nM and the maximal velocity was 56.7 pmol cGMP.mg protein-1.h-1. After CNP stimulation, approximately 60% of the total synthesized cGMP was preferentially exported from the polarized epithelial cells across the basolateral membrane by a probenecid-sensitive process. Isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) export revealed a similar export pattern and probenecid sensitivity, although a lower efficiency of export (27% of total cAMP was exported). Consistent with previous reports, export of neither cyclic nucleotide was saturable at the concentrations tested. We conclude that the respiratory epithelium expresses a soluble guanylate cyclase, a CNP-specific receptor, and a novel vectorial cyclic nucleotide export mechanism.
Hydrogen peroxide stimulates sodium-potassium pump activity in cultured pulmonary arterial endothelial cells
Authors: Meharg J. V. , McGowan-Jordan J. , Charles A. , Parmelee J. T. , Cutaia M. V. , Rounds S.
Published: December 2017

Oxidant injury to pulmonary vascular endothelium is an important factor in the pathogenesis of acute lung injury. Oxidant injury to other cell types has been reported to alter the function of Na-K-adenosinetriphophatase (ATPase) an enzyme important in maintenance of cellular ionic homeostasis and in transport of ions across biological membranes. We investigated the effect of H2O2 (0.001-10 mM) or xanthine (X) (15.2 micrograms/ml) plus xanthine oxidase (XO) (0.0153 U/ml) on the Na-K pump activity of cultured bovine pulmonary arterial endothelial cells (PAECs). We used a functional assay, using 86RbCl as a tracer for K+ and expressing Na-K pump activity as ouabain-inhibitable K+ uptake. Our results demonstrate that H2O2 and X/XO stimulate Na-K pump activity of bovine PAECs, an effect prevented by catalase. In addition, we assessed the affinity, number, and turnover of [3H]ouabain binding sites on intact endothelial monolayers and found that H2O2 increased affinity to [3H]ouabain, decreased the number of binding sites, and increased the rate of pump turnover. Influx of 22Na increased in response to a nonlytic concentration of H2O2. Cell injury, as assessed by 51Cr release, adherent cell number, and phase-microscopic morphology, was not observed after 30-min incubations with the lowest dose (1 mM) of H2O2 effective in stimulating Na-K pump activity, or after incubation with X/XO. Na-K pump inhibition by ouabain significantly increased the 51Cr release caused by H2O2 or by X/XO, suggesting that the increase in Na-K pump activity may be a compensatory response to the cellular alterations produced by H2O2. Incubation with H2O2 decreased cell ATP content, an effect which was not prevented by coincubation with ouabain. In summary, these results show that H2O2 increases Na-K pump activity of PAECs, an effect mediated, at least in part, by increased intracellular [Na] and by an increased rate of pump turnover. It is possible that the increased pump activity may be an early marker of endothelial cell perturbation.
Growth factors alter neonatal type II alveolar epithelial cell proliferation
Authors: Ryan R. M. , Mineo-Kuhn M. M. , Kramer C. M. , Finkelstein J. N.
Published: December 2017

The type II alveolar epithelial cell plays a critical role in the repair of lung injury by repopulating the entire damaged alveolar epithelium. We report our studies of the effects of known growth factors on the in vitro proliferation of isolated neonatal rabbit type II cells. Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) increased [3H]thymidine incorporation, cell number, and labeling index above control. Transforming growth factor-beta (TGF-beta) decreased [3H]thymidine incorporation, cell number, and labeling index compared with control. When added simultaneously, TGF-beta blocked the stimulatory effect of TGF-alpha or EGF. If TGF-alpha is added before TGF-beta, the ability of TGF-beta to block the mitogenic effect of TGF-alpha was diminished the later in time TGF-beta was added. If TGF-beta was added first, later addition of TGF-alpha had no effect. The current work demonstrates that specific growth factors, including some known to be produced by other lung cells, alter the proliferation in vitro of isolated neonatal rabbit type II alveolar epithelial cells.
Interleukin-1-induced lung neutrophil accumulation and oxygen metabolite-mediated lung leak in rats
Authors: Leff J. A. , Baer J. W. , Bodman M. E. , Kirkman J. M. , Shanley P. F. , Patton L. M. , Beehler C. J. , McCord J. M. , Repine J. E.
Published: December 2017

We found that intratracheal administration of recombinant interleukin-1 alpha (IL-1) into rats rapidly (< 5 h) increased neutrophils in lung lavages and caused an acute edematous lung injury which was reflected by lung albumin accumulation (lung leak) and histological abnormalities (perivascular cuffing). These IL-1-dependent processes were inhibited by prior administration of recombinant IL-1 receptor antagonist and did not occur following administration of heated IL-1. Several lines of evidence suggested that neutrophil-derived oxygen metabolites contributed to lung leak. First, lung leak did not occur in rats rendered neutropenic by vinblastine treatment 4 days before IL-1 administration but did occur in neutrophil-replete rats given vinblastine 1 day before IL-1 administration and control rats given IL-1. Second, treatment with a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO) or a superoxide anion scavenger, manganese superoxide dismutase, decreased lung leak, lung lavage neutrophils, and histological abnormalities in rats given IL-1 intratracheally. Third, intratracheal IL-1 administration increased lung oxidized glutathione (GSSG) levels and expired H2O2 concentrations, and these two indices of oxidative stress were decreased by dimethyl sulfoxide or manganese superoxide dismutase treatment. We conclude that intratracheal administration of IL-1 increases neutrophils in the lung and causes a neutrophil and oxygen metabolite-dependent acute edematous lung injury.
Protection against oxygen toxicity by tracheal insufflation of endotoxin: role of Mn SOD and alveolar macrophages
Authors: Tang G. , Berg J. T. , White J. E. , Lumb P. D. , Lee C. Y. , Tsan M. F.
Published: December 2017

Endotoxin and the cytokines, tumor necrosis factor and interleukin-1, are known to protect adult rats against O2 toxicity. However, whether the effect of endotoxin is mediated through its direct effect on lung cells or through cytokines is not clear. In this study, we demonstrated that endotoxin at a dosage of 5 micrograms/rat (14-20 micrograms/kg) attenuated O2-induced pulmonary injury and markedly prolonged the survival of rats exposed to 100% O2. Endotoxin was more protective when given by intratracheal insufflation or intravenous injection than by intraperitoneal injection. The endotoxin-induced O2 tolerance was associated with a selective enhancement of pulmonary manganese superoxide dismutase, but not Cu,Zn SOD, mRNA. In addition, depletion of 84% rat alveolar macrophages by liposome-encapsulated dichloromethylene diphosphonate, resulted in a marked reduction (86%) of endotoxin-induced release of tumor necrosis factor into the alveolar space. However, endotoxin was still protective in these alveolar macrophage-depleted animals.
Changes in cytosolic cGMP and calcium in airway smooth muscle relaxed by 3-morpholinosydnonimine
Authors: Jones K. A. , Lorenz R. R. , Warner D. O. , Katusic Z. S. , Sieck G. C.
Published: December 2017

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.