bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of inhibitors of EDRF and EDHF on vasoreactivity of perfused rat lungs
Authors: Hasunuma K. , Yamaguchi T. , Rodman D. M. , O'Brien R. F. , McMurtry I. F.
Published: December 2017

Recent studies indicate that the endothelium of isolated rat pulmonary arteries releases two different factors, endothelium-derived relaxing factor (EDRF) and hyperpolarizing factor (EDHF), which participate in histamine- and acetylcholine-induced relaxation. There is evidence for EDRF vasoreactivity in perfused lungs, but a role for EDHF, which hyperpolarizes vascular smooth muscle by activating membrane K+ channels, has not been reported. We used the inhibitors of EDRF, 20 microM hemoglobin, 200 microM NG-mono-methyl-L-arginine, and 2 mM L-canavanine, the nonselective blocker of K+ channels, 10 mM tetraethylammonium (TEA), and the inhibitor of ATP-sensitive K+ channels, 20 microM glibenclamide, to compare the roles of EDRF and EDHF in the vasoregulation of meclofenamate-treated, salt solution-perfused rat lungs. The three EDRF inhibitors had little or no effect on baseline perfusion pressure, but each potentiated the peak pressor response to airway hypoxia. Neither of them inhibited the pulmonary vasodilation to 5 microM histamine. TEA, but not glibenclamide, increased baseline pressure and potentiated the peak hypoxic response. Both K+ channel blockers, but not the EDRF inhibitors, also prolonged the hypoxic response by reducing the rate of spontaneous vasodilation. TEA, but not glibenclamide, inhibited histamine vasodilation. These results suggest roles for both EDRF and EDHF in the control of rat pulmonary vascular reactivity. EDRF is apparently not responsible for the low vascular tone of the normoxic lung and does not mediate the vasodilation to histamine, but it does modulate the hypoxic pressor response. The exact role of EDHF is uncertain, but it may also modulate hypoxic vasoconstriction and mediate at least part of the histamine vasodilation.
Rat alveolar epithelial cells concomitantly express plasminogen activator inhibitor-1 and urokinase
Authors: Gross T. J. , Simon R. H. , Kelly C. J. , Sitrin R. G.
Published: December 2017

There is considerable evidence to suggest that intra-alveolar plasminogen activation is instrumental in many aspects of inflammatory lung injury and subsequent tissue repair. Rat alveolar epithelial cells produce large quantities of urokinase-type plasminogen activator (uPA) in vitro, and uPA expression is modulated in association with cellular differentiation and exposure to inflammatory mediators. We now report that these cells also secrete heat-stable PA inhibitory activity having the characteristics of PA inhibitor type 1 (PAI-1). In particular, immunoreactive PAI-1 was demonstrable in conditioned media, cell lysates, and extracellular matrix from epithelial cell cultures. As alveolar epithelial cells differentiated in vitro, secreted PA inhibitor activity increased significantly from 104 +/- PAI U/ml (n = 5, mean +/- SE) on day 2 to 442 +/- 150 on day 7 in parallel with increases in secreted and matrix-associated immunoreactive PAI-1. PAI-1 mRNA expression decreased over this same period suggesting posttranscriptional regulation. The levels of both newly synthesized antigen and PAI-1 mRNA were increased by exposure to lipopolysaccharide and tumor necrosis factor-alpha. Thus, by the coexpression of uPA and PAI-1, the alveolar epithelium may actively regulate the generation of plasmin in both the normal and injured alveolus.
Endotoxin-induced exposure of pulmonary glycoproteins in an intact animal
Authors: Carley W. W. , Merker M. , Gillis C. N.
Published: December 2017

A method has been developed to radioiodinate luminally disposed endothelial proteins in an in situ perfused lung system without causing obvious vascular changes. The spectrum of endothelial cell proteins labeled in control animals and those treated with endotoxin for 45 min were compared. No changes in gross tissue morphology or in the distribution of radiolabel (125I-s-SHPP) were detected in control or treated lungs. Lectin affinity purification was applied to a lung membrane fraction to isolate labeled proteins, which were in turn resolved by gel electrophoresis and autoradiography. Comparisons of gel autoradiographs from control and treated lungs identified eight glycoproteins, the labeling of which was enhanced in endotoxin-treated animals. A similar lectin affinity analysis of radiolabeled effluent blood cells from the lungs identified only two proteins, neither of which were consistently changed by endotoxin pretreatment. A glycoprotein response can, therefore, be measured at the pulmonary endothelial surface on endotoxin administration to the whole animal without causing obvious lung injury.
Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia
Authors: Fleming R. E. , Whitman I. P. , Gitlin J. D.
Published: December 2017

Pages L68–L74: R. E. Fleming, I. P. Whitman, and J. D. Gitlin. “Induction of ceruloplasmin gene expression in rat lung during inflammation and hyperoxia.” The vertical axes of Figs. 2, 3B, and 5C should read: % over control.
Immunohistochemistry of central respiratory neurotransmitters
Authors: Holtman J. R.
Published: December 2017

Significant progress has been made in identifying the nuclei and pathways involved in central control of respiration in mammals. This knowledge provides a framework from which to focus on the question of which putative neurotransmitters may be involved in central respiratory control. Application of chemical neuroanatomical methods, particularly immunohistochemistry, is a powerful approach to this important question. This commentary discusses how immunohistochemical approaches have been used so far to extend our knowledge of central respiratory neurotransmitters and also how they can be further applied to gain new information. The results of immunohistochemical studies provide a rationale for subsequent physiological and pharmacological studies of respiratory neurons where specific actions of putative neurotransmitters can be evaluated.
Identification and structural characterization of Fc gamma-receptors on pulmonary alveolar macrophages
Authors: Kindt G. C. , van de Winkel J. G. , Moore S. A. , Anderson C. L.
Published: December 2017

Little is known about the structure of the cell surface receptors for the Fc portion of immunoglobulin G (Fc gamma R) on tissue macrophages. Studies on leukocytes indicate the existence of three classes of Fc gamma R, denoted I, II, and III. The purpose of this study was to structurally characterize Fc gamma R on alveolar macrophages obtained by bronchoalveolar lavage, comparing them with Fc gamma R of monocytes, neutrophils, and U937 cells. Flow-cytometric evaluation, utilizing anti-Fc gamma R class-specific monoclonal antibodies, showed that alveolar macrophages expressed three Fc gamma R classes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunopurified Fc gamma R molecules revealed the following apparent molecular mass for each Fc gamma R class: Fc gamma RI, 70 kDa; Fc gamma RII, 44 kDa; and Fc gamma RIII, 57-65 kDa. RNA blot analysis demonstrated a 1.7-kb transcript for Fc gamma RI, 2.5 and 1.6 kb transcripts for Fc gamma RII, and a 2.2 kb mRNA for Fc gamma RIII. Fc gamma RII displayed the high-responder/low-responder polymorphism. Fc gamma RIII did not express the neutrophil antigen-type specific structural polymorphism of the deglycosylated Fc gamma R molecule and appeared to be a transmembrane molecule.
Heterogeneity in superoxide production as measured by nitro blue tetrazolium reduction from individual PAM
Authors: DiGregorio K. A. , Cilento E. V. , Lantz R. C.
Published: December 2017

Heterogeneity in superoxide (O2-.) production (as determined by the reduction of nitro blue tetrazolium to a diformazan precipitate), cell volume, and cell spreading were measured from single rat pulmonary alveolar macrophages (PAM). Of the 330 cells that produced O2-. due to stimulation by adherence, the maximum diformazan produced (MAX) varied between 1.8 and 47.2 fmol and the maximum (initial) rate of diformazan production (R) varied between 2.2 and 81.7 X 10(-3) fmol/s. Only six PAM of 336 analyzed failed to produce O2-. suggesting that O2-. production is not a specific function of a small subpopulation of PAM but rather that all PAM are capable of producing O2-.. Importantly, O2-. production by single cells showed extensive heterogeneity that did not correlate (r2 less than 0.12) with individual cell volumes, suggesting that the observed heterogeneity may not be due to differences in maturation and/or differentiation. However, when the single cell data were grouped into cell volume subfractions small but increasing linear trends (r2 greater than 0.81) in MAX and R were found. This discrepancy suggests that PAM heterogeneity in O2-. fraction cannot be estimated adequately by measurements on subpopulations of cells.
ADP ribosylation of type II pulmonary epithelial cell G proteins
Authors: Rybin V. O. , Uhal B. D. , Russo L. A. , Rannels D. E.
Published: December 2017

Secretion of pulmonary surfactant by type II pulmonary epithelial cells (T2P) is regulated by receptor-mediated mechanisms. In other systems, coupling of receptor-linked signals to intracellular events involves guanine nucleotide-binding proteins (G proteins), but the specific role of G proteins in T2P signaling pathways is poorly defined. The present studies begin to address the role of G proteins in transmembrane signaling in these pneumocytes. Membrane preparations from purified T2Ps demonstrated ADP ribosylation of specific substrates by pertussis, cholera, and botulinum toxins (PT, CT, and BT, respectively). Toxin-dependent T2P substrate labeling from 32P-labeled NAD was dependent on time and membrane protein concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed ADP ribosylation of membrane substrates of the following molecular masses: PT, 40/41 kDa; CT, 47/51 kDa; BT, 22 kDa. BT-dependent ADP ribosylation of a 22-kDa cytosolic substrate was also observed. Pretreatment of cultured T2P with the individual toxins led to ADP ribosylation of their respective specific substrates in a time-dependent fashion. In cells pretreated with PT or CT, substrates for the complimentary toxins remained available for subsequent ADP ribosylation in vitro. This result supports the specificity of the toxin effects. Basal secretion of the major phospholipid of pulmonary surfactant, disaturated phosphatidylcholine (DSPC) was unaffected in T2P treated with PT, but was stimulated in cells exposed to CT or BT. Neither CT nor BT altered release of lactate dehydrogenase. In cells treated with AMP or with isoproterenol DSPC secretion was stimulated six- to eightfold; preexposure of the cells to CT reduced the response to either agonist by 70%.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of albumin on hydraulic conductivity of pulmonary artery endothelial monolayers
Authors: McCandless B. K. , Powers M. R. , Cooper J. A. , Malik A. B.
Published: December 2017

We tested the hypothesis that albumin reduces the vascular wall hydraulic conductivity by an interaction with the endothelium. The system consisted of luminal and abluminal chambers separated by a microporous filter onto which was grown a confluent monolayer of ovine pulmonary artery endothelial cells. The abluminal chamber filtrate was collected for timed periods during increases in transendothelial pressures of 5, 10, 15, and 20 cmH2O. The transendothelial water flux was linearly related to the hydrostatic pressure. Hydraulic conductivity (Lp) was determined from the slope of this relationship per unit surface area. In the absence of albumin, Lp of the endothelium and the filter was 14.8 +/- 3.8 x 10(-5) cm.s-1.cmH2O-1. The addition of either 2.5 or 5.0 mg/ml ovine serum albumin to the medium reduced Lp values similarly to 2.0 +/- 0.3 x 10(-5) and 2.5 +/- 1.1 x 10(-5) cm.s-1.cmH2O-1, respectively. Removal of albumin from the media reversed the effect of albumin on Lp. The filter Lp value of 3.2 +/- 0.3 x 10(-3) cm.s-1.cmH2O-1 was unaffected by albumin. Endothelial Lp value did not decrease with 5.0 mg/ml of 70-kDa neutral dextran. Albumin decreased Lp in the presence of epsilon-amino-caproic acid to the same extent as albumin alone, suggesting that the positively charged lysine sites on albumin did not mediate the effect. The results indicate that albumin decreases Lp due to an interaction between albumin and the endothelial cell.
Enhanced fatty acid biosynthesis and normal surfactant secretion in hypertrophic rat type II cells
Authors: Rami J. , Sasic S. M. , Rooney S. A.
Published: December 2017

Silica instillation causes lung surfactant accumulation as well as hyperplasia and hypertrophy of type II pneumocytes. Two populations of type II cells can be isolated from silica-treated rats: type IIA, which are similar to type II cells from normal animals and type IIB, which are larger and have a higher rate of phosphatidylcholine biosynthesis. We have compared fatty acid biosynthesis and phosphatidylcholine secretion in types IIA and IIB cells and in type II cells from control rats. The cells were isolated by elastase digestion and panning on immunoglobulin G-coated plates and fractionated into types IIA and IIB by centrifugal elutriation. Type IIB cells contained more phospholipid and had an enhanced rate of [3H]choline incorporation into phosphatidylcholine. The activity of choline-phosphate cytidylyltransferase was elevated in the type IIB cells and the extent of the increase was diminished when phosphatidylglycerol was included in the assay, suggesting that the enhanced activity was due to enzyme activation rather than protein synthesis. The basal rate of phosphatidylcholine secretion was the same in all three groups as was the response to a variety of secretagogues. Incorporation of [3H]acetate into fatty acids was elevated in type IIB cells and the activity of fatty acid synthase was eightfold greater than in control cells. These data show that de novo fatty acid biosynthesis is increased in hypertrophic type II cells and that surfactant secretion is not elevated.
Microperfusion of sheep bronchioles
Authors: al-Bazzaz F. J. , Tarka C. , Farah M.
Published: December 2017

We isolated segments of sheep bronchioles (length 406 +/- 46 microns, diameter 227 +/- 53 microns, n = 27). These segments were placed in a 37 degree C bath and cannulated at both ends with glass micropipettes. The proximal end was cannulated and held by three concentrically arranged micropipettes that delivered perfusion solution by gravity. The distal end was cannulated and held by two concentrically arranged micropipettes for fluid collection. When both the lumen and the bath contained oxygenated Krebs-Henseleit buffer (pH 7.40), spontaneous potential difference (PD) was 2.46 +/- 0.39 mV, lumen negative. KCN in the bath diminished PD by 1.48 +/- 0.29 mV (P = 0.00003, n = 25). The isolated bronchiole was depolarized when the luminal solution was Na free (delta PD -1.78 +/- 0.11 mV, P = 0.00007, n = 5) or when the submucosal bath was Cl free (delta PD -2.63 +/- 0.81, P = 0.018, n = 7). In preparations pretreated with 10 microM indomethacin, 1 microM isoproterenol raised PD by 0.75 +/- 0.29 mV (P = 0.03, n = 8), whereas 0.1 mM submucosal bumetanide reduced PD by 0.21 +/- 0.04 mV (P = 0.0005, n = 8). The data show that microperfusion technique is applicable for investigating ion transport by distal bronchioles and that the bronchiolar epithelium probably has both Na and Cl conductive pathways.
Neutrophil chemoattractant production by cultured serotonin-stimulated bovine and human endothelial cells
Authors: Charles A. , Rounds S. , Farber H. W.
Published: December 2017

Recent studies have demonstrated that serotonin (5-HT) is avidly taken up and metabolized by vascular endothelial cells (EC) and have suggested that 5-HT may contribute to inflammatory responses. Because EC can produce neutrophil cytokines among their biologically active molecules, we hypothesized that the interaction of 5-HT and EC might cause production of such a cytokine. Using a modified Boyden chamber assay, we found that cultured bovine aortic (BA), bovine pulmonary arterial (BPA), and human umbilical vein (HUV) EC incubated with 5-HT produced a neutrophil chemoattractant (NCA). The NCA was predominantly chemotactic, was not stored in an active form, appeared within 5 min of incubation with 5-HT, and required de novo protein synthesis for its appearance. The mechanism of NCA production was different in the three types of EC examined. Elaboration of NCA and BAEC and HUVEC was apparently mediated by 5-HT1 receptors and did not require uptake of 5-HT, whereas its elaboration from BPAEC required 5-HT uptake and was apparently mediated by 5-HT2 receptors. Incubation with three different lipoxygenase inhibitors blocked production of NCA, whereas incubation with a cyclooxygenase inhibitor did not. Further characterization of the NCA demonstrated that it was a mixture of several different chemotactic lipids and was distinct from other lipid or phospholipid neutrophil chemoattractants. These studies suggest that the interaction of the platelet-release product, 5-HT, with the adjacent endothelium results in the production of a chemoattractant that could affect neutrophil accumulation at sites of inflammation.
Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans
Authors: Karlinsky J. B. , Bucay P. J. , Ciccolella D. E. , Crowley M. P.
Published: December 2017

Incorporation of [3H]glucosamine and 35S into glycosaminoglycan (GAG) was measured in hamster lung explant cultures at 0, 1, 4, and 24 h after a single endotracheal instillation of Escherichia coli endotoxin. Lung content of GAG was measured in a second group of treated animals over an 8-day period. Albumin was detected after endotoxin treatment in bronchoalveolar lavage fluid at 24 h but was not found in lavage fluid 7 days later or in lavage fluid of saline-treated animals. Over the initial 24 h, increasing amounts of radiolabeled precursor molecules were incorporated into all classes of GAG. Proportionally more radiolabel was incorporated into hyaluronic acid and chondroitin sulfate, and less was incorporated into heparan sulfate. The proportion of radiolabel incorporated into dermatan sulfate did not change. Total lung content of hyaluronate and chondroitin sulfate was elevated at 24 h but was returning to baseline by 8 days. The lung content of dermatan sulfate was increased at 8 days; lung content of heparan sulfate did not change over the 8-day study period. Elevations in the amount of explant heparan sulfate that bound to antithrombin III (AT III) were found at 1 h after both saline and endotoxin treatment. Radiosulfated heparan sulfates were found in blood from hamsters treated with endotoxin 1 h previously; these heparan sulfates did not bind to AT III. However, blood contained heparin-like activity. We conclude that endotoxin differentially alters the metabolism of each class of hamster lung glycosaminoglycans and that metabolic changes begin very rapidly after endotoxin exposure. The relation of pulmonary endothelial injury to the presence of heparin-like activity in blood is not yet clear.
Regulation of the contractile element of airway smooth muscle
Authors: Gerthoffer W. T.
Published: December 2017

Smooth muscle of the mammalian airways controls airway diameter and resistance to airflow. Smooth muscle tone is in turn controlled by a variety of external signals that are transduced to useful work by contractile proteins. The protein components of the contractile element of airway smooth muscle are similar to those found in other smooth muscles and include actin, myosin, tropomyosin, caldesmon, and calponin. There has been significant recent progress in studies of contractile system regulation of airway smooth muscle. Regulation of myosin light chain kinase, identification of the sites phosphorylated on the regulatory myosin light chains, and the effect of myosin phosphorylation on stress development and crossbridge cycling rates have all been studied in some detail. We infer from these studies that besides myosin phosphorylation there is an important role for a thin filament Ca(2+)-dependent regulatory mechanism. The potentially important thin filament proteins caldesmon and calponin are present in tracheal smooth muscle and may be phosphorylated during contraction. The use of intracellular Ca2+ indicators to estimate changes in intracellular Ca2+ ([Ca2+]i) and the development of several skinned fiber preparations have broadened the scope of physiological studies with airway smooth muscle and have suggested that the contractile element sensitivity to Ca2+ is not fixed but might be modulated by undefined messengers or excitation-contraction pathways. This adds an additional challenge to the continuing effort to define the messengers and regulatory proteins that couple activation of membrane receptors to the contractile element in airway smooth muscle.
Regulation of secretion in cultured tracheal serous cells by protein kinases A and C
Authors: Paul A. , Mergey M. , Veissiere D. , Hermelin B. , Cherqui G. , Picard J. , Basbaum C. B.
Published: December 2017

We recently reported that cultured gland serous cells release chondroitin sulfate proteoglycans (CSPGs) in response to beta-adrenergic agonists. In this study, we analyzed this regulatory pathway and other cellular mechanisms responsible for CSPG secretion. We show the following. 1) Isoproterenol increased CSPG secretion in a concentration-dependent manner, with maximal stimulation (50%) obtained at 10(-5) M; at this concentration, the beta-agonist also stimulated protein kinase A (PKA) by 50%, whereas it increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 300%. 2) Phenylephrine (10(-5) M), 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (1.6 x 10(-7) M), and A23187 (10(-6) M) also stimulated CSPG secretion; this stimulation was concomitant with protein kinase C (PKC) translocation from cytosol to membrane, was blocked by sphingosine (2 x 10(-5) M), and was additive with that elicited by isoproterenol. 3) All PKC activators potentiated the isoproterenol-induced increased in cAMP accumulation without modifying the activation of PKA elicited by the beta-agonist. Our results indicate that although the signaling pathways triggered by alpha- and beta-adrenergic agonists converge at the level of adenylate cyclase in tracheal serous cells, PKA and PKC independently regulate CSPG secretion.
Endothelin stimulates chloride secretion across canine tracheal epithelium
Authors: Plews P. I. , Abdel-Malek Z. A. , Doupnik C. A. , Leikauf G. D.
Published: December 2017

The endothelins (ET) are a group of isopeptides produced by a number of cells, including canine tracheal epithelial cells. Because these compounds are endogenous peptides that may activate eicosanoid metabolism, we investigated the effects of ET on Cl secretion in canine tracheal epithelium. Endothelin 1 (ET-1) was found to produce a dose-dependent change in short-circuit current (Isc) that increased slowly and reached a maximal value within 10-15 min. When isopeptides of ET were compared, 300 nM ET-1 and ET-2 produced comparable maximal increases in Isc, whereas ET-3 produced smaller changes in Isc (half-maximal concentrations of 2.2, 7.2, and 10.4 nM, respectively). Ionic substitution of Cl with nontransported anions, iodide and gluconate, reduced ET-1-induced changes in Isc. Furthermore, the response was inhibited by the NaCl cotransport inhibitor, furosemide. In paired tissues, ET-1 significantly increased mucosal net 36Cl flux without significant effect on 22Na flux. The increase in Isc induced by ET was diminished by pretreatment with indomethacin. The second messengers mediating the increase in Isc were investigated in cultured canine tracheal epithelial cells. ET-1 stimulated the release of [3H]arachidonate from membrane phospholipids, increased intracellular Ca2+ (occasionally producing oscillations), and increased adenosine 3',5'-cyclic monophosphate accumulation. The latter was diminished by indomethacin. Thus ET is a potent agonist of Cl secretion (with the isopeptides having the following potency: ET-1 greater than or equal to ET-2 greater than ET-3) and acts, in part, through a cyclooxygenase-dependent mechanism.
Surfactant protein A is opsonin in phagocytosis of herpes simplex virus type 1 by rat alveolar macrophages
Authors: van Iwaarden J. F. , van Strijp J. A. , Ebskamp M. J. , Welmers A. C. , Verhoef J. , van Golde L. M.
Published: December 2017

In the present study we used flow cytometry to investigate the phagocytosis of fluorescein isothiocyanate-labeled herpes simplex virus type 1 (FITC-HSV-1) by rat alveolar macrophages and the effects of surfactant protein A (SP-A) on this process. The phagocytosis of FITC-HSV-1 by alveolar macrophages, which was studied as a model for virus phagocytosis in general, was strongly enhanced in the presence of SP-A. The SP-A-mediated phagocytosis was time and concentration dependent, reaching a maximal level after 15 min of incubation and at an SP-A concentration of 5 micrograms/ml. Using a fluorescence quenching technique, we could show that at least 65% of the viruses were indeed internalized by the macrophages. The addition of SP-A to the system was sufficient for the phagocytosis of FITC-HSV-1 by the alveolar macrophages, suggesting that SP-A acts as an opsonin. This hypothesis was further strengthened by the observation that F(ab')2 fragments of immunoglobulin G directed against SP-A could abolish FITC-HSV-1 phagocytosis by alveolar macrophages preincubated with SP-A. Comparing the opsonic capacity of serum and SP-A, SP-A proved to be twice as potent as serum in stimulating phagocytosis of FITC-HSV-1 by alveolar macrophages. Complement factor C1q, which is known to possess a similar collagen-like domain as SP-A, did not stimulate phagocytosis of FITC-HSV-1 by alveolar macrophages nor did it inhibit SP-A-mediated HSV-1 phagocytosis. This study demonstrates that SP-A may play an important role in the antiviral defenses of the lung.
Factors affecting distribution of alveolar surfactant during resting ventilation
Authors: Oyarzun M. J. , Iturriaga R. , Donoso P. , Dussaubat N. , Santos M. , Schiappacasse M. E. , Lathrop M. E. , Larrain C. , Zapata P.
Published: December 2017

Few studies have been done to establish the ventilatory factors affecting alveolar surfactant under resting conditions. Experiments in which ventilatory variables were recorded for 4 h were performed in 12 adult cats breathing spontaneously under pentobarbital sodium anesthesia. After the animals were killed and bronchoalveolar lavages (BALs) were performed, the resulting fluid was subjected to differential centrifugation and determinations of proteins, phospholipids (PL), and disaturated phosphatidylcholine (DSPC). Pellet P1+2 was obtained by two centrifugations at 140 g; the supernatant was centrifuged at 1,000 g to obtain pellet P3 and subsequently at 60,000 g to obtain P4. Pellets P3 and P4 had the higher contents of PL and DSPC. Bivariate and multivariate correlational analyses indicate that 1) total PL in BAL was not related to any of the ventilatory variables studied, 2) PL in P3 and P4 fractions was directly correlated to the physiological range of variations in the frequency of large spontaneous gasps (fL), and 3) PL in P1+2 fraction was inversely related to fL. Cats subjected to bilateral section of carotid nerves, although presenting reduced chemosensory drive and ventilatory chemoreflexes, did not exhibit significant differences in resting ventilatory variables nor in alveolar surfactant components. Present results indicate that the total content of alveolar surfactant is not modified by ventilatory variations within physiological range but that the spontaneous occurrence of large gasps increases the proportion of more active forms of alveolar surfactant. This may mediate the role of augmented breaths in keeping lung compliance and preventing atelectasis under resting ventilatory conditions.
Mechanism underlying ozone-induced in vitro hyperresponsiveness in canine bronchi
Authors: Janssen L. J. , O'Byrne P. M. , Daniel E. E.
Published: December 2017

The effects of ozone inhalation on the contractility of canine bronchi and the mechanisms underlying any changes observed were investigated. Ozone inhalation caused acetylcholine airway hyperresponsiveness in vivo. Isolated segments of bronchi (3rd to 5th order) from dogs previously exposed to ozone (or normal air) were mounted in organ baths. Contractile responses to electrical field stimulation (FS) in all tissues decreased in magnitude over time (measured at 10, 120, 210, and 300 min after mounting tissues in the organ baths); responses to 150 mM KCl did not show such a progressive decrease. Ozone inhalation produced a dramatic enhancement of FS responses, a smaller enhancement of responses to carbachol (10(-8)-10(-4) M), and no change in responses to KCl (150 mM). In the presence of 10(-5) M indomethacin, all responses were enhanced and tended to increase in magnitude over time; indomethacin also abolished the differences in responsiveness between the control and ozone-exposed tissues. In the presence of the thromboxane receptor antagonist L-670,596 (10(-8) M), the magnitudes of all FS responses showed a progressive decrease over time while KCl responses did not; again, the differences in FS responses between control and ozone-exposed tissues were decreased or made insignificant. In conclusion, ozone produced airway hyperresponsiveness in vivo and hyperresponsiveness in canine bronchi in vitro via decreased prejunctional and postjunctional inhibition (likely mediated by prostaglandin E2) and increased prejunctional excitation mediated by thromboxane A2. However, direct measurements of mediator release should be carried out to reach a firm conclusion.
Effects of bradykinin on intracellular calcium regulation in human ciliated airway epithelium
Authors: Paradiso A. M. , Cheng E. H. , Boucher R. C.
Published: December 2017

The Ca(2+)-mobilizing action of bradykinin (BK) was investigated in ciliated human nasal epithelial (HNE) cells utilizing fura-2 fluorescence and microspectrofluorimetry. In ciliated cells, basal intracellular Ca2+ concentration ([Ca2+]i) was 123 +/- 3 nM (n = 142). BK caused [Ca2+]i to increase (spike) rapidly (within 6 s) to greater than 550 nM by releasing Ca2+ from intracellular pools. The mean effective dose for the process was 1.5 x 10(-7) M BK. The spike was due to the activation of a beta 2-receptor. The spike was unaffected by inhibitors of either cyclooxygenase or voltagegated Ca2+ channels. After the spike, [Ca2+]i decreased to a plateau level (120-250 nM). This plateau persisted (up to 10 min) until the addition of La3+ (0.3 x 10(-3) M) or until the removal of either extracellular Ca2+ or the agonist. No changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels were detected after BK exposure. Additional studies revealed that indomethacin (10(-6) M), isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (1 mM) had no effect on [Ca2+]i in ciliated HNE cells. In summary, these data suggest that 1) BK mediates both Ca2+ release from internal pools and Ca2+ entry into the cytoplasm from the extracellular space, and 2) unlike the response to cultured dog airway epithelia, the release of [Ca2+]i in response to BK does not appear to be mediated by either cyclooxygenase pathway or adenylate cyclase-cAMP systems.
Regulation of ATP-dependent surfactant secretion and activation of second-messenger systems in alveolar type II cells
Authors: Voyno-Yasenetskaya T. A. , Dobbs L. G. , Williams M. C.
Published: December 2017

Several different classes of agonists are known to stimulate exocytosis in type II cells. These agonists cause increases in second messengers, such as adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+, and/or stimulate protein kinase C. We studied generation of cAMP and phosphoinositide (PI) turnover in monolayer cultures of type II cells and measured [Ca2+]i in single cultured cells. ATP [10-4 M], which stimulates secretion of phosphatidylcholine (PC) and increases cellular cAMP, also stimulated PI turnover and increased [Ca2+]i. 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulates PC secretion and activates protein kinase C, did not increase [Ca2+]i. Pretreatment of type II cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the PC secretion induced by ATP and TPA and blocked the increase in PI turnover caused by ATP. ATP-dependent surfactant secretion and stimulation of PI turnover could also be inhibited by pretreatment of the cells with pertussis toxin. We used the fluorescent probe indo-1 to measure [Ca2+]i in single cultured type II cells. ATP produced rapid transient increases in [Ca2+]i, which could be prevented by pretreatment of the cells with either TPA or W-7. Our data suggest that pertussis toxin-sensitive G protein(s) are involved in ATP-dependent activation of PI turnover and in secretion of surfactant in type II cells. Activation of protein kinase C blocks the ATP-stimulated increase in [Ca2+]i. Finally, calmodulin may be involved in the regulation of ATP-dependent increase in [Ca2+]i, the activation of PI turnover, and the secretion of surfactant in type II cells.

lung; exocytosis; cell calcium; G proteins; phosphoinositide turnover
Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome
Authors: Idell S. , Koenig K. B. , Fair D. S. , Martin T. R. , McLarty J. , Maunder R. J.
Published: December 2017

We studied the changes of coagulation and fibrinolysis in bronchoalveolar lavage (BAL) and plasma obtained serially at intervals after the onset of adult respiratory distress syndrome (ARDS). BAL procoagulant activity was increased at 3 days and tended to decrease thereafter. Tissue factor associated with factor VII was the major BAL procoagulant. Fibrinopeptide A was increased, indicating increased thrombin-mediated conversion of fibrinogen to fibrin. Fibrinolytic activity was usually undetectable in BAL at 3 days post-ARDS and remained depressed for up to 14 days despite unchanged concentrations of urokinase and variably detectable tissue plasminogen activator. Depressed fibrinolytic activity was associated with increased antiplasmin activity and plasminogen activator inhibitor 1 (PAI-1) while PAI-2 concentrations approximated those of control samples and did not change during evolving ARDS. Evidence of systemic coagulopathy and increased systemic fibrin degradation were commonly found in serial ARDS plasma samples, consistent with accelerated vascular and/or extravascular fibrin deposition in these patients. The data indicate that intra-alveolar as well as systemic derangements of fibrin turnover are common features of evolving ARDS. Concurrent local abnormalities of both coagulation and fibrinolytic pathways favor persistence of alveolar fibrin for up to 14 days after clinical recognition of ARDS.
Influence of fetal pulmonary epithelium on thrombin activity
Authors: O'Brodovich H. , Berry L. , D'Costa M. , Burrows R. , Andrew M.
Published: December 2017

Epithelial injury and intra-alveolar fibrin are present in lung injury. To determine whether healthy fetal and neonatal lung epithelium could regulate thrombin activity (hence fibrin formation) we collected amniotic and postnatal endotracheal tube fluids from humans and directly sampled lung and amniotic fluids from fetal guinea piglets, rabbit pups, and lambs. The coagulant properties of the cell surface and media conditioned by rat fetal type II alveolar epithelium were assessed. All fluids contained glycosaminoglycans (GAGs), but mass and biological assays demonstrated only some (heparan sulfate and to a lesser extent dermatan sulfate) had antithrombin activity. The presence of proteoglycans (greater than 1,000 kDa) yielding active GAGs with less than 100 kDa after base elimination were demonstrated by Sepharose CL4B chromatography. Epithelial-derived fluids contained a factor VII-dependent procoagulant activity, but concentrated conditioned media overlying primary cultures of type II epithelium demonstrated a net antithrombin effect. These studies demonstrate that the lungs of human and nonprimate mammalian fetuses and fetal type II epithelium secrete GAGs, some of which possess antithrombin activity, which would oppose intra-alveolar fibrin formation.
Control of growth in the neonatal pig heart
Authors: Beinlich Cathy J. , Baker Kenneth M. , White Gloria J. , Morgan Howard E.
Published: December 2017

The newborn heart is an excellent model in which to study cardiac growth because the neonatal period is a normal situation in which the left ventricle (LV) grows rapidly and the right ventricle grows slowly. Accelerated LV growth is in response to mechanical, neural, and endocrine changes at birth. Faster growth of the LV is accounted for by greater capacity for protein synthesis, as evidenced by greater RNA content. At 18 h of life, ribosomes are formed in preference to total heart protein, but at 48 h of life, faster rates of both ribosome formation and total protein synthesis are observed. In the LV of hearts from 2-day-old pigs, these rates are insensitive to the addition of glucagon, 1-methyl-3-isobutylxanthine, or a combination of norepinephrine and propranolol. These observations could result because of maximal growth stimulation already present in the LV of the newborn heart. To restrain LV growth in the neonatal period, we treated pigs with enalapril maleate, an angiotensin II-converting enzyme inhibitor. Enalapril blocked growth of the LV as well as the increase in RNA content. When hearts from enalapril-treated pigs were perfused in vitro, rates of protein synthesis and ribosome formation in the LV were lower. These studies suggest that angiotensin II is an important factor accounting for rapid growth of the neonatal heart in response to pressure overload at birth.

adenosine 3',5'-cyclic monophosphate; angiotensin II; cardiac hypertrophy; glucagon; norepinephrine; protein synthesis; ribosome formation
Effects of immunization against VIP on neurotransmission in cat trachea
Authors: Hakoda H. , Xie Z. Q. , Aizawa H. , Inoue H. , Hirata M. , Ito Y.
Published: December 2017

To examine the possible role of vasoactive intestinal polypeptide (VIP) in excitatory and inhibitory neurotransmission and physiological function of coexistence VIP and acetylcholine at vagus nerve terminals in the cat trachea, we immunized five cats each against conjugate of VIP-bovine serum albumin (BSA) and BSA, respectively. A booster injection of VIP-BSA given 3 wk after the primary one elevated antibodies against VIP, as detected by enzyme-linked immunosorbent assay, but not in the control cats identically injected with BSA. After immunization, in vitro, summation phenomena observed in the amplitude of contractions and excitatory junction potentials of the trachea evoked by repetitive field stimulation were markedly enhanced, whereas the amplitude of phasic relaxation evoked by repetitive field stimulation in the presence of serotonin, atropine, and guanethidine was markedly reduced. In contrast, in vivo, the change in pulmonary resistance evoked by vagal stimulation was not affected in the immunized cats. We conclude that antibodies against neurotransmitter VIP are a potential modulatory factor in excitatory and inhibitory neurotransmission in airway smooth muscle at tissue or cellular levels. The lack of correlation between in vivo and in vitro conditions was discussed in relation to VIP autoantibodies in normal and asthmatic humans.
Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice
Authors: Glasser S. W. , Korfhagen T. R. , Wert S. E. , Bruno M. D. , McWilliams K. M. , Vorbroker D. K. , Whitsett J. A.
Published: December 2017

Transgenic mice bearing chimeric genes consisting of 5'-sequences derived from the human surfactant protein C (SP-C) gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were generated. Analysis of CAT activity was utilized to demonstrate tissue-specific and developmental expression of chimeric genes containing 3.7 kb of sequences from the human SP-C gene. Lung-specific expression of the 3.7 SP-C-CAT transgene was observed in eight distinct transgenic mouse lines. Expression of the 3.7 SP-C-CAT transgene was first detected in fetal lung on day 11 of gestation and increased dramatically with advancing gestational age, reaching adult levels of activity before birth. In situ hybridization demonstrated that expression of 3.7 SP-C-CAT mRNA was confined to the distal respiratory epithelium. Antisense CAT hybridization was detected in bronchiolar and type II epithelial cells in the adult lung of the 3.7 SP-C-CAT transgenic mice. In situ hybridization of four distinct 3.7 SP-C-CAT transgenic mouse lines demonstrated bronchiolar-alveolar expression of the chimeric CAT gene, although the relative intensity of expression at each site varied within the lines studied. Glucocorticoids increased murine SP-C mRNA in fetal lung organ culture. Likewise, expression of 3.7 SP-C-CAT transgene increased during fetal lung organ or explant culture and was further enhanced by glucocorticoid in vitro. The 5'-regions of human SP-C conferred developmental, lung epithelial, and glucocorticoid-enhanced expression of bacterial CAT in transgenic mice. The increased expression of SP-C accompanying prenatal lung development and exposure to glucocorticoid is mediated, at least in part, at the transcriptional level, being influenced by cis-active elements contained within the 5'-flanking region of the human SP-C gene.
Thyroid control over membrane processes in rat heart
Authors: Seppet Enn K. , Kadaya Lumme Y. , Hata Tomoji , Kallikorm Ants P. , Saks Valdur A. , Vetter Roland , Dhalla Naranjan S.
Published: December 2017

We have studied the effects of hypo- and hyperthyroidism on sarcolemmal (SL) and sarcoplasmic reticular (SR) ion transport processes and mitochondrial energy production in rat heart. The following conclusions were derived. 1) Compared with euthyroid state, hyperthyroidism led to increased SR Ca+-accumulation. In SL, the activities of Ca2+-stimulated adenosine triphosphatase (ATPase), ATP-dependent Ca+ pumping, and Na+-Ca2+ exchanger were not affected; but ouabain-sensitive Na+-K+-ATPase activity was enhanced. 2) Hypothyroidism resulted in depressed activities of Ca2+ pumps both in SL and SR. In SL, the Na+-K+-ATPase activity was decreased, but Na+-Ca2+ exchange was unaltered. 3) Thus slower relaxation of the hypothyroid myocardium may be attributed to depressed functioning of Ca2+ pumps in SR and SL, whereas faster relaxation of the hyperthyroid heart may be based on increased Ca2+-pumping activity of SR. 4) Hyperthyroidism and hypothyroidism, respectively, led to enhanced and decreased rates of mitochondrial phosphocreatine synthesis. The thyroid state appears to control the functional coupling between mitochondrial creatine kinase and ATP-ADP translocase: the energy of oxidative phosphorylation was transformed into phosphocreatine more effectively in mitochondria from hypothyroid hearts than in those from hyperthyroid hearts.

euthyroid; hyperthyroid; hypothyroid; mitochondrial creatine kinase; myocardium; oxidative phosphorylation; phosphocreatine synthesis; sarcolemma; sarcoplasmic reticulum
Cellular adaptation during chronic neonatal hypoxic pulmonary hypertension
Authors: Stenmark Kurt R. , Aldashev Almas A. , Orton Ernest C. , Durmowicz A. G. , Badesch D. B. , Parks W. C. , Mecham R. P. , Voelkel N. F. , Reeves J. T.
Published: December 2017

Newbor animals develop more severe hypoxic pulmonary hypertension than do adults, their vascular changes are greater, and both the hypertension and vascular changes occur more rapidly. We hypothesize that this differential developmentally controlled response may arise from either a difference in the type or quantity of endogenously secreted mediators in response to a given injury or a difference in the replicative and/or matrix-producing response of the vascular cells to physical or chemical stimuli. We investigated the effect of chronic hypoxia (14 days) on the proliferative and matrix-producing phenotype of the neonatal (14-day-old) pulmonary artery smooth muscle cell (SMC) and examined the heterogeneity and potential mechanisms responsible for this response. In situ hybridization studies demonstrated a remarkable change in the distribution of cells hybridizing with a tropoelastin cRNA probe after 14 days of hypoxia. Studies also demonstrated a population of SMC that did not hybridize with the elastin or collagen probes, indicating that the pulmonary artery contains SMC of multiple phenotypes and that the response to hypoxic and hemodynamic stress is not uniform for the various types. Bromodeoxyuridine labeling experiments indicated a large increase in DNA synthesis in hypertensive vessels, which, again, was not uniform either across or along the arterial wall. In vitro experiments with neonatal SMC suggested that hypoxia alone could not be responsible for the proliferative or matrix changes. These observations were supported by in vivo experiments in which coarctation of the left pulmonary artery, which markedly decreased pressure and flow to the left lung in hypoxic animals (14 days), resulted in significant decreases in collagen and elastin message levels in the left pulmonary artery distal to the coarctation compared with location-matched vessels from the right lung. Finally, we noted marked decreases in B-receptor density and adenyl cyclase activity in right atrial and pulmonary artery tissue from the chronically hypoxic animals. Decreases in the ability of the cell to produce adenosine 3',5'-cyclic monophosphate could significantly affect both the proliferative and matrix-producing potential of the SMC. We conclude that in vivo adaptation of the pulmonary artery SMC to chronic hypoxia includes changes in protein synthesis, cell proliferation, receptor expression, and enzyme activity. Further, there is a marked heterogeneity of these responses both across and along the arterial wall.

hypoxia; phenotype; signal transductions; smooth muscle cells
Modulation of bFGF in lung fibroblasts by TGF-beta and PDGF
Authors: Goldsmith K. T. , Gammon R. B. , Garver R. I.
Published: December 2017

Growth factors produced by alveolar macrophages are thought to promote the fibroblast proliferation within interstitial spaces of fibrotic lungs. This study investigated the possibility that the macrophage-produced growth factors might modulate the expression of basic fibroblast growth factor (bFGF) by lung fibroblasts. To evaluate this question, bFGF gene expression and protein production were evaluated in normal adult human lung fibroblast cell lines. Under normal culture conditions, the fibroblasts expressed the bFGF gene as two major transcripts (7.1, 3.7 kb). The addition of fetal calf serum (FCS) to serum-starved fibroblasts caused a 5- to 10-fold increase in bFGF expression. Steady-state bFGF expression was increased 108% by platelet-derived growth factor (PDGF) and 602% by transforming growth factor-beta (TGF-beta). Insulin-like growth factor-1 had no significant effect on bFGF expression. Nuclear runoff studies demonstrated that both PDGF and TGF-beta increased the relative rates of bFGF transcription in the fibroblasts. Western blot analysis of lysates from fibroblasts treated with either PDGF or TGF-beta had no detectable increase in bFGF protein above unstimulated controls. However, the simultaneous addition of PDGF and TGF-beta, or FCS, produced a marked increase in bFGF. These experiments show that two growth factors present in the alveolar airspace compartment of fibrotic lungs can promote the expression of bFGF within lung fibroblasts.