bluuebunny/crossref_metadata_2025 huggingface dataset:
Pre- and postjunctional effects of a thromboxane mimetic in canine bronchi
Authors: Janssen L. J. , Daniel E. E.
Published: December 2017

Thromboxane A2 is believed to participate in causation of hyperreactivity of airway smooth muscle (ASM). We therefore investigated the effects of a thromboxane mimetic (U-46619) on isolated ring segments of canine bronchial ASM (3rd to 5th order). U-46619 (10(-9)-10(-5) M) was found to elicit tonic contraction, membrane depolarization, and oscillations in membrane potential. These effects were sensitive to blockade of thromboxane receptors (using 10(-8) M L 670, 596); the mechanical response was insensitive to 3 x 10(-8) M atropine, 10(-7) M nitrendipine, or exposure to Ca(2+)-free media [(containing 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)]. U-46619 also potentiated electrical field stimulation-elicited contractions and excitatory junction potentials without altering the sensitivity to carbachol. This potentiation was also sensitive to L 670,596 but not to blockade of adrenoceptors (using phentolamine plus propranolol). We conclude that canine bronchial ASM possesses both prejunctional and postjunctional thromboxane receptors. The former potentiate cholinergic neurotransmission through a mechanism not involving adrenoceptors. The latter excite the ASM through a mechanism not involving muscarinic receptors and that utilizes intracellular Ca2+ (rather than extracellular Ca2+). Excitation-contraction coupling was found to be pharmacomechanical in nature (rather than electromechanical).
Ozone reduces murine alveolar and peritoneal macrophage phagocytosis: the role of prostanoids
Authors: Canning B. J. , Hmieleski R. R. , Spannhake E. W. , Jakab G. J.
Published: December 2017

Continuous ozone exposure (0.5 ppm, 1–14 days) reduced the phagocytic activity of murine alveolar and peritoneal macrophages. The response of peritoneal macrophages to ozone was virtually indistinguishable from the response of alveolar macrophages. When added exogenously, prostaglandin E2 (PGE2) inhibited alveolar and peritoneal macrophage phagocytosis. To test the hypothesis that prostanoids mediated the effects of ozone on macrophages, PGE levels of bronchoalveolar lavage fluid (BALF) and the phagocytic activity of macrophages from ozone-exposed mice pretreated with cyclooxygenase inhibitors were measured. PGE levels in BALF were increased following ozone exposure, with high levels of PGE associated with large decreases in phagocytic activity. Pretreatment with indomethacin and d-naproxen completely inhibited ozone-induced increases in PGE recovered by BAL and the suppression of peritoneal macrophage phagocytic activity. The inactive enantiomer of naproxen, l-naproxen, was without effect. Indomethacin partially inhibited ozone-induced suppression of alveolar macrophage phagocytic activity. These observations suggest that prostanoids play a key role in the response to ozone.
Fibrinogen degradation product fragment D increases endothelial monolayer permeability
Authors: Ge M. , Ryan T. J. , Lum H. , Malik A. B.
Published: December 2017

We assessed the effects of the two primary high-molecular-weight fibrinogen degradation products (FDP), fragments D and E, on the pulmonary vascular endothelial barrier function. Fragments D and E were purified to homogeneity by QAE Sephadex chromatography followed by gel filtration. Incubation of bovine pulmonary artery endothelial monolayers with 0.5–2.0 microM fragment D for 2 h caused a doubling of transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability). Fragment E only produced a 0.6-fold increase in 125I-albumin clearance rate at concentration of 4.0 microM. Both FDP remained active in incubating media with serum. The permeability-increasing effect of fragment D was reversible and was not due to cell detachment or lysis. The fragment-D effect was time dependent and was associated with redistribution of endothelial F-actin microfilaments. The effect was independent of the carboxy-terminal sequence on gamma-chain of fragment D. Fragments D and E binding to pulmonary artery endothelial cells was specific and reversible, but fragment D binding was three-fold greater than fragment E, which may account for the greater permeability increase mediated by fragment D. The results indicate that FDP, especially fragment D, increase endothelial permeability to albumin. The response involves specific binding of fragment D to endothelial cells and redistribution of intracellular actin.
Upregulation of rat lung Na-K-ATPase during hyperoxic injury
Authors: Nici L. , Dowin R. , Gilmore-Hebert M. , Jamieson J. D. , Ingbar D. H.
Published: December 2017

A major function of the alveolar epithelium is to keep the airspace free of fluid and preserve gas exchange. Since Na-K-ATPase is believed to be important in this process, we hypothesized that Na-K-ATPase in the rat lung would increase in response to acute lung injury with pulmonary edema. Na-K-ATPase localization, mRNA expression, and protein levels were determined in hyperoxic lung injury. Adult male rats were exposed to greater than 97% oxygen for 60 h followed by recovery in room air. At 60 h of hyperoxia, the wet-to-dry lung weights increased, consistent with edema. Within the alveolar capillary region, the sodium pump remained localized to the type II cell basolateral membrane by immunocytochemistry. By Northern blot analysis, the level of total lung mRNA expression of the alpha 1- and beta-subunits of Na-K-ATPase increased three- to fourfold during hyperoxia compared with unexposed rats. Total lung Na-K-ATPase membrane protein, visualized with a Western blot technique, appeared to increase by 24 h of hyperoxic insult when compared with levels in unexposed animals. The increase in sodium pump gene expression that occurs during hyperoxic insult, followed by an increase in sodium pump membrane protein, suggests that type II cells increase their Na-K-ATPase synthesis as an early response to pulmonary edema and/or hyperoxia.
Energy-linked functional alterations in experimental cardiomyopathies
Authors: Kapelko V. I. , Veksler V. I. , Popovich M. I. , Ventura-Clapier R.
Published: December 2017

Changes in high-energy phosphate content and cardiac contractile function of isolated rat hearts as well as changes in Ca2+ sensitivity and mitochondrial respiration of myocardial skinned fibers were assessed in hereditary cardiomyopathies and in cardiomyopathies induced by chronic treatment with adriamycin or norepinephrine, by autoimmunization, by diabetes, or by creatine deficiency. The sum of ATP and phosphocreatine contents as well as cardiac output at standard load conditions was substantially lower in almost all groups. The common features of cardiac pump failure were mild bradycardia, elevated left ventricular (LV) diastolic pressure, and stiffness that limited cardiac contractile adaptation to volume or resistance loads. The LV diastolic stiffness at maximal functional load was inversely correlated with high-energy phosphate content. Increased myofibrillar sensitivity to Ca2+ and defective function of mitochondrial creatine kinase were found in skinned myocardial fibers. These results suggested that both increased myofibrillar Ca2+ sensitivity and energy deficiency within myofibrils may contribute to increased myocardial stiffness. Increased stiffness limits LV filling but facilitates pressure development, which partly compensates for decreased contractility of cardiomyopathic hearts.

cardiac contractile function; high-energy phosphates; isolated heart; myocardial stiffness
Control of cardiac energy turnover by cytoplasmic phosphates: 31P-NMR study
Authors: Kupriyanov V. V. , Lakomkin V. L. , Korchazhkina O. V. , Steinschneider A. Ya. , Kapelko V. I. , Saks V. A.
Published: December 2017

Energy flux, estimated from the cardiac work index (pressure-rate product) and the rate of oxygen consumption, was varied in different ways; and the free concentrations of cytosolic phosphates were detected by the31P-nuclear magnetic resonance method. A reversible decrease in phosphocreatine (PCr) and concomitant increase in [ADP] at nearly constant Pi were induced by 2-deoxyglucose (2-DG) treatment and its subsequent washout and were followed by a reversible suppression of pressure-rate product and elevation of end-diastolic pressure. 2-DG treatment also resulted in an irreversible and severe reduction of the cytosolic adenine nucleotide pool (to approximately one-third of control value) that did not recover during 2-DG washout. Reduction of the energy turnover rate, either by suppression of the PCr shuttle with iodoacetamide or by inhibition of the respiratory chain with amytal, was associated with a drop of PCr level, an elevation of both [ADP] and [Pi], and a rise of end-diastolic pressure. In contrast, a decrease in energy flux by reduction of perfusate Ca2+ led to a PCr rise and a fall in [ADP] and [Pi]. Most of these experimental groups were exposed to two types of loads, isoproterenol stimulation and coronary flow (CF) elevation. Iodoacetamide-treated hearts showed a poor mechanical response to both types of loads compared with other groups. The metabolic response to isoproterenol was uniform in all groups and was associated with some decrease in PCr and increase of [ADP] and [Pi], implying limitations in the respiratory chain. In contrast, activation of energy flux by a CF rise in control and amytal-inhibited hearts did not affect the concentrations of PCr, ADP, and Pi. Thus cytosolic phosphates can serve as a feedback signal for energy production and utilization when the single control point is located in oxidative phosphorylation pathway or contractile system. If control of energy metabolism is equally distributed among producing and utilizing systems (CF variation), other regulatory mechanisms seem to be involved, e.g., cytosolic [Ca2+].

contraction-relaxation; creatine kinase; cytosolic phosphates; isolated rat heart; oxygen consumption
Targeting of macromolecular carriers and liposomes by antibodies to myosin heavy chain
Authors: Klibanov Alexander L. , Khaw Ban An , Nossiff Naseem , O'Donnell Sean M. , Huang Leaf , Slinkin Mikhail A. , Torchilin Vladimir P.
Published: December 2017

Macromolecular carriers and liposomes were covalently coupled to monoclonal antibodies against cardiac myosin heavy chain. Deferoxamine-modified polymers bound tightly with 67Ga and68 Ga radioisotopes. Ternary deferoxamine-polylysine antibody conjugates specifically targeted the radioisotopes to a myosin-coated microplate. Scatchard analysis revealed a high affinity of the conjugate for the target with a Kas of ≈108 M-1. Liposomes that contained immobilized antimyosin antibodies were targeted specifically to the myosin-coated plate. Additional coating of these liposomes with polyethylene glycol reduced specific binding to the target in vitro. However, because of the presence of polyethylene glycol on the surface of liposomes, these liposomes had a long half-life and slowly cleared from the bloodstream after intravenous injection. These immunoliposomes showed up to 16- to 18-fold specific localization to the necrotic areas of the myocardium in rabbits with experimental infarction.

antimyosin; chelating polymer; liposome; monoclonal antibody; myocardial infarction; radioimmunoimaging
Phenotypic changes of human aortic smooth muscle cells during development and in the adult vessel
Authors: Glukhova Marina A. , Frid Maria G. , Koteliansky Victor E.
Published: December 2017

To characterize phenotypic expression of human aortic smooth muscle cells (SMCs), we have studied the content of cytodifferentiation-related cytoskeletal proteins, and of fibronectin (FN) variants in the samples of media from the fetal, child, and adult aorta and in the subendothelial intima of the normal and atherosclerotic aorta. Mature SMCs from the adult aortic media contained high amounts of agr-SM-actin, SM-myosin heavy chains, meta-vinculin, and 150 kDa caldesmon. Cytokeratin 8 and extra domain-containing variants of FN (A-FN and B-FN) were not found in these cells. The SMCs from the aortic media of 10-wk-old fetus contained low amounts of the SM markers, expressed cytokeratin 8, A-FN, and B-FN. In 25-wk-old fetus, as well as in 2- and 6-mo-old child, aortic medial SMCs expressed an intermediate phenotype, and only in 18-mo-old child were the cells found to be similar to those from adult media. SMCs from the normal adult subendothelial intima contained reduced amounts of meta-vinculin and of 150 kDa caldesmon, and they expressed A-FN. In addition, the SMCs from atherosclerotic fibrous plaque contained a decreased proportion of agr-SM-actin and of SM-myosin heavy chains, whereas cytokeratin 8 was found. Therefore we conclude that the SMCs from intimal thickenings appear to express a less mature phenotype than that of the medial cells from adult aorta. Rather, these SMCs contain reduced amounts of the SM markers and express proteins typical of the fetal SMC phenotype, A-FN and cytokeratin 8.

cytodifferentiation; cytoskeletal proteins; fibronectin variant forms
Free radical metabolites in myocardium during ischemia and reperfusion
Authors: Ruuge Enno K. , Ledenev Alexander N. , Lakomkin Vladimir L. , Konstantinov Alexander A. , Ksenzenko Marina Yu.
Published: December 2017

Low-temperature electron paramagnetic resonance (EPR) spectroscopy and spin traps were used to measure paramagnetic species generation in rat hearts and isolated mitochondria. The hearts were freeze-clamped at 77 K during control perfusion by the Langendorff procedure, after 20–30 min of normothermic ischemia or 10–30 s of reperfusion with oxygenated perfusate. All EPR spectra measured at 4.5–50 K exhibited signals of both mitochondrial free radical centers and FeS proteins. The analysis of spectral parameters measured at 243 K showed that free radicals in heart tissue were semiquinones of coenzyme Q10 and flavins. The appearance of a typical “doublet” signal at g = 1.99 in low-temperature spectra indicated that a part of ubisemiquinones formed a complex with a high potential FeS protein of succinate dehydrogenase. Ischemia decreased the free radical species in myocardium ≈50%; the initiation of reflow of perfusate resulted in quick increase of the EPR signal. Mitochondria isolated from hearts during control perfusion and after 20–30 min of ischemia were able to produce superoxide radicals in both the NADH-coenzyme Q10 reductase and the bc1 segments of the respiratory chain. The rate of oxyradical generation was significantly higher in mitochondria isolated from ischemic heart.

electron paramagnetic resonance; oxygen paradox; oxyradicals; rat heart; semiquinones
Fractal geometry: a design principle for living organisms
Authors: Weibel E. R.
Published: December 2017

Fractal geometry allows structures to be quantitatively characterized in geometric terms even if their form is not even or regular, because fractal geometry deals with the geometry of hierarchies and random processes. The hypothesis is explored that fractal geometry serves as a design principle in biological organisms. The internal membrane surface of cells, or the inner lung surface, are difficult to describe in terms of classical geometry, but they are found to show properties describable by fractal geometry, at least sectionwise and within certain bounds set by deterministic design properties. Concepts of fractal geometry are most useful in characterizing the structure of branching trees, such as those found in pulmonary airways and in blood vessels. This explains how the large internal gas exchange surface of the lung can be homogeneously and efficiently ventilated and perfused at low energetic cost. It is concluded that to consider fractal geometry as a biological design principle is heuristically most productive and provides insights into possibilities of efficient genetic programming of biological form.
Effect of protein kinase C activating agents on respiratory glycoconjugate release from feline airways
Authors: Rieves R. D. , Lundgren J. D. , Logun C. , Wu T. , Shelhamer J. H.
Published: December 2017

Abnormal regulation of airway glycoprotein secretion may underlie many respiratory diseases. Experimental activation of the protein kinase C (PKC) family of cytosolic enzymes has been shown to induce a secretory response in many tissues. To estimate the effect of PKC activation on airway secretion, alteration in the amount of radiolabeled respiratory glycoconjugate (RGC) released into culture media was determined following feline airway explant exposure to PKC activating agents. Exposure to two known activators of PKC, phorbol 12-myristate 13-acetate (PMA) and mezerein (MEZ), resulted in profound increases in respiratory glycoconjugate release over a seven day experimental period. The response evolved over several hours and was dose dependent. Maximal RGC release, 90% above control, occurred 2 days after exposure to either PMA or MEZ. Pharmacological inhibition of the PKC effect using two PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and sphingosine, resulted in dose-dependent antagonism of the maximal PMA (10(-7) M)-stimulated RGC release, suggesting altered PKC activity was responsible for augmenting RGC release. Since altered arachidonic acid metabolism has been implicated in mediating some PKC effects, eicosanoids were assayed in airway explant supernatants following PMA exposure. Enhanced release of both cyclooxygenase and lipoxygenase pathway products was detected by radioimmunoassay. Cotreatment of explants with PMA and an inhibitor of oxidative arachidonic acid metabolism, nordihydroguaiaretic acid, blocked RGC release. These data demonstrate prolonged augmentation of respiratory glycoconjugate release from airway explants following exposure to PKC-activating agents.
Spatial and temporal differences in fibroblast behavior in fetal rat lung
Authors: Caniggia I. , Tseu I. , Han R. N. , Smith B. T. , Tanswell K. , Post M.
Published: December 2017

Fibroblast-epithelial interactions were investigated in cells from late-gestation fetal rat lung. Fibroblasts from the pseudoglandular stage of lung development stimulated epithelial cell proliferation, whereas fibroblasts from the saccular stage promoted epithelial cell differentiation. The developmental switch from proliferation to differentiation seemed to be controlled by both cell types. Fibroblast-derived epithelial cell growth-promoting activity, evident in cells from the pseudoglandular period, decreased during development and almost disappeared in cells from the saccular stage. Interestingly, the response of epithelial cells to this growth-promoting activity declined with advancing gestational age as epithelial cells became more responsive to fibroblast-derived differentiation factor(s). Production of differentiation factor(s) by fibroblasts increased during the canalicular stage of lung development. Platelet-derived growth factor (PDGF) and low concentrations of transforming growth factor-beta (TGF-beta) stimulated epithelial cell proliferation. PDGF did not affect differentiation, whereas TGF-beta was inhibitory. Dependent on their proximity to the epithelium, two subpopulations of fibroblasts that differed in their ability to promote epithelial cell proliferation or differentiation were isolated. Fibroblasts in close proximity to the epithelium mainly produced differentiation factors, whereas more distant fibroblasts primarily stimulated proliferation.
Blockade of N-methyl-D-aspartate receptors has no effect on certain inspiratory reflexes
Authors: Karius D. R. , Ling L. M. , Speck D. F.
Published: December 2017

Previous studies have indicated that excitatory amino acids are involved in many afferent pathways. This study investigated the effects of intravenous MK-801 [an N-methyl-D-aspartate (NMDA) receptor-associated channel blocker] on several well-known respiratory reflexes elicited by afferent stimulation of the superior laryngeal (SLN), the intercostal (ICN), and the phrenic (PN) nerves. Control responses to stimulation were obtained from recordings of phrenic nerve activity in decerebrate, paralyzed cats. Inspiratory termination elicited by the delivery of stimulus trains to either the SLN or the ICN persisted after MK-801. The onset latency or duration of the short-latency excitations produced by SLN or ICN stimulation were unchanged. The transient inhibitions produced by SLN, ICN, PN, or medullary stimulation showed no significant changes in threshold, onset latency, or duration. Withholding lung inflation produced apneusis after administration of MK-801, indicating a central effect of the drug. Higher doses of MK-801 did not alter the parameters of these reflexes. These data indicate that NMDA-dependent neurotransmission is not required for the production of these reflexes.
Ion transport by cultures of human tracheobronchial submucosal glands
Authors: Yamaya M. , Finkbeiner W. E. , Widdicombe J. H.
Published: December 2017

Acini of human tracheobronchial submucosal glands were isolated by enzymatic disaggregation, and, when plated on flasks coated with human placental collagen (HPC) in media containing Ultroser G serum substitute (USG) and a variety of growth factors (GF), they became confluent after 14–20 days. The cells were then isolated by trypsinization and replated in media containing USG and GF at 10(6) cells/cm2 on porous-bottomed inserts coated with HPC. Confluent monolayers formed on day 1 after replating and were studied on day 10. Transepithelial resistance and short-circuit current (Isc) were 578 +/- 89 omega.cm2 and 12.9 +/- 1.9 microA/cm2 (means +/- SE, n = 23 cell sheets). The potency sequence for stimulation of Isc by mediators was methacholine greater than bradykinin greater than isoproterenol approximately or equal to phenylephrine. Amiloride decreased baseline Isc by 42 +/- 9% (n = 6 cell sheets) but had little effect on the Isc response to mediators. Diphenylamine-2-carboxylic acid, however, had no effect on baseline Isc but markedly inhibited the Isc response to all mediators. These results show that submucosal gland cells from human trachea can be grown in culture to produce epithelial sheets of high resistance, which secrete Cl in response to bradykinin and alpha- and beta-adrenergic and cholinergic agents.
Autonomic innervation to feline tracheal submucosal glands for mucus glycoprotein secretion
Authors: Shimura S. , Sasaki T. , Ishihara H. , Sato M. , Sasaki H. , Takishima T.
Published: December 2017

We examined the effect of electrical field stimulation (FS) on radiolabeled glycoconjugate released from feline tracheal isolated glands. Trichloroacetic acid precipitable [3H]-glycoconjugates released into the culture medium were counted, divided by the dry weight of sample, and data were expressed as percent of the release from the same animal that was unstimulated but otherwise received identical treatment. FS produced an increase in the glycoconjugate release that was dependent on the duration of stimulation, reaching a maximum response of 215% of nonstimulated sample for 30 min stimulation (10 Hz, 10 V). FS-evoked secretion was abolished by 10(-7) M tetrodotoxin but not altered by 10(-5) M hexamethonium. Atropine (10(-6) M) alone abolished the response to FS for 3 min or less. By contrast, a mixture of 10(-6) M atropine, 10(-5) M propranolol, and 10(-5) M phentolamine blocked only part of the response to FS for 15 min or more. The mixture of three antagonists reduced the response to FS for 30 min to 159% of control, which was significantly higher than control. Furthermore, 10(-6) M atropine, 10(-5) M propranolol, or 10(-5) M phentolamine significantly reduced the response to FS for 30 min to 162, 193, and 195%, respectively, from 215% of sample in the absence of blockers. Serotonin (10(-5) M) augmented FS-evoked response, which was abolished by 10(-6) M atropine or by 10(-5) M methysergide.(ABSTRACT TRUNCATED AT 250 WORDS)
Neutrophil elastase stimulates tracheal submucosal gland secretion that is inhibited by ICI 200,355
Authors: Schuster A. , Ueki I. , Nadel J. A.
Published: December 2017

To investigate whether human neutrophil elastase (HNE) stimulates airway submucosal gland secretion, we studied the effect of purified HNE on secretion of 35S-labeled macromolecules from isolated tracheal tissues from ferrets, dogs, and humans. HNE stimulated secretion in a concentration-dependent fashion, the secretory response being most pronounced in ferret tissues with a maximal response of 1,498 +/- 384% above baseline at 10(-5) M. In dog tissues, maximal secretory responses (509 +/- 169%) to HNE were much greater than to bethanechol (80 +/- 26%). Human tissues obtained several hours postmortem still responded to HNE (179 +/- 48% at 10(-5) M) significantly more than to the combination of isoproterenol, phenylephrine, and bethanechol (23 +/- 10%). Morphometric analysis of canine tracheal tissues showed degranulation of submucosal gland cells after HNE. A specific inhibitor of HNE (ICI 200,355) potently inhibited secretory responses in a concentration-dependent fashion. We suggest that HNE in airways of patients may cause hypersecretion and that treatment with ICI 200,355 may provide a strategy for therapeutic intervention.
Paraquat-induced lung injury: prevention by N-tert-butyl-alpha-phenylnitrone, a free-radical spin-trapping agent
Authors: Sata T. , Kubota E. , Misra H. P. , Mojarad M. , Pakbaz H. , Said S. I.
Published: December 2017

The herbicide paraquat causes lung injury that is believed to be oxygen-radical mediated. To further characterize this injury and explore new methods of preventing it, we used the spin-trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) to identify the paraquat radical in lung tissue and to reduce the injury resulting from the subsequent generation of reactive oxygen species. The formation of a paraquat free radical by guinea pig lung was detected under anaerobic conditions by electron paramagnetic resonance spectrometry. Infused (25, 50, or 100 mg/kg) into guinea pig lungs (perfused at constant flow with Krebs solution containing 4% bovine serum albumin and ventilated with 95% O2-5% CO2), paraquat produced dose-dependent increases in peak airway pressure (Paw), mean pulmonary arterial perfusion pressure (Ppa), and wet-to-dry (W/D) lung weight ratio. At 100 mg/kg, paraquat increased Paw by 589.6 +/- 59.8% (mean +/- SE, n = 8) and W/D ratio from 5.33 +/- 0.07 to 6.29 +/- 0.11 (P less than 0.001). Pulmonary vascular leak index increased from 0.40 +/- 0.09 to 1.96 +/- 0.45 (P less than 0.02), without changes in pulmonary microvascular pressure. Perfusate concentrations of thromboxane B2 and 6-ketoprostaglandin F1 alpha increased, but indomethacin did not reduce the injury. PBN (2.3 mM) markedly attenuated all evidence of lung injury, which was also reduced by catalase, mannitol, ethanol, and vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium-dependent regulation of Cl secretion in tracheal epithelium
Authors: Hartmann T. , Kondo M. , Mochizuki H. , Verkman A. S. , Widdicombe J. H.
Published: December 2017

In primary cultures of dog tracheal epithelium, isoproterenol produced a transient increase in short-circuit current (Isc) (duration 30 s; maximal increase, 32 +/- 5 microA/cm2). This was followed by a more slowly developing sustained increase (9 +/- 3 microA/cm2), which mimicked the response to N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP). The transient and sustained responses had dissociation constants for isoproterenol of 2 x 10(-8) and 2 x 10(-9) M, respectively. Bradykinin (in the presence of indomethacin), substance P, histamine, and thrombin produced only transient increases in Isc. The time courses of these transients closely paralleled changes in concentration of intracellular Ca ([Ca2+]i) as measured with fura 2. For different mediators, there was a significant correlation between the maximal transient increase in Isc and the maximal increase in [Ca2+]i. The transients in Isc were not associated with elevation of adenosine 3',5'-cyclic monophosphate (cAMP) and were unaffected by pretreatment with DBcAMP, which abolishes the steady-state increase in response to isoproterenol. Both the transient increases in Isc and [Ca2+]i were inhibited by pretreatment with the Ca chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate abolished the transient increases in [Ca2+]i and Isc in response to isoproterenol but not to bradykinin. These results provide evidence that 1) isoproterenol and bradykinin elevate [Ca2+]i by different mechanisms, and 2) Ca elevation is associated with a transient increase in Isc, whereas increased cAMP is associated with a smaller sustained increase.
Antioxidant activity of bronchoalveolar lavage fluid in the adult respiratory distress syndrome
Authors: Lykens M. G. , Davis W. B. , Pacht E. R.
Published: December 2017

Bronchoalveolar lavage (BAL) fluid from normal subjects is a potent inhibitor of lipid peroxidation. This antioxidant activity (AOA) of BAL fluid is primarily due to the serum proteins transferrin and ceruloplasmin. In the adult respiratory distress syndrome (ARDS), there is an influx of protein-rich edema fluid into the alveolar space that may increase antioxidant activity and provide protection against further oxidant-mediated lung injury. To test this hypothesis, the AOA of BAL fluid was measured in patients with ARDS (n = 11) and normal subjects (n = 12). When compared with normal subjects, BAL fluid from ARDS patients had a significantly higher concentration of total protein (2,536.8 +/- 408.2 micrograms/ml vs. 77.3 +/- 7.0 micrograms/ml, P less than 0.005). When compared at several volumes, BAL fluid from ARDS patients was a more potent inhibitor of lipid peroxidation than BAL fluid from normals. In addition, when AOA was determined on equal milligram amounts of BAL fluid protein from ARDS patients and normal subjects, ARDS BAL fluid protein had a significantly higher AOA. Consistent with its higher AOA, ARDS BAL fluid contained increased concentrations of both transferrin (77.8 +/- 15.3 micrograms/ml vs. 2.78 +/- 0.3 micrograms/ml, P less than 0.05) and ceruloplasmin (36.5 +/- 5.6 micrograms/ml vs. 0.26 +/- 0.02 micrograms/ml, P less than 0.005) compared with normal subjects. The importance of both ceruloplasmin and transferrin in the enhanced AOA of ARDS BAL fluid was further demonstrated by studies that showed a significant decrement in AOA when the antioxidant functions of these two proteins were selectively blocked.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in permeability of dog tracheal epithelium in response to hydrostatic pressure
Authors: Kondo M. , Finkbeiner W. E. , Widdicombe J. H.
Published: December 2017

We tested the hypothesis that, in asthma, the airway epithelial damage and leakage of blood proteins into the lumen are the result of edema and raised submucosal hydrostatic pressure. Sheets of dog tracheal epithelium were mounted in Ussing chambers, and the effects of transepithelial hydrostatic pressure differences (delta P) on conductance (G), [3H]mannitol flux (Jman), and fluorescein isothiocyanate-albumin flux (Jalb) were determined. delta P values of 20 cmH2O directed from the mucosal to submucosal side of the tissue (m----s) had no significant effects on G, Jman, Jalb, or tissue ultrastructure. delta Ps----;m caused increases in conductance (G) with a maximal effect at approximately 20 cmH2O. delta Ps----m of 20 cmH2O significantly (P less than 0.05) increased G (4.3 +/- 0.6 to 10.6 +/- 1.6 mS/cm2), Jman s—m (18 +/- 5 to 411 +/- 54 nmol.cm-2.h-1), J(alb)s----m (0.3 +/- 0.1 to 6.0 +/- 2.0 micrograms.cm-2.h-1), and J(alb)m—s (0.7 +/- 0.3 to 1.8 +/- 0.4 micrograms.cm-2.h-1). Jman m----s was not affected. On removal of delta P, G and Jman s----m returned to preexposure values, though J(alb)s----m remained slightly elevated at 1.1 +/- 0.3 micrograms.cm-2.h-1. Morphologically, delta Ps----m caused dilation of lateral intercellular spaces, disruption of tight junctions, and submucosal edema. The large increases in s----m fluxes of albumin and mannitol are consistent with bulk flow of fluid toward the lumen via the areas of epithelial damage.
Ontogeny of shortening velocity in porcine trachealis
Authors: Ikeda K. , Mitchell R. W. , Guest K. A. , Seow C. Y. , Kirchhoff C. F. , Murphy T. M. , Leff A. R.
Published: December 2017

We examined the effect of maturation on force-velocity (F-V) parameters in porcine tracheal smooth muscle (TSM) to determine the relationship between maximal isometric contractile force (Po) and maximal velocity of shortening (Vmax). Strips of TSM excised from 1-day-old neonatal swine (neo; n = 8), 2-wk-old swine (2ws; n = 7), and 10-wk-old swine (10ws; n = 7) were tethered to an electromagnetic lever system for F-V analysis of contractility. TSM strips were activated by electrical field stimulation at optimal resting tension, voltage, and length (Lo) so that maximal reproducible contractile force (Po) was elicited. Velocities were measured at the early phase of isometric contraction (3.1 +/- 0.1 s for neo, 2.9 +/- 0.1 s for 2ws, and 3.1 +/- 0.1 s for 10ws; P = NS). Shortening velocity increased progressively with maturation; Vmax was 0.164 +/- 0.011 Lo/s for neo, 0.194 +/- 0.013 Lo/s for 2ws (P less than 0.05 vs. neo), and 0.260 +/- 0.024 Lo/s for 10ws (P less than 0.01 vs. neo; P less than 0.05 vs. 2ws). Maximal isometric force generation increased substantially during the first 2 wk of postnatal life and thereafter returned to neonatal levels; Po was 71.5 +/- 2.1 mN/mm2 for neo, 95.4 +/- 7.0 mN/mm2 for 2ws, and 74.7 +/- 6.2 mN/mm2 for 10ws (P less than 0.05, 2ws vs. neo and 10ws). In separate studies, we also determined whether differences in Vmax occurred during the normal cycling phase of the cross bridge (3 s) or during the slowly cycling phase of the latch bridge (8 s) in tissue from 12 additional animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Mixtures of synthetic peptides and dipalmitoylphosphatidylcholine as lung surfactants
Authors: McLean L. R. , Krstenansky J. L. , Jackson R. L. , Hagaman K. A. , Olsen K. A. , Lewis J. E.
Published: December 2017

Synthetic peptides that differ in their lipid-peptide interactions were combined with dipalmitoylphosphatidylcholine (DPPC) and tested in an adult rat lavaged lung model in vitro for efficacy as totally synthetic lung surfactants. The putative amphipathic alpha-helical region of the major lung surfactant apoprotein (SP-A81-102), an analogue with increased amphipathic alpha-helical potential ([Lys88,97,Glu99,Trp102]-SP-A81-102]), and the hydrophobic peptide gramicidin D were all ineffective. Three water-soluble lipid-binding peptides that contain amphipathic alpha-helical regions were also tested. Of these, only a 24-residue amphipathic alpha-helical peptide (18As) based on the lipid-binding sequences of the plasma apolipoproteins was effective. Melittin and glucagon were ineffective. Mixtures of 18As and DPPC also restored gas exchange in an in vivo lavaged guinea pig lung model to 90-95% of its prelavage value and maintained it for at least 3 h. Mixtures of DPPC and 18As are also surface active (gamma min less than 4 mN/m in the pulsating bubble). These data demonstrate the efficacy of a combination of a single lipid and a small, water-soluble, nonhemolytic, synthetic peptide containing an amphipathic alpha-helical structure and a sequence unrelated to any of the reported lung surfactant apoprotein sequences.
Glutathione protection in alveolar type II cells from fetal and neonatal rabbits
Authors: Brown L. A. , Bai C. , Jones D. P.
Published: December 2017

Previous studies have demonstrated a correlation between intracellular glutathione (GSH) pools and sensitivity to oxidative injury. In the present study, we demonstrated that de novo GSH synthesis or GSH uptake could increase intracellular GSH by 7- and 19-fold, respectively, in type II cells from neonatal rabbits. This suggested that the rate of GSH uptake was against a concentration gradient and greater than the synthetic rate. This increased intracellular GSH was associated with protection from oxidant injury by paraquat or 80% O2. A relationship between GSH uptake and protection was further supported by blockage of both processes by gamma-L-glutamyl-L-glutamate, a GSH analogue. With a greater oxidative burden, both de novo synthesis and GSH uptake were required to maintain protection. Although the transport rate was only 6% of that for neonatal cells, cells from fetal animals transported GSH and were protected from oxidative injury. From these results we conclude there was a causal relationship between GSH transport and protection from oxidative injury in type II cells from neonatal and fetal animals.
Modulation of alveolar type II cell differentiated function in vitro
Authors: Shannon J. M. , Jennings S. D. , Nielsen L. D.
Published: December 2017

We have investigated whether the loss of differentiated function observed in adult rat alveolar type II cells cultured on a substratum that promotes cell spreading and flattening represents a reversible phenotypic change. Cells were cultured for 4 and 8 days in association with fetal rat lung fibroblast feeder layers on either attached collagen gels, which promote the loss of differentiated function, or on floating collagen gels, which support differentiation. A fifth group of cultures were maintained as attached gels for 4 days, then detached and cultured as floating gels for the remaining 4 days. Expression of mRNAs for surfactant proteins A, B, and C, patterns of phospholipid biosynthesis, rates and patterns of protein synthesis, and cell morphology were evaluated as markers of differentiation. Without exception, detaching the gels after 4 days in culture resulted in significant recovery of differentiated characteristics, demonstrating that type II cells modulate differentiated function in response to the culture environment. The results are discussed in relation to the importance of normal cell architecture to normal cell function and to the possible in vitro progression of type II cells to type I cells.
Expression and transport of rabbit surfactant protein A in COS-1 cells
Authors: Alcorn J. L. , Chen Q. , Boggaram V. , Mendelson C. R.
Published: December 2017

SV40-transformed green monkey kidney (COS-1) cells were transfected with expression plasmids that contained either the structural gene or cDNA for surfactant protein A (SP-A), a major protein of rabbit lung surfactant. The transfected COS-1 cells synthesized several isoforms of SP-A that were found to be less acidic than those produced in rabbit lung tissue. SP-A species with apparent molecular weight (M(r)) approximately equal to 29,000–33,000 were detected in the transfected cells, whereas glycosylated forms with apparent M(r) approximately equal to 33,000–38,000 were detectable only in the culture medium. Analysis of transfected cells by indirect immunofluorescence revealed that SP-A was localized in punctate bodies throughout the cytoplasm. Expressed SP-A was not detectable on the cell surface nor was there evidence that secreted SP-A was endocytosed by COS-1 cells. After subcellular fractionation of the transfected COS-1 cells, SP-A was found to be localized predominantly in the 5,000- and 18,000-g pellet fractions; little or no immunoreactive SP-A was detectable in cytosolic fractions. Treatment of transfected cells with the glycosylation inhibitor tunicamycin prevented secretion of SP-A into the medium, suggesting a role of glycosylation in secretion of SP-A. On the other hand, treatment of transfected cells with inhibitors of proline hydroxylation, which may cause destabilization of the collagen-like domain of SP-A, reduced but did not prevent secretion of SP-A into the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)
A23187 stimulates translocation of 5-lipoxygenase from cytosol to membrane in human alveolar macrophages
Authors: Pueringer R. J. , Bahns C. C. , Monick M. M. , Hunninghake G. W.
Published: December 2017

Human alveolar macrophages stimulated with the calcium ionophore A23187 selectively release large amounts of leukotriene B4 (LTB4) and (+/-)-5-hydroxy-(6E, 8Z-11Z, 14Z)-eicosatetraenoic acid. To determine whether LTB4 release by human alveolar macrophages following A23187 stimulation required the de novo production of 5-lipoxygenase, alveolar macrophages were stimulated under conditions that would preclude a role for new enzyme production. We found that A23187-stimulated alveolar macrophages release LTB4 within 10 min following stimulation, that LTB4 release is not inhibited by protein synthesis inhibitors, and that release of LTB4 does not correlate with the de novo synthesis of the first committed enzyme, 5-lipoxygenase. In contrast, LTB4 release correlated with the translocation of 5-lipoxygenase from the cytosol to the membrane fraction of the cells following A23187 stimulation and was inhibited by MK-886. These findings show that A23187 stimulation of alveolar macrophages results in translocation of a preexistent 5-lipoxygenase from the cytosol to the membrane fraction of the cell and that this translocation of 5-lipoxygenase is associated with release of LTB4 from the cells.
Nitric oxide mediates the neural nonadrenergic, noncholinergic relaxation of pig tracheal smooth muscle
Authors: Kannan M. S. , Johnson D. E.
Published: December 2017

In pig tracheal smooth muscle, the isometric tension responses to electrical field stimulation (EFS) were studied after raising the tone with carbamylcholine chloride (carbachol). EFS induced frequency-dependent relaxations that were nonadrenergic, noncholinergic (NANC) in nature. Addition of NG-nitro-L-arginine (L-NOArg), an inhibitor of nitric oxide (NO) synthesis from L-arginine, resulted in concentration-dependent inhibition of the relaxation response to EFS. Pretreatment of the tissues with L-arginine (1 mM) prevented the inhibitory effect of L-NOArg on the EFS-induced relaxations at the frequencies studied. However, in the presence of D-arginine, EFS-induced relaxations were inhibited by L-NOArg. L-Arginine, D-arginine, and L-NOArg had no significant effects on basal tone of the muscle. In the presence of L-NOArg, vasoactive intestinal polypeptide (3 x 10(-7) M), the nicotinic agonist dimethylphenyl piperazinium bromide (100 microM), and isoproterenol (1 microM) relaxed carbachol-induced tone. The concentration-dependent selective inhibition of neural relaxation by L-NOArg and its reversal by L-arginine in a stereospecific manner are consistent with NO-mediated NANC relaxation of pig tracheal smooth muscle.
Human tryptase as a potent, cell-specific mitogen: role of signaling pathways in synergistic responses
Authors: Hartmann T. , Ruoss S. J. , Raymond W. W. , Seuwen K. , Caughey G. H.
Published: December 2017

Mast cells are hypothesized to participate in processes leading to tissue fibrosis in human lung and skin. To explore the possible involvement of mast cell mediators in fibrogenesis, the mitogenic activity of mast cell tryptase from human lung was examined in vitro. The results indicate that human tryptase is a potent inducer of DNA synthesis in fibroblasts from multiple sources, including human lung. As demonstrated by mitogenic responses in fibroblasts, but not in vascular smooth muscle cells, tryptase is a mitogen with target cell specificity. Additionally, specificity is demonstrated by the differences in mitogenic activity of tryptase in comparison with thrombin, a structurally related mitogenic proteinase. Examination of the mitogenic effects of tryptase in the presence of other mitogens reveals synergy with mitogens that act through receptors coupled to intrinsic tyrosine kinases (insulin, epidermal growth factor, and basic fibroblast growth factor) or to G proteins (thrombin and serotonin). In the latter case, studies in Chinese hamster lung fibroblasts using specific receptor agonists and antagonists or receptor-transfected cell lines reveal a requirement for the activation of a G protein (Gi) negatively coupled to adenylate cyclase to act synergistically with tryptase. These data establish that human tryptase is a potent and specific mitogen in vitro and suggest that mitogenic signals generated by tryptase can interact synergistically with signals generated by both tyrosine kinase-coupled and G protein-coupled growth factor receptors.
Comparative responses of brain stem and hippocampal neurons to O2 deprivation: in vitro intracellular studies
Authors: Donnelly D. F. , Jiang C. , Haddad G. G.
Published: December 2017

Most mammalian neurons are known to be sensitive to oxygen availability, but the nature of the sensitivity is not well understood. Previous results have suggested that brain stem neurons may respond differently than cortical neurons during oxygen deprivation. We pursued this hypothesis by examining the time course of change in membrane potential (Vm) and input resistance (Rn) during periods of reduced oxygen availability in a tissue slice preparation. Since extracellular potassium is an important factor determining resting membrane potential, extracellular K+ activity, (K+o), was also measured. Adult rat neurons from three regions were recorded: hippocampal CA1 region, hypoglossal nucleus (XII), and dorsal vagal motor nucleus (DMNX). At the end of a 5-min hypoxic exposure, all neurons depolarized and this depolarization was greatest in XII (28.8 +/- 3.2 mV) compared with DMNX (17.8 +/- 3.7 mV) and CA1 (6.7 +/- 4.4 mV). K+o increased in all regions and was larger in DMNX (7.1 +/- 2.6 mM) and XII (5.3 +/- 2.1 mM) compared with CA1 (2.2 +/- 1.4 mM). During more severe oxygen deprivation (anoxia), neurons also depolarized at different rates with XII greater than DMNX greater than CA1. K+o increased markedly (28–36 mM) by 5 min into anoxia, and no statistical difference was observed between regions. From these results we conclude that 1) all cells tested were depolarized after 5 min of hypoxia; however, regional variability exists in the sensitivity to hypoxia; brain stem neurons depolarize faster than cortical neurons; 2) during anoxia, all brain stem and cortical neurons show a major depolarization, and 3) these differences in membrane potential cannot be solely attributed to changes in extracellular K+.
p-Aminohippurate transport in airways: competitive inhibition
Authors: Cloutier M. M. , Guernsey L.
Published: December 2017

p-Aminohippurate (PAH) transport in canine tracheal epithelium occurs by a HCO3- -PAH exchange process that is located on the luminal membrane and is inhibited by stilbene derivatives. The effects of increasing concentrations of other organic anions, including probenecid (10–250 microM), dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP; 10–1,000 microM), phenol red (10–250 microM), and urate (25–500 microM), and the organic cation tetraethylammonium bromide (TEA; 250 microM) on PAH transport were examined in canine tracheal epithelium mounted in Ussing chambers. Neither phenol red, urate, nor TEA had any effect on electrophysiological properties or unidirectional or net PAH fluxes. In contrast, beginning at 10 microM, both probenecid and cAMP produced significant decreases in unidirectional and net PAH absorption without change in unidirectional PAH secretion. The initial change in net PAH absorption occurred in the absence of any change in electrophysiological properties. Higher concentrations of both probenecid and cAMP produced further decreases in net PAH absorption and significant changes in electrophysiological properties. Probenecid and cAMP increased the apparent Michaelis constant for PAH absorption without affecting maximum transport rate. The inhibitory constant for probenecid was 1.01 +/- 0.06 x 10(-4) M (mean +/- SE) and for cAMP was 5.18 +/- 0.20 x 10(-4) M. We conclude that PAH transport in canine tracheal epithelium demonstrates competitive inhibition by other organic anions and substrate specificity. We also conclude that the affinity of the exchange transport system is higher for probenecid than for PAH and cAMP.