bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate utilization in the perinatal lung
Authors: Patterson C. E. , Rhoades R. A.
Published: December 2017

Lung cellular metabolism is fundamental to both respiratory and nonrespiratory function. The lung has very little energy reserve and is highly dependent on circulating substrates. The pattern of substrate utilization is determined primarily substrate availability, competition between certain substrates, and the ability of lung cells for uptake and metabolism. The lung uses a number of substrates (glucose, lactate, fatty acids, choline, ketone bodies, and amino acids) as basic building blocks for synthesis, as energy-providing fuels, to provide NADPH for lipid biosynthesis, and for glutathione production. Since the availability of substrates, the cellular profile, the hormonal environment, and the physiological state of the lung are drastically altered during perinatal lung development, this review focuses on current knowledge of lung substrate utilization during this critical period. Because development of the surfactant system has been specifically linked to infant respiratory distress syndrome, the majority of investigations relate to substrate utilization for phospholipid synthesis. It is hoped that this review will outline basic principles for interpretation of information on perinatal substrate utilization, collate available data, and provide a stimulus for future research.
Spermidine uptake by type II pneumocytes: interactions of amine uptake pathways
Authors: Rannels D. E. , Kameji R. , Pegg A. E. , Rannels S. R.
Published: December 2017

Uptake of exogenous spermidine by type II pulmonary epithelial cells in primary culture is inhibited by several amines. The present studies further detail the interaction of those alternative substrates with the pathway for spermidine (SPD) transport. Transport activity was measured on the first day of primary culture in type II cells isolated by elastase digestion, followed by density gradient centrifugation and differential adherence in vitro. Spermidine uptake was inhibited in a concentration-dependent manner by the polyamines putrescine (PUTR) and spermine (SPM), by the drug methylglyoxal bis-(guanylhydrazone) (MGBG), and by the herbicide paraquat (PQ). The order of effectiveness of these competitors was SPM greater than PUTR approximately MGBG much greater than PQ. The kinetics of inhibition by SPM were mixed, with an increase in the apparent Michaelis constant and a reduction in the maximal velocity of the SPD uptake pathway. Cellular uptake of SPD and PUTR was inhibited by replacement of extracellular sodium with choline or lithium (PUTR greater than SPD), but SPM uptake was unaffected. PQ appeared to interact with the polyamine transport pathway with low affinity. Compared with SPD and SPM, PQ was most effective as an inhibitor of PUTR uptake, and like PUTR, uptake of the herbicide was sharply inhibited as extracellular sodium was reduced. These observations suggest the presence of diverse pathways for uptake of exogenous polyamines by type II pulmonary epithelial cells and indicate that PQ probably enters the cells by the sodium-dependent transport system favored by PUTR.
Local mediator release after antigen challenge of a bronchial segment in allergic dogs
Authors: Reiss T. F. , Rubinstein I. , Emery D. L. , Gold W. M. , Boushey H. A.
Published: December 2017

To investigate the local intraluminal bronchial response to an antigenic stimulus, we developed a bronchoscopic double-balloon system to challenge and lavage a segment of the left main-stem bronchus. We studied whether fluid from above or below the occlusion balloons leaked into the bronchial segment. Lavage was performed before and after placement of red and blue pigments proximal and distal to the inflated balloons, respectively, and the recovered lavage fluid was analyzed visually and spectrophotometrically in three experiments. There was no evidence for pigment leakage into the segment. In six anesthetized ragweed-allergic dogs, local ragweed antigen challenges were performed. After balloon inflation in the left main-stem bronchus, we performed two baseline lavages of the interballoon segment, introduced a ragweed antigen solution, and performed two postchallenge lavages. The recovered fluid was analyzed for the concentrations of prostaglandin D2 (PGD2; radioimmunoassay) and histamine (fluorometric technique) and for total and differential cell counts. Antigen challenge was associated with a significant increase in PGD2 concentration in the recovered fluid, rising from a median of 178 pg/ml (range, 157-647) before to 919 pg/ml (range, 149-2,452) after challenge. Median histamine concentrations were 3.1 ng/ml (range, 1-5.4 ng/ml) before and 5.6 ng/ml (range, 1-16.2) after challenge (P = not significant). In four dogs, a control challenge with the antigen vehicle alone showed no change in either mediator. Changes in cell counts after challenge were inconsistent.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in gene expression in hyperoxia-induced neonatal lung injury
Authors: Horowitz S. , Shapiro D. L. , Finkelstein J. N. , Notter R. H. , Johnston C. J. , Quible D. J.
Published: December 2017

Exposure to high concentrations of oxygen (hyperoxia) can result in lung injury. The biochemical basis of this injury is poorly understood, but it is likely to include alterations in gene expression. Hyperoxia-induced (H-I) cDNAs have been molecularly cloned (Horowitz et al. J. Biol. Chem. 264: 7092-7095, 1989) from the lungs of an adult rabbit exposed to toxic levels of oxygen. One of them (H-I 1) was identified as encoding the tissue inhibitor of metalloproteinases (TIMP), a key regulatory protein of extracellular matrix turnover. Here we identify another clone (H-I 3) as encoding pulmonary surfactant apoprotein A (SP-A). We also show that in neonatal rabbits exposed to 100% oxygen for 96 h, the mRNAs corresponding to TIMP, SP-A, and another H-I gene are increased. These studies have begun to explore specific changes in gene expression associated with neonatal hyperoxic lung injury.
Adenosine A1 receptor-mediated inhibition of surfactant secretion in rat type II pneumocytes
Authors: Gobran L. I. , Rooney S. A.
Published: December 2017

Phosphatidylcholine secretion in type II pneumocytes has been reported to be stimulated by P1 and P2 purinoceptor agonists. P1 receptors are divided into A1 and A2 subtypes with opposite effects on the levels of adenosine 3',5'-cyclic monophosphate (cAMP). Stimulated secretion in type II cells is mediated by the A2 receptor and accompanied by an increase in cAMP concentration. We now report evidence suggesting the existence of an A1 receptor-inhibiting secretion in type II cells from adult rats. The rate of phosphatidylcholine secretion was approximately doubled by 5'(N-ethylcarboxyamido) adenosine (NECA), terbutaline, and forskolin, all of which increase cAMP levels. Adenosine deaminase increased the stimulatory effect of these agonists to approximately three-fold but it had not effect on secretion stimulated by agonists which do not increase cAMP levels. The effect of adenosine deaminase on terbutaline-stimulated secretion was antagonized by selective adenosine A1 receptor agonists, N6-cyclopentyladenosine (CPA) and 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCA). The maximum inhibitory effects of CPA and DCCA were achieved at 10(-9) M and 10(-11) M, respectively. At these concentrations CPA and DCCA had no effect on the rate of basal secretion or on terbutaline-stimulated secretion in the absence of adenosine deaminase. We suggest that adenosine deaminase stimulates phosphatidylcholine secretion by removing adenosine that occupies A1 receptors, thus reversing inhibition of cAMP-mediated secretion.
Virus-induced alterations in macrophage production of tumor necrosis factor and prostaglandin E2
Authors: Panuska J. R. , Midulla F. , Cirino N. M. , Villani A. , Gilbert I. A. , McFadden E. R. , Huang Y. T.
Published: December 2017

The cellular immune response to respiratory syncytial virus (RSV) is felt to contribute to viral clearance and/or the inflammation accompanying pulmonary infections with this virus. Both tumor necrosis factor (TNF) and prostaglandin E2 (PGE2) are important regulatory mediators of the cellular immune response. We examined the production of these mediators from purified human alveolar and blood mononuclear phagocytes (MP) after RSV infection in vitro and compared production induced by virus with that induced by lipopolysaccharide (LPS). RSV infection of alveolar MP did not alter PGE2 production but increased expression of TNF alpha mRNA paralleled by increased secretion of immunoreactive and biologically active TNF. TNF production by alveolar MP was dependent on the infectious dose of virus and occurred early in the viral replication cycle. In contrast, RSV had minimal effects on blood MP production of TNF and PGE2. However, blood MP (and not alveolar MP) infected with RSV and costimulated with LPS demonstrated a 1.7-fold increase in PGE2 levels compared with LPS alone (P less than 0.001). Therefore, RSV has differential effects on human alveolar and blood MP production of these immunoregulatory molecules.
Evidence that Ca2(+)-dependent activation of airway epithelia Cl- channels is not dependent on phosphorylation
Authors: Clancy J. P. , McCann J. D. , Welsh M. J.
Published: December 2017

Previous studies have shown that an increase in the cytosolic Ca2+ concentration [( Ca2+]c) activates Cl- channels in airway epithelia but that the effect is indirect. Because adenosine 3',5'-cyclic monophosphate (cAMP) and phorbol myristate acetate (PMA) activate Cl- channels via phosphorylation by cAMP-dependent protein kinase and protein kinase C, respectively, we asked whether Ca2(+)-dependent Cl- channel activation is phosphorylation dependent. We measured 125I- efflux as an assay of Cl- channel activation in the intact cell. We found that depletion of cellular ATP prevented cAMP- and PMA-induced activation but did not alter activation produced by the Ca2+ ionophore A23187. Moreover, addition of high concentrations of staurosporine (5 microM), to nonspecifically inhibit kinase activity, blocked cAMP- and PMA-stimulated 125I- efflux but had no effect on A23187-induced efflux. These results suggest that elevation of [Ca2+]c does not activate Cl- channels via phosphorylation.
Use of antibodies in the study of protein structure and function in lung diseases
Authors: Miller E. J. , Cohen A. B.
Published: December 2017

Proteins normally fold in a variety of three-dimensional structures. This variety of forms accounts for a multiplicity of functions. One of the major undertakings of modern biochemistry is to determine the structure of a given protein and in doing so learn about its function. Much of the effort in the field of protein chemistry is currently focused on defining areas or domains within the molecule that are responsible for its function. Antibodies specific for functional domains on a protein have been powerful tools in these studies and have provided a great deal of the current knowledge related to the location and function of well-defined structural areas within the protein molecules. The use of antibodies in the study of proteins has considerably advanced our knowledge of both the structure of proteins and their interaction with other molecules. In reviewing the use of antibodies for structure and/or function analysis, we have tried not only to review the techniques and applications involving antibodies but also to show how useful antibody reagents may be designed and prepared. In this review, some of the newer uses of antibodies in modern biology will be described, and a few illustrations of each will be provided.
Effect of in vitro preconditioning on tracheal smooth muscle responsiveness
Authors: Mitchell R. W. , Kelly E. , Leff A. R.
Published: December 2017

We evaluated the effect of preconditioning of the isometric contractile response of canine tracheal smooth muscle (TSM) in vitro. Strips of epithelium-free TSM (n = 90) were excised from 16 anesthetized dogs and fixed isometrically in tissue perfusion chambers. Experiments were performed using methods previously reported in which the following parameters were investigated: 1) quiescent equilibration time in the perfusion chamber (0-120 min); 2) effect of repeated exchange of perfusate; 3) method of determining the optimal resting length (Lmax) for presetting of resting tension (RT); 4) effect of precontraction during the equilibration phase on the contractile response to agonists administered subsequently; and 5) method of determining RT on the response to muscarinic stimulation. When other variables were uniform, neither equilibration time nor perfusate exchange affected potency or efficacy of the response generated subsequently to acetylcholine (ACh). However, both potency (range of EC50: -5.71 +/- 0.14 log M to -6.52 +/- 0.24 log M; P less than 0.02) and efficacy (range of maximal active tension: 1,143 +/- 268 g/cm2 to 2,878 +/- 151 g/cm2; P less than 0.001) of ACh were altered substantially as a result of the method used to estimate Lmax. Repeated precontraction by electrical field stimulation or with 127 mM KCl did not alter potency or efficacy of contraction elicited by ACh. Maximal active tension generated with 10(3) M ACh was 2,878 +/- 151 g/cm2 after 12–15 tetanizing field stimulations and 2,696 +/- 198 g/cm2 after 5–7 contractions with 127 mM KCl (P = NS).(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of maturational changes in myosin content and morphometry on airway smooth muscle contraction
Authors: Murphy T. M. , Mitchell R. W. , Halayko A. , Roach J. , Roy L. , Kelly E. A. , Munoz N. M. , Stephens N. L. , Leff A. R.
Published: December 2017

We studied the relationship of airway morphometry, the content of myosin heavy-chain and isoform stoichiometry, and the distribution of bronchoconstrictor responses in the airways of maturing swine. Lungs were excised in 2-wk-old farm swine (2ws; n = 13) and 10-wk-old swine (10ws; n = 13), and tracheal smooth muscle strips and bronchial rings from generations 2–5 were fixed for in vitro isometric measurement of force generation. Split samples were placed in formaldehyde solution or glutaraldehyde for light- or electron-microscopic morphometry or frozen for analysis of tissue myosin content. The rank order of force generation elicited by both receptor- and nonreceptor-dependent mechanisms for both 2ws and 10ws was generation 4 greater than 3 greater than or equal to 2. For all matched airway generations, contractile force was 257#x2013;100% greater in 2ws than 10ws. Differences in force generation were not related to morphometric differences in smooth muscle mass content among airways. The relative cross-sectional area of smooth muscle derived by computerized morphometry was 5.5–7% for each airway generation and did not change with age. Electron-microscopic morphometry demonstrated comparable myocyte content within muscle bundles for all airways in both age groups. In generation 4 airways, myocyte size in 2ws (27.3 +/- 0.8 nuclei/2,500 microns2) hypertrophied approximately 15% in 10ws (20.4 +/- 0.6 nuclei/2,500 microns2; P less than 0.01). Tissue content of myosin measured by computerized laser densitometry of gel electrophoresis of homogenates was greater in trachea from 2ws than 10ws (135 +/- 10 vs. 90 +/- 4 micrograms/g tissue; P less than 0.01); homology of 200- and 205-kDa isoforms was confirmed by Western blot against polyclonal myosin antibody and Cleveland digest analysis of each band. Differences in contractile forces between generations in 2ws and 10ws were not correlated to functional myosin isoform content. We demonstrate a maturational downregulation of contractile forces in maturing swine. This response is independent of smooth muscle receptor distribution and is not related to morphological changes in airways muscle mass, cellularity, changes in content of nonmyocyte tissues, or tissue content of functional myosin isoform.
Supplemental fatty acids alter lipid peroxidation and oxidant injury in endothelial cells
Authors: Hart C. M. , Tolson J. K. , Block E. R.
Published: December 2017

The purpose of this study was to determine the effects of supplemental fatty acids on oxidant injury in cultured endothelial cells. Porcine pulmonary artery endothelial cells (PAEC) in monolayer culture were incubated in culture medium supplemented with 0.1 mM fatty acid or with fatty acid vehicle alone for 3 h. Monolayers were then exposed to oxidant stress (100 microM H2O2 in buffer) or to control conditions (buffer alone) for 30 min. Supplementation with stearic acid (18:0) or oleic acid [18:1(n-9)] reduced H2O2-induced PAEC injury measured as release of intracellular lactate dehydrogenase (LDH). In contrast, supplementation with linolenic acid [18:3(n-6)] or eicosatrienoic acid [20:3(n-3)] enhanced H2O2-induced injury to PAEC. Both supplemental cis-vaccenic acid [18:1(n-7)] and 18:1(n-9) reduced the production of lipid peroxidation products in oxidant-stressed PAEC, whereas supplementation with 18:3(n-6) enhanced lipid peroxidation. Supplementation with 18:1(n-9) protected PAEC from H2O2 as long as 72 h after supplementation despite the intracellular redistribution of [18:1(n-9)] from triglycerides to phospholipids. Saturated and monounsaturated supplemental fatty acids protected PAEC from oxidant injury, but polyunsaturated fatty acids enhanced oxidant injury. These results support the hypothesis that supplemental fatty acids replace resident fatty acids, alter the oxidant reactivity of the cellular lipids, and thereby modify the oxidant susceptibility of PAEC.
Generation of an endothelium-derived relaxing factor-like substance in bovine tracheal smooth muscle
Authors: Sheng H. , Ishii K. , Murad F.
Published: December 2017

The mechanism of the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) induced by carbachol was investigated in bovine tracheal smooth muscle preparations. The effects of carbachol on cGMP were prevented with methylene blue, hemoglobin, or atropine, indicating that the increase of cGMP induced by carbachol is due to the activation of muscarinic cholinergic receptors and is mediated by the stimulation of soluble guanylyl cyclase. The elevation in cGMP induced by carbachol was also inhibited by the superoxide anion-generating compound pyrogallol (100 microM). This inhibition was reversed by superoxide dismutase (100 U/ml). Furthermore, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (3 microM) inhibited cGMP accumulation induced by carbachol, and this was reversed by the addition of L-arginine (1 mM). These results suggest that an endothelium-derived relaxing factor-like substance is generated in the course of carbachol-induced cGMP accumulation in bovine tracheal smooth muscle and that the precursor of this substance is L-arginine.
A2 and P2 purine receptor interactions and surfactant secretion in primary cultures of type II cells
Authors: Griese M. , Gobran L. I. , Rooney S. A.
Published: December 2017

Phosphatidylcholine secretion in type II pneumocytes can be stimulated by P1 (adenosine) and P2 (ATP) purinoceptor agonists. The effect of adenosine is mediated by the A2 subtype of the P1 receptor. The A1 subtype is inhibitory. We examined the influence of ATP and the A2 agonist 5'-(N-ethylcarboxyamido)adenosine (NECA) on phosphatidylcholine secretion in primary cultures of rat type II cells. The stimulatory effects of ATP and NECA were less than additive, suggesting a common mechanism of action. NECA and ATP both caused a rapid increase in cAMP, and the combination enhanced this even further. ATP promoted inositol trisphosphate (IP3) formation, whereas NECA did not. The effect of ATP on adenosine 3',5'-cyclic monophosphate (cAMP) but not on IP3 was abolished by a P1 antagonist, and such antagonists diminished its effect on secretion by as much as 75%. The potency orders of ATP analogues in increasing formation of cAMP and IP3 were different. The effects of the ATP analogues on phosphatidylcholine secretion were also inhibited by the P1 antagonists, with the greatest degree of inhibition being observed with the analogue that increased cAMP to the greatest extent. The effect of ATP on secretion was not diminished by either adenosine deaminase (previous data) or AMP deaminase showing that the effects of ATP were not mediated by its metabolism to the P1 agonists adenosine or AMP. These data show that ATP acts at both A2 and P2 receptors but that most of its effects on phosphatidylcholine secretion are mediated by the A2 receptor.
Amplification of LTB4 generation in AM-PMN cocultures: transcellular 5-lipoxygenase metabolism
Authors: Grimminger F. , Sibelius U. , Seeger W.
Published: December 2017

The generation of arachidonic acid (AA) metabolites by human polymorphonuclear leukocytes (PMN) and by rabbit alveolar macrophages (AM) was investigated and compared with that produced under conditions of coculture. Incubation of PMN with the calcium ionophore A23187 resulted in rapid generation of leukotriene (LT) B4 and its omega-oxidation products, paralleled by substantial secretion of 5-hydroxyeicosatetraenoic acid (HETE) and intact LTA4. Rapid LTA4 decay to nonenzymatic hydrolysis products in the extracellular space ensued. Exogenous AA, offered simultaneously with the ionophore, markedly increased 5-lipoxygenase product formation. Incubation of AM with A23187 evoked protracted generation of LTB4 in the absence of omega-oxidation, with concomitant liberation of 5-HETE, 15-HETE, free AA, and minor amounts of AA cyclooxygenase products. Exogenously offered LTA4 was avidly taken up and converted into LTB4 by these cells. Costimulation of AM and PMN with the ionophore resulted in an approximately 2.5-fold increase in the generation of LTB4 and its metabolites (compared with the summed amounts of the isolated cell experiments), whereas 5-HETE and nonenzymatic LTA4, hydrolysis product formation were markedly reduced. This change in metabolite profile was dependent on the AM-to-PMN ratio. Acetylsalicylic acid increased 5-lipoxygenase product formation in the coculture studies but not in the isolated cell experiments. AA prelabeling of either PMN or AM resulted in radioactivity detection in all AA lipoxygenase products except for 15-HETE.(ABSTRACT TRUNCATED AT 250 WORDS)
Neuropeptides and nasal secretion
Authors: Baraniuk J. N. , Kaliner M.
Published: December 2017

The nasal mucosa is innervated by the sensory, parasympathetic, and sympathetic nervous systems. Nociceptive sensory nerves are stimulated by mucosal injury, inhalation of irritants, or mast cell degranulation and release of the calcitonin gene-related peptide, the tachykinins substance P and neurokinin A, and other peptides by the axon response mechanism. Sensory nerve stimulation initiates systemic reflexes, such as the sneeze, and central parasympathetic reflexes which release acetylcholine, vasoactive intestinal peptide, and other peptides and lead to glandular secretion. In concert, these proinflammatory neural responses lead to vasodilation, vascular permeability, and glandular secretion. Sympathetic nerves release neuropeptide Y and norepinephrine, potent vasoconstrictors which act to decompress the nasal mucosa and produce nasal patency. The balance between the effects of parasympathetic and sympathetic neurotransmitters may regulate nasal homeostasis, whereas the nociceptive sensory system may be held in reserve as a defense mechanism. Dysfunction of these systems may lead to pathological nasal syndromes. In the future, specific neuropeptide agonists and antagonists may be useful for the treatment of human rhinitic diseases.
The sugared path to a normal lung: soluble beta-galactoside-specific lectins
Authors: Powell J. T. , Harrison F. L.
Published: December 2017

The diverse functions of a family of soluble beta-galactoside-binding proteins in lung are discussed. The smallest and most abundant member, a 14-kDa lectin, appears to be a negative modulator of cell growth. Larger members of this family include RL29, a protein involved in the regulation of cytoplasmic RNA levels, and one subunit of the cell surface elastin receptor involved in the regulation of elastic fibrillogenesis. The structure and function of other members of this family remain to be elucidated, but probably homologous carboxy-terminal domains will specify the galactose-binding function and varied amino-terminal domains will specify the diverse biological functions. Although currently these functions are being described at the cellular level, these beta-galactoside-binding proteins appear to have important roles in regulating lung physiology.
Ontogenic expression of acetylcholinesterase activity in trachealis of young swine
Authors: Murphy T. M. , Mitchell R. W. , Phillips I. J. , Leff A. R.
Published: December 2017

Previous investigations have demonstrated that cholinergic contraction of porcine tracheal smooth muscle (TSM) decreases between the second and tenth weeks of life. In this investigation, we hypothesized that the greater contractile response to acetylcholine (ACh) in TSM of 2-wk-old swine (2ws) vs. 10-wk-old swine (10ws) was the result of a relative decrease in activity of acetylcholinesterase (AChase). To examine this hypothesis, we assessed AChase activity directly in homogenates of TSM from eight 2ws and seven 10ws using a newly adapted method that measures the rate of cleavage of acetylthiocholine; enzyme activity was expressed as absorbance units per minute per milligram protein. The AChase from tissues of both age groups saturated at approximately 3 mM substrate. However, maximal AChase activity (Vmax) was significantly greater in 10ws than 2ws. Eadie-Hofstee analysis of enzyme kinetics revealed similar Michaelis-Menten constants for 2ws and 10ws. The concentration of physostigmine (PS), an inhibitor of cholinesterase, that elicited half-maximal inhibition of AChase activity also was similar for 2ws and 10ws. In separate studies, contraction of TSM strips was assessed in vitro at optimal resting length and expressed as a function of maximal force generation to potassium chloride. Strips of TSM from 2ws contracted with greater force than those of 10ws. After pretreatment with 10(-8) M PS, contractile forces were similar in 2ws and 10ws. We conclude that AChase activity measured directly in muscle homogenates is significantly reduced in TSM of 2ws vs. 10ws and that this may result in augmented contraction to ACh under conditions of zero-order kinetics.
Muscarinic receptor subtypes in guinea pig airways
Authors: Haddad E. B. , Landry Y. , Gies J. P.
Published: December 2017

The muscarinic receptors present in the guinea pig tracheobronchial tree were characterized using ligand-binding studies and functional approaches. The binding constants of four selective antagonists, pirenzepine,[11-([2-[(diethylamino) methyl]-1-piperidinyl]acetyl)-5,11-dihydro-6H-pyrido(2,3) (1,4) benzodiazepine-6-on] (AF-DX 116), methoctramine, and 4-diphenylacetoxy-Nmethylpiperidine methobromide(4-DAMP) were examined. Heterogeneity in the airway muscarinic receptors population was revealed by competitive binding experiments against [N-methyl3H]scopolamine with the M2 muscarinic antagonists AF-DX 116 and methoctramine. In guinea pig lung and trachea, AF-DX 116 and methoctramine recognized 86–88% and 50–60% of total receptors with high affinity, respectively. These receptors exhibit binding constants for these two compounds similar to those of the M2 subtype. The low-affinity M2 antagonist binding constants were close to those reported for M3 receptors. In lung and trachea, we found no evidence for a high-affinity [N-methyl-3H]pirenzepine binding sites. In functional studies, pirenzepine, methoctramine, and 4-DAMP inhibited the methacholine-induced contraction of lung parenchymal, main bronchial, and tracheal strips with affinities characteristic of smooth muscle M3 receptors. These results are consistent with the presence of M2 and M3 receptors in guinea pig airways. Throughout the airways, the muscarinic receptors mediating smooth muscle contraction are of the M3 subtype.
Expression of cytokeratin 8 in human aortic smooth muscle cells
Authors: Glukhova Marina A. , Shekhonin Boris V. , Kruth Howard , Koteliansky Victor E.
Published: December 2017

An immunofluorescence method was used to study the expression of cytokeratin 8 in human aortic smooth muscle cells (SMCs) during prenatal development and in atherosclerotic plaques. Aortic SMCs from a 10-wk-old fetus contained cytokeratin 8 in additional to vimentin and a small amount of desmin, whereas, in the cells from a 25-wk-old fetus, cytokeratin 8 was not detected. Cytokeratin 8 was found in the SMCs from intimal thickenings, fatty streaks, and atherosclerotic fibrous plaques. Clusters of cytokeratin 8-positive cells were more abundant in rather advanced lesions (fibrous plaques) that contained at least some amount of lipid. Expression of cytokeratin 8 in the cells of human atherosclerotic lesions probably reflects general rearrangement of gene expression in the intimal cells.

cytodifferentiation; fetal phenotype; lipid accumulation
Alveolar uptake of lipid and protein components of surfactant
Authors: Fisher Aron B. , Dodia Chandra , Chander Avinash
Published: December 2017

Pages L334–L340: Aron B. Fisher, Chandra Dodia, and Avinash Chander.”;Alveolar uptake of lipid and protein components of surfactant.” Page L337, Table 5, contains an incorrect value. The table should appear as follows (the corrected value appears in boldface):

(See PDF)
Alterations in surfactant protein gene expression associated with premature birth and exposure to hyperoxia
Authors: Minoo P. , Segura L. , Coalson J. J. , King R. J. , DeLemos R. A.
Published: December 2017

Steady-state levels of mRNAs for the three surfactant-associated proteins, SP-A, SP-B, and SP-C, were measured in a primate model of premature birth and survival. These values were determined by Northern and quantitative slot blot analyses of total lung RNA during both in utero and extrauterine development of the fetus as well as in response to hyperoxic exposure. The composition and surface properties of surfactant were also analyzed to determine the effect of differential expression of the surfactant proteins on the overall composition and function of surfactant. The data clearly demonstrate that the regulation of surfactant mRNA levels in the premature fetus is under complex physiological control. Interruption of in utero development by premature birth results in increased levels of all three surfactant mRNAs, presumably in response to precocious initiation of air breathing. Within the first 24 h after parturition both SP-B and SP-C mRNA levels are increased beyond the levels found in the full-term fetal controls. Expression of mRNA for these genes peaks on day 2 and thereafter drops to levels below that found on day 1. However, response of the SP-A gene to premature birth is slow and transcripts from this gene lag considerably behind values found in the full-term fetus. Furthermore, exposure of the premature fetus to hyperoxia results in an increase in the steady-state levels of SP-B and SP-C mRNA without significant changes in SP-A. Defects in the ability of the SP-A gene to respond to extrauterine exposure and hyperoxia may be contributing to development of bronchopulmonary dysplasia, a common clinical complication of premature birth in humans.
Hyperoxia increases plasminogen activator activity of cultured endothelial cells
Authors: Phillips P. G. , Birnby L. , Di Bernardo L. A. , Ryan T. J. , Tsan M. F.
Published: December 2017

Confluent calf pulmonary artery endothelial monolayers exposed to 95% oxygen for 1, 2, or 3 days exhibit a time-dependent increase in adherence to substratum, which closely parallels changes in actin cytoarchitecture and the distribution of focal contact proteins vinculin and talin. Oxygen exposure also resulted in elevated plasminogen activator (PA) activity in conditioned media (CM) and in cytoskeletal protein- and focal contact protein-enriched fractions, with highest levels achieved in the latter two fractions at 48 h after oxygen exposure. PAs have been shown to participate in dismantling of extracellular matrix in a number of physiological and pathological situations. Immunocytochemical studies demonstrated extensive restructuring of matrix proteins collagen IV, laminin, and fibronectin, which correlated temporally with elevated PA levels. Further, when protease-containing cell fractions were used to study degradation of isolated matrices, those obtained from hyperoxia-exposed cells were substantially more active than those from normoxia-exposed cells. Our data suggest that hyperoxia-induced production of PA (and perhaps other proteases) may be partly responsible for degradation of the extracellular matrix of endothelial cells.
Assessment of signal transduction mechanisms regulating airway smooth muscle contractility
Authors: Schramm C. M. , Grunstein M. M.
Published: December 2017

Agonist-receptor interactions regulate airway smooth muscle tone through activation of guanine nucleotide binding proteins (G proteins) which are coupled to second-messenger pathways that mediate changes in the tissue's contractile state. Various methods have been applied to identify the structure/function characteristics of G proteins and their role in signal transduction in airway smooth muscle, including the use of exotoxins, nonhydrolyzable analogs of guanosine-triphosphate (GTP), antibodies to purified G proteins, and membrane reconstitution studies. In elucidating mechanisms of airway smooth muscle relaxation, considerable progress has been made in identifying the molecular basis for receptor/G protein coupling and other regulatory processes leading to both the activation and down-regulation of the adenylate cyclase/adenosine 3' 5'-cyclic monophosphate system. Further, with respect to airway smooth muscle contraction, various approaches have been used to evaluate the role of membrane phosphoinositide turnover and the mechanisms of action of the bifurcating signal transduction pathways associated with the production and metabolism of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, and activation of protein kinase C. This review identifies much of the information gained to date on the above signal transduction pathways, with an emphasis placed on various methodological approaches used to determine membrane and transmembrane signaling processes in airway smooth muscle.
Acceleration of alveolar type II cell differentiation in fetal rhesus monkey lung by administration of EGF
Authors: Plopper C. G. , St George J. A. , Read L. C. , Nishio S. J. , Weir A. J. , Edwards L. , Tarantal A. F. , Pinkerton K. E. , Merritt T. A. , Whitsett J. A. , et al.
Published: December 2017

To examine the effect of epidermal growth factor (EGF) on lung parenchymal maturation in fetal rhesus monkey, recombinant human EGF was administered intraperitoneally (IP) at 66 mg/kg body wt over a 7-day period into the fetal peritoneal cavity alone or IP and into the amniotic fluid (AF) simultaneously. The saline carrier was injected IP and AF into control (CO) fetuses. The body weights of the IP + AF group were significantly larger than CO. Overall lung growth, measured as wet lung weight or fixed volume of the right cranial lobe, was unchanged. Fixed lung volume per gram body weight was significantly lower for both IP + AF and IP compared with CO. Morphogenesis of lung parenchyma, measured as percent parenchymal airspace or airspace size, was unchanged. Alveolar type II cell ultrastructure was significantly altered by EGF treatment; volume fraction of cytoplasmic glycogen was 50% less and lamellar bodies threefold greater for IP + AF and IP groups compared with CO. Total phospholipid content of AF was not altered, but relative percentages of different phospholipids were changed by EGF treatments; phosphatidylinositol was significantly reduced, and phosphatidylglycerol was significantly elevated. The lecithin-to-sphingomyelin ratio was unchanged. Surfactant apoprotein A concentration in AF was significantly elevated and was detected by immunoperoxidase in more cuboidal alveolar cells in EGF-treated animals when compared with CO. We conclude that exogenous EGF administered in the last trimester of pregnancy accelerates structural and functional cytodifferentiation of the alveolar type II cell in fetal primates. These maturational changes occur in the absence of significant alterations in overall lung growth or morphogenesis of the gas exchange area.
Clara cell secretory protein gene expression in bronchiolar epithelium
Authors: Hackett B. P. , Shimizu N. , Gitlin J. D.
Published: December 2017

To determine the mechanisms of Clara cell secretory protein (CCSP) gene expression, a cDNA clone was isolated and used in RNA blot analysis. A single 600 bp CCSP specific transcript was detected in the developing rat lung on fetal day 18. This transcript increased in abundance during late fetal life such that adult levels were attained within 2 wk postpartum. CCSP gene expression was tissue specific, being confined to lung and trachea at all developmental stages. The abundance of CCSP mRNA in lung tissue was unchanged after the induction of lung injury in adult rats either with lipopolysaccharide or prolonged exposure to hyperoxia. In situ hybridization of lung tissue revealed that CCSP gene expression is localized to the nonciliated epithelial (Clara) cells of the bronchiolar epithelium throughout fetal and postnatal development. Taken together the results indicate that the gene for CCSP is abundantly expressed in a cell-specific fashion in the lung and suggest that analysis of such expression will be useful in elucidating the role of Clara cells in the growth and development of the bronchiolar epithelium.
Specific binding of surfactant apoprotein SP-A to rat alveolar macrophages
Authors: Pison U. , Wright J. R. , Hawgood S.
Published: December 2017

Surfactant protein A (SP-A) influences the function of alveolar macrophages in vitro. In this study the characteristics of the binding of 125I-labeled SP-A to rat alveolar macrophages has been investigated. The binding of SP-A to alveolar macrophages at 4 degrees C was saturable with half-maximal binding at a SP-A concentration of 4 micrograms/ml. Bound SP-A was rapidly displaced by an excess of unlabeled SP-A. The binding of labeled SP-A to the alveolar macrophages was blocked in a dose-dependent fashion by unlabeled SP-A, the collagen-like protein C1q and type V collagen but not by bovine serum albumin. These results suggest that a component of the interaction between SP-A and alveolar macrophages is mediated through the collagen-like domain of SP-A and that the characteristics of this interaction are consistent with there being a specific receptor for SP-A on the surface of alveolar macrophages.
Transient cholinergic glycoconjugate secretion from swine tracheal submucosal gland cells
Authors: Dwyer T. M. , Szebeni A. , Diveki K. , Farley J. M.
Published: December 2017

By inference, muscarinic stimulation of glycoconjugate release from tracheal submucosal gland cells appears to be a transient, nonequilibrium process (J. M. Farley and T. M. Dwyer, Life Sci. 48: 59–67, 1991). To directly characterize the release kinetics of glycoconjugate, we developed an enzyme-linked lectin assay (ELLA) of much improved precision and resolution. To collect secreted products with an improved time resolution, freshly isolated swine tracheal submucosal gland cells were continuously superfused with medium 199 at 37 degrees, buffered with N-2-hydroxyethylpiperazine-N$#x0027-2-ethanesulfonic acid (HEPES) and CO2 or bicarbonate; fractions were collected every 15 s to 2 min. A 30-s pulse of 30 nM acetylcholine (ACh) increased the rate of glycoconjugate release by 10- to 25-fold for 2–3 min. The peak response averaged 14.2 +/- 9.0 ng protein.ml-1.min-1. 4 million cells-1 or 3.6 +/- 2.3 fg.cell-1.min-1 for 30 nM ACh and 16.2 +/- 3.0 ng protein.ml-1.min-1 for 100 nM ACh. There was no significant glycoconjugate release following a 30-s pulse of either 10 nM or 1 microM ACh. A second pulse after 7 min had no measurable effect on glycoconjugate release but a full response was obtained after 30 min. A continuous superfusion begun 1 min following the 30-s pulse resulted in a greater release of glycoconjugate than the pulse alone, but the response was not sustained, falling to twice basal levels within 5 min. We conclude that a brief muscarinic stimulation causes a triggered release of mucus glycoprotein followed by a relative refractory period.
Tricarboxylic acid cycle activity in perfused rat lungs after O2 exposure
Authors: Bassett D. J. , Reichenbaugh S. S.
Published: December 2017

O2-induced impairment of mitochondrial energy generation was examined in intact lungs isolated from rats after 18-30 h exposure to either air or 100% O2 in vivo. Mitochondrial metabolic rates were determined by separate measurements of 14CO2 production from [1-14C]pyruvate and [U-14C]palmitate, perfused under normal and stimulated metabolic conditions brought about by perfusion with the uncoupler of oxidative phosphorylation, 2,4-dinitrophenol (DNP). In the absence of DNP, O2 exposure did not significantly alter 14CO2 productions from either substrate. DNP increased lung pyruvate and palmitate catabolism to CO2 twofold in air-exposed lungs but did not alter 14CO2 production in lungs isolated from O2-exposed rats. These data demonstrated an O2-induced impairment of maximal mitochondrial metabolism of both pyruvate and palmitate that could not be explained by alterations in tissue free coenzyme A or by loss of pyridine nucleotides. However, comparisons of the steady-state levels of tricarboxylic acid cycle intermediates between O2- and air-exposed lungs did identify isocitrate dehydrogenase as a possible site of O2-induced enzyme inactivation.
Impairment of sodium-coupled uptakes by hydrogen peroxide in alveolar type II cells: protective effect of d-alpha-tocopherol
Authors: Clerici C. , Friedlander G. , Amiel C.
Published: December 2017

Hydrogen peroxide (H2O2) is likely to play an important role in oxidant alveolar epithelium injury. We investigated the effect of H2O2 on uptake of phosphate, alanine in cultured rat alveolar type II cells. H2O2 induced inhibition of Na-dependent component of phosphate and alanine uptakes in time- and concentration-dependent manner. Twenty minutes exposure to 2.5 mM H2O2 decreased the maximum velocity (Vmax) of phosphate and alanine uptake by 50 and 62%, respectively, whereas Michaelis constant (Km) values were unchanged. H2O2 also decreased Na-K-ATPase activity, measured by ouabain-sensitive rubidium influx, and this effect was independent of H2O2-induced ATP depletion. A lipid-soluble antioxidant, d-alpha-tocopherol (20 microM, 24 h), prevented H2O2-induced decrease in Na-coupled uptake and Na-K-ATPase activity. These results indicate that H2O2 affects Na-dependent phosphate and alanine uptakes and suggest that this effect may be related at least, in part, to a decrease in Na transmembrane gradient, since H2O2 also affects Na-K-ATPase activity. The protective effect of d-alpha-tocopherol suggests that peroxidation of the membrane lipids is likely to be involved in the observed effects.
Extracellular matrix synthesis and turnover by type II pulmonary epithelial cells
Authors: Rannels D. E. , Dunsmore S. E. , Grove R. N.
Published: December 2017

Both type I and type II pulmonary epithelial cells contact the extracellular matrix (ECM). Type II cell-ECM interactions are bidirectional; they involve matrix-mediated modulation of type II cell differentiation, as well as cellular synthesis and deposition of ECM components. The present experiments examine the kinetics of accumulation of newly synthesized proteins in cell and matrix fractions from primary cultures of type II pneumocytes. Cycloheximide-sensitive incorporation of [3H]leucine into total protein of both the cell and ECM fractions was linear for 24–30 h, when steady-state labeling was reached and maintained to at least day 8. Over this interval, the cells enlarged but did not divide. Newly synthesized proteins recovered in the matrix fraction averaged 1–2% of those in the cells. Relative rates of radiolabeling of matrix proteins peaked at culture day 2 and increased in the absence of serum. In short-pulse studies, initial rates of protein synthesis were equal on culture days 1 and 3; this suggested that the steady-state labeling kinetics above reflected protein turnover. This was supported by rapid loss of radioactivity from the ECM after fresh type II cells were seeded on a prelabeled, cell-free matrix surface. Fresh or conditioned Dulbecco's modified Eagle's medium containing 10% fetal calf serum had little effect on matrix stability. These results demonstrate regulated deposition and turnover of a complex ECM by type II cells and provide a basis for further investigations of factors that control these processes.
Bumetanide-sensitive NaCl uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and hypertonicity
Authors: Liedtke C. M.
Published: December 2017

Loop diuretic-sensitive NaCl(K) cotransport plays a fundamental role in absorption and secretion of electrolytes in epithelial tissues. Cotransport activity was measured as uptake of 22Na, 36Cl, or 86Rb at 27 degrees C in isolated rabbit tracheal epithelial cells. Uptake of radiotracer was linear from 1 to 2 min after initiation of radiotracer transport. Bumetanide at 10 microM final concentration did not affect tracer uptake. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agent clonidine increased sodium and chloride uptake at least 5.5-fold. Bumetanide blocked sodium uptake by 85% and chloride uptake by 72%. 86Rb uptake was not affected by l-epinephrine, clonidine, or bumetanide. The alpha 2-adrenergic antagonist yohimbine blocked the effects of l-epinephrine and clonidine on 22Na and 36Cl uptake. In Ca(2+)-depleted transport medium, baseline levels of sodium and chloride uptake increased 3.8- and 2.4-fold, respectively, in a bumetanide-independent manner. Nevertheless, l-epinephrine and clonidine induced a net stimulation of sodium and chloride uptake similar to that found in Ca(2+)-replete medium. This response was reduced by bumetanide and yohimbine. The Ca(2+)-elevating agent ionomycin increased bumetanide-sensitive sodium and chloride uptake 7.2- and 6.2-fold, respectively. Replacement of chloride with gluconate or sodium with N-methyl-D-glucamine in the extracellular medium inhibited l-epinephrine and clonidine-stimulated bumetanide-sensitive sodium and chloride uptake, respectively. Osmotic shrinkage in hyperosmotic (500 mM NaCl with all other electrolytes at normal concentration) transport medium markedly increased bumetanide-inhibitable sodium and chloride uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
Bioelectric properties of cultured epithelial monolayers from distal lung of 18-day fetal rat
Authors: Barker P. M. , Stiles A. D. , Boucher R. C. , Gatzy J. T.
Published: December 2017

In vivo studies of fetal sheep suggest that the liquid present in the lumen of the lung throughout fetal life is derived from Cl- secretion by the pulmonary epithelium. Monolayer preparations of enriched epithelial cells from distal fetal rat (18-day gestation) lung, grown in serum-free media, were histologically similar to acinar (prealveolar) structures of fresh tissue. In Ussing chambers, basal transepithelial potential difference (PD), calculated equivalent short-circuit current (Ieq), and transepithelial resistance (R) were 4.4 +/- 0.3 mV (matrix positive), 35.6 +/- 1.6 microA/cm2, and 120.0 +/- 4.0 omega cm2, respectively. Ouabain (10(-3) M) eliminated 57% of basal Ieq within 30 min, amiloride (10(-4) M) induced a 13% fall in Ieq, and phlorizin (10(-4) M) had no effect on bioelectric properties. Diphenylamine-2-carboxylate (DPC, 3 x 10(-3) M) inhibited Ieq by 50%. Bumetanide had no effect on baseline bioelectric parameters. The hyperpolarization that accompanied apical or bilateral replacement of Cl- and was enhanced by terbutaline suggested an apical Cl- permselectivity. Effects of Na+ replacement on amiloride-pretreated monolayers were consistent with Na(+)-dependent Cl- secretion or amiloride-insensitive pathways. Under these growth conditions, this preparation exhibits bioelectric characteristics that are compatible with Cl- secretion and Na+ absorption. The mechanism of Cl- secretion may be similar to that of airways but is uniquely bumetanide insensitive.