bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of airway inflammation on smooth muscle shortening and contractility in guinea pig trachealis
Authors: Mitchell R. W. , Ndukwu I. M. , Arbetter K. , Solway J. , Leff A. R.
Published: December 2017

We studied the effect of either 1) immunogenic inflammation caused by aerosolized ovalbumin or 2) neurogenic inflammation caused by aerosolized capsaicin in vivo on guinea pig tracheal smooth muscle (TSM) contractility in vitro. Force-velocity relationships were determined for nine epithelium-intact TSM strips from ovalbumin-sensitized (OAS) vs. seven sham-sensitized controls and TSM strips for seven animals treated with capsaicin aerosol (Cap-Aer) vs. eight sham controls. Muscle strips were tethered to an electromagnetic lever system, which allowed isotonic shortening when load clamps [from 0 to maximal isometric force (Po)] were applied at specific times after onset of contraction. Contractions were elicited by supramaximal electrical field stimulation (60 Hz, 10-s duration, 18 V). Optimal length for each muscle was determined during equilibration. Maximal shortening velocity (Vmax) was increased in TSM from OAS (1.72 +/- 0.46 mm/s) compared with sham-sensitized animals (0.90 +/- 0.15 mm/s, P < 0.05); Vmax for TSM from Cap-Aer (0.88 +/- 0.11 mm/s) was not different from control TSM (1.13 +/- 0.08 mm/s, P = NS). Similarly, maximal shortening (delta max) was augmented in TSM from OAS (1.01 +/- 0.15 mm) compared with sham-sensitized animals (0.72 +/- 0.14 mm, P < 0.05); delta max for TSM from Cap-Aer animals (0.65 +/- 0.11 mm) was not different from saline aerosol controls (0.71 +/- 0.15 mm, P = NS). We demonstrate Vmax and delta max are augmented in TSM after ovalbumin sensitization; in contrast, neurogenic inflammation caused by capsaicin has no effect on isolated TSM contractility in vitro. These data suggest that airway hyperresponsiveness in vivo that occurs in association with immunogenic or neurogenic inflammation may result from different effects of these types of inflammation on airway smooth muscle.
Mechanisms of peroxynitrite-induced injury to pulmonary surfactants
Authors: Haddad I. Y. , Ischiropoulos H. , Holm B. A. , Beckman J. S. , Baker J. R. , Matalon S.
Published: December 2017

Activated alveolar macrophages secrete both nitric oxide and superoxide in the alveolar lining fluid which combine rapidly to form peroxynitrite, a potent oxidizing agent capable of damaging lipids and proteins in biological membranes. Peroxynitrite (1 mM) plus 100 microM Fe3+EDTA inhibited calf lung surfactant extract (CLSE) from reaching a minimum surface tension below 10 mN/m on dynamic compression. Peroxynitrite and its by-products reacted with the unsaturated lipid components of CLSE, as evidenced by the appearance of conjugated dienes and thiobarbituric acid products, and damaged all surfactant proteins. A mixture of the hydrophobic proteins [surfactant protein B (SP-B) and surfactant protein C (SP-C)] exposed to peroxynitrite became incapable of lowering phospholipid minimum surface tension on dynamic compression. Exposure of SP-A to peroxynitrite decreased its ability to cause lipid aggregation and to act synergistically with SP-B and SP-C in lowering surface tension of surfactant lipids. Western blot analysis of SP-A exposed to peroxynitrite was consistent with fragmentation and polymerization of the 28- to 36-kDa triplet band, and amino acid analysis revealed the presence of significant levels of 3-nitro-L-tyrosine. We conclude that peroxynitrite and its reactive intermediates inhibit pulmonary surfactant function by lipid peroxidation and damaging surfactant proteins.
Secretion of surfactant protein A from rat type II pneumocytes
Authors: Rooney S. A. , Gobran L. I. , Umstead T. M. , Phelps D. S.
Published: December 2017

Secretion of surfactant phosphatidylcholine has been extensively studied and there is evidence that it is a regulated process that can be influenced by a variety of physiological factors and pharmacological agents. In contrast, secretion of the major surfactant protein, surfactant protein A (SP-A), has been investigated to much lesser extent. It is not known whether SP-A secretion is constitutive or regulated and, if regulated, whether its regulation is similar to that of phosphatidylcholine. To address those questions we measured SP-A secretion in primary cultures of type II pneumocytes under conditions identical to those used to study phosphatidylcholine secretion. Freshly isolated cells from adult rats were cultured overnight, washed, and then incubated in fresh medium in the presence and absence of surfactant phospholipid secretagogues. As previously reported for phosphatidylcholine, SP-A secretion was linear with time for up to 4 h. However, the rate of SP-A secretion, approximately 6% of total SP-A (cells+medium) released into the medium per hour, was more than sixfold greater than that of the lipid. Although freshly isolated cells contained 70% more SP-A than cells that were cultured overnight, the rate of SP-A secretion was not significantly different. Secretion of SP-A by freshly isolated or cultured type II cells was not increased by a combination of ATP, terbutaline, the adenosine A2 receptor agonist 5'(N-ethylcarboxyamido)adenosine, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin at concentrations that optimally stimulated phosphatidylcholine secretion. We conclude that secretion of the major lipid and protein components of surfactant are independently regulated.
Pulmonary carbonic anhydrase IV: developmental regulation and cell-specific expression in the capillary endothelium
Authors: Fleming R. E. , Crouch E. C. , Ruzicka C. A. , Sly W. S.
Published: December 2017

To investigate the pulmonary expression of membrane-associated carbonic anhydrase (CA IV), the full-length rat lung CA IV cDNA was isolated and characterized. The 1,205-bp cDNA was found to encode a 309-amino acid protein, including a 27-amino acid hydrophobic COOH-terminal extension, presumably cleaved prior to transfer of the protein to its glycosyl-phosphatidylinositol membrane anchor. The tissue-specific pattern of CA IV gene expression was found to be distinct from that of other CAs, with highest expression in lung, colon, kidney, brain, and heart. Pulmonary CA IV expression was found to be developmentally regulated, with a marked postnatal increase in mRNA content and immunoreactive protein concentration. CA IV localized by immunohistochemistry to the luminal side of the alveolar capillary endothelium, but was absent from endothelium of larger pulmonary vessels. Selective expression of CA IV in the alveolar capillaries provides evidence for specialized differentiation of this endothelial cell subpopulation and places CA IV in a strategic location to participate in carbon dioxide exchange and local pH regulation.
Upregulation of sodium conductive pathways in alveolar type II cells in sublethal hyperoxia
Authors: Haskell J. F. , Yue G. , Benos D. J. , Matalon S.
Published: December 2017

We investigated whether exposure of rats to sublethal hyperoxia (85% O2 for 7 days) raises the levels of proteins antigenically related to Na+ channels in alveolar type II (ATII) cells and, if so, whether this rise was accompanied by an increase in conductive Na+ transport in vitro. ATII cells were isolated from the lungs of these rats at the end of the exposure period. In Western blot studies, a polyclonal antibody raised against Na+ channel protein (NaAb), recognized in a specific manner a 135 +/- 10 kDa polypeptide in plasma membrane vesicles of ATII cells from both control and oxygen-exposed rats. However, higher levels of immunoreactivity were seen in ATII cells from oxygen-exposed rats. When ATII cells were patched in the whole cell mode using symmetrical solutions (150 mM Na(+)-glutamate), outward rectified Na+ currents were observed. When corrected for cell capacitance, both inward and outward currents of ATII cells from rats exposed to hyperoxia were significantly higher than control. Addition of either 1 microM amiloride or 1 microM 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride in the bath solution decreased the magnitude of outward currents of both control and hyperoxic ATII cells by approximately 50%. Taken together, these results indicate that exposure of rats to sublethal hyperoxia results in upregulation of ATII cell conductive pathways with low affinity to amiloride and increased Na+ transport. This may be an early adaptive response that limits the degree of alveolar edema in injured lungs.
Contrasting effects of alveolar macrophages and neutrophils on asbestos-induced pulmonary epithelial cell injury
Authors: Kamp D. W. , Dunn M. M. , Sbalchiero J. S. , Knap A. M. , Weitzman S. A.
Published: December 2017

Pulmonary toxicity from asbestos may be due in part to oxidant-mediated mechanisms. The purpose of this study was to determine whether alveolar macrophages (AM) contribute to asbestos-induced alveolar epithelial cell injury by oxidant-dependent mechanisms similar to that previously described for polymorphonuclear leukocytes (PMN). We assessed 51Cr release from cultured rat alveolar epithelial cells (RAEC) and transformed human pulmonary epithelial-like cell lines (rat L2 and human WI-26: HPEC). Amosite asbestos caused dose-dependent injury to both RAEC and L2 cells after an 18-h incubation period. Rat PMN increased asbestos-induced injury to RAEC (11 vs. 20% 51Cr release). In contrast, rat AM diminished asbestos-induced injury to RAEC and L2 cells by 60-80%. Human monocytes cultured for 72 h also attenuated asbestos-induced HPEC damage. Asbestos stimulated more H2O2 release from PMN than from AM isolated from the same rats (5.3 +/- 0.6 vs. 0.3 +/- 0.1 nmol x 10(6) cells-1 x 2h-1). The protective effect of rat AM, as opposed to PMN, was not due to differences in asbestos-induced toxicity to each cell type, since > 90% of AM and PMN were nonviable after 18 h. Transmission electron microscopy demonstrated comparable uptake of asbestos by AM and PMN after a 2-h incubation period. However, after an 18-h exposure period, the PMN were completely lysed, whereas over 90% of the AM contained fibers, despite morphologic evidence of cytotoxicity. These results demonstrate that AM, unlike PMN, can reduce alveolar epithelial cell injury in this model.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of cigarette smoke and its condensates on alveolar epithelial cell injury in vitro
Authors: Lannan S. , Donaldson K. , Brown D. , MacNee W.
Published: December 2017

The oxidant-antioxidant balance in the airspaces of the lungs may be critical in protecting the lungs from the effects of cigarette smoke. We studied the effect of cigarette smoke and its condensates on the detachment, attachment, and proliferation of the A549 human alveolar epithelial cell line, in an in vitro model of cell injury and regeneration and the protective effects of antioxidants. Whole and vapor phase cigarette smoke decreased 51Cr-labeled A549 cell attachment, increased cell detachment, and decreased cell proliferation, as assessed by [3H]thymidine uptake. Freshly isolated rat type II alveolar epithelial cells showed an enhanced susceptibility to smoke-induced cell lysis when compared with the A549 cell line. Reduced glutathione (GSH) (400 microM) protected against the effects of cigarette smoke exposure on cell attachment, proliferation, and detachment. Depletion of intracellular GSH with buthionine sulfoxamine enhanced the epithelial cell detachment injury produced by smoke condensates. We conclude that cigarette smoke and its condensates cause an oxidant-induced injury to A549 human type II alveolar epithelial cells. Both intra- and extracellular GSH have important roles in protecting epithelial cells from the injurious effects of cigarette smoke.
Pseudomonas siderophore pyochelin enhances neutrophil-mediated endothelial cell injury
Authors: Britigan B. E. , Rasmussen G. T. , Cox C. D.
Published: December 2017

Pyochelin, a siderophore secreted by Pseudomonas aeruginosa, binds iron in a form which can catalyze the formation of hydroxyl radical (.OH) from neutrophil-derived superoxide (O2-.) and hydrogen peroxide (H2O2). Ferripyochelin induced a concentration-dependent increase in endothelial cell injury (51Cr release) resulting from exposure to H2O2, a xanthine/xanthine oxidase O2-./H2O2 generating system, or stimulated neutrophils. This process was dependent on the presence of iron. Formation of .OH was confirmed using spin trapping. Although a slight (13%) increase in neutrophil O2-. production in the presence of ferripyochelin was observed, this did not appear to account for the extent of endothelial cell injury observed. The antioxidants dimethylthiourea and catalase decreased endothelial cell injury, whereas dimethyl sulfoxide and superoxide dismutase were without effect. Fe-nitrilotriacetic acid and Fe-EDTA, which are also .OH catalysts, did not augment endothelial cell injury resulting from exposure to the above oxidant systems. In contrast to results with the endothelial cells, killing of P. aeruginosa by O2-./H2O2 derived from the reaction of xanthine and xanthine oxidase was not increased by ferripyochelin. These data are consistent with the possibility that the interaction of Pseudomonas- and phagocyte-derived secretory products could contribute to local tissue injury at sites of P. aeruginosa infection by causing the generation of .OH.
Role of potassium channels in hypoxic relaxation of porcine bronchi in vitro
Authors: Lindeman K. S. , Fernandes L. B. , Croxton T. L. , Hirshman C. A.
Published: December 2017

To elucidate the mechanism of hypoxic relaxation of airway smooth muscle in vitro, we investigated the role of adenosine triphosphate-sensitive potassium (KATP) channels in this response. Second- and third-order porcine bronchial rings were suspended in 10-ml organ baths containing Krebs-Henseleit solution. To demonstrate the presence of KATP channels in this tissue, bronchial rings were contracted with carbachol (1 microM) in the presence of glibenclamide (100 microM), a KATP channel blocker, or the vehicle dimethyl sulfoxide (DMSO) (0.1 ml), and dose-response curves to levcromakalim (a KATP channel opener) or isoproterenol were constructed. In separate experiments, either glibenclamide or DMSO was added to the chamber and rings were contracted with carbachol (1 microM) in the presence of 95% O2-5% CO2. At the plateau, airways were relaxed with either isoproterenol (0.1 or 0.3 microM) or hypoxia (50, 28, or 0% O2, with constant 5% CO2). Glibenclamide, when compared with DMSO, shifted the dose-response curve to levcromakalim, but not to isoproterenol. Glibenclamide attenuated hypoxic relaxation in rings exposed to 50% O2 (from 35 +/- 4% to 23 +/- 3%, n = 6, P < 0.001) and increased the time to 63% relaxation in rings exposed to 50% O2 or to 28% O2. Responses in rings exposed to 0% O2 or to isoproterenol (0.1 or 0.3 microM) were not significantly altered. The ability of glibenclamide to attenuate the maximum response to 50% O2 and to increase the time to 63% relaxation during exposure to 50 or 28% O2 suggests that one component of hypoxic bronchodilation during moderate degrees of hypoxia is opening of KATP channels.
TNF-alpha-induced transendothelial neutrophil migration is IL-8 dependent
Authors: Smart S. J. , Casale T. B.
Published: December 2017

The early phases of airway inflammation include complex interactions between leukocytes, vascular endothelium, and inflammatory cytokines. Therefore, we examined tumor necrosis factor-alpha (TNF-alpha)-induced neutrophil migration through polycarbonate filters and human umbilical vein endothelial (HUVE) cells cultured as monolayers on these filters. TNF-alpha induced both dose- and time-dependent migration of neutrophils across both barriers. At 10(-11)-10(-9) M TNF-alpha, neutrophil migration across HUVE monolayers was more than twofold greater than that observed across naked filters. Modified checkerboard experiments indicated that neutrophils crossed naked filters as a chemokinetic rather than chemotactic response. Supernatants of TNF-alpha (10(-9) M)-stimulated HUVE monolayers induced threefold greater migration of neutrophils across naked filters than 10(-9) M TNF-alpha itself, suggesting release of soluble chemotactic factor(s). Pretreatment of HUVE monolayers with actinomycin D inhibited both TNF-alpha-induced production of soluble chemotactic factors and transendothelial neutrophil migration by > 90%. Supernatants from TNF-alpha-treated HUVE cells contained significant concentrations of interleukin 8 (IL-8), and coincubation of these supernatants with anti-IL-8 decreased supernatant-induced chemotaxis. Finally, coincubation of TNF-alpha with anti-IL-8 during transmigration experiments nearly completely inhibited the increase in neutrophil migration measured across HUVE monolayers. In contrast, WEB 2086, a platelet-activating factor receptor antagonist, had no effect. Therefore, endothelial cells greatly facilitate TNF-alpha-induced transcellular migration of neutrophils. This facilitation is dependent on TNF-alpha-stimulated production of IL-8. These data further support the active role of vascular endothelium in recruiting leukocytes to sites of inflammation.
Constitutive and stimulated MCP-1, GRO alpha, beta, and gamma expression in human airway epithelium and bronchoalveolar macrophages
Authors: Becker S. , Quay J. , Koren H. S. , Haskill J. S.
Published: December 2017

Constitutive expression of mRNAs for GRO alpha, GRO beta, GRO gamma, and MCP-1, belonging to the chemokine family of 8- to 10-kDa cytokines with chemotactic properties for granulocytes and monocytes, has been identified in freshly isolated human nasal and bronchial epithelium, and in bronchoalveolar macrophages (AM). Expression of GRO alpha, GRO gamma, and MCP-1, but not GRO beta, was found in airway epithelial cells. AM expressed all three GRO genes in addition to MCP-1. On reverse transcription, chemokine mRNAs yielded 0.5-30 cDNA molecules/cell, depending on the chemokine and cell type, as determined by a semiquantitative reverse transcriptase-polymerase chain reaction technique. When chemokine mRNA expression in AM and bronchial epithelium from healthy nonatopic individuals was compared, AM expressed more GRO alpha, but similar levels of GRO gamma, MCP-1, and interleukin-8 (IL-8), as in the bronchial epithelial cells. Modulation of chemokine expression by tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) or endotoxin [lipopolysaccharide (LPS), 100 ng/ml] exposure was studied in primary nasal epithelial cell and alveolar macrophage cultures. In epithelial cells, LPS did not induce chemokine expression but GRO alpha, IL-8, and MCP-1 were upregulated approximately 100-fold by TNF alpha; GRO gamma expression was elevated only 1.5- to 4-fold. In AM cultures, all three GROs were strongly induced by LPS with peak mRNA expression 24 h after stimulation (approximately 50- to 100-fold increase compared with control cultures). MCP-1 mRNA expression, on the other hand, was not increased by LPS in AM. GRO protein was present in supernatants of stimulated epithelial cells and AM.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain natriuretic peptide: possible role in the modulation of hypoxic pulmonary hypertension
Authors: Hill N. S. , Klinger J. R. , Warburton R. R. , Pietras L. , Wrenn D. S.
Published: December 2017

To test the hypothesis that brain natriuretic peptide (BNP) plays a role similar to that of atrial natriuretic peptide (ANP) in modulating pulmonary vascular responses to hypoxia, we measured the vasodilator potency of ANP and BNP in rat pulmonary artery (PA) and thoracic aorta (TA) rings and in isolated rat lungs. We also measured the effect of chronic hypoxia on plasma levels and cardiac gene expression of both peptides. BNP had a vasorelaxant effect equipotent to that of ANP on preconstricted TA and PA rings, but was less potent than ANP in relaxing the vasoconstrictor response to hypoxia in isolated lungs [mean 50% inhibitory concentration (IC50) 10(-7) vs. 10(-6) M for ANP and BNP, respectively]. Plasma BNP levels were 30-fold lower than ANP, but both peptides increased approximately 70% during chronic hypoxia. In the right atrium, hypoxia lowered BNP mRNA slightly, but had no effect on ANP mRNA or tissue levels of either peptide. However, hypoxia increased right ventricular content and mRNA levels of both peptides by three- to fourfold. We conclude that BNP and ANP have similar pulmonary vasodilator effects and are upregulated proportionally during chronic hypoxia. These results support a role for BNP in modulating the pulmonary hypertensive response to chronic hypoxia.
Transgenic models for the study of lung biology and disease
Authors: Ho Y. S.
Published: December 2017

Transgenic models provide a means of understanding the molecular mechanisms for the temporal, spatial, and stimulus-responsive regulation of gene expression in vivo and importantly the pathophysiological consequences of the altered expression of a normal or mutated gene. To facilitate the application of transgenic models in lung research, this review describes several practical considerations in generation of transgenic mice. The potential of transgenic models in lung research is also illustrated by depicting the current models in lung research including those for understanding lung gene regulation, tumorigenesis, mutation detection, antioxidant defense, emphysema, fibrosis, and hypertension. The impact of important new development of producing transgenic mice carrying large fragments of DNA contained in yeast artificial chromosomes to achieve proper control of transgene expression and gene targeting technology is also discussed. It is anticipated that transgenic models will provide invaluable information in future lung research.
Activation of G proteins may inhibit or stimulate surfactant secretion in rat alveolar type II cells
Authors: Pian M. S. , Dobbs L. G.
Published: December 2017

To investigate how G proteins regulate surfactant secretion, we subjected rat alveolar type II cells to conditions known to activate or to inactivate G proteins. AlF-4, which activates G proteins, inhibited secretion in intact cells. Guanosine-5'-O-(3-thiotriphosphate), which activates G proteins in permeabilized cells, stimulated secretion at basal cytosolic [Ca2+], but inhibited secretion at higher [Ca2+]. In contrast, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates G proteins, stimulated secretion at each [Ca2+] tested. Because treatment with GDP beta S stimulated secretion at basal cytosolic [Ca2+], surfactant secretion appears to be subject to G protein-regulated tonic inhibition. Pertussis toxin (PTX) inhibited terbutaline- and ionomycin-stimulated secretion in intact cells, but did not inhibit secretion stimulated by either forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Inhibition by PTX of terbutaline-stimulated, but not 8-bromoadenosine 3',5'-cyclic monophosphate- or forskolin-stimulated secretion, suggests that PTX-sensitive G proteins regulate beta-adrenergic-stimulated surfactant secretion proximal to second messenger generation. Inhibition of ionomycin-stimulated secretion, however, suggests that PTX-sensitive G proteins may also regulate non-receptor-mediated secretory events.
SP-A, SP-B, and SP-C in surfactant subtypes around birth: reexamination of alveolar life cycle of surfactant
Authors: Baritussio A. , Alberti A. , Quaglino D. , Pettenazzo A. , Dalzoppo D. , Sartori L. , Pasquali-Ronchetti I.
Published: December 2017

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.
Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle
Authors: Nakayama D. K. , Geller D. A. , Di Silvio M. , Bloomgarden G. , Davies P. , Pitt B. R. , Hatakeyama K. , Kagamiyama H. , Simmons R. L. , Billiar T. R.
Published: December 2017

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)
The beta 2-adrenergic receptor agonist formoterol reduces microvascular leakage by inhibiting endothelial gap formation
Authors: Baluk P. , McDonald D. M.
Published: December 2017

beta 2-Adrenergic receptor agonists inhibit the increase in vascular permeability produced by a variety of inflammatory mediators. The anti-edema effect of beta 2-agonists is assumed to result from a direct action on endothelial cells, but such a mechanism has not been demonstrated in vivo. The aim of this study was to determine whether beta 2-agonists exert their anti-edema effect by inhibiting the formation of endothelial gaps at sites of plasma leakage. Vascular permeability in the rat trachea was increased by electrical stimulation of the vagus nerve or by intravenous injection of substance P (5 micrograms/kg iv). Plasma leakage was quantified by using Monastral blue and Evans blue as tracers. Endothelial gaps were made visible for light microscopy by staining the borders of endothelial cells with silver nitrate. The experiments showed that the selective beta 2-agonist formoterol, which is known to have anti-edema effects, reduced the plasma leakage produced by either stimulus. The effect was dose dependent, with a formoterol dose of 10 micrograms/kg iv producing maximal reduction of Monastral blue leakage (64 +/- 14%). The amounts of extravasation of Monastral blue and Evans blue were closely correlated (r2 = 0.76, P < 0.01). After the injection of substance P, there were 15.3 +/- 1.0 gaps/endothelial cells in postcapillary venules of vehicle-pretreated rats, but only 5.0 +/- 0.2 gaps/cell in formoterol-pretreated (10 micrograms/kg iv) rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of endocytosis inhibitors on alveolar clearance of albumin, immunoglobulin G, and SP-A in rabbits
Authors: Hastings R. H. , Wright J. R. , Albertine K. H. , Ciriales R. , Matthay M. A.
Published: December 2017

Protein in the alveolar space may be cleared by endocytosis and degradation inside alveolar epithelial cells, by transcytosis across the alveolar epithelium, or by restricted diffusion through the epithelium. The relative contributions of these three pathways to clearance of large quantities of protein from the air spaces is not known. This study investigated the effects of monensin and nocodazole, agents which inhibit endocytosis in cell culture, on alveolar epithelial protein transport in anesthetized rabbits. There was evidence that monensin and nocodazole inhibited endocytosis by the alveolar epithelium in vivo. Nocodazole increased the number of vesicles in the alveolar epithelium and capillary endothelium. Monensin increased vesicle density in the endothelium. These results suggested that the inhibitors disrupted microtubules or interrupted cellular membrane traffic in the lung. Both inhibitors decreased lung parenchymal uptake of immunoreactive human albumin from the air spaces. Monensin and nocodazole inhibited albumin uptake in cultured alveolar type II cells. Monensin increased the amount of 125I-labeled surfactant protein A associated with the lungs, compared with the quantity remaining in the air space 2 h after instillation. Although the drugs decreased alveolar epithelial protein uptake, they did not decrease alveolar clearance of 125I-labeled immunoglobulin G or 131I-labeled albumin in anesthetized rabbits. Thus monensin- and nocodazole-sensitive protein-uptake pathways do not account for most alveolar protein clearance when the distal air spaces are filled with a protein solution.
Increased endothelin receptor gene expression in hypoxic rat lung
Authors: Li H. , Elton T. S. , Chen Y. F. , Oparil S.
Published: December 2017

Our previous studies demonstrated that exposure to hypoxia increases pulmonary artery pressure and plasma endothelin-1 (ET-1) levels and selectively enhances ET-1 gene expression in rat lung. The current study examined the effects of hypoxia (48 h, 10% O2, 1 atm) on ET-1 and endothelin A (ETA) and ETB receptor steady-state mRNA levels in lung, heart, pulmonary artery, thoracic aorta, superior vena cava, kidney, spleen, and liver of the rat. In lung, hypoxic exposure was associated with significant increases in ET-1 mRNA (4.1-fold), ET-1 peptide (1.5-fold) and ETA mRNA (2.3-fold) levels; ETB mRNA levels were unchanged. ET-1 mRNA was increased in response to hypoxia in pulmonary artery but not in aorta; both ETA and ETB receptor steady-state mRNA levels were increased in thoracic aorta, left atrium, and right ventricle, and tended to be increased in right atrium of hypoxia-exposed rats, compared with air controls. ETB but not ETA receptor steady-state mRNA levels were increased in pulmonary artery of hypoxia-exposed rats. No change in expression of either ET receptor steady-state mRNA levels was seen in organs perfused by the systemic vascular bed. In no case were ET receptor mRNA levels in hypoxic rats reduced below air control levels, despite elevations in local and/or circulating ET-1. These findings are consistent with a role for ET-1, acting through ETA receptors, in the pathogenesis of hypoxia-induced pulmonary hypertension.
Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology
Authors: Dwyer L. D. , Miller A. C. , Parks A. L. , Jaken S. , Malkinson A. M.
Published: December 2017

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.
Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats
Authors: Olivera W. , Ridge K. , Wood L. D. , Sznajder J. I.
Published: December 2017

Active Na+ transport and lung edema clearance were studied in a model of lung injury caused by sublethal oxygen exposure. Rats exposed to 85% O2 for 7 days were studied at 0, 7, 14, and 30 days after removal from the hyperoxic chamber and compared with room air controls. In the isolated-perfused, fluid-filled rat lung, albumin flux from the perfusate into the air spaces increased after oxygen exposure and returned to control values after 7 days of recovery. However, permeability to small solutes (Na+ and mannitol) normalized only after 30 days of recovery from hyperoxia. Active Na+ transport increased immediately after oxygen exposure and returned to control values 7 days after removal from hyperoxic chamber. Na-K-adenosinetriphosphatase (ATPase) activity, and protein expression in alveolar epithelial type II cells obtained at the end of the isolated lung experiments increased significantly after the oxygen exposure compared with controls in association with the increased active Na+ transport. We conclude that active Na+ transport and lung liquid clearance are increased in the subacute hyperoxic phase of lung injury in rats, due in part to the upregulation of alveolar epithelial Na-K-ATPases. Conceivably, this behavior protects against the effects of lung injury by allowing the injured lung to clear edema more effectively. Accordingly, this upregulation may be targeted as a strategy to diminish edema in patients with lung injury.
Roles of Ca2+ influx and intracellular Ca2+ release in agonist-induced contractions in guinea pig trachea
Authors: Cuthbert N. J. , Gardiner P. J. , Nash K. , Poll C. T.
Published: December 2017

The contribution of receptor-operated Ca2+ channels (ROCs), voltage-operated Ca2+ channels (VOCs), and intracellular Ca2+ release to contractions induced by a range of stimuli in the guinea pig isolated trachea has been evaluated. In the presence of physiological Ca2+ (1.3 x 10(-3) M), tissue pretreatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (4 x 10(-3) M for 5 min) markedly inhibited (> 90%) the contractile responses to a range of agonists. Therefore, under physiological Ca2+ concentration, Ca2+ mobilization from internal stores appeared to make little contribution to maximum contractions. Nifedipine (10(-7) M) or verapamil (10(-5) M) abolished KCl-induced contractions but produced variable inhibition of contractions induced by other agonists. The ROC (and VOC) blocker, SK&F 96365 (10(-5)-10(-4) M), inhibited both KCl-induced contractions and the nifedipine-insensitive component of contractions induced by acetylcholine (ACh), U46619, or leukotriene D4 [half maximal inhibitory concentration (IC50) values 1.7-3.8 x 10(-5) M]. Ni2+, which has ROC- and VOC-blocking actions, also abolished nifedipine-insensitive contractions induced by ACh. When Ca2+ was replaced with Ba2+, the contraction induced by ACh was blocked by nifedipine. Also, under these conditions, ACh did not increase the KCl maximum contraction. These data are consistent with there being distinct ROC and VOC influx pathways in guinea pig trachea and with ROCs playing a significant role in smooth muscle contraction.
Platelet-derived growth factor and its receptor in lungs from patients with asthma and chronic airflow obstruction
Authors: Aubert J. D. , Hayashi S. , Hards J. , Bai T. R. , Pare P. D. , Hogg J. C.
Published: December 2017

The airway walls of patients who have asthma or chronic obstructive pulmonary disease (COPD) are thickened by an increase in the amount of smooth muscle and connective tissue. Platelet-derived growth factor (PDGF) is a candidate cytokine for this increase because it can produce smooth muscle proliferation in vitro. The present study was designed to examine the expression of PDGF and its receptor (PDGFR) in lungs from six asthmatics, six patients with COPD, and six patients with normal lung function. PDGF was immunolocalized to tissue macrophages, but the number of PDGF-positive cells was similar in all three groups. PDGFR-beta was rarely expressed on interstitial cells, and, occasionally, on bronchial epithelium. Northern blotting, performed on tissue from the same groups, showed a positive correlation of PDGF(B) with PDGFR-beta mRNA level (r = 0.74, P < 0.001) and a higher abundance of PDGF(B) and PDGFR-beta mRNA in the asthmatics vs. the COPD (P < 0.05). We conclude that PDGF and its receptor are expressed in human lungs but do not correlate closely with the structural changes in diseased airways.
Retinoids control surfactant phospholipid biosynthesis in fetal rat lung
Authors: Fraslon C. , Bourbon J. R.
Published: December 2017

Vitamin A (retinol) may play an important role in lung maturation: 1) premature delivery is simultaneously a source of vitamin A deficiency and increased risk of neonatal respiratory distress syndrome and subsequent bronchopulmonary dysplasia (BPD), due to deficit in pulmonary surfactant; 2) neonatal supplementation with retinol reduces the risk of BPD; and 3) fetal rat lung stores retinol in late gestation just before the onset of surfactant synthesis. To test the hypothesis of an implication of retinoids in the control of pulmonary surfactant synthesis, experiments were designed in the pregnant rat, aiming either at enhancing fetal lung vitamin A stores, bringing the active metabolite of vitamin A, retinoic acid (RA), or inhibiting the conversion of retinol to RA with aid of citral. Maternal administration of a single dose of 50,000 IU of retinyl palmitate on day 16 (term 22 days) increased 22 and 29%, respectively, the total phospholipid (TPL) and disaturated fraction of phosphatidylcholine (PC) in extracted fetal surfactant on day 19 but did not change surfactant protein (SP) A concentration. Chronic administration of retinyl palmitate to the mother from day 16 through 20 increased disaturated PC content on day 21 but decreased SP-A concentration. Fetal lung surfactant phospholipids were increased by chronic administration of RA and considerably reduced by citral (-31 and -35% for TPL and PC concns, respectively). RA also enhanced labeled choline incorporation into fetal lung PC on day 20. Given once on day 17, it accelerated the appearance of surfactant precursors on day 18.(ABSTRACT TRUNCATED AT 250 WORDS)
Increased expression of CTP:phosphocholine cytidylyltransferase in maturing type II cells
Authors: Hogan M. , Zimmermann L. J. , Wang J. , Kuliszewski M. , Liu J. , Post M.
Published: December 2017

We previously reported that phosphatidylcholine synthesis increased in fetal rat lung type II cells with advancing gestation. This increase was accompanied by an increase in CTP:phosphocholine cytidylyltransferase activity, which catalyses a rate regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. To determine whether this increase in cytidylyltransferase activity is due to an increase in cytidylyltransferase protein levels, the gene and protein expression of cytidylyltransferase was investigated in maturing type II cells. The cytidylyltransferase cDNA was cloned from fetal rat type II cells and showed 99% sequence homology with rat liver cDNA. The cDNA detected two mRNA transcripts (1.8 and 7.5 kb) in fetal rat lung. By reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, cytidyltransferase mRNA content increased three-fold in fetal type II cells with advancing gestation, whereas cytidylyltransferase mRNA levels in fibroblasts remained constant. An antibody against rat liver cytidylyltransferase was used to assess cytidylyltransferase protein. Western blotting revealed that cytidylyltransferase protein content increased threefold in the microsomal fraction of type II cells with advancing gestation. The enzyme protein levels in the cytosolic fraction did not significantly change with development. Enzyme activity studies confirmed these latter observations. We conclude that the increase in surfactant phosphatidylcholine synthesis by type II cells at late fetal gestation is due in part to an increase in the amount of cytidylyltransferase protein.
Alteration of cellular cytosolic calcium and chemotactic peptide binding by an inhibitor of neutrophil function
Authors: Cooper J. A. , Bridges T. A. , Kennedy J. I. , Culbreth R.
Published: December 2017

The lung is frequently exposed to particulate material that can potentially stimulate release of factors that attract polymorphonuclear neutrophils (PMN). However, few PMN are noted in the airways of normal subjects, suggesting there is some mechanism to dampen influx of these cells. We have isolated from bronchial lavage a peptide that inhibits PMN chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP). In the present study we examined effects of this molecule on 1) chemotaxis to other agonists, 2) FMLP-stimulated PMN superoxide production, 3) PMN calcium fluxes, and 4) binding of FMLP. Our results show that purified inhibitor attenuates PMN chemotaxis to C5a and leukotriene B4. This molecule also inhibits PMN superoxide release in response to FMLP. Exposure to this inhibitor causes an abrupt rise in cytosolic calcium concentration due to a pertussis toxin-sensitive shift of intracellular calcium and attenuates subsequent influx of extracellular calcium in response to FMLP. Binding studies demonstrate the inhibitor induces increased FMLP binding at 37 degrees C but has no effects at 4 degrees C. Inhibition of chemotaxis and increased FMLP binding mediated by this molecule are attenuated by buffering PMN calcium transients. These studies suggest an inhibitor of neutrophil function present in the bronchial environment alters PMN through effects on calcium homeostasis.
Regulation of low-amiloride-affinity sodium channels in alveolar type II cells
Authors: Yue G. , Shoemaker R. L. , Matalon S.
Published: December 2017

We determined the mechanisms by which beta-agonists increase sodium (Na+) currents across rat alveolar type II (ATII) cells grown in primary culture. When ATII cells were patched in the cell-attached mode using symmetrical Na+ solutions (150 mM Na(+)-glutamate), single-channel currents were observed for holding potentials between -80 and 30 mV (referenced to the pipette solution) with a single-channel conductance of 27 +/- 3 pS, a mean open time (tau 1) of 3.3 +/- 0.15 ms and an open probability (Po) of 0.36 +/- 0.06 (n = 7). Addition of 10 microM terbutaline into the bath increased tau 1 to 6.43 +/- 0.5 ms and Po to 0.62 +/- 0.06 (n = 7) without affecting channel conductance. Single-channel currents with a conductance of 25 +/- 2 pS were also recorded across ATII cells patched in the inside-out mode. Addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 in the bath solution (150 mM Na(+)-glutamate) increased the single channel tau 1 from 3.26 +/- 0.15 to 7.38 +/- 0.38 and Po from 0.41 +/- 0.06 to 0.72 +/- 0.07 (n = 6) without altering conductance. Addition of 1 microM amiloride or ethylisopropylamiloride (EIPA) in the pipette solution (150 mM Na(+)-glutamate) blocked single-channel activity almost completely. Ionic substitution experiments showed the relative permeability of Na+ to K+ and Na+ to Cl- to be 7:1 and 8:1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Interleukin-8 is a major component of pleural liquid chemotactic activity in a rabbit model of endotoxin pleurisy
Authors: Boylan A. M. , Hebert C. A. , Sadick M. , Wong W. L. , Chuntharapai A. , Hoeffel J. M. , Hartiala K. T. , Broaddus V. C.
Published: December 2017

Gram-negative endotoxin induces production of the potent chemotactic factor interleukin-8 (IL-8) in vitro; however, the importance of IL-8 in endotoxin-induced inflammation in vivo is unknown. We asked whether IL-8 is an important contributor to chemotactic activity in acute inflammatory liquids formed in response to endotoxin, and, if present, what concentrations of IL-8 antigen are generated. For these studies, we cloned and expressed rabbit recombinant IL-8 (rrIL-8), developed specific anti-rabbit IL-8 monoclonal antibodies (mAb), and then used these reagents to develop assays to detect rabbit IL-8 bioactivity and measure rabbit IL-8 antigen. Escherichia coli endotoxin (20 ng/ml, n = 4, or 2,000 ng/ml, n = 4) was instilled into the pleural space of eight rabbits for 6 h. Rabbit IL-8 bioactivity in the endotoxin pleurisy samples was assayed by measuring the migration of rabbit neutrophils toward the pleural liquid under two different conditions: 1) after addition of an anti-IL-8 neutralizing mAb and 2) after desensitization of the neutrophils to rrIL-8. Addition of the anti-IL-8 mAb decreased neutrophil migration toward the pleural liquid by 65 +/- 13 and 75 +/- 22% (mean +/- SE, after 20 and 2,000 ng/ml endotoxin, respectively; P < 0.01 compared with a control mAb). Desensitization of neutrophils to rrIL-8 decreased their migration toward the pleural liquid by 72 +/- 5% (P = 0.03, compared with exposure of neutrophils to buffer alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Apically localized IP3 receptors control chloride current in airway gland acinar cells
Authors: Sasaki T. , Shimura S. , Wakui M. , Ohkawara Y. , Takishima T. , Mikoshiba K.
Published: December 2017

We examined the role of inositol 1,4,5-trisphosphate (IP3) receptors in acetylcholine (ACh)-induced chloride (Cl-) current in acinar cells of human and feline airway submucosal glands, using whole cell patch-clamp analysis. ACh (10 nM-1 microM) induced an initial Cl- current followed by a K+ current, and lower doses of ACh (1-10 nM) often induced oscillations of both currents, which were mimicked by the application of intracellular IP3. Neither isoproterenol (-10 microM) nor raising intracellular adenosine 3,5-cyclic monophosphate induced any current. Caffeine (20-50 mM) and intracellular ryanodine (1-100 microM) induced a K+ current alone without Cl- current. Monoclonal antibodies to the IP3 receptor abolished both ACh-induced K+ and Cl- currents. Immunohistochemical analysis revealed the localization of IP3 receptors on both the cytosol and some regions of the endoplasmic reticulum beneath the apical membrane of acinar cells. These results indicate that apically localized IP3 receptors control Cl- secretion from airway submucosal gland cells.