bluuebunny/crossref_metadata_2025 huggingface dataset:
Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs
Authors: Boggaram Vijayakumar , Margana Ramgopal K.
Published: December 2017

Pages L634–L644: Vijayakumar Boggaram and Ramgopal K. Margana. “Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs.” Page L634, left-hand column, in the abstract, the sentence beginning on line 22 should read: Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene. Page L634, left-hand column, paragraph 2, line 4 should read: SP-A [relative molecular weight (Mr) ≈35,000] and SP-D (Mr≈43,000) are hydrophilic proteins that are similar in having collagenous amino-terminus domain and noncollagenous lectinlike carboxy-terminus domain. SP-B (Mr ap8,000) and SP-C (Mr ap5,000) are hydrophobic proteins derived from larger precursor proteins by amino- and carboxy-terminus proteolytic processing.
Peptidase inhibitors reverse steroid-induced suppression of neutrophil adhesion in rat tracheal blood vessels
Authors: Katayama M. , Nadel J. A. , Piedimonte G. , McDonald D. M.
Published: December 2017

Exposure of the tracheal mucosa of rats to capsaicin evokes neurogenic inflammation, one manifestation of which is the adherence of neutrophils to the endothelium of venules. In the present experiments, with the use of aerosolized capsaicin, we determined whether this neutrophil adhesion is inhibited by dexamethasone and whether the effect of dexamethasone can be reversed by inhibiting endopeptidase 24.11 (neutral endopeptidase, NEP) and kininase II (angiotensin-converting enzyme, ACE), which degrade the neuropeptides that mediate neurogenic inflammation. Adult male pathogen-free F344 rats were treated for 2 days with dexamethasone or with vehicle (controls) and were then exposed for 2 min to aerosolized capsaicin. Neutrophils adhering to the endothelium of venules in tracheal whole mounts were stained histochemically for myeloperoxidase and then counted. Sites of increased vascular permeability were localized with Monastral blue. In the control rats, aerosolized capsaicin (10(-8)-10(-3) M) increased in a concentration-dependent fashion the number of adherent neutrophils and the amount of Monastral blue labeling of blood vessels. Dexamethasone in doses of 0.5, 1, 2, or 4 mg.kg-1.day-1 reduced by 49 63, 80, and 93%, respectively, the number of adherent neutrophils in capsaicin-exposed rats and caused similar reductions in the amount of Monastral blue labeling. When given alone, neither phosphoramidon, an inhibitor of NEP, nor captopril, an inhibitor of ACE, completely reversed this effect of dexamethasone, but when the two drugs were administered together, adherent neutrophils were as numerous in the dexamethasone-pretreated rats (112 +/- 9 neutrophils/mm2) as in controls (109 +/- 10 neutrophils/mm2). The amount of Monastral blue labeling was also similar in these two groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of gastrin-releasing peptide and analogues on guinea pig nasal mucosal secretion
Authors: Gawin A. Z. , Baraniuk J. N. , Lundgren J. D. , Kaliner M.
Published: December 2017

The effects of gastrin-releasing peptide (GRP), bombesin, GRP-(1–16) and GRP-(21–27) on guinea pig nasal mucosal secretion were studied in vivo. GRP, bombesin, and GRP-(21–27) induced significant secretion of total protein, albumin, and alkaline phosphatase. GRP induced significant secretion at lower concentrations (10(-11) and 10(-10) M) than were required for bombesin and GRP-(21–27) (10(-7) M). GRP-(1–16) did not stimulate secretion, indicating that the COOH-terminal region of GRP contained the secretagogic principle. Capsaicin, a stimulant of nociceptive sensory nerves, stimulated GRP release into nasal secretions. These data suggest that GRP is present in guinea pig nasal mucosa and that the COOH-terminal region of GRP may regulate mucosal macromolecule secretion.
Recycling of surfactant lipid and apoprotein-A studied by electron microscopic autoradiography
Authors: Young S. L. , Fram E. K. , Larson E. , Wright J. R.
Published: December 2017

Recycling of lipid and apoprotein components by lung epithelium appears to be a key part of the metabolism of surfactant, but many details of this process are poorly understood, including its efficiency and which intracellular pathways participate. We chose electron microscopic autoradiography to track time-dependent changes in the intracellular location of [methyl-3H]-choline dipalmitoyl phosphatidylcholine or 125I-labeled apoprotein-A that were incorporated into liposomes and instilled intratracheally into rats or added to cultures of freshly isolated rat lung type II cells. At times from 2 to 120 min after the start of labeling, lungs or cells were fixed and processed for autoradiography. We found a time-dependent uptake of lipid and apoprotein by type II cells and by macrophages but not by other lung cell types. There was a time-dependent incorporation of lipid and apoprotein label into lamellar bodies, but the labels appeared in other intracellular compartments without a convincing time-dependent relationship to lamellar bodies. Although all type II cell organelles received some lipid-bound label, the apoprotein-bound label was not found within electron-dense multivesicular bodies (d-MVB). Selective lack of apoprotein label in d-MVB indicated a segregation of lipid and apoprotein labels and suggested a unique role for the d-MVB, possibly as a degradation pathway. Our results also provided conclusive morphological evidence of surfactant lipid and apoprotein recycling by type II cells.
Modulation of vasoactive intestinal peptide pulmonary relaxation by NO in tracheally superfused guinea pig lungs
Authors: Lilly C. M. , Stamler J. S. , Gaston B. , Meckel C. , Loscalzo J. , Drazen J. M.
Published: December 2017

The mechanism of vasoactive intestinal peptide (VIP)-induced pulmonary relaxation in tracheally perfused guinea pig lungs was defined with the use of inhibitors of nitric oxide synthase (NOS) and by direct measurement of nitric oxide (NO) equivalents recovered from lung perfusion fluid. Lungs treated with 200 microM NG-nitro-L-arginine were resistant to the relaxant effects of VIP in these lungs; the 50% inhibitory dose (ID50) for VIP was 32 nmol/kg (95% confidence interval, 16–79), which was approximately 100-fold greater than the ID50 of control lungs which was 0.39 nmol/kg, (0.16–0.79, P < 0.0001). This inhibitory effect could be overcome with excess L- but not D-arginine. In contrast, VIP-induced relaxation of isolated guinea pig trachea was not modified by inhibitors of NOS. To confirm that VIP infusion resulted in NO generation in whole lungs, we measured NO equivalents in lung effluent by two distinct technologies. We found that VIP injection caused a significant increase in NO equivalents from 0.11 +/- 0.04 microM to 0.78 +/- 0.15 microM (P < 0.05) and that this increase preceded VIP-induced pulmonary relaxation. Lungs pretreated with the putative guanylyl cyclase inhibitor methylene blue were less responsive to VIP [ID50 4.0 nmol/kg (1.5–10), P < 0.005 compared with control lungs], consistent with a physiologically significant guanosine 3',5'-cyclic monophosphate-dependent mechanism. Our data demonstrate that VIP has the capacity to relax whole lungs in part by stimulating the generation of NO.
Degradation of surfactant protein B by alveolar type II cells
Authors: Bates S. R. , Fisher A. B.
Published: December 2017

Surfactant protein B (SP-B) metabolism was studied in primary cultures of alveolar type II cells. Iodinated SP-B reconstituted with surfactant was incorporated rapidly into lung pneumocytes and degraded to trichloroacetic acid (TCA)-soluble products after a lag period of 1 h. Cellular degradation of SP-B occurred whether or not phospholipid liposomes or surfactant was added to the phospholipid-poor SP-B. Uptake and degradation of SP-B at 37 degrees C showed a linear increase up to 3 micrograms SP-B/ml after which the slope of the curve became less steep with increasing concentrations of SP-B in the media. After 4 h of incubation with SP-B, 35% of the SP-B processed was recovered as degradation products. Ninety-six percent of the degradation products were in the media and only 4% were recovered in the cell. The bulk of the breakdown of SP-B occurred inside the type II cells since degradation did not occur at 4 degrees C, showed a 1-h lag period, was proportional to the SP-B protein internalized by the cells, was inhibited 47% by ammonium chloride, was unaffected by the addition of protease inhibitors to the medium, and cell-conditioned medium produced only limited SP-B degradation. Alveolar macrophages also degraded SP-B, whereas other cell types degraded SP-B to a lesser extent. Thus the specificity of the metabolism of SP-B may be through the capability of lung cells to degrade SP-B.(ABSTRACT TRUNCATED AT 250 WORDS)
Involvement of neurogenic inflammation in antigen-induced bronchoconstriction in guinea pigs
Authors: Bertrand C. , Geppetti P. , Graf P. D. , Foresi A. , Nadel J. A.
Published: December 2017

The role of tachykinins released from sensory nerves in bronchoconstriction induced by antigen was studied in sensitized guinea pigs anesthetized with pentobarbital sodium and pretreated with atropine. The combination of NK2 (SR-48968) and NK1 (CP-96,345) tachykinin-receptor antagonists abolished the increase in total pulmonary resistance (RL) evoked by intravenous capsaicin but did not affect the response evoked by intravenous histamine. A small dose of aerosolized ovalbumin (OVA, 0.1%) produced a small increase in RL that was further increased and markedly prolonged by the neutral endopeptidase (NEP) inhibitor phosphoramidon; this bronchoconstrictor effect of OVA was markedly reduced by the NK2-receptor antagonist and was abolished by the combination of the NK1 and NK2-receptor antagonists together. When a larger dose of OVA (0.5%) was used, a maximal bronchoconstrictor response was obtained. Phosphoramidon did not potentiate this response significantly. The combination of NK1- and NK2-receptor antagonists blunted the response at 5 min only slightly but markedly attenuated the later (10–20 min) response. These results show that tachykinins released from sensory nerves play a significant role in antigen-induced bronchoconstriction in guinea pigs. This effect is exaggerated when the normal modulation of neuropeptides by NEP is inhibited and is mediated predominantly by NK2-receptor activation, with a smaller contribution by NK1 receptors.
Dilator effect of endothelins in pulmonary circulation: changes associated with chronic hypoxia
Authors: Eddahibi S. , Springall D. , Mannan M. , Carville C. , Chabrier P. E. , Levame M. , Raffestin B. , Polak J. , Adnot S.
Published: December 2017

To investigate dilator effects of endothelins (ETs) on the pulmonary circulation and possible changes induced by chronic hypoxia, we examined vascular responses to ET-1 and ET-3 as well as ET binding to receptor subtypes ETA and ETB in the lungs from rats exposed to either room air (controls), hypoxia (10% O2) for 3 wk (3 WH), or 3 WH followed by recovery to room air (3 WH+R). In controls, both ETA and ETB receptor binding was present in smooth muscle of airways and vessels. Infusion of ET-1 or ET-3 (3-100 pM) to isolated perfused lungs preconstricted by U-46619 produced dose-dependent vasodilation with a greater potency of ET-3 (P < 0.01). The vasodilator responses to ET-1 and ET-3 were potentiated by the cyclooxygenase blocker meclofenamate (3 x 10(-6) M) or by the thromboxane synthetase inhibitor R-68070. In meclofenamate-treated lungs, the vasodilator responses to ET-1 and ET-3 remained unaffected by the inhibitor of nitric oxide synthesis, NG-monomethyl-L-arginine (5 x 10(-4) M) or by the guanylate cyclase inhibitor, methylene blue (10(-4) M). Conversely, the K+ channel blockers glibenclamide (10(-4) M) and tetraethylammonium (10(-4) M) attenuated the vasodilator responses to both ET-1 and ET-3. The selective ETA receptor antagonist BQ-123 did not alter ET-induced vasodilation, whereas it attenuated ET-induced vasoconstriction. Vasodilation to both ET-1 and ET-3 was abolished in lungs from 3 WH rats (P < 0.01) but was fully restored in lungs from 3 WH+R rats. Pulmonary vasodilation induced by the K+ channel opener pinacidil, which was suppressed by glibenclamide, did not differ between controls and 3 WH rat lungs. We found no change in ETA and ETB receptor binding from pulmonary vessels in H rat lungs compared with controls. In conclusion, endothelin-induced pulmonary vasodilation which may involve activation of K+ channels is abolished during chronic hypoxia. This abolition does not appear to be related to alterations in ET-receptor subtypes or to unresponsiveness of K+ channels in the pulmonary circulation.
Airway vasodilation by bradykinin is mediated via B2 receptors and modulated by peptidase inhibitors
Authors: Yamawaki I. , Geppetti P. , Bertrand C. , Chan B. , Nadel J. A.
Published: December 2017

We studied the effect of exogenous bradykinin on blood flow in the airway microcirculation of anesthetized F344 rats in vivo. We made three successive determinations of airway blood flow and cardiac output using a modification of the reference sample microsphere technique. Injection of bradykinin into the left ventricle increased airway blood flow in a dose-related manner. Pretreatment with the bradykinin B2 receptor antagonist, Hoe 140, completely abolished bradykinin-, but not histamine-induced vasodilation. A bradykinin B1 receptor agonist, [des-Arg9]bradykinin, did not affect airway blood flow. We also studied the effect of inhibitors of angiotensin-converting enzyme (captopril) and neutral endopeptidase (phosphoramidon) on bradykinin-induced vasodilation. Pretreatment with captopril, but not phosphoramidon, potentiated the bradykinin-induced vasodilation. However, the addition of phosphoramidon further potentiated the effect of captopril. We conclude that injection of bradykinin into the left ventricle produces a dose-related vasodilation in the airway microcirculation mediated via B2 receptors, an effect that is modulated primarily by angiotensin-converting enzyme and, to a lesser extent, by neutral endopeptidase.
Ontogeny and effects of exogenous surfactant treatment on SP-A, SP-B, and SP-C mRNA expression in rabbit lungs
Authors: Ohashi T. , Polk D. , Ikegami M. , Ueda T. , Jobe A.
Published: December 2017

The aims of the present study were to first characterize the ontogeny of mRNAs encoding the principal surfactant proteins SP-A, SP-B, and SP-C in rabbit lung tissues, followed by an evaluation of the effect of exogenous surfactant administration on endogenous expression of mRNA levels. Lung tissues were collected from New Zealand White rabbits on days 19, 21, 24, 26, 28, 30 of gestation, 1 day and 10 days after birth, and as adults. SP-A, SP-B, and SP-C mRNAs were first detected on gestational days 26, 24, and 19, respectively. The specific levels of these mRNAs generally increased to gestational day 28 and decreased transiently on 1 day of life. Adult values remained lower than those noted at 30 days gestation. For the second study, 29-day-gestation preterm rabbits were given either one or two treatment doses of natural surfactant or an organic solvent extract of natural surfactant, and mRNA levels were measured 10 or 24 h later. No effect of surfactant treatments, type of surfactant, or dosage (1 or 2 treatments) was noted on SP-A, SP-B, or SP-C mRNA expression. These data indicate that the mRNA expression for the three major surfactant proteins was highest late in gestation in developing rabbits and that exogenous surfactant treatment was not associated with significant changes in SP-A, SP-B, or SP-C mRNA levels.
Hypoxia induces the synthesis of tropomyosin in cultured porcine pulmonary artery endothelial cells
Authors: Rao U. J. , Denslow N. D. , Block E. R.
Published: December 2017

The present study examined the effect of hypoxia on protein synthesis by porcine pulmonary artery endothelial cells (PAEC). Hypoxia decreased protein synthesis in PAEC, but two-dimensional gel electrophoresis of [35S]methionine-labeled PAEC proteins demonstrated the increased synthesis of a set of proteins having molecular masses (M(r)) of 35, 36.5, 45, 116, and 205 kDa. The synthesis of the 35-, 36.5-, and 45-kDa proteins was increased in preconfluent and postconfluent cells. The 35- and 45-kDa proteins were not induced by hyperthermia, whereas the 36.5-kDa protein was induced slightly by hyperthermia. Induction of the 36.5- and 45-kDa proteins required a minimum of 8 h of hypoxia, whereas induction of the 35-kDa protein required only 4 h of exposure to hypoxia. The upregulated synthesis of the 35-, 36.5-, and 45-kDa proteins was reversible with return to normoxia. Actinomycin D, an inhibitor of transcription, did not block the hypoxic induction of the 35- and 36.5-kDa proteins but did block induction of the 45-kDa protein. The partial amino acid sequence of the 35-kDa protein obtained from cyanogen bromide cleavage of the molecule was Asp-Ala-Ile-Lys-Lys-Lys-Met-Gln-Met-Leu-Lys-Leu-Asp-Lys-Glu. This partial sequence of the 35-kDa protein identically matches the sequence of tropomyosin. Amino acid composition data and the isoelectric point (4.8) were also typical of tropomyosin. Finally, specific cross-reactivity was detected between the 35-kDa protein and a monoclonal antibody to chicken gizzard tropomyosin on immunoblot. Thus hypoxia induces the synthesis of tropomyosin, a major microfilament-associated protein, in porcine PAEC in monolayer culture.
Glutathione transport by type II cells in perfused rat lung
Authors: Bai C. , Brown L. A. , Jones D. P.
Published: December 2017

Glutathione (GSH) is an antioxidant that protects the lung against oxidative-injury. Most cells rely on synthesis of GSH to maintain intracellular supply and only a few cell types take up intact GSH. Although isolated type II cells from rat have a Na(+)-dependent uptake system that transports GSH into the cells against a concentration gradient, it is not known whether this occurs from the vasculature in the intact lung or whether other cell types in the lung also transport GSH. Based on the knowledge that gamma-glutamyl analogues of GSH are also transported by the Na(+)-GSH transporter, a method was developed and used to study the cell specificity of GSH uptake in perfused lung. A stable, fluorescent GSH S-conjugate (GSH-I14) was synthesized and separated from the original dye as analyzed by high-performance liquid chromatography. Studies with isolated alveolar type II cells showed that uptake of GSH-I14 was Na+ dependent and inhibited by GSH. In addition, uptake of GSH by the type II cells was inhibited by GSH-I14. After perfusion of the isolated rat lung with GSH-I14, the conjugate accumulated primarily in the alveolar type II cell as observed by fluorescence microscopy. This was confirmed by isolation of type II cells and measurement of GSH-I14 content. Thus these results show that specificity of GSH transport can be studied with the fluorescent derivative, GSH-I14, and that in the isolated perfused lung type II cells can transport and concentrate GSH-I14 from the perfusate. Quantitative fluorescence microscopy will be required to further determine relative transport activities by other cell types.
Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes
Authors: Bates S. R. , Dodia C. , Fisher A. B.
Published: December 2017

We have investigated the internalization of surfactant by type II cells in primary culture. Previously, we demonstrated that type II cells cultured on microporous membranes [Transwell membranes (TM)] maintained the morphological characteristics of lung pneumocytes and took up surfactant protein and phospholipid in a fashion similar to that described for the uptake of surfactant by whole lung. In the present study, cells cultured on TM and exposed to equivalent amounts of phospholipid (PL) as either natural surfactant or liposomes incorporated fivefold greater amounts of natural surfactant PL. The evidence supported an important role for surfactant protein A (SP-A), as the incorporation of surfactant into type II cells on TM was reduced by anti-SP-A antisera and by pretreatment of the surfactant with beta-mercaptoethanol. In addition, the uptake of liposomes into type II cells on TM was augmented by SP-A. With the use of iodinated bovine SP-A reconstituted in rat surfactant, cells cultured on TM showed a 2.6-fold increase in binding and a 3.2-fold stimulation in uptake of SP-A over that seen with cells cultured on plastic. The change in the slope of the total binding curve of 125I-labeled bovine SP-A reconstituted with bovine surfactant to type II cells on the two substrates occurred at the same concentration of SP-A (17 micrograms/mg), but maximal binding was approximately eight times higher for cells on TM than for cells on plastic dishes. Thus, in a more physiological environment, i.e., SP-A in surfactant and cells on microporous membranes, SP-A plays an important role in the uptake of phospholipids by alveolar pneumocytes.
Smoke-induced emphysema in guinea pigs is associated with morphometric evidence of collagen breakdown and repair
Authors: Wright J. L. , Churg A.
Published: December 2017

We have previously shown that chronic cigarette smoke exposure produces emphysema and airflow obstruction in the guinea pig. To further examine the changes in the connective tissue matrix associated with emphysema in this model, we used ultrastructural morphometry to determine the volume proportions of collagen and elastin in the alveolar walls of animals exposed to smoke or air (control) for 1, 3, 6, and 12 mo. After 1 mo of smoke exposure, there was a statistically significant (P < 0.001) decrease in the volume proportion of collagen in the smoke-exposed animals, whereas by 6 and 12 mo of smoke exposure, the proportion of collagen had significantly (P < 0.02, P < 0.03, respectively) increased. The volume proportion of elastin was increased in the smoke-exposed animals at the 12-mo time period. While our results do not exclude reorganization of elastin within the alveolar wall, we conclude that, in this model, cigarette smoke-induced emphysema appears to be associated with collagen breakdown and repair. We suggest that the currently accepted proteolysis-antiproteolysis theory is too narrow in its focus on elastin destruction as the major contributor to emphysema and should be broadened to the concept that smoke-induced emphysema reflects breakdown and resynthesis (possibly overproduction in the form of scarring) of a variety of connective tissue proteins in addition to elastin.
Bioelectric properties of cultured monolayers from epithelium of distal human fetal lung
Authors: Barker P. M. , Boucher R. C. , Yankaskas J. R.
Published: December 2017

Throughout intrauterine life, Cl(-)-rich liquid is secreted by the pulmonary epithelium. To evaluate the role of the most distal epithelium in liquid secretion, we measured bioelectric properties of monolayers composed of epithelial cells from acinar structures of postmortem human fetal lung (mean gestation, 22.3 wk; range, 18-24 wk). These monolayers formed high-resistance (R) barriers (mean R = 363 Ohm/cm2) when cultured in hormone-supplemented, serum-free medium. The transepithelial electrical potential difference (4.0 mV, lumen negative), was similar to that of whole fetal sheep lung in vivo. Equivalent short-circuit current (Ieq) was inhibited by apical amiloride (-20%), 5-(N-ethyl-N-isopropyl)-amiloride (-33 to -49%), or diphenylamine-2-carboxylate (DPC; -26%), and by basolateral ouabain (-77%), whereas apical 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) had no effect. Bumetanide added to the basolateral bath did not affect resting Ieq, but inhibited Ieq (-19%) in monolayers pretreated with apical amiloride, basolateral terbutaline, and apical ATP, and also inhibited Ieq (-22%) of monolayers pretreated with basolateral amiloride and DIDS. Ieq was stimulated by terbutaline (90–128%), ATP (70–186%), and ionomycin (141%). Stimulation of Ieq by these agents is compatible with induction of Cl- secretion through two pathways: channels that are opened by a rise in adenosine 3',5'-cyclic monophosphate, and channels that are opened by a rise in intracellular Ca2+. Inhibition of Ieq by apical DPC implies that Cl- secretion may contribute to basal Ieq.
Transcription and mRNA stability regulate developmental and hormonal expression of rabbit surfactant protein B gene
Authors: Margana R. K. , Boggaram V.
Published: December 2017

Surfactant protein B (SP-B), a hydrophobic protein of pulmonary surfactant, is essential for the surface tension-reducing properties of surfactant. In the present study, we isolated and characterized cDNAs encoding rabbit SP-B, and used transcription run-on assays and Northern blot analysis to investigate the role of transcriptional and posttranscriptional mechanisms in the developmental and cAMP and dexamethasone induction of SP-B mRNA. We found two forms of SP-B cDNAs that differed by an insertion of 69 nucleotides in the 3' untranslated regions. We found that transcription across the SP-B gene is nonequimolar and the 3' end of the gene has high levels of antisense transcription. SP-B gene transcription and SP-B mRNA levels increased during fetal lung development. However, increased SP-B mRNA levels could not be accounted for primarily on the basis of increased transcription. These results suggested that enhanced SP-B gene transcription and enhanced SP-B mRNA stability mediate developmental induction of SP-B gene. In rabbit fetal lung in vitro, both dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and dexamethasone increased SP-B mRNA levels. DBcAMP-dependent increase in SP-B mRNA levels resulted from increased SP-B gene transcription, whereas dexamethasone-dependent increase resulted from combined effects on increased SP-B gene transcription and SP-B mRNA stability. In tissues treated with dexamethasone the half-life (t1/2) of SP-B mRNA increased > 2.5-fold (t1/2 control = 9 h; t1/2 dex-treated = 25 h). These data show that both transcription and mRNA stability regulate induction of SP-B gene expression during fetal lung development and by cAMP and dexamethasone in fetal lung in vitro.
Human respiratory syncytial virus affects nonadrenergic noncholinergic inhibition in cotton rat airways
Authors: Colasurdo G. N. , Hemming V. G. , Prince G. A. , Loader J. E. , Graves J. P. , Larsen G. L.
Published: December 2017

A dysfunction of the nonadrenergic noncholinergic inhibitory (NANCi) system has been invoked as a possible mechanism underlying or contributing to altered airway function. In the present study we assessed whether human respiratory syncytial virus (HRSV) infection affects the airways' neurally mediated contractile and relaxant (NANCi) responses in vitro. NANCi responses were studied on tracheal smooth muscle (TSM) segments obtained from young adult cotton rats, a well-established model for HRSV infection. To assess NANCi responses, TSM segments were removed and placed in tissue baths containing modified Krebs-Henseleit, atropine (1 x 10(-6) M) and propranolol (5 x 10(-6) M). After contraction with neurokinin A (1 x 10(-5) M), electrical field stimulation (EFS) was applied at stimulation frequencies ranging from 5 to 30 Hz. The NANCi responses were measured and expressed as the mean (+/- SE) percent relaxation. To evaluate neurally mediated contractile responses, full frequency response curves (0.5-30 Hz) to EFS were also performed. We found significantly decreased NANCi responses in TSM segments obtained from infected cotton rats (n = 12) compared with control animals (n = 9) (P < 0.002). Furthermore, the contractile responses to EFS were increased in infected animals compared with the control group (P = 0.0001). These findings demonstrate that HRSV infection leads to an enhanced contractile response to EFS and a significant decrease in NANCi response in cotton rat airways in vitro. This disruption of the neural control of airways may lead to the development of altered airway function.
Alterations in pulmonary artery tone during repeated episodes of hypoxia
Authors: Yang B. C. , Mehta J. L.
Published: December 2017

To examine the basis of pulmonary constriction during chronic hypoxia, rat pulmonary artery rings were precontracted and exposed to multiple episodes of hypoxia. The first hypoxic episode resulted in a transient contraction, followed by potent relaxation, and then a slow sustained contraction. Repeated hypoxic exposure resulted in stronger initial contraction and attenuated relaxation. Prolongation of the normoxic interval between hypoxic episodes reversed the attenuation of hypoxic relaxation. Pulmonary artery rings that were deendothelialized or treated with the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester or oxyhemoglobin displayed only hypoxic relaxation without initial or late contractions and no attenuation of relaxation during repeated hypoxia. Pretreatment of rings with indomethacin or adenosine or endothelin receptor antagonists had no effect on the hypoxia-mediated alterations. Thus repetitive exposure to hypoxia results in an increase in initial contraction and a decrease in relaxation of pulmonary artery rings. Frequency of hypoxic episodes and endothelial integrity determine pulmonary tone during repeated hypoxia. However, cyclooxygenase products, adenosine, or endothelin do not play a role in hypoxia-mediated changes in rat pulmonary artery tone.
In situ localization of sodium-potassium ATPase mRNA in developing mouse lung epithelium
Authors: Crump R. G. , Askew G. R. , Wert S. E. , Lingrel J. B. , Joiner C. H.
Published: December 2017

The ontogeny of Na(+)-K(+)-adenosinetriphosphatase (ATPase) mRNA in the mouse lung was examined, using alpha- and beta-isoform-specific probes in Northern blot assays and for in situ hybridization analysis. Northern blot assays demonstrated an increase in Na(+)-K(+)-ATPase expression in the perinatal period, peaking at birth (D1), with alpha 1- and beta 1-isoform levels reaching six to eight times adult levels. In situ alpha 1-isoform hybridization signals were localized primarily to developing airway epithelium and were most intense on D1. Postnatally, alpha 1-isoform hybridization signals persisted in airway epithelium, although progressively diminishing in intensity relative to perinatal levels. In developing alveolar regions, alpha 1-isoform hybridization signals remained slightly above background during this period. beta 1-Isoform hybridization signals increased dramatically during the perinatal period in both developing airway and alveolar epithelia. Postnatally, beta 1-isoform hybridization signals declined slightly in airway epithelium and developed a punctate pattern in alveolar epithelium. These data indicate that the perinatal increase in Na(+)-K(+)-ATPase expression observed in the developing mouse lung is localized primarily to epithelial structures and is therefore likely to be related to the changes in transepithelial ion and fluid transport known to occur in the perinatal period.
In vivo lipopolysaccharide pretreatment inhibits cGMP release from the isolated-perfused rat lung
Authors: Kurrek M. M. , Zapol W. M. , Holzmann A. , Filippov G. , Winkler M. , Bloch K. D.
Published: December 2017

Administration of bacterial lipopolysaccharide (LPS) to rats stimulates synthesis of nitric oxide (NO), a free radical molecule that activates soluble guanylate cyclase, thereby increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration and inducing systemic vasodilatation. To investigate the effect of endotoxemia on the pulmonary NO/cGMP signal transduction system, we measured the release of cGMP by isolated-perfused lungs of rats that received an intraperitoneal injection of LPS (1 mg/kg) or saline 2 days earlier. Over 90 min, 1.4 +/- 0.78 and 0.079 +/- 0.016 nmol cGMP accumulated in pulmonary perfusates of saline- and LPS-treated rats, respectively (P < 0.05). Despite addition to the perfusate of Zaprinast, superoxide dismutase, or A23187, markedly less cGMP was released from the lungs of rats exposed to LPS than from the lungs of control rats. In contrast, after ventilation with 100 parts per million NO gas, cGMP accumulating in the perfusate of the lungs of both groups of rats was markedly increased, and the quantity of cGMP released from the lungs of LPS-treated rats was similar to that released by control rat lungs (2.8 +/- 0.57 vs. 3.3 +/- 0.88 nmol, P = NS). With the use of immunoblot techniques, equal concentrations of constitutive endothelial NO synthase were detected in the lungs of rats treated with saline or LPS. These results demonstrate that the NO/cGMP signal transduction system is abnormal in the lungs of rats exposed to LPS, at least in part, at the level of endothelial NO synthase activation.
Impact of angiotensin-converting enzyme substrate conformation on fractional hydrolysis in lung
Authors: Merker M. P. , Armitage I. M. , Audi S. H. , Kakalis L. T. , Linehan J. H. , Maehl J. R. , Roerig D. L. , Dawson C. A.
Published: December 2017

We examined the hydrolysis kinetics of benzoyl-phenylalanyl-glycyl-proline (BPGP) in the isolated perfused lung and in vitro for evidence of preferential hydrolysis of the trans isomer by angiotensin-converting enzyme (ACE). Nuclear magnetic resonance spectroscopy showed that BPGP exists as cis and trans isomers in a ratio of 44:56. After a single pass through the perfused rabbit lung over a wide range of infused BPGP concentrations, 42% of the BPGP was not hydrolyzed. In single-pass bolus-injection studies, 41% of the injected BPGP was not hydrolyzed, and very little further hydrolysis occurred in a second passage of the bolus through the lungs. In rat lung recirculation and in vitro studies of BPGP hydrolysis by ACE, approximately 60% of the substrate was hydrolyzed rapidly compared with the remaining approximately 40%, and the peptidyl-prolyl cis-trans isomerase cyclophilin increased the rate of the slower phase of the reaction in both kinds of experiments. We conclude that the rapid hydrolysis phase represents primarily the hydrolysis rate of the trans isomer and the slower phase the cis-trans isomerization rate, suggesting that the trans isomer of BPGP is preferentially hydrolyzed by ACE in the perfused lung and in vitro.
Regulation of ion transport across porcine distal bronchi
Authors: Inglis S. K. , Corboz M. R. , Taylor A. E. , Ballard S. T.
Published: December 2017

Distal airways comprise the vast majority of total human airway surface area, yet little is known about transepithelial ion transport in these tissues. Pathways that regulate ion transport in porcine small bronchi (3.62 +/- 0.04 mm outer diameter) were studied by measuring the effects of secretogogues that stimulate Cl- secretion in proximal airways. Resting potential difference (PD), shortcircuit current (ISC), and resistance (Rt) across the distal bronchi were 3.4 +/- 0.1 mV (lumen negative), 40.8 +/- 1.7 microA/cm2, and 92.1 +/- 5.0 omega.cm2, respectively. Isoproterenol (10 microM), acetylcholine (ACh; 10 microM), or ATP (100 microM) induced immediate significant (P < 0.05) increases in PD and ISC. The Ca2+ ionophore A23187 (1 microM) induced a slow increase in PD that was accompanied by a significant 2.4-fold increase in Rt and no change in ISC. The responses to isoproterenol, ACh, and ATP were maintained in the presence of 10 microM amiloride. Pretreatment with 10 microM bumetanide to block Cl- secretion inhibited responses to isoproterenol and ATP but not to ACh. The ACh effect was inhibited only after both Cl- and HCO3- secretion were blocked. These data suggest that isoproterenol, ACh, and ATP stimulate anion secretion. Isoproterenol and ATP specifically stimulate Cl- secretion, whereas ACh can stimulate both Cl- and HCO3- secretion. A23187 has no effect on active transepithelial ion transport but increases barrier resistance in intact distal bronchial epithelium.
Sensory denervation by neonatal capsaicin treatment exacerbates Mycoplasma pulmonis infection in rat airways
Authors: Bowden J. J. , Baluk P. , Lefevre P. M. , Schoeb T. R. , Lindsey J. R. , McDonald D. M.
Published: December 2017

Mycoplasma pulmonis infection in rats results in life-long disease, characterized by chronic inflammation of the airway mucosa with widespread accumulation of lymphoid tissue, mucous cell hyperplasia, and mucosal thickening. In addition, there is angiogenesis and increased sensitivity of mucosal blood vessels to substance P (SP), so tachykinins released from sensory nerve fibers cause an abnormally large amount of plasma leakage. We sought to learn whether the sensory nerves influence the severity of the chronic inflammatory response of M. pulmonis infection. Our strategy was to destroy the nerves by capsaicin pretreatment at birth, infect the rats with M. pulmonis at 8 wk of age, and then study the animals 6 wk later. We found that capsaicin pretreatment increased the severity of the infection, exaggerated the pathological changes in the tracheal mucosa, and increased the amount of SP-induced plasma leakage, as quantified with Monastral blue. The thickness of the tracheal mucosa in these infected rats was 80% greater than in their vehicle-pretreated counterparts and 200% greater than in the pathogen-free controls. The area density of Monastral blue-labeled blood vessels averaged 20% in the infected rats pretreated with capsaicin, which represented a 40-fold increase over the leakage in the pathogen-free group. By comparison, the amount of Monastral blue labeling was only 13% in rats pretreated with vehicle (P<0.05), which was a 22-fold increase over the corresponding pathogen-free group. The number of SP-immunoreactive nerve fibers was reduced both by neonatal capsaicin and by infection (87 and 63% reductions, respectively); but when the two conditions were combined, their effects were not additive (79% reduction), perhaps because of nerve regrowth. We conclude that destruction of sensory nerves increases the severity of infection- induced chronic inflammation in the airway mucosa, with exaggerated mucosal thickening, angiogenesis, plasma leakage, and nerve remodeling.
Eosinophil VLA-4 binding to fibronectin augments bronchial narrowing through 5-lipoxygenase activation
Authors: Munoz N. M. , Rabe K. F. , Neeley S. P. , Herrnreiter A. , Zhu X. , McAllister K. , Mayer D. , Magnussen H. , Galens S. , Leff A. R.
Published: December 2017

We examined the effect of ligation of human eosinophils activated by platelet-activating factor (PAF) to soluble human fibronectin (FN) on the augmented contractile response of human bronchial explants. Styrene microplate wells were FN-coated and eosinophils were allowed to adhere in the presence of 1) buffer control, 2) 20 micrograms/ml monoclonal antibody (HP2/1) to the alpha 4 beta 1 ligand (VLA-4) on the eosinophils, 3) 20 micrograms/ml anti-CD18 R15.7, 4) 20 micrograms/ml anti-CD16 3G8, or 5) 10(-6) M A63162, a 5-lipoxygenase inhibitor. Sixty minutes later, treated cells were activated with either buffer or 10(-6) M PAF. Airway luminal diameter was assessed by computerized videomicrometry as a function of pixel number, and activation of eosinophils was confirmed by measurement of leukotriene C4 (LTC4) secretion. Ligation with FN caused an increase in PAF-stimulated LTC4 secretion from 276 +/- 75.6 pg/10(6) cell at baseline to 606 +/- 90.2 pg/10(6) cell (P < 0.01). This corresponded to augmented luminal narrowing of human bronchial explants from 25.3 +/- 9.39% (PAF activation alone) to 42.9 +/- 8.0% (PAF-activated eosinophils + FN) (P < 0.01). Both augmented airway luminal narrowing and increased LTC4 secretion caused by PAF-activated cells after FN ligation were blocked completely by anti-VLA-4 MAb (P < 0.05 vs. control). Pretreatment with 10(-6) MA63162 inhibited completely the PAF-stimulated LTC4 secretion to baseline level ( P < 0.001). Inhibition of 5-lipoxygenase similarly blocked luminal narrowing caused by eosinophils stimulated by PAF by > 95% (P < 0.001). We demonstrate that the binding of human eosinophils to the matrix protein FN causes augmented secretion of LTC4 which, in turn, causes augmented luminal narrowing of explanted human bronchi in vitro. We also demonstrate that the augmented activity is blocked selectively by pretreatment with specific monoclonal antibody against VLA-4 and blockade of eosinophil 5-lipoxygenase inhibits both LTC4 secretion and airway narrowing after PAF-stimulation.
Ion transport properties of fetal sheep alveolar epithelial cells in monolayer culture
Authors: Tessier G. J. , Lester G. D. , Langham M. R. , Cassin S.
Published: December 2017

Alveolar type II cells were isolated from late-term fetal sheep to investigate ion transport across fetal distal lung epithelium. In Ussing chambers, basal transepithelial potential difference (PD; reference apical side), equivalent short-circuit current (Ieq), and resistance were -0.10 +/- 0.05 mV, 0.10 +/- 0.08 microA/cm2, and 821.5 +/- 38.8 omega .cm2, respectively. Epinephrine (100 nM) increased PD from -0.13 +/- 0.19 to -1.37 +/- 0.20 mV and Ieq from 0.18 +/- 0.26 to 1.47 +/- 0.28 microA/cm2. Propranolol (100 nM) inhibited responses to epinephrine. Forskolin (10 microM) increased PD to -0.81 +/- 0.08 mV and Ieq to 1.02 +/- 0.12 microA/cm2. Mucosal amiloride (200 microM) and serosal bumetanide (10 microM) decreased the forskolin-stimulated PD by 23.42 +/- 4.73 and 25.57 +/- 3.9%, respectively. We conclude that in fetal sheep distal lung epithelium amiloride-inhibitable sodium absorption and bumetanide-sensitive chloride secretion are stimulated by forskolin and that epinephrine effects on ion transport are mediated by beta-adrenergic receptors.
T cell activation in a murine model of asthma
Authors: Krinzman S. J. , De Sanctis G. T. , Cernadas M. , Kobzik L. , Listman J. A. , Christiani D. C. , Perkins D. L. , Finn P. W.
Published: December 2017

To determine the mechanisms by which inhaled antigens produce pulmonary inflammation and bronchial hyperreactivity, we have developed a murine model of asthma. BALB/c mice are sensitized and challenged with ovalbumin (OVA). Compared with mice treated with phosphate-buffered saline (PBS), OVA-treated mice developed increased lung resistance, decreased dynamic compliance, and greater methacholine reactivity. Bronchoalveolar lavage fluid revealed significant increases in the proportion of neutrophils and eosinophils. Tissue sections of OVA-treated mice demonstrated goblet cell metaplasia and focal perivascular and peribronchial infiltrates composed of lymphocytes, neutrophils, and eosinophils. Analysis of thoracic lymphocytes via flow cytometry revealed an expansion of both CD4+ and B cell populations, with increased expression of interleukin-2 receptor on CD4+ T cells, indicated increased activation. There was also increased expression of CD44 on CD4+ and CD8+ lymphocytes, suggesting an expansion of the local memory cell population. These findings support the hypothesis that activation of T lymphocytes mediates allergic pulmonary inflammation and bronchial reactivity in asthma.
Effects of inflammation and acute beta-agonist inhalation on beta 2-AR signaling in human airways
Authors: Penn R. B. , Shaver J. R. , Zangrilli J. G. , Pollice M. , Fish J. E. , Peters S. P. , Benovic J. L.
Published: December 2017

Although alterations in beta 2-adrenergic receptor (AR) responsiveness may in part explain reports linking deterioration of asthma control with beta-agonist treatment of asthmatics, few data exist on beta 2-AR regulation in human airway cells. We have employed a bronchoscopy model to examine inflammation- and beta-agonist-induced alterations in human bronchial epithelial cell beta 2-AR density and responsiveness. Allergic asthmatic subjects participated in 2-day protocols examining airways before and 24 h after segmental antigen challenge (SAC) with ragweed. To assess the effect of acute beta-agonist exposure, bronchoscopies were performed both with (+ beta-Ag) and without (-beta-Ag) inhalation of beta-agonist 30 min before the procedure. Measurements of inflammatory cell infiltration were obtained by analysis of bronchoalveolar lavage fluid, and beta 2-AR density and responsiveness were examined in bronchial epithelial cells obtained by bronchoscopic brushing. Neither SAC nor acute beta-agonist administration alone significantly affected epithelial cell beta 2-AR density. beta-Agonist-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) generation was significantly lower in the + beta-Ag groups compared with the-beta-Ag group, demonstrating acute agonist-specific beta 2-AR desensitization in vivo. SAC caused a small, statistically insignificant reduction in beta-Agonist-stimulated cAMP production in both -beta-Ag or + beta-Ag groups. These lata suggest that acute beta-agonist inhalation, but not airway inflammation, significantly reduces maximal beta 2-AR responsiveness in airway cells.
Leukotrienes are indicated as mediators of hyperoxia-inhibited alveolarization in newborn rats
Authors: Boros V. , Burghardt J. S. , Morgan C. J. , Olson D. M.
Published: December 2017

We investigated the role of leukotrienes (LT) in hyperoxia-induced changes in lung parenchyma in neonatal rat pups. Rat pups were exposed to 21% O(2) (air) or >95% O(2) from days 4 to 14 after birth and were administered the 5-lipoxygenase (5-LO) inhibitor and LTD4 receptor antagonist Wy-50295, 5-LO-activating protein inhibitor MK-0591, or vehicle from days 3 to 14. All measurements were done on days 12-14. There was a significant (P < 0.05) increase in peptido-LT output from lung slices of animals exposed to O(2) compared with air-exposed animals. Both Wy-50295 and MK-0591 significantly lowered (P < 0.05) peptido-LT output in O(2)-exposed animals. The 6-ketoprostaglandin F(1alpha) output was increased similarly in both vehicle- and drug-treated O(2)-exposed animals. O(2) exposure also caused a significant increase in bronchoalveolar lavage fluid protein and extravascular lung water that could not be ameliorated by Wy-50295 or MK-0591. Hyperoxia-induced inhibition of alveolarization, indicated by a significantly (P < 0.05) lower parenchymal tissue density, specific internal surface area, and airspace perimeter-to-area ratio, and a significantly (P < 0.05) higher mean linear intercept and airspace unit volume than air-exposed animals, was prevented by both Wy-50295 and MK-0591. Although hyperoxia had no effect on septal thickness, Wy-50295 caused significant thickening in both air- and O(2)-exposed pups. Our studies provide evidence that hyperoxia-induced peptido-LT may mediate O(2)-induced inhibition of alveolarization and that this is not caused by an arachidonic acid shunt to cyclooxygenase.