bluuebunny/crossref_metadata_2025 huggingface dataset:
Solubilization of active receptors for glucagon-like peptide-1(7-36)amide from rat lung membranes
Authors: Goke R. , Kolligs F. , Richter G. , Lankat-Buttgereit B. , Goke B.
Published: December 2017

We report on a protocol that allows the solubilization of active glucagon-like peptide (GLP)-1-(7–36)amide receptors from rat lung membranes. Digitonin-solubilized GLP-1(7–36)amide binding proteins from lung membranes most effectively, whereas (3-[(3-cholamidopropyl)- dimethylamino]-1-propane-sulfonate was less affective, and octyl-beta-glucoside, Triton X-100 and Lubrol PX were almost ineffective. Solubilization of binding activity was optimal at a digitonin concentration of 1%, a protein-to-detergent ratio of 1:10, and a pH between 7.0 and 8.0. Binding of GLP-1(7–36)amide to solubilized receptors was dependent on the concentration of solubilized protein. The presence of certain mono- and divalent cations was crucial for binding of GLP-1(7–36)amide to solubilized receptors. Scatchard analysis of the binding data revealed a single class of binding sites with dissociation and maximum binding constant values of 0.40 +/- 0.20 nM and 80.0 +/- 26.0 fmol/mg protein for membrane bound and 7.0 +/- 0.6 microM and 12.0 +/- 6.0 nmol/mg protein for solubilized receptors, respectively. In cross-linking experiments 125I-labeled GLP-1(7-36)amide was covalently attached to GLP-1(7–36)amide receptors on lung membranes. The apparent molecular mass of the solubilized receptor was 55,000 Da. This was proven in another experiment when receptor was consecutively cross-linked after solubilization. Nonhydrolyzable GTP analogues (GTP gamma S or GDP beta S) were unable to reduce GLP-1(7–36)amide-binding at solubilized receptors. This argues that the receptor is solubilized as a single protein and not as a receptor-G protein complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Tubule formation by human surface respiratory epithelial cells cultured in a three-dimensional collagen lattice
Authors: Benali R. , Tournier J. M. , Chevillard M. , Zahm J. M. , Klossek J. M. , Hinnrasky J. , Gaillard D. , Maquart F. X. , Puchelle E.
Published: December 2017

Human surface respiratory epithelial (HSRE) cells from nasal polyps have been cultured within collagen lattices in a serum-free defined medium. Cell growth observed over a period of 12 days showed a population doubling time of 36 h. Under these culture conditions, we observed a contraction of the lattices. Phase-contrast light microscopy and transmission electron microscopy demonstrated that the HSRE cells formed tubular ductlike structures. Lumens formed by HSRE cells were surrounded by cuboidal-shaped polarized cells with numerous ciliated cells, secretory cells, and undifferentiated cells. Epidermal growth factor (EGF) was observed to stimulate the tubule formation and the contraction of the lattices. Videomicroscopic observations and analysis of the ciliary beating frequency (CBF) demonstrated that the cilia were homogeneously distributed on the whole apical surface of the ciliated cells and that their movement was well coordinated, with a CBF similar to that observed in outgrowth cells from cultured human nasal and tracheal epithelia. Immunofluorescent staining of basement membrane components synthesized and secreted by cells revealed the presence of type III collagen around the tubules. Type IV collagen and laminin were present in the cytoplasm and at the periphery of the cells. The biotin-streptavidin-gold immunocytochemical technique with monoclonal anti-mucin antibody showed intracellular localization of mucins in secretory granules of the secretory cells. With the use of substrate gel electrophoresis polyacrylamide gels impregnated with gelatin, collagenase activity was detected in the conditioned medium of the cultured HSRE cells. These results suggest that both three-dimensional collagen gel and soluble factors such as EGF regulate tubule formation by HSRE cells. Moreover, the capacity of the epithelial cells to contract the gel suggests they may be involved in the wound healing process.
Quantitative evaluation of gene expression in freshly isolated human respiratory epithelial cells
Authors: Trapnell B. C.
Published: December 2017

Recent advances in molecular biology have led to development of methods to evaluate the in vivo expression of specific genes in epithelial cells of the human respiratory tract. These methods involve collection of small samples of respiratory epithelium from the human airway with a cytology brush, together with sensitive techniques to enumerate cells and to detect and quantify RNA using the polymerase chain reaction (PCR). Thus, using the typical kinds of tissue samples obtainable in a clinical setting, one can now evaluate in vivo expression of specific genes in human airway epithelium. Specifically, mRNA transcript levels can be determined on a copy number per cell basis even when the level of expression is in the range of 1-10 mRNA transcripts/cell. The currently available techniques for measuring mRNA transcript levels in small tissue samples are reviewed with emphasis on the use of quantitative PCR assays.
Pertussis toxin attenuates platelet-activating factor-induced pulmonary hemodynamic alterations in pigs
Authors: Olson N. C. , Kruse-Elliott K. T. , Whorton A. R. , Dodam J. R.
Published: December 2017

To determine whether platelet-activating factor (PAF)-induced release of cyclooxygenase products might be dependent on G proteins in vivo, we administered pertussis toxin (PTX) (9.7-10.0 micrograms/kg iv) to conscious pigs approximately 48 h before bolus infusions of PAF (10 ng/kg). Autoradiography of ADP-ribosylated lung cell membrane proteins from PTX-treated pigs demonstrated marked reduction in the amount of radiolabel ([32P]NAD) incorporated, indicating that PTX induced ADP-ribosylation of G proteins in vivo. PAF, infused at hourly intervals from 0-4 h, caused increases in plasma concentrations of thromboxane B2 (TxB2) concomitant with pulmonary hypertension and vasoconstriction in anesthetized pigs. These physiological changes were blocked or markedly attenuated by indomethacin, indicating they were dependent on cyclooxygenase products. In PTX-treated pigs, the PAF-induced pulmonary hypertension and vasoconstriction were modestly attenuated, whereas the increases in plasma TxB2 were markedly attenuated. PTX prevented PAF-induced aggregation of platelets in vivo as evidenced by blockade of thrombocytopenia. However, in vitro, PAF-induced aggregation of platelets was independent of PTX. Moreover, incubation of platelet-rich plasma with 50 microM PAF failed to increase TxB2 levels. These findings suggested that a PTX-sensitive cell other than the platelet was responsible for triggering release of TxA2 and thrombocytopenia in vivo. We conclude that PAF-induced release of TxA2, pulmonary vasoconstriction, and thrombocytopenia in anesthetized pigs are dependent on a PTX-sensitive G protein; however, the residual hemodynamic effects indicate involvement of a PTX-insensitive G protein, or alternatively, G protein independent pathways.
Low-conductance chloride channels in IEC-6 and CF nasal cells expressing CFTR
Authors: Bijman J. , Dalemans W. , Kansen M. , Keulemans J. , Verbeek E. , Hoogeveen A. , De Jonge H. , Wilke M. , Dreyer D. , Lecocq J. P. , et al.
Published: December 2017

The properties of the cystic fibrosis gene product (CFTR) were studied by expression of cloned cDNA in different cell systems. Infection of both simian fibroblast (Vero) cells and immortalized CF nasal polyp cells (NCF3A) with a vaccinia virus encoding CFTR induced forskolin-induced Cl- permeability and low-conductance (8 pS) Cl- channels. By stable transfection of the rat intestinal crypt-derived cell line IEC-6 we have isolated a clone, IEC-CF7, which expresses CFTR mRNA and antigen. IEC-CF7 cells, but not IEC-6, display forskolin-induced Cl- permeability and multiple linear low-conductance (+/- 8 pS) Cl- channels in cell-attached membrane patches. In excised patches of IEC-CF7 cells, low-conductance Cl- channels could be activated by addition of the catalytic subunit of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) plus ATP. During bath fluid replacement studies, the activated low-conductance channel remained active in the absence of ATP at room temperature and showed saturation kinetics. Rectifying (32 pS) Cl- channels were not observed in either IEC-6 cells or IEC-CF7 cells, indicating that there is no relation between CFTR expression and the incidence of this channel. Our data strongly support the conclusion that CFTR can act as a low-conductance Cl- channel, gated by PKA. The IEC-6-derived cell line IEC-CF7 may prove to be a useful model in the study of CFTR function because of the absence of 32-pS Cl- channel activity and its potential for differentiation.
Biphasic glucocorticoid regulation of pulmonary SP-A: characterization of inhibitory process
Authors: Iannuzzi D. M. , Ertsey R. , Ballard P. L.
Published: December 2017

Pulmonary surfactant, which is necessary for normal lung function, is under both developmental and hormonal regulation. Glucocorticoids induce all components of surfactant and have a unique biphasic effect on surfactant protein A (SP-A), either stimulating or inhibiting accumulation in cultured fetal lung depending on dose and time of exposure. In this study we further characterized glucocorticoid inhibition of SP-A in cultured explants of human fetal lung. Decreased content of SP-A mRNA was the dominant response to dexamethasone added either early or later during culture. Inhibition occurred at < or = 1 nM dexamethasone on prolonged exposure, was blocked by RU 486, and was observed with other glucocorticoids but not sex steroids. When cortisol was removed from the culture medium, inhibition was rapidly reversed. The immediate inhibitory effect of 100 nM dexamethasone on SP-A mRNA content was completely blocked in the presence of cycloheximide. SP-A gene transcription, measured by nuclear elongation assay, was decreased by 60% after 4- to 8-h exposure to 100 nM dexamethasone. Stability of SP-A mRNA, determined both by addition of actinomycin D and by label-chase experiments, was transiently decreased immediately after adding dexamethasone (t1/2 approximately 3 h). In tissue treated with dexamethasone for > or = 8 h the stability of SP-A mRNA in control and treated explants was not different (t1/2 approximately 8 h). Our findings indicate that inhibition of SP-A is the dominant response to glucocorticoid. This effect is receptor mediated and apparently involves induction of a labile protein(s) that decreases gene transcription and transiently reduces mRNA stability.
Increased pulmonary vascular permeability in rats with biliary cirrhosis: role of thromboxane A2
Authors: Chang S. W. , Ohara N.
Published: December 2017

Rats with liver cirrhosis exhibit arterial hypoxemia and loss of hypoxic pulmonary vasoconstriction similar to some patients with end-stage liver disease. We hypothesized that the pulmonary circulatory dysfunction in cirrhosis results from vascular endothelial cell injury and interstitial lung edema. To investigate this hypothesis, we compared the extravascular lung albumin leak, lung ultrastructural changes, and tissue eicosanoid levels in control and cirrhotic rats. In comparison to sham-operated controls, rats with biliary cirrhosis, 6 wk after ligation of the common bile duct, had increased lung albumin leak index (1.46 +/- 0.12 vs. 0.80 +/- 0.04, P < 0.001) and bloodless lung wet-to-dry weight ratio (4.94 +/- 0.05 vs. 4.78 +/- 0.03, P < 0.05). Electron-microscopic sections of lungs from cirrhotic rats demonstrated infiltration with intravascular macrophage-like cells, endothelial cell injury, and interstitial edema. In addition, lung tissue thromboxane B2 was significantly increased in cirrhotic rats, and pretreatment with a thromboxane synthase inhibitor, dazoxiben, reduced lung thromboxane B2 level and attenuated extravascular lung albumin leak (control 1.03 +/- 0.07, cirrhotic 2.29 +/- 0.06, cirrhotic plus dazoxiben, 1.57 +/- 0.17). In contrast, WEB 2086, a platelet-activating factor antagonist, had no effect on lung albumin leak. We conclude that pulmonary vascular permeability is increased in rats with biliary cirrhosis and that thromboxane A2 contributes to the pulmonary circulatory abnormalities in cirrhosis.
Increased surfactant internalization by rat type II cells cultured on microporous membranes
Authors: Chinoy M. R. , Dodia C. , Fisher A. B.
Published: December 2017

We evaluated the influence of various substrata in maintaining internalization of biosynthesized natural surfactant by type II pneumocytes in primary culture. Cells were incubated for 2 h with 3H, 35S-labeled surfactant, prepared by perfusing isolated rat lungs with [3H]-choline + [35S]methionine, and analyzed for trypsin-resistant radiolabel. At 24 h of culture, uptake of [3H]phosphatidylcholine (PC) was 71.9 +/- 6.1 micrograms/mg protein and 35S-protein was 12.4 +/- 2.4 micrograms/mg cell protein for cells cultured on Transwell membranes. Uptake with this substratum was significantly greater than for cells cultured on plastic, Primaria, or Transwell-COL and was maintained for 72 h of culture. [3H]PC/35S-protein (micrograms/micrograms) for cells on Transwell was 5.8 +/- 0.7 compared with 3.4 +/- 0.1 in isolated surfactant. Results were similar at 72 h of culture. 8-Bromoadenosine 3',5'-cyclic monophosphate or terbutaline significantly stimulated the uptake of both surfactant components for cells on Transwell, but not by cells cultured on plastic. By light and electron microscopy, cells cultured on Transwell maintained characteristics of granular pneumocytes, including cuboidal shape, lamellar bodies, and microvilli. We conclude that the use of Transwell microporous membrane for primary culture of rat type II cells maintains their differentiated characteristics as assessed by the uptake of lung surfactant and its response to beta-adrenergic agonists.
Dexamethasone modulation of ion transport and fluid movement across airway epithelium
Authors: Satoh M. , Shimura S. , Ishihara H. , Yamada K. , Masuda T. , Sasaki T. , Sasaki H. , Takishima T.
Published: December 2017

Electrolyte or fluid is secreted across airway mucosa by both superficial epithelium and submucosal glands. To understand the effect of glucocorticoid on fluid movement across airway mucosa, we examined the effects of dexamethasone (Dex) on bioelectric properties of canine and feline tracheal epithelium and on 22Na efflux from isolated feline tracheal submucosal glands. Potential difference (PD) and short-circuit current (SCC) across tracheal epithelium were measured using an Ussing chamber, and conductance (G) was calculated as the ratio SCC/PD. Isolated glands were loaded with 22Na, and the rate constant (RC) of Na2+ efflux was calculated by measuring the radioactivity of each effluent sample. After treatment with 10(-9) to 10(-5) M Dex for up to 6 h, the epithelium and isolated glands were stimulated with isoproterenol (ISP) and methacholine (MCh), respectively. Dex treatment did not alter significantly baseline values of PD, SCC, or RC. However, Dex treatment produced a dose-dependent attenuation of ISP-evoked epithelial PD and SCC and of MCh-evoked glandular RC. In canine epithelium, pretreatment with 10(-5) M Dex for 6 h reduced by 40% the ISP (10(-6) M)-evoked rise in PD and SCC, whereas G remained unchanged. After 10(-5) M Dex treatment for 6 h, MCh (10(-5) M)-evoked RC in isolated glands was significantly less than in control glands (MCh alone) by 23%. These findings suggest that glucocorticoid decreases the fluid secretion across the airway mucosa, especially when the mucosa are stimulated.
Heparin inhibits histamine release from canine mast cells
Authors: Inase N. , Schreck R. E. , Lazarus S. C.
Published: December 2017

To determine the role of heparin in mast cell exocytosis, we studied the effect of heparin on histamine release induced by compound 48/80 or calcium ionophore A23187 in canine mastocytoma cells (BR). Heparin caused concentration-dependent inhibition of compound 48/80-induced histamine release from mast cells (n = 4; P < 0.05) with a mean inhibitory concentration of 0.14 +/- 0.01 U/ml (mean +/- SE). Mean maximal inhibition was 69.3 +/- 2.0%. In contrast, heparin had no effect on calcium ionophore A23187-induced histamine release. Although benzyl alcohol, a preservative of pharmaceutical heparin, had no effect, purified heparin produced a similar inhibitory effect on compound 48/80-induced histamine release (n = 4; P < 0.05). The inhibitory effect of heparin on histamine release was rapid and was eliminated by washing cells. Dextran sulfate, a polysaccharide with negative charge density, produced a similar inhibitory effect on compound 48/80-induced histamine release (n = 4; P < 0.05). We conclude that heparin inhibits compound 48/80-induced exocytosis in mast cells probably by its negative charge density.
Phosphodiesterase isozymes modulating inherent tone in human airways: identification and characterization
Authors: Rabe K. F. , Tenor H. , Dent G. , Schudt C. , Liebig S. , Magnussen H.
Published: December 2017

The effects of the nonselective phosphodiesterase (PDE)-inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the selective PDE inhibitors SKF 94120 (type III), rolipram (type IV), zardaverine (type III/IV), and zaprinast (type V) on inherent tone in human airways were investigated. Substantial relaxation was achieved by IBMX [concentration eliciting 50% of maximum response (EC50): 2.9 microM, n = 14] and SKF 94120 (EC50: 1.4 microM, n = 15); rolipram and zaprinast were almost ineffective. Zardaverine (EC50: 0.31 microM, n = 8), and the combination of SKF 94120 and rolipram (1 microM; EC50: 0.41 microM) were effective relaxants. Biochemical studies revealed the presence of PDE isozymes I, III, IV, and V in the cytosolic and particulate phase of airway homogenates, whereas PDE II was present only in the cytosol. Partial inhibition of total PDE adenosine 3',5'-cyclic monophosphate-hydrolyzing activity was achieved with rolipram and a selective type III inhibitor, whereas there was almost complete inhibition of total PDE activity with either zardaverine or the combination of type III and IV inhibitors. We conclude that all five PDE isozyme families are present in crude preparations of human peripheral airways. Inherent tone in this tissue is most effectively relaxed through selective type III/IV PDE inhibitors.
Role of serine esterase in A23187-mediated activation of phospholipase A2 in pulmonary endothelium
Authors: Chakraborti S. , Michael J. R.
Published: December 2017

To test the hypothesis that an endothelial cell membrane-associated serine esterase is involved in regulating phospholipase A2 (PLA2), we studied the effect of the calcium ionophore A23187 on intracellular PLA2 activity and arachidonic acid (AA) release in bovine pulmonary arterial endothelial cells. Exposure of these cells to A23187 causes a concentration-dependent increase in PLA2 activity and [14C]AA release. In addition to increasing PLA2 activity and AA release, A23187 enhances the activity of endothelial cell membrane-associated serine esterase that acts on the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester. Serine esterase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, prevent the A23187-mediated increase in serine esterase activity, PLA2 activity, and AA release. Pretreatment of the cells with actinomycin D or cycloheximide does not prevent the A23187-mediated increase in AA release, serine esterase activity, or PLA2 activity. The membrane-associated serine esterase activity directly correlates with membrane PLA2 activity. These results suggest that a membrane-associated serine esterase plays a pivotal role in regulating PLA2 activity after exposure to A23187.
N-type, omega-conotoxin-sensitive Ca2+ channels mediate electrically evoked release of ACh in guinea pig trachea
Authors: Baker D. G. , Don H. F. , Brown J. K.
Published: December 2017

To determine whether N- or L-type Ca2+ channels mediate acetylcholine (ACh) release from airway parasympathetic nerve endings, we compared the effects of omega-conotoxin (N-type inhibitor) and nifedipine (L-type inhibitor) on electrically evoked release of ACh in guinea pig trachea. Reconnected segments of guinea pig trachea were mounted in organ baths containing Krebs-Henseleit buffer, indomethacin (10 microM) to inhibit cyclooxygenase, neostigmine (1 microM) to inhibit acetylcholinesterase, and atropine (0.3 microM) to inhibit muscarinic autoreceptors, as well as phentolamine and propranolol to inhibit adrenergic receptors. After electrical field stimulation (EFS), aliquots of buffer were removed, and ACh was measured directly by high-performance liquid chromatography with electrochemical detection. Tracheas were stimulated for 10-min periods at a frequency of 5 Hz, and ACh release was measured for five separate periods (S1-S5) after treatment with increasing concentrations of omega-conotoxin or vehicle alone (acetic acid). Thirty minutes was allowed between stimulation periods. We found that EFS-evoked release of ACh was inhibited by omega-conotoxin in a concentration-dependent manner [mean effective concentration (EC50) approximately 8 nM] but was unaffected by vehicle treatment. In other experiments, ACh release was measured for two separate periods (S1 and S2), and between periods tracheas were treated with omega-conotoxin (1 microM), nifedipine (10–100 microM), tetrodotoxin (TTX), or buffer containing low (0.8 mM) Ca2+. ACh release was 12 +/- 2 (mean +/- SE) and 3 +/- 0.3 pmol.mg protein-1.min-1 before and during omega-conotoxin (n = 5, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
EDRF suppresses an unidentified vasoconstrictor mechanism in hypertensive rat lungs
Authors: Oka M. , Hasunuma K. , Webb S. A. , Stelzner T. J. , Rodman D. M. , McMurtry I. F.
Published: December 2017

To test whether endothelium-derived relaxing factor (EDRF) plays a role in regulating the hypertensive pulmonary vascular bed, we compared effects of the inhibitor of EDRF production, N omega-nitro-L-arginine (L-NNA), on resting vascular tone in lungs and conduit pulmonary arteries isolated from control and chronically hypoxic rats. In contrast to no effect on normoxic vascular tone in salt solution-perfused normotensive lungs, 100 microM L-NNA caused a marked, L-arginine-sensitive, precapillary vasoconstriction in unstimulated hypertensive lungs. Bioassay of hypertensive lung perfusate did not detect a circulating vasoconstrictor, and L-NNA vasoconstriction was not inhibited by blockers of cyclooxygenase, 5-lipoxygenase, platelet-activating factor receptors, alpha-adrenoceptors, and serotonin 5-HT2 receptors or by scavengers of superoxide anion and H2O2. Inhibitors of endothelin-1 (ET-1) production and vasoconstriction tended to blunt the response, but accumulation of perfusate ET-1 was not increased in hypertensive lungs. L-NNA vasoconstriction was blocked by Ca(2+)-free plus ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid perfusion but not by nifedipine. Quiescent, endothelium-intact hypertensive but not normotensive conduit pulmonary artery rings were markedly constricted by 200 microM L-NNA. The onset but not the peak of the response was blunted by meclofenamate. The response was reduced slightly by the ETA receptor antagonist, BQ 123. L-NNA had little effect on denuded hypertensive arteries, and treatment with dilators showed they had constricted spontaneously. Both the L-NNA and the spontaneous constrictions were readily inhibited by nifedipine. These results indicate that in rat hypertensive pulmonary arteries, the basal release of EDRF suppresses vasoconstrictor mechanisms which are not expressed in normotensive arteries.
Effects of ozone on transepithelial potential of mouse trachea
Authors: Takahashi M. , Kleeberger S. R. , Croxton T. L.
Published: December 2017

The effects of ozone on tracheal electrical potential were investigated in inbred strains of mice that are differentially susceptible to ozone-induced inflammation. In male mice (9–13 wk), a tracheostomy was made under pentobarbital anesthesia for spontaneous breathing and tracheal potential was measured in the cephalad portion of the bisected trachea using Hanks' salt/agar-capped KCl bridges connected to a pair of calomel half cells. The mean tracheal potentials of five different strains of mice (C3H/HeJ, DBA/2J, C57BL/6J, BALB/cJ, and 129/J) were approximately 10 mV (lumen negative) with no significant interstrain difference. Amiloride reduced mouse tracheal potentials by approximately 70% in both C3H/HeJ and C57BL/6J mice, indicating that sodium absorption is the predominant ion transport across this tissue. Relative to air-exposed controls, acute ozone exposure (2 ppm for 3 h) significantly attenuated tracheal potential of inflammation-susceptible C57BL/6J mice by approximately 50% at 6 h and 40% at 24 h postexposure but had no effect immediately after exposure. The mean tracheal potential of C3H/HeJ mice was not changed by ozone. The differential effect of acute ozone exposure on tracheal potential in C57BL/6J and C3H/HeJ mice is consistent with differential susceptibility to ozone-induced increases in epithelial permeability in these strains.
Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats
Authors: Hershenson Marc B. , Aghili Shahriar , Punjabi Naresh , Hernandez Claudia , Ray Daniel W. , Garland Allan , Glagov Seymour , Solway Julian
Published: December 2017

Pages L263–L269: Hershenson, Marc B., Shahriar Aghili, Naresh Punjabi, Claudia Hernandez, Daniel W. Ray, Allan Garland, Seymour Glagov, and Julian Solway. “Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats.” Page 264: right-hand column, 2nd paragraph in the results section, the first sentence, with corrected values, should read: There was no difference in baseline Rrs between air-exposed and O2-exposed rats (control, n = 20, 0.348 ± 0.212 cmH2O·ml-1·s; hyperoxia, n = 19, 0.252 ± 0.078 cmH2O·ml-1·s; NS). Since all of our other calculations were based on changes in respiratory system resistance relative to baseline, the major findings and conclusions of the article are not altered by this calibration error.
Age-related changes in pulmonary muscarinic receptor binding properties
Authors: Wills-Karp M.
Published: December 2017

The goal of this study was to elucidate mechanisms responsible for age-related reductions in responsiveness to cholinergic muscarinic stimulation in guinea pigs, by examining the binding properties of muscarinic receptors and their coupling to guanine nucleotide regulatory proteins as a function of animal age. In addition, the binding constants of three selective muscarinic receptor antagonists pirenzepine, [11-((2-[(diethylamino)methyl]-1-piperidinyl)-acetyl)-5, 11-dihydro-6H-pyrido(2,3)(1,4)benzodiazepine-6-on], and 4-diphenylacetoxy-N-methylpiperidine methobromide were examined. We found that there were no changes in either the receptor density or the affinity of the muscarinic receptor with age. There was a significant reduction in the affinity constant for the high-affinity agonist binding site in the old tissues (7.63 +/- 0.08) compared with the young tissues (8.31 +/- 0.10). Guanine nucleotides lowered agonist affinity for the receptor in young lungs, however, they had no effect on agonist binding in old tissues. Antagonist competition binding curves in young tissues revealed that 73% of the receptors are of the M2 type, with 27% being of the M3 subtype. In contrast, antagonist competition binding curves in the old tissues revealed that 37% of the receptors were of the M2 subtype, 30% were M3, and 33% were of the M1 subtype. Our studies provide evidence that the loss of sensitivity to cholinergic muscarinic stimulation in the senescent lung may be due to changes in both muscarinic receptor subtypes and receptor coupling to G proteins.
Type II pneumocytes secrete vitamin E together with surfactant lipids
Authors: Rustow B. , Haupt R. , Stevens P. A. , Kunze D.
Published: December 2017

Lung surfactant is exposed to strongly oxidizing conditions. We examined the hypothesis that in lung, lipophilic antioxidants are secreted together with surfactant to counteract the peroxidation of surfactant constituents. Lung lavage and the subfractions of the alveolar surfactant contain the lipophilic antioxidants vitamin E, vitamin A, and plasmalogens. The specific radioactivity of vitamin E isolated from serum, lung homogenate, lamellar bodies, and lung lavage increased linearly up to 3 h after intraperitoneal application of [3H]tocopherol. [3H]tocopherol was secreted in situ together with [14C]palmitic acid-labeled phospholipid in response to isoproterenol. Type II cells cultured in presence of [3H]tocopherol or of [3H]cholecalciferol and [14C]palmitic acid responded to isoproterenol by a time-dependent increase in secretion of [3H]tocopherol and of 14C-labeled phospholipids but not of [3H]cholecalciferol. The isoproterenol-stimulated secretion of [3H]tocopherol and of 14C-labeled phospholipids by type II cells is inhibited by surfactant protein A. We conclude that the alveolar surfactant contains lipophilic antioxidants as integral constituents. [3H]tocopherol seems to be secreted together with surfactant.
Glucocorticoid regulation of fatty acid synthase gene expression in fetal rat lung
Authors: Xu Z. X. , Stenzel W. , Sasic S. M. , Smart D. A. , Rooney S. A.
Published: December 2017

There are developmental and glucocorticoid-induced increases in the rate of fatty acid biosynthesis and in the activity of fatty acid synthase in late gestation fetal lung. We have now measured mRNA levels of fatty acid synthase and of two other enzymes of fatty acid biosynthesis, ATP citrate lyase and acetyl-CoA carboxylase, in developing fetal and postnatal rat lung and in fetal lung explants cultured with and without dexamethasone. There was a developmental increase in the mRNA for fatty acid synthase with the maximum level being reached on fetal day 21 (term is fetal day 22). This profile was similar to that reported for de novo fatty acid synthesis and fatty acid synthase activity. There was a similar but less pronounced developmental increase in the mRNA for ATP citrate lyase and a corresponding increase in its activity. There was no developmental change in the mRNA for acetyl-CoA carboxylase. Dexamethasone increased the level of fatty acid synthase mRNA approximately threefold but had no effect on those for ATP citrate lyase and acetyl-CoA carboxylase. The effect of dexamethasone on fatty acid synthase mRNA was rapid, biphasic, and partly inhibited by actinomycin D and cycloheximide. We conclude that glucocorticoids increase expression of the gene for fatty acid synthase in fetal lung. The effect of the hormone appears to be due to increased transcription and post-transcriptional events and is dependent on protein synthesis.
Incorporation of fibronectin into matrix decreases TNF-induced increase in endothelial monolayer permeability
Authors: Wheatley E. M. , McKeown-Longo P. J. , Vincent P. A. , Saba T. M.
Published: December 2017

Plasma fibronectin, a dimeric adhesive protein in blood, incorporates into the subendothelial and interstitial matrix in the lung especially during vascular injury. Fibronectin in the matrix is believed to influence cell-cell interaction and endothelial cell adhesion to the collagen-rich extracellular matrix. We previously observed that addition of purified soluble human plasma fibronectin (hFn) to cultured pulmonary endothelial monolayers attenuates the increase in protein permeability of such monolayers exposed to tumor necrosis factor-alpha (TNF-alpha). In the current study, we determined the specificity of this permeability response to fibronectin by comparing hFn to two other purified adhesive proteins in human plasma, i.e., vitronectin (Vn) and fibrinogen (Fg). We also determined whether matrix incorporation was essential for this hFn-mediated protective response by comparing normal intact hFn to either hFn alkylated with N-ethylmaleimide (NEM) or to purified 160/180-kDa hFn fragments, since these alternate forms of fibronectin are believed to exhibit limited ability to incorporate into matrix. Calf pulmonary artery endothelial (CPAE) monolayers (3-4 days postseeding) were exposed to human recombinant TNF-alpha for 18 h at a medium concentration of 200 U/ml followed by assessment of protein permeability using transendothelial 125I-labeled albumin clearance. Dimeric hFn (600 micrograms/ml) significantly (P < 0.05) reduced the TNF-induced increase in endothelial monolayer permeability. Vn or Fg, added at equal molar concentrations to the hFn, were unable to attenuate endothelial permeability. Immunofluorescent analysis utilizing antibodies specific to either hFn, human Vn, or human Fg revealed incorporation of the exogenous hFn into the extracellular matrix, but no matrix incorporation of Vn or Fg. Both NEM-treated dimeric hFn as well as purified 160/180-kDa fragments of hFn, which cannot incorporate into the matrix, were also unable to prevent the TNF-induced increase in protein permeability. Thus the ability for soluble hFn to reduce the TNF-induced increase in lung endothelial monolayer permeability was specific and dependent on its incorporation into the extracellular matrix.
Lung CTP:choline-phosphate cytidylyltransferase: activation of cytosolic species by unsaturated fatty acid
Authors: Mallampalli R. K. , Salome R. G. , Hunninghake G. W.
Published: December 2017

CTP:choline-phosphate cytidylyltransferase is the principal rate-limiting enzyme required for surfactant phosphatidylcholine synthesis. We examined the in vitro effect of unsaturated fatty acids on the expression of the two cytosolic forms of this enzyme in fetal and adult rat lung. In the adult, a substantial portion of cytidylyltransferase is expressed as the active form (H form). By contrast, the majority of enzyme mass in the fetus is in an inactive form (L form). Oleic acid, or its esterified derivative, oleoyl-CoA, each stimulated the inactive form (L form) in vitro. However, the addition of oleoyl-CoA directly to the active form (H form) resulted in a dose-dependent decrease in H-form activity, suggesting feedback inhibition. Further, exposure of the enzyme in fetal lung cytosol to either fatty acid increased the mass of the enzyme, consistent with a shift from the inactive form (L form) to the active species (H form). These observations support a key role for unsaturated fatty acids in the developmental regulation of this enzyme.
Sulfhydryl-depleting agents, but not deferoxamine, modulate EDRF action in cultured pulmonary arterial cells
Authors: Marczin N. , Ryan U. S. , Catravas J. D.
Published: December 2017

The potential role of intracellular sulfhydryls and iron on the biological activity of endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial (BPAE) cells was investigated in a cultured cell bioassay system, by measuring N omega-nitro-L-arginine-sensitive guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rabbit pulmonary arterial smooth muscle (SM) cells. The role of sulfhydryls in the biosynthesis of EDRF was studied by selectively exposing the endothelial cells to thiol-depleting agents. Both N-ethylmaleimide (NEM) and maleic acid diethyl ester (DEM) inhibited EDRF-induced cGMP accumulation in a dose-dependent manner. To study the requirement of SM thiols in the metabolism of EDRF to a stimulator of cGMP formation, SM were selectively exposed to NEM and DEM before bioassay with control, untreated BPAE. DEM and NEM inhibited cGMP formation in response to EDRF by 30 and 68%, respectively. The requirement of SM sulfhydryls was further investigated in the stimulation of SM cGMP accumulation elicited by nitrosothiols [S-nitroso-L-cysteine, S-nitroso-mercaptoproprionic acid, and sodium nitroprusside (SNP)]. NEM pretreatment of SM cells abolished cGMP responses to all vasodilators; DEM did not affect the nitrosothiol responses but reduced by 30% the cGMP accumulation to SNP. The role of iron in the endothelial synthesis of EDRF was assessed by chelating endothelial low-molecular-weight iron compounds. Exposure of BPAE to deferoxamine mesylate had no effect on cGMP accumulation in SM, suggesting that deferoxamine-available iron is not necessary for the endothelial stimulation of SM cGMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Receptor mechanism of thrombin-mediated pulmonary vasodilation in neonates
Authors: Pinheiro J. M. , Andersen T. T. , Malik A. B.
Published: December 2017

A recently identified peptide sequence exposed after proteolytic cleavage of the NH2-terminus of the thrombin receptor mimics some cellular effects of alpha-thrombin. To determine whether a proteolytic action of thrombin is required for vasoactivity, we examined the vascular effects of modified thrombins and synthetic NH2-terminus peptide sequences of the thrombin receptor (TRPs) in isolated piglet lungs. Lungs of piglets 1-6 days old were perfused with recirculating Ringer-albumin solution at a constant flow of 60 ml/min. We measured the pulmonary artery pressure (Ppa) and segmental distribution of pulmonary vascular resistance (using the double occlusion method) in response to injections of human alpha-thrombin, modified thrombins, and TRP-14 and TRP-7 (i.e., 14 and 7 amino acid NH2-terminus peptides of the cleaved thrombin receptor). alpha-Thrombin produced a rapid and transient decrease in Ppa; the magnitude and duration [time for one-half recovery (t1/2 R)] of the vasodilation responses were concentration dependent [t1/2 R values of 1.4 +/- 0.1 and 3.3 +/- 2.4 min (mean +/- SE) at concentrations of 10(-10) and 10(-9) M, respectively]. The vasodilation was due primarily to a decrease in precapillary resistance. Proteolytically active, but binding-impaired gamma-thrombin was a less potent vasodilator and proteolytically inactive D-phenylalanyl-prolyl-arginine-chloromethyl ketone (PPACK)-alpha-thrombin did not induce vasodilation. TRP-14 was also a pulmonary vasodilator with a t1/2R value of 0.8 +/- 0.09 min at a concentration of 10(-7) M; both TRP-14 and TRP-7 were approximately 3-log less potent than equimolar alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Corticosteroids differentially regulate secretion of IL-6, IL-8, and G-CSF by a human bronchial epithelial cell line
Authors: Levine S. J. , Larivee P. , Logun C. , Angus C. W. , Shelhamer J. H.
Published: December 2017

Human airway epithelial cells play an active role in modulating airway inflammation by elaborating a variety of proinflammatory molecules, including cytokines. The purpose of this study was to define the role of corticosteroids in the regulation of cytokine gene transcription and secretion by human bronchial epithelial cells. In particular, we assessed whether dexamethasone was capable of inhibiting the tumor necrosis factor-alpha (TNF-alpha)-mediated secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), and granulocyte colony-stimulating factor (G-CSF) by a human bronchial epithelial cell line (BEAS-2B). Stimulation with 20 ng/ml of TNF-alpha resulted in significant increases in secretion of immunoreactive IL-6, IL-8, and G-CSF that were maximal at 24 h. TNF-alpha-mediated IL-6, IL-8, and G-CSF secretion was concentration dependent and specific. In addition, stimulation with TNF-alpha resulted in significant increases in the quantity of IL-6, IL-8, and G-CSF mRNA as detected by reverse-transcription polymerase chain reaction. Dexamethasone preconditioning significantly inhibited both the secretion of immunoreactive IL-6 and the accumulation of IL-6 mRNA. Although dexamethasone appeared to reduce both the secretion of immunoreactive IL-8 and accumulation of IL-8 mRNA, the inhibitory effects did not reach statistical significance. Finally, dexamethasone did not inhibit either the secretion of immunoreactive G-CSF or the accumulation of G-CSF mRNA. In summary, our results suggest that corticosteroids have a differential effect on the regulation of cytokine secretion by human bronchial epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in lung expansion alter pulmonary DNA synthesis and IGF-II gene expression in fetal sheep
Authors: Hooper S. B. , Han V. K. , Harding R.
Published: December 2017

Our aim was to determine the effect of short-term (7 days) alterations in fetal lung liquid volume on pulmonary DNA synthesis rates and insulin-like growth factor-II (IGF-II) mRNA levels. Fifteen chronically catheterized fetal sheep were divided into three groups. In one, the trachea was obstructed, in another lung liquid was drained by gravity, and the third group served as controls. After 7 days, [3H]thymidine was injected into each fetus and 8 h later fetal tissues were collected. Fetal lung-to-body weight ratios and total lung DNA contents were greatly increased in fetuses with tracheal obstruction compared with control fetuses, whereas the drainage of lung liquid did not affect these measurements. DNA synthesis rates in pulmonary tissue were significantly reduced from a mean control value of 153.3 +/- 25.1 disintegrations per minute (dpm)/microgram DNA to 57.2 +/- 8.6 dpm/microgram DNA by lung liquid drainage (P < 0.05) and were significantly increased to 236.0 +/- 24.0 dpm/microgram DNA by tracheal obstruction (P < 0.05). Following tracheal obstruction, lung IGF-II mRNA levels were increased to 177.0 +/- 18.2% (P < 0.05) of the mean value for control fetuses, whereas they were reduced to 56.1 +/- 7.1% of control in lung liquid-drained fetuses. We conclude that altering fetal lung expansion has a potent and rapid effect on pulmonary DNA synthesis and that this effect may, in part, be mediated by an alteration in IGF-II gene expression.
Radioligand binding methods: practical guide and tips
Authors: Bylund D. B. , Toews M. L.
Published: December 2017

Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.
ATP and UTP increase secretion of bronchial inhibitor by human tracheal gland cells in culture
Authors: Merten M. D. , Breittmayer J. P. , Figarella C. , Frelin C.
Published: December 2017

The effects of ATP and UTP on intracellular Ca2+ levels and on the secretion of the bronchial inhibitor and high-molecular-weight glycoproteins were studied in cultures of human bronchotracheal gland cells. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP increased intracellular Ca2+ levels in a manner that was partially dependent on the presence of extracellular Ca2+. Other nucleotides (ADP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, and 2-methylthio ATP) and adenosine were ineffective, thus suggesting the presence of a “nucleotide” receptor specific for ATP and UTP. At concentrations similar to those that raised intracellular Ca2+ concentration, ATP, UTP, and ATP gamma S stimulate the secretion of the bronchial inhibitor. ATP and UTP also increase the production of sulfated high-molecular-weight glycoproteins. These results indicate the presence in human tracheal gland cells of a nucleotide receptor that mediates intracellular Ca2+ mobilization and controls the secretion of macromolecules.
Elevated lung G protein levels and muscarinic receptor affinity in a mouse model of airway hyperreactivity
Authors: Gavett S. H. , Wills-Karp M.
Published: December 2017

A genetic model of airway hyperreactivity has been described in which strains of mice are hyperresponsive (A/J) or hyporesponsive (C3H/HeJ) to intravenous acetylcholine challenge. To determine the mechanism of this differential responsiveness, we compared beta 2-adrenergic and muscarinic cholinergic receptor properties and their coupling to guanine nucleotide binding proteins (G proteins) in peripheral lung membrane fractions from these strains. No significant differences were found between the strains with regard to beta 2-adrenergic or muscarinic receptor density or antagonist affinity. No strain difference was found in beta 2-adrenergic receptor affinity for isoproterenol in the presence or absence of the nonhydrolyzable guanine nucleotide 5'-guanylimidodiphosphate [Gpp(NH)p]. In contrast, affinity of the high-affinity carbachol binding site of muscarinic receptors was threefold greater in A/J lung compared with C3H/HeJ lung (pKH = 7.34 +/- 0.16 vs. 6.79 +/- 0.06, respectively, P < 0.05). In the presence of Gpp(NH)p, this affinity was decreased sevenfold in A/J lung but was not significantly affected in C3H/HeJ lung, suggesting that muscarinic receptors in A/J lung are more effectively coupled to G proteins. Levels of Gs alpha and Gi alpha proteins in peripheral lung were significantly greater in the A/J strain compared with the C3H/HeJ strain (40 and 20% greater, respectively). These studies suggest that airway hyperreactivity in A/J mice is not associated with alterations in beta 2-adrenoceptors, but may be a result of enhanced muscarinic receptor signal transduction due to increased agonist affinity for muscarinic receptors and upregulation of G protein levels.
Effect of gas composition on liquid secretion by explants of distal lung of fetal rat in submersion culture
Authors: Barker P. M. , Gatzy J. T.
Published: December 2017

Throughout gestation, fetal lung is filled with liquid secreted by the pulmonary epithelium. Factors responsible for secretion and for induction of liquid absorption by postnatal lung are poorly understood. We studied effects of “fetal” (3% O2-8% CO2) and “postnatal” (21% O2-5% CO2) gas tensions and of low hormone concentrations [media with 10% charcoal-stripped fetal bovine serum (stFBS) or unstripped FBS] on water content and morphology of distal lung explanted from 14-, 20-, and 22-day fetal (term, 22 days) and 2-day neonatal rats and incubated in submersion culture. Water-to-dry weight ratios of freshly excised fetal whole lung at 20 (6.2) or 22 days (7.0) were greater than that of 2-day postnatal lung (4.7). Culture conditions did not affect water-to-dry weight ratio of cyst-forming 14-day explants (10.8) or acystic 2-day postnatal explants (2.4). Fetal gases and stFBS supported cyst formation in [and high water/dry weight (9.2, 12.6) of] 20- and 22-day explants. Cysts also formed in 20-day explants exposed to postnatal gases and stFBS (water/dry weight = 6.5). Other conditions resulted in minimal cyst formation by 20- and 22-day explants and in water/dry weight similar to that of freshly excised and drained distal lung from 22-day fetuses (2.1). Cysts were lined with cuboidal and thin epithelial cells. No cells were ciliated. We conclude that 1) secretion dominates liquid flow across epithelia of fetal rat lung until birth, 2) alveolar epithelium contributes to this secretion, and 3) liquid secretion by fetal distal lung late in gestation is regulated by gas composition.