bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostanoids mediate IL-1β-induced β-adrenergic hyporesponsiveness in human airway smooth muscle cells
Authors: Laporte Johanne D. , Moore Paul E. , Panettieri Reynold A. , Moeller Winfried , Heyder Joachim , Shore Stephanie A.
Published: December 2017

We have previously reported that pretreatment of cultured human airway smooth muscle (HASM) cells with interleukin-1β (IL-1β) results in decreased β-adrenergic responsiveness. The purpose of this study was to determine whether prostanoids released as a result of cyclooxygenase-2 (COX-2) induction by IL-1β contribute to this effect of the cytokine. Confluent serum-deprived HASM cells were studied in passages 4–7. IL-1β (20 ng/ml for 22 h) reduced the ability of the β-agonist isoproterenol (Iso) to decrease stiffness of HASM cells as measured by magnetic twisting cytometry. The effect of IL-1β on Iso-induced changes in cell stiffness was abolished by nonselective [indomethacin (Indo), 10−6M] and selective (NS-398, 10−5M) COX-2 inhibitors. Indo and NS-398 also inhibited both the increased basal cAMP and the decreases in Iso-stimulated cAMP production induced by IL-1β. IL-1β (20 ng/ml for 22 h) caused an increase in both basal (15-fold) and arachidonic acid (AA)-stimulated (10-fold) PGE2release. Indo blocked basal and AA-stimulated PGE2release in both control and IL-1β-treated cells. NS-398 also markedly reduced basal and AA-stimulated PGE2release in IL-1β-treated cells but had no significant effect on AA-stimulated PGE2release in control cells. Western blot analysis confirmed the induction of COX-2 by IL-1β. Exogenously administered PGE2(10−7M, 22 h) caused a significant reduction in the ability of Iso to decrease cell stiffness, mimicking the effects of IL-1β. Cycloheximide (10 μg/ml for 24 h), an inhibitor of protein synthesis, also abolished the effects of IL-1β on Iso-induced cell stiffness changes and cAMP formation. In summary, our results indicate that IL-1β significantly increases prostanoid release by HASM cells as a result of increased COX-2 expression. The prostanoids appear to contribute to β-adrenergic hyporesponsiveness, perhaps by heterologous desensitization of the β2receptor.
Nitric oxide inhalation transiently elevates pulmonary levels of cGMP, iNOS mRNA, and TNF-α
Authors: Brady Todd C. , Crapo James D. , Mercer Robert R.
Published: December 2017

The initial pulmonary vasodilation that occurs during nitric oxide (⋅ NO) inhalation does not appear to be maintained chronically in many cases. ⋅ NO may acutely relax vascular smooth muscle by increasing levels of guanosine 3′,5′-cyclic monophosphate (cGMP), tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) while decreasing levels of lipid peroxidation. It was hypothesized that the acute ⋅ NO-induced changes in cGMP, TNF-α, iNOS, and lipid peroxidation, all of which may mediate vasodilation, are transient rather than sustained. Lungs from rats kept in chambers containing 6 parts/million ⋅ NO for 1 h, 1 day, or 1 wk were analyzed for levels of ⋅ NO-induced vasodilatory mediators. Pulmonary cGMP, iNOS mRNA, and TNF-α were increased 1 h after ⋅ NO exposure but decreased to control values at later times. Levels of malonyl dialdehyde, an indicator of lipid peroxidation, were decreased at all times during ⋅ NO inhalation. As a whole, the data suggest that in lungs the vasodilatory mediators cGMP, iNOS, and TNF-α are only acutely and transiently elevated during inhalation of ⋅ NO, consistent with the initially positive clinical response to inhaled ⋅ NO that deteriorates over time.
Ionic mechanisms underlying electrical slow waves in canine airway smooth muscle
Authors: Janssen Luke J. , Hague Chris , Nana Roopung
Published: December 2017

In canine bronchial smooth muscle (BSM), spasmogens evoke oscillations in membrane potential (“slow waves”). The depolarizing phase of the slow waves is mediated by voltage-dependent Ca2+ channels; we examined the roles played by Cl− and K+ currents and Na+-K+-ATPase activity in mediating the repolarizing phase. Slow waves were evoked using tetraethylammonium (25 mM) in the presence or absence of niflumic acid (100 μM; Cl− channel blocker) or ouabain (10 μM; block Na+-K+-ATPase) or after elevating external K+concentration ([K+]) to 36 mM (to block K+ currents); curve fitting was performed to quantitate the rates of rise/fall and frequency under these conditions. Slow waves were markedly slowed, and eventually abolished, by niflumic acid but were unaffected by ouabain or high [K+]. Electrically evoked slow waves were also blocked in similar fashion by niflumic acid. We conclude that the repolarization phase is mediated by Ca2+-dependent Cl− currents. This information, together with our earlier finding that the depolarizing phase is due to voltage-dependent Ca2+ current, suggests that slow waves in canine BSM involve alternating opening and closing of Ca2+ and Cl− channels.
Surfactant protein B processing in human fetal lung
Authors: Guttentag Susan H. , Beers Michael F. , Bieler Bert M. , Ballard Philip L.
Published: December 2017

Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8(Phe201–Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145–Leu160, Gln186–Gln200, and Gly284–Ser304). Western blot analysis revealed a novel intermediate at ∼9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of ∼10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.
Prolonged high intermittent positive-pressure ventilation induces airway remodeling and reactivity in young rats
Authors: Fukunaga Tetsu , Davies Paul , Zhang Leilei , Hashida Yoshie , Motoyama Etsuro K.
Published: December 2017

We postulated that prolonged exposure to intermittent positive-pressure ventilation (IPPV) with high pressure (HIPPV) alone without hyperoxia promotes the development of airway hyperresponsiveness and remodeling. To test this hypothesis, young rats were ventilated under halothane anesthesia with HIPPV (maximum inspiratory pressure at 32–35 cmH2O in 70% nitrous oxide and 30% O2) for 3.5–4 h daily for 6 days. Control rats were ventilated with low IPPV (maximum inspiratory pressure < 13 cmH2O) during the same time period with the same gas mixture. With the use of tracheal rings isolated from these rats and a setup in tissue baths, contractile responses to carbachol (10−6 to 10−2 mM), 5-hydroxytryptamine (5-HT; 10−9 to 10−5 mM) and KCl (1–100 mM) were examined isometrically. In tracheal rings from HIPPV rats compared with low-pressure IPPV rats, the concentration tension curves showed a significantly enhanced response to all agonists ( P < 0.005). Sensitivity to carbachol, 5-HT, and KCl was also significantly increased ( P < 0.05) compared with control rats as evidenced by decreases in EC50. Maximum tension (reactivity) to 5-HT and KCl in the HIPPV group increased significantly ( P < 0.05), and there was a trend ( P = 0.07) toward increased reactivity to carbachol in this group as well. Histological examinations of tracheal rings demonstrated epithelial squamous metaplasia in the HIPPV group. Morphometric studies demonstrated tracheal smooth muscle thickening ( P < 0.05) without changes in the thickness of the mucosa or the lamina propria. When contractile responses were normalized for the smooth muscle cross-sectional area (i.e., stress), reactivity to all contractile agents was reduced, whereas reactivity to 5-HT still demonstrated significant increase ( P < 0.005). Sensitivity of tracheal segments to all three agents was not affected by this normalization. These findings suggest that prolonged exposure to HIPPV without hyperoxia and the resultant overdistension of lung tissues (volutrauma) induced airway remodeling and airway hyperreactivity.
Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1
Authors: Moore Timothy M. , Brough George H. , Babal Paul , Kelly John J. , Li Ming , Stevens Troy
Published: December 2017

Activation of Ca2+entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+that was dependent on a membrane current having a net inward component of 5.45 ± 0.90 pA/pF at −80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential ( trp) gene have been proposed to form Ca2+entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.
Osmotic stress induces both secretion and apoptosis in rat alveolar type II cells
Authors: Edwards Yasmin S. , Sutherland Leanne M. , Power John H. T. , Nicholas Terence E. , Murray Andrew W.
Published: December 2017

The aim of this study was to analyze the effects of osmotic shock and secretagogues such as ATP and 12- O-tetradecanoylphorbol 13-acetate (TPA) on various intracellular signaling pathways in primary cultures of alveolar type II cells and examine their potential role in regulating events such as secretion and apoptosis in these cells. Sorbitol-induced osmotic stress caused the sustained release of [3H]phosphatidylcholine ([3H]PC) from primary cultures of rat alveolar type II cells prelabeled with [3H]choline chloride. This release was not dependent on protein kinase C because downregulation of the major protein kinase C isoforms (α, βII, δ, and η) expressed in alveolar type II cells had no effect on [3H]PC secretion. Sorbitol, as well as the known secretagogues TPA and ATP, activated extracellular signal-regulated kinase. Although an inhibitor of the extracellular signal-regulated kinase cascade, PD-98059, blocked this activation, it had no effect on the release of [3H]PC. Sorbitol and ultraviolet C radiation, but not TPA or ATP, were also found to activate both p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Furthermore, both sorbitol and ultraviolet C radiation induced apoptosis in alveolar type II cells as demonstrated by Hoechst 33258 staining of the condensed nuclei, the generation of DNA ladders, and the activation of caspases. The data indicate that multiple signaling pathways are activated by traditional secretagogues such as TPA and ATP and by cellular stresses such as osmotic shock and that these may be involved in regulating secretory and apoptotic events in alveolar type II cells.
O2-induced ENaC expression is associated with NF-κB activation and blocked by superoxide scavenger
Authors: Rafii Bijan , Tanswell A. Keith , Otulakowski Gail , Pitkänen Olli , Belcastro-Taylor Rose , O’Brodovich Hugh
Published: December 2017

Cultured rat fetal distal lung epithelial cells (FDLEs), when switched from fetal (3%) to postnatal (21%) O2concentrations, have increased epithelial Na+channel (ENaC) mRNA levels and amiloride-sensitive Na+transport [O. Pitkänen, A. K. Tanswell, G. Downey, and H. O’Brodovich. Am. J. Physiol. 270 ( Lung Cell. Mol. Physiol. 14): L1060–L1066, 1996]. The mechanisms by which O2mediates these effects are unknown. After isolation, FDLEs were kept at 3% O2overnight, then switched to 21% O2(3–21% O2group) or maintained at 3% O2(3–3% O2group) for 48 h. The amiloride-sensitive short-circuit current ( Isc) in the 3–21% O2group was double that in the 3–3% O2group. Amiloride-sensitive Isccould not be induced by medium conditioned by 21% O2-exposed FDLEs but was reversed by returning the cells to 3% O2. Neither the cyclooxygenase inhibitor ibuprofen, liposome-encapsulated catalase, nor hydroperoxide scavengers (U-74389G or Trolox) blocked the O2-induced amiloride-sensitive Isc. In contrast, the cell-permeable superoxide scavenger tetramethylpiperidine- N-oxyl (TEMPO) eliminated the O2-induced increases in amiloride-sensitive Iscand ENaC mRNA levels. The switch from 3 to 21% O2induced the transcription factor nuclear factor-κB, which could also be blocked by TEMPO. We conclude that 1) the O2-induced increase in amiloride-sensitive Iscis reversible and 2) the O2-induced increase in amiloride-sensitive Iscand ENaC mRNA levels is associated with activation of nuclear factor-κB and may be mediated, at least in part, by superoxide.
Role of Ca2+/calmodulin-dependent phosphatase 2B in thrombin-induced endothelial cell contractile responses
Authors: Verin Alexander D. , Cooke Clare , Herenyiova Maria , Patterson Carolyn E. , Garcia Joe G. N.
Published: December 2017

Thrombin-induced Ca2+mobilization, activation of Ca2+/calmodulin-dependent myosin light chain (MLC) kinase (MLCK), and increased phosphorylation of MLCs precede and are critical to endothelial cell (EC) barrier dysfunction. Net MLC dephosphorylation after thrombin is nearly complete by 60 min and involves type 1 phosphatase (PPase 1) activity. We now report that thrombin does not alter total PPase 1 activity in EC homogenates but rather decreases myosin-associated PPase 1 activity. The PPase 1 inhibitor calyculin fails to prevent thrombin-induced MLC dephosphorylation. However, thrombin significantly increased the activity of Ca2+-dependent PPase 2B in EC homogenates (∼1.5- to 2-fold), with PPase 2B activation correlating with phosphorylation of the PPase 2B catalytic subunit. Western immunoblotting revealed PPase 2B to be present in cytoskeletal EC fractions, with specific PPase 2B inhibitors such as cyclosporin (200 nM) and deltamethrin (100 nM to 1 μM) attenuating thrombin-induced cytoskeletal protein dephosphorylation, including EC MLC dephosphorylation. These results suggest a model whereby thrombin-inducible contraction is determined by the phosphorylation status of EC MLC regulated by the balance between EC MLCK, PPase 1 (constitutive), and PPase 2B (inducible) activities.
Gαi-2is required for carbachol-induced stress fiber formation in human airway smooth muscle cells
Authors: Hirshman Carol A. , Togashi Hideaki , Shao Dan , Emala Charles W.
Published: December 2017

To determine which heterotrimeric G protein couples muscarinic receptors to stress fiber formation [measured by an increase in the filamentous (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (ASM) cells, cultured human ASM cells expressing the M2muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 μM antisense oligonucleotides designed to specifically bind to the mRNA encoding Gαi-2, Gαi-3, or Gqα. A randomly scrambled oligonucleotide served as a control. F- to G-actin ratios were measured with dual-fluorescence labeling after 5 min of carbachol exposure, which is known to increase the F- to G-actin ratio. Cells in parallel wells were harvested for immunoblot analysis of G protein α-subunit expression. Oligonucleotide antisense treatment decreased protein expression of the respective G protein α-subunit. Antisense depletion of the Gαi-2protein but not of Gαi-3or Gqα protein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the Gαi-2protein couples muscarinic receptors to stress fiber formation in ASM.
An electrogenic amino acid transporter in the apical membrane of cultured human bronchial epithelial cells
Authors: Galietta Luis J. V. , Musante Luciana , Romio Leila , Caruso Ubaldo , Fantasia Annarita , Gazzolo Andrea , Romano Luca , Sacco Oliviero , Rossi Giovanni A. , Varesio Luigi , Zegarra-Moran Olga
Published: December 2017

We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolaterall-arginine (10–1,000 μM) increased the short-circuit current. Maximal effect and EC50were ∼3.5 μA/cm2and 80 μM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor ( NG-nitro-l-arginine methyl ester) did not decrease the arginine-dependent current. Apicall-lysine,l-alanine, andl-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50values ranging from 26 to 971 μM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.
Shedding of L-selectin and PECAM-1 and upregulation of Mac-1 and ICAM-1 on neutrophils in RSV bronchiolitis
Authors: Wang Shan-Ze , Smith Peter K. , Lovejoy Melanie , Bowden Jeffrey J. , Alpers John H. , Forsyth Kevin D.
Published: December 2017

Bronchiolitis is characterized histologically by epithelial necrosis and peribronchial infiltration of leukocytes, with a high percentage of neutrophils in the airways. We investigated the expression of adhesion molecules (CD11a, CD11b, CD18, CD31, CD54, and CD62L) on neutrophils from nasopharyngeal aspirates (NPAs) and peripheral blood (PB) of infants with respiratory syncytial virus (RSV)-induced bronchiolitis. The expression of CD31 and CD62L on neutrophils from NPAs is decreased and the expression of CD11b, CD18, and CD54 on neutrophils from NPAs is increased compared with cells from PB of RSV-infected infants. The expression of CD18 and CD54 on neutrophils from PB of RSV-infected infants is also increased compared with cells from PB of control infants. Shedding of CD31 and CD62L on neutrophils in RSV infection may contribute to the neutrophil emigration from blood to airways; the upregulation of Mac-1 (CD11b/CD18) and CD54 on neutrophils may help explain the high percentage of neutrophils in the airways of RSV bronchiolitis; and the upregulation of Mac-1 may be involved in the increased neutrophil-airway epithelial adhesion in RSV infection.
Effects of initial length on intrinsic tone in guinea pig tracheal smooth muscle
Authors: Bard Martin , Salmeron Sergio , Coirault Catherine , Blanc Francois-Xavier , Lecarpentier Yves
Published: December 2017

In the guinea pig, tracheal smooth muscle (TSM) exhibits intrinsic tone (IT). The active nature of IT suggests that it could be influenced by muscle length and load. In the guinea pig, IT is entirely suppressed by the cyclooxygenase inhibitor indomethacin. IT could be measured as the difference between resting tone before and after indomethacin addition. We examined, in electrically stimulated TSM strips ( n= 9), the influence of initial muscle length ( Li) on IT, the relationship between IT and the maximum extent of relaxation (ΔF1), and the influence of indomethacin on active isometric force. When Lidecreased from 100 to 75% of optimal Li, there was a significant decrease in IT (from 12.0 ± 0.2 to 5.3 ± 0.1 mN; P < 0.001). Over the range of Listudied, ΔF1underestimated the amount of IT, but there was a close linear relationship between ΔF1and IT ( r = 0.9). Compared with the basal state, indomethacin increased active isometric force (from 9.5 ± 1.0 to 19.7 ± 2.0 mN at optimal Li; P < 0.001) and induced its length dependency. In guinea pig TSM, Liwas an important determinant of IT.
Effects of hypoxia on energy state and pH in resting pulmonary and femoral arterial smooth muscles
Authors: Leach R. M. , Sheehan D. W. , Chacko V. P. , Sylvester J. T.
Published: December 2017

To determine the effects of hypoxia on energy state and intracellular pH (pHi) in resting pulmonary and systemic arterial smooth muscles, we used31P nuclear magnetic resonance spectroscopy and colorimetric and enzymatic assays to measure pHi; intracellular concentrations of ATP, phosphocreatine, creatine, and Pi; and phosphorylation potential in superfused tissue segments from porcine proximal intrapulmonary and superficial femoral arteries. Under baseline conditions ([Formula: see text] 467 ± 12.1 mmHg), energy state and total creatine (phosphocreatine + creatine) concentration were lower and pHiwas higher in pulmonary arteries. During hypoxia ([Formula: see text] 23 ± 2.4 mmHg), energy state deteriorated more in femoral arteries than in pulmonary arteries. pHifell in both tissues but was always more alkaline in pulmonary arteries. Reoxygenation reversed the changes induced by hypoxia. These results suggest that production and/or elimination of ATP and H+was different in resting pulmonary and systemic arterial smooth muscles under baseline and hypoxic conditions. Because energy state and pHiaffect a wide variety of cellular processes, including signal transduction, contractile protein interaction, and activities of ion pumps and channels, further investigation is indicated to determine whether these differences have functional significance.
Differential regulation of centrin genes during ciliogenesis in human tracheal epithelial cells
Authors: LeDizet Michel , Beck James C. , Finkbeiner Walter E.
Published: December 2017

Centrins are small calcium-binding proteins found in a variety of cell types, often in association with microtubule-organizing centers. Here we present results regarding the expression of centrins during the in vitro differentiation of human tracheal epithelial cells. When grown at an air-liquid interface, these cells differentiate into mucus-secreting cells or undergo ciliogenesis. In immunofluorescence and immunoelectron microscopy experiments, an anti-centrin antibody stained exclusively the basal bodies of the ciliated cells. There was no staining over the axonemes or the striated rootlets. Northern blots and RT-PCR analysis of the three known human centrin genes showed that these genes have distinct patterns of expression during the growth and differentiation of human tracheal epithelial cells. Centrin-1 is never transcribed. Centrin-2 mRNA is present at all times, and its concentration increases when ciliogenesis occurs. Centrin-3 mRNA is found at a constant level throughout the entire process. This differential regulation suggests that centrins are not interchangeable but instead have unique functions.
cAMP-dependent absorption of chloride across airway epithelium
Authors: Uyekubo S. N. , Fischer H. , Maminishkis A. , Illek B. , Miller S. S. , Widdicombe J. H.
Published: December 2017

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell85: 229–236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.
Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils
Authors: Cai Guang-Zuan , Griffin Gail L. , Senior Robert M. , Longmore William J. , Moxley Michael A.
Published: December 2017

Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to α-d-glucosyl residues. It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain (“neck” region), and a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic for inflammatory cells. To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric. Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10−10M equal to the positive control [formyl-Met-Leu-Phe (fMLP) at 10−8M]. The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10−10vs. 10−11M). The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD. These preincubations did not affect chemotaxis to fMLP. These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D.
Roles for C-X-C chemokines and C5a in lung injury after hindlimb ischemia-reperfusion
Authors: Bless Nicolas M. , Warner Roscoe L. , Padgaonkar Vaishalee A. , Lentsch Alex B. , Czermak Boris J. , Schmal Hagen , Friedl Hans P. , Ward Peter A.
Published: December 2017

We evaluated the roles of the C-X-C chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) as well as the complement activation product C5a in development of lung injury after hindlimb ischemia-reperfusion in rats. During reperfusion, CD11b and CD18, but not CD11a, were upregulated on neutrophils [bronchoalveolar lavage (BAL) and blood] and lung macrophages. BAL levels of CINC and MIP-2 were increased during the ischemic and reperfusion periods. Treatment with either anti-CINC or anti-MIP-2 IgG significantly reduced lung vascular permeability and decreased lung myeloperoxidase content by 93 and 68%, respectively ( P < 0.05). During the same period, there were significant increases in serum C5a-related neutrophil chemotactic activity. Treatment with anti-C5a decreased lung vascular permeability, lung myeloperoxidase, and BAL CINC by 51, 58, and 23%, respectively ( P < 0.05). The data suggest that the C-X-C chemokines CINC and MIP-2 as well as the complement activation product C5a are required for lung neutrophil recruitment and full induction of lung injury after hindlimb ischemia-reperfusion in rats.
Evidence for a role of protein kinase C in hypoxic pulmonary vasoconstriction
Authors: Weissmann Norbert , Voswinckel Robert , Hardebusch Thorsten , Rosseau Simone , Ghofrani Hossein Ardeschir , Schermuly Ralph , Seeger Werner , Grimminger Friedrich
Published: December 2017

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation, thus optimizing gas exchange. NADPH oxidase-related superoxide anion generation has been suggested as part of the signaling response to hypoxia. Because protein kinase (PK) C activation can occur during hypoxia and PKC activation is known to be critical for NADPH oxidase stimulation in different cell types, we probed the role of PKC in hypoxic vasoconstriction in intact rabbit lungs. Control vasoconstrictor responses were elicited by angiotensin II (ANG II) and the stable thromboxane analog U-46619. Portions of the experiments were performed while NO synthesis and prostanoid generation were blocked with NG-monomethyl-l-arginine and acetylsalicylic acid to avoid confounding effects due to interference with these vasoactive mediators. The PKC inhibitor H-7 (10–50 μM) caused dose-dependent inhibition of HPV, but this agent lacked specificity because ANG II- and U-46619-induced vasoconstrictions were correspondingly suppressed. In contrast, low concentrations of the specific PKC inhibitor bisindolylmaleimide I (BIM; 1–15 μM) strongly inhibited the hypoxic vasoconstriction without any interference with the responses to the pharmacological agents. Superimposable dose-inhibition curves were also obtained for BIM when lung NO synthesis and prostanoid generation were blocked throughout the experiments. Under either condition, BIM did not affect normoxic vascular tone. The PKC activator farnesylthiotriazole (FTT), ascertained to stimulate rabbit NADPH oxidase by provocation of alveolar macrophage superoxide anion generation in vitro, caused rapid-onset, transient pressor responses in normoxic lungs. After FTT, the hypoxic vasoconstrictor response was totally suppressed, in contrast to the largely maintained pressor responses to ANG II and U-46619. The lungs became refractory even to delayed hypoxic challenges after FTT application. In conclusion, these data support the concept that activation of PKC is involved in the transduction pathway forwarding pulmonary vasoconstriction in response to alveolar hypoxia.
Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep
Authors: Lines A. , Nardo L. , Phillips I. D. , Possmayer F. , Hooper S. B.
Published: December 2017

Obstruction of the fetal trachea is a potent stimulus for fetal lung growth, and it has been suggested that this procedure may be used therapeutically to reverse lung growth deficits in human fetuses with lung hypoplasia. However, little is known about the effects of increased lung expansion on other aspects of lung development. Our aim was to determine the effect of increased and decreased lung expansion on the mRNA levels encoding surfactant protein (SP) A, SP-B, and SP-C in ovine fetal lungs. Lung tissue samples were collected from fetuses exposed to 2, 4, or 10 days of increased lung expansion caused by tracheal obstruction. The mRNA levels for SP-A, SP-B, and SP-C were determined by Northern blot analysis with specific ovine cDNA probes; SP-A protein levels were determined by Western blot analysis. Compared with age-matched (128-day gestational age) control fetuses, SP-A, SP-B, and SP-C mRNA levels in fetal lung tissue were significantly reduced at 2 days of tracheal obstruction and remained reduced at 4 and 10 days. However, SP-A protein levels were not reduced at 2 days of tracheal obstruction, tended to be reduced at 4 days, and were almost undetectable at 10 days. In contrast to tracheal obstruction, 7 days of lung liquid drainage significantly increased SP-C, but not SP-A, mRNA levels in fetal lung tissue compared with age-matched control fetuses. Our results demonstrate that increases in fetal lung expansion, induced by obstruction of the fetal trachea, cause large simultaneous reductions in SP-A, SP-B, and SP-C mRNA levels in the fetal lung as well as a decrease in SP-A protein levels. These data suggest that expression of the genes encoding SPs in the fetal lung are specifically responsive to the degree of lung expansion.
Production of interferon-γ by lung lymphocytes in HIV-infected individuals
Authors: Twigg Homer L. , Spain Blake A. , Soliman Diaa M. , Knox Kenneth , Sidner Richard A. , Schnizlein-Bick Carol , Wilkes David S. , Iwamoto Gary K.
Published: December 2017

A CD8+lymphocytic alveolitis occurs in up to 60% of asymptomatic human immunodeficiency virus (HIV)-infected individuals. Early in HIV infection, lymphocytes consist predominantly of cytotoxic T lymphocytes directed against HIV-infected targets. As HIV disease progresses, they are replaced by CD8+CD57+suppressor cells. Virus-specific cytotoxic T lymphocytes secrete interferon-γ (IFN-γ), an important cytokine in upregulating immune responses, primarily through macrophage activation. We examined the ability of lung and blood lymphocytes from HIV-positive patients at various stages of HIV infection to secrete IFN-γ spontaneously and in response to phytohemagglutinin A. IFN-γ production and secretion were determined with ELISA, Western blot, immunoprecipitation, and Northern blot techniques. Lung lymphocytes from HIV-infected individuals secreted large amounts of IFN-γ. However, this ability was lost in patients with late-stage disease. Correlation between blood and lung lymphocyte IFN-γ secretion was poor, suggesting regional differences in lymphocyte function. These data suggest that lung levels of IFN-γ are high until late in HIV disease. These findings support the concept of administering exogenous IFN-γ to patients with late-stage HIV disease and opportunistic infections.
Airway synthesis of 20-hydroxyeicosatetraenoic acid: metabolism by cyclooxygenase to a bronchodilator
Authors: Jacobs Elizabeth R. , Effros Richard M. , Falck John R. , Reddy K. Malla , Campbell William B. , Zhu Daling
Published: December 2017

Rabbit airway tissue is a particularly rich source of cytochrome P-4504A protein, but very little information regarding the effect(s) of 20-hydroxyeicosatetraenoic acid (20-HETE) on bronchial tone is available. Our studies examined the response of rabbit bronchial rings to 20-HETE and the metabolism of arachidonic acid and 20-HETE from airway microsomes. 20-HETE (10−8to 10−6M) produced a concentration-dependent relaxation of bronchial rings precontracted with KCl or histamine but not with carbachol. Relaxation to 20-HETE was blocked by indomethacin or epithelium removal, consistent with the conversion of 20-HETE to a bronchial relaxant by epithelial cyclooxygenase. A cyclooxygenase product of 20-HETE also elicited relaxation of bronchial rings. [14C]arachidonic acid was converted by airway microsomes to products that comigrated with authentic 20-HETE (confirmed by gas chromatography-mass spectrometry as 19- and 20-HETE) and to unidentified polar metabolites. [3H]20-HETE was metabolized to indomethacin-inhibitable products. These data suggest that 20-HETE is an endogenous product of rabbit airway tissue and may modulate airway resistance in a cyclooxygenase-dependent manner.
EMAP II: a modulator of neovascularization in the developing lung
Authors: Schwarz Margaret , Lee Matt , Zhang Fangrong , Zhao Jingsong , Jin Yangsun , Smith Susan , Bhuva Janki , Stern David , Warburton David , Starnes Vaughn
Published: December 2017

Neovascularization is a key regulatory process in fetal growth and development. Although factors promoting growth and development of the pulmonary vasculature have been investigated, nothing is known regarding the molecular mechanisms that may counteract these stimuli. Endothelial monocyte-activating polypeptide (EMAP) II has recently been identified as an antiangiogenic factor in tumor vascular development. We postulated that EMAP II is a putative negative modulator of lung vascular growth. EMAP II mRNA and protein decrease fivefold ( P < 0.01) as the developing lungs in the fetal mouse progress from having poor vascularization ( day 14) to having complete vascular development at term ( day 18.5). EMAP II protein expression continues to remain low throughout postnatal life and into adulthood, with the exception of a surge that correlates with microvascular maturation. Furthermore, through the use of in situ hybridization and immunohistochemistry, EMAP II is localized throughout the lung, with significant expression in the submyoepithelial area during the early stages of lung development when there is minimal vascular development. In contrast, EMAP II is distributed around the large vessels during the end of vascular development, suggesting that EMAP II modulates the neovascularization process. We speculate that EMAP II is a director of neovascularization in the developing lung.
TNF-α upregulates Giα and Gqα protein expression and function in human airway smooth muscle cells
Authors: Hotta Kunihisa , Emala Charles W. , Hirshman Carol A.
Published: December 2017

Chronic inflammation is a characteristic feature of asthma. Multiple inflammatory mediators are released within the asthmatic lung, some of which may have detrimental effects on signal transduction pathways in airway smooth muscle. The effects of tumor necrosis factor (TNF)-α on the expression and function of muscarinic receptors and guanine nucleotide-binding protein (G protein) α-subunits were examined in human airway smooth muscle cells. Cultured human airway smooth muscle cells were incubated in serum-free culture medium for 72 h in the presence and absence of 10 ng/ml of TNF-α, after which the cells were lysed and subjected to electrophoresis and Gαi-2, Gqα, and Gsα protein subunits were detected by immunoblot analysis with specific antisera. TNF-α treatment for 72 h significantly increased the expression of Gαi-2and Gqα proteins and enhanced carbachol (10−7M)-mediated inhibition of adenylyl cyclase activity and inositol phosphate synthesis. These data provide new evidence demonstrating that TNF-α not only increases expression of Gαi-2and Gqα proteins but also augments the associated signal transduction pathways that would facilitate increased tone of airway smooth muscle.
Facilitatory β2-adrenoceptors on cholinergic and adrenergic nerve endings of the guinea pig trachea
Authors: de Haas Jan Roelof A. , Terpstra J. Saskia , van der Zwaag Monica , Kockelbergh Pieter G. E. , Roffel Ad F. , Zaagsma Johan
Published: December 2017

Using electrical field stimulation of epithelium-denuded intact guinea pig tracheal tube preparations, we studied the presence and role of prejunctional β2-adrenoceptors by measuring evoked endogenous acetylcholine (ACh) and norepinephrine (NE) release directly. Analysis of ACh and NE was through two HPLC systems with electrochemical detection. Electrical field stimulation (150 mA, 0.8 ms, 16 Hz, 5 min, biphasic pulses) released 29.1 ± 2.5 pmol ACh/g tissue and 70.2 ± 6.2 pmol NE/g tissue. Preincubation for 15 min with the selective β2-adrenoceptor agonist fenoterol (1 μM) increased both ACh and NE overflow to 178 ± 28 ( P < 0.01) and 165 ± 12% ( P < 0.01), respectively, of control values, increases that were abolished completely by the selective β2-adrenoceptor antagonist ICI-118551 (1 μM). Further experiments with increasing fenoterol concentrations (0.1–100 μM) and different preincubation periods (1, 5, and 15 min) showed a strong and concentration-dependent facilitation of NE release, with maximum response levels decreasing (from nearly 5-fold to only 2.5-fold of control value) with increasing agonist contact time. In contrast, sensitivity of facilitatory β2-adrenoceptors on cholinergic nerves to fenoterol gradually increased when the incubation period was prolonged; in addition, a bell-shaped concentration-response relationship was found at 15 min of preincubation. Fenoterol concentration-response relationships (15-min agonist preincubation) in the presence of atropine and yohimbine (1 μM each) were similar in the case of NE release, but in the case of ACh release, the bell shape was lost. The results indicate a differential capacity and response time profile of facilitatory prejunctional β2-adrenoceptors on adrenergic and cholinergic nerve terminals in the guinea pig trachea and suggest that the receptors on adrenergic nerves are more susceptible to desensitization.
Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions
Authors: Clark Howard , Allen Lennell , Collins Erin , Barr Frederick , Dobbs Leland , Putz Gunther , Goerke Jon , Hawgood Samuel
Published: December 2017

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222–230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L404–L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.
Aerosolized GM-CSF ameliorates pulmonary alveolar proteinosis in GM-CSF-deficient mice
Authors: Reed Jacquelyn A. , Ikegami Machiko , Cianciolo Eli R. , Lu Wei , Cho Patricia S. , Hull William , Jobe Alan H. , Whitsett Jeffrey A.
Published: December 2017

Surfactant proteins and phospholipids accumulate in the alveolar spaces and lung tissues of mice deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF), with pathological findings resembling the histology seen in the human disease pulmonary alveolar proteinosis (PAP). Previous metabolic studies in GM-CSF-deficient [GM(−/−)] mice indicated that defects in surfactant clearance cause the surfactant accumulation in PAP. In the present study, GM(−/−) mice were treated daily or weekly with recombinant mouse GM-CSF by aerosol inhalation or intraperitoneal injection for 4–5 wk. Lung histology, alveolar macrophage differentiation, and surfactant protein B immunostaining returned toward normal levels in the GM-CSF aerosol-treated mice. Alveolar and lung tissue saturated phosphatidylcholine and surfactant protein B concentrations were significantly decreased after treatment with aerosolized GM-CSF. Cessation of aerosolized GM-CSF for 5 wk resulted in increased saturated phosphatidylcholine pool sizes that returned to pretreatment levels. In contrast, PAP did not improve in GM(−/−) mice treated daily for 5 wk with larger doses of systemic GM-CSF. Aerosolized GM-CSF improved PAP in the GM(−/−) mice, demonstrating that surfactant homeostasis can be influenced by local administration of GM-CSF to the respiratory tract.
Giα but not Gqα is linked to activation of p21rasin human airway smooth muscle cells
Authors: Emala Charles W. , Liu Feng , Hirshman Carol A.
Published: December 2017

Airway smooth muscle hypertrophy contributes to the narrowing of asthmatic airways. Activation of the mitogen-activated protein kinases is an important event in mediating cell proliferation. Because the monomeric G protein p21rasis an important intermediate leading to activation of mitogen-activated protein kinases, we questioned which heterotrimeric G protein-coupled receptors were linked to the activation of p21rasin cultured human airway smooth muscle and which of the heterotrimeric G protein subunits (α or βγ) transmitted the activation signal. Carbachol and endothelin-1 increased GTP-bound p21rasin a pertussis toxin-sensitive manner [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): control, 30 ± 1.7; 3 min of 1 μM carbachol, 39 ± 1.1; 3 min of 1 μM endothelin-1, 40 ± 1.2], whereas histamine, bradykinin, and KCl were without effect. Transfection of an inhibitor of the G protein βγ-subunit [the carboxy terminus (Gly495–Leu689) of the β-adrenoceptor kinase 1] failed to inhibit the carbachol-induced activation of p21ras. These data suggest that Gi- but not Gq-coupled receptors activate p21rasin human airway smooth muscle cells, and this effect most likely involves the α-subunit.
Hypoxia inhibits increased ETB receptor-mediated NO synthesis in hypertensive rat lungs
Authors: Sato Koichi , Rodman David M. , McMurtry Ivan F.
Published: December 2017

Although hypertensive lungs of chronically hypoxic rats express increased levels of nitric oxide (NO) synthases (NOSs) and produce increased amounts of NO-containing compounds (NOx) during normoxic ventilation, the level of NO production during hypoxic exposure is unclear. Because hypoxia inhibits NO synthesis in normotensive lungs, we investigated whether hypoxic ventilation inhibited NO synthesis in isolated hypertensive lungs and chronically hypoxic rats. Measurement of perfusate NOx concentration in hypertensive lungs from male rats exposed to 4 wk of hypobaric hypoxia showed that basal NOx production was reduced during hypoxic (0% O2) vs. normoxic (21% O2) ventilation. Similarly, plasma NOxconcentration was lower in chronically hypoxic rats breathing 10% O2 than in those breathing 21% O2. Hypoxic inhibition of lung NOx production was not prevented by supplementaryl-arginine or tetrahydrobiopterin and was not mimicked by inhibition of Ca2+ influx. However, it was mimicked by inhibition of constitutive NOS with N G-monomethyl-l-arginine and chelation of intracellular Ca2+. The endothelin type B-receptor antagonist BQ-788 prevented the increases in NOx production associated with normoxic ventilation in both isolated hypertensive lungs and intact chronically hypoxic rats. These results suggest that a reduced supply of the cosubstrate molecular O2 to NOS counteracts an endothelin type B receptor-mediated stimulation of NO synthesis in hypertensive rat lungs. Thus, despite increased NOS protein in the lungs and pulmonary arteries of chronically hypoxic rats, direct hypoxic inhibition of NO production may contribute to the development of pulmonary hypertension.