bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection of perfused lung from oxidant injury by inhibitors of anion exchange
Authors: Nozik-Grayck E. , Piantadosi C. A. , van Adelsberg J. , Alper S. L. , Huang Y. C.
Published: December 2017

Hyperoxic lung injury is enhanced in isolated perfused lungs (IPL) in the presence of L-arginine. Reactive O2 species such as superoxide anion (O2-.) produced during hyperoxia are known to react with nitric oxide to form the strong oxidant species peroxynitrite. The appearance of O2-. in red blood cell membranes in vitro and in buffer-perfused lung preparations can be inhibited by the stilbene compound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) DIDS also inhibits anion exchange across the cell membrane regulated by a family of anion exchange proteins (AE). In this study, we hypothesized that anion exchange inhibitors would prevent lung injury from hyperoxia and L-arginine (O2 + L-Arg) by decreasing O2-. flux into the vascular space of the IPL. We found that both DIDS and a structurally distinct anion transport blocker, dipyridamole, protected the rabbit IPL from pulmonary hypertension and edema produced by O2 + L-Arg. The protective effect was associated with increased nitrite concentrations in the perfusate. Protection also was conferred when sodium bicarbonate in the perfusion buffer was replaced with either sodium thiosulfate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In lungs perfused with thiosulfate or HEPES-containing buffer, protection from O2 and L-arginine was also associated with diminished detection of reducing activity consistent with O2-. in the vascular space. Western blot analysis of lung protein and immunocytochemical staining of lung sections using antibodies against rabbit red blood cell AE1 and mouse gastric AE2 peptide showed that lung contains membrane protein antigenically similar to gastric AE2. These data suggest the possibility that inhibition of AE or other anion transporters may play an important role in mediating oxidative lung injury.
Upregulation of alveolar liquid clearance after fluid resuscitation for hemorrhagic shock in rats
Authors: Modelska K. , Matthay M. A. , McElroy M. C. , Pittet J. F.
Published: December 2017

The primary objective of this study was to test the hypothesis that a catecholamine-dependent mechanism would upregulate alveolar liquid clearance after fluid resuscitation from 15 min of hemorrhagic shock. Anesthetized rats were hemorrhaged to a mean arterial pressure of 35 mmHg for 15 min and were resuscitated with a 4% albumin solution. Alveolar liquid clearance was measured 5 h later by the concentration of protein in the distal airspaces over 1 h after instillation of a 5% albumin solution into one lung. Hemorrhaged rats developed a severe metabolic acidosis that was associated with a significant rise-in plasma epinephrine levels throughout the study. There was a 60% increase in alveolar liquid clearance in hemorrhaged and resuscitated rats compared with control rats. Amiloride (10(-4) or 10(-6) M), propranolol (10(-4) M), or bilateral adrenalectomy inhibited the increase in alveolar liquid clearance. This effect was reproduced by the intravenous administration of epinephrine in adrenalectomized and hemorrhaged rats. Thus these data provide evidence for a catecholamine-dependent regulation of sodium transport that protects the airspaces against flooding several hours after fluid resuscitation from hemorrhagic shock.
NO elicits prolonged relaxation of bovine pulmonary arteries via endogenous peroxynitrite generation
Authors: Davidson C. A. , Kaminski P. M. , Wolin M. S.
Published: December 2017

We previously reported that acute exposure of endothelium-removed bovine pulmonary arteries (BPA) to high levels (0.1 mM) of peroxynitrite (ONOO-) caused a prolonged guanosine 3',5'-cyclic monophosphate-related relaxation that appeared to be mediated through a thiol-dependent generation of nitric oxide (NO). In this study, we examined the importance of endogenous ONOO- formation in the regulation of BPA force generation by elevated physiological levels of NO. Exposure of BPA precontracted with 30 mM KCl to approximately 50 nM NO for 2 min caused a subsequent prolonged relaxation of KCl-induced force and an increased release of NO (measured in head space gas after a 5-min deoxygenation with 95% N2-5% CO2). This subsequent release of NO was reduced after depletion of tissue glutathione with diethyl maleate (DEM). Also, the NO-elicited prolonged relaxation of BPA was reversed by post-NO treatment with 10 microM methylene blue (MB; which inhibits guanylate cyclase stimulation by NO) or 1 microM oxyhemoglobin (which traps NO). Furthermore, inhibiting the biosynthesis of endogenous superoxide anion (O2-.) with 1 microM diphenyliodonium (DPI) or scavenging O2-. with 10 mM Tiron also promoted reversal of the NO-elicited prolonged relaxation seen in BPA after NO gas exposure. During exposure of BPA smooth muscle to approximately 50 nM NO gas, there appears to be a marked increase in ONOO- formation as detected by a DPI- and Tiron-inhibitable prominent increase in luminol-dependent chemiluminescence and a decrease in O2-. levels as detected by a reduction in lucigenin-dependent chemiluminescence during exposure to NO. Thus, during exposure to elevated physiological levels of NO, BPA appear to produce ONOO-, a species that seems to participate in prolonging the initial relaxation to NO through a thiol-dependent trapping and/or regeneration of NO.
Pulmonary artery smooth muscle cell [Ca2+]i and contraction: responses to diphenyleneiodonium and hypoxia
Authors: Zhang F. , Carson R. C. , Zhang H. , Gibson G. , Thomas H. M.
Published: December 2017

To investigate mechanisms of inhibition of hypoxic pulmonary vasoconstriction (HPV), we studied pulmonary artery smooth muscle cell (PASMC) responses to hypoxia, utilizing diphenyleneiodonium (DPI), which blocks HPV. We measured cell contraction in primary cultures of rat PASMC grown on collagen gels and cytosolic free Ca2+ concentration ([Ca2+]i) in PASMC grown on glass. DPI (5 and 20 microM) caused contraction of PASMC and increased [Ca2+]i. Omission of extracellular Ca2+ diminished the DPI-induced PASMC contraction and greatly reduced the increase in [Ca2+]i. DPI substantially inhibited KCl-induced PASMC contraction (1 microM DPI) and the increase in [Ca2+]i (5 microM DPI). Severe hypoxia contracted PASMC and quadrupled [Ca2+]i. DPI, 1 microM, substantially inhibited hypoxic contraction, but neither 1 nor 5 microM DPI diminished the hypoxia-induced increase in [Ca2+]i, which was greatly attenuated by 20 microM DPI. These data show 1) that DPI increases [Ca2+]i, accounting for DPI-induced PASMC contraction and 2) that 1 and 5 microM DPI inhibit the hypoxia-induced contraction but not the hypoxia-induced increase in [Ca2+]i, suggesting that DPI inhibits hypoxic PASMC contraction downstream of the Ca2+ signal by desensitizing the contractile apparatus and indicating a potential control point for modulation of HPV.
Integrin activation protects pulmonary endothelial cells from the genotoxic effects of bleomycin
Authors: Hoyt D. G. , Rizzo M. , Gerritsen M. E. , Pitt B. R. , Lazo J. S.
Published: December 2017

Integrin activation promotes the survival of endothelial cells undergoing diverse forms of stress. Here we determined the ability of integrins to inhibit DNA strand breakage by bleomycin (BLM), a DNA-cleaving antitumor antibiotic that causes acute endothelial injury and subsequent pulmonary fibrosis. We found that BLM produced DNA breakage in cultured murine lung endothelial cells (MLEC) within 45 min of treatment as measured by DNA sedimentation and in situ labeling of 3'-OH by nick translation (ISNT). Two hours after the removal of BLM, we found a marked but incomplete reduction in DNA strand breakage as measured by ISNT, indicating that the damage was reversible. DNA sedimentation and ISNT demonstrated that strand breakage due to BLM was inhibited in MLEC cultured on fibronectin, and no evidence of breakage was found 2 h after removal of the drug in ISNT experiments. Gelatin, type IV collagen, laminin, and the integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro, but not the inactive Gly-Arg-Ala-Asp-Ser-Pro peptide, also inhibited DNA strand breakage. Activation of integrins, either by coating surfaces with antibodies to alpha 5-, beta 1-, or beta 3-integrin subunits or by receptor clustering with the soluble antibodies, inhibited BLM-induced DNA breakage. Inhibition of BLM-induced DNA strand breakage by soluble beta 1-integrin antibody increased with increasing antibody concentration and duration of receptor clustering before BLM treatment. Thus integrin activation protects pulmonary endothelial cells from the genotoxic effects of BLM.
Increased expression of the interleukin-10 gene by alveolar macrophages in interstitial lung disease
Authors: Martinez J. A. , King T. E. , Brown K. , Jennings C. A. , Borish L. , Mortenson R. L. , Khan T. Z. , Bost T. W. , Riches D. W.
Published: December 2017

Idiopathic pulmonary fibrosis (IPF) and bronchiolitis obliterans with organizing pneumonia (BOOP) are interstitial lung diseases of unknown pathogenesis. Alveolar macrophages play a major role in the regulation of the inflammatory response in these diseases through their ability to produce cytokines that modify the inflammatory response. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) exhibit proinflammatory and anti-inflammatory actions, respectively, and thus an imbalance in the expression of these cytokines may contribute to the pathogenesis of IPF and BOOP. Therefore, we quantified IL-10 and TNF-alpha mRNA levels in alveolar macrophages obtained by bronchoalveolar lavage (BAL) from patients with IPF and BOOP and in normal healthy volunteers. The level of TNF-alpha mRNA in macrophages obtained from IPF and BOOP patients was not significantly different from normal healthy subjects. However, macrophages from patients with IPF and BOOP expressed increased levels of IL-10 mRNA compared with healthy controls. In addition, stimulation of alveolar macrophages with lipopolysaccharide in the presence of a neutralizing anti-IL-10 antibody augmented the production of TNF-alpha over that seen in the absence of anti-IL-10 antibody, suggesting that the increased expression of IL-10 by alveolar macrophages may act to control the expression of TNF-alpha. Paradoxically, measurement of IL-10 protein in cell-free BAL fluid revealed lower amounts of the protein in patients with IPF and BOOP compared with healthy controls.
Exosurf enhances adenovirus-mediated gene transfer to alveolar type II cells
Authors: Manuel S. Machelle , Guo Yi , Matalon Sadis
Published: December 2017

We assessed the role of surfactant replacement mixtures in the enhancement of adenovirus-mediated gene transfer to pulmonary epithelial cells both in vitro and in vivo. A549 cells, a pulmonary epithelium-derived adenocarcinoma cell line, were incubated with either media alone or media containing 10 μg phospholipid/ml Exosurf or Infasurf for 50 min followed by addition of a replication-deficient adenovirus (E1-deleted) expressing the luciferase reporter gene [AdCMV-Luc; 10 plaque-forming units (PFU)/cell] for 4 h. Pretreatment with Exosurf, but not Infasurf, at 37°C, but not at 4°C, enhanced luciferase activity in A549 cells 24 h later by 156% ( P < 0.01). Intratracheal instillation of AdCMV-Luc (2 × 109PFU) into rats resulted in luciferase expression mainly in alveolar macrophages and to a smaller extent in alveolar type II (ATII) cells 24 h later. However, when the AdCMV-Luc instillation was preceded by Exosurf (250 μl; 25 mg/ml), a 10-fold increase in ATII cell luciferase activity was noted. Preincubation of cultured ATII cells with Exosurf also enhanced their transfection by AdCMV-Luc by 515% ( P < 0.001). The results of these studies provide a new strategy for targeting ATII cells for gene delivery.
Biosynthesis of sulfated extracellular matrices by alveolar type II cells increases with time in culture
Authors: Sannes Philip L. , Khosla Jody , Peters Barry P.
Published: December 2017

The aim of this study was to determine the extent to which sulfate incorporated into biosynthesized basement membrane (BM) components increased as isolated type II cells progress toward a more type I cell-like phenotype from 7 to 21 days in culture. Specific sulfate cytochemistry, using high iron diamine, showed that type I-like cells in 21-day cultures deposited a more highly sulfated extracellular matrix. Biosynthetic labeling experiments using [35S]cysteine or [35S]sulfate as precursors confirmed the increased capacity of 21-day type I-like cells to biosynthesize sulfated BM components compared with type II-like cells in 7-day cultures, including a novel sulfated laminin. These biochemical changes in sulfation of BM components coincide with the established phenotypic transition from type II to type I cells during prolonged culture. More importantly, the data suggest that regulation of sulfation constitutes a potential mechanism by which type I and type II cells alter their environment in such a manner as to stabilize phenotype and modulate responses to growth factors.
Pulmonary dysfunction in neonatal SP-B-deficient mice
Authors: Tokieda Keisuke , Whitsett Jeffrey A. , Clark Jean C. , Weaver Timothy E. , Ikeda Kazushige , McConnell Keith B. , Jobe Alan H. , Ikegami Machiko , Iwamoto Harriet S.
Published: December 2017

Pulmonary function was assessed in newborn wild-type and homozygous and heterozygous surfactant protein B (SP-B)-deficient mice after birth. SP-B+/+ and SP-B+/− mice became well oxygenated and survived postnatally. Although lung compliance was decreased slightly in the SP-B+/− mice, lung volumes and compliances were decreased markedly in homozygous SP-B−/− mice. They died rapidly after birth, failing to inflate their lungs or oxygenate. SP-B proprotein was absent in the SP-B−/− mice and was reduced in the SP-B+/− mice, as assessed by Western analysis. Surfactant protein A, surfactant proprotein C, surfactant protein D, and surfactant phospholipid content in lungs from SP-B+/− and SP-B−/− mice were not altered. Lung saturated phosphatidylcholine and precursor incorporation into saturated phosphatidylcholine were not influenced by SP-B genotype. Intratracheal administration of perfluorocarbon resulted in lung expansion, oxygenation, and prolonged survival of SP-B−/− mice and in reduced lung compliance in SP-B+/+ and SP-B+/− mice. Lack of SP-B caused respiratory failure at birth, and decreased SP-B protein was associated with reduced lung compliance. These findings demonstrate the critical role of SP-B in perinatal adaptation to air breathing.
rhs-TM prevents ET-induced increase in pulmonary vascular permeability through protein C activation
Authors: Uchiba Mitsuhiro , Okajima Kenji , Murakami Kazunori , Johno Masayoshi , Mohri Mitsunobu , Okabe Hiroaki , Takatsuki Kiyoshi
Published: December 2017

We have previously demonstrated that recombinant human soluble (rhs) thrombomodulin (TM) inhibits the endotoxin (ET)-induced increase in pulmonary vascular permeability by inhibiting leukocyte activation. In the present study, we examined whether rhs-TM could inhibit the ET-induced increase in pulmonary vascular permeability in rats by activating protein C. rhs-TM did not inhibit ET-induced increases in pulmonary vascular permeability when its protein C activation ability was selectively inhibited by a monoclonal antibody (MAb) against rhs-TM (MAb R5G12). Histological examination revealed that neutrophil infiltration in lung tissues after ET administration was significantly reduced by rhs-TM, but infiltration was not reduced by MAb R5G12-pretreated rhs-TM. ET-induced intravascular coagulation was prevented by rhs-TM and by MAb R5G12-pretreated rhs-TM. However, ET-induced coagulation was not prevented by rhs-TM that had been treated with MAb F2H5, which cannot bind thrombin or activate protein C. These observations strongly suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes through thrombin binding and the subsequent protein C activation and may prevent ET-induced intravascular coagulation through thrombin binding.
Airway surface fluid composition in the rat determined by capillary electrophoresis
Authors: Cowley Elizabeth A. , Govindaraju Karuthapillai , Lloyd David K. , Eidelman David H.
Published: December 2017

The apical surface of respiratory epithelial cells is covered by a thin layer of low-viscosity fluid termed airway surface fluid (ASF), about which relatively little is known. We collected samples of ASF from anesthetized rats, which were then analyzed using capillary electrophoresis, a method that enables extremely small quantities of fluid to be analyzed. We found values for Na+ (40.57 ± 3.08 mM), K+ (1.74 ± 0.36 mM), and Cl− (45.16 ± 1.81 mM), indicating that this fluid is hypotonic compared with rat plasma. In contrast, the concentrations of nitrite and nitrate within ASF were higher than reported plasma values. Additionally, intravenous administration of the cholinergic agonist methacholine (MCh) resulted in a dose-dependent increase in the concentration of Na+ and Cl− within the ASF. This increase is ∼50% in these ions after a dose of 100 ng MCh/g body wt. This animal model, together with this microanalytical technique, may be useful for investigating the in vivo regulation of ASF composition.
In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP
Authors: Kelley Thomas J. , Cotton Calvin U. , Drumm Mitchell L.
Published: December 2017

Inhibitors of guanosine 3′,5′-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl−transport across the nasal epithelia of cystic fibrosis mice carrying the ΔF508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF/ΔF)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and ΔF508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(−/−)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3′,5′-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl−secretion across the nasal epithelium of CFTR(ΔF/ΔF) mice. No effect on TEPD was seen with this combination when used on CFTR(−/−) mice, implying that the CNP-induced change in TEPD in CFTR(ΔF/ΔF) mice is CFTR dependent.
Glycosylation differences between a cystic fibrosis and rescued airway cell line are not CFTR dependent
Authors: Jiang Xiaosui , Hill Warren G. , Pilewski Joseph M. , Weisz Ora A.
Published: December 2017

Altered glycosylation of mucus and membrane glycoconjugates could explain reported differences in binding of bacterial pathogens to cystic fibrosis (CF) versus normal tissue. However, because bacteria can alter cell surface glycoconjugates, it is not possible to assess the role of cystic fibrosis transmembrane conductance regulators (CFTR) in glycosylation in these studies. To address this issue, we have developed quantitative lectin binding assays to compare cell surface glycosylation in well-matched immortalized CF cells and rescued cell lines. The CF airway bronchial epithelial cell line IB3-1 consistently bound more peanut agglutinin (PNA) than its clonal derivative S9, which stably expresses functional wild-type CFTR. Pretreatment with neuraminidase increased PNA binding and abolished the difference between the two cell lines. However, infection of the IB3-1 cells with a replication-deficient recombinant adenovirus encoding CFTR restored CFTR function but did not alter PNA binding to cells. In contrast, treatment with the weak base ammonium chloride increased PNA binding to both cell lines as expected. Our data show that even clonally related CF and rescued cells can exhibit significant differences in carbohydrate processing. Although the differences that we found are consistent with the proposed role for CFTR in modulating intraorganellar pH, our data strongly suggest that they are CFTR independent. These studies add a cautionary note to the interpretation of differences in glycosylation between CF and normal primary tissues and immortalized cells.
KATP channels contribute to β- and adenosine receptor-mediated pulmonary vasorelaxation
Authors: Sheridan Brett C. , McIntyre Robert C. , Meldrum Daniel R. , Fullerton David A.
Published: December 2017

ATP-sensitive K+(KATP) channels have been implicated in the regulation of vasomotor tone in aortic, mesenteric, and pulmonary vascular smooth muscle. Several investigators have described an association between KATP channels and isoproterenol (Iso)-stimulated relaxation responses. To study the relationship between receptor-dependent pulmonary vasorelaxation and KATP channels, we examined the response to agonists that generate adenosine 3′,5′-cyclic monophosphate at two distinct levels of the signal transduction pathway after inhibition or activation of KATP channels in isolated rat pulmonary artery rings. Cumulative concentration responses to β-adrenergic receptor stimulation (Iso), purinergic receptor stimulation [adenosine (Ado)], and direct stimulation of adenylate cyclase [forskolin (FSK)] were studied with and without concurrent inhibition of KATP channels (glibenclamide or tolbutamide). In addition, the effect of direct KATP channel activation (cromakalim) on the response to β-adrenergic and purinergic receptor stimulation was determined. Last, we investigated the influence of KATP channel inhibition on endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation linked to guanosine 3′,5′-cyclic monophosphate production. KATPchannel inhibition impaired the response to Iso and Ado. Activation of KATP channels caused a leftward shift in the dose responses of Iso and Ado, with a significant decrease in the 50% effective concentration for each agent. KATP channel inhibition did not impair the pulmonary arterial vasorelaxation response to FSK, acetylcholine, or sodium nitroprusside. KATP channels appear to contribute to β-adrenergic and purinergic receptor-stimulated vasorelaxation in rat pulmonary arteries.
Myogenic and neurogenic mechanisms and arachidonate metabolites in bronchial muscle response to allergen
Authors: Janssen L. J. , McGrogan I. , Wattie J. , O’Byrne P. M. , Daniel E. E.
Published: December 2017

We investigated allergen-induced airway hyperresponsiveness (AH) in bronchial tissues obtained from dogs that inhaled Ascaris suum leading to AH (RESP) in vivo or that exhibited no change (NON-RESP) as well as from dogs that inhaled saline (SHAM). RESP tissues were not hyperresponsive to KCl or to carbachol, whereas contractions to electrical field stimulation (EFS) were reduced. This reduction was reversed partially by indomethacin and completely by replacement of the bathing fluid. Radioimmunoassay revealed marked elevation of prostaglandin (PG) E2 generation in RESP tissues compared with SHAM and NON-RESP tissues. EFS-evoked contractions were often followed by a slowly developing secondary contraction in RESP tissues but not in SHAM or NON-RESP tissues. However, indomethacin unmasked such secondary contractions in many SHAM and NON-RESP tissues and markedly enhanced those in RESP tissues, whereas L-655,240 (thromboxane A2/PGD2receptor antagonist) abolished such contractions in all groups. We were unable to detect thromboxane using radioimmunoassay. We conclude that allergen-induced AH involves altered generation of cyclooxygenase metabolites of arachidonic acid (particularly PGE2) as well as of a nonprostanoid inhibitory factor; as such, the responsiveness of the tissue in vitro is dependent on the relative levels of inhibitory and excitatory metabolites.
Effect of hypercarbia on surface proteins of cultured bovine endothelial cells
Authors: Rounds Sharon , Piggott Damani , Dawicki Doloretta D. , Farber Harrison W.
Published: December 2017

Hypercarbia is a common complication of respiratory failure, and the technique of “permissive hypercapnia” is used to ventilate individuals with increased peak airway pressures on mechanical ventilators, resulting in elevated arterial [Formula: see text]. We studied the effects of hypercarbia on cultured bovine aortic and main pulmonary artery endothelial cell surface proteins, assessing cell surface iodination using lactoperoxidase bound to latex microspheres. We found that 4 h of exposure to 10% CO2increased the display of substances of apparent molecular masses of 27, 47, and 52 kDa. This effect was not mimicked by acidotic media. Western blots of detergent extracts of main pulmonary artery endothelial cell monolayers did not show increased expression of carbonic anhydrase IV (molecular mass = 52 kDa) after incubation under hypercarbic conditions. Hypercarbia did not change the pattern of [35S]methionine incorporation into endothelial cell proteins. We conclude that hypercarbia of 4-h duration changes iodinated endothelial cell surface proteins. We speculate that this effect may be related to changes in secretion or display of apical cell membrane-associated proteins.
Hyperoxic effects on alveolar sodium resorption and lung Na-K-ATPase
Authors: Carter Ethan P. , Wangensteen O. Douglas , Dunitz Jordan , Ingbar David H.
Published: December 2017

Active Na+transport by the alveolar epithelium keeps alveoli relatively dry. Hyperoxia increases epithelial permeability, resulting in pulmonary edema. We sought to determine whether active Na+resorption from the air spaces and Na-K-ATPase activity increased in rats exposed to >95% O2for 60 h. The permeability × surface area products for unidirectional resorption of alveolar [14C]sucrose ( PSsucrose) and22Na+([Formula: see text]) were measured in isolated, perfused rat lungs immediately after hyperoxia and after 3 and 7 days of recovery in room air. At 60 h of hyperoxia, the mean PSsucroseand[Formula: see text]increased from 6.71 ± 0.8 × 10−5to 12.6 ± 1.6 × 10−5cm3/s ( P = 0.029) and from 23.6 ± 1.1 × 10−5to 31.0 ± 1.6 × 10−5cm3/s ( P < 0.008), respectively. However, the values in individual rats ranged widely from no change to nearly a fourfold increase. Subgroup analysis revealed that benzamil- or amiloride-sensitive (transcellular)[Formula: see text]was significantly reduced in the exposed lungs with normal PSsucrosebut was maintained in the lungs with high PSsucrose. By day 3of recovery, mean Na+and sucrose fluxes returned to values similar to control. Na-K-ATPase membrane hydrolytic maximal velocity ( Vmax) activity fell significantly immediately after hyperoxic exposure but recovered to normal values by day 3 of recovery. The Na-K-ATPase β1-subunit antigenic signal did not significantly change, whereas the α1-subunit levels increased during recovery. In summary, there was a heterogeneous response of different rats to acute hyperoxia. Hyperoxia led to complex, nonparallel changes in Na+pump antigenic protein, hydrolytic activity, and unidirectional active Na+resorption. Active Na+transport was differentially affected, depending on degree of injury, but permeability and transport normalized by day 3 of recovery.
Calcium-stimulated Cl−secretion in Calu-3 human airway cells requires CFTR
Authors: Moon Samina , Singh Meetpaul , Krouse Mauri E. , Wine Jeffrey J.
Published: December 2017

Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl−-dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl−channels. Typically, CF disrupts adenosine 3′,5′-cyclic monophosphate (cAMP)-mediated Cl−secretion but spares Ca2+-mediated secretion. However, in CF airway glands, Ca2+-mediated secretion is also greatly reduced. To determine the basis of Ca2+-mediated Cl−secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current ( Isc) and transepithelial conductance ( Gte) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (Δ) in Iscand Gte, whereas forskolin stimulated variable and smaller increases. Δ Iscwas decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 4,4′-dinitrostilbene-2,2′-disulfonic acid, and calixarene. Iscwas measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl−conductance ( GCl) that was decreased by DPAC but not by DIDS. Apical GClcould be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive GClpathway in the apical membrane and display ∼60% of maximum conductance at rest. Thus elevated [Ca2+]iincreases K+conductance to force Cl−through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.
Wound-induced calcium waves in alveolar type II cells
Authors: Hinman Lee E. , Beilman Greg J. , Groehler Kristine E. , Sammak Paul J.
Published: December 2017

Alveolar type II epithelial (ATII) cells repopulate the alveolus after acute lung injury. We hypothesized that injury would initiate signals in nearby survivors. When rat ATII monolayers were wounded, elevations in intracellular free Ca2+concentration ([Ca2+]i) began at the edge of the wound and propagated outward as a wave for at least 300 μm. The [Ca2+]iwave was due to both influx of extracellular Ca2+and release of intracellular Ca2+stores. Reducing Ca2+influx with brief treatments of ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid or Gd3+reduced both the amplitude and the apparent speed. Draining intracellular Ca2+stores by pretreatment with cyclopiazonic acid eliminated the [Ca2+]iwave. Therefore, the [Ca2+]iwave depended critically on intracellular Ca2+stores. [Ca2+]ielevations propagated over a break in the monolayer, suggesting that extracellular pathways were involved. Furthermore, extracellular factors from injured cells elevated [Ca2+]iin uninjured cultures. We conclude that wounding produces a [Ca2+]iwave in surviving cells and part of this response is mediated by soluble factors released into the extracellular space during injury.
Peroxisome proliferators alter lipid acquisition and elastin gene expression in neonatal rat lung fibroblasts
Authors: McGowan Stephen E. , Jackson Sheila K. , Doro Melissa M. , Olson Paula J.
Published: December 2017

During the alveolar stage of lung development, lipid droplet-laden interstitial cells are present at the base of elongating alveolar septa. These cells that have been named lipid interstitial cells or lipofibroblasts (LFs) may supply lipids for surfactant production, the synthesis of membrane phospholipids, and/or energy metabolism. They also have myofibroblastic characteristics and participate in the generation of the interstitial elastic fiber network, that is, in the pulmonary alveolar septum. To understand how this cell regulates its lipid-storing and elastin-producing properties, we have examined the effects of peroxisome proliferators on the expression of the genes that are associated with an elastin-producing myofibroblastic phenotype or an adipocyte-like phenotype. Two known ligands for peroxisome proliferator-activated receptors, 5,8,11,14-eicosatetraynoic acid (ETYA) and 15-deoxy-Δ-12,14-prostaglandin J2(15-dPGJ2), decrease elastin gene transcription and the steady-state levels of tropoelastin (TE) and α-smooth muscle actin mRNAs in cultured LFs. Concurrently, cultured LFs increase the expression of adipocyte lipid binding protein, which is regarded as an adipocyte-specific protein, and accumulate lipid droplets. Their abilities to store lipids and express desmin intermediate filaments, α-smooth muscle actin, and smooth muscle myosin heavy chain in contractile filaments in vitro illustrate similarities among the pulmonary LF, the hepatic lipocyte, and the contractile interstitial cell, which contribute to the repair reaction in the lung after pulmonary injury.
Selected isozymes of PKC contribute to augmented growth of fetal and neonatal bovine PA adventitial fibroblasts
Authors: Das Mita , Stenmark Kurt R. , Ruff Laura J. , Dempsey Edward C.
Published: December 2017

We sought to determine which isozymes of protein kinase C (PKC) contribute to the increased proliferation of immature bovine pulmonary artery (PA) adventitial fibroblasts. Seven were identified in lysates of neonatal PA fibroblasts by Western blot: three Ca2+dependent (α, βI, and βII) and four Ca2+independent (δ, ε, ζ, and μ). Four isozymes (γ, η, θ, and ι) were not detected in fibroblasts isolated at any developmental stage. Of the seven detected isozymes, only PKC-α and -βII protein levels were higher in fetal and neonatal cells compared with adult fibroblasts. Their role in the enhanced growth of immature fibroblasts was then evaluated. The isozyme nonselective PKC inhibitor Ro-31-8220 was first compared with GF-109203X, a structural analog of Ro-31-8220 with relative specificity for the Ca2+-dependent isozymes of PKC. GF-109203X selectively inhibited the growth of immature cells and was nearly as potent as Ro-31-8220. Go-6976, a more specific inhibitor of the Ca2+-dependent isozymes, mimicked the antiproliferative effect of GF-109203X. PKC downregulation with 1 μM phorbol 12-myristate 13-acetate had the same selective antiproliferative effect on immature fibroblasts as GF-109203X and Go-6976. The protein levels of PKC-α and -βII, but not of PKC-βI, were completely degraded in response to phorbol 12-myristate 13-acetate pretreatment. These results suggest that PKC-α and -βII are important in the augmented growth of immature bovine PA adventitial fibroblasts.
Prostaglandin E2 integrates the effects of fluid distension and glucocorticoid on lung maturation
Authors: Torday J. S. , Sun H. , Qin J.
Published: December 2017

Both glucocorticoids and alveolar fluid distension affect the rate of fetal lung maturation, possibly representing a common cellular pathway. In an explant culture, there is a spontaneous increase in triglyceride incorporation into saturated phosphatidylcholine over time. This mechanism is stimulated by prostaglandin (PG) E2, blocked by both bumetanide and indomethacin, and overridden by exogenous PGE2. Type II cells synthesized and produced PGE2 between days 16 and 21 postconception, increasing fourfold between days 19 and 21. Fetal rat lung fibroblasts released triglyceride in response to PGE2, increasing 10- to 14-fold between days 19 and 21 postconception; phloretin (1 × 10−5 M) completely blocked this effect of PGE2 on triglyceride release. Dexamethasone stimulated both type II cell PGE2 synthesis (threefold) and fibroblast triglyceride release in response to PGE2 (60%) by day 20 cells. Stretching type II cells also increased PGE2 synthesis (∼100% at 1, 2, and 3 h vs. static cultures). Recombination of [3H]triglyceride-labeled fibroblasts with type II cells in an organotypic culture resulted in progressive incorporation of label into saturated phosphatidylcholine by type II cells. This process was also blocked by the addition of indomethacin and overridden by exogenous PGE2. These data suggest that the combined effects of alveolar fluid dilatation and glucocorticoids may coordinate the timely transfer of triglyceride from fibroblasts to type II cells for augmented surfactant production through their effects on PGE2 production and action as term approaches.
Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP
Authors: Allen Corrie B. , White Carl W.
Published: December 2017

To determine whether glucose depletion is a principal determinant of hyperoxic cell death in vitro, human lung epithelial-like cells (A549) were exposed to hyperoxia (95% O2) in either 10, 30, or 50 ml of medium (Ham’s F-12K). Glucose was depleted in the medium after 36, 60, or 96 h, respectively. Medium lactate dehydrogenase (LDH) activity increased only after glucose was depleted. To confirm that glucose depletion was critical to cell death, cells exposed to 95% O2were supplemented with glucose at regular intervals to reestablish initial medium glucose concentrations. Other cells received no supplements. Without supplementation, glucose was depleted within 48 h, followed within 12 h by an almost complete loss of cell ATP and elevated medium LDH activity. Glucose-supplemented cells appeared normal microscopically and did not release LDH activity despite an extracellular pH of 6.5 due to fermentation. Additional experiments at sea-level pressure confirmed that glucose supplementation prevents extensive cell death in hyperoxia in cultured A549 cells.
Cloning of rat eotaxin: ozone inhalation increases mRNA and protein expression in lungs of Brown Norway rats
Authors: Ishii Yukio , Shirato Manabu , Nomura Akihiro , Sakamoto Tohru , Uchida Yoshiyuki , Ohtsuka Morio , Sagai Masaru , Hasegawa Shizuo
Published: December 2017

The C-C chemokine eotaxin is thought to be important in the selective recruitment of eosinophils to the site of inflammation in guinea pigs, mice, and humans. We isolated the rat eotaxin gene to determine whether a similar molecule might play a role in the pulmonary infiltration of eosinophils during acute inflammation in the rat. The cDNA for rat eotaxin encoded a 97-amino acid protein containing a 74-amino acid mature eotaxin protein with 97.3% identity to mouse eotaxin. The recombinant protein encoded by this gene displayed specific chemotactic activity for eosinophils when analyzed with a microchemotactic chamber. The expression of eotaxin mRNA increased ∼1.6-fold immediately after exposure to ozone and was 4-fold higher after 20 h. The number of lavageable eosinophils at the same time points were 3- and 15-fold greater, respectively, than control eosinophils. Immunocytochemistry revealed that alveolar macrophages and bronchial epithelial cells were positive for eotaxin. These results suggest that eotaxin may be involved in the recruitment of eosinophils into the air spaces during certain inflammatory conditions in rats.
Ontogeny of cyclooxygenase-1 and cyclooxygenase-2 gene expression in ovine lung
Authors: Brannon Timothy S. , MacRitchie Amy N. , Jaramillo Marina A. , Sherman Todd S. , Yuhanna Ivan S. , Margraf Linda R. , Shaul Philip W.
Published: December 2017

Prostacyclin is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in whole lung increases during that period. The rate-limiting enzyme in prostacyclin synthesis in the developing lung is cyclooxygenase (COX). We investigated the ontogeny and cellular localization of COX-1 (constitutive) and COX-2 (inducible) gene expression in lungs from late-gestation fetal lambs, 1-wk-old newborn lambs (NB1), and 1- to 4-mo-old newborn lambs (NB2). COX-1 mRNA abundance rose progressively from fetal to NB1 to NB2, increasing 12-fold overall. In parallel, immunoblot analysis revealed a progressive increase in COX-1 protein, rising fourfold from fetal lambs to NB2. COX-2 mRNA levels increased fivefold from fetal to NB1 but were similar in NB1 and NB2. However, COX-2 protein was not detectable by immunoblot analysis in any age group. Immunohistochemistry for COX-1 showed intense immunostaining in endothelial cells at all ages. COX-1 was also expressed in airway epithelium at all ages, with a greater number of epithelial cells staining positively in NB2 compared with fetal and NB1 groups. In addition, COX-1 was expressed in airway smooth muscle from NB1. COX-2 immunostaining was absent in all age groups. These findings indicate that there is differential expression of COX-1 and COX-2 in the developing lung and that the enzymes are expressed in a cell-specific manner. The developmental upregulation in COX-1 may optimize the capacity for prostaglandin-mediated vasodilation, bronchodilation, and surfactant synthesis in the newborn lung.
Influence of the cytoskeleton on surfactant protein gene expression in cultured rat alveolar type II cells
Authors: Shannon John M. , Pan Tianli , Edeen Karen E. , Nielsen Larry D.
Published: December 2017

We have investigated the role of the cytoskeleton in surfactant protein gene expression. Cytochalasin D (CD), colchicine (Col), or nocodazole (Noco) were tested on primary cultures of adult rat alveolar type II cells. Treatment with any of the drugs did not result in dramatic cell shape changes, but ultrastructural examination revealed that the cytoplasm of cells treated with CD was markedly disorganized; cells treated with Col did not exhibit such changes. Treatment with any of the three drugs resulted in a reduction in surfactant protein (SP) mRNAs. These decreases were not the result of cell toxicity, since overall protein synthesis was unimpaired by drug treatment. Washing the cells followed by an additional 2 days of culture resulted in a reaccumulation of SP mRNAs in CD-treated cells but not in Col-treated cells. Washing of Noco-treated cultures resulted in partial recovery. SP mRNA stability was estimated in the presence or absence of cytoskeleton-disrupting drugs. Disruption of either microfilaments or microtubules significantly affected the half-lives of mRNAs for SP-A, SP-B, and SP-C. These data support a role for the cytoskeleton in the maintenance of type II cell differentiation and suggest that the role of the cytoskeleton is at least in part to stabilize SP mRNAs.
Alveolar liquid clearance in the anesthetized ventilated guinea pig
Authors: Norlin Andreas , Finley Neelu , Abedinpour Parisa , Folkesson Hans G.
Published: December 2017

Alveolar liquid clearance was examined in ventilated, anesthetized guinea pigs. An isosmolar 5% albumin solution was instilled into the lungs. Alveolar liquid clearance was studied over 1 h and was measured from the increase in alveolar protein concentration as water was reabsorbed. Basal alveolar liquid clearance was 38% of instilled volume. The high basal alveolar liquid clearance was not secondary to endogenous catecholamine release. Compared with control animals, epinephrine and the general β-adrenergic agonist isoproterenol increased alveolar liquid clearance to ∼50% of instilled volume ( P < 0.05), whereas the β2-adrenergic agonist terbutaline was without effect. The stimulation of alveolar liquid clearance by epinephrine or isoproterenol was completely inhibited by the addition of the general β-adrenergic inhibitor propranolol or the β1-adrenergic inhibitor atenolol. Alveolar liquid clearance was inhibited by the sodium-channel inhibitor amiloride by 30–40% in control animals and in animals treated with epinephrine or isoproterenol. Isoproterenol and epinephrine, but not terbutaline, increased adenosine 3′,5′-cyclic monophosphate in in vitro incubated lung tissue. The results suggest that alveolar liquid clearance in guinea pigs is mediated partly through amiloride-sensitive sodium channels and that alveolar liquid clearance can be increased by stimulation of primarily β1-adrenergic receptors.
Evidence for functional ANP receptors in cultured alveolar type II cells
Authors: Tharaux Pierre-Louis , Dussaule Jean-Claude , Couette Sylvianne , Clerici Christine
Published: December 2017

Because atrial natriuretic peptide (ANP) is considered to play a role in lung physiology and pathology, our aim was to characterize natriuretic peptide receptors in cultured rat alveolar type II (ATII) cells. Guanylate cyclase A- and B-receptor but not clearance-receptor mRNAs were detected by reverse transcription-polymerase chain reaction. The absence of clearance-receptor expression in ATII cells was confirmed by competitive inhibition of ANP binding; ANP (0.1–100 nM) decreased the binding of125I-ANP, whereas C-ANP-(4—23), a specific ligand of clearance receptors, was ineffective. ANP induced a dose-dependent increase in guanosine 3′,5′-cyclic monophosphate (cGMP) production, with a threshold of 0.1 nM, whereas the response to C-type natriuretic peptide was weak and was observed only at high concentrations (100 nM). In ATII cells cultured on filters, 1) ANP receptors were present on both the apical and basolateral surfaces and 2) cGMP egression was polarized, as indicated by the greater ANP-induced cGMP accumulation in the basolateral medium, and was partially inhibited by probenecid, an organic acid transport inhibitor. Influx studies demonstrated that ANP decreased the amiloride-sensitive component of22Na influx but did not change ouabain-sensitive86Rb influx. In conclusion, ATII cells behave as a target for ANP. ANP activation of guanylate cyclase A receptors produces cGMP, which is preferentially extruded on the basolateral side of the cells and inhibits the amiloride-sensitive Na-channel activity.
Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages
Authors: Hickman-Davis Judy M. , Lindsey J. Russell , Zhu S. , Matalon S.
Published: December 2017

Mycoplasma pneumoniae is a leading cause of pneumonia and exacerbates other respiratory diseases in humans. We investigated the potential role of surfactant protein (SP) A in antimycoplasmal defense using alveolar macrophages (AMs) from C57BL/6NCr (C57BL) mice, which are highly resistant to infections of Mycoplasma pulmonis. C57BL AMs, activated with interferon (IFN)-γ and incubated with SP-A (25 μg/ml) at 37°C, produced significant amounts of nitric oxide (⋅ NO; nitrate and nitrite production = 1.1 μM ⋅ h−1⋅ 105AMs−1) and effected an 83% decrease in mycoplasma colony-forming units (CFUs) by 6 h postinfection. Preincubation of AMs with the inducible nitric oxide synthase inhibitor NG-monomethyl-l-arginine abolished ⋅ NO production and SP-A-mediated killing of mycoplasmas. No decrease in CFUs was seen when IFN-γ-activated macrophages were infected with mycoplasmas in the absence of SP-A despite significant ⋅ NO production (nitrate and nitrite production = 0.6 μM ⋅ h−1⋅ 105AMs−1). These results demonstrate that SP-A mediates killing of mycoplasmas by AMs, possibly through an ⋅ NO-dependent mechanism.
Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II
Authors: Damron Derek S. , Nadim Hany S. , Hong Sung Jin , Darvish Ahmad , Murray Paul A.
Published: December 2017

Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+concentration in response to ANG II. Six isoforms (α, δ, ε, ζ, ι/λ, and μ) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (α, δ, ζ, ι/λ, and μ) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-α from the cytosol to the nuclear envelope and of PKC-δ to the myofilaments. In contrast, cytosolic PKC-ζ did not translocate, but nuclear PKC-ζ was upregulated. Translocation of PKC-α and PKC-δ and upregulation of PKC-ζ in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The α and ζ isoforms may act to regulate nuclear events, whereas PKC-δ may be involved in modulating contraction via actions on the myofilaments.