bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined effects of nitric oxide and hyperoxia on surfactant function and pulmonary inflammation
Authors: Robbins C. G. , Davis J. M. , Merritt T. A. , Amirkhanian J. D. , Sahgal N. , Morin F. C. , Horowitz S.
Published: December 2017

NO and its derivative ONOO- are potent free radicals that can cause cell damage, especially in the presence of O2. To determine the potential pulmonary toxicities of nitric oxide (NO) and peroxynitrite (ONOO-) in vitro, Survanta (2.5 mg/ml) was exposed to ONOO- (0.3-8 mM) in the presence of two different buffering systems (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and phosphate buffer) and minimum surface tension (MST) was determined with an oscillating bubble surfactometer. Significant increases in MST were seen only with exposure to 8 mM ONOO-, indicating that in vitro, high concentrations of ONOO- can inhibit natural surfactant function. The in vivo effects of NO and hyperoxia were then studied in four groups of newborn piglets ventilated for 48 h with 21% O2, 100% O2, 21% O2 and 100 ppm NO, or with 90% O2 and 100 ppm NO. Five animals served as an untreated control group. Bronchoalveolar lavage fluid (BAL) obtained at 48 h was subjected to centrifugation and the surfactant pellet was reconstituted to 5 mg phospholipid/ml. Significant increases in MST were seen in surfactant from piglets ventilated with NO and 90% O2, compared with either untreated controls or piglets ventilated with 21% O2 for 48 h (P < 0.05, analysis of variance). Significant increases in neutrophil chemotactic activity (NCA) of BAL were also found in the NO and O2 group (P < 0.05), with significant positive interaction between NO and O2 found (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Sequence of rat surfactant protein A gene and functional mapping of its upstream region
Authors: Smith C. I. , Rosenberg E. , Reisher S. R. , Li F. , Kefalides P. , Fisher A. B. , Feinstein S. I.
Published: December 2017

Surfactant protein A (SP-A) is the major pulmonary surfactant protein. We have isolated a rat SP-A genomic clone and determined the sequence from 2.9 kilobases upstream of the transcriptional start through the termination of translation. The exon-intron structure of the rat gene has been determined and compared with the mouse, rabbit, and human genes. We have localized the major transcriptional start site in adult rat lung to nucleotide 30 downstream from the start of the TATA box. Functional mapping indicates that a DNA fragment containing 163 nucleotides upstream of the transcriptional start (-163) can function as a promoter of transcription of a reporter gene in both lung and nonlung derived cell lines. However, the function of this element is weaker in cells of nonlung origin. DNA elements located between -2902 and -163 silence the promoter activity in both lung and nonlung cells. Because the SP-A gene promoter region exhibits limited tissue specificity, the results suggest the existence of other DNA elements which overcome the silencer and confer further lung specificity.
Tachykinins induce gelatinase production by guinea pig alveolar macrophages: involvement of NK2 receptors
Authors: D'Ortho M. P. , Jarreau P. H. , Delacourt C. , Pezet S. , Lafuma C. , Harf A. , Macquin-Mavier I.
Published: December 2017

To determine whether tachykinins induce gelatinase production by guinea pig alveolar macrophages (AM), and to characterize the mechanism involved, we incubated AM with substance P (SP), neurokinin A (NKA), or the NH2-terminal fragment of SP, SP(1-7). The effects of increasing concentrations of selective NK1 and NK2 agonists on tachykinin-induced gelatinase production were also evaluated, as were the effects of a selective NK2 antagonist. Gelatinase activity in conditioned culture media (CCM) was assessed by zymography and quantified by image analysis. SP increased 92-kDa gelatinase activity in CCM of AM in a concentration-dependent manner, with a maximum increase at 10(-4) M. NKA, the NH2-terminal fragment of SP, and an NK1-selective agonist had no effect. In contrast, a selective NK2 agonist induced a concentration-dependent increase in gelatinase activity. The increase in this activity induced by SP and the selective NK2 agonist was inhibited by a selective NK2 antagonist. We conclude that SP induces gelatinase production by AM through NK2 receptor activation. The release of gelatinase may constitute one mechanism through which SP contributes to the epithelial lesions observed in bronchial hyperreactivity and asthma.
Human lung cell beta 2-adrenergic receptors desensitize in response to in vivo administered beta-agonist
Authors: Turki J. , Green S. A. , Newman K. B. , Meyers M. A. , Liggett S. B.
Published: December 2017

Few studies have addressed whether target tissue adrenergic receptors in humans undergo desensitization in response to agonist administration. To determine whether lung cell beta 2-adrenergic receptors (beta 2-AR) undergo such desensitization, we harvested bronchial epithelial cells and alveolar macrophages via bronchoscopy from eight normal subjects before and after inhalation of six doses of the beta-agonist metaproterenol given over 24 h. After metaproterenol inhalation, beta 2-AR expression as determined by 125I-labeled cyanopindolol binding decreased approximately 70% on bronchial epithelial cells, from 6.3 +/- 0.7 to 2.0 +/- 0.2 fmol/mg (P < 0.001) and to a similar extent on macrophages from 13.3 +/- 0.4 to 3.9 +/- 0.6 fmol/mg (P < 0.001). Agonist inhalation also resulted in impairment of beta 2-AR function in both cell types. With bronchial epithelial cells, maximal isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation decreased from 9.5 +/- 1.8 to 4.9 +/- 1.2 pmol/10(6) cells (P = 0.003), which amounts to a 48 +/- 6% desensitization. Isoproterenol-stimulated cAMP accumulation in alveolar macrophages decreased from 39.5 +/- 9.0 to 2.9 +/- 0.3 pmol/10(6) cells (P = 0.007), equivalent to 86 +/- 5% desensitization. The cAMP response to forskolin in both cell types was unaffected by metaproterenol inhalation. Thus administration of inhaled beta-agonists results in substantial downregulation and functional desensitization of lung cell beta 2-AR. This supports the concept of a dynamically regulated beta 2-AR in humans, the function of which can be attenuated in relevant target tissues by administration of standard doses of beta-agonist.
Posttranslational processing of surfactant protein C in rat type II cells
Authors: Vorbroker D. K. , Voorhout W. F. , Weaver T. E. , Whitsett J. A.
Published: December 2017

Pulmonary surfactant consists of phospholipids and proteins that form a stable monolayer at the surface of the alveoli to prevent lung collapse. Surfactant protein C (SP-C) is a hydrophobic 4-kDa palmitoylated protein derived from a 21-kDa precursor. We determined the membrane insertion, proteolytic processing, and subcellular location of 21-kDa proSP-C. In vitro, proSP-C associated with canine microsomes, and the NH2-terminal of proSP-C was protected from digestion with proteinase K, suggesting that proSP-C was inserted in a type III transmembrane configuration. Treatment of freshly isolated rat type II cells with cerulenin blocked acylation of the 21-kDa precursor. Pulse-chase labeling of type II cells demonstrated proSP-C processing intermediates of 19, 16, and 13 kDa that contained the NH2-terminal of proSP-C. Proteolytic processing of proSP-C was inhibited by incubation at 20 degrees C, suggesting that processing of proSP-C begins in a late Golgi or post-Golgi compartment. Immunogold labeling of rat lung with an antiserum to the NH2-terminal of proSP-C identified proSP-C in the trans-Golgi and multivesicular bodies but not in lamellar bodies. These findings suggest that proSP-C processing takes place in the trans-Golgi and multivesicular bodies before SP-C is incorporated into lamellar bodies.
Composition of extracellular matrix of type II pulmonary epithelial cells in primary culture
Authors: Dunsmore S. E. , Martinez-Williams C. , Goodman R. A. , Rannels D. E.
Published: December 2017

Type II pulmonary epithelial cells in primary culture synthesize and deposit an extracellular matrix which has reciprocal biological effects on cellular differentiation. The present work establishes conditions for metabolic labeling of matrix constituents and for separation of cells from the associated matrix; it also defines matrix composition, which does not appear to change qualitatively between days 2 and 6 of primary culture. Type II cells synthesize and deposit a spectrum of radiolabeled components on the culture surface. These include fibronectin, laminin, type IV collagen, and plasminogen activator inhibitor-1, along with additional unidentified proteins. Few radiolabeled proteins in medium conditioned by type II cells bind nonspecifically to the culture surface in the absence of cells. Fibroblasts and macrophages, which may contaminate the primary cultures, do not appear to contribute substantially to the type II cell matrix. These results demonstrate that type II cells synthesize and deposit a complex multicomponent extracellular matrix. The work provides a basis for further investigations of bidirectional interactions between type II cells and the extracellular matrix.
Turnover of fibronectin and laminin by alveolar epithelial cells
Authors: Dunsmore S. E. , Martinez-Williams C. , Goodman R. A. , Rannels D. E.
Published: December 2017

Type II pulmonary epithelial cells in primary culture synthesize and assemble a multicomponent extracellular matrix which exhibits biological activity in vitro. Simultaneously, the pneumocytes degrade components of the underlying matrix, such that matrix composition may be determined by the balance of synthesis and turnover. The present work defines turnover of the specific matrix glycoproteins, fibronectin and laminin, both in the type II cell and in its extracellular matrix. Pulse-chase experiments demonstrate that both fibronectin and laminin, identified by immunoprecipitation, turn over rapidly in the cell and extracellular matrix compartments, with half-lives < 10 h. In the cell compartment, initial rates of laminin turnover are more rapid than those of fibronectin on culture day 2, but these rates are similar on day 6. Matrix fibronectin also turns over rapidly, with similar rates on day 2 and day 6. During the chase interval, small but increasing amounts of immunoprecipitable fibronectin are detected in the medium, suggesting that a portion of the glycoprotein may be released to the extracellular compartment, rather than degraded. Alternatively, release of immunoreactive glycoprotein may involve ongoing processing and secretion of residual radiolabeled fibronectin by the cells. The results suggest that matrix composition may be determined by turnover, as well as synthesis, of its components.
Cholinergic regulation of voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells
Authors: Yamakage M. , Hirshman C. A. , Croxton T. L.
Published: December 2017

To investigate cholinergic regulation of voltage-dependent Ca2+ channels (VDCs) in airway smooth muscle, we measured inward currents through VDCs in enzymatically dispersed porcine tracheal smooth muscle cells using conventional (10 mM Ca2+ as charge carrier) and nystatin-perforated (5 mM Ba2+ as charge carrier) whole cell patch clamp techniques. Carbachol (CCh) had significant and dose-dependent inhibitory effects on inward currents (12% with 10(-7) M and 42% with 10(-6) M) in perforated whole cell clamp experiments, but had no effect on currents in conventional whole cell experiments. CCh also shifted the steady-state inactivation curve to more negative potentials. Further experiments tested the hypothesis that CCh inhibits VDCs in part by the activation of protein kinase C (PKC). Phorbol 12,13-diacetate, an exogenous PKC activator, inhibited currents through VDCs. and calphostin C, a specific PKC inhibitor, antagonized the inhibitory effect of CCh. Furthermore, intracellular exposure to the activating PKC fragment 530-558, using a pipette perfusion technique, also inhibited currents through VDCs. We conclude that cholinergic receptor stimulation can inhibit inward Ca2+ currents through VDCs of porcine tracheal smooth muscle and that this effect may be mediated in part by activation of PKC.
Activation of L-type Ca2+ channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2)
Authors: Dietl P. , Haller T. , Wirleitner B. , Volkl H. , Friedrich F. , Striessnig J.
Published: December 2017

In the alveolar epithelium, ATP increases the intracellular Ca2+ concentration ([Ca2+]i) and stimulates the secretion of surfactant. We investigated the effects of extracellular ATP on the membrane potential (Vm), the whole cell current, and [Ca2+]i in a cloned rat alveolar epithelial cell line (L2). In microelectrode experiments, ATP caused a sustained depolarization of Vm, resulting from the activation of cation and Cl- conductances, as revealed by ion replacements. The depolarizing phase of the Vm shift was superimposed by Ca(2+)-dependent depolarizing spikes. Spikes were also induced by depolarizing Vm with charybdotoxin or maitotoxin. Replacement of bath Ca2+ with Ba2+ or Sr2+ also evoked repetitive spikes. Ca2+ (Ba2+, Sr2+)-induced spikes were unaffected by pretreatment with ionomycin or thapsigargin. They were, however, completely abolished by (+)-isradipine (100 nM) and stimulated by BAY K 8644 (100 nM). Whole cell L-type Ca2+ (Ba2+, Sr2+) currents were similarly abolished by (+)-isradipine and enhanced by BAY K 8644. L-type Ca2+ channels were further confirmed by demonstrating high-affinity dihydropyridine receptors stereoselectively labeled by (+)-[3H]-isradipine, apparent dissociation constant < 1 nM. In fura 2 experiments, ATP evoked a transient elevation of [Ca2+]i in the absence of Ca2+ and a biphasic sustained elevation in the presence of Ca2+, indicating intracellular Ca2+ release and Ca2+ entry. The ATP-induced fura 2 signals were unaffected by (+)-isradipine. We conclude that in L2 cells, L-type Ca2+ channels are activated after purinoceptor stimulation by ATP. The overall [Ca2+]i response is, however, mediated by Ca2+ entry through and (+)-isradipine-insensitive mechanism and by intracellular Ca2+ release.
Mob-1 expression in IL-2-induced ARDS: regulation by TNF-alpha
Authors: Neville L. F. , Abdullah F. , McDonnell P. M. , Young P. R. , Feuerstein G. Z. , Rabinovici R.
Published: December 2017

We have recently established an animal model of adult respiratory distress syndrome (ARDS)-like microvascular lung injury elicited by infusion of human interleukin-2 (IL-2). Based on the pronounced, transcriptional upregulation of multiple pro-inflammatory mediators in IL-2-induced ARDS, differential display was applied to search for potentially novel genes in this paradigm of lung injury. Differential display on total lung RNA derived from IL-2-challenged rats presented a highly reproducible 3'-UTR fragment profile in which a band (approximately 250 bp), termed B1, was strongly induced. B1 cDNA sequence exhibited 99.14% homology to the 3'-UTR of mob-1, a recently cloned gene belonging to the C-X-C chemokine superfamily. Furthermore, Northern blot analysis showed that IL-2-induced pulmonary mob-1 mRNA was expressed at time points before the onset of lung injury and suppressed after TNF-alpha inhibition. These data imply that lung mob-1 is a novel, highly inducible gene in a clinically relevant model of ARDS and, based on its identification as a chemokine, could participate in the development of lung injury.
Alveolar macrophages modulate the epithelial cell response to coal dust in vitro
Authors: Lee Y. C. , Rannels D. E.
Published: December 2017

The response of the alveolar epithelium to coal dust exposure is poorly understood. Coal or other dusts may act on the epithelium directly or indirectly through nearby alveolar macrophages (AM) that produce cytokines and other soluble products. AM and type II pneumocytes (T2P) were thus exposed to dust in coculture to evaluate their possible interactions. Anthracite coal dust PSOC 867 increased synthesis of extracellular matrix (ECM) components by T2P. AM alone did not produce ECM. Similarly, coculture of T2P with AM (3.75:1) had little effect on epithelial ECM synthesis. In contrast, coculture of T2P with AM significantly increased PSOC 867 effects on T2P rates of ECM synthesis, ECM fibronectin content, and T2P levels of fibronectin mRNA. AM-conditioned medium did not change the PSOC 867 effect on T2P. Neither control nor PSOC 867-treated AM on Falcon culture inserts (0.45-micron pore size) over T2P stimulated ECM synthesis by either untreated or dust-exposed epithelium. Thus AM-mediated changes in ECM synthesis by PSOC 867-treated T2P require close cell-cell interactions, suggesting a role for cell-cell contact or for short-lived soluble mediators of the AM effects.
Mechanisms of smooth muscle contraction elicited by cationic proteins in guinea pig trachealis
Authors: Strek M. E. , Williams F. S. , Gleich G. J. , Leff A. R. , White S. R.
Published: December 2017

Cationic proteins elicit contraction of airway smooth muscle, but the mechanisms by which this occurs are not completely understood. We studied potential mechanisms by which eosinophil major basic protein (MBP) and the synthetic cationic proteins poly-L-lysine (PL) and poly-L-arginine (PA) cause contraction of isolated guinea pig tracheal smooth muscle (TSM) in vivo. Topical application of 10(-8) mol/cm2 of each protein to an isolated tracheal segment elicited TSM contraction with potency PL > MBP > PA. Pretreatment with atropine blocked the subsequent response to MBP but did not block the response to either PL or PA. Pretreatment with indomethacin blocked the subsequent response to both MBP and PL but did not block the response to PA. We demonstrate that MBP causes contraction of guinea pig TSM both through stimulation of the parasympathetic nervous system and secretion of a cyclooxygenase mediator. Neither PL nor PA, while of similar molecular weight and charge as MBP, cause TSM contraction via the parasympathetic nervous system, though some cationic proteins may act via a prostanoid mediator. Thus the cationic charge of MBP is not solely responsible for its effects on TSM in the guinea pig.
Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung
Authors: Le Cras T. D. , Xue C. , Rengasamy A. , Johns R. A.
Published: December 2017

The effect of chronic hypoxia-induced pulmonary hypertension on nitric oxide synthase (NOS) in the lung is controversial. To clarify the regulation of endothelial and inducible NOS (eNOS and iNOS) expression in the chronically hypoxic lung, Northern and Western blot analyses were performed on mRNA and total protein from lungs of rats exposed to 3 wk of hypoxia (10% O2, normobaric) or normoxia. Expression of the mRNA and protein for eNOS was significantly increased (1.6-fold and 2.1-fold, respectively) by hypoxia. Immunohistochemistry with an isoform-specific antibody demonstrated de novo expression of eNOS in the endothelium of resistance vessels in the pulmonary vasculature of the hypoxic rats. eNOS was detected in the endothelium of large vessels in both normoxic and hypoxic rat lungs. The level of mRNA and protein for iNOS was also found to be significantly increased (1.9-fold and 1.4-fold, respectively). In addition to the 4.4-kilobase (kb) iNOS mRNA species, a novel 4.0-kb species was also induced by hypoxia. We conclude that expression of eNOS and iNOS was increased in the lungs of rats subjected to chronic hypoxia, and that there was de novo expression of eNOS protein in the microvascular endothelium.
Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion
Authors: Bloemen P. G. , Van den Tweel M. C. , Henricks P. A. , Engels F. , Van de Velde M. J. , Blomjous F. J. , Nijkamp F. P.
Published: December 2017

It has become clear that the bronchial epithelium is not just a passive barrier but plays an active role in inflammation. It can produce several inflammatory mediators and does express cell adhesion molecules of which intercellular adhesion molecule (ICAM)-1 can be upregulated by cytokines like interferon (IFN)-gamma. In the present study, we analyzed in detail the interaction of neutrophils with human bronchial epithelial cells, both primary cultured cells and the bronchial epithelial cell line BEAS-2B. Confluent monolayers of epithelial cells were incubated with freshly isolated 51Cr-labeled neutrophils for 30 min at 37 degrees C; then the nonadherent cells were removed by washing gently. Stimulation of the epithelial cells with IFN-gamma or the combination of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) (which doubles the ICAM-1 expression) increased neutrophil adhesion. Activation of the neutrophils themselves with N-formylmethionyl-leucyl-phenylalanine (fMLP), platelet-activating factor, or TNF-alpha also caused a profound enhancement of the adhesion. A significant additional increase was found when the epithelial cells had been exposed to IFN-gamma and the neutrophils were stimulated with fMLP simultaneously. This effect was even more pronounced with epithelium preincubated with IFN-gamma and TNF-alpha. With the use of monoclonal antibodies against CD18 and ICAM-1, it was demonstrated that the increased adhesion was mainly mediated by the ICAM-1/beta 2-integrin interaction. This study highlights that both the activation state of the bronchial epithelial cells and the activation state of the neutrophils are critical for their interactive adhesion.
Developmental expression of NOS isoforms in fetal rat lung: implications for transitional circulation and pulmonary angiogenesis
Authors: Xue C. , Reynolds P. R. , Johns R. A.
Published: December 2017

To better understand the role of nitric oxide (NO) in fetal lung development, specifically in the transition of the fetal circulation at birth, we studied the timing of cell-specific expression of NO synthase (NOS) isoforms from formation of lung buds (13th day of gestation) to 7 days postnatal. Expression of NOS was studied using immunohistochemical labeling with antibodies against the three known NOS isoforms and the NADPH diaphorase technique (NADPH-d). Endothelial NOS (eNOS) immunoreactivity was found in the cells of the 14-day fetal lung. As gestation proceeded, the quantity of these immunopositive cells increased, and they coalesced to form an inner (endothelial) layer of pulmonary vessels. This process of angiogenesis marked by eNOS-positive cells was seen from 15 days of gestation to at least 7 days postnatal. A majority of the eNOS immunoreactivity appeared densely in one focal spot in the cytoplasm, indicating that during development the eNOS may be primarily located in a cytoplasmic organelle. Epithelial cells of the rat airway from the same developmental period were positively stained with both brain NOS antibody (bNOS) and NADPH-d at the beginning of 13 days of gestation. Then the intensity of stainings began to decrease and reached the lowest level in the 16-day fetal lung. However, the NOS stainings of the epithelium, especially in small canalicular structures of the airways, began to increase at 18 days of gestation and was dramatically elevated at 20 days of gestation (term is 22 days). Postnatally, NOS in epithelium was decreased in distal airways in conjunction with the formation of alveolar structure. Inducible NOS (iNOS) immunoreactivity was also found in the epithelium of rat lung airways after 16 days of gestation. Unlike the bNOS staining, iNOS immunoreactivity exhibited a pattern of a small dot-like staining within epithelial cytoplasm during gestation and the first day postnatal, then changed to a pattern of diffuse cytoplasmic staining by the 7th postnatal day. This study concludes that 1) expression of three isoforms of NOS is present and regulated during lung development; 2) markedly increased NOS in epithelium near term supports a role for NO in mediating the pulmonary transition from fetal to neonatal life; and 3) eNOS immunohistochemistry serves as an effective marker to follow the process of pulmonary angiogenesis and suggests the concept of in situ formation of endothelial vesicles in developing mesenchyme.
The epidermal growth factor receptor network in type 2 pneumocytes exposed to hyperoxia in vitro
Authors: Nici L. , Medina M. , Frackelton A. R.
Published: December 2017

Hyperoxia is a well-characterized model of injury and repair of the lung. Type 1 cell damage is followed by type 2 cell proliferation and differentiation which restore normal structure and function. The epidermal growth factor receptor (EGFR) network is known to be a potent modulator of epithelial cell growth. Here we examine the EGFR network on isolated rat type 2 cells and SV40T-T2, a type 2 cell line, under normoxic conditions, after 24 and 48 h of in vitro hyperoxia, and after 24 h of normoxic recovery. EGF induces tyrosine phosphorylation of EGFRs in type 2 cells and SV40T-T2 cells, which decreases with hyperoxia and increases above normoxic levels in recovering cells, suggesting biphasic changes in receptor number or function with injury. The EGFR appears to be stimulated in an autocrine fashion in these cells. There is decreased DNA synthesis and proliferation in SV40T-T2 and isolated type 2 cells treated with tyrphostin B56, a specific EGFR inhibitor. Pretreatment with suramin, which binds to growth factor, results in increased EGFR tyrosine phosphorylation after stimulation, suggesting disruption of normal autocrine receptor downregulation. We have also identified transforming growth factor-alpha (TGF-alpha) in conditioned media (CM) from normoxic and hyperoxic SV40T-T2 and type 2 cells. Finally, we show increased EGF bioactivity in both bronchoalveolar lavage (BAL) from hyperoxic rats and CM from hyperoxic cells compared with normoxic controls. These findings support an integral role for an autocrine EGFR network in the type 2 cell response to injury.
Inhaled nitric oxide injures the pulmonary surfactant system of lambs in vivo
Authors: Matalon S. , DeMarco V. , Haddad I. Y. , Myles C. , Skimming J. W. , Schurch S. , Cheng S. , Cassin S.
Published: December 2017

Nitric oxide (.NO) is a free radical, and as such may damage the pulmonary surfactant system. To determine the potential toxicity of .NO in vivo, we exposed 35 newborn lambs to 0, 20, 80 or 200 ppm .NO in either 21 or 60% O2 for 6 h. At the end of the exposure, lambs had normal values of arterial Po2, Pco2, and pH; total protein concentration in the bronchoalveolar lavage was also at normal levels. There were no differences in the surface properties of surfactant among the air or 60% O2 groups. Pulmonary surfactant samples, isolated from the bronchoalveolar lavage of lambs breathing air or 20 ppm .NO and reconstituted at a lipid concentration of 3 mg/ml, reached a low minimum surface tension (Tmin < 3 mN/m) in a pulsating bubble surfactometer. On the other hand, abnormal surface properties were observed in 36 and 60% of surfactant samples isolated from lungs of lambs that breathed 80 or 200 ppm .NO, respectively. These findings were confirmed using a captive bubble surfactometer. Surfactant protein A, isolated from the lungs of lambs that breathed 200 ppm .NO, exhibited decreased ability to aggregate lipids in vitro. These data are consistent with injury to the surfactant apoproteins during inhalation of either 80 or 200 ppm .NO for 6 h.
Nitration of surfactant protein A results in decreased ability to aggregate lipids
Authors: Haddad I. Y. , Zhu S. , Ischiropoulos H. , Matalon S.
Published: December 2017

We assessed the extent to which nitration of surfactant protein (SP) A, isolated from the bronchoalveolar lavage of patients with alveolar proteinosis, alters its ability to enhance lipid aggregation, bind lipids, and act synergistically with surfactant apoproteins B and C (SP-B, SP-C) in lowering the surface activity of surfactant lipids. SP-A was treated with various concentrations of tetranitromethane (TNM) at pH 6, 7.4, 8, or 10. Depending on the pH, TNM acts either as a nitrating (pH > or = 7.4) or an oxidizing agent (pH < or = 6). Exposure of SP-A to TNM (0.1-1 mM) at pH 7.4 or 8 for 30 min resulted in dose-and pH-dependent increases in nitrotyrosine, detected by Western blotting, enzyme-linked immunosorbent assay, and direct amino acid analysis. Treatment of SP-A with 0.5 mM TNM decreased its ability to aggregate lipids by 30% at pH 7.4, and 90% at pH 8, but had no effect on the disulfide-dependent oligomeric state of SP-A. In contrast, SP-A exposed to 1 mM TNM at pH 6 had background levels of nitrotyrosine and exhibited normal lipid aggregation properties. TNM, but not a hydroxyl radical-generating system, resulted in a pH-dependent loss of SP-A fluorescence, suggesting that tryptophan also may have been nitrated. Nitration of SP-A did not affect its ability to bind lipids. In addition, SP-A (1-3% by weight), treated with 0.25-0.5 mM TNM at pH 8, restored the surface-active properties of calf lung surfactant extract, previously damaged by exposure to peroxynitrite. We conclude that tyrosine nitration selectively inhibits the SP-A-mediated lipid aggregation without affecting its ability to bind lipids.
Lung alveolar epithelial cell migration in vitro: modulators and regulation processes
Authors: Lesur O. , Arsalane K. , Lane D.
Published: December 2017

After acute lung injury, altered bronchioloalveolar epithelia must be repaired quickly in order to restore lung function. During reepithelialization, type II cells initially appear to migrate and spread over a remodeled matrix; then a secondary proliferative phase occurs. It was hypothesized that 1) type II cells can develop locomotion in vitro that is modulated by growth factors, proinflammatory cytokines, and substrate adhesion molecules and 2) migration and proliferation of type II cells can occur as distinctive processes. Chemotaxis assays were elaborated using short term cultures of rat type II pneumocytes. Epidermal growth factor (EGF), transforming growth factor-alpha, laminin, fibronectin were found to be the main attractants for type II cells with respective increases of approximately 8.5-, 10.5-, 8-, and 7-fold in cell migration (P<0.05 vs. control). Laminin induced gradient-dependent and random cell migration. Addition of laminin with EGF had a synergistic effect in promoting cell migration (approximately 30-fold increase over control, P<0.05). Interferon-gamma and interleukin-6 inhibited EGF-induced type II cell migration, whereas tumor necrosis factor-alpha and interleukin-1beta acted as primers for type II cell migration (approximately 1.5-fold increase over control, P<0.05. Type II cells did not need to be in a proliferative phase in order to exhibit motility. New insights regarding the regulatory processes for type II cell migration are especially relevant in our understanding of early events occurring during epithelial repair after acute lung injury.
Serine proteinase inhibitors influence the stability of tropoelastin mRNA in neonatal rat lung fibroblast cultures
Authors: McGowan S. E. , Liu R. , Harvey C. S. , Jaeckel E. C.
Published: December 2017

Elastin, an elastic extracellular structural protein, is a polymer comprised of soluble tropoelastin (TE) monomers that are joined by covalent cross-links and become insoluble. In cultured vascular smooth muscle cells, the steady-state level of TE mRNA is influenced by soluble elastin moieties in the culture medium, either TE or its fragmentation products. We have hypothesized that an enzyme-mediated proteolytic event may modulate the quantities of TE and its fragmentation products in the culture medium of mesenchymal cells, and thereby indirectly regulate the steady-state level of TE mRNA. Neonatal rat lung fibroblasts were cultured in the presence or absence of the serine proteinase inhibitor, aprotinin, and the quantities of soluble elastin and TE mRNA were analyzed. Exposures to aprotinin lasting up to 12 h increased the soluble elastin content of the culture medium. The increase in the soluble elastin content did not reflect an increase in TE mRNA, which diminished after exposures for 12 h or longer. The decrease in TE mRNA resulted from a decrease in its half-life, rather than a decrease in the rate of TE gene transcription. Aprotinin did not reduce TE mRNA in plasminogen-depleted cultures, but the effect of aprotinin was evident when purified plasminogen was added back to the cultures. Therefore, a serine proteinase, possibly plasmin, may participate in a feedback mechanism and modulate the quantity of TE in lung fibroblast cultures. This mechanism may help ensure that intracellular TE synthesis occurs in tandem with extracellular elastin deposition and cross-linking.
Glutathione supplements protect preterm rabbits from oxidative lung injury
Authors: Brown L. A. , Perez J. A. , Harris F. L. , Clark R. H.
Published: December 2017

The main objective of this study was to determine if glutathione (GSH) supplementation attenuated hyperoxic lung injury. Preterm (29 days) rabbits were delivered and exposed for 24 h to 1) room air, 2) room air and GSH, 3) 95% oxygen and GSH. GSH supplements (1 mM) were delivered in the nutritional support of 10% dextrose and saline through a peritoneal catheter. Animals assigned to oxygen had decreased lung volumes at 35 cmH2O, decreased lung compliance, increased edema, decreased cell viability, and decreased lung tissue and lavage-reduced/oxidized GSH levels, compared with control animals. Despite exposure to hyperoxia, animals supplemented with GSH were not different from room air controls with respect to lung mechanics, edema, cell viability, or tissue and lavage GSH. These studies suggest that GSH supplementation maintains normal lavage and lung tissue GSH levels in preterm animals exposed to hyperoxia and attenuates the changes in lung mechanics associated with oxygen-induced lung injury.
In vitro analysis of rat surfactant protein A gene expression
Authors: Stuempfle K. J. , Koptides M. , Quinn P. G. , Floros J.
Published: December 2017

Previous findings suggest that the rat proximal promoter segment (rPPS) of the SP-A gene is important in the regulation of the lung-specific expression of the gene. In this report, two regions within the rPPS containing thyroid transcription factor-1 (TTF-1) binding sites are identified that form strong lung- and thyroid- specific DNA-protein complexes. These regions bind nuclear polypeptides with similar apparent molecular mass to TTF-1, suggesting that TTF-1 binds to these regions. Two regions within the rPPS that form weaker lung-specific DNA-protein complexes are also identified. The transcription start site is mapped, and a functional analysis shows that the sequences of the 5' flanking region are sufficient to support in vitro transcription but are not sufficient to reproduce tissue-specific expression. Together, these results show that lung- and thyroid-specific, as well as lung-specific, DNA-protein interactions occur within the rPPS but are not sufficient for the lung-specific expression of the rat SP-A gene to be duplicated in vitro.
Hyperoxia-responsive proteins in rat pulmonary microvascular endothelial cells
Authors: Visner G. A. , Fogg S. , Nick H. S.
Published: December 2017

Exposure to high partial pressures of oxygen are toxic to the lung, and much of the damage observed is related to injury of the pulmonary microvasculature. In this study, we evaluated the response of the pulmonary microvascular endothelial cell to high oxygen concentrations, using two-dimensional protein gel electrophoresis as a direct molecular assay of differences between cells exposed to room air or hyperoxia. We observed a differential expression of five specific proteins within 24 h of a hyperoxic insult that we termed hyperoxia-responsive proteins. After 4 h of hyperoxia there was a decrease in two of the proteins. From 8 to 24 h we observed a repression of a third and an induction of the other two proteins. One of the induced proteins was also increased by heat shock and hydrogen peroxide and has characteristics similar to heat shock protein (HSP) 32 (heme oxygenase 1). Western analysis using an antibody specific to rat heme oxygenase 1 verified that this oxygen-responsive protein is heme oxygenase 1. The response of the other four hyperoxia-responsive proteins appears to be specific to oxygen and not a general stress response, since they were not changed in response to heat shock or hydrogen peroxide. Based on RNA inhibitor and pulse chase experiments, these changes may result from transcriptional/posttranscriptional mechanisms or hyperoxia-dependent protein turnover.
Cytoskeletal architecture in mouse lung epithelial cells is regulated by protein-kinase C-alpha and calpain II
Authors: Dwyer-Nield L. D. , Miller A. C. , Neighbors B. W. , Dinsdale D. , Malkinson A. M.
Published: December 2017

Brief exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a uniformly flattened population of mouse lung epithelial cells to become more heterogeneous; some cells rounded up, and others detached to overlap with flatter cells. Actin stress fiber organization was disrupted, and F-actin accumulated in lemellipodia. Vinculin dissociated from the focal adhesion plaques to diffuse throughout the cytoplasm. Inhibition of protein kinase C (PKC) activity blocked these effects of TPA. After 8 h of TPA exposure, actin filaments reassembled and vinculin again localized to the cell periphery. Calpain inhibition attenuated the decrease of PKC-alpha protein and PKC activity from the membrane fraction, and prevented the redistribution of cytoskeletal elements. Talin immunostaining was widespread throughout control cells but was localized to the periphery 8 h after treatment with TPA or with inhibitors of PKC and calpain. Both vinculin and talin concentrations increased with prolonged TPA treatment. PKC-zeta and calpain II were not appreciably affected by TPA exposure. Translocation of PKC-alpha to the membrane, followed by its calpain-induced downmodulation, is apparently required for the reversible pattern of cytoskeletal changes caused by TPA.
Type I receptor serine-threonine kinase preferentially expressed in pulmonary blood vessels
Authors: Panchenko M. P. , Williams M. C. , Brody J. S. , Yu Q.
Published: December 2017

Type II and type I receptor serine-threonine kinases (RSTK) are important components of the transmembrane signaling machinery that allow cells to respond to the transforming growth factor-beta (TGF-beta) superfamily of cytokines. We have cloned from rat lung and report here a 3,935-base pair (bp) cDNA encoding a type I RSTK previously identified as R-3 (rat) or ALK-1 (human). Northern blot analysis reveals that the R-3 mRNA is more abundant in lung than in other adult rat tissues. With the use of in situ hybridization, the R-3 transcripts are found exclusively in the pulmonary vessels of all sizes, as well as in aorta, vena cava, and certain blood vessels of kidney, spleen, heart and intestine. In most blood vessels, a higher level of gene expression is found in endothelium than in adjacent smooth muscle. The R-3 transcripts are also found in splenic macrophages, as well as within cells of marginal zone of the splenic lymphoid tissue. In fetal rat lung, the expression of R-3 transcripts differs from the expression patterns of two other type 1 RSTK. The R-3 is expressed in vessels; the activin type IB receptor (R-2) is preferentially expressed in putative developing airways, whereas the TGF-beta type I receptor (R-4) transcripts appear to be ubiquitous. Our data suggest that in vivo R-3 may propagate signaling of TGF-beta in selected cell types. The differential expression of multiple type I receptors within different cell lineages may therefore define cell specific responses to TGF-beta.
Expression of LAR-PTP2 in rat lung is confined to proliferating epithelia lining the airways and air sacs
Authors: Kim H. , Yeger H. , Han R. , Wallace M. , Goldstein B. , Rotin D.
Published: December 2017

The LAR family tyrosine phosphatase LAR-PTP2B (RPTP sigma) was previously shown to be expressed in the central and peripheral nervous system. Here we show that LAR-PTP2, the larger alternatively spliced form of the gene, is expressed in proliferating undifferentiated lung epithelia in a developmentally regulated manner: Using in situ hybridization and parallel immunostaining with proliferating cell nuclear antigen to detect proliferating cells, we demonstrate that LAR-PTP2 is expressed exclusively in the undifferentiated epithelial cell layer lining the bronchi, bronchioles, and air sacs in late fetal development and in the neonatal lung. These cells correspond to Clara and fetal alveolar type II cells, as determined by parallel immunostaining with antibodies to surfactant proteins A and B. LAR-PTP2 expression declined progressively with postnatal development, and by adult stage there was no detectable expression in the airways or in the distal (type I and II) mature nonproliferating alveolar epithelial cells. These results suggest that LAR-PTP2 may be involved in the regulation of epithelial cell proliferation/differentiation during lung development.
Rabbit surfactant protein B gene: structure and functional characterization of the promoter
Authors: Margana R. K. , Boggaram V.
Published: December 2017

Surfactant protein B (SP-B) is essential for physiological function of pulmonary surfactant. In the present investigation, we isolated rabbit SP-B gene and determined its nucleotide sequence, transcription start site, and exon/intron organization. The coding region of rabbit SP-B gene is comprised of 6.8 kb of sequence and is organized into 10 introns and 11 exons. By deletion analysis we determined that a region of SP-B gene extending from -236 to +39 nucleotides is sufficient for high-level expression of CAT reporter gene in a cell type-specific manner in the pulmonary adenocarcinoma cell line NCI-H441. Deletion of 5'-flanking sequence to -140 and -71 nucleotides significantly reduced SP-B promoter activity, suggesting that the -236 to +39 region contains cis-DNA elements required for cell type-specific expression. The proximal promoter region of SP-B gene contained DNA sequence motifs for binding thyroid transcription factor 1 (TTF-1) (-113 to -97) and hepatocyte nuclear factor 3(HNF-3) (-91 to -81). Although SP-B gene sequence -140 to +39 did not support high level expression of CAT gene in NCI-H441 cells, it was capable of activation by TTF-1 in HeLa cells, suggesting that the -236 to -140 sequence plays an important role in cell type-specific activation of SP-B promoter.
Clinical surfactant preparations mediate SOD and catalase uptake by type II cells and lung tissue
Authors: Nieves-Cruz B. , Rivera A. , Cifuentes J. , Pataki G. , Matalon S. , Carlo W. A. , Tanswell A. K. , Freeman B.
Published: December 2017

Pulmonary surfactant mixtures are rapidly taken up by alveolar type II cells and thus may serve as vectors for the pulmonary delivery of antioxidant enzymes to the alveolar epithelium. We prepared emulsions of Survanta with superoxide dismutase (CuZn-SOD) and catalase and quantified their cellular uptake both in vitro and in vivo. Incubations of fetal lung epithelial cells with an emulsion of Survanta plus SOD and catalase mixtures resulted in significant augmentation of SOD and catalase activities (12.8 +/- 4.6 U SOD/microgram DNA; 7.49 +/- 2.21 U catalase/microgram DNA). These numbers were significantly greater than those obtained in controls (1.8 U SOD/microgram DNA; 0.55 +/- 0.52 U catalase/microgram DNA, Survanta alone (0.43 U SOD/microgram DNA; 0.16 U catalase/microgram DNA), and SOD and catalase alone (3.47 +/- 5.2 U SOD/microgram DNA; 4.24 +/- 3.0 U catalase/microgram DNA). Intratracheal instillation of the Survanta plus SOD and catalase mixture resulted in significant augmentation of enzymes by the rat lung homogenates. Confocal microscopic analysis revealed the presence of antioxidant enzymes in the cytoplasm of epithelial cells. We concluded that Survanta supplementation, in addition to replenishing surfactant stores, can also enhance the delivery of antioxidant enzymes to alveolar epithelium both in vitro and in vivo.
Alveolar type II cell growth on a pulmonary endothelial extracellular matrix
Authors: Adamson I. Y. , Young L.
Published: December 2017

Most of the alveolar epithelium overlies a fused basement membrane produced by epithelial and endothelial cells. To determine how this type of matrix influences type II cell growth and function, we studied the effects of culturing isolated rat alveolar type II cells on an extracellular matrix (ECM) freshly produced by pulmonary vascular endothelial cells grown 5 days in culture. Type II cells from the same rats were cultured on plastic or Matrigel for comparison. A large increase in mitotic activity was seen in type II cells grown on the endothelial ECM at 2 days only; thereafter cells spread rapidly to confluence and lost their lamellar bodies. Cells grown on Matrigel remained cuboidal with lamellar bodies but grew more slowly, as judged by [3H]thymidine uptake and cell numbers. Incorporation of labeled choline into disaturated phosphatidylcholine (DSPC) was used as a marker of surfactant synthesis. After the rapid, brief burst of proliferation, type II cells on endothelial ECM showed a sudden decline in DSPC-DNA by day 4 compared with cells grown on matrigel. Binding of the lectin Bauhinia purpurea (BPA) indicated that after a phase of division, cells on endothelial ECM developed as type I epithelium by 4 days of culture, when > 70% of cells stained positively for BPA binding, whereas few cuboidal cells on Matrigel were stained. The results indicate that type II cells respond briefly to growth factors in pulmonary endothelial ECM; then this type of matrix promotes cell spreading with loss of type II function as cells subsequently resemble type I epithelium.
Inhibition of alveolar type II cell ATP and surfactant synthesis by nitric oxide
Authors: Haddad I. Y. , Zhu S. , Crow J. , Barefield E. , Gadilhe T. , Matalon S.
Published: December 2017

Alveolar type II (ATII) cells, are often exposed to increased concentration of endogenous and exogenous nitric oxide (.NO). Exposure of freshly isolated rat ATII cells for 2 h to 1-3 microM .NO, generated by S-nitroso-N-penicillamine (SNAP), spermine NONOate, or 3-morpholino-sydnonimine (SIN-1) in the presence of superoxide dismutase, resulted in approximately 60% decrease in the rate of surfactant synthesis, as measured by the rate of incorporation of [methyl-3H]choline into phosphatidylcholine, and 60-80% inhibition of cellular ATP levels, as determined by bioluminescence. Similar results were obtained after incubation of ATII cells with authentic peroxynitrite (0.5 mM) but not SIN-1, a putative generator of peroxynitrite. Addition into the medium of oxyhemoglobin (20 microM), which scavenged .NO, or enhancement of ATII glutathione levels by preincubation with glutathione ester (5 mM) totally prevented the NONOate (100 microM) inhibition of cellular ATP. In contrast to the in vitro findings, normal levels of ATP and lipid synthesis were measured in ATII cells isolated from the lungs of rats that breathed .NO gas (80 ppm) in 21% O2 for 2 h (n = 4). This lack of effect may be due either to the presence of various antioxidants (such as glutathione) in the epithelial lining fluid or to the relatively low concentrations of .NO reaching the alveolar epithelium. We conclude that .NO and peroxynitrite, at concentrations likely to be encountered in vivo during inflammation, decrease ATII cell energy stores and surfactant synthesis, which may lead to derangement of important physiological functions.