bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic intrauterine pulmonary hypertension impairs endothelial nitric oxide synthase in the ovine fetus
Authors: Villamor E. , Le Cras T. D. , Horan M. P. , Halbower A. C. , Tuder R. M. , Abman S. H.
Published: December 2017

Endothelial (e) nitric oxide synthase (NOS) activity modulates pulmonary vascular tone in the normal fetus and decreases pulmonary vascular resistance (PVR) at birth. Mechanisms contributing to sustained elevations of PVR and the failure of postnatal adaptation at birth are uncertain but may include decreased eNOS activity. To test this hypothesis, we studied the effects of chronic intrauterine pulmonary hypertension on lung eNOS content and NOS activity in an ovine model of perinatal pulmonary hypertension and in normal lambs. We measured eNOS mRNA and protein content by Northern and Western blot analyses, respectively. Calcium-dependent and total NOS activities were determined by assaying the conversion of L-[14C]arginine to L-[14C]citrulline from lung homogenates. To determine the effects of intrauterine hypertension on lung eNOS content, fetal lung tissue was harvested 8-12 days after intrauterine closure of the ductus arteriosus (DA) performed at 125-128 days of gestation (term = 147 days). Although positive immunostaining for eNOS persisted in lung vascular endothelium, eNOS protein content was reduced by 48%, as measured by Western analysis (P < 0.001). Chronic hypertension reduced lung eNOS mRNA content by 30% (P < 0.05). Compared with age-matched controls, Ca(2+)-dependent NOS activity was decreased after DA ligation by 75% (P < 0.01). We conclude that chronic intrauterine pulmonary hypertension decreases eNOS in the fetal lung. We speculate that decreased NO production contributes to failure of postnatal adaptation in this experimental model of persistent pulmonary hypertension of the newborn.
Endothelin-1 mediates nitro-L-arginine vasoconstriction of hypertensive rat lungs
Authors: Muramatsu M. , Rodman D. M. , Oka M. , McMurtry I. F.
Published: December 2017

Inhibition of endothelium-derived nitric oxide (NO) synthesis by L-arginine analogs such as nitro-L-arginine (L-NNA) elicits marked precapillary vasoconstriction in lungs from rats with chronic hypoxia-induced pulmonary hypertension. To investigate the role of endogenous endothelin (ET)-1 in L-NNA-induced vasoconstriction, we tested, in salt solution-perfused hypertensive lungs isolated from chronically hypoxic (3-4 wk at barometric pressure = 410 mmHg) adult male rats, if the pressor responses to L-NNA and exogenous ET-1 were inhibited by either separate or combined ETA and ETB receptor blockade. Whereas only combined pretreatment with 5 microM BQ-123 (selective ETA receptor blocker) and 5 microM BQ-788 (selective ETB receptor blocker) inhibited the response to 100 microM L-NNA, the response to 10 nM ET-1 was reduced by both BQ-123 alone and the combined blockers. Because exogenous ET-1 causes postcapillary vasoconstriction in salt solution-but not blood-perfused normotensive rat lungs, we next compared effects of ETA and ETB receptor blockade on L-NNA and ET-1 vasoconstrictions in blood-perfused hypertensive lungs. In this case, the combined but not the separate effects of BQ-123 and BQ-788 inhibited the responses to both L-NNA and ET-1. The last experiment showed that the use of BQ-788 to inhibit ETB receptor-mediated clearance of circulating ET-1 resulted in greater accumulation of endogenous ET-1 in the perfusate of hypertensive than of normotensive lungs. There was no difference between L-NNA-treated and vehicle control hypertensive lungs in accumulation of ET-1. These results suggest that increased endogenous levels of ET-1 acting through stimulation of both ETA and ETB receptors contribute to the vasoconstriction unmasked by inhibition of NO synthesis in hypertensive rat lungs. The increased ET-1 is apparently not due to the inhibition of NO synthesis, but, instead, its underlying stimulation of smooth muscle cell contraction is counteracted by NO activity.
Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts
Authors: Partridge C. A. , Phillips P. G. , Niedbala M. J. , Jeffrey J. J.
Published: December 2017

The cell-surface localization and site of activation of type IV collagenases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonary microvascular endothelial (BPMVE) cells was examined. Sucrose density centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalization with beta 1-integrin, indicating incorporation into the focal contacts. The focal contacts were extracted with saponin, and associated proteolytic activity was examined by zymography. The focal contacts contained latent MMP-2, and stimulation of the cells with cytochalasin D or with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-methylxanthine increased both latent and activated MMP-9 in the focal contacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact extracts was derived from previously secreted enzyme. The activated metalloproteinase degraded extracellular matrix collagens and was inhibited by 1,10-phenanthroline. These findings indicate that endothelial cells release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.
Activation of alveolar macrophages and lung host defenses using transfer of the interferon-gamma gene
Authors: Lei D. , Lancaster J. R. , Joshi M. S. , Nelson S. , Stoltz D. , Bagby G. J. , Odom G. , Shellito J. E. , Kolls J. K.
Published: December 2017

Interferon-gamma (IFN-gamma) is a critical cytokine in pulmonary host defenses against both intracellular and extracellular pathogens. To investigate whether this cytokine could be used therapeutically, we constructed an E1-deleted recombinant adenovirus encoding murine IFN-gamma. After intratracheal inoculation in rats, this vector resulted in prolonged expression of functional cytokine in vivo, as demonstrated by increased alveolar macrophage class II major histocompatibility complex expression, enhanced release of tumor necrosis factor in response to lipopolysaccharide, and enhanced host defenses against Pseudomonas aeruginosa. We postulate that this vector may be useful to study the role of exogenous IFN-gamma in a variety of pulmonary intracellular and extracellular pathogens.
Glucocorticoids increase fatty-acid synthase mRNA stability in fetal rat lung
Authors: Xu Z. X. , Rooney S. A.
Published: December 2017

Fatty-acid synthase (FAS) is a critical enzyme in surfactant biosynthesis. In fetal lung, glucocorticoids increase synthesis of phosphatidylcholine, the principal lipid component of surfactant, and there is evidence that this effect is mediated by increased expression of the FAS gene. Dexamethasone increases FAS activity, mass, mRNA content, and rate of transcription in cultured explants of fetal rat lung. As previous experiments with actinomycin D suggested that dexamethasone may also increase FAS mRNA content by a posttranscriptional mechanism, we examined the effect of the hormone on FAS mRNA stability. Explants of 19-day fetal rat lungs were cultured for 44 h with and without 100 nM dexamethasone. Some explants were harvested at that point, and others were cultured further with 60 microM 5,6-dichlororibofuranosylbenzimidazole (DRB), an inhibitor of transcription. RNA was then extracted, and FAS mRNA levels were measured by Northern analysis, mRNA stability was assessed by comparing the amount remaining after culture with DRB with the initial level before addition of the inhibitor. The apparent half-life of FAS mRNA was 4 h in control explants cultured without hormone. FAS mRNA stability was increased 84% in the explants cultured with dexamethasone for 44 h and by 40% in those cultured with the hormone for 5 h. We conclude that glucocorticoids enhance expression of the FAS gene in fetal lung by increasing mRNA stability in addition to stimulating transcription.
Effects of lung injury on pulmonary surfactant aggregate conversion in vivo and in vitro
Authors: Veldhuizen R. A. , Ito Y. , Marcou J. , Yao L. J. , McCaig L. , Lewis J. F.
Published: December 2017

Within the alveolar space pulmonary surfactant is converted from the surface active large aggregates (LA) to the inactive small aggregates (SA). This conversion is affected by a change in surface area, lung injury, breathing pattern, and protease activity. This study examined the effect of N-nitroso-N-methylurethane-induced acute lung injury on aggregate conversion in mechanically ventilated and spontaneously breathing rabbits. Both the in vitro surface area cycling techniques and the in vivo technique of intratracheally injecting radiolabeled LA were used for analyzing aggregate conversion. Mechanical ventilation of injured lungs resulted in increased aggregate conversion and increased surfactant aggregate ratios compared with controls. Spontaneously breathing injured animals had aggregate conversion and aggregate ratios that were not significantly different from controls. In vitro aggregate conversion was slower for LA obtained from injured animals compared with normal animals. We conclude that the mechanical stress of mechanical ventilation results in increased aggregate conversion and aggregate ratios. Furthermore, in vitro conversion of isolated LA does not necessarily reflect the conversion of aggregates within the alveoli.
Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells
Authors: Kelsen S. G. , Anakwe O. O. , Aksoy M. O. , Reddy P. J. , Dhanesekaran N. , Penn R. , Benovic J. L.
Published: December 2017

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.
Hypoxia inhibits the induction of argininosuccinate synthetase by endotoxin in lung endothelial cells
Authors: Su Y. , Block E. R.
Published: December 2017

Pulmonary artery endothelial cells (PAEC) possess a two-step pathway for synthesizing L-arginine from L-citrulline. The first and rate-limiting step is catalyzed by argininosuccinate synthetase (AS). We have previously shown that hypoxia inhibits synthesis of L-arginine from L-citrulline in PAEC. In this study, we examined the effect of hypoxia on the induction of AS in PAEC. Porcine PAEC were incubated with or without endotoxin under normoxia (air-5% CO2) or hypoxia (0% O2-95% N2-5% CO2) for 24 h, and then AS activity and AS mRNA content were determined. Incubation with endotoxin resulted in increases in AS activity and mRNA, and the latter was blocked by actinomycin D. Exposure to hypoxia for 24 h decreased AS activity and mRNA content and stability, and it also abolished the increases in AS activity and mRNA induced by endotoxin. These results indicate that hypoxia inhibits endotoxin-mediated induction of AS. This inhibition might reduce the availability of intracellular L-arginine and thereby limit immunostimulant-induced nitric oxide production by lung endothelial cells.
Role of calcium-activated chloride current in regulating pulmonary vasomotor tone
Authors: Yuan X. J.
Published: December 2017

Many agonists induce vasoconstriction by releasing intracellularly stored Ca2+ and promoting Ca2+ influx. Activation of Ca(2+)-activated Cl- (ClCa) channels may be a critical mechanism by which a rise in intracellular free Ca2+ concentration ([Ca2+]i) causes membrane depolarization that serves to sustain the elevated [Ca2+]i and maintain vascular tone. In this study the biophysical and pharmacological properties of ClCa currents [ICKCa] were characterized in rat pulmonary artery (PA) smooth muscle cells, and their relationship to the regulation of pulmonary vascular tone was determined. When K+ currents were eliminated by using Cs(+)-containing internal solution, depolarization elicited an inward Ca2+ current followed by a time-dependent outward Cl- current that reversed near Cl- equilibrium potential. Repolarizing voltage steps produced a large inward tail Cl- current that also reversed at a potential close to Cl- equilibrium potential. Replacement of extracellular Ca2+ with Ba2+ significantly augmented the Ca2+ current but abolished the Cl- currents. The Cl- channel blocker niflumic acid (10-50 microM) diminished the time-dependent outward Cl- current and the inward tail Cl- current, decreased serotonin-induced membrane depolarization, and inhibited agonist-induced PA contraction. In the absence of extracellular Ca2+, cyclopiazonic acid, which releases Ca2+ from sarcoplasmic reticulum, elicited an inward Cl- current at a holding potential of -70 mV. These results indicate that rat PA myocytes possess ClCa channels that are activated by depolarization-induced Ca2+ influx and agonist-induced Ca2+ release. This Cl- current contributes to agonist-induced pulmonary vasoconstriction via membrane depolarization.
SP-A enhances uptake of bacillus Calmette-Guerin by macrophages through a specific SP-A receptor
Authors: Weikert L. F. , Edwards K. , Chroneos Z. C. , Hager C. , Hoffman L. , Shepherd V. L.
Published: December 2017

Surfactant-associated protein A (SP-A) is a C-type lectin that is involved in surfactant metabolism as well as host defense functions in the lung. We have recently identified a receptor on macrophages [specific 210-kDa SP-A receptor (SPR210)] that binds SP-A. In the current study we have investigated the role of SP-A in mediating uptake of bacillus Calmette-Guerin (BCG) by rat macrophages and human monocytes and have examined the role of the macrophage SPR210 in this process. 125I-labeled SP-A bound BCG in a Ca(2+)-, carbohydrate-, and dose-dependent manner. To examine association of SP-A-BCG complexes with macrophages, BCG were opsonized with SP-A and were incubated with rat bone marrow-derived macrophages (RBMM), rat alveolar macrophages (RAM), or human monocytes at a 1-to-1 ratio for 4 h. The cells were washed, fixed in formalin, and stained with auramine-rhodamine. Cell-associated organisms were enumerated by fluorescent microscopy. The percentage of cells with one or more associated BCG was increased by SP-A from 27% of RBMM with BCG alone to 54% with SP-A-BCG complexes; 1-16% in RAM; and 39-67% in human monocytes. This enhanced uptake was dependent on the dose of SP-A, with maximal increases seen with 10 micrograms/ml. Electron microscopic analysis supported the conclusion that organisms were ingested by and not simply bound to the macrophages. Inclusion of SPR210 antibodies blocked association of SP-A-BCG complexes, suggesting a role for SPR210 in mediating the interaction of SP-A-BCG with the macrophages. This was further supported by the finding that modulation of SPR210 activity resulted in altered SP-A-BCG uptake. These results demonstrate that SP-A binds to BCG and that uptake of these SP-A-BCG complexes is mediated in part by the SPR210 on rat macrophages and human monocytes.
Adenovirus E1A upregulates interleukin-8 expression induced by endotoxin in pulmonary epithelial cells
Authors: Keicho N. , Elliott W. M. , Hogg J. C. , Hayashi S.
Published: December 2017

Adenovirus E1A DNA and proteins are frequently detected in lungs of patients with chronic obstructive pulmonary disease. Because adenovirus E1A can regulate host gene expression by interacting with cellular transcription factors, we postulate that E1A enhances synthesis of inflammatory mediators. To examine this possibility, we measured the expression of inflammatory cytokines in E1A-producing A549 human pulmonary epithelial cells and control cells. Interleukin (IL)-8 mRNA was markedly induced by lipopolysaccharide (LPS) in E1A-producing cells but not in controls. IL-8 protein levels were elevated in parallel. In both cell types, monocyte chemotactic and activating factor mRNA induced by LPS was low, and transforming growth factor-beta 1 mRNA was not affected. IL-1 beta, IL-6, granulocyte macrophage colony-stimulating factor, and granulocyte colony-stimulating factor mRNAs were too low to show any effect of E1A. We conclude that the LPS responsiveness of A549 pulmonary epithelial cells is altered by adenoviral E1A by upregulating IL-8. We speculate that this mechanism may be important in the amplification of the inflammatory process of lungs to other irritants.
5-Lipoxygenase products are necessary for ovalbumin-induced airway responsiveness in mice
Authors: Irvin C. G. , Tu Y. P. , Sheller J. R. , Funk C. D.
Published: December 2017

To determine the role of 5-lipoxygenase products in the development of airway reactivity that follows antigen exposure, we sensitized mice by intraperitoneal injection of ovalbumin and aluminum hydroxide and serial exposure to aerosols of ovalbumin. Mice lacking a functioning 5-lipoxygenase enzyme were produced by targeted gene disruption. They and their wild-type controls had measurements of lung resistance (RL) made in response to intravenous methacholine; bronchoalveolar lavage fluid cell counts and serum immunoglobulin concentrations were also measured. Wild-type mice developed striking increases in cholinergic responsiveness; 5-lipoxygenase-deficient mice manifested minimal alterations in methacholine responsiveness (RL at the highest methacholine dose was 9.9 +/- 2.4 cmH2O.ml-1.s-1 under control conditions vs. 27.6 +/- 4.6 cmH2O.ml-1.s-1 after ovalbumin in wild-type mice; 5.9 +/- 0.9 vs. 7.01 +/- 2.2 cmH2O.ml-1.s-1 in 5-lipoxygenase-deficient mice). Ovalbumin provoked airway eosinophilia and increased immunoglobulins in wild-type mice, which were present to a significantly lesser degree in 5-lipoxygenase-deficient mice. We conclude that 5-lipoxygenase products are essential for the production of nonspecific airway reactivity in mice and suggest that 5-lipoxygenase products may be important in immunoglobulin formation.
PAMP is a novel inhibitor of the tachykinin release in the airway of guinea pigs
Authors: Kanazawa H. , Kamoi H. , Kawaguchi T. , Shoji S. , Fujii T. , Kudoh S. , Hirata K. , Yoshikawa J.
Published: December 2017

Proadrenomedullin NH2-terminal 20 peptide (PAMP), a newly identified hypotensive peptide, may play physiological roles in airway and cardiovascular controls. This study was designed to determine the mechanism responsible for the bronchoprotective effects of PAMP on capsaicin-induced bron-choconstriction in anesthetized guinea pigs. PAMP (10(-8)-10(-6) M) significantly inhibited capsaicin-induced bronchoconstriction in a dose-dependent manner. The bronchoprotective effect of PAMP (10(-6) M) was as large as that of isoproterenol (10(-7) M) and lasted > 10 min. The concentration of immunoreactive substance P (SP) in bronchoalveolar lavage fluid after administration of capsaicin (4 x 10(-6) M) was 120 +/- 10 fmol/ml. PAMP significantly inhibited the release of immunoreactive SP in a dose-dependent manner (60 +/- 6 fmol/ml for (10(-6) M PAMP, P < 0.01; 84 +/- 6 fmol/ml for 10(-7) M PAMP, P < 0.01; and 95 +/- 6 fmol/ml for 10(-8) M PAMP, P < 0.05). PAMP (10(-6) M) did not significantly affect exogenous neurokinin A (NKA) or NKA + SP-induced bronchoconstriction, whereas isoproterenol (10(-7) M) significantly inhibited exogenous tachykinin-induced bronchoconstriction. These findings suggest that the bronchoprotective effects of PAMP are mainly due to inhibition of the release of tachykinins at airway C-fiber endings.
Extracellular ATP stimulates K+ secretion across cultured human airway epithelium
Authors: Clarke L. L. , Chinet T. , Boucher R. C.
Published: December 2017

Extracellular ATP applied to the luminal side of human airway epithelium (HAE) activates an apical membrane Cl- conductance and transepithelial Cl- secretion. However, in some HAE preparations, we have found that luminal ATP induces a change in short-circuit current (Isc), consistent with K+ secretion. Using intracellular microelectrodes and radioisotopic flux studies, we investigated whether extracellular ATP regulates transepithelial K+ secretion in primary HAE cultures. In physiological Ringer solution, HAE had a negligible electrochemical driving force for Cl- secretion (DFCl), and luminal ATP induced a change in Isc opposite in polarity to Cl- secretion. Intracellular microelectrode measurements indicated that the "reversed" Isc was associated with activation of a hyperpolarizing (K+) conductance in the apical membrane. Radioisotope studies of HAE pretreated with amiloride to induce a favorable DFCl revealed that luminal ATP stimulates a small 42K secretory flux concurrently with Cl- secretion. In ion-substituted Ringer solution, luminal ATP stimulated both the outward (K+) current and the inward (Cl-) current with approximately equal potency (approximately 10(-6) M). We conclude that luminal ATP activates an apical membrane K+ conductance and transepithelial K+ secretion across HAE.
Nitric oxide activates chloride currents in human lung epithelial cells
Authors: Kamosinska B. , Radomski M. W. , Duszyk M. , Radomski A. , Man S. F.
Published: December 2017

Epithelial Cl- channels are regulated by various physiological factors, including guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP mediates many of the physiological actions of nitric oxide (NO), we have studied both the presence of endogenous NO and the effects of exogenous NO on Cl- currents in A549 human lung epithelial cells. We have detected Ca(2+)-dependent NO synthase activity in A549 cells. Using the perforated patch-clamp technique, we have shown that inhibition of this enzyme by NG-monomethyl-L-arginine decreased Cl- current, an effect that was reversed by the NO donor S-nitrosoglutathione (GSNO). In addition, the NO donors GSNO and S-nitroso-N-acetyl-D,L-penicillamine increased whole-cell Cl- currents in A549 cells. This stimulatory effect of the NO donors was sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) are involved in the action of NO on A549 cells. In addition, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, decreased NO-mediated stimulation of Cl- currents. Our results suggest that, in lung epithelial cells, NO regulates a non-CFTR Cl- conductance acting via a cGMP-dependent mechanism.
Expression of functional gap junctions in cultured pulmonary alveolar epithelial cells
Authors: Lee Y. C. , Yellowley C. E. , Li Z. , Donahue H. J. , Rannels D. E.
Published: December 2017

Recent observations suggest that cell-cell interactions may modulate the response of the alveolar epithelium to injury. Expression and function of gap junctions were thus evaluated in isolated alveolar type II cells. Freshly isolated (day 0) type II cells expressed mRNAs for gap junctional connexins 26, 32, and 43. Whereas connexin 26 mRNA declined approximately 40% in cultured cells, connexin 32 message decreased rapidly and was not detectable on day 1. In contrast, connexin 43 expression increased 10-fold by day 3 compared with day 0. Western blot confirmed a 30-fold elevation in connexin 43 protein. Connexin 45 mRNA was not detected. Functional gap junction-mediated calcium signal propagation was monitored using fura 2, a calcium-sensitive dye. Membrane deformation in a single type II cell increased intracellular calcium; the signal spread rapidly to neighboring cells by octanol-sensitive pathways. Transfer of Lucifer yellow between cells also was inhibited by octanol. These observations demonstrate functional gap junctions between cultured alveolar epithelial cells and suggest that gap junctional expression and gap junction-mediated cell-cell coupling are regulated with time in culture.
Pulmonary cell kinetics and morphometry after ozone exposure: day versus night and dose response in rats
Authors: Witschi H. , Espiritu I. , Pinkerton K. E.
Published: December 2017

Male Sprague-Dawley rats were exposed to increasing concentrations of ozone as follows: 0.12, 0.24, 0.36, 0.6, or 0.8 ppm. Controls were kept in chambers ventilated with filtered air. One-half of the animals in ozone was exposed for 12 h a day during daytime hours, and the other one-half of the animals was exposed for 12 h during nighttime. Cumulative labeling indexes were measured after 4 and 7 days in the terminal bronchioles, large intrapulmonary airways, trachea, and nasal epithelia. The penetration of the lesions from the bronchiole-alveolar junction into the alveolar zone was measured with quantitative morphometry. After 4 days of exposure, the extent of injury was dose dependent. Labeling indexes in the terminal bronchioles were 15-20% higher in animals exposed during nighttime compared with the animals exposed during daylight hours. On the other hand, depth of penetration of ozone lesions into the centriacinar region was not significantly different in animals exposed during the night compared with animals exposed during daytime. Labeling indexes in the large airways, trachea, or nasal cavity were not influenced by time of exposure. Between days 4 and 7, the lesions in the terminal bronchioles progressed only to a minimal degree (10%). It was concluded that the pattern of centriacinar tissue remodeling 1) followed a gradient based on ozone concentration and 2) was essentially complete after only 4 days of ozone exposure. Although a difference between daytime and nighttime exposure was observed, it was not considered to be large enough to invalidate conclusions drawn from studies in which animals are exposed to ozone during daylight hours.
Hypoxia inhibits nitric oxide synthesis in isolated rabbit lung
Authors: Kantrow S. P. , Huang Y. C. , Whorton A. R. , Grayck E. N. , Knight J. M. , Millington D. S. , Piantadosi C. A.
Published: December 2017

Nitric oxide (NO.) has been proposed to modulate hypoxic vasoconstriction in the lung. The activity of nitric oxide synthase (NOS) can be inhibited by hypoxia because molecular oxygen is a necessary substrate for the enzyme. On the basis of this mechanism, we hypothesized that NOS activity has a key role in regulation of pulmonary vascular tone during hypoxia. We measured oxidation products of NO. released into the vasculature of isolated buffer-perfused rabbit lung ventilated with normoxic (21% O2), moderately hypoxic (5% O2), or anoxic (0% O2) gas using two methods. Mean PO2 in perfusate exiting the lung was 25 Torr during anoxic ventilation and 47 Torr during moderately hypoxic ventilation. We found that the amount of the NO. oxidation product nitrite released into the perfusate was suppressed significantly during ventilation with anoxic but not moderately hypoxic gas. During normoxic ventilation, nitrite release was inhibited by pretreatment with NG-monomethyl-L-arginine, a competitive inhibitor of NOS. To confirm that changes in nitrite concentration reflected changes in NO. release into the perfusate, major oxidation products of NO. (NOx) were assayed using a method for reduction of these products to NO. by vanadium(III) Cl. Release of NOx into the perfusate was suppressed by severe hypoxia (anoxic ventilation), and this effect was reversed by normoxia. Pulmonary vasoconstriction was observed during severe but not moderate hypoxia and was related inversely to the rate of nitrite release. These observations provide evidence that decreased NO. production contributes to the pulmonary vasoconstrictor response during severe hypoxia.
Peripheral hypertension and alterations in pulmonary vascular regulation
Authors: Burke-Wolin T. , Pino P. , Itani M. , Talerico M. , Pucci M. , Benson D. , Fayngersh R.
Published: December 2017

We have recently reported in normal isolated-perfused rat lungs that low basal tone appears to be regulated by nitric oxide (NO)-dependent and -independent mechanisms of soluble guanylate cyclase activation. In this study, we examined the role of NO in the regulation of pulmonary artery (PA) tone from rats with renin-dependent hypertension. Rats were made hypertensive by ligating the abdominal aorta above the left and below the right renal artery (aortic coarctation, AC). Mean arterial pressure significantly increased from 119 +/- 8.4 mmHg in control animals to 156 +/- 15 mmHg 7-14 days after AC surgery. PA pressures, however, remained unchanged (8.5 +/- 3.4 mmHg in control animals vs. 11 +/- 3.3 mmHg in AC animals). Hypoxic contractions in U-46619 precontracted isolated small PA (160-260 microns diameter) were significantly increased from 51 +/- 13 mg in the control group to 142 +/- 38 mg (P < or = 0.05) in AC animals. Nitro-L-arginine (NLA; 100 microM) contractions were also enhanced in the AC animal. The enhanced NLA response may correlate with an increase in endothelial cell NO synthase (NOS) as detected by Western blotting (132 +/- 28% of control; P < 0.05). These data suggest that, in this renin-dependent model of systemic hypertension, there is increased endothelial cell NOS activity that maintains low PA tone, preventing the lung from developing increased pressures.
CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain
Authors: Wilkinson D. J. , Strong T. V. , Mansoura M. K. , Wood D. L. , Smith S. S. , Collins F. S. , Dawson D. C.
Published: December 2017

To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (KA) for activation by 3-isobutyl-1-methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.
Integrin mediation of alveolar epithelial cell migration on fibronectin and type I collagen
Authors: Kim H. J. , Henke C. A. , Savik S. K. , Ingbar D. H.
Published: December 2017

Acute lung injury leads to type I alveolar epithelial cell (AEC) death, denudation of the alveolar basement membrane, and formation of an alveolar provisional matrix from fibronectin, fibrinogen, and type I collagen. The provisional matrix provides a scaffold for alveolar repair. To restore normal lung architecture, surviving type II AECs must reepithelialize denuded alveoli. We examined whether AECs migrate on provisional matrix proteins and whether integrins mediate this migration using a Boyden chemotaxis chamber. Cultured AECs migrated on fibronectin-coated filters by haptotaxis (defined as movement on a solid-phase substrate) more than one type I collagen-coated filters, and they did not migrate on fibrinogen-coated filters. Soluble fibronectin augmented migration on type I collagen-coated filters, but not on fibronectin-coated filters. Anti-alpha v beta 3-integrin monoclonal antibody (MAb) inhibited migration on substrate-bound fibronectin by 62-77%, whereas anti-beta 1-integrin MAb inhibited migration by 48%. Anti-alpha 2-integrin MAb almost completely inhibited migration on substrate-bound type I collagen, but not on fibronectin. The novel findings in this study are as follows: 1) AECs migrate by haptotaxis more effectively on substrate-bound fibronectin than on type I collagen; 2) alpha v beta 3- and beta 1-integrins partially mediate AEC haptotaxis on fibronectin; and 3) the alpha 2 beta 1-integrin mediates AEC migration on type I collagen. These results support the importance of type II cell migration on provisional matrix proteins during repair of lung injury.
Association of granular exocytosis with Ca(2+)-activated K+ channels in human eosinophils
Authors: Saito M. , Sato R. , Munoz N. M. , Herrnreiter A. , Oyaizu M. , Kasugai H. , Narahashi T. , Leff A. R.
Published: December 2017

We studied the mechanism of degranulation caused by Ca(2+)-activated K+ channels (KCa channels) in eosinophils isolated from mildly atopic donors using negative immunoselection. Stimulation of eosinophils with 0.1 microM platelet-activating factor (PAF) caused activation of single channels as recorded by the cell-attached patch-clamp technique. These channels were selectively permeable to K+ because the reversal potential was close to the equilibrium potential for K+. However, the channels were not permeable to Na+ or Cl- as demonstrated by ion substitution experiments. The calcium ionophore A-23187, at 1 microM, increased the K+ channel activity in the presence of Ca2+ in the external perfusate but did not induce channel activity in the absence of Ca2+. Similar results were obtained with another calcium ionophore, ionomycin (1 microM), and the Ca(2+)-releasing agent thapsigargin (10 microM). K+ channels activated by PAF and A-23187 had similar characteristics: two levels of single-channel conductances were observed, 10 +/- 1.5 and 22 +/- 1.7 pS as induced by PAF and 11 +/- 1.3 and 24 +/- 1.9 pS by A-23187; the mean open times of the large-conductance channels were 1.45 +/- 0.3 ms as induced by PAF and 1.26 +/- 0.5 ms by A-23187. These results indicate that PAF activates KCa channels. Both KCa currents and major basic protein release caused by A-23187 were blocked by quinidine. It is suggested that KCa channels are associated with granule secretion in human eosinophils.
Mechanisms of ionomycin-induced endothelial cell barrier dysfunction
Authors: Garcia J. G. , Schaphorst K. L. , Shi S. , Verin A. D. , Hart C. M. , Callahan K. S. , Patterson C. E.
Published: December 2017

Myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+)- calmodulin-dependent MLC kinase (MLCK) is critical to thrombin-mediated endothelial cell gap formation and barrier dysfunction. We have tested the hypothesis that the Ca2+ ionophore ionomycin stimulates MLCK-dependent endothelial cell contraction and permeability. Ionomycin significantly increased albumin clearance and decreased electrical resistance across confluent bovine pulmonary microvascular and macrovascular endothelial cell monolayers in a concentration-dependent manner that was temporally similar to that produced by thrombin. In contrast, however, ionomycin produced a significant Ca(2+)-dependent reduction in the levels of phosphorylated MLC with evidence of serine/threonine phosphatase activation. Potential MLCK-independent mechanisms of endothelial cell permeability were examined with little evidence to support a role for stimulated nitric oxide synthase or phospholipase A2 activities. Importantly, ionomycin produced 1) reductions in the activities of the barrier protective adenylate cyclase and the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A, 2) dramatic dose- and time-dependent inhibition of endothelial cell tyrosine kinase activities, and 3) marked decreases in the phosphotyrosine content of the p125 focal adhesion kinase. These data indicate that ionomycin produces endothelial cell barrier dysfunction by mechanisms that are independent of MLCK activation and may involve reductions in endothelial cell tethering forces via inhibition of protein kinase A and tyrosine kinase activities, especially the p125 focal adhesion kinase.
Systemic blood loss affects NF-kappa B regulatory mechanisms in the lungs
Authors: Moine P. , Shenkar R. , Kaneko D. , Le Tulzo Y. , Abraham E.
Published: December 2017

The nuclear regulatory factor (NF)-kappa B is activated in the lungs of patients with acute respiratory distress syndrome (ARDS). In experimental models of acute lung injury, activation of NF-kappa B contributes to the increased expression of immunoregulatory cytokines and other proinflammatory mediators in the lungs. Because of the important role that NF-kappa B activation appears to play in the development of acute lung injury, we examined cytoplasmic and nuclear NF-kappa B counterregulatory mechanisms in lung mononuclear cells, using a murine model in which inflammatory lung injury develops after blood loss. Sustained activation of NF-kappa B was present in lung mononuclear cells over the 4-h period after blood loss. The activation of NF-kappa B after hemorrhage was accompanied by alterations in levels of the NF-kappa B regulatory proteins I kappa B alpha and Bcl-3. Cytoplasmic and nuclear I kappa B alpha were increased and nuclear Bcl-3 was decreased during the first hour after blood loss, but, by 4 h posthemorrhage, cytoplasmic and nuclear I kappa B alpha levels were decreased and nuclear levels of Bcl-3 were increased. Inhibition of xanthine oxidase activity in otherwise unmanipulated unhemorrhaged mice resulted in increased levels of I kappa B alpha and decreased amounts of Bcl-3 in nuclear extracts from lung mononuclear cells. No changes in the levels of nuclear I kappa B alpha or Bcl-3 occurred after hemorrhage when xanthine oxidase activity was inhibited. These results demonstrate that blood loss, at least partly through xanthine oxidase-dependent mechanisms, produces alterations in the levels of both I kappa B alpha and Bcl-3 in lung mononuclear cell populations. The effects of hemorrhage on proteins that regulate activation of NF-kappa B may contribute to the frequent development of inflammatory lung injury in this setting.
Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells
Authors: Abdullah L. H. , Conway J. D. , Cohn J. A. , Davis C. W.
Published: December 2017

Airway goblet cells secrete mucin in response to ATP and uridine 5'-triphosphate (UTP), but the underlying signal transduction pathways are poorly understood. Cultures of SPOC1 cells (L. H. Abdullah, S. W. Davis, L. Burch, M. Yamauchi, S. H. Randell, P. Nettesheim, and C. W. Davis. Biochem. J. 316: 943-951, 1996) secreted mucin on exposure to phorbol 12-myristate 13-acetate (PMA) [apparent affinity (K0.5) approximately 100 nM] and ionomycin (K0.5 approximately 5 microM) almost fivefold over baseline. Thapsigargin also elicited secretion (K0.5 approximately 20 nM). Ionomycin and PMA together elicited approximately twice the secretion of either agent alone. Overnight exposure to half-maximal PMA abolished the response to maximal doses of UTP and PMA, whereas ionomycin was fully effective. Protein kinase C (PKC) activity in the membrane fraction was increased by maximal doses of PMA and UTP, whereas ionomycin had no effect. PKC inhibitors were relatively ineffective against PMA- and UTP-induced mucin secretion. Human and canine goblet cells in epithelial explants, by video microscopy, underwent exocytosis with ionomycin (1 microM) and PMA (0.1 or 1 microM). SPOC1 cell mucin secretion was not stimulated by forskolin, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate. Cystic fibrosis transmembrane conductance regulator was not detected in SPOC1 cells by Western blotting, and its mRNA was detected by reverse transcriptase polymerase chain reaction (PCR) only as a very weak band and after 55 PCR cycles. Multidrug resistance (MDR1), however, was readily detected by Western blotting, and its mRNA was detected as a major band after 35 PCR cycles. Thus airway goblet cell mucin secretion, distal to receptor activation, may be regulated independently by Ca(2+)- and PKC-dependent pathways. Cystic fibrosis transmembrane conductance regulator and cyclic nucleotides, however, may not play a major role in this secretion.
Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells
Authors: Ridge K. M. , Rutschman D. H. , Factor P. , Katz A. I. , Bertorello A. M. , Sznajder J. L.
Published: December 2017

Lung Na-K-ATPase has been shown to contribute to vectorial Na+ transport and edema clearance. The alpha 1- and beta 1-Na-K-ATPase subunits have been localized to alveolar type II (ATII) cells, and the alpha 2-Na-K-ATPase has been reported in rat lung homogenates. Expression of Na-K-ATPase alpha 1-, alpha 2-, and beta 1-subunits was investigated in rat ATII cells cultured for 7 days, a period during which they lose their phenotypic markers and differentiate to an alveolar type I (ATI)-like cell phenotype. Differentiation of ATII cells to an ATI-like phenotype resulted in a decrease of alpha 1- and an increase of alpha 2-mRNA and protein abundance without changes in the beta 1-subunit. Thus ATI-like cells exhibited a mixture of alpha 1- and alpha 2-isoforms. Nuclear run-on analysis suggests that these changes were transcriptionally regulated. The existence of the distinct functional classes of Na-K-ATPase in ATII and ATI-like cells was confirmed by ouabain inhibition of Na-K-ATPase activity. Ouabain inhibition of ATII cells was consistent with expression of the alpha 1-isozyme [50% inhibitory concentration (IC50) = 4 x 10(-5) M], whereas, in ATI-like cells, it was consistent with the presence of both alpha 1- and alpha 2-isozymes (IC50 = 9.0 x 10(-5) and 1.5 x 10(-7) M, respectively); [3H]ouabain binding studies corroborated these findings. Our results indicate that, during ATII cell cytodifferentiation with time in culture, there is a shift in isoform composition that may reflect physiological functions of alveolar epithelial cells.
Clearance of intra-amniotic lung surfactant: uptake and utilization by the fetal rabbit lung
Authors: Hallman M. , Lappalainen U. , Bry K.
Published: December 2017

To investigate the metabolism of intra-amniotic surfactant, surfactant containing double-labeled dipalmitoylphosphatidylcholine (DPPC) was injected in amniotic fluid on days 23-27 of gestation. Within 44 h, DPPC was distributed to the gastrointestinal tract (45.9%), fetal membranes and placenta (8.2%), fetal lung (6.6%), and liver (1.9%). DPPC uptake was higher in the upper than in the lower lung lobes. The mixture of phosphatidylglycerol and DPPC increased the uptake of DPPC that was not saturable (range 15-60 mg phospholipid). There was no detectable metabolism of DPPC taken up by the fetal lung. Surfactant protein A, originating from intra-amniotic heterplogous surfactant, was detected immunohistochemically in alveolar epithelium. Intra-amniotic surfactants did not affect the expression of surfactant protein mRNAs. Intra-amniotic surfactant (1,500-2,000 mg/kg on day 25.3) improved lung compliance of ventilated 27.0-day premature rabbits less than intratracheal surfactant at birth (75-100 mg/kg). Reutilization by the alveolar epithelium of surfactant secreted to future airspaces, airways, and amniotic fluid may be a mechanism that increases intracellular surfactant pool before birth.
Endothelin receptor changes in hypoxia-induced pulmonary hypertension in the newborn piglet
Authors: Gosselin R. , Gutkowska J. , Baribeau J. , Perreault T.
Published: December 2017

Endothelin (ET)-1, a potent vasoconstrictor and mitogen, acts through ETA and ETB receptors and may be involved in the pathogenesis of persistent pulmonary hypertension of the newborn. We hypothesized that hypoxia-induced pulmonary hypertension in the newborn is associated with increased ET-1 release and modified ET receptor characteristics leading to vasoconstriction and vascular remodeling. Therefore, we studied 1-day-old piglets exposed for 3 or 14 days to hypoxia (fraction of inspired O2 = 0.10) or normoxia (controls). ET-1 circulating levels in pulmonary artery and vein were measured. Pulmonary vascular reactivity to ET-1 was evaluated using isolated-perfused lungs. ET binding characteristics were examined in microsomes from pulmonary arteries (down to 100 microns). ET-1 circulating levels are low and are not altered by hypoxia. The magnitude of the initial dilator response to ET-1 decreases after 3 days of hypoxia (P < 0.05), whereas the number of ETB receptors is reduced by 40% in the pulmonary arteries (P < 0.05). ETA receptors are predominant (65-90%) in pulmonary arteries. ETA receptors decrease by 50% after 14 days of exposure to hypoxia (P < 0.05), whereas the constrictor response to ET-1 remains unchanged. The fact that the reduction in vasodilator response parallels the decrease in ETB receptors suggests a decrease in receptor expression. We speculate that the maintenance of the vasoconstrictor response to ET-1 despite a reduction in the number of binding sites is likely due to receptor occupancy. In conclusion, in the newborn piglet pulmonary vasculature, ETA and ETB receptors may be affected differently by hypoxia.
Adrenal hormone regulation of fibronectin synthesis by type II pulmonary epithelial cells
Authors: Dunsmore S. E. , Lee Y. C. , Rannels D. E.
Published: December 2017

Previous work suggested an association between changes in the alveolar extracellular matrix and epithelial cell growth in lungs of adrenalectomized rats in vivo. Other studies demonstrated that adrenal hormones modulate extracellular matrix synthesis by type II pulmonary epithelial cells in vitro. Adrenal hormone regulation of type II cell fibronectin synthesis was thus examined. Fibronectin synthesis was quantitated by immunoprecipitation of the metabolically labeled molecule from cells, extracellular matrix, and culture medium. On day 1 of primary culture, synthesis of matrix fibronectin by type II cells isolated from the lungs of adrenalectomized animals was more than twice that by cells isolated from control rats. Adrenalectomy elevated steady-state fibronectin mRNA levels in primary isolates of type II cells cultured for 1 or 3 days. These results suggest that altered fibronectin synthesis and deposition into the extracellular matrix accompany changes in type II cell phenotype that occur after adrenalectomy.