RESUMEN
We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/fisiología , Cardiolipinas/biosíntesis , Células Endoteliales/fisiología , Animales , Cardiolipinas/sangre , Bovinos , Células Cultivadas , Impedancia Eléctrica , Hidrólisis , Ratones Endogámicos C57BL , Oxidación-Reducción , Infecciones por Pseudomonas/sangre , Transducción de SeñalRESUMEN
Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Metalotioneína/metabolismo , Superóxido Dismutasa/metabolismo , Zinc/metabolismo , Animales , Femenino , Hiperoxia , Inflamación/inmunología , Metalotioneína/genética , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/análisis , Mucosa Respiratoria/metabolismoRESUMEN
A role in signal transduction for a vanishingly small labile pool of intracellular zinc ([Zn](i)) has been inferred by the sensitivity of various physiological pathways to zinc chelators such as N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and/or associations with changes in nonprotein-bound zinc-sensitive fluorophores. Although we (44) reported that LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) was exacerbated by TPEN, 1) we did not detect acute (30 min) changes in [Zn](i), and 2) it is unclear from other reports whether LPS increases or decreases [Zn](i) and whether elevations or decreases in [Zn](i) are associated with cell death and/or apoptosis. In the present study, we used both chemical (FluoZin-3 via live cell epifluorescence microscopy and fluorescence-activated cell sorting) and genetic (luciferase activity of a chimeric reporter encoding zinc-sensitive metal-response element and changes in steady-state mRNA of zinc importer, SLC39A14 or ZIP14) techniques to show that LPS caused a delayed time-dependent (2-4 h) decrease in [Zn](i) in SPAEC. A contributory role of decreases in [Zn](i) in LPS-induced apoptosis (as determined by caspase-3/7 activation, annexin-V binding, and cytochrome c release) in SPAECs was revealed by mimicking the effect of LPS with the zinc chelator, TPEN, and inhibiting LPS- (or TPEN)-induced apoptosis with exogenous zinc. Collectively, these are the first data demonstrating a signaling role for decrease in [Zn](i) in pulmonary endothelial cells and suggest that endogenous levels of labile zinc may affect sensitivity of pulmonary endothelium to the important and complex proapoptotic stimulus of LPS.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Espacio Intracelular/metabolismo , Lipopolisacáridos/farmacología , Arteria Pulmonar/citología , Zinc/metabolismo , Animales , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Genes Reporteros , Espacio Intracelular/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ovinos , Transducción de Señal/efectos de los fármacos , Espectrometría de Fluorescencia , Regulación hacia Arriba/efectos de los fármacosRESUMEN
S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H(2)S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N(G)-nitro-L-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H(2)S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H(2)S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N(G)-nitro-L-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H(2)S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.
Asunto(s)
Citoprotección/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Pulmón/irrigación sanguínea , Compuestos Nitrosos/farmacología , Albúmina Sérica Bovina/farmacología , Animales , Apoptosis/efectos de los fármacos , Arterias/citología , Células Cultivadas , Endocitosis/efectos de los fármacos , Células Endoteliales/enzimología , Humanos , Sulfuro de Hidrógeno/farmacología , Microvasos/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Necrosis/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitrosación/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Ratas , terc-Butilhidroperóxido/farmacologíaRESUMEN
The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 microm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT(-/-) mice. Data from NO-sensitive fluorescence resonance energy transfer-based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT(-/-) mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.
Asunto(s)
Hipoxia/fisiopatología , Óxido Nítrico/fisiología , Arteria Pulmonar/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos , Zinc/fisiología , Animales , Aorta/efectos de los fármacos , Tamaño de la Célula , Células Cultivadas , Quelantes/farmacología , Células Endoteliales/efectos de los fármacos , Etilenodiaminas/farmacología , Técnicas In Vitro , Metalotioneína/efectos de los fármacos , Metalotioneína/fisiología , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Nitrosación , Especificidad de Órganos , Oxígeno/farmacología , Ratas , Ratas Sprague-Dawley , Ovinos , Vasoconstricción/efectos de los fármacosRESUMEN
We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release.
Asunto(s)
Bronquios/metabolismo , Cloruros/toxicidad , Células Epiteliales/efectos de los fármacos , Metalotioneína/metabolismo , Níquel/toxicidad , Compuestos de Zinc/toxicidad , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Separación Celular/métodos , Células Cultivadas , Quelantes/farmacología , Cloruros/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Etilaminas/farmacología , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Metalotioneína/genética , Ratones , Ratones Noqueados , Compuestos Policíclicos , Piridinas , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Transfección , Regulación hacia Arriba , Compuestos de Zinc/metabolismo , Factor de Transcripción MTF-1RESUMEN
Low-molecular-weight S-nitrosothiols are found in many tissues and affect a diverse array of signaling pathways via decomposition to *NO or exchange of their -NO function with thiol-containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of D- and L-stereoisomers of S-nitrosocysteine ethyl ester (SNCEE) on fluorescence resonance energy transfer (FRET)-based reporters that are targets for the following NO-related modifications: (a) S-nitrosation, via the cysteine-rich protein metallothionein (FRET-MT), and (b) nitrosyl-heme-Fe, via guanosine 3',5'-cyclic monophosphate (cygnet-2). Conformational changes consistent with S-nitrosation of FRET-MT were specific to l-SNCEE. In addition, they were reversed by dithiothreitol (DTT) but unaffected by exogenous oxyhemoglobin. In contrast, d- and l-SNCEE had comparable effects on cygnet-2, likely via activation of soluble guanylyl cyclase (sGC) by *NO as they were sensitive to the sGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-alpha] quinoxalin-1-one and exogenous oxyhemoglobin. These data demonstrate the utility of spectral laser scanning confocal imaging in revealing subtle aspects of NO signal transduction in live cells. Stereoselective transnitrosation of MT emphasizes the specificity of posttranslational modification as a component of NO signaling.
Asunto(s)
Cisteína/análogos & derivados , Microscopía Fluorescente/métodos , Óxido Nítrico/metabolismo , Espectrometría de Fluorescencia/métodos , Compuestos de Sulfhidrilo/química , Animales , Células Cultivadas , GMP Cíclico/metabolismo , Cisteína/química , Ditiotreitol/química , Endotelio Vascular/citología , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/farmacología , Radicales Libres , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Guanilato Ciclasa , Hemo/química , Óxido Nítrico/química , Compuestos Nitrosos/química , Péptidos/química , Conformación Proteica , Proteínas/química , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes de Fusión/metabolismo , Ovinos , Transducción de Señal , Guanilil Ciclasa SolubleRESUMEN
Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1(-/-) mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1(-/-) mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1(-/-) MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.
Asunto(s)
Albúminas/metabolismo , Caveolas/fisiología , Endocitosis , Endotelio Vascular/metabolismo , Pulmón/irrigación sanguínea , Animales , Secuencia de Bases , Caveolina 1/genética , Caveolina 1/metabolismo , Células Cultivadas , Cartilla de ADN , Endotelio Vascular/citología , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Pinocitosis , ARN Interferente Pequeño/genética , RatasRESUMEN
We have shown that zinc-thiolate moieties of the metal binding protein metallothionein (MT) are critical targets for nitric oxide (NO) with resultant increases in intracellular labile zinc. Such an NO-MT-Zn signaling pathway appears to participate in important cardiovascular functions associated with biosynthesis of NO including hypoxic vasoconstriction in the lung. Although downstream effector signaling molecules and critical contractile targets remain unclear, current investigations reveal a contributory role for zinc dependent protein kinases and cytoskeletal proteins in mediating hypoxic induced constriction of pulmonary endothelial cells.
Asunto(s)
Endotelio/citología , Endotelio/fisiología , Homeostasis/fisiología , Pulmón/citología , Pulmón/fisiología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , Zinc/fisiología , Animales , Comunicación Celular/fisiología , Muerte Celular/fisiología , Endotelio/metabolismo , Humanos , Pulmón/química , Pulmón/metabolismo , Metalotioneína/fisiología , Óxido Nítrico/química , Zinc/químicaRESUMEN
Oxidation of two anionic phospholipids--cardiolipin (CL) in mitochondria and phosphatidylserine (PS) in extramitochondrial compartments--is important signaling event, particularly during the execution of programmed cell death and clearance of apoptotic cells. Quantitative analysis of CL and PS oxidation products is central to understanding their molecular mechanisms of action. We combined the identification of diverse phospholipid molecular species by ESI-MS with quantitative assessments of lipid hydroperoxides using a fluorescence HPLC-based protocol. We characterized CL and PS oxidation products formed in a model system (cyt c/H(2)O(2)), in apoptotic cells (neurons, pulmonary artery endothelial cells) and mouse lung under inflammatory/oxidative stress conditions (hyperoxia, inhalation of single walled carbon nanotubes). Our results demonstrate the usefulness of this approach for quantitative assessments, identification of individual molecular species and structural characterization of anionic phospholipids that are involved in oxidative modification in cells and tissues.
Asunto(s)
Apoptosis , Cardiolipinas/química , Cardiolipinas/inmunología , Fosfatidilserinas/química , Fosfatidilserinas/inmunología , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Células Cultivadas , Células Endoteliales/química , Células Endoteliales/citología , Células Endoteliales/inmunología , Peróxidos Lipídicos/química , Peróxidos Lipídicos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Neuronas/química , Neuronas/citología , Neuronas/inmunología , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , OvinosRESUMEN
We hypothesized that: (a) S-nitrosylation of metallothionein (MT) is a component of pulmonary endothelial cell nitric oxide (NO) signaling that is associated with an increase in labile zinc; and (b) NO mediated increases in labile zinc in turn reduce the sensitivity of pulmonary endothelium to LPS-induced apoptosis. We used microspectrofluorometric techniques to show that exposing mouse lung endothelial cells (MLEC) to the NO-donor, S-nitrosocysteine, resulted in a 45% increase in fluorescence of the Zn2+-specific fluorophore, Zinquin, that was rapidly reversed by exposure to the Zn2+ chelator, NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine; TPEN). The absence of a NO-mediated increase in labile Zn2+ in MLEC from MT-I and -II knockout mice inferred a critical role for MT in the regulation of Zn2+ homeostasis by NO. Furthermore, we found that prior exposure of cultured endothelial cells from sheep pulmonary artery (SPAEC), to the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP) reduced their sensitivity to lipopolysaccharide (LPS) induced apoptosis. The anti-apoptotic effects of NO were significantly inhibited by Zn2+ chelation with low doses of TPEN (10 microM). Collectively, these data suggest that S-nitrosylation of MT is associated with an increase in labile (TPEN chelatable) zinc and NO-mediated MT dependent zinc release is associated with reduced sensitivity to LPS-induced apoptosis in pulmonary endothelium.