RESUMEN
Despite some theoretical concern about cancer-enhancing effects ofhyperbaric oxygen (HBO2) therapy, it is frequently administered to cancer patients. We evaluated the growth of murine breast cancer cells in the lung after hyperbaric oxygen treatment in an experimental metastasis assay. Young nu/nu mice were injected intravenously with 3 x 10(3) 4T1-GFP tumor cells per g body weight followed by lung isolation, perfusion, and intact organ epifluorescence microscopy 1 to 37 days after injection. A group of animals (n=32) was exposed once daily for 5 days a week to 45 min of 2.8 ATA hyperbaric oxygen (HBO2) in a research animal HBO2 chamber. Control animals (n=31) were not subjected to HBO2 treatment, but received similar intravenous administration of 3 x 10(3) 4T 1-GFP tumor cells. Single tumor cells and colonies were counted in the subpleural vessels in areas of about 0.5 cm2 of lung surface. HBO2 treatment did not lead to an increase in the number of the large or small colonies in the lungs. Rather, a significant reduction in the number of the large colonies was observed at 1 and 16 to 21-day periods of measurements after hyperbaric treatment. However, most importantly, there was a significant decrease in large colony size in the HBO2 group during all periods of observation. The results indicate that HBO2 is not prometastatic for breast cancer cells; rather it restricts the growth of large tumor cell colonies.
Asunto(s)
Oxigenoterapia Hiperbárica/efectos adversos , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/terapia , Animales , Recuento de Células/métodos , Línea Celular Tumoral , Ratones , Ratones Desnudos , Trasplante de Neoplasias/métodos , Trasplante de Neoplasias/patologíaRESUMEN
Ischemia in the intact ventilated lung (oxygenated ischemia) leads to endothelial generation of reactive oxygen species (ROS) and nitric oxide (NO). This study investigated the signaling pathway for NO generation with oxygenated ischemia in bovine pulmonary artery endothelial cells (BPAEC) that were flow adapted in vitro. BPAECs were cultured in an artificial capillary system and subjected to abrupt cessation of flow (ischemia) under conditions where cellular oxygenation was maintained. Immunoblotting and dichlorofluorescein/triazolofluorescein fluorescence were used to assess extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and ROS/NO generation, respectively. ERK1/2 phosphorylation significantly increased during ischemia, whereas total ERK1/2 did not change. ERK1/2 phosphorylation was suppressed by an inhibitor of tyrosine phosphorylation (genestein), cholesterol-binding reagents (filipin or cyclodextrin), or inhibitors of ROS (diphenyleneiodonium, N-acetylcysteine, or catalase), suggesting a role for both membrane cholesterol and ROS in ERK1/2 activation. Ischemia resulted in a 1.8-fold increase in NO generation that was suppressed by inhibitors of ERK1/2 activation (PD-98059 or U-0126). A calmodulin inhibitor (calmidizolium) or removal of Ca2+ from the medium also blocked NO generation, indicating that endothelial NO synthase (eNOS) is the activated isoform. These results indicate ischemia induces NO generation (possibly through a membrane cholesterol-sensitive flow sensor), the ERK1/2 cascade mediates signaling from the sensor to eNOS, and ROS are required for ERK activation.
Asunto(s)
Endotelio Vascular/metabolismo , Isquemia/metabolismo , Óxido Nítrico/biosíntesis , Transducción de Señal/fisiología , Animales , Bovinos , Células Cultivadas , Colesterol/metabolismo , Endotelio Vascular/citología , Fluoresceína , Indicadores y Reactivos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Arteria Pulmonar/citología , Especies Reactivas de Oxígeno/metabolismo , Estrés MecánicoRESUMEN
Abrupt cessation of lung perfusion induces a rapid endothelial response that is not associated with anoxia but reflects loss of normal shear stress. This response includes membrane depolarization, H(2)O(2) generation, and increased intracellular Ca(2+). We evaluated these parameters immediately upon nonhypoxic ischemia using fluorescence videomicroscopy to image in situ endothelial cells in isolated, ventilated rat lungs. Lungs labeled with 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS; a membrane potential probe), Amplex Red (an extracellular H(2)O(2) probe), or fluo 3-AM (a Ca(2+) indicator) were subjected to control perfusion followed by global ischemia. Endothelial di-8-ANEPPS fluorescence increased significantly within the first second of ischemia and stabilized at 15 s, indicating membrane depolarization by approximately 17 mV; depolarization was blocked by preperfusion with the K(+) channel agonist lemakalim. Increased H(2)O(2), inhibitable by catalase, was detected in the vascular space at 1-2 s after the onset of ischemia. Increased intracellular Ca(2+) was detected 10-15 s after the onset of ischemia; the initial increase was inhibited by preperfusion with thapsigargin. Thus the temporal sequence of the initial response of endothelial cells in situ to loss of shear stress (i.e., ischemia) is as follows: membrane depolarization, H(2)O(2) release, and increased intracellular Ca(2+).
Asunto(s)
Endotelio Vascular/metabolismo , Isquemia/metabolismo , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Animales , Calcio/metabolismo , Comunicación Celular/fisiología , Colorantes Fluorescentes , Técnicas In Vitro , Masculino , Microcirculación/fisiología , Potasio/metabolismo , Compuestos de Piridinio , Ratas , Ratas Sprague-Dawley , Estrés MecánicoRESUMEN
Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.
Asunto(s)
Endotelio Vascular/enzimología , Isquemia/metabolismo , Óxido Nítrico/biosíntesis , Estallido Respiratorio/fisiología , Adaptación Fisiológica/fisiología , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Potenciales de la Membrana/fisiología , Microscopía Fluorescente , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Consumo de Oxígeno/fisiología , Arteria Pulmonar/citología , Flujo Pulsátil/fisiología , Estrés Mecánico , Superóxidos/metabolismoRESUMEN
Whereas large numbers of cells from a primary tumor may gain access to the circulation, few of them will give rise to metastases. The mechanism of elimination of these tumor cells, often termed "metastatic inefficiency," is poorly understood. In this study, we show that apoptosis in the lungs within 1-2 days of introduction of the cells is an important component of metastatic inefficiency. First, we show that death of transformed, metastatic rat embryo cells occurred via apoptosis in the lungs 24-48 h after injection into the circulation. Second, we show that Bcl-2 overexpression in these cells inhibited apoptosis in culture and also conferred resistance to apoptosis in vivo in the lungs 24-48 h after injection. This inhibition of apoptosis led to significantly more macroscopic metastases. Third, comparison between the extent of apoptosis by a poorly metastatic cell line to that by a highly metastatic cell line 24 h after injection in the lungs revealed more apoptosis by the poorly metastatic cell line. These results indicate that apoptosis, which occurs at 24-48 h after hematogenous dissemination in the lungs is an important determinant of metastatic inefficiency. Although prior work has shown an association between apoptosis in culture and metastasis in vivo, this work shows that apoptosis in vivo corresponds to decreased metastasis in vivo.
Asunto(s)
Apoptosis/fisiología , Neoplasias Pulmonares/secundario , Células Neoplásicas Circulantes/patología , Animales , Línea Celular Transformada , Transformación Celular Neoplásica/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Fibrosarcoma/secundario , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Melanoma Experimental/secundario , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Ratas , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patología , Sarcoma Experimental/secundarioRESUMEN
Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca(2+). In situ endothelial generation of NO and changes in intracellular Ca(2+) in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca(2+) probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca(2+) due to both influx of extracellular Ca(2+) and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca(2+)-, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.
Asunto(s)
Calcio/metabolismo , Endotelio Vascular/metabolismo , Isquemia/metabolismo , Pulmón/irrigación sanguínea , Óxido Nítrico/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Endotelio Vascular/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The hypothesis that NAD(P)H oxidase may serve as an oxygen sensor was tested using the mice deficient (knock-out) in gp91phox subunit of NAD(P)H oxidase enzyme complex and compared with wild-type (C57BL/6J) strain measuring the ventilatory and glomus cell intracellular calcium ([Ca(2+)](i)) responses of carotid body to hypoxia. The hypoxic ventilatory responses as well as the [Ca(2+)](i) were preserved in the NAD(P)H oxidase knock-out mice. NAD(P)H oxidase, though a major source of oxygen radical production, is not the oxygen sensor in mice carotid body.
Asunto(s)
Calcio/metabolismo , Cuerpo Carotídeo/metabolismo , Enfermedad Granulomatosa Crónica/metabolismo , Hipoxia/metabolismo , Glicoproteínas de Membrana/deficiencia , NADPH Oxidasas/genética , Animales , Cuerpo Carotídeo/citología , Enfermedad Granulomatosa Crónica/genética , Líquido Intracelular/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , Consumo de Oxígeno/fisiología , Presión Parcial , Ventilación Pulmonar/fisiología , Volumen de Ventilación Pulmonar/fisiologíaRESUMEN
We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Vasoconstricción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Aorta Torácica/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Calcio/metabolismo , Cartilla de ADN/genética , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
This study evaluated the roles of endothelial cell membrane potential and reactive oxygen species (ROS) in the increase of tissue free iron during lung ischemia. Oxygenated ischemia was produced in the isolated rat lung by discontinuing perfusion while ventilation with O2 was maintained. We have shown previously that tissue oxygenation is maintained in this model of ischemia and that biochemical changes are the result of an abrupt reduction in endothelial shear stress. With 1 hr oxygenated ischemia, generation of ROS, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 8.0-fold, lung thiobarbituric acid reactive substances (TBARS) increased 3.4-fold, and lung protein carbonyl content increased 2.4-fold. Lung tissue free iron, measured in the lung homogenate with a fluorescent desferrioxamine derivative, increased 4.0-fold during ischemia. Pretreatment of lungs with thapsigargin abolished the increase in free iron with ischemia indicating that this effect is dependent on Ca2+ release from intracellular stores. Perfusion of lungs with high (25 mM) K+ to depolarize the endothelium also led to a significant increase in tissue free iron. Pretreatment of lungs with 35 microM cromakalim, a K+-channel agonist, significantly inhibited both ischemia-induced tissue oxidant injury and the increase in free iron with ischemia or with high K+ perfusion. A similar increase in free iron was observed when lungs were ventilated with either O2 or N2 during the ischemic period or were pre-perfused with an inhibitor of ROS production (diphenyleneiodonium). These results indicate that ROS generation is not required for ischemia-mediated iron release. Thus, ROS generation and iron release with ischemia are independent although both are subsequent to endothelial cell membrane depolarization.
Asunto(s)
Endotelio Vascular/metabolismo , Hierro/metabolismo , Pulmón/metabolismo , Estrés Oxidativo , Animales , Broncodilatadores/farmacología , Calcio/metabolismo , Carbono/metabolismo , Cromakalim/farmacología , Inhibidores Enzimáticos/farmacología , Isquemia , Peroxidación de Lípido , Masculino , Microscopía Fluorescente , Nitrógeno/farmacología , Oxígeno/metabolismo , Oxígeno/farmacología , Perfusión , Potasio/farmacología , Bloqueadores de los Canales de Potasio , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Tapsigargina/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factores de TiempoRESUMEN
Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.
Asunto(s)
Endotelio Vascular/patología , Fibrosarcoma/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia , Animales , Adhesión Celular , Femenino , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/fisiopatología , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/fisiopatología , Ratones , Ratones Desnudos , Microcirculación/patología , Circulación Pulmonar , Ratas , Ratas Sprague-Dawley , Transfección , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the "ischemic" period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-kappaB-specific or AP-1-specific (32)P-labeled oligonucleotides, and (3)H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm(2) shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-kappaB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with N-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of (3)H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-kappaB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling.
Asunto(s)
Endotelio Vascular/metabolismo , Isquemia/fisiopatología , Arteria Pulmonar/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Animales , Bovinos , División Celular , Células Cultivadas , ADN/biosíntesis , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Isquemia/metabolismo , Isquemia/patología , FN-kappa B/metabolismo , Perfusión , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Estrés Mecánico , Timidina/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
We propose that generation of reactive oxygen species (ROS) during ischemia is associated with an increase in intracellular calcium ([Ca2+]i) in pulmonary capillary endothelial cells. We used an isolated rat lung model and epifluorescence microscopy to evaluate [Ca2+]i in subpleural microvascular endothelial cells in situ by ratio imaging of the fluorophores, Calcium Green and Fura Red (CG/FR). Lungs were ventilated continuously under control (continuously perfused) or global ischemia (no perfusion) and thus remained adequately oxygenated even with ischemia. Ischemia for 5 min led to increase in CG/FR, indicating increase in [Ca2+]i in endothelial cells in situ; CG/FR remained elevated during a subsequent 10 min of ischemia. Ca(2+)-free perfusion and gadolinium (100 microM) inhibited the increase in [Ca2+]i, while thapsigargin (250 nM) had no effect. These results indicate that increase in endothelial cell [Ca2+]i with ischemia was due to influx from the extracellular medium. Perfusion with N-acetyl-L-cysteine (20 mM) or diphenyleneiodonium chloride (10 microM) prevented the ischemia-mediated [Ca2+]i increase, suggesting a role for ROS in the Ca2+ changes with ischemia. Membrane depolarization by perfusion with high potassium (K+) or glyburide also resulted in increased [Ca2+]i whereas the K(+)-channel agonist cromakalim, inhibited ischemia-mediated Ca2+ influx. We conclude that increased ROS generation with 'oxygenated' lung ischemia is associated with influx of Ca2+ and an increase in endothelial cell cytosolic calcium concentration.
Asunto(s)
Calcio/metabolismo , Endotelio Vascular/metabolismo , Líquido Intracelular/metabolismo , Isquemia/metabolismo , Pulmón/irrigación sanguínea , Pleura/irrigación sanguínea , Animales , Endotelio Vascular/fisiopatología , Técnicas In Vitro , Pulmón/metabolismo , Pulmón/fisiopatología , Masculino , Microcirculación/metabolismo , Microcirculación/fisiopatología , Microscopía Fluorescente , Perfusión , Pleura/metabolismo , Pleura/fisiopatología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoAsunto(s)
Lesión Pulmonar , Pulmón/metabolismo , Daño por Reperfusión/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Mediadores de Inflamación/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/fisiología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/etiologíaRESUMEN
We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.
Asunto(s)
Endotelio Vascular/enzimología , Isquemia/metabolismo , Pulmón/efectos de los fármacos , NADPH Oxidasas/metabolismo , Potasio/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Técnicas In Vitro , Pulmón/irrigación sanguínea , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Oxidantes/metabolismo , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
We hypothesize that lung ischemic injury is related to cessation of flow leading to endothelial cell membrane depolarization and activation of oxidant-generating systems. Cell membrane potential was assessed in isolated, oxygen ventilated, Krebs-Ringer bicarbonate buffer-dextran-perfused rat lungs by lung surface fluorescence after infusion of bis-oxonol or 5,5',6,6'-tetrachloro-1, 1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), voltage-sensitive dyes. Surface fluorometry showed increased bis-oxonol fluorescence (34.7 +/- 3.3% above baseline) and decreased JC-1 fluorescence (24.5 +/- 4.5% below baseline) with ischemia, compatible with membrane depolarization. Fluorescence change was initiated within 1-2 min of the onset of ischemia and was rapidly reversible with reperfusion. Fluorescence changes varied with perfusion flow rate; maximal increase occurred with the transition from 1.8 ml/min to zero flow. Elevation of static intravascular pressure resulted in only a minor increase of bis-oxonol fluorescence. In situ subpleural fluorescence microscopy showed that endothelial cells are the major site of the increased bis-oxonol fluorescence signal with ischemia. These results indicate that endothelial cell membrane depolarization represents an early event with lung ischemia. Since the adenosine triphosphate content of lung was unchanged with ischemia in the O2-ventilated lungs, we postulate that membrane depolarization results from elimination of shear stress, possibly via inactivation of flow-sensitive K+-channels.
Asunto(s)
Isquemia/fisiopatología , Pulmón/irrigación sanguínea , Pulmón/fisiología , Oxígeno/farmacología , Adenosina Trifosfato/fisiología , Animales , Compuestos de Bario/farmacología , Capilares/química , Capilares/citología , Capilares/fisiología , Cloruros/farmacología , Endotelio Vascular/química , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Colorantes Fluorescentes , Pulmón/efectos de los fármacos , Masculino , Potenciales de la Membrana/fisiología , Potasio/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio/agonistas , Canales de Potasio/fisiología , Circulación Pulmonar , Ratas , Ratas Sprague-Dawley , Tetraetilamonio/farmacología , TiobarbitúricosRESUMEN
Normoxic lung ischemia/reperfusion (I/R) leads to oxidative injury of the pulmonary tissue. We analyzed angiotensin-converting enzyme (ACE) in perfused rat lungs upon I/R in order to assess the endothelial injury produced. I/R led to a time-dependent increase in ACE activity in the perfusate, from 145+/-14 mU to 252+/-1 mU, and to reduction of ACE activity in the lung tissue homogenate, from 29.7+/-2.3 U to 22.7+/-1.7 U. About 80% of ACE activity in control and I/R rat lungs was associated with an aqueous phase of extracted perfusates, thus indicating that I/R accelerates shedding of the hydrophilic form of ACE from the plasma membrane. To specifically assess ACE localized on the luminal surface of the pulmonary endothelium, we perfused rat lungs with a radiolabeled monoclonal antibody (mAb) to ACE (anti-ACE mAb 9B9). Pulmonary uptake of mAb 9B9 with I/R was reduced from 32.1+/-1.7% to 24.8+/-0.9%. In contrast, I/R led to a marked increase in the pulmonary uptake of nonspecific [125I]IgG, from 0.17+/-0.02% to 0.67+/-0.04%. Lung wet weight was equal to 0.78+/-0.08% of body weight in the I/R group versus 0.57+/-0.02% at the control level. The observed increase in [125I]IgG uptake and wet lung weight indicate that I/R causes an increase in lung vascular permeability. These results indicate that normoxic lung I/R induces injury to the pulmonary vascular endothelium.
Asunto(s)
Endotelio Vascular/enzimología , Pulmón/irrigación sanguínea , Peptidil-Dipeptidasa A/metabolismo , Arteria Pulmonar/enzimología , Daño por Reperfusión/enzimología , Animales , Anticuerpos Monoclonales , Permeabilidad Capilar , Membrana Celular/enzimología , Modelos Animales de Enfermedad , Técnicas In Vitro , Pulmón/citología , Masculino , Ratones , Oxígeno/sangre , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Respiración ArtificialRESUMEN
Surface fluorometry with 40 microM hydroethidine (HE) as a probe was used to detect oxidant generation in isolated, ventilated rat lungs during lung ischemia. Ethidium fluorescence due to HE oxidation was continuously monitored with 470 nm excitation and 610 nm emission. Fluorescence increased with ischemia in O2-ventilated lungs [0.98 +/- 0.08 arbitrary fluorescence units (AFU)/min vs. 0.58 +/- 0.07 with control perfusion]. HE oxidation during ischemia was prevented by N2 ventilation but was unaltered by preperfusion with superoxide dismutase. Ethidium fluorescence in homogenate prepared from lungs subjected to 1 h of nonhypoxic ischemia was increased (16.8 +/- 1.5 vs. 9.8 +/- 0.4 AFU/mg protein in control) but was unchanged in lungs that had been N2 ventilated. Microfluorographs of HE perfused and fixed lung sections demonstrated marked generalized increases in ethidium fluorescence with ischemia compared with control perfusion. Ischemia resulted in significant increases in tissue thiobarbituric acid reactive substance (176 +/- 13 vs. 44 +/- 3 pmol/mg protein for control) and in lung conjugated dienes (0.90 +/- 0.07 vs. 0.48 +/- 0.06 U/mg protein for control), indicating peroxidation of lung lipids. These results indicate that lung ischemia leads to intracellular oxidant generation that can be continuously monitored by surface fluorometry.
Asunto(s)
Membranas Intracelulares/metabolismo , Isquemia/metabolismo , Circulación Pulmonar , Especies Reactivas de Oxígeno/metabolismo , Animales , Fluorometría , Técnicas In Vitro , Peróxidos Lipídicos/metabolismo , Masculino , Fenantridinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
This study evaluated whether cell membrane depolarization can induce oxidant generation in the isolated perfused rat lung as has been demonstrated with bovine pulmonary artery endothelial cells. Depolarization was produced by perfusing the lungs with high [K+] or with glyburide and was evaluated with bis-oxonol lung surface fluorometry. Lung surface bis-oxonol fluorescence increased above baseline (at 5.9 mM K+) by 18.5% with 24 mM K+, 35% with 48 mM K+, and 67% with 96 mM K+, indicating graded membrane depolarization, and by 75% during perfusion with 10 microM glyburide. Oxidant generation was evaluated with hydroethidine lung surface fluorometry, and with assay of tissue thiobarbituric acid reactive substance (TBARS), conjugated dienes, and perfusate H2O2. Depolarization by high K+ or glyburide led to significant increases in generation of tissue oxidants and lipid peroxidation. Bodipy-FL-glyburide microfluorography showed localization of glyburide binding primarily to vascular endothelial cells vascular and airway smooth muscle cells, alveolar type II cells, and to nonciliated cells of the airway epithelium. These results indicate that cellular depolarization is associated with oxidant generation by the lung and suggests a role for K(+)-channels in these events.
Asunto(s)
Pulmón/metabolismo , Oxidantes/metabolismo , Potasio/farmacología , Animales , Colorantes Fluorescentes/metabolismo , Fluorometría , Gliburida/metabolismo , Gliburida/farmacología , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Peroxidación de Lípido , Pulmón/citología , Pulmón/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Microscopía Confocal , Perfusión , Fenantridinas/metabolismo , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Tiobarbitúricos/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
Oxidant generation in anoxia-reoxygenation and ischemia-reperfusion was compared in isolated rat lungs. Anoxia-reoxygenation was produced by N2 ventilation followed by O2 ventilation. After anoxia, lung ATP content was decreased by 59%. Oxygenated ischemia was produced by discontinuing perfusion while ventilation with O2 was maintained. With anoxia-reoxygenation, oxidant generation, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 3.6-fold, lung thiobarbituric acid reactive substances (TBARS) increased 342%, conjugated dienes increased 285%, and protein carbonyl content increased 46%. Pretreatment of lungs with 100 microM allopurinol inhibited the reoxygenation-mediated increase in lung fluorescence by 75% and TBARS by 69%. Oxygenated ischemia resulted in an approximately eightfold increase in lung H2DCF oxidation and a fourfold increase in TBARS, but allopurinol had no effect. On the other hand, 100 microM diphenyliodonium (DPI) inhibited the ischemia-mediated increase in lung fluorescence by 69% and lung TBARS by 70%, but it had no effect on the increase with anoxia-reoxygenation. Therefore, both ischemia-reperfusion and anoxia-reoxygenation result in oxidant generation by the lung, but a comparison of results with a xanthine oxidase inhibitor (allopurinol) and a flavoprotein inhibitor (DPI) indicate that the pathways for oxidant generation are distinctly different.