RESUMEN
Asthma is characterized by oxidative stress and inflammation of the airways. Although proinflammatory lipids are involved in asthma, therapies targeting them remain lacking. Ac-DWFKAFYDKVAEKFKEAFNH(2) (4F) is an apolipoprotein (apo)A-I mimetic that has been shown to preferentially bind oxidized lipids and improve HDL function. The objective of the present study was to determine the effects of 4F on oxidative stress, inflammation, and airway resistance in an established murine model of asthma. We show here that ovalbumin (OVA)-sensitization increased airway hyperresponsiveness, eosinophil recruitment, and collagen deposition in lungs of C57BL/6J mice by a mechanism that could be reduced by 4F. OVA sensitization induced marked increases in transforming growth factor (TGF)ß-1, fibroblast specific protein (FSP)-1, anti-T15 autoantibody staining, and modest increases in 4-hydroxynonenal (4-HNE) Michael's adducts in lungs of OVA-sensitized mice. 4F decreased TGFß-1, FSP-1, anti-T15 autoantibody, and 4-HNE adducts in the lungs of the OVA-sensitized mice. Eosinophil peroxidase (EPO) activity in bronchial alveolar lavage fluid (BALF), peripheral eosinophil counts, total IgE, and proinflammatory HDL (p-HDL) were all increased in OVA-sensitized mice. 4F decreased BALF EPO activity, eosinophil counts, total IgE, and p-HDL in these mice. These data indicate that 4F reduces pulmonary inflammation and airway resistance in an experimental murine model of asthma by decreasing oxidative stress.
Asunto(s)
Apolipoproteína A-I , Asma/tratamiento farmacológico , Hipersensibilidad/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Péptidos/farmacología , Neumonía/tratamiento farmacológico , Sistema Respiratorio/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Asma/sangre , Asma/inmunología , Asma/metabolismo , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Materiales Biomiméticos/uso terapéutico , Recuento de Células , HDL-Colesterol/metabolismo , Colágeno/metabolismo , Modelos Animales de Enfermedad , Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Hipersensibilidad/sangre , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Inmunoglobulina E/sangre , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/química , Péptidos/uso terapéutico , Neumonía/sangre , Neumonía/inmunología , Neumonía/metabolismo , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismoRESUMEN
Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/biosíntesis , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Infecciones por Adenoviridae/enzimología , Animales , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Western Blotting , Bovinos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , ReologíaRESUMEN
BACKGROUND/PURPOSE: The molecular and cellular events that regulate inflammatory lung injury, a major cause of morbidity in surgical patients, remain unclear. The authors hypothesize that nitric oxide (NO) plays an important role in regulating polymorphonuclear cell (PMN)-induced acute lung injury, and further, that attenuated expression of inducible nitric oxide synthase (iNOS) and therefore decreased production of NO by lung microvascular endothelial cells (LMVEC), accelerates inflammation and injury. METHODS: LMVEC and aortic EC (AEC) from rat and human were stimulated with lipopolysaccharide (LPS) and cytokines; changes in iNOS mRNA expression and iNOS activity were determined. The role of NO in mediating inflammatory responses was evaluated by determining PMN adherence to LMVEC and lung tissue slices in the presence and absence of NOS inhibitors and NO donors. Human LMVEC and AEC were assessed by FACS analysis for ICAM-1 expression, because this is thought to be a critical determinant of PMN adherence. RESULTS: When stimulated with endotoxin and cytokines, rat AEC monolayers express nearly 3-fold more iNOS mRNA than rat LMVEC. The low levels of LMVEC iNOS expression are associated with a 4-fold lower nitrite and nitrate production. Similar trends are seen in human endothelial cells. When iNOS activity was blocked, PMN adherence to tumor necrosis factor alpha (TNFalpha)/LPS-stimulated LMVEC was markedly increased. In contrast, adding a nitric oxide donor to endotoxin/cytokine-stimulated LMVEC monolayers reduced PMN adherence to near background levels. Similar responses were observed in vivo. Human lung microvascular endothelial cells show a substantially increased level of ICAM-1 upregulation when compared with similarly stimulated human aortic macrovascular endothelial cells. CONCLUSIONS: These data indicate that LMVEC express less iNOS and produce less NO than AEC. This lower expression and activity of iNOS in LMVEC may be linked to increased expression of ICAM-1. Because ICAM-1 has been shown to be essential for tight PMN adherence, these data suggest that relatively low iNOS expression in LMVEC may contribute to a propensity for the lung to be injured by activated PMNs.
Asunto(s)
Endotelio Vascular/enzimología , Molécula 1 de Adhesión Intercelular/análisis , Pulmón/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Northern Blotting , Células Cultivadas , Citocinas/farmacología , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Citometría de Flujo , Humanos , Lipopolisacáridos/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
The balance of nitric oxide (.NO) and superoxide anion (O(2)) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates.NO. As eNOS is capable of generating both.NO and O(2), we hypothesized that Hsp90 might also mediate eNOS-dependent O(2) production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 microg/ml; 17.8 microm) and then stimulated with the calcium ionophore, (5 microm). GA significantly decreased -stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased -stimulated eNOS-dependent O(2) production (p < 0.001, n = 8). increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent.NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O(2) production.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Calcimicina/farmacología , Bovinos , Células Cultivadas , Ionóforos/farmacología , Óxido Nítrico Sintasa de Tipo IIIRESUMEN
Nitric oxide synthase (NOS) is believed to play an important role in protecting the myocardium against ischemia. Chronic hypoxia from birth increases NOS activity in the myocardium resulting in enhanced nitric oxide production and increased resistance to ischemia. We examined the effects of chronic hypoxia on NOS gene and protein expression and on NOS protein association with caveolin-3. Rabbits were raised from birth in a normoxic (F(I)O(2) = 0.21) or a hypoxic (F(I)O(2) = 0.12) environment for 9 d, and then the hearts were isolated. Ribonuclease protection assays revealed that chronic hypoxia did not alter NOS transcript levels for NOS1, NOS2, or NOS3. The most abundant transcript was NOS3. Western analysis revealed NOS3 was the only isoform detected. Immunoblots of NOS3 immunoprecipitates showed that chronic hypoxia increases NOS3 protein by 2.0 +/- 0.4-fold and decreases the amount of caveolin-3 that can be coprecipitated with NOS3 by 5.5 +/- 0.9-fold. Immunoblots of normoxic and hypoxic hearts showed that chronic hypoxia decreases the amount of caveolin-3 in heart homogenates by 2. 2 +/- 0.5-fold. These data suggest that a decrease in caveolin-3 plays a role in the mechanisms by which chronic hypoxia increases NOS3 activity in the myocardium.
Asunto(s)
Caveolinas/metabolismo , Hipoxia/metabolismo , Miocardio/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Caveolina 3 , Enfermedad Crónica , Cartilla de ADN/genética , Hipoxia/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevención & control , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , ARN Mensajero/genética , ARN Mensajero/metabolismo , ConejosRESUMEN
Occupational, airborne pollutants, such as heavy metals, are recognized for inducing injury and cytotoxicity. Chromium(VI) is a redox cycling heavy metal that has been strongly implicated in the initiation of cancer. Its proinflammatory effects, however, have not been systematically examined. In our study, we found that potassium dichromate [Cr(VI)] treatment of human umbilical vein endothelial cells (HUVEC) increased intracellular adhesion molecule (ICAM) expression at the message level. ICAM message levels remained elevated for 12-24 hours after exposure and increased with time and concentration. Cr(VI) increased the release of superoxide anion without affecting the ability of endothelial cultures to produce nitric oxide. However, Cr(VI) decreased cGMP in HUVEC, suggesting that the nitric oxide produced was scavenged intracellularly. Cr(VI) also increased nitrotyrosine in HUVEC cultures. These data are consistent with the idea that exposure to Cr(VI) increases the production of superoxide anion, which scavenges nitric oxide to increase the formation of peroxynitrite. The loss in nitric oxide activity and increased formation of peroxynitrite likely enhance endothelial cell expression of ICAM-1. Cr(VI)-induced increases in the adhesive properties of the endothelium may play a critical role in the initiation and progression of tissue injury through increased recruitment of proinflammatory white blood cells.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Óxido Nítrico/biosíntesis , Dicromato de Potasio/toxicidad , Tirosina/análogos & derivados , Células Cultivadas , Endotelio Vascular/metabolismo , Radicales Libres/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Superóxidos/metabolismo , Tirosina/biosíntesisRESUMEN
Adrenal zona glomerulosa (ZG) cells do not contain nitric oxide (NO) synthase (NOS). We conferred endothelial NOS activity onto adrenal ZG cells through transduction with a recombinant adenovirus encoding the endothelial NOS gene (AdeNOS) to determine the effect of endogenous NO on aldosterone synthesis. A 135-kDa protein band immunoreactive to anti-endothelial NOS antibody was observed in Western blots of AdeNOS-transduced ZG cells but not in control cells or cells transduced with adenovirus encoding the beta-galactosidase gene (AdbetaGal). Nitrate/nitrite production in AdeNOS-transduced ZG cells increased from 0.15+/-0.01 to 0.27+/-0.01 micromol/L after stimulation with 1 nmol/L angiotensin II. The treatment of AdeNOS-transduced cells with 30 micromol/L L-nitro-arginine decreased angiotensin II-stimulated nitrite production from 0.27+/-0. 01 to 0.17+/-0.01 micromol/L. Basal and angiotensin II-stimulated nitrite production was not increased in AdbetaGal-transduced or control cells. AdeNOS-transduced cells demonstrated diaminofluorescein-2 diacetate fluorescence, which was blocked by pretreatment with L-nitro-arginine. Angiotensin II-stimulated aldosterone synthesis decreased from 5123+/-177 pg/mL in AdbetaGal-transduced ZG cells to 72+/-27 pg/mL in AdeNOS-transduced cells. Treatment with the NOS inhibitor thiocitrulline (30 micromol/L) increased angiotensin II-stimulated aldosterone synthesis to 2158+/-45 pg/mL after AdeNOS transduction. These data demonstrate that adenovirus-mediated gene transfer of eNOS in ZG cells results in the expression of active endothelial NOS enzyme and that this endogenous NO production by ZG cells decreases aldosterone synthesis.
Asunto(s)
Aldosterona/biosíntesis , Óxido Nítrico/metabolismo , Zona Glomerular/citología , Zona Glomerular/enzimología , Adenoviridae/genética , Infecciones por Adenoviridae/metabolismo , Animales , Western Blotting , Bovinos , Células Cultivadas , Fluoresceínas , Regulación Enzimológica de la Expresión Génica , Técnicas de Transferencia de Gen , Nitratos/análisis , Nitratos/metabolismo , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Nitritos/análisis , Nitritos/metabolismo , Zona Glomerular/virologíaRESUMEN
The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.
Asunto(s)
Biopterinas/análogos & derivados , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Superóxidos/metabolismo , Animales , Arginina/metabolismo , Biopterinas/metabolismo , Bovinos , Citrulina/biosíntesis , Espectroscopía de Resonancia por Spin del Electrón , Modelos Químicos , NADP/metabolismo , Óxido Nítrico Sintasa de Tipo I , Oxígeno/metabolismoRESUMEN
Chronic exposure to hypoxia from birth increased the tolerance of the rabbit heart to subsequent ischemia compared with age-matched normoxic controls. The nitric oxide donor GSNO increased recovery of post-ischemic function in normoxic hearts to values not different from hypoxic controls, but had no effect on hypoxic hearts. The nitric oxide synthase inhibitors L-NAME and L-NMA abolished the cardioprotective effect of hypoxia. Message and catalytic activity for constitutive nitric oxide synthase as well as nitrite, nitrate, and cGMP levels were elevated in hypoxic hearts. Inducible nitric oxide synthase was not detected in normoxic or chronically hypoxic hearts. Increased tolerance to ischemia in rabbit hearts adapted to chronic hypoxia is associated with increased expression of constitutive nitric oxide synthase.
Asunto(s)
Adaptación Fisiológica/fisiología , Hipoxia/fisiopatología , Isquemia Miocárdica/fisiopatología , Óxido Nítrico/biosíntesis , Animales , Enfermedad Crónica , GMP Cíclico/metabolismo , Hipoxia/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Nitritos/metabolismo , ARN Mensajero/metabolismo , ConejosRESUMEN
NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EAred). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.
Asunto(s)
Óxido Nítrico Sintasa/análisis , Superóxidos/análisis , Acridinas , Animales , Espectroscopía de Resonancia por Spin del Electrón/métodos , Radicales Libres , Humanos , Óxido Nítrico Sintasa/química , Detección de Spin , Superóxidos/químicaRESUMEN
In this article, we address the mechanism of superoxide formation from constitutive nitric oxide synthases (NOS). Merits and drawbacks of the various superoxide detection assays are reviewed. One of the most viable techniques for measuring superoxide from NOS is electron spin resonance (ESR) spin-trapping using a novel phosphorylated spin trap. Implications of superoxide and peroxynitrite formation from NOS enzymes in cardiovascular and cerebrovascular disorders are discussed.
Asunto(s)
Doxorrubicina/farmacología , Espectroscopía de Resonancia por Spin del Electrón/métodos , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Superóxidos/metabolismo , Animales , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Humanos , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa de Tipo II , Oxidación-Reducción , Marcadores de Spin , Superóxidos/análisis , Superóxidos/químicaRESUMEN
The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.
Asunto(s)
NADP/metabolismo , Óxido Nítrico Sintasa/metabolismo , Superóxidos/metabolismo , Animales , Arginina/farmacología , Biopterinas/análogos & derivados , Biopterinas/farmacología , Calcio/metabolismo , Calmodulina/metabolismo , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III , Marcadores de SpinAsunto(s)
Corazón/fisiopatología , Hipoxia/fisiopatología , Isquemia Miocárdica/fisiopatología , Reperfusión Miocárdica , Miocardio/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico/metabolismo , Choque Séptico/fisiopatología , Envejecimiento , Animales , GMP Cíclico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Corazón/fisiología , Técnicas In Vitro , Nitratos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Nitritos/metabolismo , Conejos , Choque Séptico/metabolismo , Estrés Fisiológico/fisiopatología , Transcripción Genética , Función Ventricular Izquierda/fisiología , Función Ventricular Derecha/fisiologíaRESUMEN
Adriamycin (or doxorubicin) is an active and broad spectrum chemotherapeutic agent. Unfortunately, its clinical use is severely restricted by a dose-limiting cardiotoxicity which has been linked to the formation of superoxide. Enzymatic one-electron reduction of adriamycin forms adriamycin semiquinone radical, which rapidly reacts with oxygen to form superoxide and adriamycin. In this way, adriamycin provides a kinetic mechanism for the one-electron reduction of oxygen by flavoenzymes such as NADPH-cytochrome P450 reductase and mitochondrial NADH dehydrogenase. We demonstrate here that the endothelial isoform of nitric oxide synthase (eNOS) reduces adriamycin to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. Adriamycin binds to eNOS with a Km of approximately 5 microM, as calculated from both eNOS-dependent NADPH consumption and superoxide generation. Adriamycin stimulated superoxide formation is not affected by calcium/calmodulin and is abolished by the flavoenzyme inhibitor, diphenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. A consequence of eNOS-mediated reductive activation of adriamycin is the disruption of the balance between nitric oxide and superoxide. This may lead eNOS to generate peroxynitrite and hydrogen peroxide, potent oxidants implicated in several vascular pathologies.
Asunto(s)
Doxorrubicina/metabolismo , Endotelio Vascular/enzimología , Isoenzimas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Superóxidos/metabolismo , Doxorrubicina/toxicidad , Espectroscopía de Resonancia por Spin del Electrón , Flavoproteínas/metabolismo , Isoenzimas/antagonistas & inhibidores , Modelos Químicos , NADP/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Oxidación-ReducciónRESUMEN
Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.
Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Estrés Mecánico , Acetilcolina/farmacología , Animales , Aorta , Fenómenos Biomecánicos , Bovinos , Células Cultivadas , Constricción Patológica/enzimología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/enzimología , Regulación de la Expresión Génica , Masculino , Óxido Nítrico Sintasa/metabolismo , Norepinefrina/farmacología , Regiones Promotoras Genéticas , Arteria Pulmonar/enzimología , Conejos , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , TransfecciónRESUMEN
Lucigenin (LC2+) is frequently used as a superoxide probe. To detect superoxide, lucigenin must be reduced to the lucigenin cation radical (LC.+). We show, using the phosphorylated spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), that lucigenin stimulates NADPH-dependent superoxide production by endothelial nitric oxide synthase (eNOS). The formation of the DEPMPO-superoxide adduct is calcium/calmodulin independent. DEPMPO-superoxide adduct formation is inhibited by diphenyleneiodonium and is abolished by superoxide dismutase. It is likely that eNOS/NADPH can reduce lucigenin to LC.+ which reduces oxygen to superoxide. Consequently, lucigenin cannot be used to measure superoxide formation.
Asunto(s)
Acridinas/metabolismo , Superóxidos/metabolismo , Acridinas/química , Animales , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Sondas Moleculares , NADP/metabolismo , Óxido Nítrico Sintasa/aislamiento & purificación , Óxido Nítrico Sintasa/metabolismo , Oxidación-Reducción , Superóxidos/químicaRESUMEN
A variety of cell types, including endothelial cells, oxidize low-density lipoprotein (LDL). To investigate the mechanisms by which endothelial cells modulate LDL oxidation states, endothelial cell cultures were incubated with LDL (240 mg cholesterol/dL) for 24 hours in M199 supplemented with fetal bovine serum (FBS, 16.7%). These conditions were not toxic to endothelial cells over the time frame of the study. Changes in LDL oxidation were monitored by measuring thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxide (LOOH), and conjugated dienes (A234nm). LDL medium incubated in the absence of endothelial cells contained higher TBARS than did LDL medium incubated with endothelial cells (0.35 +/- 0.08 versus 0.23 +/- 0.08 nmol MDA/mg, respectively). LOOHs were higher in LDL medium incubated without endothelial cells than in LDL medium incubated with endothelial cells (6.8 +/- 4.4 versus 0.49 +/- 0.89 nmol/mg, respectively). Conjugated diene formation, based on changes in absorbance at 234 nm, increased to a greater extent in LDL medium incubated in the absence of endothelial cells than when endothelial cells were present. To increase oxidative stress on the endothelial cell cultures, increasing concentrations of Cu2+ (0 to 4 mumol/L) were added to LDL medium. Endothelial cells prevented LOOH accumulation until the concentration of Cu2+ exceeded 0.75 mumol/L. At 1.5 and 4 mumol/L Cu2+, endothelial cells enhanced LOOH formation nearly 3 and 2.5 times the LOOH values in the corresponding medium incubated in the absence of endothelial cells. This loss of protective function however, was not permanent. Endothelial cells, preincubated for 24 hours with Cu(2+)-containing LDL medium, were still able to prevent LOOH accumulation in fresh LDL medium. Endothelial cells prevented LOOH accumulation even when exposed to LDL medium that contained low concentrations of LOOHs (< 22 nmol/mg). However, endothelial cells accelerated the accumulation of LOOHs in LDL when exposed to LDL medium that contained slightly higher concentrations of preexisting LOOHs (approximately equal to 33 nmol/mg). These data indicate that endothelial cells have a limited capacity for preventing LOOH formation and that small increases in LOOHs may play a critical role in enhancing the potential of endothelial cells for oxidative modification of LDL.
Asunto(s)
Endotelio Vascular/metabolismo , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/metabolismo , Antioxidantes/farmacología , Hidroxitolueno Butilado/farmacología , Células Cultivadas , Cobre/química , Humanos , Oxidación-ReducciónRESUMEN
Native LDL (n-LDL) increased human umbilical vein endothelial cell (EC) adherence of mononuclear cells. Such phenotypic changes suggest that n-LDL alters the usual expression of cell adhesion molecules to enhance the adhesive properties of the endothelium. To investigate n-LDL mechanisms governing adherence, ECs were exposed to n-LDL in concentrations up to 240 mg/dL for 2 and 4 days. n-LDL-treated ECs bound nearly threefold more phorbol myristate acetate (PMA)-stimulated U937 cells than control ECs but did not bind unstimulated U937 cells. Anti-cellular adhesion molecule-1 (ICAM-1) antibodies blocked PMA-stimulated U937 cell binding to control and n-LDL-treated ECs by more than 80%, suggesting that increases in ICAM-1 may be involved in this increased adherence. Although increases in PMA-stimulated U937 cell binding developed with respect to time and concentration, statistically significant increases were achieved only when n-LDL concentrations exceeded 180 mg cholesterol/dL at day 4. n-LDL increased endothelial adherence of freshly isolated human monocytes more than twofold and neutrophils by almost twofold. Fluorescent-linked immunoassays revealed that n-LDL increased ICAM-1 protein expression by twofold, which corresponded with increased ICAM-1 message levels. n-LDL also appeared to increase E-selectin and vascular cell adhesion molecule-1 message levels, but these changes did not translate into statistically significant differences in protein levels. Taken together, these data indicate that n-LDL increases ICAM-1 expression to enhance the adhesive properties of the endothelium. Such perturbations in EC function likely represent a proinflammatory response to protracted n-LDL exposure and one of the early steps in atherogenesis.
Asunto(s)
Endotelio Vascular/fisiología , Molécula 1 de Adhesión Intercelular/metabolismo , Lipoproteínas LDL/fisiología , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Células Cultivadas/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Lipoproteínas LDL/farmacología , Sondas Moleculares/genética , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O2-) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O2- by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [3H]citrulline assay. N omega-Nitro-L-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O2- by > 300% but decreases LDL-treated EC (LDL-EC) release by > 95%. L-Arginine inhibits the release of O2- from LDL-ECs by > 95% but did not effect C-EC release of O2-. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O2- production by approximately 50% and 30%, respectively. Thus, n-LDL increases O2- and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO-), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO-, and tyrosine by approximately 50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aminoácido Oxidorreductasas/fisiología , Endotelio Vascular/efectos de los fármacos , Lipoproteínas LDL/farmacología , Superóxidos/metabolismo , Arginina/análogos & derivados , Arginina/farmacología , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , NG-Nitroarginina Metil Éster , Óxido Nítrico/fisiología , Óxido Nítrico SintasaRESUMEN
This study examines the effects of oxidized low density lipoprotein (ox-LDL) on mononuclear cell arachidonic acid (AA) metabolism. U937 and HL60 cells, employed as models for mononuclear cells and promyelocytic cells, respectively, were exposed to ox-LDL (20 micrograms protein/ml) for 24 hours. HPLC analysis of 1-14C-AA metabolites indicates that ox-LDL increases U937 and HL60 cell production of 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Northern analysis indicates that ox-LDL increases U937 cell FLAP transcript levels 10-times control levels but did not appear to alter 5-lipoxygenase (5-LO) mRNA levels. In contrast, ox-LDL increases HL60 cell transcript levels for FLAP and 5-LO 1.5 times and 10 times control levels, respectively. Thus, we propose that ox-LDL plays an important role in the up-regulation of the 5-LO pathway in mononuclear cells. Such activation may explain, in part, the mechanisms by which ox-LDL promotes atherogenesis.