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1.
Free Radic Biol Med ; 79: 69-77, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25452141

RESUMO

In isolated rat lung perfused with a physiological saline solution (5.5mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (Kf), effects that are overcome using an amphipathic quinone (CoQ1) [Free Radic. Biol. Med.65:1455-1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8nmol/mg protein, depolarized the mitochondrial membrane potential (Δψm) from -129.0±3.7 (mean±SEM) to -92.8±5.5mV, and decreased O2 consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC-dextran (~40kDa) continually over a 6 h time course. When CoQ1 was present with rotenone, normal ATP (17.4±1.4nmol/mg protein), O2 consumption (1.5±0.1nmol/min/mg protein), Δψm (-125.2±3.3mV), and permeability (PS) were maintained. Protective effects of CoQ1 on rotenone-induced changes in ATP, O2 consumption rate, Δψm, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol-mitochondria electron shuttle [Free Radic. Biol. Med.65:1455-1463; 2013]. Key rotenone effects without and with CoQ1 were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2mM.


Assuntos
Trifosfato de Adenosina/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Endotélio Vascular/efeitos dos fármacos , Pulmão/irrigação sanguínea , Ubiquinona/farmacologia , Animais , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Ratos
2.
Redox Biol ; 2: 884-91, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25180165

RESUMO

Mitochondrial dysfunction is a fundamental abnormality in the vascular endothelium and smooth muscle of patients with pulmonary arterial hypertension (PAH). Because coenzyme Q (CoQ) is essential for mitochondrial function and efficient oxygen utilization as the electron carrier in the inner mitochondrial membrane, we hypothesized that CoQ would improve mitochondrial function and benefit PAH patients. To test this, oxidized and reduced levels of CoQ, cardiac function by echocardiogram, mitochondrial functions of heme synthesis and cellular metabolism were evaluated in PAH patients (N=8) in comparison to healthy controls (N=7), at baseline and after 12 weeks oral CoQ supplementation. CoQ levels were similar among PAH and control individuals, and increased in all subjects with CoQ supplementation. PAH patients had higher CoQ levels than controls with supplementation, and a tendency to a higher reduced-to-oxidized CoQ ratio. Cardiac parameters improved with CoQ supplementation, although 6-minute walk distances and BNP levels did not significantly change. Consistent with improved mitochondrial synthetic function, hemoglobin increased and red cell distribution width (RDW) decreased in PAH patients with CoQ, while hemoglobin declined slightly and RDW did not change in healthy controls. In contrast, metabolic and redox parameters, including lactate, pyruvate and reduced or oxidized gluthathione, did not change in PAH patients with CoQ. In summary, CoQ improved hemoglobin and red cell maturation in PAH, but longer studies and/or higher doses with a randomized placebo-controlled controlled design are necessary to evaluate the clinical benefit of this simple nutritional supplement.


Assuntos
Suplementos Nutricionais , Hipertensão Pulmonar/dietoterapia , Ubiquinona/administração & dosagem , Ubiquinona/farmacologia , Administração Oral , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ubiquinona/metabolismo , Adulto Jovem
3.
Free Radic Biol Med ; 65: 1455-1463, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23912160

RESUMO

Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 µM). Compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 µmol · g(-1) dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 to 38.62 ± 3.14 µmol · 15 min(-1) perfusion · g(-1) dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone coenzyme Q1 (CoQ1; 50 µM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H-quinone oxidoreductase 1 or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 to 0.156 ± 0.037 ml · min(-1) · cm H2O(-1) · g(-1) dry lung, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate:pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low-molecular-weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency.


Assuntos
Complexo I de Transporte de Elétrons/antagonistas & inibidores , Pulmão/irrigação sanguínea , Mitocôndrias/metabolismo , Rotenona/farmacologia , Ubiquinona/farmacologia , Trifosfato de Adenosina/análise , Animais , Antibacterianos/farmacologia , Barreira Alveolocapilar/efeitos dos fármacos , Permeabilidade Capilar , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Endotélio Vascular/metabolismo , Metabolismo Energético , Glicólise , Ácido Láctico/análise , Lesão Pulmonar , Masculino , NAD(P)H Desidrogenase (Quinona)/farmacologia , Oxirredução/efeitos dos fármacos , Piridinas/farmacologia , Ácido Pirúvico/análise , Ratos , Ratos Sprague-Dawley , Reperfusão , Desacopladores/farmacologia
4.
Am J Physiol Lung Cell Mol Physiol ; 302(9): L949-58, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22268123

RESUMO

Previous studies showed that coenzyme Q(1) (CoQ(1)) reduction on passage through the rat pulmonary circulation was catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex I, but that NQO1 genotype was not a factor in CoQ(1) reduction on passage through the mouse lung. The aim of the present study was to evaluate the complex I contribution to CoQ(1) reduction in the isolated perfused wild-type (NQO1(+/+)) and Nqo1-null (NQO1(-)/(-)) mouse lung. CoQ(1) reduction was measured as the steady-state pulmonary venous CoQ(1) hydroquinone (CoQ(1)H(2)) efflux rate during infusion of CoQ(1) into the pulmonary arterial inflow. CoQ(1)H(2) efflux rates during infusion of 50 µM CoQ(1) were not significantly different for NQO1(+/+) and NQO1(-/-) lungs (0.80 ± 0.03 and 0.68 ± 0.07 µmol·min(-1)·g lung dry wt(-1), respectively, P > 0.05). The mitochondrial complex I inhibitor rotenone depressed CoQ(1)H(2) efflux rates for both genotypes (0.19 ± 0.08 and 0.08 ± 0.04 µmol·min(-1)·g lung dry wt(-1) for NQO1(+/+) and NQO1(-/-), respectively, P < 0.05). Exposure of mice to 100% O(2) for 48 h also depressed CoQ(1)H(2) efflux rates in NQO1(+/+) and NQO1(-/-) lungs (0.43 ± 0.03 and 0.11 ± 0.04 µmol·min(-1)·g lung dry wt(-1), respectively, P < 0.05 by ANOVA). The impact of rotenone or hyperoxia on CoQ(1) redox metabolism could not be attributed to effects on lung wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total venous effluent CoQ(1) recoveries, the latter measured by spectrophotometry or mass spectrometry. Complex I activity in mitochondria-enriched lung fractions was depressed in hyperoxia-exposed lungs for both genotypes. This study provides new evidence for the potential utility of CoQ(1) as a nondestructive indicator of the impact of pharmacological or pathological exposures on complex I activity in the intact perfused mouse lung.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Pulmão/enzimologia , Mitocôndrias/enzimologia , NAD(P)H Desidrogenase (Quinona)/genética , Ubiquinona/metabolismo , Animais , Biomarcadores/metabolismo , Hipóxia Celular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidroquinonas/metabolismo , Técnicas In Vitro , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredução , Perfusão , Cianeto de Potássio/farmacologia , Ubiquinona/fisiologia
5.
Am J Physiol Lung Cell Mol Physiol ; 300(5): L773-80, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21296895

RESUMO

The quinones duroquinone (DQ) and coenzyme Q(1) (CoQ(1)) and quinone reductase inhibitors have been used to identify reductases involved in quinone reduction on passage through the pulmonary circulation. In perfused rat lung, NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the predominant DQ reductase and NQO1 and mitochondrial complex I as the CoQ(1) reductases. Since inhibitors have nonspecific effects, the goal was to use Nqo1-null (NQO1(-)/(-)) mice to evaluate DQ as an NQO1 probe in the lung. Lung homogenate cytosol NQO1 activities were 97 ± 11, 54 ± 6, and 5 ± 1 (SE) nmol dichlorophenolindophenol reduced·min(-1)·mg protein(-1) for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs, respectively. Intact lung quinone reduction was evaluated by infusion of DQ (50 µM) or CoQ(1) (60 µM) into the pulmonary arterial inflow of the isolated perfused lung and measurement of pulmonary venous effluent hydroquinone (DQH(2) or CoQ(1)H(2)). DQH(2) efflux rates for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs were 0.65 ± 0.08, 0.45 ± 0.04, and 0.13 ± 0.05 (SE) µmol·min(-1)·g dry lung(-1), respectively. DQ reduction in NQO1(+/+) lungs was inhibited by 90 ± 4% with dicumarol; there was no inhibition in NQO1(-/-) lungs. There was no significant difference in CoQ(1)H(2) efflux rates for NQO1(+/+) and NQO1(-/-) lungs. Differences in DQ reduction were not due to differences in lung dry weights, wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total DQ recoveries. The data provide genetic evidence implicating DQ as a specific NQO1 probe in the perfused rodent lung.


Assuntos
NAD(P)H Desidrogenase (Quinona)/metabolismo , Animais , Benzoquinonas/metabolismo , Benzoquinonas/farmacologia , Dicumarol/farmacologia , Pulmão/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/deficiência , NAD(P)H Desidrogenase (Quinona)/genética , Oxirredução , Circulação Pulmonar/efeitos dos fármacos , Ubiquinona/metabolismo , Ubiquinona/farmacologia
6.
Am J Physiol Lung Cell Mol Physiol ; 300(5): L762-72, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21239539

RESUMO

Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K(+) concentration ([K(+)]), used to manipulate contributions of membrane potentials, attenuated decreases in [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 µl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.


Assuntos
Células Endoteliais/fisiologia , Corantes Fluorescentes , Potenciais da Membrana/fisiologia , Mitocôndrias/fisiologia , Compostos Organometálicos , Artéria Pulmonar/metabolismo , Rodamina 123 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Bovinos , Células Cultivadas , Hiperóxia/metabolismo
7.
Free Radic Biol Med ; 50(8): 953-62, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21238579

RESUMO

Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5µM, 24h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP(+) in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP(+) ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the K(m) for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold.


Assuntos
Endotélio Vascular/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Tiocianatos/farmacologia , Animais , Western Blotting , Bovinos , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Isotiocianatos , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologia , Sulfóxidos
8.
Free Radic Biol Med ; 46(1): 25-32, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18848878

RESUMO

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.


Assuntos
Células Endoteliais/enzimologia , Glicólise/fisiologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADP/metabolismo , Androsterona/farmacologia , Animais , Bovinos , Células Cultivadas , Citosol , Desoxiglucose/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Iodoacetatos/farmacologia , Ácido Láctico/metabolismo , NADP/análogos & derivados , NADP/química , Oxirredução , Artéria Pulmonar/citologia , Especificidade por Substrato
9.
J Appl Physiol (1985) ; 105(4): 1114-26, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18703762

RESUMO

The objective was to evaluate the pulmonary disposition of the ubiquinone homolog coenzyme Q(1) (CoQ(1)) on passage through lungs of normoxic (exposed to room air) and hyperoxic (exposed to 85% O(2) for 48 h) rats. CoQ(1) or its hydroquinone (CoQ(1)H(2)) was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of CoQ(1)H(2) and CoQ(1) were measured. CoQ(1)H(2) appeared in the venous effluent when CoQ(1) was infused, and CoQ(1) appeared when CoQ(1)H(2) was infused. In normoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 58 and 33% in the presence of rotenone (mitochondrial complex I inhibitor) and dicumarol [NAD(P)H-quinone oxidoreductase 1 (NQO1) inhibitor], respectively. Inhibitor studies also revealed that lung CoQ(1)H(2) oxidation was via mitochondrial complex III. In hyperoxic lungs, CoQ(1)H(2) efflux rates when CoQ(1) was infused decreased by 23% compared with normoxic lungs. Based on inhibitor effects and a kinetic model, the effect of hyperoxia could be attributed predominantly to 47% decrease in the capacity of complex I-mediated CoQ(1) reduction, with no change in the other redox processes. Complex I activity in lung homogenates was also lower for hyperoxic than for normoxic lungs. These studies reveal that lung complexes I and III and NQO1 play a dominant role in determining the vascular concentration and redox status of CoQ(1) during passage through the pulmonary circulation, and that exposure to hyperoxia decreases the overall capacity of the lung to reduce CoQ(1) to CoQ(1)H(2) due to a depression in complex I activity.


Assuntos
Hiperóxia/enzimologia , Pulmão/enzimologia , Oxirredutases/metabolismo , Circulação Pulmonar , Ubiquinona/sangue , Animais , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Hiperóxia/fisiopatologia , Cinética , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Mitocôndrias/enzimologia , Modelos Cardiovasculares , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredução , Oxirredutases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley
10.
Am J Physiol Lung Cell Mol Physiol ; 293(3): L809-19, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17601793

RESUMO

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.


Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Células Endoteliais/enzimologia , Hiperóxia/metabolismo , Mitocôndrias/enzimologia , Artéria Pulmonar/citologia , Artéria Pulmonar/enzimologia , Ubiquinona/metabolismo , Aerobiose/efeitos dos fármacos , Animais , Benzoquinonas/farmacologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ferricianetos/farmacologia , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Espectrofotometria , Cloreto de Tolônio/farmacologia , Ubiquinona/análise , Ubiquinona/farmacologia
11.
Am J Physiol Lung Cell Mol Physiol ; 290(3): L607-19, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16243901

RESUMO

The objective of this study was to examine the impact of chronic hyperoxic exposure (95% O2 for 48 h) on intact bovine pulmonary arterial endothelial cell redox metabolism of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ). DQ or durohydroquinone (DQH2) was added to normoxic or hyperoxia-exposed cells in air-saturated medium, and the medium DQ concentrations were measured over 30 min. DQ disappeared from the medium when DQ was added and appeared in the medium when DQH2 was added, such that after approximately 15 min, a steady-state DQ concentration was approached that was approximately 4.5 times lower for the hyperoxia-exposed than the normoxic cells. The rate of DQ-mediated reduction of the cell membrane-impermeant redox indicator, potassium ferricyanide [Fe(CN)6(3-)], was also approximately twofold faster for the hyperoxia-exposed cells. Inhibitor studies and mathematical modeling suggested that in both normoxic and hyperoxia-exposed cells, NAD(P)H:quinone oxidoreductase 1 (NQO1) was the dominant DQ reductase and mitochondrial electron transport complex III the dominant DQH2 oxidase involved and that the difference between the net effects of the cells on DQ redox status could be attributed primarily to a twofold increase in the maximum NQO1-mediated DQ reduction rate in the hyperoxia-exposed cells. Accordingly, NQO1 protein and total activity were higher in hyperoxia-exposed than normoxic cell cytosolic fractions. One outcome for hyperoxia-exposed cells was enhanced protection from cell-mediated DQ redox cycling. This study demonstrates that exposure to chronic hyperoxia increases the capacity of pulmonary arterial endothelial cells to reduce DQ to DQH2 via a hyperoxia-induced increase in NQO1 protein and total activity.


Assuntos
Benzoquinonas/química , Endotélio Vascular/metabolismo , Hidroquinonas/química , Hiperóxia/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Artéria Pulmonar/metabolismo , Animais , Benzoquinonas/metabolismo , Bovinos , Citosol/enzimologia , Ferricianetos , Genes Dominantes , Humanos , Hidroquinonas/metabolismo , Oxirredução , Consumo de Oxigênio , Circulação Pulmonar
12.
Am J Physiol Lung Cell Mol Physiol ; 289(5): L788-97, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15994278

RESUMO

NAD(P)H:quinone oxidoreductase 1 (NQO1) plays a dominant role in the reduction of the quinone compound 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) to durohydroquinone (DQH2) on passage through the rat lung. Exposure of adult rats to 85% O2 for > or =7 days stimulates adaptation to the otherwise lethal effects of >95% O2. The objective of this study was to examine whether exposure of adult rats to hyperoxia affected lung NQO1 activity as measured by the rate of DQ reduction on passage through the lung. We measured DQH2 appearance in the venous effluent during DQ infusion at different concentrations into the pulmonary artery of isolated perfused lungs from rats exposed to room air or to 85% O2. We also evaluated the effect of hyperoxia on vascular transit time distribution and measured NQO1 activity and protein in lung homogenate. The results demonstrate that exposure to 85% O2 for 21 days increases lung capacity to reduce DQ to DQH2 and that NQO1 is the dominant DQ reductase in normoxic and hyperoxic lungs. Kinetic analysis revealed that 21-day hyperoxia exposure increased the maximum rate of pulmonary DQ reduction, Vmax, and the apparent Michaelis-Menten constant for DQ reduction, Kma. The increase in Vmax suggests a hyperoxia-induced increase in NQO1 activity of lung cells accessible to DQ from the vascular region, consistent qualitatively but not quantitatively with an increase in lung homogenate NQO1 activity in 21-day hyperoxic lungs. The increase in Kma could be accounted for by approximately 40% increase in vascular transit time heterogeneity in 21-day hyperoxic lungs.


Assuntos
Benzoquinonas/metabolismo , Hiperóxia/metabolismo , Pulmão/metabolismo , Animais , Doença Crônica , Técnicas In Vitro , Cinética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredução , Perfusão , Circulação Pulmonar , Ratos , Ratos Sprague-Dawley
13.
Free Radic Biol Med ; 37(1): 86-103, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15183197

RESUMO

The study objective was to use pulmonary arterial endothelial cells to examine kinetics and mechanisms contributing to the disposition of the quinone 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) observed during passage through the pulmonary circulation. The approach was to add DQ, durohydroquinone (DQH2), or DQ with the cell membrane-impermeant oxidizing agent, ferricyanide (Fe(CN)6(3)-), to the cell medium, and to measure the medium concentrations of substrates and products over time. Studies were carried out under control conditions and with dicumarol, to inhibit NAD(P)H:quinone oxidoreductase 1 (NQO1), or cyanide, to inhibit mitochondrial electron transport. In control cells, DQH2 appears in the extracellular medium of cells incubated with DQ, and DQ appears when the cells are incubated with DQH2. Dicumarol blocked the appearance of DQH2 when DQ was added to the cell medium, and cyanide blocked the appearance of DQ when DQH2 was added to the cell medium, suggesting that the two electron reductase NQO1 dominates DQ reduction and mitochondrial electron transport complex III is the predominant route of DQH2 oxidation. In the presence of cyanide, the addition of DQ also resulted in an increased rate of appearance of DQH2 and stimulation of cyanide-insensitive oxygen consumption. As DQH2 does not autoxidize-comproportionate over the study time course, these observations suggest a cyanide-stimulated one-electron DQ reduction and durosemiquinone (DQ*-) autoxidation. The latter processes are apparently confined to the cell interior, as the cell membrane impermeant oxidant, ferricyanide, did not inhibit the DQ-stimulated cyanide-insensitive oxygen consumption. Thus, regardless of whether DQ is reduced via a one- or two-electron reduction pathway, the net effect in the extracellular medium is the appearance of DQH2. These endothelial redox functions and their apposition to the vessel lumen are consistent with the pulmonary endothelium being an important site of DQ reduction to DQH2 observed in the lungs.


Assuntos
Benzoquinonas/metabolismo , Endotélio Vascular/fisiologia , Animais , Bovinos , Células Cultivadas , Cinética , Ácido Linoleico/metabolismo , Modelos Biológicos , Oxirredução , Artéria Pulmonar
14.
Am J Physiol Lung Cell Mol Physiol ; 285(5): L1116-31, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12882764

RESUMO

The lungs can substantially influence the redox status of redox-active plasma constituents. Our objective was to examine aspects of the kinetics and mechanisms that determine pulmonary disposition of redox-active compounds during passage through the pulmonary circulation. Experiments were carried out on rat and mouse lungs with 2,3,5,6-tetramethyl-1,4-benzoquinone [duroquinone (DQ)] as a model amphipathic quinone reductase substrate. We measured DQ and durohydroquinone (DQH2) concentrations in the lung venous effluent after injecting, or while infusing, DQ or DQH2 into the pulmonary arterial inflow. The maximum net rates of DQ reduction to DQH2 in the rat and mouse lungs were approximately 4.9 and 2.5 micromol. min(-1).g dry lung wt(-1), respectively. The net rate was apparently the result of freely permeating access of DQ and DQH2 to tissue sites of redox reactions, dominated by dicumarol-sensitive DQ reduction to DQH2 and cyanide-sensitive DQH2 reoxidation back to DQ. The dicumarol sensitivity along with immunodetectable expression of NAD(P)H-quinone oxidoreductase 1 (NQO1) in the rat lung tissue suggest cytoplasmic NQO1 as the dominant site of DQ reduction. The effect of cyanide on DQH2 oxidation suggests that the dominant site of oxidation is complex III of the mitochondrial electron transport chain. If one envisions DQ as a model compound for examining the disposition of amphipathic NQO1 substrates in the lungs, the results are consistent with a role for lung NQO1 in determining the redox status of such compounds in the circulation. For DQ, the effect is conversion of a redox-cycling, oxygen-activating quinone into a stable hydroquinone.


Assuntos
Benzoquinonas/farmacocinética , Artéria Pulmonar/fisiologia , Circulação Pulmonar/fisiologia , Animais , Benzoquinonas/administração & dosagem , Biotransformação , Hidroquinonas/farmacocinética , Injeções Intra-Arteriais , Cinética , Pulmão/fisiologia , Camundongos , Modelos Biológicos , Músculo Liso Vascular/fisiologia , Oxirredução , Ratos , Distribuição Tecidual
15.
Free Radic Biol Med ; 34(7): 892-907, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12654478

RESUMO

The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.


Assuntos
Artérias/citologia , Endotélio Vascular/metabolismo , Pulmão/citologia , Oxirredução , Ubiquinona/metabolismo , Animais , Biotina/farmacologia , Biotinilação , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Elétrons , Radicais Livres , Modelos Químicos , NADP , Fatores de Tempo
16.
Am J Physiol Lung Cell Mol Physiol ; 282(1): L36-43, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11741813

RESUMO

Pulmonary arterial endothelial cells possess transplasma membrane electron transport (TPMET) systems that transfer intracellular reducing equivalents to extracellular electron acceptors. As one aspect of determining cellular mechanisms involved in one such TPMET system in pulmonary arterial endothelial cells in culture, glycolysis was inhibited by treatment with iodoacetate (IOA) or by replacing the glucose in the cell medium with 2-deoxy-D-glucose (2-DG). TPMET activity was measured as the rate of reduction of the extracellular electron acceptor polymer toluidine blue O polyacrylamide. Intracellular concentrations of NADH, NAD(+), NADPH, and NADP(+) were determined by high-performance liquid chromatography of KOH cell extracts. IOA decreased TPMET activity to 47% of control activity concomitant with a decrease in the NADH/NAD(+) ratio to 34% of the control level, without a significant change in the NADPH/NADP(+) ratio. 2-DG decreased TPMET activity to 53% of control and decreased both NADH/NAD(+) and NADPH/NADP(+) ratios to 51% and 55%, respectively, of control levels. When lactate was included in the medium along with the inhibitors, the effects of IOA and 2-DG on both TPMET activity and the NADPH/NADP(+) ratios were prevented. The results suggest that cellular redox status is a determinant of pulmonary arterial endothelial cell TPMET activity, with TPMET activity more highly correlated with the poise of the NADH/NAD(+) redox pair.


Assuntos
Endotélio Vascular/fisiologia , Membranas Intracelulares/metabolismo , Artéria Pulmonar/fisiologia , Animais , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Desoxiglucose/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Endotélio Vascular/citologia , Iodoacetatos/farmacologia , Ácido Láctico/farmacologia , NAD/metabolismo , Oxirredução , Artéria Pulmonar/citologia
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