Forum: oxidative stress statusDetection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques
Introduction
In the late sixties, McCord and Fridovich showed that superoxide free radical anion (O2•−) could be produced enzymatically in mammalian tissues, and demonstrated that superoxide dismutase (SOD) catalyzed the dismutation of O2•− [1]. The role of O2•− in nonspecific host defence has been recognized for a long time, and more recently in the signal transduction of physiological communications as well as in the pathophysiological mechanisms of various processes [2]. Often, the implication of reactive oxygen species (ROS) in these processes is indirectly demonstrated through the use of antioxidant molecules. Indeed, the half-life of ROS as O2•− is very short and the assessment of its production is not an easy task, particularly in nonphagocytic cells such as endothelium. However, endothelium-derived ROS appears to play a crucial role in the pathophysiology of many processes, such as aging or atherosclerosis [3].
Four techniques were independently used to assess the relatively low production of O2•− by endothelium. The simplest and easiest technique consisted of following spectrophotometrically the reduction of ferricytochrome c by O2•−. This technique was used to evaluate basal and stimulated O2•− generation from cultured endothelial cells [2], [4], [5], [6], [7] (to quote only the initial reports). In contrast, only very few studies employed electron spin resonance (ESR) and the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) to detect the endothelial production of O2•−. Uncoupling of cellular reductase by menadione [8] and anoxia followed by reoxygenation [9] were the two conditions allowing detection of O2•− from cultured endothelial cells. Using ESR, we recently were able to detect O2•− from cultured bovine aortic endothelial cells (BAEC) using the calcium ionophore A23187 as a stimulus [10]. Finally, the most sensitive techniques for detecting the low O2•− production from endothelial cells seem to be fluorescence and lucigenin-enhanced chemiluminescence. Hydroethidine fluorescence has been applied as a detector of intracellular O2•− in endothelial cells [11], but is less frequently used than the lucigenin chemiluminescence technique, which has wide application in the assessment of extra- and intracellular O2•− production [12], [13], [14], [15]. However, this technique was recently questioned because reduced lucigenin can itself generate O2•− [16], [17], [18], [19], although others have rehabilitated lucigenin luminescence [20], [21], [22].
Thus, it would seem urgent to compare these techniques for assessing O2•− when its production is low, as in endothelial cells. In the present study, we applied ferricytochrome c reduction, ESR spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O2•− production in cultured BAEC. We focused our study on extracellular O2•− production because the specificity of the signal is provided from the use of SOD, and this control cannot be obtained intracellularly. The calcium ionophore A23187 was used to stimulate O2•− production and the effect of cell confluency on O2•− production was determined. As an NAD(P)H oxidase is suspected to be one of the major sources of extracellular O2•− production, and as these electron donors are often used to enhance O2•− production [23], [24], we investigated the effect of NADH and NADPH on the signals.
Section snippets
Cell culture and materials
BAEC were obtained as described previously [25] and cultured in Dulbecco’s modified Eagle’s (DME) medium supplemented with 10% heat-inactivated calf serum (CS) at 37°C and 1 ng/ml basic fibroblast growth factor (bFGF) under a 10% CO2 humidified atmosphere. The cells used in this study were between the fifth and fifteenth passage. To avoid confounding effects produced by differences in cell density upon initial seeding, all passages were made using a splitting ratio of 1:6. This ensured that the
Characterization of the reduction of cytochrome c signals
As various concentrations of cytochrome c have been reported in the literature (70, 20, 140, 80, and 100 μm) [4], [5], [6], [7], [30], respectively, and as different preparations of cytochrome c are commercialy available (purified with trichloroacetic acid (TCA) or not, acetylated or not), we first compared three preparations of cytochrome c at two different concentrations (20 and 100 μm) to detect O2•− generated by postconfluent BAEC (D0+6). We found that, at the concentration of 20 μm, C2506
Acknowledgements
This work was supported by INSERM, Fondation de France, and ARC (national grant 5358).
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