Forum: oxidative stress status
Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques

https://doi.org/10.1016/S0891-5849(00)00336-1Get rights and content

Abstract

Endothelium produces oxygen-derived free radicals (nitric oxide, NOradical dot; superoxide anion, O2•−) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O2•− production, particularly due to the difficulty in assessing O2•− when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O2•− assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O2•− production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O2•− production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O2•− production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 μm rather than at 100 μm appears more suitable to detect O2•−. However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O2•−. Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O2•− production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.

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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