Production of extracellular superoxide by human lymphoblast cell lines: Comparison of electron spin resonance techniques and cytochrome C reduction assay
Introduction
Superoxide (O2−) production plays an important role in redox cell signaling and development of pathophysiological conditions, such as hypertension, ischemia-reperfusion injury, inflammation and atherosclerosis [1]. However, detection of O2− is still a challenging problem. One of the most sensitive and definitive methods of O2− detection is electron spin resonance (ESR) [2], [3]. The ESR spin-trapping technique has been used to detect O2− radicals induced by inflammation via neutrophil NADPH oxidase in cellular systems in vitro [4]. However, the commonly used nitrone spin traps have a very low efficacy for trapping of O2− radicals (Fig. 1) [5]. Thus, formation of the radical adduct is limited by slow kinetics of O2− trapping and obstruction by antioxidants. Furthermore, superoxide radical adducts suffer from decomposition to hydroxyl (OH)-radical adducts by glutathione (GSH) peroxidase [6]. Finally, both O2− and OH radical adducts can be reduced to ESR silent hydroxylamines by ascorbate, transition metals, or flavin enzymes (Fig. 1) [7].
Recently, cyclic hydroxylamines were found to be effective scavengers of O2− radicals [8], [9]. Hydroxylamine probes 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PPH) and 1-hydroxy-3-carboxy-pyrrolidine (CPH) have been previously used for quantitative detection of extracellular O2−. The advantage of hydroxylamine probes is that they are effective scavengers of O2− and produce a stable nitroxide radical [8]. Previously, we reported the activity of the phagocytic NADPH oxidase in neutrophils from healthy subjects using CPH, and measuring O2− as SOD-inhibitable formation of 3-carboxyproxyl [10].
In the current investigation, we studied superoxide production in cultured lymphoblast cell lines at baseline and upon stimulation with phorbol 12-myristate 13-acetate (PMA) by three methods: (a) superoxide dismutase (SOD)-inhibitable cytochrome C reduction, (b) spin trapping of superoxide with 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), and (c) using ESR with the cell-permeable spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Fig. 1) [11]. Reaction of O2− with CMH is much faster (1.2 × 104 M−1 s−1) than with nitrone spin traps, thereby enabling the hydroxylamines to compete with cellular antioxidants and react with both extra- and intracellular O2−.
Our study describes a new technique for O2− measurement in cultured human lymphoblasts using ESR and CMH.
Section snippets
Reagents
Spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) and spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) were purchased from Alexis Corporation (San Diego, USA). Polyethylene-glycol-conjugated superoxide dismutase (PEG–SOD), and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma–Aldrich (St. Louis, MO). All other reagents were obtained from Sigma–Aldrich.
Establishment of immortalized cell lines
In collaboration with the
Measurement of O2− in stimulated and unstimulated lymphoblasts using CMH
Incubation of human lymphoblasts with the spin probe CMH resulted in the generation of the ESR signal of 3-methoxycarbonyl-proxyl nitroxide (CM) (Fig. 2A). Time scan of the low-field component of the ESR signal showed linear accumulation of CM in unstimulated cells. Stimulation of cells by PMA (10 μM) led to several-fold increase in the slope of CM kinetics (Fig. 2B).
CMH detects both extra- and intracellular O2−[11], [15]. Extracellular O2− can be quantified by inhibition of the ESR signal by
Discussion
In the present study, we observed that the cell-permeable spin probe CMH provided the most quantitative measurement of O2− generation in human lymphoblast cell lines. As shown in Table 1, cytochrome C and the spin traps EMPO and DEPMPO detected two to four times less O2− compared to CMH. Higher reactivity of CMH with O2− and the stability of the CM-nitroxide (product of CMH and O2− reaction) are likely responsible for higher O2− detection by CMH. In addition, we show that human lymphoblasts
Acknowledgements
This research was supported by National Institutes of Health grants PO-1 HL058000-05 and PO-1 HL075209, the Emory University Research Committee, the Emory University General Clinical Research Center (M01-RR00039), Atlanta, GA, and by National Institutes of Health cardiovascular training grant T-32 HL07745 to PM and SSW.
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