Original ContributionsATP converts necrosis to apoptosis in oxidant-injured endothelial cells
Introduction
Reactive oxygen species including hydrogen peroxide (H2O2) generated by neutrophils and macrophages are important mediators of cellular injury during acute inflammation [1], [2], [3], [4]. Cells exposed to H2O2 undergo a series of biochemical events that contribute to their demise including: reduction in ATP levels through the inhibition of glyceraldehyde-3-phosphate dehydrogenase [5], [6], [7], [8], DNA strand breakage following depletion of nicotinamide adenine dinucleotide (NAD) in association with activation of poly-ADP-ribose polymerase [9], [10], alterations in the cytoskeleton [11], [12], [13], and activation of the glutathione redox cycle with oxidation or loss of glutathione [12], [14].
H2O2 exposure at high concentrations (>1 mM), in many cellular models results in cell death with a morphologic pattern consistent with necrosis. The necrotic pattern of cell death is manifested by swelling and disruption of internal organelles and plasma membrane lysis resulting in the liberation of denatured proteins, DNA fragments, lysosomal contents, and other cellular debris from the cytoplasm into the extracellular space. The dispersion of cellular debris into the extracellular space invokes an acute inflammatory response [15]. H2O2 at micromolar concentrations, however, has been shown to induce an apoptotic pattern of cell death [16]. In contrast to necrosis, cells undergoing apoptosis exhibit intact plasma membranes and cytoplasmic organelles (e.g., mitochondria). During the late phases of apoptosis, the internal contents including fragmented DNA are packaged into membrane-bound apoptotic bodies. Apoptotic bodies are subsequently released, and then undergo phagocytosis by surrounding cells and macrophages before their contents are released thus minimizing any inflammatory response [15]. >
Recently, several groups have demonstrated that apoptosis in T cells and T cell lines induced by a variety of treatments (i.e., anti-Fas monoclonal antibody CH11, Ca++ ionophore, etoposide, dexamethasone, staurosporin, or CD95 stimulation) requires ATP [17], [18], [19]. In many of these models, depletion of ATP early in the apoptotic process can switch the predominant form of cell death from apoptosis to necrosis. Kass et al. [20] have also demonstrated (in an in vitro model of the nuclear events of apoptosis) that the nuclear morphologic events of apoptosis (e.g., chromatin condensation) require ATP, whereas DNA fragmentation does not.
In earlier work from our laboratory, we noted that the amino acid glutamine, which can enter the Krebs cycle via α-ketoglutarate, can act as an alternative fuel substrate in endothelial cells lethally injured with H2O2 [8], [21]. The glutamine bypassed the blockade of glucose-dependent ATP synthesis induced by the H2O2-mediated inhibition of the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase [5], [8]. The partial restoration of ATP levels by glutamine was inhibitable by the mitochondrial inhibitor, oligomycin [8]. At a concentration of glutamine (2 mM), which maximally enhanced ATP levels in the injured cells, the amino acid did not act as a scavenger of H2O2, as demonstrated in a fluorometic assay [21], Partial restoration of ATP levels in the cells with glutamine administration restored organic anion transport by the cells and markedly prolonged cell survival [8], [21], [22].
In this report, we have tested the hypothesis that ATP can act as a switch to determine the pattern of cell death induced by H2O2, either apoptosis or necrosis. We have used a model already well characterized in our laboratory [8], [21], [22], [23] in which ATP levels in H2O2-injured endothelial cells can be modulated positively by glutamine administration or negatively by metabolic inhibition to demonstrate the dependence of H2O2-induced apoptosis on ATP and to define the threshold level of ATP required for apoptosis to proceed.
Section snippets
Cells and culture
Bovine pulmonary artery endothelial cells from the National Institute of Aging, Aging Cell Culture Repository Center (Camden, NJ) were cultured in RPMI 1640 medium supplemented with 2 mM glutamine (GIBCO), 10% heat-inactivated fetal bovine serum (Whittaker, MA Bioproducts), 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO). Cells were cultured under 5% CO2/95% air at 37°C in 75- or 150-cm2 flasks (Corning, Corning, NY).
Pattern of cell death in endothelial cells induced by H2O2
Endothelial cells were exposed to varying concentrations of H2O2 (0 to 5 mM) and then evaluated over a 4-h time course. Fluorescence microscopy with AO-EB staining demonstrated two distinct patterns of cell death; necrosis and apoptosis. The uninjured control demonstrated only a minimal loss of viability throughout the time course with only a small percentage undergoing apoptosis (4%) and necrosis (8.7%) (Fig. 1). Cells exposed to concentrations of H2O2 ≤ 1 mM demonstrated the nuclear features
Discussion
In this study, we have been able to confirm and extend to oxidant injury earlier observations [17], [18], [19] that ATP is required for apoptosis and that it can act as a switch determining the pattern of cell death-necrosis vs. apoptosis. When a concentration of H2O2 (100 μM), which induced apoptosis in the endothelial cells without altering cellular ATP levels, was combined with the independent reduction of ATP levels to ≤10% of control by metabolic inhibition, the apoptotic pattern of
Acknowledgements
This work was supported by the U.S. Army Medical Research and Materiel Command under Agreement No. 93-MM-3571 and also in part by the Department of Veterans Affairs. The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation.
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