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ATP converts necrosis to apoptosis in oxidant-injured endothelial cells
Free Radical Biology & Medicine, Vol. 25, No. 6, pp. 694 –702, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98 $19.00 1 .00
PII S0891-5849(98)00107-5
Original Contribution ATP CONVERTS NECROSIS TO APOPTOSIS IN OXIDANT-INJURED ENDOTHELIAL CELLS JOSEPH L. LELLI, JR.,* LAUREN L. BECKS,† MILENA I. DABROWSKA,†
and
DANIEL B. HINSHAW†
*Sections of Pediatric and General Surgery, Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, USA, and †Department of Veterans Affairs Medical Center, Ann Arbor, MI, USA (Received 19 December 1997; Revised 14 April 1998; Accepted 23 April 1998)
Abstract—Cell death due to necrosis results in acute inflammation, while death by apoptosis generally does not. The effect of adenosine triphosphate (ATP) on the pattern of cell death induced by oxidants was examined in bovine endothelial cells. ATP levels were altered by hydrogen peroxide (H2O2), glutamine (Gln), and metabolic inhibition (MI), to determine if necrosis can be shifted to apoptosis during oxidant injury. The form of cell death was determined by fluorescence microscopic techniques and the pattern of DNA degradation on agarose gels. ATP levels were measured using the luciferase–luciferin assay. Apoptosis occurred with 100 mM H2O2 without an alteration in ATP levels. ATP was significantly lowered with 5 mM H2O2, and necrosis occurred. MI, in combination with 100 mM H2O2, decreased ATP and resulted in necrosis. MI alone, however, did not cause cell death. Gln partially restored ATP levels in cells injured with 5 mM H2O2 and resulted in a significant increase in apoptosis. DNA laddering on agarose gels confirmed the apoptotic changes seen by fluorescence microscopy. In summary, a threshold level of ATP 25% of basal levels is required for apoptosis to proceed after oxidant stress, otherwise necrosis occurs. Agents like glutamine that enhance ATP levels in oxidant-stressed cells may be potent means of shifting cell death during inflammation to the noninflammatory form of death—apoptosis. © 1998 Elsevier Science Inc. Keywords—ATP, Necrosis, Apoptosis, Glutamine, Hydrogen peroxide, Endothelial cells, Free radical
INTRODUCTION
phologic 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
Reactive oxygen species including hydrogen peroxide (H2O2) generated by neutrophils and macrophages are important mediators of cellular injury during acute inflammation [1– 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– 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 [11–13], 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 morAddress correspondence to: Daniel B. Hinshaw, M.D., VA Medical Center, 2215 Fuller Road (11), Ann Arbor, MI 48105, USA; Tel: (313) 761-7903; Fax: (313) 769-7412; E-Mail: [email protected] 694
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treatments (i.e., anti-Fas monoclonal antibody CH11, Ca11 ionophore, etoposide, dexamethasone, staurosporin, or CD95 stimulation) requires ATP [17–19]. 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 a-ketoglutarate, can act as an alternative fuel substrate in endothelial cells lethally injured with H2O2 [8,21]. The glutamine bypassed the blockade of glucosedependent 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 [8,21–23] 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. MATERIALS AND METHODS
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-2ethanesulfonic acid (HEPES), 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO). Cells were cultured under 5% CO2/95% air at 37°C in 75- or 150-cm2 flasks (Corning, Corning, NY). Experiments were carried out using cells from passages 2–9, suspended in modified Geys buffer (MGB) containing, in mM, 147 NaCl, 5
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KCl, 0.22 KH2PO4, 1.0 MgCl, 0.28 MgSO4, 10 HEPES, 1.5 CaCl2, and 5.5 glucose at pH 7.4. In experiments where oligomycin was used, glucose was omitted from the MGB. In experiments in which glutamine supplementation occurred, a final concentration of 2 mM glutamine was present in the MGB [6,21]. Injury protocol with hydrogen peroxide Upon reaching confluence, endothelial cells were suspended by exposure to 0.05% trypsin and 0.02% EDTA for 10 –15 min. The cells were washed in MGB and resuspended in MGB 1 glucose at a concentration of 2 3 106/ml. H2O2 was added at final concentrations of 100 mM, 1 mM, and 5 mM, and the cells were observed over a 4-h time course. Endothelial cells in suspension were utilized throughout the experiments to be consistent with our earlier work [8,21,22]. Selected experiments were also done with adherent endothelial cells that demonstrated similar findings to those reported here, except that the adherent endothelial cells were somewhat more resistant to a given concentration of H2O2. For example, whereas 40% of suspended endothelial cells were apoptotic 4 h after exposure to 100 mM H2O2 (see Fig. 2A below), 19 6 9.9% (n 5 6) of adherent endothelial cells 4 h after exposure to 100 mM H2O2 were apoptotic. By 6 h, the adherent cells were 34 6 25.6% (n 5 5) apoptotic after exposure to 100 mM H2O2, demonstrating the great variability in the timing of apoptosis in adherent endothelial cells. These findings are consistent with the observation of others that cellular suspension itself may promote cell death signaling [24] and, in this experimental model, may account for a more rapid and synchronous apoptotic process. Treatment with glutamine and metabolic inhibition Endothelial cells contain high levels of glutaminase activity [25], suggesting they possess an enhanced ability to utilize glutamine for anabolic and potentially catabolic purposes. Attempts to use other mitochondrial substrates (e.g., acetoacetate and succinate) have demonstrated no significant enhancement of ATP levels in endothelial cells injured with H2O2 (data not shown). In our earlier work, we were able to enhance ATP levels in the injured endothelial cells in a dose-dependent manner achieving a maximal effect with 2 mM glutamine [21]. Thus, in experiments in which glutamine was used, endothelial cells were suspended in MGB containing 2 mM glutamine (Sigma) and then exposed to H2O2 (0 –5 mM). In other experiments in which the cells were metabolically inhibited, the cells were treated with 650 nM oligomycin (Sigma), an inhibitor of the mitochondrial Fo
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ATP-synthase, in the absence of glucose [23]. In samples in which H2O2 injury was combined with metabolic inhibition, the oligomycin was added 30 min after addition of H2O2 to allow for H2O2-mediated effects on cell death signaling to be initiated before full metabolic inhibition occurred. Samples for ATP measurement were taken 1– 4 h after H2O2 addition. Fluorescence microscopic assay of apoptotic nuclear features [26] The nuclear morphology and staining pattern of suspended endothelial cells was examined using a Nikon optiphot fluorescence microscope at each time point following exposure of the cells to the dyes acridine orange (AO) and ethidium bromide to determine the percentage of cells undergoing nuclear changes characteristic of apoptosis [26] A dye mixture made up of 10 mM acridine orange and 10 mM ethidium bromide prepared in phosphate-buffered saline was used. Acridine orange (a fluorescent DNA-binding dye) intercalates into DNA, making it appear green, and binds to RNA, staining it red-orange. Ethidium bromide is taken up only by the nuclei of nonviable cells, and its fluorescence overwhelms that of the acridine orange, making the chromatin of lysed cells appear orange [26]. At each time point, 25 ml of cell sample was mixed with 1 ml of the dye mix and 15 ml placed on a microscope slide with coverslip. Two hundred cells per sample were examined by fluorescence microscopy, according to the following criteria: (1) viable cells with normal nuclei (fine reticular pattern of green stain in the nucleus and red-orange granules in the cytoplasm); (2) viable cells with apoptotic nuclei (green chromatin which is highly condensed or fragmented and uniformly stained by the acridine orange); (3) nonviable cells with normal nuclei (bright orange chromatin with organized structure); and (4) nonviable cells with apoptotic nuclei (bright orange chromatin that is highly condensed or fragmented). The combination of cells meeting criteria 2 and 4 represented the total apoptotic cell population, because apoptotic cells eventually lose plasma membrane integrity in vitro. Criterion 3 was used to define the population of cells undergoing necrosis without evidence of apoptosis.
for 1 h. Then, an equal amount of cold isopropanol was added and the samples were mixed by inversion until formation of precipitate. At this point, the DNA was centrifuged for 30 min at 12,000 3 g, the supernatant removed, the pellet of DNA air dried for 30 min, and then resuspended in 150 ml of TE buffer (10 mM Tris, 1.0 mM EDTA, pH 7.6) with 40 mg/ml RNase (Sigma) and left to incubate at room temperature overnight. Sample buffer (0.25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol dissolved in deionized H2O) was added to the RNase treated samples, which were then run on a 1.6% agarose gel containing 7 mg ethidium bromide, at 120 mV for 1.5 h. The gel was then photographed under ultraviolet light, to visualize the DNA for evidence of endonucleolytic cleavage (“DNA laddering”), characteristic of apoptosis [26].
ATP measurement Cellular ATP levels were assayed as previously reported using the luciferase–luciferin method [8]. Endothelial cells were suspended at a concentration of 2.0 3 106 cells/ml and exposed to the experimental conditions. Measurements of ATP were performed using the luciferase–luciferin assay [8] at the different experimental time points.
Statistics Analysis of variance (one-way ANOVA) and paired t-tests were used for comparisons between experimental and control observations. Results are presented as means 6 SEM for five to eight experiments unless otherwise noted.
Measurement of endonucleolytic DNA cleavage [27] At each experimental time point, 2 3 106 suspended endothelial cells were harvested by centrifugation at 4000 3 g for 4 min, the supernatant removed, and the cells were resuspended in 500 ml of lysis buffer (100 mM TRIS, 5 mM EDTA, 200 mM NaCl, 0.2% SDS, pH 8.5). Next, 100 mg/ml Proteinase K (Sigma) was added to the samples and they were left to shake in a 37°C water bath
Fig. 1. Viability of uninjured control endothelial cells. Each bar represents the mean 1 SEM of N 5 8 separate determinations. Apoptotic and necrotic cells were differentiated using AO-EB staining [26]. Note the small background level of spontaneous apoptosis and necrosis occurring in the cells.
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Fig. 2. Viability of endothelial cells after exposure to different concentrations of H2O2: (A) 100 mM H2O2; (B) 1 mM H2O2; and (C) 5 mM H2O2. Each bar represents the mean 1 SEM of n 5 5– 8 separate determinations. Open bars—percentage of apoptotic cells; solid bars—percentage of necrotic cells determined using AO-EB staining [26]. *p , .05 compared to control in Fig. 1. Note the predominantly apoptotic pattern of endothelial cell death seen with H2O2 concentrations , 1 mM.
RESULTS
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 following staining with AO-EB characteristic for apoptotic cell death as early as 1 h, and demonstrated significant increases in apoptotic cells by 2 h after H2O2 exposure (Fig. 2A and B). At concentrations of H2O2 # 1 mM, there was no significant increase in the percentage of cells dying by necrosis as compared to the control. The percentage of cells undergoing apo-
ptosis after exposure to concentrations of H2O2 # 1 mM was significantly higher than that of the control cells (p , .05). At the highest concentration of H2O2 (5 mM), the pattern of cell death was predominantly necrotic (Fig. 2C). Compared to control cells and to the lower concentrations of H2O2, there was no significant increase in the percentage of apoptotic cells with 5 mM H2O2. At 4 h, 5 mM H2O2 caused a significant increase (p , .05) in the percentage of necrotic cells compared to the control and concentrations of H2O2 # 1 mM (Figs. 1 and 2). In longer time course experiments (data not shown), the dose-related demarcation in the pattern of cell death, necrosis, or apoptosis, remained consistent for 8 h. The AO-EB staining demonstrated that apoptotic bodies were formed with viable membranes containing fragmented DNA that excluded ethidium bromide. Agarose gel electrophoresis of DNA isolated from the endothelial cells confirmed the presence of apoptosis in the injured cell population (Fig. 3). Exposure to either 100 mM or 1 mM H2O2 resulted in the endonucleolytic
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Fig. 3. Agarose gel electrophoresis of DNA extracted from endothelial cells. Lane 1: DNA molecular size marker (Hind III/Hae III; GIBCO); lanes 2–5: DNA from control cells at 1, 2, 3, and 4 h, respectively; lanes 6 –9: DNA from cells exposed to 100 mM H2O2 at 1, 2, 3, and 4 h, respectively; lanes 10 –13: DNA from cells exposed to 1 mM H2O2 at 1, 2, 3, and 4 h, respectively; and lanes 14 –17: DNA from cells exposed to 5 mM H2O2 for 1, 2, 3, and 4 h, respectively. Note the distinct presence of “DNA ladders” by 3 h following exposure to H2O2 concentrations #1 mM. Changes in the DNA of cells injured with 5 mM H2O2 (lanes 16 and 17) were more typical of DNA smearing seen with necrosis.
cleavage of DNA into multiples of the 180 base-pair nucleosomes, “DNA laddering,” as early as 2 h after injury. Injury with 5 mM H2O2 was associated with only slight evidence of endonuclease activity (Fig. 3). Effects of H2O2 on cellular ATP levels Endothelial cells exposed to the different concentrations of H2O2 (0 –5 mM) were assayed for ATP using the luciferase–luciferin assay [8]. Previous work demonstrated an inhibitory effect of H2O2 on glycolysis in endothelial cells [8] secondary to inhibition of glyceraldehyde 3-phosphate dehydrogenase [5], with a resulting decline in cellular ATP levels. In this study, control cells showed no change in ATP levels over the time course (Fig. 4). Exposure to 100 mM H2O2 did not significantly alter ATP levels over the 4-h time course. Exposure to 1 mM H2O2 resulted in a moderate reduction of ATP levels from 8.0 (control level at time of injury) to 4.13 nmol/2 3 106 cells by 4 h, representing a decline in ATP of 48% (Fig. 4). H2O2 (5 mM) caused a rapid and dramatic reduction in ATP levels (from 8.0 to 0.77nmol/2 3 106 cells), which was significantly lower (p , .05) than the levels observed after exposure to both 100 mM and 1 mM H2O2 (Fig. 4). The reduction in ATP levels following exposure to 1 mM H2O2 was statistically significant only after 4 h (Fig. 4). Thus, a H2O2 concentration (100 mM) associated with no measurable reduction in endothelial ATP levels was almost exclusively correlated with apoptosis, whereas a concentration of H2O2 (5 mM) that caused profound reduction of endothelial ATP levels (#10% of control) was correlated with the appearance of a necrotic pattern of cell death. H2O2 (1 mM), which had an intermediate
effect on endothelial ATP levels, was correlated primarily with apoptosis. Modeling the effect of ATP depletion and repletion during H2O2 injury We hypothesized that ATP depletion, per se, did not account for the cell death signal associated with H2O2 exposure, because 100 mM H2O2 was sufficient to induce cell death without reduction of cellular ATP levels. Rather, ATP might act as a “switch” determining which pattern of cell death would ensue. Oligomycin, an inhibitor of the mitochondrial Fo ATP synthase in combination with glucose deprivation [23], was used to inhibit ATP synthesis without delivering an exogenous oxidant stress to the cells. Endothelial cells were incubated with 650 nM oligomycin in the absence of glucose, and the effects of this metabolic inhibition on ATP and cell viability were determined. ATP levels were assayed using the luciferase–luciferin assay [8]. Metabolic inhibition (MI) caused a rapid
Fig. 4. The effect of 0 –5 mM H2O2 on endothelial ATP levels over a 4-h time course. ATP levels were measured with the luciferin–luciferase assay [8]. Each bar represents the mean 1 SEM of n 5 5– 8 separate determinations. C 5 control; 0.1 5 0.1 mM (100 mM) H2O2, 1 5 1 mM H2O2, and 5 5 5 mM H2O2. *p , .05 compared to control.
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Fig. 5. Modeling ATP depletion and partial repletion during H2O2 injury. ATP levels were measured with the luciferin–luciferase assay [8]. Each bar represents the mean 1 SEM of n 5 5– 8 separate determinations. (A) Comparison of the effect of metabolic inhibition with 650 nM oligomycin in the absence of glucose with 5 mM H2O2 on endothelial cell ATP levels. *p , .05 compared to control. (B) The effect of supplementation with 2 mM glutamine on endothelial ATP levels during injury with 5 mM H2O2 (5 mM 1 glutamine) vs. the effect of 5 mM H2O2 alone (5 mM). *p , .05 compared to 5 mM.
decrease in cellular ATP levels from 8.0 to 0.13 nmol/2 3 106 cells, a decrease similar to that seen with H2O2 concentrations (e.g., 5 mM) associated with necrosis (Fig. 5A). Fluorescence microscopy using the AO-EB staining criteria was used to characterize and quantitate cell death under the conditions of metabolic inhibition. At 4 h, 16% (compared to 12.7% in controls) of the cells undergoing metabolic inhibition alone with oligomycin minus glucose died by necrosis (data not shown). The cells dying after metabolic inhibition alone were predominantly necrotic with little apoptosis occurring. There was no significant increase in cell death compared to controls. ATP depletion with metabolic inhibition alone in the absence of an oxidant stress did not induce the significant cell death seen with 5 mM H2O2, nor did it induce apoptosis. H2O2 interferes with glycolysis by inhibiting glyceraldehyde-3-phosphate dehydrogenase, which is a proximal event in glycolysis prior to entering into the tricarboxyclic acid (TCA) cycle [5,8]. Glutamine enters into the TCA cycle as an intermediate metabolite (i.e., a-ketoglutarate), and has been shown to partially restore ATP production in cells that have undergone injury with H2O2 [8,21]. Endothelial cells were incubated with 2 mM glutamine [8,21] prior to exposure to H2O2 at varying concentrations. Using the luciferase–luciferin assay [8], ATP levels were determined throughout the four hour time course. Control cell ATP levels were not altered by the addition of glutamine (data not shown). ATP levels that fell from 8.0 to 0.77 nmol/2 3 106 cells following exposure to 5 mM H2O2 were partially restored to 2.0 nmol/2 3 106 cells, a statistically significant (p , .05) increase at 1, 2, and 4 h after injury (Fig. 5B).
Effect of modulating ATP levels by glutamine supplementation or metabolic inhibition on the pattern of cell death following exposure to H2O2 Fluorescence microscopy with the AO-EB staining assay was used to determine the viability and cell death pattern of endothelial cells that were incubated in 2 mM glutamine and then injured with a range of concentrations of H2O2. Control cells demonstrated a minimal death rate over the 4-h time course that was not affected by the addition of glutamine. The 100 mM and 1 mM concentrations of H2O2 were not associated with any significant benefit of glutamine on the amount or pattern of cell death-necrosis vs. apoptosis (Fig. 6A and B). Even though ATP levels following exposure to 100 mM and 1 mM H2O2 were increased slightly with 2 mM glutamine present, no significant effect on cell death occurred (data not shown). The pattern of cell death following 5 mM H2O2 injury, however, was significantly altered by the addition of glutamine. A progressive increase in the number of cells dying by apoptosis was seen over the 4-h time course when cellular ATP levels were partially restored by glutamine (Fig. 6C). By 4 h, the percentage of apoptotic cells had increased from 20 to 46%, which was a significant (p , .05) increase, while the necrotic percentage at 4 h decreased from 24 to 14% (Fig. 6C). The lower concentrations of H2O2 (100 mM and 1 mM) induced primarily an apoptotic form of cell death. Endothelial ATP levels were reduced by metabolic inhibition to the range of ATP reduction associated with 5 mM H2O2 in combination with exposure to 100 mM or 1 mM H2O2 to determine if the low ATP levels would shift the pattern of cell death from apoptosis to necrosis. Endothelial ATP levels declined to 0.06 nmol/2 3 106 cells when metabolic inhibition was combined with ex-
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Fig. 6. The effect of modulating ATP levels by glutamine supplementation or metabolic inhibition on the pattern of endothelial cell death following exposure to H2O2. In each panel, hatched bars represent the mean percentage of apoptotic cells and solid bars represent the mean percentage of necrotic cells differentiated by AO-EB staining [25]; n 5 5– 8 separate determinations. (A) Comparison of the effect on cell survival of supplementation with 2 mM glutamine (2) or metabolic inhibition with 650 nM oligomycin in the absence of glucose (3) during injury with 100 mM H2O2 with endothelial cell death after exposure to 100 mM H2O2 alone (1). C 5 control. *p , .05 compared to C (control) and #p , .05 compared to 1 (100 mM H2O2 alone). (B) Comparison of the effect on cell survival of supplementation with 2 mM glutamine (2) or metabolic inhibition with 650 nM oligomycin in the absence of glucose (3) during injury with 1 mM H2O2 with endothelial cell death after exposure to 1 mM H2O2 alone (1). C 5 control. *p , .05 compared to C (Control) and #p , .05 compared to 1 (1 mM H2O2 alone). (C) The effect of supplementation with 2 mM glutamine (2) on endothelial cell survival during injury with 5 mM H2O2. 5 mM H2O2 alone 5 1 and C 5 control. *p , .05 compared to C (control) and #p , 0.05 compared to 1 (5 mM H2O2 alone). Note the conversion from an apoptotic to necrotic pattern of cell death in A and B when ATP depletion was combined with concentrations of H2O2 #1 mM. Also note the shift from necrosis to apoptosis in C when ATP levels were partially repleted by glutamine supplementation in cells injured with 5 mM H2O2.
posure to 100 mM H2O2. H2O2 (1 mM) injury in combination with metabolic inhibition reduced ATP levels to 0.04 nmol/2 3 106 cells. The pattern of cell death, when assessed by fluorescence microscopy of AO-EB stained cells, showed a significant shift from a primarily apoptotic pattern of death to one dominated by necrosis (Fig. 6A and B). Thus, it appears that oxidant stress induces
apoptosis, but the apoptotic pattern of death requires ATP to proceed or necrosis ensues. DISCUSSION
In this study, we have been able to confirm and extend to oxidant injury earlier observations [17–19] that ATP is
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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 mM), 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 endothelial cell death was replaced by necrosis. This was very similar to the pattern of death following exposure to 5 mM H2O2, which provided an oxidant challenge while reducing ATP levels to a similar extent as seen with metabolic inhibition alone. Reduction of ATP alone without oxidant exposure did not significantly alter the background pattern of cell death seen in control cells over the 4-h time course; thus, oxidant exposure appears to be the critical signal for inducing endothelial death in this model. Glutamine supplementation during exposure to the necrosis-inducing concentration of H2O2 (5 mM) increased ATP levels in the lethally injured cells to ;25% of control levels and was sufficient to significantly increase the number of cells entering apoptosis following exposure to this high concentration of the oxidant. Although 1 mM H2O2 reduced cellular ATP to ;50% of control levels, this level of ATP was associated with an apoptotic pattern of death in the endothelial cells. Thus, the approximate threshold level of endothelial ATP required to support significant apoptotic death following exposure to H2O2 is ;25% of the control level, whereas ;50% of the control ATP level is sufficient to fully support apoptosis. This study clarifies earlier observations from our laboratory demonstrating that glutamine via mitochondrial synthesis of ATP prolongs survival of oxidant-injured cells [8,21]. The “prolongation” of survival is a reflection of the shift from a predominantly necrotic to apoptotic pattern of death. Interestingly, glutamine-mediated increases in cellular ATP restore organic anion transport in oxidant-injured cells [22]. Inhibition of organic anion transport alone can cause rapid cell lysis [22]. Alterations in intracellular pH have been associated with both inhibitory and stimulatory effects on the apoptotic process [28,29]. It is possible that failure of organic anion transport in ATP-depleted cells following high level oxidant exposure may lead to a change in intracellular pH inhibitory to apoptosis and, thus, be an important factor in driving the cells toward a necrotic pattern of death. There has been much interest in recent years regarding the nature of the differences between apoptosis and necrosis. Are they separate and distinct pathways of cell death, or are they more closely interconnected? The pattern of DNA degradation on agarose gels (“DNA laddering”) and specific morphologic changes have been commonly used markers to differentiate between apoptosis and necrosis. In two recent studies, Crawford et. al.
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[30,31] have demonstrated that degradation of mitochondrial 16S rRNA is a very early event associated with apoptosis induced by a variety of agents including H2O2. Indeed, if degradation of 16S rRNA is not found to occur during necrosis, it may be the earliest and most sensitive marker for apoptosis yet identified. Hockenbery et. al. [32] and recently our laboratory [33] in different models of cell death have been able to demonstrate the utility of the redox active agent, N-acetyl-L-cysteine, to selectively inhibit apoptosis and support the hypothesis that the two patterns of cell death are distinct. This study in an oxidant-mediated model of endothelial cell death further clarifies this relationship as does other recent work [17– 19], demonstrating that at least in some models the two patterns of cell death might share common initiators (e.g., oxidant stress) and that ATP requirements will ultimately define the final pattern exhibited by the dying cells. Leist and Nicotera [34] have recently reviewed the relationship between necrosis and apoptosis and suggest that they may represent only the extremes of a range of possible biochemical and morphologic patterns of cell death. That the same initiating agent (depending on dose) can induce either apoptosis or necrosis, and that the level of cellular ATP may define which pattern of death occurs, is seen as strong evidence supporting this perspective [34]. Thus, it appears that the necrosis following H2O2 exposure may represent an incomplete execution of the apoptotic program that will otherwise occur if sufficient ATP is present. Apoptosis serves an important role in the economy of tissues by eliminating cells without the attendant risks of an acute inflammatory response associated with necrosis [15]. Oxidant-mediated cell death is a common feature and byproduct of acute inflammation. Agents like glutamine, which may enhance ATP levels in cells and tissues experiencing oxidant stress during acute inflammation, may help to diminish ongoing inflammation and potentially improve function by shifting the predominant pattern of cell death to the noninflammatory apoptosis. Acknowledgements—This work was supported by the U.S. Army Medical Research and Materiel Command under Agreement No. 93-MM3571 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.
ABBREVIATIONS
AO-EB—acridine orange and ethidium bromide ATP—adenosine triphosphate Gln— glutamine H2O2— hydrogen peroxide MGB— modified Grey’s buffer
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MI—metabolic inhibition TCA—tricarboxyclic acid cycle [17] REFERENCES [1] Simon, R. H.; Scoggin, C. H.; Patterson, D. Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals. J. Biol. Chem. 256:7181; 1981. [2] Weiss, S. J.; Young, J.; LoBuglio, A. F.; Slivka, A.; Nimeh, N. H. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. Clin. Invest. 68:714; 1981. [3] Nathan, C. F.; Silversteing, S. L.; Breckner, L. H.; Cohn, Z. A. Extracellular cytolysis by activated macrophages and granuloctyes. II. Hydrogen peroxide as a mediator of cytotoxicity. J. Exp. Med. 149:100; 1979. [4] Martin, W. J., II. Neutrophils kill pulmonary endothelial cells by a hydrogen peroxide dependent pathway. Am. Rev. Respir. Dis. 130:209; 1984. [5] Hyslop, P. A.; Hinshaw, D. B.; Halsey, W. A., Jr.; Schraufstatter, I. U.; Sauerheber, R. D.; Spragg, R. G.; Jackson, J. H.; Cochrane, C. G. Mechanisms of oxidant-mediated cell injury: the glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J. Biol. Chem. 263:1665; 1988. [6] Brodie, A. E.; Reed, D. J. Reversible oxidation of glyceraldehyde 3-phosphate dehydrogenase thiols in human lung carcinoma cells by hydrogen peroxide. Biochem. Biophys. Res. Commun. 148: 120; 1987. [7] Baker, M. S.; Feigan, J.; Lowther, D. A. The mechanism of chondrocyte hydrogen peroxide damage. Depletion of intracellular ATP due to suppression of glycolysis caused by oxidation of glyceraldehyde 3-phosphate dehydrogenase. J. Rheumatol. 16:7; 1989. [8] Hinshaw, D. B.; Burger, J. M.; Delius, R E.; yslop, P.A. Mechanism of protection of oxidant-injured endothelial cells by glutamine. Surgery 108:298 –304; 1990. [9] Schraufstatter, I. U.; Hinshaw, D. B.; Hyslop, P. A.; Spragg, R. G.; Cochrane, C. G. Oxidant injury of cells: DNA strand breaks activate poly-(ADP-ribose) polymerase and lead to depletion of nicotinamide adenine dinucleotide. J. Clin. Invest. 77:1312; 1986. [10] Schraufstatter, I. U.; Hyslop, P. A.; Hinshaw, D. B.; Spragg, R. G.; Sklar, L. A.; Cochrane, C. G. Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly-(ADPribose) polymerase. Proc. Natl. Acad. Sci. USA 83:4908; 1986. [11] Hinshaw, D. B.; Armstrong, B. C.; Burger, J. M.; Beals, T. F.; Hyslop, P. A. ATP and microfilaments in cellular oxidant injury. Am. J. Pathol. 132:479 – 488; 1988. [12] Hinshaw, D. B.; Burger, J. M.; Armstrong, B. C.; Hyslop, P. A. Mechanism of endothelial cell shape change in oxidant injury. J. Surg. Res. 46:3390-349; 1989. [13] Mirabelli, F.; Salis, A.; Marinoni, V.; Finardi, G.; Bellomo, G.; Thor, H.; Orrenius, S. Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin. Arch. Biochem. Biophys. 264:261–269; 1988. [14] Schraufstatter, I. U.; Hinshaw, D. B.; Hyslop, P. A.; Spragg, R. G.; Cochrane, C. G. Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells. J. Clin. Invest. 76:1131–1139, 1985. [15] Kroemer, G.; Petit, P.; Zamzami, N.; Vayssiere, J. L.; Mignotte, B. The biochemistry of programmed cell death. FASEB J. 9:1277–1287; 1995. [16] deBono, D. P.; Yang, W. D. Exposure to low concentrations of
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hydrogen peroxide causes delayed endothelial cell death and inhibits proliferation of surviving cells. Atherosclerosis 114:2350-245; 1995. Leist, M.; Single, B.; Castoldi, A. F.; Kuhnle, S.; Nicotera, P. Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J. Exp. Med. 185:1481–1486; 1997. Eguchi, Y.; Shimizu, S.; Tsujmoto, Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57:1835–1840; 1997. Stefanelli, C.; Bonavita, F.; Stanie, I.; Farruggia, G.; Falcieri, E.; Robuffo, I.; Pignatti, C.; Muscari, C.; Rossoni, C.; Guarnieri, C.; Caldarerra, C. M. ATP depletion inhibits glucocorticoid-induced thymocyte apoptosis. Biochem. J. 322:909 –917; 1997. Kass, G. E. N.; Eriksson, J. E.; Weis, M.; Orrenius, S.; Chow, S. C. Chromatin condensation during apoptosis requires ATP. Biochem. J. 318:749 –752; 1996. Hinshaw, D. B.; Burger, J. M. Protective effect of glutamine on endothelial cell ATP in oxidant injury. J. Surg. Res. 49:222–227; 1990. Hinshaw, D. B.; Burger, J. M.; Delius, R. E.; Hyslop, P. A.; Omann, G. M. Inhibition of organic anion transport in endothelial cells by hydrogen peroxide. Arch. Biochem. Biophys. 298:464 – 470; 1992. Hinshaw, D. B.; Burger, J. M.; Miller, M. T.; Adams, J. A.; Beals, T. F.; Omann, G. M. ATP depletion induces an increase in the assembly of a labile pool of polymerized actin in endothelial cells. Am. J. Physiol. 264:C1171–C1179; 1993. Ruoslathi, E.; Reed, J. C. Anchorage dependence, integrins, and apoptosis. Cell 77:477– 478; 1994. Leighton, B.; Cari, R.; Hussein, A.; Newsholme, E. A. Maximum activities of some key enzymes of glycolysis, glutaminolysis, Krebs cycle and fatty acid utilization in bovine pulmonary endothelial cells. FEBS Lett. 225:93–96; 1987. Duke, R. C.; Cohen, J. J. Morphology and biochemical assays of apoptosis. Curr. Protein Immunol. 17(Suppl. 3):1–16; 1992. Laird, P. W.; Zijderveld, A.; Linders, K.; Rudnicki, M. A.; Jaenisch, R.; Berns, A. Simplified mammalian DNA isolation procedure. Nucleic Acid. Res. 19:4293; 1991. Perez-Sala, D.; Collado-Escobar, D.; Mollinedo, F. Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease. J. Biol. Chem. 270:6235– 6242; 1995. Zhu, W.-H.; Loh, T.-T. Effects of Na1/H1 antiport and intracellular pH in the regulation of HL-60 cell apoptosis. Biochim. Biophys. Acta 1269:122–128; 1995. Crawford, D. R.; Wang, Y.; Schools, G. P.; Kochheiser, J.; Davies, K. J. A. Down-regulation of mammalian mitochondrial RNAs during oxidative stress. Free Radic. Biol. Med. 22:551– 559; 1997. Crawford, D. R.; Lanzon, R. J.; Wang, Y.; Mazurkiewicz, J. E.; Schools, G. P.; Davies, K. J. A. 16S mitochondrial ribosomal RNA degradation is associated with apoptosis. Free Radic. Biol. Med. 22:1295–1300; 1997. Hockenbery, D. M.; Altvai, Z. N.; Yin, X. M.; Milliman, C. L.; Korsmeyer, S. J. BC1-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251; 1993. Dabrowska, M. I.; Becks, L. L.; Lelli, J. L.; Levee, M. G.; Hinshaw, D. B. Sulfur mustard induces apoptosis and necrosis in endothelial cells. Toxicol. Appl. Pharmacol. 141:568 –583; 1996. Leist, M.; Nicotera, P. The shape of cell death. Biochem. Biophys. Res. Commun. 236:1–9; 1997.
PII S0891-5849(98)00107-5
Original Contribution ATP CONVERTS NECROSIS TO APOPTOSIS IN OXIDANT-INJURED ENDOTHELIAL CELLS JOSEPH L. LELLI, JR.,* LAUREN L. BECKS,† MILENA I. DABROWSKA,†
and
DANIEL B. HINSHAW†
*Sections of Pediatric and General Surgery, Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, USA, and †Department of Veterans Affairs Medical Center, Ann Arbor, MI, USA (Received 19 December 1997; Revised 14 April 1998; Accepted 23 April 1998)
Abstract—Cell death due to necrosis results in acute inflammation, while death by apoptosis generally does not. The effect of adenosine triphosphate (ATP) on the pattern of cell death induced by oxidants was examined in bovine endothelial cells. ATP levels were altered by hydrogen peroxide (H2O2), glutamine (Gln), and metabolic inhibition (MI), to determine if necrosis can be shifted to apoptosis during oxidant injury. The form of cell death was determined by fluorescence microscopic techniques and the pattern of DNA degradation on agarose gels. ATP levels were measured using the luciferase–luciferin assay. Apoptosis occurred with 100 mM H2O2 without an alteration in ATP levels. ATP was significantly lowered with 5 mM H2O2, and necrosis occurred. MI, in combination with 100 mM H2O2, decreased ATP and resulted in necrosis. MI alone, however, did not cause cell death. Gln partially restored ATP levels in cells injured with 5 mM H2O2 and resulted in a significant increase in apoptosis. DNA laddering on agarose gels confirmed the apoptotic changes seen by fluorescence microscopy. In summary, a threshold level of ATP 25% of basal levels is required for apoptosis to proceed after oxidant stress, otherwise necrosis occurs. Agents like glutamine that enhance ATP levels in oxidant-stressed cells may be potent means of shifting cell death during inflammation to the noninflammatory form of death—apoptosis. © 1998 Elsevier Science Inc. Keywords—ATP, Necrosis, Apoptosis, Glutamine, Hydrogen peroxide, Endothelial cells, Free radical
INTRODUCTION
phologic 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
Reactive oxygen species including hydrogen peroxide (H2O2) generated by neutrophils and macrophages are important mediators of cellular injury during acute inflammation [1– 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– 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 [11–13], 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 morAddress correspondence to: Daniel B. Hinshaw, M.D., VA Medical Center, 2215 Fuller Road (11), Ann Arbor, MI 48105, USA; Tel: (313) 761-7903; Fax: (313) 769-7412; E-Mail: [email protected] 694
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treatments (i.e., anti-Fas monoclonal antibody CH11, Ca11 ionophore, etoposide, dexamethasone, staurosporin, or CD95 stimulation) requires ATP [17–19]. 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 a-ketoglutarate, can act as an alternative fuel substrate in endothelial cells lethally injured with H2O2 [8,21]. The glutamine bypassed the blockade of glucosedependent 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 [8,21–23] 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. MATERIALS AND METHODS
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-2ethanesulfonic acid (HEPES), 100 U/ml penicillin, and 100 mg/ml streptomycin (GIBCO). Cells were cultured under 5% CO2/95% air at 37°C in 75- or 150-cm2 flasks (Corning, Corning, NY). Experiments were carried out using cells from passages 2–9, suspended in modified Geys buffer (MGB) containing, in mM, 147 NaCl, 5
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KCl, 0.22 KH2PO4, 1.0 MgCl, 0.28 MgSO4, 10 HEPES, 1.5 CaCl2, and 5.5 glucose at pH 7.4. In experiments where oligomycin was used, glucose was omitted from the MGB. In experiments in which glutamine supplementation occurred, a final concentration of 2 mM glutamine was present in the MGB [6,21]. Injury protocol with hydrogen peroxide Upon reaching confluence, endothelial cells were suspended by exposure to 0.05% trypsin and 0.02% EDTA for 10 –15 min. The cells were washed in MGB and resuspended in MGB 1 glucose at a concentration of 2 3 106/ml. H2O2 was added at final concentrations of 100 mM, 1 mM, and 5 mM, and the cells were observed over a 4-h time course. Endothelial cells in suspension were utilized throughout the experiments to be consistent with our earlier work [8,21,22]. Selected experiments were also done with adherent endothelial cells that demonstrated similar findings to those reported here, except that the adherent endothelial cells were somewhat more resistant to a given concentration of H2O2. For example, whereas 40% of suspended endothelial cells were apoptotic 4 h after exposure to 100 mM H2O2 (see Fig. 2A below), 19 6 9.9% (n 5 6) of adherent endothelial cells 4 h after exposure to 100 mM H2O2 were apoptotic. By 6 h, the adherent cells were 34 6 25.6% (n 5 5) apoptotic after exposure to 100 mM H2O2, demonstrating the great variability in the timing of apoptosis in adherent endothelial cells. These findings are consistent with the observation of others that cellular suspension itself may promote cell death signaling [24] and, in this experimental model, may account for a more rapid and synchronous apoptotic process. Treatment with glutamine and metabolic inhibition Endothelial cells contain high levels of glutaminase activity [25], suggesting they possess an enhanced ability to utilize glutamine for anabolic and potentially catabolic purposes. Attempts to use other mitochondrial substrates (e.g., acetoacetate and succinate) have demonstrated no significant enhancement of ATP levels in endothelial cells injured with H2O2 (data not shown). In our earlier work, we were able to enhance ATP levels in the injured endothelial cells in a dose-dependent manner achieving a maximal effect with 2 mM glutamine [21]. Thus, in experiments in which glutamine was used, endothelial cells were suspended in MGB containing 2 mM glutamine (Sigma) and then exposed to H2O2 (0 –5 mM). In other experiments in which the cells were metabolically inhibited, the cells were treated with 650 nM oligomycin (Sigma), an inhibitor of the mitochondrial Fo
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ATP-synthase, in the absence of glucose [23]. In samples in which H2O2 injury was combined with metabolic inhibition, the oligomycin was added 30 min after addition of H2O2 to allow for H2O2-mediated effects on cell death signaling to be initiated before full metabolic inhibition occurred. Samples for ATP measurement were taken 1– 4 h after H2O2 addition. Fluorescence microscopic assay of apoptotic nuclear features [26] The nuclear morphology and staining pattern of suspended endothelial cells was examined using a Nikon optiphot fluorescence microscope at each time point following exposure of the cells to the dyes acridine orange (AO) and ethidium bromide to determine the percentage of cells undergoing nuclear changes characteristic of apoptosis [26] A dye mixture made up of 10 mM acridine orange and 10 mM ethidium bromide prepared in phosphate-buffered saline was used. Acridine orange (a fluorescent DNA-binding dye) intercalates into DNA, making it appear green, and binds to RNA, staining it red-orange. Ethidium bromide is taken up only by the nuclei of nonviable cells, and its fluorescence overwhelms that of the acridine orange, making the chromatin of lysed cells appear orange [26]. At each time point, 25 ml of cell sample was mixed with 1 ml of the dye mix and 15 ml placed on a microscope slide with coverslip. Two hundred cells per sample were examined by fluorescence microscopy, according to the following criteria: (1) viable cells with normal nuclei (fine reticular pattern of green stain in the nucleus and red-orange granules in the cytoplasm); (2) viable cells with apoptotic nuclei (green chromatin which is highly condensed or fragmented and uniformly stained by the acridine orange); (3) nonviable cells with normal nuclei (bright orange chromatin with organized structure); and (4) nonviable cells with apoptotic nuclei (bright orange chromatin that is highly condensed or fragmented). The combination of cells meeting criteria 2 and 4 represented the total apoptotic cell population, because apoptotic cells eventually lose plasma membrane integrity in vitro. Criterion 3 was used to define the population of cells undergoing necrosis without evidence of apoptosis.
for 1 h. Then, an equal amount of cold isopropanol was added and the samples were mixed by inversion until formation of precipitate. At this point, the DNA was centrifuged for 30 min at 12,000 3 g, the supernatant removed, the pellet of DNA air dried for 30 min, and then resuspended in 150 ml of TE buffer (10 mM Tris, 1.0 mM EDTA, pH 7.6) with 40 mg/ml RNase (Sigma) and left to incubate at room temperature overnight. Sample buffer (0.25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol dissolved in deionized H2O) was added to the RNase treated samples, which were then run on a 1.6% agarose gel containing 7 mg ethidium bromide, at 120 mV for 1.5 h. The gel was then photographed under ultraviolet light, to visualize the DNA for evidence of endonucleolytic cleavage (“DNA laddering”), characteristic of apoptosis [26].
ATP measurement Cellular ATP levels were assayed as previously reported using the luciferase–luciferin method [8]. Endothelial cells were suspended at a concentration of 2.0 3 106 cells/ml and exposed to the experimental conditions. Measurements of ATP were performed using the luciferase–luciferin assay [8] at the different experimental time points.
Statistics Analysis of variance (one-way ANOVA) and paired t-tests were used for comparisons between experimental and control observations. Results are presented as means 6 SEM for five to eight experiments unless otherwise noted.
Measurement of endonucleolytic DNA cleavage [27] At each experimental time point, 2 3 106 suspended endothelial cells were harvested by centrifugation at 4000 3 g for 4 min, the supernatant removed, and the cells were resuspended in 500 ml of lysis buffer (100 mM TRIS, 5 mM EDTA, 200 mM NaCl, 0.2% SDS, pH 8.5). Next, 100 mg/ml Proteinase K (Sigma) was added to the samples and they were left to shake in a 37°C water bath
Fig. 1. Viability of uninjured control endothelial cells. Each bar represents the mean 1 SEM of N 5 8 separate determinations. Apoptotic and necrotic cells were differentiated using AO-EB staining [26]. Note the small background level of spontaneous apoptosis and necrosis occurring in the cells.
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Fig. 2. Viability of endothelial cells after exposure to different concentrations of H2O2: (A) 100 mM H2O2; (B) 1 mM H2O2; and (C) 5 mM H2O2. Each bar represents the mean 1 SEM of n 5 5– 8 separate determinations. Open bars—percentage of apoptotic cells; solid bars—percentage of necrotic cells determined using AO-EB staining [26]. *p , .05 compared to control in Fig. 1. Note the predominantly apoptotic pattern of endothelial cell death seen with H2O2 concentrations , 1 mM.
RESULTS
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 following staining with AO-EB characteristic for apoptotic cell death as early as 1 h, and demonstrated significant increases in apoptotic cells by 2 h after H2O2 exposure (Fig. 2A and B). At concentrations of H2O2 # 1 mM, there was no significant increase in the percentage of cells dying by necrosis as compared to the control. The percentage of cells undergoing apo-
ptosis after exposure to concentrations of H2O2 # 1 mM was significantly higher than that of the control cells (p , .05). At the highest concentration of H2O2 (5 mM), the pattern of cell death was predominantly necrotic (Fig. 2C). Compared to control cells and to the lower concentrations of H2O2, there was no significant increase in the percentage of apoptotic cells with 5 mM H2O2. At 4 h, 5 mM H2O2 caused a significant increase (p , .05) in the percentage of necrotic cells compared to the control and concentrations of H2O2 # 1 mM (Figs. 1 and 2). In longer time course experiments (data not shown), the dose-related demarcation in the pattern of cell death, necrosis, or apoptosis, remained consistent for 8 h. The AO-EB staining demonstrated that apoptotic bodies were formed with viable membranes containing fragmented DNA that excluded ethidium bromide. Agarose gel electrophoresis of DNA isolated from the endothelial cells confirmed the presence of apoptosis in the injured cell population (Fig. 3). Exposure to either 100 mM or 1 mM H2O2 resulted in the endonucleolytic
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Fig. 3. Agarose gel electrophoresis of DNA extracted from endothelial cells. Lane 1: DNA molecular size marker (Hind III/Hae III; GIBCO); lanes 2–5: DNA from control cells at 1, 2, 3, and 4 h, respectively; lanes 6 –9: DNA from cells exposed to 100 mM H2O2 at 1, 2, 3, and 4 h, respectively; lanes 10 –13: DNA from cells exposed to 1 mM H2O2 at 1, 2, 3, and 4 h, respectively; and lanes 14 –17: DNA from cells exposed to 5 mM H2O2 for 1, 2, 3, and 4 h, respectively. Note the distinct presence of “DNA ladders” by 3 h following exposure to H2O2 concentrations #1 mM. Changes in the DNA of cells injured with 5 mM H2O2 (lanes 16 and 17) were more typical of DNA smearing seen with necrosis.
cleavage of DNA into multiples of the 180 base-pair nucleosomes, “DNA laddering,” as early as 2 h after injury. Injury with 5 mM H2O2 was associated with only slight evidence of endonuclease activity (Fig. 3). Effects of H2O2 on cellular ATP levels Endothelial cells exposed to the different concentrations of H2O2 (0 –5 mM) were assayed for ATP using the luciferase–luciferin assay [8]. Previous work demonstrated an inhibitory effect of H2O2 on glycolysis in endothelial cells [8] secondary to inhibition of glyceraldehyde 3-phosphate dehydrogenase [5], with a resulting decline in cellular ATP levels. In this study, control cells showed no change in ATP levels over the time course (Fig. 4). Exposure to 100 mM H2O2 did not significantly alter ATP levels over the 4-h time course. Exposure to 1 mM H2O2 resulted in a moderate reduction of ATP levels from 8.0 (control level at time of injury) to 4.13 nmol/2 3 106 cells by 4 h, representing a decline in ATP of 48% (Fig. 4). H2O2 (5 mM) caused a rapid and dramatic reduction in ATP levels (from 8.0 to 0.77nmol/2 3 106 cells), which was significantly lower (p , .05) than the levels observed after exposure to both 100 mM and 1 mM H2O2 (Fig. 4). The reduction in ATP levels following exposure to 1 mM H2O2 was statistically significant only after 4 h (Fig. 4). Thus, a H2O2 concentration (100 mM) associated with no measurable reduction in endothelial ATP levels was almost exclusively correlated with apoptosis, whereas a concentration of H2O2 (5 mM) that caused profound reduction of endothelial ATP levels (#10% of control) was correlated with the appearance of a necrotic pattern of cell death. H2O2 (1 mM), which had an intermediate
effect on endothelial ATP levels, was correlated primarily with apoptosis. Modeling the effect of ATP depletion and repletion during H2O2 injury We hypothesized that ATP depletion, per se, did not account for the cell death signal associated with H2O2 exposure, because 100 mM H2O2 was sufficient to induce cell death without reduction of cellular ATP levels. Rather, ATP might act as a “switch” determining which pattern of cell death would ensue. Oligomycin, an inhibitor of the mitochondrial Fo ATP synthase in combination with glucose deprivation [23], was used to inhibit ATP synthesis without delivering an exogenous oxidant stress to the cells. Endothelial cells were incubated with 650 nM oligomycin in the absence of glucose, and the effects of this metabolic inhibition on ATP and cell viability were determined. ATP levels were assayed using the luciferase–luciferin assay [8]. Metabolic inhibition (MI) caused a rapid
Fig. 4. The effect of 0 –5 mM H2O2 on endothelial ATP levels over a 4-h time course. ATP levels were measured with the luciferin–luciferase assay [8]. Each bar represents the mean 1 SEM of n 5 5– 8 separate determinations. C 5 control; 0.1 5 0.1 mM (100 mM) H2O2, 1 5 1 mM H2O2, and 5 5 5 mM H2O2. *p , .05 compared to control.
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Fig. 5. Modeling ATP depletion and partial repletion during H2O2 injury. ATP levels were measured with the luciferin–luciferase assay [8]. Each bar represents the mean 1 SEM of n 5 5– 8 separate determinations. (A) Comparison of the effect of metabolic inhibition with 650 nM oligomycin in the absence of glucose with 5 mM H2O2 on endothelial cell ATP levels. *p , .05 compared to control. (B) The effect of supplementation with 2 mM glutamine on endothelial ATP levels during injury with 5 mM H2O2 (5 mM 1 glutamine) vs. the effect of 5 mM H2O2 alone (5 mM). *p , .05 compared to 5 mM.
decrease in cellular ATP levels from 8.0 to 0.13 nmol/2 3 106 cells, a decrease similar to that seen with H2O2 concentrations (e.g., 5 mM) associated with necrosis (Fig. 5A). Fluorescence microscopy using the AO-EB staining criteria was used to characterize and quantitate cell death under the conditions of metabolic inhibition. At 4 h, 16% (compared to 12.7% in controls) of the cells undergoing metabolic inhibition alone with oligomycin minus glucose died by necrosis (data not shown). The cells dying after metabolic inhibition alone were predominantly necrotic with little apoptosis occurring. There was no significant increase in cell death compared to controls. ATP depletion with metabolic inhibition alone in the absence of an oxidant stress did not induce the significant cell death seen with 5 mM H2O2, nor did it induce apoptosis. H2O2 interferes with glycolysis by inhibiting glyceraldehyde-3-phosphate dehydrogenase, which is a proximal event in glycolysis prior to entering into the tricarboxyclic acid (TCA) cycle [5,8]. Glutamine enters into the TCA cycle as an intermediate metabolite (i.e., a-ketoglutarate), and has been shown to partially restore ATP production in cells that have undergone injury with H2O2 [8,21]. Endothelial cells were incubated with 2 mM glutamine [8,21] prior to exposure to H2O2 at varying concentrations. Using the luciferase–luciferin assay [8], ATP levels were determined throughout the four hour time course. Control cell ATP levels were not altered by the addition of glutamine (data not shown). ATP levels that fell from 8.0 to 0.77 nmol/2 3 106 cells following exposure to 5 mM H2O2 were partially restored to 2.0 nmol/2 3 106 cells, a statistically significant (p , .05) increase at 1, 2, and 4 h after injury (Fig. 5B).
Effect of modulating ATP levels by glutamine supplementation or metabolic inhibition on the pattern of cell death following exposure to H2O2 Fluorescence microscopy with the AO-EB staining assay was used to determine the viability and cell death pattern of endothelial cells that were incubated in 2 mM glutamine and then injured with a range of concentrations of H2O2. Control cells demonstrated a minimal death rate over the 4-h time course that was not affected by the addition of glutamine. The 100 mM and 1 mM concentrations of H2O2 were not associated with any significant benefit of glutamine on the amount or pattern of cell death-necrosis vs. apoptosis (Fig. 6A and B). Even though ATP levels following exposure to 100 mM and 1 mM H2O2 were increased slightly with 2 mM glutamine present, no significant effect on cell death occurred (data not shown). The pattern of cell death following 5 mM H2O2 injury, however, was significantly altered by the addition of glutamine. A progressive increase in the number of cells dying by apoptosis was seen over the 4-h time course when cellular ATP levels were partially restored by glutamine (Fig. 6C). By 4 h, the percentage of apoptotic cells had increased from 20 to 46%, which was a significant (p , .05) increase, while the necrotic percentage at 4 h decreased from 24 to 14% (Fig. 6C). The lower concentrations of H2O2 (100 mM and 1 mM) induced primarily an apoptotic form of cell death. Endothelial ATP levels were reduced by metabolic inhibition to the range of ATP reduction associated with 5 mM H2O2 in combination with exposure to 100 mM or 1 mM H2O2 to determine if the low ATP levels would shift the pattern of cell death from apoptosis to necrosis. Endothelial ATP levels declined to 0.06 nmol/2 3 106 cells when metabolic inhibition was combined with ex-
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Fig. 6. The effect of modulating ATP levels by glutamine supplementation or metabolic inhibition on the pattern of endothelial cell death following exposure to H2O2. In each panel, hatched bars represent the mean percentage of apoptotic cells and solid bars represent the mean percentage of necrotic cells differentiated by AO-EB staining [25]; n 5 5– 8 separate determinations. (A) Comparison of the effect on cell survival of supplementation with 2 mM glutamine (2) or metabolic inhibition with 650 nM oligomycin in the absence of glucose (3) during injury with 100 mM H2O2 with endothelial cell death after exposure to 100 mM H2O2 alone (1). C 5 control. *p , .05 compared to C (control) and #p , .05 compared to 1 (100 mM H2O2 alone). (B) Comparison of the effect on cell survival of supplementation with 2 mM glutamine (2) or metabolic inhibition with 650 nM oligomycin in the absence of glucose (3) during injury with 1 mM H2O2 with endothelial cell death after exposure to 1 mM H2O2 alone (1). C 5 control. *p , .05 compared to C (Control) and #p , .05 compared to 1 (1 mM H2O2 alone). (C) The effect of supplementation with 2 mM glutamine (2) on endothelial cell survival during injury with 5 mM H2O2. 5 mM H2O2 alone 5 1 and C 5 control. *p , .05 compared to C (control) and #p , 0.05 compared to 1 (5 mM H2O2 alone). Note the conversion from an apoptotic to necrotic pattern of cell death in A and B when ATP depletion was combined with concentrations of H2O2 #1 mM. Also note the shift from necrosis to apoptosis in C when ATP levels were partially repleted by glutamine supplementation in cells injured with 5 mM H2O2.
posure to 100 mM H2O2. H2O2 (1 mM) injury in combination with metabolic inhibition reduced ATP levels to 0.04 nmol/2 3 106 cells. The pattern of cell death, when assessed by fluorescence microscopy of AO-EB stained cells, showed a significant shift from a primarily apoptotic pattern of death to one dominated by necrosis (Fig. 6A and B). Thus, it appears that oxidant stress induces
apoptosis, but the apoptotic pattern of death requires ATP to proceed or necrosis ensues. DISCUSSION
In this study, we have been able to confirm and extend to oxidant injury earlier observations [17–19] that ATP is
ATP/necrosis/apoptosis
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 mM), 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 endothelial cell death was replaced by necrosis. This was very similar to the pattern of death following exposure to 5 mM H2O2, which provided an oxidant challenge while reducing ATP levels to a similar extent as seen with metabolic inhibition alone. Reduction of ATP alone without oxidant exposure did not significantly alter the background pattern of cell death seen in control cells over the 4-h time course; thus, oxidant exposure appears to be the critical signal for inducing endothelial death in this model. Glutamine supplementation during exposure to the necrosis-inducing concentration of H2O2 (5 mM) increased ATP levels in the lethally injured cells to ;25% of control levels and was sufficient to significantly increase the number of cells entering apoptosis following exposure to this high concentration of the oxidant. Although 1 mM H2O2 reduced cellular ATP to ;50% of control levels, this level of ATP was associated with an apoptotic pattern of death in the endothelial cells. Thus, the approximate threshold level of endothelial ATP required to support significant apoptotic death following exposure to H2O2 is ;25% of the control level, whereas ;50% of the control ATP level is sufficient to fully support apoptosis. This study clarifies earlier observations from our laboratory demonstrating that glutamine via mitochondrial synthesis of ATP prolongs survival of oxidant-injured cells [8,21]. The “prolongation” of survival is a reflection of the shift from a predominantly necrotic to apoptotic pattern of death. Interestingly, glutamine-mediated increases in cellular ATP restore organic anion transport in oxidant-injured cells [22]. Inhibition of organic anion transport alone can cause rapid cell lysis [22]. Alterations in intracellular pH have been associated with both inhibitory and stimulatory effects on the apoptotic process [28,29]. It is possible that failure of organic anion transport in ATP-depleted cells following high level oxidant exposure may lead to a change in intracellular pH inhibitory to apoptosis and, thus, be an important factor in driving the cells toward a necrotic pattern of death. There has been much interest in recent years regarding the nature of the differences between apoptosis and necrosis. Are they separate and distinct pathways of cell death, or are they more closely interconnected? The pattern of DNA degradation on agarose gels (“DNA laddering”) and specific morphologic changes have been commonly used markers to differentiate between apoptosis and necrosis. In two recent studies, Crawford et. al.
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[30,31] have demonstrated that degradation of mitochondrial 16S rRNA is a very early event associated with apoptosis induced by a variety of agents including H2O2. Indeed, if degradation of 16S rRNA is not found to occur during necrosis, it may be the earliest and most sensitive marker for apoptosis yet identified. Hockenbery et. al. [32] and recently our laboratory [33] in different models of cell death have been able to demonstrate the utility of the redox active agent, N-acetyl-L-cysteine, to selectively inhibit apoptosis and support the hypothesis that the two patterns of cell death are distinct. This study in an oxidant-mediated model of endothelial cell death further clarifies this relationship as does other recent work [17– 19], demonstrating that at least in some models the two patterns of cell death might share common initiators (e.g., oxidant stress) and that ATP requirements will ultimately define the final pattern exhibited by the dying cells. Leist and Nicotera [34] have recently reviewed the relationship between necrosis and apoptosis and suggest that they may represent only the extremes of a range of possible biochemical and morphologic patterns of cell death. That the same initiating agent (depending on dose) can induce either apoptosis or necrosis, and that the level of cellular ATP may define which pattern of death occurs, is seen as strong evidence supporting this perspective [34]. Thus, it appears that the necrosis following H2O2 exposure may represent an incomplete execution of the apoptotic program that will otherwise occur if sufficient ATP is present. Apoptosis serves an important role in the economy of tissues by eliminating cells without the attendant risks of an acute inflammatory response associated with necrosis [15]. Oxidant-mediated cell death is a common feature and byproduct of acute inflammation. Agents like glutamine, which may enhance ATP levels in cells and tissues experiencing oxidant stress during acute inflammation, may help to diminish ongoing inflammation and potentially improve function by shifting the predominant pattern of cell death to the noninflammatory apoptosis. Acknowledgements—This work was supported by the U.S. Army Medical Research and Materiel Command under Agreement No. 93-MM3571 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.
ABBREVIATIONS
AO-EB—acridine orange and ethidium bromide ATP—adenosine triphosphate Gln— glutamine H2O2— hydrogen peroxide MGB— modified Grey’s buffer
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