Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

Abstract

Hypoxia/reoxygenation induces cellular injury by promoting oxidative stress. Reversible oxidation of methionine in proteins involving the enzyme peptide methionine sulfoxide reductase type A (MSRA) is postulated to serve a general antioxidant role. Therefore, we examined whether overexpression of MSRA protected cells from hypoxia/reoxygenation injury. Brief hypoxia increased the intracellular reactive oxygen species (ROS) level in PC12 cells and promoted apoptotic cell death. Adenovirus-mediated overexpression of MSRA significantly diminished the hypoxia-induced increase in ROS and facilitated cell survival. Measurements of the membrane potentials of intact mitochondria in PC12 cells and of isolated rat liver mitochondria showed that hypoxia induced depolarization of the mitochondrial membrane. The results demonstrate that MSRA plays a protective role against hypoxia/reoxygenation-induced cell injury and suggest the therapeutic potential of MSRA in ischemic heart and brain disease.

Reactive oxygen species (ROS) promote oxidative damage to many cellular constituents, including amino acids, lipids, and nucleic acids, and play critical roles in aging and senescence-associated disorders (1–3). ROS are also likely mediators of acute cellular injury events caused by ischemia/hypoxia (4). Reperfusion after an ischemic/hypoxic episode dramatically increases the overall cellular oxidant level (4). In addition, the oxidant level may increase at least transiently during ischemia/hypoxia before reperfusion (5, 6). To protect against the oxidative insults induced by a variety of causes, including ischemia/reperfusion, cells contain multiple “antioxidant” mechanisms (1, 2). For example, superoxide dismutase, catalase, and glutathione peroxidase scavenge the superoxide anion and H₂O₂ to prevent ROS-induced damages. Nonenzymatic ROS scavengers, such as vitamins E and C, also contribute to the total antioxidant capacity (7).

The amino acid methionine, both free and in peptide linkage, is readily oxidized by ROS, leading to the formation of the R and S epimers of methionine sulfoxide (met-O) (8). Reduction of the S form of met-O in proteins is catalyzed by the enzyme peptide methionine sulfoxide reductase A (MSRA) (9–11), whereas the R form is reduced by methionine sulfoxide reductase B (MSRB) (11–15). At least one major variant of human MSRA is preferentially localized in mitochondria, and its N terminus is important in this subcellular localization (16).

Oxidation of selected methionine residues in some proteins, including K⁺ channels (17) and calmodulin (18), drastically alters their function, suggesting that methionine oxidation and MSRA may have a role in cellular signal transduction (19). Methionine oxidation in other proteins, such as glutamine synthetase, however, does not cause any noticeable functional change (20). This observation led to the speculation that a reversible oxidation–reduction cycle of methionine involving MSRA may also act as a general antioxidant mechanism, functioning as a sink for ROS to protect other cellular components (20).

The importance of ROS in ischemia/hypoxia-induced cellular injury and the postulated antioxidant potential of MSRA suggest that overexpression of MSRA may protect cells from hypoxia/reoxygenation-mediated cell injury. We tested this hypothesis by inducing hypoxia in PC12 cells overexpressing MSRA.

Materials and Methods

PC12 Cells. PC12 cells were cultured at 37°C in 10% CO₂ without any added nerve growth factor as described (21). The cell culture medium contained Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% FBS.

Overexpression of MSRA. Enhanced green fluorescent protein (EGFP), bovine MSRA (bMSRA) (22), and EGFP–bMSRA, where EGFP is fused to the N terminus of bMSRA (23), were overexpressed by using the adenovirus-mediated gene transfer method (24). The gene coding sequences were inserted by PCR into the shuttle plasmid vector pacAd5CMV with a cytomegalovirus promoter (24). The gene coding sequences were verified. The recombinant virus particles were prepared by the University of Iowa Gene Transfer Vector Core. Functional overexpression of MSRA in PC12 cells was confirmed by using an assay for MSRA based on the reduction of N-[³H]acetyl methionine sulfoxide (25). Controls contained the virus lacking an insert.

Determination of met-O Levels. PC12 cells were treated with 90% N₂:10% CO₂ (hypoxia), 90% O₂:10% CO₂ (hyperoxia), or 90% air:10% CO₂ in a commercially available gas-control chamber (Billups-Rothenberg, Del Mar, CA) for 10 min. The cells were allowed to recover in the standard growth condition for 24 h, and then they were harvested. The cells were then suspended in 20 mM Tris·Cl, pH 7.4, and disrupted by freezing and thawing three times. The cell suspension was centrifuged at 12,000 × g, and the supernatant was removed. An aliquot of the supernatant was incubated with 4 μg of Pronase for 16 h at 37°C. The mixture was heated at 100°C for 1 min and then centrifuged to remove insoluble material. The supernatant was analyzed for its amino acid composition by a Beckman 7300 amino acid analyzer. The two epimers of met-O were the first amino acids to emerge from the column and readily resolve from the other amino acids. The percentage of met-O present in the samples was calculated as pmol met-O/(pmol methionine + met-O). The estimated values would include any free met-O that might be present in the cytoplasm.

Trypan Blue Cell Viability Assay. The cells in the culture medium (21) were challenged with hypoxia (90% N₂:10% CO₂), hyperoxia (90% O₂:10% CO₂), or normoxia (90% air:10% CO₂) for 5 min. The cell culture medium was removed 24 h later, and 30 μl of trypan blue solution [0.4% in PBS (Sigma)] was applied to the cells on glass coverslips for 5 min. PBS (1 ml) was then added, and the number of stained and nonstained cells in four to five randomly selected fields on each coverslip was counted. Typically, 300–500 cells were counted in each group. Ethanol (1%) was dissolved with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL, Calbiochem), the solution of which was present for 1 h before the start of the experimental treatments and then washed out after the treatments.

ROS Measurements. The fluorescent dye dihydrorhodamine (DHR) 123 (Calbiochem) was used to estimate the ROS level (21). The excitation and emission wavelengths were 500 nm and 530 nm, respectively. PC12 cells harvested from a confluent flask were resuspended in the standard saline solution (see above), and DHR123 (10 mM stock in DMSO) was added to achieve a final concentration of 10 or 20 μM. To measure the ROS production during normoxia/hyperoxia/hypoxia, the cells were loaded with DHR123 for 5 min at room temperature, centrifuged, resuspended in a 3-ml cuvette containing the solution that had been bubbled with air, O₂, or N₂, and immediately placed in the spectrofluorometer (FP-750, JASCO, Tokyo). Because multiple ROS convert DHR123 to the stable fluorescent derivative DHR (26), the slope of signal at 530 nm during the first 4 min was used to estimate the overall ROS production rate. Cells were perfused with the standard saline solution [(in mM) 140 NaCl/5 KCl/1 MgCl₂/1.5 CaCl₂/5 glucose/10 Hepes, pH 7.4] that had been bubbled with 100% N₂, O₂, or air for at least 30 min to induce hypoxia, hyperoxia, or normoxia, respectively. The O₂ pressure level of the solution bubbled with N₂ in the recording chamber was ≈25 mmHg (1 mmHg = 133 Pa) as measured by an oxygen meter (ISO2, WPI, Sarasota, FL). To measure the ROS level on reoxygenation after hypoxia, the cells were subjected to hypoxia for 5 min, washed with the normoxia solution, and placed in the spectrofluorometer. The measurements presented here represent the ROS levels during the first 2–6 min after reoxygenation.

Apoptosis and Necrosis Assays. Fractions of the cells undergoing apoptosis and necrosis were determined by FACS with a commercially available kit that uses Annexin-V-FLUOS and propidium iodide (PI) (Roche Applied Science). The cells in the culture medium were challenged with hypoxia (90% N₂:10% CO₂), hyperoxia (90% O₂:10% CO₂), or normoxia (90% air:10% CO₂) for 10 min, allowed to recover, and then harvested 24 h later. Approximately 10⁶ cells in each condition were washed with PBS and centrifuged for 2 min. The cell pellet was resuspended in 100 μl of Annexin-V-FLUOS- and PI-labeling solutions and incubated for 10–15 min at 15–20°C. The mixture was analyzed by a FACScan flow cytometer (University of Pennsylvania Cancer Center Flow Cytometry and Cell Sorter Shared Resource) with a 488-nm excitation and a 515-nm (30-nm bandwidth) filter for Annexin-V-FLUORS and a 585-nm (42-nm bandwidth) filter for PI.

Intact Mitochondria Imaging. PC12 cells were plated on glass coverslips coated with poly-l-lysine (Sigma) the day before the experiments. The culture medium was replaced with the standard saline solution containing a potential-dependent mitochondria-targeted J-aggregate-forming fluorescent dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine for 15 min (10 μg/ml; JC-1, Molecular Probes). The cells were treated with the saline solution that had been previously bubbled with air, O₂,orN₂. Fluorescence imaging was performed at room temperature as described (27) by using a Nikon Eclipse TE200, Lambda DG-4 filter changer with a 175-W xenon lamp (Sutter Instruments, Novato, CA), a ×100 super fluorescence objective (Nikon, Melville, NY), dichroic mirror Quad Set 84000, and a S490/20× filter for the green signal and a S555/28× filter for the red signal (Chroma Technology, Brattleboro, VT). Fluorescence was captured with a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI), and image collection and analysis was performed by using metamorph software (Universal Imaging, West Chester, PA). The fluorescent emission signals at 590 and 535 nm in response to excitation at 555 and 490 nm, respectively, were recorded, and the bandwidths for the two signals were 10 nm and 20 nm. The fluorescent signal intensity at 590 nm (red) was divided by the intensity at 535 nm (green), and this red/green ratio was used to estimate the mitochondrial membrane potential (ΔΨ_m). Larger JC-1 red/green ratio values reflect more negative ΔΨ_m. To quantify JC-1 data from individual mitochondria, the JC-1 red/green ratio signals were compiled by using the intensity linescan function of metamorph. Briefly, four 2-pixel-wide lines were randomly drawn through each acquired image, and the peak red/green ratio signal was calculated for each mitochondrion intersected by the lines.

Mitochondria Isolation. Male Sprague–Dawley rats were killed with i.p. injections of pentobarbital (15 mg/kg), and decapitated according to a protocol approved by the Institutional Animal Care and Use Committee. Mitochondria were isolated from rat liver as described (28). The isolation medium contained (in mM) 225 mannitol, 75 sucrose, 1 EGTA, and 10 KH₂PO₄ (pH 7.2). The mitochondria were resuspended in the recording medium [(in mM) 120 KCl/succinate/5 Na pyruvate/10 Mops/0.1 MgCl₂/0.5 Mg ATP/0.1 EGTA, pH 7.2], at ≈10 mg/ml protein. The mitochondria were kept on ice until immediately before use.

JC-1 Signals from Isolated Mitochondria. The isolated mitochondrial potential ΔΨ_m was measured by using JC-1 (27). Before each experiment, mitochondria in the recording medium (see above) were incubated with the dye for 5 min in the dark in a 3-ml recording cuvette at 35°C. The emission signals at 590 and 527 nm elicited by excitation at 485 nm were measured with a spectrofluorometer (FP-750, JASCO). The ratio of the signal at 590 nm over that at 527 nm (red/green ratio) was calculated to estimate ΔΨ_m. The recording chamber was magnetically stirred, and the measurements were carried out at 35°C. To induce hypoxia and hyperoxia, the mitochondria were placed in the above solution that had been bubbled with 100% N₂ and 100% O₂, respectively, for at least 30 min. The normoxia solution was bubbled with air. The pH values of the solutions after bubbling were verified.

Statistical Analysis. All results are reported as the mean ± SEM. Comparisons between the control and MSRA overexpression groups in the normoxic, hyperoxic, and hypoxic conditions were made by using ANOVA followed by the least significant difference post hoc test (DataDesk, DataDescription, Ithaca, NY). Statistical significance was assumed at P ≤ 0.05.

Results

Cellular responses to hypoxia have been extensively studied by using dopamine-containing PC12 cells (29). These cells can be efficiently infected with adenovirus particles to induce gene expression. After treatment with EGFP-bMSRA adenovirus particles, virtually every cell showed EGFP fluorescence (data not shown). This near 100% efficiency by using the viral method allowed us to use fluorescence measurements in populations of cells as described below. The in vitro enzymatic assay for MSRA activity involving the formation of N-acetyl methionine (25) showed that the MSRA virus treatment led to a >10-fold increase in the enzyme activity. In a typical experiment, the reductase activity was ≈14–21 pmol of N-acetyl methionine formed per μg of protein in the untreated control cells and in the cells treated with empty virus particles and ≈250 pmol/μg protein in the cells treated with the MSRA virus particles. These results corroborate the fluorescence measurements and show that the infection with adenovirus leads to an increase in the functional level of MSRA.

Hypoxia Increases ROS. Oxidative stress is implicated in reperfusion after an ischemic episode (30), and hypoxia may enhance production of ROS before reoxygenation (5, 6, 31). We used two groups of PC12 cells, one group treated with control adenovirus particles containing no insert and the other treated with MSRA virus particles, and challenged them with normoxia, hyperoxia, and hypoxia (Fig. 1A). The ROS production was measured by using the ROS-sensitive dye DHR123 (21), which has been used as a general indicator of cellular ROS production (32, 33). Multiple ROS convert DHR123 into a highly fluorescent form (26), such that the rate of the fluorescent signal increase reflects the ROS production rate. DHR123 is reported to be more sensitive in detecting ROS than other dyes tested (34). In the control group, treatment with hypoxia significantly changed the mean DHR123 signal (P < 0.00001), increasing it by ≈100%. In contrast, hyperoxia failed to alter the DHR123 signal (P = 0.82). This observation affirms that hypoxia rapidly increases the overall ROS production before reperfusion in PC12 cells.

Fig. 1.

Hypoxia increases ROS production and promotes cell death. (A) Hypoxia enhances ROS production in PC12 cells. The slope of the DHR123 signal in the first 4 min of the treatment was used to estimate the ROS level. The results obtained from the cells treated with blank control adenovirus particles, bMSRA adenovirus particles, TEMPOL (5 mM), and bMSRA particles and TEMPOL (5 mM) together are shown. The cells were treated with the virus particles 24 h before the measurements and then subjected to normoxia (open bar), hyperoxia (shaded bar), and hypoxia (filled bar) (10 μM DHR123; n = 4–10). (B) ROS production during reoxygenation is inhibited by MSRA overexpression. The cells were treated with hypoxia, and the DHR123 signals were measured as in A (20 μM DHR123; n = 3). (C) MSRA overexpression protects cells from hypoxia-induced cell death. Data regarding nonviable fractions in the groups treated with no virus, bMSRA adenovirus particles, control EGFP adenovirus particles, and TEMPOL (5 mM) are shown. The cells were challenged with normoxia (open bar), hyperoxia (shaded bar), or hypoxia (filled bar) for 5 min. The cell viability assay with trypan blue was performed 24 h later. The effect of ethanol (1%) on cell viability was indistinguishable from that of control (data not shown).

MSRA Overexpression Lowers ROS. The reversible oxidation–reduction of methionine/met-O involving MSRA has been postulated to serve as a ROS sink (20). This idea predicts that overexpression of MSRA to facilitate the reduction process should decrease the overall ROS level. Consistent with this prediction, overexpression of MSRA decreased the ROS level (Fig. 1 A). Whether the cells were subjected to normoxia, hyperoxia, or hypoxia, the mean DHR123 signal was significantly smaller in the cells treated with bMSRA virus particles than in those treated with blank control virus particles (P < 0.00001). Within the cells treated with bMSRA adenovirus particles, hyperoxia increased the mean DHR123 signal significantly when compared with the normoxia-treated cells (P = 0.035), but the increase by hypoxia was not significant (P = 0.15). The mean DHR123 signals measured during hyperoxia and hypoxia in the MSRA virus-treated cells were significantly smaller than in the control cells receiving the same treatments (P = 0.002 and P < 0.00001, respectively). The results show that overexpression of MSRA is effective in reducing the overall ROS level irrespective of the oxygen concentration. It should be noted that the most striking effect of MSRA virus treatment was observed during hypoxia; the DHR123 signal in the MSRA group was only 25% of that in the control group during hypoxia. The membranepermeable ROS scavenger TEMPOL also decreased the ROS production (P < 0.00001). The lowest levels of ROS were observed in the cells treated with both TEMPOL and MSRA virus particles (Fig. 1 A).

Reoxygenation after hypoxia increased the ROS level to ≈9 times that found during hypoxia, and this increase was significantly lower in the cells treated with bMSRA adenovirus particles (Fig. 1B). The fractional decrease in the ROS level during reoxygenation associated with MSRA overexpression (Fig. 1B) was smaller than that during hypoxia (filled bars in Fig. 1 A).

MSRA Prevents Cell Death After Hypoxia. Hypoxia/reoxygenation promotes cell death (35). We hypothesized that the decrease in the ROS level caused by the overexpression of MSRA might protect cells against hypoxia/reoxygenation-induced cell death. Thus, we assayed the viability of PC12 cells 24 h after brief normoxia, hyperoxia, and hypoxia treatments by using the trypan blue exclusion cell viability assay (Fig. 1C). In untreated control cells or cells treated with EGFP adenovirus particles, hypoxia (10 min) increased the mean nonviable stained fraction by ≈100% (P < 0.0001). In contrast, hypoxia failed to alter the nonviable fraction in the MSRA overexpression group (P = 0.43), confirming that the protective effect was specific to MSRA expression. Unlike hypoxia, hyperoxia did not alter the nonviable cell fraction in any of the groups examined (P = 0.60). Consistent with the finding that the ROS scavenger TEMPOL reduced ROS production (Fig. 1 A), hypoxia failed to alter the nonviable fraction in the presence of TEMPOL (Fig. 1C).

MSRA Prevents Apoptotic Cell Death. To examine whether MSRA overexpression protects PC12 cells against apoptosis or necrosis, flow cytometry measurements were performed by using Annexin-V-FLUOS and PI (36, 37). The Annexin-V-FLUOS assay measures the translocation of phosphatidylserine to the external surface of the plasma membrane early in apoptosis, whereas the PI assay primarily detects late apoptotic and necrotic cells (37, 38). Representative flow cytometry dot plots are shown in Fig. 2A, where the dots in the bottom right quadrant in each plot represent the Annexin-V-positive early apoptotic cells. Hypoxia (10 min) increased the number of early apoptotic PC12 cells in the control group when measured 24 h later but not in the MSRA overexpression group (P = 0.04; Fig. 2B). The number of necrotic cells remained unaltered (Fig. 2C).

Fig. 2.

MSRA overexpression preferentially prevents apoptosis. (A) Flow cytometric analysis of PC12 cells double-stained with Annexin-V-FLOUS and PI. Cells were treated with normoxia, hyperoxia, or hypoxia for 10 min, and the measurements were made 24 h later. In each of the six plots, the bottom left quadrangle indicates viable cells that are negative for both Annexin–V binding and PI uptake; the bottom right quadrangle includes apoptotic cells positive for Annexin–V binding but negative for PI uptake; and the top right quadrangle primarily includes necrotic cells positive for both Annexin–V binding and PI uptake. (Upper) Representative dot plots from the control cells treated with normoxia (Left), hyperoxia (Center), and hypoxia (Right). (Lower) Representative dot plots from the cells overexpressing bMSRA. (B) The fractional increases in the number of apoptotic Annexin-V-positive cells. The results from the control and MSRA overexpressing cells are shown. In each cell group, the results after normoxia (open bar), hyperoxia (shaded bar), and hypoxia (filled bar) treatments are shown (n = 3). The measurements were made as in A, and the average percentage of cells undergoing apoptosis without any treatment (20.8 ± 3.7%) has been subtracted. (C) The fractional increases in the number of necrotic PI-positive cells. The average percentage of necrotic cells (1.7 ± 0.1%) was subtracted (n = 3). (D) Flow cytometric cell death analysis of PC12 cells after 24-hr treatments. Cells were treated with normoxia or hypoxia and immediately assayed for cell death. (E) MSRA overexpression partially inhibits the increase in the number of apoptotic cells after 24-hr hypoxia treatment. The measurements are made as in A and B. The average percentage of apoptotic cells in the group treated with normoxia (8.9%) was subtracted (n = 3). (F) MSRA overexpression does not alter the number of necrotic cells. The measurements are made as in A and B. The average percentage of necrotic cells in the group treated with normoxia (2.7%) was subtracted (n = 3).

Hypoxia itself increases the ROS level (Fig. 1), but a burst of ROS is also created on reperfusion (30). To infer whether reoxygenation is required to trigger cell death, the Annexin-V-FLUOS assay was performed immediately after 24-h hypoxia. This prolonged hypoxia treatment preferentially promoted apoptotic cell death similar to what was observed with short hypoxia (Fig. 2D). The fraction of apoptotic cells after 24-h hypoxia was 18.4 ± 4.48% compared with 5.37 ± 2.19% after 24-h normoxia. MSRA overexpression at least partially protected the cells against apoptosis after 24-h hypoxia (Fig. 2E). The necrotic fraction was unchanged by MSRA overexpression (Fig. 2F).

Cellular met-O Contents After Hypoxia. The finding that the mean ROS level was lower in the cells treated with the MSRA virus particles suggests that the met-O level in the MSRA group may also be lower. This hypothesis was tested by analyzing the met-O levels in the proteins of the cells treated with normoxia, hyperoxia, and hypoxia. There was no significant difference in the level of met-O among the three groups when expressed as a percentage of the total methionine (see Materials and Methods). The levels of met-O ranged from ≈9.5% to 13%. The increased levels of ROS in the hypoxic cells did not lead to a global detectable increase in the levels of met-O in these cells, nor did the presence of higher levels of MSRA decrease the total amounts of met-O in proteins. The reasons for the high levels of met-O are unclear, but the observation may indicate that selective oxidation of methionine residues in proteins may be critical and that the majority of met-O in these cell preparations may be functionally inaccessible to MSRA.

Depolarization of Mitochondria. Depolarization of the inner mitochondrial membrane potential facilitated by the opening of large mitochondrial permeability transition pores is regarded as one of the signs of cell death (39). To investigate whether MSRA impedes the effect of hypoxia to promote cell death by preserving the mitochondrial membrane potential, we measured inner mitochondrial membrane potentials by using the fluorescent voltage-sensitive dye JC-1, which preferentially localizes across the inner mitochondrial membrane (27, 40). JC-1 was selected because its emission signals could be analyzed in a ratiometric manner and interpreted semiquantitatively (41). Depolarization of the inner mitochondrial membrane decreases the emission signal intensity at 595 nm (red) and increases the signal intensity at 535 nm (green), such that it reduces the red/green ratio.

Representative JC-1 red/green ratio signals recorded in two groups of cells, control cells treated with control virus particles and the cells treated with bMSRA virus particles, are shown in Fig. 3A. In the control cells treated with normoxia, robust JC-1 red/green ratio signals were observed in a punctate manner, representing healthy polarized mitochondria. We found a clear main effect of hypoxia on the JC-1 red/green ratio signal (Fig. 3 A and B; P < 0.0001). In both the control and bMSRA groups, hypoxia decreased the JC-1 red/green ratio signal. Importantly, the mean JC-1 red/green ratio signal in the MSRA group during hypoxia was greater than that in the control group (Fig. 3 A Bottom and B; P < 0.0001). Application of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (Fig. 3C) reduced the JC-1 red/green ratio signals, confirming that the signals did reflect ΔΨ_m. The magnitude of the inhibition of the JC-1 signal by FCCP was markedly greater than that by hypoxia (Fig. 3 B and C).

Fig. 3.

Membrane potentials of intact mitochondria in PC12 cells estimated by JC-1. (A) Control PC12 cells treated with blank control virus particles (Left) and the cells treated with bMSRA adenovirus particles (Right) were challenged with normoxia (Top), hyperoxia (Middle), and hypoxia (Bottom). The images were obtained ≈5 min after the start of the treatment. The JC-1 red/green ratio signals are represented with the color scale shown. The full scale represents the JC-1 red/green ratios of 0 and 2. Greater JC-1 red/green ratio values indicated by brighter colors reflect more polarized ΔΨ_m. The size scale bar represents 20 μm. (B) Linescan analysis of the JC-1 results. The JC-1 red/green ratio results from the mitochondria in two groups are shown. The control group received blank control virus particles, and the MSRA group received bMSRA particles (n = 19–25 in each condition). (C) JC-1 signals are attenuated by the mitochondrial uncoupler FCCP. FCCP (0.2 μM) was applied for 20 min, and the cells were imaged.

The ratio of the JC-1 emission intensity at 590 nm over that at 535 nm is commonly used to infer ΔΨ_m in a semiquantitative and ratiometric manner (27). In strong oxidizing conditions induced by experimentally applied H₂O₂, the decreases in the JC-1 emission signal at 595 nm may not reflect mitochondrial depolarization of ΔΨ_m, whereas the signal increases at 535 nm still report ΔΨ_m (42). To guard against this possibility, the fluorescent signal intensities at 535 nm alone were also compared. As found with the red/green ratio signals, hypoxia indeed increased the 535-nm signal in the control group (P < 0.0001). Furthermore, the mean 535-nm signal in the control group subjected to hypoxia was also greater than that in the MSRA group subjected to hypoxia, indicating greater depolarization in the control cells (P = 0.02). These results taken together suggest that brief hypoxia is associated with depolarization of the inner mitochondrial membrane and that overexpression of MSRA attenuates this depolarization.

We also investigated the effects of hypoxia on the JC-1 red/green ratio signals from isolated mitochondria because the interference from the cytoplasm sometimes makes interpretations of JC-1 signals in intact cells difficult (41). The use of isolated mitochondria should largely remove the contributions from the cytoplasmic redox-dependent signal transduction pathways (43). At time 0 (t = 0), the mitochondria were challenged with the normoxic, hyperoxic, or hypoxic solution (Fig. 4A). In the normoxia condition, the JC-1 red/green ratio signal did not change with time, indicating that the inner mitochondrial membrane potential was stable. Hyperoxia caused a slight decrease in the JC-1 red/green ratio signal. In contrast, hypoxia rapidly decreased the JC-1 red/green ratio signal, corresponding to depolarization of the inner mitochondrial membrane potential. The changes in the JC-1 red/green ratio signals as determined by the maximal slopes of the signals are summarized in Fig. 4B.

Fig. 4.

Membrane potentials of isolated rat liver mitochondria measured by JC-1. (A) JC-1 red/green ratio after the onset of normoxia, hyperoxia, and hypoxia. Isolated mitochondria were placed in a cuvette and challenged with normoxia, hyperoxia, or hypoxia starting at time 0. It took ≈15 s to load the cuvettes into the spectrofluorometer, and the recording could not be performed during this period (line width indicates SEM; n = 3–4 in each condition). The values shown are slightly different from those in the Fig. 3B, because the measurements here were made by using isolated mitochondria. Similar results were obtained when the emission results at 535 nm only were compared. (B) Mean JC-1 red/green ratio slope values in the normoxia, hyperoxia, and hypoxia conditions. The slopes were calculated by differentiating the JC-1 fluorescence ratio traces as shown in A.

Discussion

The results of this study show that brief hypoxia followed by reoxygenation increases the ROS level in PC12 cells and that this increase is associated with mitochondrial depolarization and more prevalent cell death. Overexpression of MSRA, which facilitates the reduction of met-O in proteins to methionine, attenuates these hypoxia/reperfusion-induced changes.

By using PC12 cells, we detected rapid changes in the overall ROS level and ΔΨ_m in response to brief hypoxia. These results are in line with those of Roy et al. (44), who demonstrated significant changes in the plasma membrane potential and ΔΨ_m by hypoxia in <30 s in rat glomus cells and PC12 cells. However, other cells may not exhibit such rapid and high sensitivity to hypoxia. For example, more severe and prolonged hypoxia treatments may be necessary to induce mitochondrial depolarization in cardiomyocytes (45). Notwithstanding, our results with dopamine-containing PC12 cells confirm the conclusions of previous studies in other systems that hypoxia depolarizes ΔΨ_m, as demonstrated by using different voltage-sensitive dyes (44, 45), and that hypoxia increases the overall cellular ROS level before the onset of reperfusion (6, 31, 46).

Many of the surface-exposed methionine residues in the enzyme glutamine synthetase can be oxidized without altering its function (20). Based on this observation, it was proposed that cyclic oxidation and reduction of methionine residues involving MSRA might function as a cellular sink for ROS (20). The lower overall ROS level observed in our study with MSRA overexpression is consistent with this idea. The ROS-scavenging hypothesis also predicts that the total met-O level may be higher with greater oxidative stress. However, the total cellular met-O level was not markedly altered by MSRA overexpression or by the oxygen concentration. Thus, there may be a specialized pool of methionine residues that specifically function as a ROS sink. The possibility that repair of specific met-O residues in selected proteins plays a critical role in enhancing the efficacy of other ROS scavenging components cannot be totally ruled out.

The MSRA virus particle construct used in this study contains a mitochondrial targeting signal in the N terminus (16). Therefore, at least some of the proteins were likely targeted toward mitochondria, but this was not directly confirmed. This mitochondrial sequence is present in many mammalian MSRA orthologs (16). Because mitochondria are likely to contribute to the hypoxia/reoxygenation-mediated increase in ROS (6, 44, 45), preferential localization of at least some MSRA in or near mitochondria is consistent with this enzyme functioning to repair any oxidative damage in this organelle.

Our finding that the lower ROS level conferred by MSRA overexpression in PC12 cells is associated with greater cell viability after both hypoxia/reoxygenation is consistent with the idea that oxidative stress is an important factor in cell injury associated with hypoxia and reoxygenation. The relative importance of ROS generation during hypoxia and on reperfusion remains to be investigated. The finding that heterologous expression of MSRA reduces the hypoxia-mediated apoptosis suggests that the endogenous antioxidant mechanism is not sufficient to handle the oxidative stress produced by hypoxia. Thus, MSRA and MSRB, which reduce the S and R forms of met-O in proteins, respectively, may be a viable therapeutic target. Given that oxidative stress is postulated to be involved in many disorders, including diabetes, atherosclerosis, and neurodegenerative diseases (4), it is plausible that modulation of the MSRA/B activity in cells may be beneficial in the treatment of these diseases.