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TOXICOLOGY

Role of Ca²⁺-Independent Phospholipase A₂ in Ca²⁺-Induced Mitochondrial Permeability Transition

Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina, (G.R.K., K.S.P., R.G.S.); and Department of Pathology, St. Louis University, St. Louis, Missouri (J.M.)

Received January 5, 2007; accepted February 15, 2007.

	Abstract

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Our laboratory previously demonstrated Ca²⁺-independent phospholipase A₂ (iPLA₂) is localized to mitochondria and that iPLA₂ inhibition blocks cisplatin-induced caspase-mediated apoptosis. Whereas the mitochondrial permeability transition (MPT) is a key control point for apoptosis, the role of mitochondrial iPLA₂ in MPT has not been established. In the present study, we addressed this issue. Ca²⁺-induced renal cortex mitochondrial (RCM) swelling was blocked by the MPT inhibitor cyclosporine A. The R-isomer of bromoenol lactone (R-BEL), which enantiospecifically inhibits iPLA₂, inhibited Ca²⁺-induced RCM MPT, whereas S-BEL (negative control) had no effect. Ca²⁺ treatment resulted in a significant increase in free arachidonic acid (AA) (>50 µM) in the RCM suspension that was blocked by pretreatment with BEL. No increases in free myristic, palmitic, stearic, oleic, linoleic, or docosahexaenoic acid were detected after Ca²⁺ treatment. The addition of AA (18 µM) to Ca²⁺-treated RCM with inhibited iPLA₂ activity restored MPT. We also determined that RCM iPLA₂ displays higher activity against plasmenylcholine with AA in the sn-2 position than oleic acid. Ca²⁺ exposure significantly increased RCM iPLA₂ activity; however, the Ca²⁺-induced activation of iPLA₂ was not the result of mitochondrial membrane potential dissipation, opening of the MPT pore, or mitochondrial swelling. Taken together these findings provide strong evidence that Ca²⁺-induced RCM MPT is mediated by iPLA₂-catalyzed AA liberation.

Mitochondrial permeability transition (MPT) has been implicated as a control point for cell death and is caused by the opening of a proteinaceous pore, which allows equilibration of cytosolic and matrix solutes up to 1.5 kDa (Lemasters et al., 2002). The MPT results in an increase in mitochondrial matrix volume (Kaasik et al., 2007) that can facilitate the release of apoptotic mediators from mitochondria. Depending on the rate at which MPT spreads through the mitochondria of a cell and the ability of the cell to generate extra-mitochondrial ATP (i.e., glycolysis), the MPT can lead to apoptosis or necrosis (Lemasters et al., 2002).

Several factors inhibit MPT by differing mechanisms, including the immunosuppressant drug cyclosporine A (CsA), which binds to cyclophilin D (Bernardi, 1999). Other inhibitors of the MPT are Ca²⁺ uniporter inhibitors (i.e., ruthenium red, ATP) and high inner mitochondrial membrane potential (Bernardi, 1999). Alternatively, several conditions can increase the probability of MPT induced by matrix Ca²⁺ accumulation, including increased reactive oxygen species, inorganic phosphate (Bernardi, 1999), and free fatty acids (Sultan and Sokolove, 2001). Unsaturated free fatty acids promote CsA-sensitive mitochondrial swelling (classic MPT) (Sultan and Sokolove, 2001), whereas saturated free fatty acids (13 to 18 carbons in length) tend to induce mitochondrial swelling that is not sensitive to CsA (nonclassic MPT) (Sultan and Sokolove, 2001). Some exceptions to the observation that unsaturated and saturated fatty acids produce different forms of MPT have been reported and may reflect differences in mitochondria from different tissues or buffer conditions (Scorrano et al., 2001; Di Paola et al., 2006).

AA, a polyunsaturated fatty acid, has been implicated in many models of cell death and tissue damage (Pompeia et al., 2003; Scorrano et al., 2001). AA has physiological and pathophysiological functions and can be enzymatically and nonenzymatically metabolized to other biologically active compounds, including prostaglandins, thromboxanes, and leukotrienes (Cook et al., 1993). Whereas an increased free AA concentration in cells can result in necrosis (Pompeia et al., 2003), it also has been implicated in apoptosis (Pompeia et al., 2003; Penzo et al., 2004; Scorrano et al., 2001). Blocking AA incorporation into cellular phospholipids, effectively elevating free AA inside cells, results in increased phosphatidylserine externalization, chromatin condensation, and poly(ADP-ribose) polymerase cleavage (Perez et al., 2006). Furthermore, the addition of AA to MH1C1 cells resulted in MPT, cytochrome c release, and apoptosis (Scorrano et al., 2001).

The identification of iPLA₂ enzymes localized in the mitochondria of different cell types supports the hypothesis that these enzymes play a role in MPT by generating free AA intramitochondrially. We have recently demonstrated that rabbit renal cortical mitochondria (RCM) possess iPLA₂ using immunoblot and inhibitor sensitivity analysis and found no evidence of iPLA₂, cPLA₂, or cPLA₂ activity (Kinsey et al., 2007). Therefore, the goals of this study were to investigate the role and mechanism of iPLA₂ in Ca²⁺-induced MPT in RCM.

	Materials and Methods

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Materials. Female New Zealand White rabbits (1.5–2.0 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). The R- and S-isomer of bromoenol lactone (R- and S-BEL, respectively) were generously provided by Brian S. Cummings (University of Georgia, Athens, GA) or purchased from Cayman Chemical Co. (Ann Arbor, MI). All other chemicals and materials were obtained from Sigma Chemical (St. Louis, MO) or reported previously (Arrington et al., 2006; Kinsey et al., 2007).

Isolation of RCM. Rabbits were euthanized by intravenous injection of 75 mg/kg pentobarbital sodium, and kidneys were removed by blunt dissection using a protocol approved by the Medical University of South Carolina Institutional Animal Care and Use Committee. Kidney cortex tissue was collected and placed on ice in mitochondrial isolation buffer containing 270 mM sucrose, 5 mM Tris-HCl, and 1 mM EGTA, pH 7.4. Rabbit RCM were isolated by differential centrifugation as described previously (Arrington et al., 2006; Kinsey et al., 2007). Only mitochondrial preparations with a respiratory control ratio (state 3:state 4) of 4 or greater were used for the experiments in this study (Schnellmann and Manning, 1990; Arrington et al., 2006).

Mitochondrial Swelling. Isolated mitochondria were suspended at a concentration of 1.2 mg protein/ml in swelling buffer (130 mM KCl, 9 mM Tris-PO₄, 4 mM Tris-HCl, and 1 mM EGTA, pH 7.4, containing 5 mM each pyruvate and malate) in 200 µl in a 96-well plate and incubated with diluent, inhibitors, or antioxidants at room temperature for 10 min. CaCl₂ was added to achieve final Ca²⁺ concentrations denoted in the text (calculated using the free Ca²⁺ calculation software available at http://www.stanford.edu/%7Ecpatton/webmaxc/webmaxclite115.htm), or diluent (swelling buffer) was added to initiate mitochondrial swelling (Arrington et al., 2006). In one set of experiments, polyethylene glycol (PEG) molecules of different molecular masses (3.4 and 0.4 kDa) were used to block the swelling associated with MPT as described previously (Pfeiffer et al., 1995). In brief, replacing 56 µl of the total 200 µl, in which the swelling experiment was carried out, with 3.4-kDa PEG (53 mM), HEPES (3 mM), and EGTA (1 mM) (pH 7.4, 300 mOsm), was sufficient to completely block Ca²⁺-induced mitochondrial swelling. For a negative control, 56 µl of the total 200 µl was replaced with 0.4-kDa PEG (247 mM), HEPES (3 mM), and EGTA (1 mM) (pH 7.4, 300 mOsm). In other experiments, mitochondria were suspended at 0.4 mg/ml for valinomycin- and FCCP-induced swelling and iPLA₂ activity measurements. Mitochondrial swelling was measured using a SpectraMax 190 spectrophotometric plate reader (Molecular Devices, Sunnyvale, CA) as the loss of optical density at 540 nm over time as described previously (Tedeschi and Harris, 1955; Kinsey et al., 2007). The initial rate of mitochondrial swelling was determined by linear regression analysis of the change in OD₅₄₀ over 5 min immediately following the addition of Ca²⁺.

Measurement of Free Fatty Acids. Mitochondria were treated exactly as described above for the swelling assay, and at 300 s after addition of Ca²⁺ or diluent, the mitochondria from each treatment group were placed on ice immediately. A modified Folch procedure was used to extract lipids (Folch et al., 1957; Broekemeier et al., 2002). In brief, to each 1-ml aliquot, 1.3 ml of methanol (containing 5 µg of heptadecanoic acid as an internal standard) was added and mixed before the addition of 2.6 ml of chloroform. The top layer was aspirated, and the bottom layer was applied to a 6 ml of LC-NH₂ solid-phase extraction tube (Supelco, Bellefonte, PA) that had been previously conditioned with 3 ml of hexane. Free fatty acids were extracted by treatment with 2% acetic acid in diethyl ether as described previously (Kaluzny et al., 1985), evaporated to dryness under nitrogen, and derivatized with BF₃-methanol (150 µl; Supelco) at 60°C for 10 min in silanized vials fitted with Teflon lined caps to produce methyl esters (FAMEs). The samples were reconstituted in heptane (30 µl), and 2 µl of sample was injected into a gas chromatograph-mass spectrometer (GC-MS, Agilent model 6890 GC-5973 MS; Agilent Technologies, Palo Alto, CA) using the pulsed splitless mode. Separations were on a 5% phenylmethylpolysiloxane GC column (30 m x0.25 mm; film 0.25 µm) with the helium carrier gas linear velocity at 55 cm/s. The oven was held at 180°C for 1.5 min, followed by a 20°C/min ramp to 280°C and held for 4.5 min. Ionization was by electron impact (70 eV) with detection by selected ion monitoring of high abundance fragment ions of the following FAMEs (m/z; retention time in minutes): myristic (m/z 242; 6.54), palmitic (m/z 270; 7.65), stearic (m/z 298; 8.65), oleic (m/z 296; 8.51), linoleic (m/z 294; 8.50), arachidonic (m/z 203; 9.27), and docosahexaenoic acid (m/z 241; 10.16). Quantitation was by peak area ratio for each FAME (analyte/internal standard) relative to high, middle, and low calibrators run in parallel to the unknowns.

Measurement of iPLA₂ Activity with Synthetic Phospholipid Substrates. PLA₂ activity was determined under linear reaction conditions in mitochondria using synthetic (16:0,[³H]18:1) or plasmenylcholine (16:0,[³H]20:4) (100 µM) in the absence of Ca²⁺ (4 mM EGTA) as described previously (McHowat and Creer, 1998; Kinsey et al., 2007). Mitochondria were incubated with diluent (DMSO) or BEL in swelling buffer for 10 min and then exposed to Ca²⁺ or diluent for 5 min, pelleted by centrifugation, and resuspended in iPLA₂ activity buffer (containing 250 mM sucrose, 10 mM KCl, 10 mM imidazole, 5 mM EDTA, and 2 mM dithiothreitol, with 10% glycerol, pH 7.8) and frozen at –80°C until activity assays were performed.

Protein Determination. Protein determination was performed using the bicinchoninic acid assay method as described by Sigma Chemical.

Statistical Analysis. Mitochondria isolated from one rabbit represent one experiment (n = 1). The appropriate analysis of variance was performed for each data set containing more than two groups, and a t test used for comparing two groups using SigmaStat statistical software (SPSS, Inc., Chicago, IL). Individual means were compared using Fisher's protected least significant difference test with P 0.05 considered as indicative of a statistically significant difference between mean values.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of iPLA2 inhibition on Ca2+-induced MPT in isolated RCM. A, RCM were exposed to different concentrations of Ca2+, and swelling was measured as the decrease in optical density at 540 nm. B, Ca2+ (1.7 µmol/mg)-induced RCM swelling in the presence and absence of the MPT inhibitor CsA (1 µM). C, Ca2+ (1.7 µmol/mg)-induced RCM swelling in presence and absence of the iPLA2 inhibitor racemic BEL (5 and 25 nmol/mg). D, correlation of the effect of BEL on Ca2+-induced swelling [mean % of initial OD540 of RCM treated with 1.7 µmol/mg Ca2+ (500 s) in the presence of 5, 25, or 50 nmol/mg diluent control or racemic BEL] (y-axis) and on iPLA2 activity (x-axis). Mitochondria were incubated with inhibitors or diluent control for 10 min at room temperature before the addition of Ca2+. Traces are representative of experiments conducted using at least three separate RCM preparations (A, B, and C). Data are presented as the mean ± S.E.M. from at least three separate RCM preparations; means with different letters in parentheses are significantly different from one another, p < 0.05 (mean % of initial OD540, % control iPLA2 activity) (D).

	Results

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To confirm that iPLA₂ inhibition is responsible for the effect of BEL on Ca²⁺-induced MPT, the R- and S-enantiomers of BEL were used. R-BEL specifically inhibits iPLA₂, whereas S-BEL specifically inhibits iPLA₂ (Jenkins et al., 2002). Pretreatment with R-BEL completely inhibited the initial phase of Ca²⁺-induced RCM swelling, whereas S-BEL had no effect (Fig. 2). During this time frame, CsA also completely inhibited Ca²⁺-induced RCM swelling. Previous studies from our laboratory demonstrated that iPLA₂ inhibition accelerated oxidant-induced CsA-insensitive RCM swelling and that oxidant-induced RCM swelling is prevented by the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) and butylated hydroxyanisole (Kinsey et al., 2007). In contrast, Ca²⁺-induced RCM swelling was not inhibited by DPPD (Fig. 2) or butylated hydroxyanisole (data not shown), demonstrating that oxidative stress is not responsible for Ca²⁺-induced RCM MPT.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Effect of iPLA2 and MPT inhibitors and antioxidants on Ca2+-induced MPT in RCM. RCM were incubated for 10 min at room temperature with the iPLA2 inhibitor R-BEL (25 nmol/mg), iPLA2 inhibitor S-BEL (25 nmol/mg), MPT inhibitor CsA (1 µM), or the antioxidant DPPD (5 µM) and then exposed to 1.7 µmol/mg Ca2+, and initial swelling rates were determined as described under Materials and Methods. Data are presented as mean ± S.E.M. initial swelling rate as a percentage of the Ca2+-induced initial swelling rate. Means with different superscripts are significantly different from one another, p < 0.05.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Changes in mitochondrial free AA induced by Ca2+ exposure. RCM-free AA content was measured by GC-MS as described under Materials and Methods. The level of AA was measured 5 min after the addition of Ca2+ (1.7 µmol/mg) in the presence and absence of racemic BEL (25 nmol/mg). Data are presented as the mean ± S.E.M. percentage of control abundance of AA from 13 separate mitochondrial preparations. *, significantly different from control, p < 0.05.

We used synthetic plasmenylcholine substrates with either [³H]oleic acid (18:1, OA) or [³H]AA (20:4) esterified at the sn-2 position to examine RCM iPLA₂ activity. Similar to our recent report (Kinsey et al., 2007), we observed iPLA₂ activity in isolated RCM using the [³H]OA-containing substrate that was significantly inhibited (60%) by treatment with BEL (Fig. 4A). The observed iPLA₂ activity increased by almost 2-fold when plasmenylcholine containing [³H]AA at the sn-2 position was used, and BEL inhibited greater than 90% of the [³H]AA liberation (Fig. 4A). iPLA₂ activity also was measured after treatment of RCM with Ca²⁺ (1.7 µmol/mg for 300 s) in the presence and absence of BEL using the [³H]AA containing plasmenylcholine substrate (Fig. 4B). In concordance with the increases in free AA induced by Ca²⁺ treatment measured by GC-MS (see above), Ca²⁺ treatment also increased the release of [³H]AA from plasmenylcholine substrates (Fig. 4B). Pretreatment with BEL completely blocked the Ca²⁺-induced increase in [³H]AA release (Fig. 4B). In summary, using exogenous substrates to measure iPLA₂ activity, RCM iPLA₂ preferentially cleaved plasmenylcholine phospholipids with AA at the sn-2 position, and exposure of RCM to Ca²⁺ significantly increased cleavage of the AA containing substrate.

View larger version (10K): [in this window] [in a new window]   Fig. 4. RCM iPLA2 activity measured using synthetic phospholipid substrates. A, iPLA2 activity measurements were made in RCM that were exposed to either diluent control or racemic BEL (25 nmol/mg) for 10 min, using synthetic plasmenylcholine with either [3H]oleic acid or [3H]AA at the sn-2 position in the presence of EGTA as described under Materials and Methods. B, iPLA2 activity measurements were made in RCM exposed to either diluent control or racemic BEL (25 nmol/mg) for 10 min and then exposed to 1.7 µmol/mg Ca2+ for 5 min, using synthetic plasmenylcholine with [3H]AA at the sn-2 position. Data are presented as the mean ± S.E.M. from at least three separate RCM preparations. Means with different superscripts are significantly different from one another, p < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of AA and BEL on Ca2+-induced MPT. Ca2+ (1.7 µmol/mg)-induced swelling in the presence of BEL (25 nmol/mg) ± AA (18 µM) ± CsA (1 µM). BEL and CsA were added 10 min before the addition of Ca2+, and AA was added immediately before Ca2+. A, trace is representative of experiments conducted using at least three separate mitochondrial preparations. B, the initial rate of RCM swelling was determined by linear regression analysis of the change in OD540 over 5 min immediately after the addition of Ca2+. Data are presented as the mean ± S.E.M. from at least three separate mitochondrial preparations. Means with different superscripts are significantly different from one another, p < 0.05.

To determine whether iPLA₂ inhibition blocks or delays RCM swelling not mediated by MPT or oxidants, the effect of BEL on valinomycin (a K⁺-selective ionophore)-induced RCM swelling (Gogvadze et al., 2004) was determined. Valinomycin induced time-dependent RCM swelling that was not inhibited by pretreatment with 1 µM CsA (data not shown) or 25 nmol/mg BEL (Fig. 6A). These results demonstrate that the inhibitory effect of BEL on Ca²⁺-induced MPT is not a nonspecific inhibitory effect on RCM swelling.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effect of non-Ca2+-induced RCM swelling and membrane potential dissipation on iPLA2 activity. RCM were incubated with diluent control or 25 nmol/mg BEL and then exposed to 3 nM valinomycin, and swelling was measured (A). RCM were exposed to 3 nM valinomycin (B), 1 µM FCCP (C), or diluent control, and iPLA2 activity was measured at different time points after exposure. iPLA2 activity was measured using synthetic plasmenylcholine (16:0,[3H]18:1) as described under Materials and Methods. Data are presented as the mean ± S.E.M. (A) and mean % control ± S.E.M. (B and C) from at least three separate mitochondrial preparations. Means with different superscripts are significantly different from one another, p < 0.05 (B and C).

PEG of different molecular weights were used to block the RCM swelling associated with Ca²⁺-induced MPT to determine whether Ca²⁺ leads to iPLA₂ activation through RCM swelling. Because the MPT pore is permeable to molecules of up to 1.5 kDa, we used a 3.4-kDa PEG to prevent mitochondrial swelling after Ca²⁺-induced MPT pore opening (Fig. 7A). A 0.4-kDa PEG was used as a negative control because this PEG will pass through the MPT pore and not inhibit Ca²⁺-induced RCM swelling. Ca²⁺-induced RCM swelling in the presence of the 0.4-kDa PEG was blocked by CsA (Fig. 7A), demonstrating the integrity of the MPT. iPLA₂ activity increased to the same extent in the presence of Ca²⁺ whether mitochondrial swelling occurred or not (Fig. 7B). No change in iPLA₂ activity was observed in the absence of Ca²⁺. These results demonstrated that the Ca²⁺-induced iPLA₂ activity was not mediated by RCM swelling.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Effect of dissociating swelling from Ca2+-induced MPT on RCM iPLA2 activity. RCM were incubated in swelling buffer containing either a 3.4-kDa PEG (300 mOsm) or 0.4-kDa PEG (300 mOsm) for 10 min in the presence and absence of 1 µM CsA and then exposed to 1.7 µmol/mg Ca2+, and swelling was measured (A). At 300 s after the addition of Ca2+, iPLA2 activity was measured using synthetic plasmenylcholine substrates as described under Materials and Methods; Data are presented as the mean % control ± S.E.M., means with different superscripts are significantly different from one another, p < 0.05 (B).

To investigate whether Ca²⁺-induced iPLA₂ activation required opening of the MPT pore, RCM were pretreated with CsA (1 µM) and then exposed to 1.7 µmol/mg calcium for 300 s, and iPLA₂ activity was measured. Ca²⁺ alone induced an increase in iPLA₂ activity (161 ± 4% control, n = 3, p < 0.05). Pretreatment with CsA resulted in an equivalent increase in Ca²⁺-induced iPLA₂ activity, which was significantly different from control (151 ± 5% control, n = 3, p < 0.05).

	Discussion

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Several novel findings are presented in this manuscript. 1) Ca²⁺-induced MPT in RCM requires the activity of iPLA₂. 2) Upon exposure to Ca²⁺, iPLA₂ is activated and specifically generates free AA. 3) The addition of exogenous free AA, at a concentration similar to that produced by RCM iPLA₂, to Ca²⁺-treated RCM produced MPT in the absence iPLA₂ activity. 4) Activation of iPLA₂ by Ca²⁺ is not the result of loss of membrane potential, opening of the MPT pore, or mitochondrial swelling. In conjunction with our recent report (Kinsey et al., 2007), these results demonstrated that iPLA₂ plays divergent roles in the same organelle. That is, iPLA₂ is RCM-protective following oxidant exposure by minimizing oxidant-induced lipid peroxidation and non-CsA-sensitive swelling (Kinsey et al., 2007). In contrast, iPLA₂ is a mediator of RCM CsA-sensitive MPT following Ca²⁺ exposure through the production of AA. Therefore, depending on the stimulus, iPLA₂ plays either a protective role (e.g., oxidative stress, presumably by removing oxidized fatty acids from the membrane) or mediates mitochondrial dysfunction (e.g., Ca²⁺-induced MPT, by generating nonoxidized AA).

An inhibitory effect of racemic BEL on MPT in rat liver mitochondria has been described recently (Gadd et al., 2006). Pfeiffer and co-workers (Gadd et al., 2006) showed that Ca²⁺-induced MPT was associated with accumulation of free fatty acids (saturated and unsaturated) over 30 min, which was decreased by pretreatment with BEL but not CsA, and the addition of palmitic acid (16:0) overcame the inhibitory effect of BEL on Ca²⁺-induced mitochondrial swelling. However, the specific fatty acids released were not reported; the CsA sensitivity of swelling induced by Ca²⁺, BEL and palmitic acid was not reported; and the rate of swelling induced by Ca²⁺, BEL, and palmitic acid was accelerated compared with Ca²⁺ alone (Gadd et al., 2006). Their data show that iPLA₂ inhibition inhibits MPT, but the iPLA₂ isoform responsible was not identified. In our study, we demonstrated BEL-sensitive AA production in the absence of other saturated or unsaturated fatty acids after Ca²⁺ exposure in rabbit RCM that resulted in MPT. Similar to Gadd et al. (2006), we determined that CsA has no effect of Ca²⁺-induced iPLA₂ activity, demonstrating that iPLA₂ activation is not dependent on MPT pore opening. Furthermore, MPT was inhibited by R-BEL but not by S-BEL, demonstrating the involvement of iPLA₂. Repletion of AA overcame the inhibitory effect of BEL on Ca²⁺-induced MPT, the swelling rate closely mimicked that of Ca²⁺ alone, and the swelling was sensitive to CsA. The differences between our study and the results of Gadd et al. (2006) may be explained by several factors. 1) Our study was conducted over a relatively short period of Ca²⁺ exposure (5 versus 30 min). 2) Mitochondria were from a different species and tissue, and 3) mitochondria were energized with different substrates (pyruvate and malate in our study versus succinate in the presence of rotenone).

Our current results, in conjunction with previous work from Bernardi and colleagues (Scorrano et al., 2001), suggest that Ca²⁺-induced MPT is the result of a direct effect of AA on MPT rather than a protonophoric decrease in mitochondrial membrane potential. This hypothesis is supported by the finding that AA, at low concentrations and in the presence of Ca²⁺, induced MPT before decreasing mitochondrial membrane potential or respiration (Scorrano et al., 2001). The mechanism of the direct effect of AA on MPT has not been determined. It is possible that AA decreases the change in mitochondrial membrane potential required to promote MPT (by way of an interaction with the putative voltage sensor of the MPT) as proposed by Scorrano et al. (2001). In contrast, AA has been reported to inhibit the ATP-dependent K⁺ channel (K_ATP) (Eddlestone, 1995; Williams and Gottlieb, 2002). The K_ATP channel blocker 5-hydroxydecanoate was shown to overcome the inhibitory effect of BEL on ischemia/reperfusion-induced infarct, suggesting that iPLA₂ mediates cell death through an inhibitory effect on the mitochondrial K_ATP channel (Williams and Gottlieb, 2002). Recently, nonspecific effects of commonly used K_ATP channel manipulators have been identified (Drose et al., 2006), demonstrating the need for more specific ways to manipulate this channel to determine its role in AA-mediated MPT.

The observation that free AA is specifically generated after Ca²⁺ treatment (measured by GC-MS) prompted us to determine the relative selectivity of RCM iPLA₂ for AA-containing phospholipids. Previous studies in RCM demonstrated that iPLA₂ preferentially cleaves plasmenylcholine compared with phosphatidylcholine phospholipids (Kinsey et al., 2007). Using plasmenylcholine with [³H]AA at the sn-2 position, the observed iPLA₂ activity in control mitochondria increased almost 2-fold over plasmenylcholine with [³H]OA at the sn-2 position. It is noteworthy that, in the presence of BEL (at a maximally effective concentration), iPLA₂ activity using the 16:0,[³H]20:4 substrate was 5% control compared with 40% using the 16:0,[³H]18:1 substrate, demonstrating that mitochondrial iPLA₂ preferentially cleaves AA containing plasmenylcholine phospholipids. These findings, in conjunction with GC-MS results, provide strong evidence that Ca²⁺ activates iPLA₂, and not cPLA₂ or secretory PLA₂, in RCM to specifically cleave AA-containing phospholipids. Furthermore, these data, along with the results from Beckett et al. (2007) demonstrating that cytosolic iPLA₂ specifically liberates AA in response to thrombin in human platelets, suggest that iPLA₂ plays a role in stimulus-induced AA generation and cell signaling, which have historically been attributed to other PLA₂ isoforms.

The mechanism by which Ca²⁺ activates iPLA₂ is not known. Gadd et al. (2006) suggested that loss of mitochondrial membrane potential activates iPLA₂ in rat liver mitochondria. We investigated the possibility that iPLA₂ was activated by a loss of RCM membrane potential or swelling of RCM (stretching of mitochondrial membranes) using valinomycin and FCCP, which induce non-CsA-sensitive Ca²⁺-independent swelling and loss of mitochondrial membrane potential, and by using PEG to prevent the swelling associated with Ca²⁺-induced MPT. In contrast to Ca²⁺ exposure, which significantly increased iPLA₂ activity, valinomycin and FCCP reduced iPLA₂ activity in RCM in the presence of mitochondrial swelling. An additional experiment further dissociated Ca²⁺-induced swelling and increased iPLA₂ activity by revealing that the Ca²⁺-induced increase in RCM iPLA₂ activity occurred in the presence and absence of RCM swelling. These results provide evidence that neither stretching of mitochondrial membranes nor loss of mitochondrial membrane potential is responsible for increased iPLA₂ activity induced by Ca²⁺. Potential reasons for the discrepancy between our current results and those of Gadd et al. (2006) have been discussed above. We have previously shown that the addition of ATP to RCM increases iPLA₂ activity in a protein kinase C inhibitor-sensitive manner, suggesting that protein kinase C-mediated phosphorylation can enhance the activity of iPLA₂ (Kinsey et al., 2007). Additional studies are required to determine whether post-translational modification of iPLA₂ or other mechanisms are responsible for Ca²⁺-induced increases in activity.

Inhibition of iPLA₂ blocks apoptosis induced by diverse toxicants in many cell types (Atsumi et al., 1998; Tithof et al., 2002; Cummings et al., 2004; Ramanadham et al., 2004). In renal proximal tubule cells (RPTC), mitochondria have been identified as early targets during cisplatin-induced apoptosis (Nowak, 2002). Approximately one-half of cisplatin-induced, p53-mediated, RPTC annexin V staining, and chromatin condensation was blocked by pretreatment with racemic BEL (Cummings et al., 2004). In human coronary artery endothelial cells, BEL inhibits the majority of 1-methylanthracene and phenanthrene-induced [³H]AA release and apoptosis (Tithof et al., 2002). Although these cellular studies demonstrate a role for iPLA₂ in apoptosis, determination of the specific role of iPLA₂ is complicated by the presence of iPLA₂ in both the mitochondria and endoplasmic reticulum (Cummings et al., 2002; Kinsey et al., 2007). In this study, we show that RCM iPLA₂ is activated by increased [Ca²⁺], which is a common trigger for apoptosis (Rizzuto et al., 2003; Penzo et al., 2004). Furthermore, our results reveal that iPLA₂ activity is required for Ca²⁺-induced RCM MPT and that inhibition of MPT may be a mechanism by which iPLA₂ inhibition attenuates apoptosis in cellular experiments.

A link between Ca²⁺, PLA₂, AA, MPT, and apoptosis has been described in liver cells (Scorrano et al., 2001; Penzo et al., 2004). Bernardi and co-workers (Penzo et al., 2004) demonstrated that exposure of the epithelial cell line MH1C1 to a Ca²⁺ ionophore resulted in increased production of AA before apoptotic cell death that required MPT (i.e., apoptosis was sensitive to CsA). Pretreatment with a general PLA₂ inhibitor, aristolochic acid, blocked the AA production, MPT, and cell death in this model (Penzo et al., 2004). The PLA₂ activity was attributed to cPLA₂ in these cells because Ca²⁺ was required for an increase in PLA₂ activity. Our studies suggest that Ca²⁺ may indirectly activate iPLA₂ in the mitochondria to specifically generate AA in situ that mediates MPT leading to cell death. It is unclear whether mitochondrial iPLA₂ is expressed in MH1C1 cells, and whole-cell experiments in RPTC are warranted to determine the role of iPLA₂ in Ca²⁺-dependent apoptosis.

In conclusion, we demonstrate that iPLA₂ activity is required for Ca²⁺-induced RCM MPT, iPLA₂ activity is increased by Ca²⁺ and specifically cleaves AA-containing phospholipids in RCM, and free AA is responsible for Ca²⁺-induced RCM MPT. Whereas the exact mechanism by which Ca²⁺-increases iPLA₂ activity has not been determined, it does not seem to be the result of loss of RCM membrane potential or induction of swelling.

	Acknowledgements

 
We thank Dr. Brian S. Cummings for generously providing the R-and S-BEL used for some of the experiments in this study.

	Footnotes

 
This research was supported by a National Institutes of Health (NIH) Grant DK-62028 (to R.G.S.). G.R.K. was supported by National Institute of Environmental Health Sciences (NIEHS), NIH Training Grant T32 ES-012878. Medical University of South Carolina animal facilities were funded by NIH Grant C06 RR015455. Its contents are solely the responsibility of the authors and do not represent the official views of the NIH.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.119545.

ABBREVIATIONS: PLA₂, cytosolic phospholipase A₂; iPLA₂, independent phospholipase A₂; cPLA₂, cytosolic phospholipase A₂; MPT, mitochondrial permeability transition; RCM, renal cortex mitochondria; BEL, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one or bromoenol lactone; RPTC, rabbit renal proximal tubular cell; CsA, cyclosporine A; AA, arachidonic acid; OA, oleic acid; DPPD, N,N'-diphenyl-p-phenylenediamine; K_ATP, ATP-dependent K⁺ channel; FCCP, carbonyl cyanide p-trifluormethoxyphenyl-hydrazone; FAME, fatty acid methyl ester; GC-MS, gas chromatograph-mass spectrometry; PEG, polyethylene glycol; LC-NH₂, liquid chromatograph amino propyl bonding.

Address correspondence to: Dr. Rick G. Schnellmann, Medical University of South Carolina, Department of Pharmaceutical Sciences, 280 Calhoun St., Charleston, SC 29425. E-mail: schnell{at}musc.edu

	References

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