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Am J Physiol Renal Physiol 292: F853-F860, 2007. First published October 17, 2006; doi:10.1152/ajprenal.00318.2006
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Identification of calcium-independent phospholipase A2{gamma} in mitochondria and its role in mitochondrial oxidative stress

Gilbert R. Kinsey,1 Jane McHowat,2 Caroline S. Beckett,2 and Rick G. Schnellmann1

1Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina; and 2Department of Pathology, St. Louis University, St. Louis, Missouri

Submitted 11 August 2006 ; accepted in final form 6 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oxidant-induced lipid peroxidation and cell death mediate pathologies associated with ischemia-reperfusion and inflammation. Our previous work in rabbit renal proximal tubular cells (RPTC) demonstrated that inhibition of Ca2+-independent phospholipase A2 (iPLA2) potentiates oxidant-induced lipid peroxidation and necrosis, implicating iPLA2 in phospholipid repair. This study was conducted to identify a RPTC mitochondrial PLA2 and determine the role of PLA2 in oxidant-induced mitochondrial dysfunction. iPLA2 activity was detected in Percoll-purified rabbit renal cortex mitochondria (RCM) and in isolated mitochondrial inner membrane fractions from rabbit and human RCM. Immunoblot analysis and inhibitor sensitivity profiles revealed that iPLA2{gamma} is the RCM iPLA2 activity. RCM iPLA2 activity was enhanced in the presence of ATP and was blocked by the PKC{epsilon} V1–2 inhibitor. Oxidant-induced mitochondrial lipid peroxidation and swelling were accelerated by pretreatment with R-BEL, but not S-BEL. Furthermore, oxidant treatment of isolated RCM resulted in decreased iPLA2{gamma} activity. These results reveal that RCM iPLA2 is iPLA2{gamma}, RCM iPLA2{gamma} is regulated by phosphorylation by PKC{epsilon}, iPLA2{gamma} protects RCM from oxidant-induced lipid peroxidation and dysfunction, and that a strategy to preserve or enhance iPLA2{gamma} activity may be of therapeutic benefit.

group VIB PLA2; lipid peroxidation

OXIDANT-INDUCED CELL DEATH is a mediator of pathologies associated with ischemia-reperfusion, toxicants, and inflammation in many different organs. Reactive oxygen species (ROS) react with several intracellular targets including DNA, proteins, and lipids. Lipid peroxidation changes the biophysical properties of membrane phospholipids, which can affect ion permeability (17) and membrane fluidity (8). Glycerophospholipids in cell and organelle membranes contain two fatty acyl chains linked to the glycerol backbone at the sn-1 and sn-2 positions. The sn-2 fatty acids have a higher degree of unsaturation than sn-1 fatty acids making them a primary target of ROS in biological membranes. Phospholipase A2 enzymes specifically catalyze the cleavage of the sn-2 bond of phospholipids and have been hypothesized to selectively cleave oxidized fatty acids from the sn-2 position to preserve membrane integrity (41).

There are more than 20 isoforms of PLA2 with different characteristics including Ca2+ requirement and subcellular localization. Six and Dennis (35) classified PLA2 enzymes into 14 groups based on their nucleotide sequence. Most PLA2 in these groups are relatively small proteins (12–19 kDa), require millimole amounts of Ca2+ for activity, and use a histidine for catalysis. These groups (I-III, V, and IX-XIV) have historically been called secreted PLA2 (sPLA2). The remaining groups consist of Ca2+-dependent cytosolic PLA2 (cPLA2; group IVA and B), platelet-activating factor acetylhydrolases (PAF-AH; groups VII and VIII), and Ca2+-independent PLA2 (iPLA2, group VI and IVC).

cPLA2{gamma} (group IVC) contains sufficient nucleotide homology to cPLA2{alpha} to be classified as a group IV enzyme, but lacks the Ca2+-dependent lipid binding domain and is anchored to endoplasmic reticulum, golgi apparatus, and mitochondrial membranes (38, 40). iPLA2beta (group VIA) is predominately cytosolic, but one of the several splice variants of iPLA2beta associates with crude membrane fractions (22). iPLA2{gamma} (group VIB) appears to be exclusively membrane bound (20, 24, 25, 44). The 63-kDa isoform of iPLA2{gamma} has been localized in peroxisomal membranes of rat liver cells (44) and higher molecular weight isoforms have been identified in microsomes of rabbit renal proximal tubular cells (RPTC) and ventricular myocytes (20) and in rat heart mitochondria (25).

One method used to identify the isoform(s) of PLA2 responsible for an observed activity is to determine the sensitivity of the activity to different PLA2 inhibitors (10, 15, 20). iPLA2beta is sensitive to methyl arachidonyl fluorophosphonate (MAFP), arachidonyl trifluoromethylketone (AACOCF3), and S-BEL, but not R-BEL at low micromolar concentrations (2, 15, 23). iPLA2{gamma} is sensitive to R-BEL, but not MAFP or S-BEL at low micromolar concentrations (15, 20). cPLA2{gamma} is inhibited by MAFP and AACOCF3, but not BEL (38). Finally, PAF-AH is potently inhibited by MAFP and not inhibited by BEL at concentrations up to 20 µM (18).

Recent studies showed that mitochondrial PLA2 activity in several different tissues and species is Ca2+ independent (6, 7, 13, 25, 40, 43). In rat liver mitochondria, the iPLA2 activity was completely inhibited by BEL but not by inhibitors of sPLA2 or cPLA2 (6). In rabbit ventricular myocytes, mitochondrial iPLA2 expression was demonstrated in the inner mitochondrial membrane (43). Several studies have utilized a commercially available iPLA2beta antibody to demonstrate iPLA2 expression in mitochondria (6, 7, 43). Recently, Gross et al. (25) reported that iPLA2{gamma} is expressed in rat heart mitochondria based on immunoblot analysis with an iPLA2{gamma} antibody. Finally, immunohistochemical analysis revealed that cPLA2{gamma} colocalizes with mitochondria in immortalized mouse lung fibroblasts (40). In summary, iPLA2{gamma}, iPLA2beta, and cPLA2{gamma} are reportedly localized to mitochondria in different tissues, but the presence, identity, and role of a mitochondrial iPLA2 in the kidney has not been examined.

Several studies have implicated PKC-mediated phosphorylation as a mechanism for activation of membrane-bound iPLA2 in the heart and kidney (11, 28, 37). Membrane-associated iPLA2 activity in ventricular myocytes and human coronary artery endothelial cells is increased in a diacylglycerol-dependent, Ca2+-independent fashion suggesting the involvement of a novel PKC isoform (28, 37). In ventricular myocytes, PKC{epsilon} is the only novel isoform detected in the membrane fraction (37). Endoplasmic reticulum iPLA2{gamma} activity in RPTC is increased by phorbol 12-myristate 13-acetate (PMA) treatment, which mimics the effect of diacylglycerol (11). To date, no studies have examined the regulation of mitochondrial iPLA2 activity by PKC.

We suggested that iPLA2{gamma} acts to repair or prevent lipid peroxidation which protects cells from oxidative stress. This hypothesis is supported by the finding that inhibition of iPLA2{gamma} activity in RPTC potentiates oxidant-induced lipid peroxidation and necrotic cell death (10). Furthermore, doxorubicin- and tert-butyl hydroperoxide (TBHP)-induced cardiomyocyte death is enhanced by inhibition of iPLA2 with BEL (27, 39). In this study, we determined that RPTC possess mitochondrial iPLA2 activity and identified the isoform responsible for the activity. Several potential mechanisms for regulation of mitochondrial iPLA2 activity were examined and the role for mitochondrial iPLA2 in oxidant-induced mitochondrial lipid peroxidation and swelling was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Female New Zealand White rabbits (1.5–2.0 kg) were purchased from Myrtle’s Rabbitry (Thompson Station, TN). R- and S-BEL were generously provided by B. S. Cummings (University of Georgia, Athens, GA) (20) or purchased from Cayman Chemical (Ann Arbor, MI). The iPLA2beta antibody was purchased from Cayman Chemical. The PKC{epsilon}-specific inhibitor PKC{epsilon} V1–2 was purchased from Biomol (Plymouth Meeting, PA). All other chemicals and materials were obtained from Sigma (St. Louis, MO) or reported previously (10, 11, 23).

Isolation of renal cortical mitochondria. 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 IACUC. Human cadaveric kidneys (International Bioresearch Solution, Tucson, AZ) that were rejected for transplant were used within 24 h of removal from the donor according to the Medical University of South Carolina IRB guidelines. Kidney cortex tissue was collected and placed on ice in mitochondrial isolation buffer containing (in mM): 270 sucrose, 5 Tris·HCl, 1 EGTA (pH 7.4). Rabbit and human renal cortical mitochondria (RCM) were isolated by differential centrifugation and purified by Percoll density gradient separation where noted (3). Mitochondrial inner membrane fractions were isolated from Percoll-purified mitochondria as described by our laboratory (3).

Immunoblot analysis. We contracted Aves Labs (Tigard, OR) to generate the anti-rabbit iPLA2{gamma} antibody using the rabbit-iPLA2{gamma}-specific peptide sequence, CENIPLDESRNEKLDQ (20). Resultant antiserum was affinity purified and used as the primary antibody. Equal amounts of mitochondrial and cytosolic protein (25 µg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with the iPLA2{gamma} or iPLA2beta antisera at a dilution of 1:1,000. Bound antibodies were visualized by chemiluminescence detection on a ChemiImager 5500 imager (Alpha Innotech, San Leandro, CA).

Mitochondrial swelling. After incubation with cis-parinaric acid and washing, isolated mitochondria were suspended at a concentration of 0.4 mg protein/ml in swelling buffer (150 mM KCl, 20 mM Tris·HCl, pH 7.4) in a 96-well plate and incubated with diluent, inhibitors, or antioxidants at room temperature for 10 min. Ferrous sulfate heptahydrate (Sigma) to achieve a final Fe2+ concentration of 10 µM or diluent (swelling buffer) was then added to initiate oxidative stress (14). 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 previously described (14). Swelling buffer was deoxygenated immediately before use to prevent immediate oxidation of Fe2+ to Fe3+ (14).

Measurement of cis-parinaric acid oxidation. Lipid peroxidation in isolated mitochondria was measured using the fluorescent lipid, cis-parinaric acid as described previously (21, 29), with modifications. Isolated mitochondria were suspended at a concentration of 1 mg protein/ml in swelling buffer and incubated on ice with cis-parinaric acid (6.4 µM) for 10 min. The mitochondria were pelleted by centrifugation, the supernatant discarded, and the mitochondria were resuspended (0.4 mg protein/ml) in deoxygenated swelling buffer. Mitochondria were added to a 96-well plate and incubated with diluent, inhibitors, or antioxidants at room temperature for 10 min and then treated with 10 µM Fe2+. Lipid peroxidation was measured as the loss of cis-parinaric acid fluorescence (excitation 320 nm, emission 405 nm) over time using a Fluoroskan Ascent fluorescent plate reader (Thermo Labsystems, Franklin, MA).

Isolation of rabbit RPTC, culture conditions, and inhibitor treatment. Rabbit RPTC were isolated using the iron oxide perfusion method and grown under improved conditions as previously described (30). Confluent monolayers were treated with R- or S-BEL or diluent control for 30 min before harvesting and isolation of mitochondria. RPTC mitochondria and cytosol were isolated by differential centrifugation as described previously (20).

Measurement of iPLA2 activity. PLA2 activity was determined under linear reaction conditions in mitochondria as described previously (10, 11, 20). Activity was measured using synthetic (16:0, [3H]18:1) plasmenyl- or phosphatidylcholine (100 µM) in the presence (1 mM CaCl2) and absence of Ca2+ (4 mM EGTA). For PLA2 activity inhibition studies, mitochondrial samples were incubated with solvent control [DMSO < 0.1% (vol/vol)], racemic, R-, or S-BEL, MAFP, or AACOCF3 for 10 min before the addition of the phospholipid substrate to start the reaction.

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

Statistical analysis. Mitochondria, cytosol, or RPTC isolated from one rabbit or human kidney represent one experiment (n = 1). The appropriate ANOVA was performed for each data set using SigmaStat statistical software. Individual means were compared using Fisher’s protected least significant difference test with P ≤ 0.05 being considered indicative of a statistically significant difference between mean values.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Rabbit RCM were assayed for PLA2 activity using synthesized plasmenylcholine and phosphatidylcholine phospholipids with palmitic acid (16:0) at the sn-1 position and [3H]oleic acid (18:1) at the sn-2 position. Percoll-purified RCM and RCM inner membrane possess PLA2 activity that is not dependent on Ca2+ and has a preference for plasmenylcholine (Fig. 1A). The effect of pH on iPLA2 activity in Percoll-purified RCM is presented in Fig. 1B. A significant increase in plasmenylcholine cleavage is observed as the pH decreases from 8 to 7.5, while a significant increase in phosphatidylcholine cleavage occurs as the pH decreases from 9 to 8. Cleavage of both substrates decreases to background levels at a pH of ~6. Ca2+-independent activity was observed in the endoplasmic reticulum (ER) and inner mitochondrial membrane of kidney cortex tissue from cadaveric human kidneys (Fig. 1C). Similar to rabbit kidney (10) there was little basal cytosolic PLA2 activity in human kidney cortex. In summary, RCM possess iPLA2 activity that prefers plasmenylcholine over phosphatidylcholine, the activity is differentially affected by pH, and human kidney cortex mitochondria exhibit a similar level of iPLA2 activity as the ER and mitochondrial inner membrane in rabbit kidney cortex.


Figure 1
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  		Fig. 1. Phospholipase A2 activity in rabbit renal cortex mitochondria and human renal cortex subcellular fractions. A: cleavage of 16:0; [3H]18:1 plasmenyl- or phosphatidylcholine substrates in the presence (1 mM CaCl2) and absence (4 mM EGTA) of calcium by whole Percoll-purified rabbit RCM and mitochondrial inner membrane fractions. B: effect of pH on the cleavage of plasmenyl- or phosphatidylcholine substrates in the absence of calcium in whole Percoll-purified rabbit RCM. C: cleavage of 16:0; [3H]18:1 plasmenyl- or phosphatidylcholine substrates in the presence (1 mM CaCl2) and absence (4 mM EGTA) of calcium by human kidney cortex ER and Percoll-purified mitochondrial inner membrane and cytosolic fractions. Values are means ± SE of 3 experiments. Means with different superscripts are significantly different from each other within groups, P < 0.05.

 
Immunoblot analysis with antibodies for iPLA2beta and iPLA2{gamma} revealed immunoreactive proteins to both antibodies in rabbit RCM (Fig. 2). While the bands are similar in size, the antigenic peptides used for both antibodies are unique for their respective proteins. The protein recognized by the Cayman iPLA2beta antibody is similar in size to iPLA2beta reported in rabbit heart mitochondria (43) and rat liver mitochondria (6). iPLA2{gamma} in RPTC mitochondria is the long isoform of iPLA2{gamma} (~88 kDa), based on the nucleotide sequence analysis of Mancuso et al. (24). Consistent with the very low cytosolic PLA2 activity, no iPLA2 expression was detected in RPTC cytosol.


Figure 2
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  		Fig. 2. Immunoblot analysis of iPLA2 expression in renal proximal tubule cell (RPTC) mitochondria. RPTC mitochondrial (Mito) and cytosolic (Cyto) proteins (25 µg) were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with iPLA2{gamma} or iPLA2beta antibodies. After incubation with anti-chicken HRP (iPLA2{gamma}) or anti-rabbit HRP (iPLA2beta) secondary, bound antibodies were visualized by chemiluminescence detection. Molecular weight markers (M) are shown in the first column of each blot. Images are representative of 3 separate immunoblots carried out on different RPTC preparations.

 
In an effort to determine which PLA2 isoform is responsible for the iPLA2 activity in RCM, the inhibitor sensitivity profile was determined. RCM iPLA2 activity was inhibited by racemic BEL, but not MAFP or AACOCF3 (Fig. 3A). This profile is consistent with iPLA2{gamma}, and not iPLA2beta or cPLA2{gamma}, both of which are inhibited by MAFP and AACOCF3 at concentrations lower than 10 µM (2, 23, 38). As an additional approach to confirm that iPLA2{gamma} is responsible for the mitochondrial PLA2 activity, the effect of the R- and S-enantiomers of BEL was investigated. R-BEL selectively inhibits iPLA2{gamma} at low micromolar concentrations while S-BEL selectively inhibits iPLA2beta (15). In Percoll-purified RCM, R-BEL was effective at inhibiting iPLA2 activity while S-BEL had no effect (Fig. 3B). In mitochondria isolated from confluent RPTC, after a 30-min incubation with increasing concentrations of inhibitors or diluent control, R-BEL was significantly more effective at inhibiting iPLA2 activity than S-BEL (Fig. 3C). These results demonstrate that iPLA2{gamma} is responsible for mitochondrial iPLA2 activity in RCM and that treatment of RPTC with R-BEL selectively decreases mitochondrial iPLA2{gamma} activity.


Figure 3
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  		Fig. 3. Inhibitor sensitivity of mitochondrial iPLA2. A: effect of the PLA2 inhibitors, racemic BEL, MAFP, and AACOCF3 on iPLA2 activity in Percoll-purified rabbit RCM. The effect of R and S enantiomers of BEL on iPLA2 activity in whole Percoll-purified mitochondria from rabbit kidney cortex (B) and mitochondria isolated from RPTC (C). B: mitochondria were incubated with solvent control, R- or S-BEL at increasing concentrations for 10 min before activity assays. C: confluent RPTC were treated with diluent or R- or S-BEL for 30 min before harvesting and isolation of mitochondria for activity assays. Activity represents cleavage of 16:0; [3H]18:1 plasmenylcholine substrates (B, C) in the presence of 4 mM EGTA. Values are means ± SE of 3 experiments. Means with different superscripts are significantly different from each other, P < 0.05.

 
The addition of ATP (1 mM) to RCM increased iPLA2 activity ~30% (Fig. 4). Previous investigators suggested that membrane-associated iPLA2{gamma} activity is regulated by PKC{epsilon}-mediated phosphorylation (11, 28, 37). and the addition of the PKC{epsilon}-specific inhibitor PKC{epsilon} V1–2 blocked ATP-induced increases in iPLA2 activity in RCM (Fig. 4A). To ensure that ATP was not activating latent iPLA2beta in the mitochondria, the effect of R-BEL and S-BEL (5 µM) on ATP-induced iPLA2 activity was determined. S-BEL had no inhibitory effect on iPLA2 activity in the presence or absence of ATP while R-BEL inhibited iPLA2 activity under both conditions, suggesting that the increase in iPLA2 activity in the presence of ATP is solely due to an increase in iPLA2{gamma} activity and not the activation of a latent iPLA2beta (Fig. 4B).


Figure 4
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  		Fig. 4. Effect of PKC{epsilon} inhibition and R- and S-BEL on ATP-induced mitochondrial iPLA2 activity. A: freshly isolated rabbit RCM were pretreated with diluent, or the PKC{epsilon}-specific inhibitor, PKC{epsilon} V1–2, for 10 min before the addition of 1 mM ATP, after 10-min mitochondria were assayed for iPLA2 activity. B: freshly isolated rabbit RCM were pretreated with diluent control or 1 mM ATP for 10 min and then exposed to R- or S-BEL 5 µM for 10 min before activity assays. Activity represents cleavage of 16:0; [3H]18:1 plasmenylcholine substrates in the presence of 4 mM EGTA. Values are means ± SE of 3 experiments. Means with different superscripts are significantly different from each other, P < 0.05.

 
To investigate the role of iPLA2{gamma} in oxidant-mediated mitochondrial damage, RCM were treated with Fe2+ to initiate oxidant stress and mitochondrial swelling (Fig. 5) (14). Mitochondrial swelling induced by 10 µM Fe2+ was completely blocked by the antioxidants butylated hydroxyanisole (25 µM BHA; Fig. 5A) and N,N'-diphenyl-1,4-benzenediamine (DPPD; 5 µM, data not shown). Pretreatment of mitochondria with R-BEL (8 µM) accelerated the onset of Fe2+-induced mitochondrial swelling, whereas pretreatment with S-BEL (8 µM) had no effect (Fig. 5A). The time required for Fe2+ to induce maximal mitochondrial swelling in the presence of R- and S-BEL was compared with the time required for Fe2+ alone to induce maximal swelling. Pretreatment with R-BEL decreased the time required for Fe2+ to initiate mitochondrial swelling by ~120 s, while S-BEL had no effect (Fig. 5B). Pretreatment with cyclosporine A (CsA; 1 µM) had no effect on Fe2+-induced mitochondrial swelling (data not shown), suggesting the Fe2+-induced swelling is not the result of mitochondrial permeability transition (MPT) pore opening. In summary, Fe2+ induced RCM swelling that is mediated by oxidative stress, but not the result of MPT, and specific iPLA2{gamma} inhibition accelerated the oxidant-induced swelling.


Figure 5
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  		Fig. 5. Effect of iPLA2 inhibition on Fe2+-induced mitochondrial swelling. Freshly isolated rabbit RCM were pretreated with diluent control, R- or S-BEL 25 nmol/mg (8 µM), or the antioxidant 25 µM BHA and then exposed to 10 µM Fe2+. Mitochondrial swelling was measured as the loss of optical density at 540 nm (A). The effect of iPLA2 inhibitors on the time required for Fe2+ to induce swelling is presented in B. Values are means ± SE of 3 experiments. Means with different superscripts are significantly different from each other, P < 0.05.

 
The fluorescent lipid cis-parinaric acid was used to measure the rate of lipid peroxidation under the same conditions as Fig. 5A. cis-Parinaric acid incorporates into membranes and loses fluorescence as it becomes oxidized (21, 29). Treatment with Fe2+ resulted in an increased rate of cis-parinaric acid oxidation compared with control mitochondria. The Fe2+-induced oxidation rate was further increased by R-BEL pretreatment; however, pretreatment with S-BEL had no effect (Fig. 6). BHA and DPPD significantly inhibited cis-parinaric acid oxidation in a concentration-dependent manner (Fig. 6 and data not shown, respectively). Similar to Fe2+-induced swelling, iPLA2{gamma} inhibition significantly accelerated the rate of oxidant-induced lipid peroxidation in RCM.


Figure 6
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  		Fig. 6. Effect of iPLA2 inhibition on Fe2+-induced mitochondrial lipid peroxidation. Freshly isolated rabbit RCM were incubated with the fluorescent lipid cis-parinaric acid and washed as described in MATERIALS AND METHODS and then pretreated with diluent control, R- or S-BEL 8 µM, or the antioxidant BHA, then exposed to 10 µM Fe2+. The loss of fluorescence (indicative of lipid peroxidation) was followed over time (A). To determine the rate of lipid peroxidation, the difference of percent initial fluorescence in each treatment group from that of control was determined and the oxidation rate was determined by linear regression analysis. Values are means ± SE of 3 experiments (B). Means with different superscripts are significantly different from each other, P < 0.05.

 
Previous studies in RPTC demonstrated that oxidants directly inactivate ER-iPLA2{gamma} (12). To determine the effect of oxidative stress on mitochondrial iPLA2{gamma} activity, RCM were incubated with diluent control or 10 µM Fe2+ and assayed for iPLA2 activity over time. Similar to ER-iPLA2{gamma}, oxidant treatment significantly inhibited iPLA2{gamma} activity in RCM (Fig. 7).


Figure 7
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  		Fig. 7. Fe2+-mediated inactivation of iPLA2{gamma} in RKC mitochondria. Freshly isolated rabbit RCM were treated with diluent control or 10 µM Fe2+ and samples were taken at the indicated time points for iPLA2 activity assays. Activity represents cleavage of 16:0; [3H]18:1 plasmenylcholine substrates in the presence of 4 mM EGTA. Values are means ± SE of 3 experiments. Means with different superscripts are significantly different from each other, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrate the novel finding that human and rabbit RCM possess iPLA2 activity. The preference of RCM iPLA2 for plasmenylcholine phospholipid substrates over phosphatidylcholine substrates is similar to iPLA2{gamma} in microsomes of RPTC and rabbit ventricular myocytes (10, 20). The localization of kidney mitochondrial iPLA2 activity to the inner mitochondrial membrane is in agreement with a previous report in rabbit heart mitochondria (43). While immunoblot analysis results suggested that iPLA2{gamma} and iPLA2beta are localized to RPTC mitochondria, inhibitor sensitivity analysis revealed that in rabbit RCM and RPTC mitochondria iPLA2 activity is due solely to the iPLA2{gamma} isoform and not iPLA2beta or cPLA2{gamma}.

The iPLA2 isoform in rabbit heart mitochondria and rat liver mitochondria was hypothesized to be iPLA2beta based on iPLA2beta immunoblot results and sensitivity of the activities to racemic BEL (6, 43). The iPLA2beta antibody also detected an immunoreactive protein of ~85 kDa in the microsomal fraction (10) and in mitochondria of RPTC. This size protein is similar to those reported by others in the mitochondria (6, 43). However, the presence of iPLA2beta in RPTC is not supported by RT-PCR (11) or inhibitor sensitivity analysis of the iPLA2 activity in RPTC microsomes (10, 20) and mitochondria. In contrast, iPLA2{gamma} expression has been confirmed at the mRNA (11) and protein level (20) (Fig. 2) and the inhibitor sensitivity profile, using racemic, R- and S-BEL, MAFP and AACOCF3, of microsomes (10, 20) and mitochondria (Figs. 3 and 4) from RPTC confirms iPLA2{gamma}. The concentrations of MAFP and AACOCF3 used (10 µM each) are well above the IC50 values reported for iPLA2beta, cPLA2{gamma}, and PAF-AH (2, 15, 18, 23). In summary, immunoblot analysis results prohibit ruling out iPLA2beta expression in RPTC, but RT-PCR and inhibitor sensitivity of the iPLA2 activity in RPTC raise questions about the identity of the protein recognized by the iPLA2beta antibody in RPTC.

Regulation of iPLA2{gamma} activity has not been extensively studied. The pH of the environment surrounding iPLA2{gamma} is a potential regulator of its activity. Our experiments revealed that mitochondrial iPLA2 activity increases toward plasmenylcholine substrates but is unchanged toward phosphatidylcholine substrates as pH decreases from 8 to 7.5. This is relevant because a significant portion of rabbit RPTC phospholipids is made up of plasmalogens (plasmenylcholine and plasmenylethanolamine) and these phospholipids are enriched with arachidonic acid (20:4) at the sn-2 position (32). Mitochondrial matrix pH is ~8 and decreases toward 7.5 as the inner mitochondrial membrane potential is lost and by agents that increase cytosolic Ca2+ (1). During mitochondrial stress (i.e., ischemia-reperfusion, oxidative stress, Ca2+ overload), the decrease in matrix pH could increase iPLA2{gamma} activity to cleave arachidonic acid containing plasmalogens, which are targets of ROS in cellular membranes (19). Changes in pH may result in conformational changes of iPLA2{gamma}, phospholipids, or both leading to the observed changes in activity.

Previous observations led to the hypothesis that microsomal iPLA2 activity in kidney, heart, and coronary artery endothelial cells is regulated by PKC-mediated phosphorylation (11, 28, 37). Our recent studies demonstrated that the microsomal iPLA2 in kidney and heart is iPLA2{gamma} (20). In support of this hypothesis, sequence analysis of iPLA2{gamma} revealed the presence of multiple potential serine/threonine phosphorylation sites (5). The addition of ATP to isolated RCM increased iPLA2{gamma} activity, and the increase in activity was blocked by pretreatment with a PKC{epsilon}-specific inhibitor, suggesting that PKC{epsilon} regulates mitochondrial iPLA2{gamma} activity. Our findings in conjunction with the observation by Nowak et al. (31) that PKC{epsilon} is present in RPTC mitochondria and that PKC{epsilon} translocation to mitochondria is increased after oxidant treatment support the hypothesis that oxidant-induced PKC{epsilon} translocation may result in upregulation of mitochondrial iPLA2{gamma} activity in response to oxidant stress in renal cells.

Previous studies from our laboratory revealed that inhibition of iPLA2 activity with BEL in rabbit RPTC-potentiated oxidant-induced lipid peroxidation and necrotic cell death (10) and inhibition of ventricular myocyte iPLA2 activity with BEL potentiated doxorubicin- and TBHP-induced cell death (27, 39). These findings led to the hypothesis that iPLA2{gamma} prevents or repairs lipid peroxidation. In this study, we used the iPLA2{gamma} inhibitor, R-BEL, to show that specific inhibition of iPLA2{gamma} in isolated RCM accelerated oxidant-induced lipid peroxidation and mitochondrial swelling. These observations support our hypothesis and demonstrate that mitochondrial iPLA2{gamma} activity protects mitochondria against oxidative stress. Our previous demonstration that iPLA2{gamma} is expressed in ER of rabbit RPTC and ventricular myocytes (20) is consistent with the idea that iPLA2{gamma} localized to these two organelles, which are routinely exposed to oxidants, serves as a defense mechanism against lipid peroxidation-induced organelle dysfunction.

A protective role for iPLA2beta in mitochondria has recently been proposed. Seleznev et al. (34) reported that overexpression of iPLA2beta in insulinoma cells and Chinese hamster ovary cells results in mitochondrial localization of the GFP-linked iPLA2beta and protects against staurosporine-induced apoptosis. Staurosporine-induced apoptosis is at least partially mediated by mitochondrial ROS production and lipid peroxidation (34). The expression and localization of different iPLA2 isoforms vary greatly among different cell types and the role of lipid peroxidation repair may be determined by the available isoforms in a specific cell or organelle.

The ER iPLA2{gamma} in RPTC is directly inactivated by diverse oxidants in a dithiothreitol-sensitive manner, implicating reduced thiols are required for iPLA2{gamma} activity and are a target of oxidants (12). Our current results demonstrate that mitochondrial iPLA2{gamma} also is inactivated by oxidants. Recently, iPLA2beta also was shown to be inactivated by oxidants and the specific oxidant-induced damage was elucidated (36). Oxidant-induced inhibition of iPLA2 may be a form of signaling (negative regulation) to prevent excessive activity. Conversely, it may represent an additional mechanism of oxidant-induced toxicity (i.e., oxidants cause lipid peroxidation and inactivate the lipid peroxidation repair enzyme). Prevention of oxidant inactivation of iPLA2{gamma} may provide protection to cells and organelles during oxidative stress.

In conclusion, we demonstrated that iPLA2{gamma} is expressed and active in RPTC mitochondria, and inhibition of iPLA2{gamma} accelerates lipid peroxidation and swelling in isolated RCM. Human kidney cortex displays significant iPLA2 activity in the inner mitochondrial membrane and ER similar to rabbit kidney cortex. Our data suggest that mitochondrial iPLA2{gamma} activity is regulated by pH and phosphorylation by PKC{epsilon}. Finally, similar to iPLA2{gamma} in RPTC ER, mitochondrial iPLA2{gamma} is inactivated by oxidant stress. Efforts to preserve and enhance mitochondrial iPLA2{gamma} activity may be useful to prevent oxidative stress-induced cell death in the proximal tubular cells of the kidney and in other tissues, including the heart.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grant DK-62028 to R. G. Schnellmann and G. R. Kinsey was supported by a training grant from the National Institute of Environmental Health Sciences (NIEHS), NIH (T32 ES-012878) and MUSC animal facilities were funded by NIH Grant no. C06 RR015455. Its contents are solely the responsibility of the authors and do not represent the official views of the NIEHS, NIH.


   ACKNOWLEDGMENTS
 
The authors thank Dr. B. S. Cummings for generously providing the R- and S-BEL used for some of the experiments in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. G. Schnellmann, Dept. of Pharmaceutical Sciences, Medical Univ. of South Carolina, 280 Calhoun St., Charleston, SC 29425(e-mail: schnell{at}musc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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