Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in oxidant-induced renal cell death

Brian S. Cummings1, Jane McHowat2, and Rick G. Schnellmann1

1 Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425; and 2 Department of Pathology, Saint Louis University, St. Louis, Missouri 63104


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phospholipase A2 (PLA2) hydrolyzes the sn-2 ester bond in phospholipids, releasing a fatty acid and a lysophospholipid. Recently, a novel 85-kDa membrane-bound-Ca2+-independent PLA2 (iPLA2) was identified in insect and bacterial cells transfected with candidate PLA2 sequences. However, few data exist demonstrating a membrane-bound-iPLA2 in mammalian cells, its subcellular localization, or its physiological role. Herein, we demonstrate the expression of an 85-kDa endoplasmic reticulum (ER)-Ca2+-iPLA2 (ER-iPLA2) in rabbit renal proximal tubule cells (RPTC) that is plasmalogen selective and is inhibited by the specific Ca2+-iPLA2 inhibitor bromoenol lactone (BEL). RPTC exposed to tert-butylhydroperoxide for 24 h exhibited 20% oncosis compared with 2% in controls. Inhibition of ER-iPLA2 with BEL before tert-butylhydroperoxide exposure resulted in 50% oncosis. To determine whether this effect was common to oxidants, we tested the ability of BEL to potentiate oncosis induced by cumene hydroperoxide, menadione, duraquinone, cisplatin, and the nonoxidant antimycin A. All oxidants tested produced oncosis after 24 h, and prior inhibition of ER-iPLA2 potentiated oncosis at least twofold. In contrast, inhibition of ER-iPLA2 did not alter antimycin A-induced oncosis. Lipid peroxidation increased from 1.4- to 5.2-fold in RPTC treated with BEL before oxidant exposure, whereas no change was seen in antimycin A-treated RPTC. These results are the first to demonstrate the expression and subcellular localization of an ER-iPLA2. These results also suggest that ER-iPLA2 functions to protect against oxidant-induced lipid peroxidation and oncosis.

kidney; oxidant-induced cell death; lipid peroxidation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PHOSPHOLIPASE A2 (pla2) IS a superfamily of enzymes that hydrolyzes the sn-2 ester bond in phospholipids, resulting in the release of a free fatty acid and a lysophospholipid (6). It is ubiquitous in nature and plays critical roles in the generation of arachidonic acid, prostaglandins, and leukotrienes (8). PLA2 is classified according to its nucleotide sequence, and to date there are at least 12 groups (I-XII) comprising 16 individual PLA2 (29). Historically, a PLA2 is classified on the basis of whether it is secreted from the cell (secretory PLA2), Ca2+ dependent and located in the cytosol (cPLA2), or Ca2+ independent (iPLA2). cPLA2 (groups IVA-B) is one of the most widely studied and is found in the cytosol of many types of cells, including the pancreas, heart, and kidney (6, 7). cPLA2 requires micromolar amounts of Ca2+ to facilitate translocation to cellular membranes on activation but does not require Ca2+ for its catalytic activity. cPLA2 range in size from 85 to 100 kDa (7). iPLA2 (groups IVC, VI, VIIB, and VIII) does not require Ca2+, ranges in molecular size from 29 to 85 kDa, and typically is found in the cytosol. A key difference between cPLA2 and iPLA2 is that iPLA2 is inhibited by the suicide substrate (E)-6-(bromoethylene)-3-(1-naphthaleny)-2H-tetrahydropyran-2-one [or bromoenol lactone (BEL)], whereas cPLA2 is not. This fact has been used in a number of studies to distinguish between cPLA2 and iPLA2 activities (1, 10). In addition, iPLA2 can be distinguished from cPLA2 activity on the basis of its selectivity for phospholipid substrates and Ca2+ requirement.

A number of iPLA2 activities have been described in a variety of tissues, including those of hamsters (32), mice (4), rats (4), and humans (14, 15). To date, the best characterized iPLA2 from all species is an 85-kDa protein that is inhibited by BEL and is present in multiple isoforms, a result of alternative splicing. This iPLA2 is commonly called iPLA2beta and is cytosolic. Recently, Mancuso et al. (16) and Tanaka et al. (31) have characterized novel membrane-associated iPLA2 that share 91% DNA sequence homology to each other, but only limited homology to iPLA2beta , confined to the ATP-binding motif and the lipase site. This iPLA2 has been termed iPLA2gamma . Mancuso et al. (16) demonstrated that translation of the DNA sequence for iPLA2gamma in insect Sf-9 cells resulted in the expression of two proteins with molecular masses of 67-77 kDa. Activity and expression of these proteins localized to membrane fractions and were inhibited by BEL. In contrast, Tanaka et al. (31) reported that translation of a candidate iPLA2gamma sequence in bacterial COS-7 cells resulted in one 85-kDa protein whose expression and activity also localized to membrane fractions. The activity of this 85-kDa iPLA2 was inhibited by BEL. The only other reported iPLA2 expressed in the membrane is an 80-kDa iPLA2 expressed in the plasma membrane of cardiomyocytes (17).

The above studies did not examine membrane-bound iPLA2 protein or activity in mammalian cells, determine the selectivity of iPLA2 for specific phospholipids, or determine the membrane location in which it was expressed. In addition, a physiological role for this iPLA2 was not addressed. In this study, we investigated an iPLA2 in renal proximal tubular cells (RPTC) with respect to expression, subcellular localization, inhibition profile, and selectivity for specific phospholipid substrates. In addition, we tested the hypothesis that iPLA2 plays a role in mediating oxidant-induced renal cell oncosis and lipid peroxidation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Female New Zealand White rabbits (1.5-2.0 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). L-Ascorbic acid-2-phosphate (magnesium salt) was obtained from Wako Chemicals (Richmond, VA). The antibody to iPLA2beta was purchased from Cayman Chemical (Ann Arbor, MI), and annexin V-FITC was obtained from R&D Systems (San Diego, CA). The antibody to the heat shock protein GRP78 was purchased from Calbiochem (La Jolla, CA), and the antibody against the peroxisomal protein PMP70 was obtained from Affinity BioReagents (Golden, CO). The endoplasmic reticulum (ER) and DNA stains (DiOC6)3 and 4,6-diamidino-2-phenylindole were obtained from Molecular Probes (Eugene, OR). Cisplatin, propidium idodide, duraquinone, menadione, antimycin A, tert-butylhydroperoxide (TBHP), cumene hydroperoxide, and all other chemicals were purchased from Sigma (St. Louis, MO).

Isolation of proximal tubules and culture conditions. Rabbit RPTC were isolated using the iron oxide perfusion method and grown in 35-mm tissue culture dishes under improved condition, as previously described (21, 22). The cell culture medium was a 1:1 mixture of DMEM/Ham's F-12 (without D-glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 µM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 µg/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 µM) were added to fresh culture medium immediately before daily media change.

Immunoblot analysis. Microsomal and cytosolic fractions were isolated from RPTC as previously described (17). Immunoblot analysis of both microsomal and cytosolic protein (20 µg) from RPTC was performed as described previously using a polyclonal rabbit anti-iPLA2 antibody that recognizes 85-kDa iPLA2beta (17).

Immunocytochemical analysis. Immunocytochemical analysis of iPLA2, ER, peroxisomes, and nuclei staining were performed using confocal laser scanning microscopy. Confluent RPTC monolayers were washed with PBS, fixed in 3.7% formaldehyde, and permeabilized with 0.1% (vol/vol) Triton X-100. Nonspecific binding was blocked using 8% BSA, and RPTC were incubated overnight with a goat anti-rabbit polyclonal antibody to 85-kDa iPLA2beta (100-fold dilution) followed by exposure to a secondary antibody (1,000-fold dilution) conjugated to either a 480/510- or 535/610-nm excitation/emission fluorochrome. For colocalization studies, RPTC were exposed to markers of the ER [(DiOC6)3 or GRP78 antibody] or the peroxisomes (PMP70 antibody). Nuclei were stained using 4,6-diamidino-2-phenylindole (350/390-nm excitation/emission). Secondary antibodies for both GRP78 and PMP70 were conjugated to a 480/510-nm excitation/emission fluorochrome. After the final wash, RPTC were covered with mounting media and coverslips. Staining was visualized with a 410 Zeiss confocal laser scanning microscope.

Measurement of PLA2 activity. PLA2 activity was determined under linear conditions in microsomes and cytosol as described previously (18). Activity was measured by using synthetic (16:0, [3H]18:1) plasmenylcholine and phosphatidylcholine substrates (100 µM) in the absence of Ca2+ (presence of 4 mM EGTA). For PLA2 activity inhibition studies, confluent RPTC were exposed to either a solvent control [DMSO <0.1% (vol/vol)] or 1-10 µM BEL for 30 min.

Measurement of oncosis. Oncosis was monitored by assessment of both annexin V (a marker for apoptosis) and propidium iodide (PI; a marker for oncosis) staining using flow cytometry. Briefly, RPTC were exposed to the oxidants TBHP (200 µM), cumene hydroperoxide (200 µM), duraquinone (400 µM), and cisplatin (400 µM) for 24 h and to menadione (10 µM) for 12 h. In addition RPTC were exposed to the nonoxidant antimycin A (0.3 µM) for 4 h. Annexin and PI staining were monitored using confocal laser scanning microscopy and flow cytometry as described previously, with modifications (9, 19, 28). After treatment, the medium was removed, and RPTC were washed twice and incubated in binding buffer [(in mM) 10 HEPES, 140 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, pH = 7.4] containing annexin V-FITC (2.5 µg/ml) and PI (25 µg/ml). Cells were washed three times in binding buffer and prepared for either flow cytometry or microscopy. For flow cytometry, RPTC were released from the monolayer by using a rubber policeman, and staining was quantified by using a Becton Dickinson FacsCalibur flow cytometer. Minimal binding of annexin V in the absence of PI staining was detected in these samples (<10%), demonstrating that the major mode of cell death was oncosis.

Measurement of lipid peroxidation. Lipid peroxidation in RPTC was monitored as the production of malondialdehyde (MDA) as described previously (26). RPTC were treated with the indicated compound for 2 h and isolated by scraping the dish with a rubber policeman. RPTC suspensions were treated with 10% trichloroacetic acid and centrifuged, the supernatant was removed, and it was combined with 2-thiobarbituric acid (0.76%), heated, cooled, and absorbance measured at 532 nm with 1,1,3,3-tetraethoxypropane as a standard.

Statistical analysis. RPTC isolated from one rabbit represent one experiment (n = 1). The appropriate ANOVA was performed for each data set by using SigmaStat statistical software. Individual means were compared with Fisher's protected least significant difference test with P <=  0.05 being considered a statistically significant difference between mean values. Linear regression was also performed by using Sigma Stat statistical software.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression and activity of iPLA2 in RPTC. Microsomal and cytosolic fractions from confluent cultures of RPTC were subjected to immunoblot analysis using a primary antibody that recognizes cytosolic 85-kDa iPLA2beta . This antibody recognized a single band in immunoblots of microsomal protein from RPTC and comigrated with purified iPLAbeta (a gift from Genetics Institute, Cambridge, MA). No band was detected in immunoblots of cytosolic RPTC protein (Fig. 1). No staining was detected in the IgG control. These data show that a protein with sequence homology to iPLA2beta , a cytosolic protein, is expressed in RPTC microsomal fractions.


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Fig. 1.   Expression of Ca2+-independent phospholipase A2 (iPLA2) in primary cultures of renal proximal tubule cells (RPTC). Confluent RPTC were separated into cytosolic and membrane fractions for immunoblot analysis. The blot is representative of at least 3 separate preparations. iPLA2, purified iPLA2 (1 µg); Micro, 20 µg of microsomal protein; Cyto, 20 µg of cytosolic protein.

Because immunoblot analysis suggested that RPTC iPLA2 was expressed in the ER, the colocalization of iPLA2 with markers of the ER, peroxisomes, and mitochondria was determined. Immunocytochemistry and confocal laser scanning microscopy of fixed RPTC revealed punctate staining as opposed to cytosolic staining, with little staining in the nucleus (Fig. 2, A-C). No staining was detected in the IgG control (data not shown). Staining of iPLA2 was similar to the staining of the ER markers (DiOC6)3 (Fig. 2, A-C) and GRP78 (Fig. 2, D-F). In contrast, the staining pattern of the peroxisomal marker protein PMP70 and iPLA2 was different, with minimal colocalization observed (Fig. 2, G-I). No staining, other than that for iPLA2, was observed in dishes incubated with primary and secondary antibodies to iPLA2 followed by secondary antibodies to GRP78 and PMP70 (data not shown). No colocalization was observed between iPLA2 and the mitochondrial stain Mitotracker green (data not shown).


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Fig. 2.   Expression and colocalization of iPLA2 in RPTC. A: staining of (DiOC6)3 in RPTC (green) demonstrating endoplasmic reticulum (ER) staining. B: staining of iPLA2 in the same RPTC (red) depicted in A. C: overlay of A and B. Yellow staining indicates colocalization of (DiOC6)3 and iPLA2, red indicates staining for iPLA2 alone, green indicates staining for ER, and blue indicates staining of nuclei with 4,6-diamidino-2-phenylindole. D: staining of the ER protein GRP78 in RPTC (green). E: staining of iPLA2 in the same RPTC (red) shown in D. F: overlay of D and E demonstrating colocalization of GRP78 and iPLA2. G: staining of the peroxisomal membrane protein PMP70 in RPTC (green). H: staining of iPLA2 in the same RPTC (red) depicted in H. I: overlay of G and H.

The activity of iPLA2 in microsomes and cytosol from RPTC was measured to further investigate the membrane localization and characterize ER-iPLA2 (Fig. 3). iPLA2 activity was measured by the release of [3H]oleic acid from phosphatidylcholine or plasmenylcholine substrates in the absence of Ca2+. Activity of ER-iPLA2 in the microsomal fraction was ~12,000 pmol · min-1 · mg protein-1 compared with ~1,000 pmol · min-1 · mg protein-1 in the cytosolic fraction. ER-iPLA2 demonstrated a preference for plasmenylcholine compared with phosphatidylcholine, whereas no preference was detected in the cytosol. Treatment of RPTC with BEL before microsomal isolation resulted in a concentration-dependent decrease in iPLA2 activity, with 3 µM BEL resulting in >50% inhibition and 5 µM BEL resulting in >90% inhibition of iPLA2 activity. In contrast, methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethyl ketone (AAOCF3), two cPLA2 inhibitors, did not inhibit ER-iPLA2 at similar concentrations. At higher concentrations, 10 µM MAFP only decreased phosphatidylcholine hydrolysis by 20% in microsomes and had no effect on plasmenylcholine hydrolysis. Similarly, AAOCF3 only decreased ER-iPLA2 activity at concentrations of 20 µM or higher. Collectively, these data demonstrate the expression and activity of an 85-kDa ER-iPLA2 in RPTC that preferentially hydrolyzes plasmalogens and is specifically inhibited by BEL.


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Fig. 3.   Activity and effect of bromoenol lactone (BEL) on iPLA2 activity in RPTC microsomes and cytosol. Primary cultures of RPTC were treated with either solvent control (DMSO) or BEL for 30 min before isolation and fractionation into microsomes and cytosol. iPLA2 activity was measured using the indicated (16:0, [3H]18:1) phospholipid substrates (100 µM) in the presence of 4 mM EGTA. Values are means ± SE of at least 4 separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.

Role of ER-iPLA2 in RPTC cell death. Little is known concerning the role of iPLA2, particularly ER-iPLA2, in cell injury and death. To test the hypothesis that ER-iPLA2 plays a role in RPTC oxidant-induced cell death, RPTC were treated with either solvent control [DMSO at < 0.1% (vol/vol)] or BEL (5 µM) before 2-, 4-, 12-, and 24-h exposures to 200 µM TBHP. TBHP caused time-dependent increases in cell death, resulting in ~20% oncosis at 24 h (Fig. 4). TBHP also induced concentration-dependent cell death, with a concentration of 500 µM resulting in 69 ± 3% oncosis after 24 h. The level of oncosis in controls was ~2-3% and remained constant over time. Confocal microscopy confirmed the time-dependent increase in oncosis (data not shown). Treatment of cells with BEL before exposure to TBHP potentiated oncosis at all time points. Linear regression analysis revealed a strong correlation (r2 = 0.98) between BEL inhibition of ER-iPLA2 activity and BEL potentiation of TBHP-induced oncosis (Fig. 5). These data suggest a direct link between ER-iPLA2 inhibition and potentiation of oxidant-induced oncosis.


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Fig. 4.   Time dependence of tert-butylhydroperoxide (TBHP)-induced RPTC oncosis in the presence and absence of BEL. RPTC were treated with either solvent control (DMSO) or BEL for 30 min before addition of TBHP (200 µM). At the indicated time points, RPTC were isolated, and the percent propidium iodide (PI) staining was determined by using flow cytometry. Values are means ± SE of at least 3 separate experiments. Means with different subscripts are significantly different from each other within a given time point, P < 0.05.



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Fig. 5.   Correlation between percent inhibition of ER-iPLA2 and the potentiation of TBHP-induced oncosis by BEL. RPTC were treated with either solvent control (DMSO) or BEL (0, 1, 3, and 5 µM) for 30 min before the addition of TBHP (200 µM) for 24 h. After treatment, the level of plasmenylcholine cleavage was determined to assess ER-iPLA2 activity and compared with the percent PI staining determined by flow cytometry in similarly treated RPTC. Values are means ± SE of at least 3 separate experiments. Linear regression analysis revealed a correlation coefficient of 0.98.

Studies were conducted to determine whether the potentiating effects of BEL on TBHP-induced oncosis occurred with other oxidants. The chemotherapeutic agent cisplatin is nephrotoxic and thought to produce renal cell death, in part, through oxidative stress (30, 36). Treatment of RPTC with cisplatin for 24 h resulted in 40% oncosis, and prior inhibition of ER-iPLA2 with BEL increased oncosis 1.5-fold (Fig. 6). The model oxidants cumene hydroperoxide, duraquinone, and menadione produced 10-50% RPTC oncosis after 24, 24, and 12 h of exposure, respectively (Fig. 6). Similar to TBHP and cisplatin, pretreatment of RPTC with BEL potentiated the degree of oncosis induced by all of these oxidants 1.5- to 3.3-fold. Antimycin A, a mitochondrial inhibitor, is used as a model of anoxia and produces RPTC oncosis without causing oxidative stress (27). Treatment of RPTC with 0.3 µM antimycin A for 4 h resulted in 50% oncosis and, unlike any of the oxidants used, pretreatment of RPTC with BEL did not increase oncosis over that of RPTC treated with antimycin A alone. Thus oncosis induced by five diverse oxidants was potentiated by inhibition of ER-iPLA2, whereas that of a nonoxidant was not.


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Fig. 6.   Effect of ER-iPLA2 inhibition on oxidant-induced RPTC oncosis. RPTC were treated with either solvent control (DMSO) or BEL for 30 min before the addition of TBHP (200 µM), cumene hydroperoxide (200 µM), duraquinone (400 µM), and cipslatin (400 µM) for 24 h, menadione (10 µM) for 12 h, or antimycin A (0.3 µM) for 4 h. After treatment, the percent PI staining was determined by flow cytometry. Values are means ± SE of at least 3 separate experiments. a and b, means that are significantly different from each other within each group, P < 0.05.

Role of ER-iPLA2 in oxidant-induced lipid peroxidation. We hypothesized that the potentiation of oncosis caused by ER-iPLA2 inhibition was the result of increased retention of peroxidized phospholipids during oxidative stress. To test this hypothesis, RPTC were pretreated with BEL (5 µM) or solvent control [DMSO at <0.1% (vol/vol)] and exposed to TBHP, cisplatin, cumene hydroperoxide, menadione, duraquinone, and antimycin A for 2 h. No cell death was evident in RPTC treated with oxidants at this time. Basal levels of MDA formation in control RPTC were 0.3 nmol/mg, and treatment of RPTC with BEL did not increase this value (Fig. 7). Treatment of RPTC with oxidants or antimycin A for 2 h did not increase MDA formation above control. Longer exposures of cells to oxidants, but not antimycin A, did result in increased MDA formation (data not shown). In contrast to oxidants alone, treatment of RPTC with BEL before oxidant exposure produced MDA formation. Treatment of RPTC with BEL before exposure to antimycin A did not increase MDA formation.


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Fig. 7.   Effect of ER-iPLA2 inhibition on oxidant-induced RPTC lipid peroxidation. RPTC were treated with either solvent control (DMSO) or BEL for 30 min before the addition of TBHP (200 µM), cumene hydroperoxide (200 µM), menadione (10 µM), duraquinone (400 µM), cipslatin (400 µM), or antimycin A (0.3 µM). After 2 h, RPTC malondialdehyde (MDA) formation was determined. Values are means ± SE of at least 3 separate experiments. a and b, means that are significantly different from each other within each group, P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the past few years, a number of PLA2 have been identified and progress has been made in elucidating the functions of many types of PLA2, especially cPLA2. However, iPLA2 are incompletely explored, and their physiological and pathological functions remain under investigation. For example, iPLA2beta is a cytosolic 85-kDa PLA2 that is expressed in the pancreas, heart, brain, and macrophages (5, 6, 14, 15, 24, 34). It also has been demonstrated to exist as multiple proteins as a result of alternative splicing (15). In normally functioning cells, iPLA2beta has been suggested to mediate phospholipid remodeling by means of mechanisms that are not fully determined (34). iPLA2beta also may mediate cell death induced by Fas ligand and hypoxia (2, 3, 35).

Recently, Mancuso et al. (16) and Tanaka et al. (31), using sequences derived from the human genome project, identified, sequenced, cloned, and expressed a novel iPLA2 (iPLA2gamma ) in insect and bacterial cells. iPLA2gamma is similiar in size to iPLA2beta and an iPLA2 found in the plasma membrane of myocytes (17) but is expressed in intracellular membranes. Neither Mancuso et al. (16) nor Tanaka et al. (31) determined the specific intracellular membrane in which iPLA2gamma is expressed, its preference for specific phospholipids, or its physiological function within the cell. We report for the first time the expression of an 85-kDa iPLA2 in primary cultures of kidney cells whose activity and expression were in microsomes, demonstrated selectivity for plasmalogen phospholipids, and were inhibited by BEL. This ER-iPLA2 appears to be constitutively expressed in microsomal membranes, because freshly isolated cells had the same iPLA2 expression and activity as primary cultures (data not shown). The ER location of RPTC iPLA2 is not the result of translocation of iPLA2beta to the membrane during isolation or culture. First, no immunoreactive band was detected in the cytosolic fractions isolated from these cells at any time point of isolation or treatment. Immunocytochemistry of untreated cells, using the same antibody used for immunoblot analysis, demonstrated that iPLA2 was expressed in a punctate manner similar to proteins localized to the ER membrane as opposed to those expressed in the cytosol. On the basis of the similarities in membrane expression, size, activity, and inhibition profile, we propose that ER-iPLA2 is the rabbit iPLA2gamma .

The majority of iPLA2 activity in RPTC was present in the microsomal fraction and displayed substrate selectivity for plasmenylcholine over phosphatidylcholine. A small amount of iPLA2 activity was detected in the cytosolic fraction (~1,000 pmol/mg), which may represent the 28-kDa cytosolic iPLA2 or a small amount of ER contamination (23). BEL resulted in 90% inhibition of plasmenylcholine hydrolysis and totally inhibited phosphatidylcholine hydrolysis by RPTC ER-iPLA2. Neither AAOCF3 nor MAFP inhibited RPTC ER-iPLA2 activity at concentrations under 10 µM and only minimally inhibited activity at higher concentrations. Thus RPTC ER-iPLA2 activity is specifically inhibited by BEL as opposed to AAOCF3 or MAFP. RPTC ER-iPLA2 decreased sensitivity to inhibition by AAOCF3 and MAFP compared with BEL is different from the inhibition profile seen for iPLA2beta (6). These differences may be a result of the membrane location of ER-iPLA2 or may reflect variations in key amino acids in the active sight of each enzyme.

The role of cPLA2 and secretory PLA2 in cellular physiology and injury is well established (3, 6, 20, 25). In comparison, the role of iPLA2 in normal cell physiology and injury is not as well characterized. In contrast to iPLA2beta , nothing is known concerning the role of ER-iPLA2 in normal cell physiology and cell injury and death. Data presented in this study suggest that ER-iPLA2 protects against oxidative-induced cell death. Inhibition of ER-iPLA2 using BEL potentiated oncosis induced by each oxidant used in this study but not that of a nonoxidant. The increase in oxidant-induced RPTC oncosis after inhibition of ER-iPLA2 correlated with increases in lipid peroxidation early in the genesis of oncosis. Finally, a strong correlation exists between the percent inhibition of ER-iPLA2 with BEL and potentiation of cell death by BEL (r2 = 0.98), suggesting that these events are linked. Thus we conclude that the potentiation of oxidant-induced oncosis in RPTC is a result of inhibition of ER-iPLA2.

Studies on the role of ER-iPLA2 in cell death were conducted with the previously characterized iPLA2-inhibitor BEL. In the absence of an alternative method to inhibit ER-iPLA2, we verified that the potentiation of oxidant-induced cell death in the presence of BEL was not the result of decreases in cellular GSH, ATP, GTP, and oxygen consumption levels (data not shown).

MDA is an end product of lipid peroxidation, which itself is a result of a free radical attack on an unsaturated double bond of a phospholipid (i.e., initiation) (33). The free radical propagates from one phospholipid to another and eventually leads to the formation of smaller lipid fragments, one of which is MDA. The reaction is stopped or terminated when the free radical is quenched or when it is removed from the site of injury. We propose that oxidant-induced lipid peroxidation results in enhanced metabolism of phospholipids by ER-iPLA2. Metabolism of peroxidized phospholipids by ER-iPLA2 would cleave and remove these damaging species before their conversion to MDA and prohibit propagation of the free radical. If this model is correct, then inhibition of ER-iPLA2 with BEL would result in an increase in lipid peroxides before increases in cell death.

In conclusion, we have identified and characterized a novel ER-iPLA2 that is similar to iPLA2gamma but distinct from iPLA2beta in that it is expressed at an intracellular membrane and inhibited specifically by BEL. Inhibition of RPTC ER-iPLA2 during oxidant-induced renal cell oncosis resulted in an increase in the level of lipid peroxides, suggesting that this enzyme may play a key role in the metabolism and removal of these damaging species during injury induced by diverse stimuli. Collectively, these data suggest that ER-iPLA2 plays a protective role during oxidative stress by removing oxidized phospholipids.


    ACKNOWLEDGEMENTS

Purified iPLA2beta was a gift from Genetics Institute, Cambridge, MA.


    FOOTNOTES

This work was supported by National Research Service Award DK-10079 (B. S. Cummings) and National Institutes of Health Grants DK-62028 (R. G. Schnellmann) and HL-54907 (J. McHowat).

Address for reprint requests and other correspondence: R. G. Schnellmann, Dept. of Pharmaceutical Sciences, Medical Univ. of South Carolina, 280 Calhoun St., PO Box 250140, 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.

April 16, 2002;10.1152/ajprenal.00022.2002

Received 16 January 2002; accepted in final form 14 April 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ackermann, EJ, Conde-Frieboes K, and Dennis EA. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem 270: 445-450, 1995[Abstract/Free Full Text].

2.   Atsumi, G, Murakami M, Kojima K, Hadano A, Tajima M, and Kudo I. Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2 alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release. J Biol Chem 275: 18248-18258, 2000[Abstract/Free Full Text].

3.   Atsumi, G, Tajima M, Hadano A, Nakatani Y, Murakami M, and Kudo I. Fas-induced arachidonic acid release is mediated by Ca2+-independent phospholipase A2 but not cytosolic phospholipase A2, which undergoes proteolytic inactivation. J Biol Chem 273: 13870-13877, 1998[Abstract/Free Full Text].

4.   Balboa, MA, Balsinde J, Jones SS, and Dennis EA. Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells. J Biol Chem 272: 8576-8580, 1997[Abstract/Free Full Text].

5.   Cirino, G. Multiple controls in inflammation. Extracellular and intracellular phospholipase A2, inducible and constitutive cyclooxygenase, and inducible nitric oxide synthase. Biochem Pharmacol 55: 105-111, 1998[ISI][Medline].

6.   Cummings, BS, McHowat J, and Schnellmann RG. Phospholipase A2s in cell injury and death. J Pharmacol Exp Ther 294: 793-799, 2000[Abstract/Free Full Text].

7.   Dennis, EA. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22: 1-2, 1997[ISI][Medline].

8.   Glaser, KB. Regulation of phospholipase A2 enzymes: selective inhibitors and their pharmacological potential. Adv Pharmacol 32: 31-36, 1995[Medline].

9.   Goldberg, JE, Sherwood SW, and Clayberger C. A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry. J Immunol Methods 222: 1-9, 1999[ISI][Medline].

10.   Hazen, SL, Zupan LA, Weiss RH, Getman DP, and Gross RW. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. J Biol Chem 266: 7227-7232, 1991[Abstract/Free Full Text].

11.   Ilic, D, Almeida EAC, Schlaepfer DD, Dazin P, and Aizawa S. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol 143: 547-560, 1998[Abstract/Free Full Text].

12.   Larsonn, PK, Claesson HE, and Kennedy BP. Multiple splice variants of the human calcium-independent phospholipase A2 and their effect on enzyme activity. J Biol Chem 273: 207-214, 1998[Abstract/Free Full Text].

13.   Longo, WE, Grossmann EM, Erickson B, Panesar N, Mazuski JE, and Kaminski DL. The effect of phospholipase A2 inhibitors on proliferation and apoptosis of murine intestinal cells. J Surg Res 84: 51-56, 1999[ISI][Medline].

14.   Ma, Z, Ramanadham S, Kempe K, Chi XS, Ladenson J, and Turk J. Pancreatic islets express a Ca2+-independent phospholipase A2 enzyme that contains a repeated structural motif homologous to the integral membrane protein binding domain of ankyrin. J Biol Chem 272: 11118-11127, 1997[Abstract/Free Full Text].

15.   Ma, Z, Xang X, Nowatzke W, Ramanadham S, and Turk J. Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1. J Biol Chem 274: 9607-9616, 1999[Abstract/Free Full Text].

16.   Mancuso, DJ, Jenkins CM, and Gross RW. The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2). J Biol Chem 275: 9937-9945, 2000[Abstract/Free Full Text].

17.   McHowat, J, and Creer MH. Calcium-independent phospholipase A2 in isolated rabbit ventricular myocytes. Lipids 33: 1203-1212, 1998[ISI][Medline].

18.   McHowat, J, Liu S, and Creer MH. Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes. Am J Physiol Cell Physiol 274: C1727-C1737, 1998[Abstract/Free Full Text].

19.   Meijerman, I, Blom WM, de Bont HJGM, Mulder GJ, and Nagelkerke JF. Induction of apoptosis and changes in nuclear G-actin are mediated by different pathways: the effect of inhibitors of protein and RNA synthesis in isolated rat hepatocytes. Toxicol Appl Pharmacol 156: 46-55, 1999[ISI][Medline].

20.   Morioka, Y, Saiga A, Yokota Y, Suzuki N, Ikeda M, Ono T, Nakano K, Fujii N, Ishizaki J, Arita H, and Hanasaki K. Mouse group X secretory phospholipase A2 induces a potent release of arachidonic acid from spleen cells and acts as a ligand for the phospholipase A2 receptor. Arch Biochem Biophys 381: 31-42, 2000[ISI][Medline].

21.   Nowak, G, and Schnellmann RG. Integrative effects of EGF on metabolism and proliferation in renal proximal tubular cells. Am J Physiol Cell Physiol 269: C1317-C1325, 1995[Abstract/Free Full Text].

22.   Nowak, G, and Schnellmann RG. L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture. Am J Physiol Cell Physiol 271: C2072-C2080, 1996[Abstract/Free Full Text].

23.   Portilla, D, and Dai G. Purification of a novel calcium-independent phospholipase A2 from rabbit kidney. J Biol Chem 271: 15451-15457, 1996[Abstract/Free Full Text].

24.   Roshak, AK, Capper EA, Stevenson-Eichman C, and Marshall LA. Human calcium-independent phospholipase A2 mediates lymphocyte proliferation. J Biol Chem 275: 35692-35698, 2000[Abstract/Free Full Text].

25.   Sapirstein, A, Spech RA, Witzgall R, and Bonventre JV. Cytosolic phospholipase A2 (PLA2), but not secretory PLA2, potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells. J Biol Chem 271: 21505-21513, 1996[Abstract/Free Full Text].

26.   Schnellmann, RG. Mechanisms of t-butyl hydroperoxide-induced toxicity to rabbit renal proximal tubules. Am J Physiol Cell Physiol 255: C28-C33, 1988[Abstract/Free Full Text].

27.   Schnellmann, RG, Yang X, and Carrick J. Arachidonic acid release in renal proximal tubule cell injuries and death. J Biochem Toxicol 9: 211-217, 1994[ISI][Medline].

28.   Schutte, B, Nuydens R, Geerts H, and Ramaekers F. Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J Neurosci Methods 86: 63-69, 1998[ISI][Medline].

29.   Six, DA, and Dennis EA. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochim Biophys Acta 1488: 1-19, 2000[ISI][Medline].

30.   Sugihara, K, Nakano S, and Gemba M. Stimulatory effect of cisplatin on production of lipid peroxidation in renal tissues. Jap J Pharmacol 43: 247-252, 1987[ISI].

31.   Tanaka, H, Takeya R, and Sumimoto H. A novel intracellular membrane-bound calcium-independent phospholipase A2. Biochem Biophys Res Commun 272: 320-326, 2000[ISI][Medline].

32.   Tang, J, Kriz RW, Wolfman N, Shaffer M, Seehra J, and Jones SS. A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs. J Biol Chem 272: 8567-8575, 1997[Abstract/Free Full Text].

33.   Timbrell, JA. Principles of Biochemical Toxicology. New York: Taylor and Francis, 1991, p. 192-284.

34.   Winstead, MV, Balsinde J, and Dennis EA. Calcium-independent phospholipase A2: structure and function. Biochim Biophys Acta 1488: 28-29, 2000[ISI][Medline].

35.   Williams, SD, and Ford DA. Calcium-independent phospholipase A2 mediates CREB phosphorylation and c-fos expression during ischemia. Am J Physiol Heart Circ Physiol 281: H168-H176, 2001[Abstract/Free Full Text].

36.   Zhang, JG, and Liundup WE. Role of mitochondria in cisplatin-induced oxidative damage exhibited by rat renal cortical slices. Biochem Pharmacol 45: 2215-2222, 1993[ISI][Medline].


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