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Late-Gestation Rat Myometrial Cells Express Multiple Isoforms of Phospholipase A2 That Mediate PCB 50-Induced Release of Arachidonic Acid wi
http://www.100md.com 《毒物学科学杂志》
     Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109–2029

    ABSTRACT

    Previous reports have shown that ortho-substituted polychlorinated biphenyls (PCBs) are uterotonic and activate phospholipase A2 to release arachidonic acid (AA) from membrane phospholipids. AA serves as the precursor to various eicosanoids, which, in addition to AA itself, are capable of modulating uterine function. To examine whether PCBs stimulate phospholipase A2 (PLA2) to mobilize arachidonic acid from late-gestation rat uterus, primary cultures of gestation day 20 (gd20) rat myometrial cells (RMC) were labeled with 0.5 μCi 3H-AA prior to a 10-, 20-, or 30-min exposure to 2,2',4,6-tetrachlorobiphenyl (PCB 50) (1–50 μM) or 0.1% DMSO (solvent control). PCB 50 stimulated the release of 3H-AA from gd20 RMC in concentration- and time-dependent manners (p < 0.05). PCB 50 stimulation of RMC was attenuated with ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) and nifedipine, suggesting that AA release was dependent on the influx of extracellular calcium through L-type voltage-operated calcium channels. PCB 50-induced release of AA from RMC was also attenuated with the PLA2-specific inhibitors methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL), and manoalide (p < 0.05). Stimulation of PLA2 enzymes in response to PCB exposure occurred via p38 mitogen activated protein kinase (MAPK) activation as indicated by the significant attenuation of PCB 50-induced AA release from RMC in the presence of SB 202190. In addition to stimulating AA release, PCB 50 induced a significant production of prostaglandins from gd20 RMC compared with controls (p < 0.05). These results suggest that myometrial cells express multiple PLA2 isoforms that may serve as a target and effector for ortho-substituted PCB-mediated stimulation of uterine function through arachidonic acid and prostaglandin release.

    Key Words: myometrium; PCBs; arachidonic acid; prostaglandins; Phospholipase A2.

    INTRODUCTION

    Phospholipase A2 (PLA2) enzymes serve as the rate-limiting step in the synthesis of bioactive lipid mediators. PLA2 cleaves glycerophospholipids localized at the sn-2 position of cell membrane phospholipids, releasing fatty acids (e.g, arachidonic acid) and lysophospholipids (Flower and Blackwell, 1976). Arachidonic acid serves as the precursor for the biosynthesis of eicosanoids, which mediate a variety of cellular processes. For example, the 2-series prostaglandins (i.e., PGE2 and PGF2) play important roles in the onset of labor through the stimulation of uterine contractions (Bygdeman et al., 1968; Karim et al., 1968), cervical ripening (Challis and Lye, 1994), and membrane rupture (Keirse, 1990). Prostaglandins also initiate luteolysis (Niswender and Nett, 1994), which contributes to the onset of parturition in the rat and other species.

    PLA2 enzymes can be characterized based on differences in structure, cellular function, localization, and catalytic activity. Cytosolic PLA2 activity includes contributions from calcium-dependent (e.g., cPLA2) as well as calcium-independent (i)PLA2 isoforms. The cellular distribution of isoform-specific PLA2 activity and requirements for calcium vary depending on the cell type, e.g., alveolar macrophages express lysosomal Ca2+-independent PLA2 activity but peritoneal macrophages lack Ca2+-independent activity in the lysosomal fraction (Abe et al., 2004). Additionally, Ca2+-independent PLA2 activity can also be expressed in the cytosol (Akiba et al., 1999) and membrane fraction (Larsson Forsell et al., 1999). Additional PLA2 activity can also be observed in the intercellular space from the contributions of the low-molecular-weight, cysteine-rich secreted PLA2 (sPLA2). One isoform of cPLA2, cPLA2, is located in the cytosol and translocates to intracellular membranes in response to nano- to micro-molar increases in intracellular calcium, thereby gaining access to phospholipid substrates (Channon and Leslie, 1990; Perisic et al., 1999). Activation of cPLA2 is regulated by phosphorylation at active site serine residues by members of the mitogen activated protein kinase (MAPK) family including extracellular signal-regulated kinase (ERK) (Nemenoff et al., 1993), p38 MAPK (Kramer et al., 1996), and Ca2+/calmodulin kinase II (Muthalif et al., 2001). Additionally, a pertussis toxin-sensitive G protein (Gi) has been reported to activate cPLA2-mediated arachidonic acid release (Burke et al., 1997). cPLA2 exerts specificity for fatty acids containing arachidonic acid in the sn-2 position, and its activity is inhibited by methyl arachidonyl fluorophosphonate (MAFP). In contrast to cPLA2, iPLA2 is catalytically active in the absence of calcium, but can serve a role in agonist-induced arachidonic acid release (Gross et al., 1995; Lehman et al., 1993) and eicosanoid production (Larsson Forsell et al., 1998). Calcium-independent PLA2 activity has been shown to be regulated by protein kinase C (PKC) (Steer et al., 2002) and p38 MAPK (Yellaturu and Rao, 2003) phosphorylation. The contribution of iPLA2 can be distinguished from other cytosolic isoforms with the inhibitor bromoenol lactone (BEL). Stimulation of sPLA2 is also associated with generation of arachidonic acid, prostaglandins, and other eicosanoids (Kudo and Murakami, 2002; Leslie, 2004). Secretory PLA2 enzymes demonstrate a wide range of phospholipid head group specificity and are catalytically active under millimolar calcium-containing conditions.

    Polychlorinated biphenyls (PCBs) are a group of environmental toxicants that were commercially produced for use as dielectric fluids for capacitors and transformers, additives in paints and plastics, and other industrial processes. Despite the ban on United States production of PCBs in the late seventies, PCBs have persisted in the environment and bioaccumulated in the food chain due to their lipophilic properties. PCBs are capable of inducing a wide range of toxicity, including adverse reproductive health effects. Previous reports have shown that PCBs activate PLA2 to release arachidonic acid from membrane phospholipids (Bae et al., 1999; Santiago et al., 2001; Tithof et al., 1995, 1996, 1998). Decreased gestation lengths have been reported in women exposed to PCBs occupationally (Taylor et al., 1984, 1989) or through the consumption of contaminated fish (Fein et al., 1984). We previously showed that in vitro exposure to the ortho-substituted congener 2,2',4,6-tetrachlorobiphenyl (PCB 50) stimulates the contraction frequency of late-gestation rat uteri (Brant and Caruso, 2003). The objective of the present study was to determine whether PCB 50 stimulates PLA2 to mobilize arachidonic acid and increase prostaglandin production from late-gestation rat myometrial cells (RMC).

    MATERIALS AND METHODS

    Chemicals, Reagents and Antibodies.

    PCB 50 was obtained from Ultra Scientific (North Kingstown, RI). The certificate of analysis stated that the purity of PCB 50 was 99.9% as determined by high resolution gas chromatography. Dimethyl sulfoxide (DMSO) and ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) were purchased from Sigma (St. Louis, MO). Cells were maintained in culture media (RPMI 1640) and fetal bovine serum were purchased from InvitrogenTM (Carlsbad, CA). The 5,6,8,9,11,12,14,15-3H-arachidonic acid was obtained from American Radiolabeled Chemical, Inc. (St. Louis, MO), and the scintillation cocktail UltimaGoldTM was purchased from PerkinElmer, Inc. (Boston, MA). Nifedipine was from Calbiochem (San Diego, CA). Cayman Chemical (Ann Arbor, MI) supplied the phospholipase inhibitor bromoenol lactone (BEL) and the anti-sPLA2-IIA antibody. Manoalide and methyl arachidonyl fluorophosphonate (MAFP) were obtained from Biomol (Plymouth Meeting, PA). Anti-iPLA2 and anti-cPLA2 antibodies were from Upstate (Lake Placid, NY) and Cell Signaling Technology (Beverly, MA), respectively.

    Animals.

    Timed-pregnant specific pathogen free (SPF) Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN) and housed at 24 ± 1°C under a 12-h light schedule. All rats were in excellent health as evaluated by trained animal technicians. Animals were provided with food (LabDiet 5001 Rodent Diet) and water ad libitum. Animal housing and handling procedures complied with institutional and NIH guidelines for care and use of laboratory animals in research.

    Myometrial cell isolation and culture.

    Late-gestation (gd20) Sprague-Dawley rats were euthanized by CO2 asphyxiation. Only those dams having a minimum of 3 pups on each side of uterine horn were selected for use. An estimated 12 dams were used to establish the myometrial cell cultures employed in these experiments. Myometrial smooth muscle cells (RMC) were isolated by methods previously described (Ohmichi et al., 1997) with slight modifications. Uteri horns were excised and removed of pups, placentae, cervical, ovarian, and decidual tissue and minced into small pieces (2 mm x 2 mm). Tissue was placed in a media bottle, transferred to a 37°C shaking water bath and digested in calcium- and magnesium-free phosphate buffered saline (CMF-PBS; 2.68 mM KCl, 1.47 mM KH2PO4, 136.89 mM NaCl, 8.1 mM Na2HPO4 at pH 7.4) containing 0.1% deoxyribonuclease I and 0.1% trypsin for 0.5 h. Collagenase was then added to the digestion medium at a final concentration of 0.1%, and digestion was continued for an additional 0.5 h. The digestion was filtered through a 200-micron pore nylon mesh to remove tissue chunks and centrifuged at 453 x g for 5 min. The cell pellet was washed two times with RPMI 1640 medium. Cells were seeded into 10-mm2 or 12-well tissue culture dishes containing RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% PenStrep, and maintained at 37°C in a 5% CO2 atmosphere. Growth medium was changed every other day. Primary cells on days 4 and 5 were used for all studies.

    Immunoblot detection of phospholipase A2.

    Unexposed rat myometrial cells grown in 10-mm2 culture dishes were collected by scraping (to minimize protein degradation) in 200 μl modified radioimmunoprecipitation (RIPA) buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% SDS, 0.5% sodium deoxycholate, 1 mM dithiothreitol, 100 μM phenylmethyl sulphonyl fluoride, 1% NP-40, and 1x protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN)) and sonicated for 6–8 s using an ultrasonic liquid processor (Misonix Sonicator 3000, Farmingdale, NY). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit from Pierce (Rockford, IL). Bovine serum albumin was used as protein standard. Cell lysates (10 μg protein/lane) were subjected to electrophoresis using 4–20% Tris-Glycine gradient gels under denaturing conditions. Proteins were transferred to polyvinyl fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA). Nonspecific protein binding sites in membranes were blocked with 5% nonfat dry milk in TBS-T (0.1% Tween-20 in Tris-buffered saline: 19.98 mM TrisHCl, pH 7.6, 13.69 mM NaCl) at room temperature for 2 h. Membranes were incubated overnight at 4°C with antibodies to iPLA2, cPLA2, or sPLA2-IIA in 1:500, 1:800, and 1:1000 dilutions, respectively, in TBS-T containing milk. After three 15-min washes with fresh exchanges of TBS-T containing milk, membranes were incubated with anti-fluorescein alkaline phosphatase (AP) conjugated secondary antibody (1:3300 dilution) for 1 h at room temperature. Membranes were then rinsed three times for 20 min in TBS-T. Protein bands were visualized using an enhanced fluorescent reagent (ECFTM Western blotting kit; Amersham Biosciences, Piscataway, NJ) on FLA 5000 imaging system (Fujifilm Medical Systems U.S.A., Inc. Stamford, CT).

    Arachidonic acid release.

    Arachidonic acid release was determined using modified procedures of previous reports (Cane et al., 1997; Kodavanti and Derr-Yellin, 2002; Tithof et al., 2000). Rat myometrial cells were labeled with 0.5 μCi/ml of 5,6,8,9,11,12,14,15-3H-arachidonic acid (specific activity 60–100 mCi/mmol) and incubated for 24 h to allow incorporation of the tritiated arachidonic acid (Bae et al., 1999; Criswell and Loch-Caruso, 1995; Wang et al., 2001). After the 24 h incubation, culture medium from each well was transferred to a vial containing 10 ml of UltimaGoldTM scintillation fluid (PerkinElmer, Boston, MA) to determine the unincorporated radioactivity by liquid scintillation counting. The labeled cells were carefully washed once with medium containing 0.1% bovine serum albumin (BSA) to remove excess label, then washed twice with CMF-PBS to remove residual unincorporated tritiated arachidonic acid. The cells were then exposed to increasing concentrations of PCB 50 (1–50 μM), 5 μM calcium ionophore A23187 [GenBank] (positive control), or 0.1% DMSO (solvent control) for 10, 20, or 30 min in serum-free RPMI 1640 containing 0.1% BSA (n = 3). Bovine serum albumin was added to the exposure medium to bind the released arachidonic acid (Spector, 1975) and prevent its metabolism into eicosanoids. After exposure to PCB 50 or solvent (controls), the medium was transferred to vials, and the amount of medium-associated radioactivity was determined by liquid scintillation counting, as follows. To obtain the cell-associated radioactivity, the reactions were terminated by adding 1 ml ice-cold methanol (Hertelendy et al., 1995), and cells were dislodged by brief incubation with a 0.25% trypsin/0.1% EDTA solution (Wang et al., 2001). The cells were collected in trypsin, and culture wells were washed again with methanol. The trypsinized cells and methanol washes were combined, transferred to vials, and the cell-associated radioactivity was determined by liquid scintillation counting. The average amount of 3H-arachidonic acid that was incorporated into rat myometrial cells was 31.73 ± 0.99%. Data are expressed as percentage of total cellular radiolabeled arachidonic acid using the following equation:

    where MT is the medium-associated radioactivity and C is the cell-associated radioactivity.

    Phospholipase A2, VOCC and p38 MAPK inhibition.

    To examine the role of calcium-dependent lipase activity in PCB 50-induced arachidonic acid release, RMC were exposed to PCB 50 in serum-free RPMI 1640 medium containing 0.1% BSA and 1 mM of the calcium chelator EGTA (n = 3–6). The role of calcium channels in PCB 50-mediated release of arachidonic acid from RMC was determined by pretreating the cells for 15 min with 10 μM of the L-type voltage operated calcium channel inhibitor nifedipine prior to PCB 50 (15, 25, and 50 μM) exposure (n = 3). For the phospholipase A2 inhibitor studies, cells were pretreated prior to PCB 50 exposure with 10 μM of the iPLA2 inhibitor BEL (IC50 7 μM (Balsinde et al., 1995)) for 30 min, 3 μM of the sPLA2-IIA inhibitor manoalide (IC50 0.02—0.2μM (Jacobson et al., 1990)) for 30 min, or 10 μM of the cytosolic (iPLA2/cPLA2) inhibitor MAFP (IC50 5 μM (Balsinde and Dennis, 1996)) for 15 min (n = 3). These concentrations of BEL, manoalide, and MAFP were chosen based on published values used to inhibit PLA2 activity (Bae et al., 1999; Balsinde and Dennis, 1996; Yellaturu and Rao, 2003). To examine the role of p38 MAPK in PCB 50-stimulated release of arachidonic acid, RMC were cotreated with 10 μM of the p38 MAPK inhibitor SB 202190 (n = 3–6).

    Prostaglandin production.

    PCB 50-induced prostaglandin production from late-gestation rat myometrial cells was measured using the Prostaglandin Screening EIA Kit (Cayman Chemical, Ann Arbor, MI) following the manufacturer's instructions. Briefly, cells grown in 12-well culture dishes (105 cells/dish) were exposed to 0 (0.1% DMSO, solvent control), 15, 25, or 50 μM PCB 50 for 30 min in serum-free RPMI 1640 medium. After the 30-min exposure, cell supernatant was collected and assayed using the EIA kit. All samples were run in triplicate at two dilutions.

    Cytotoxicity assay.

    Cell viability following exposures to PCB 50, A23187 [GenBank] , and the PLA2 inhibitors was assessed by the ability of viable cells to convert the redox dye resazurin into a fluorescent end product, resorufin, using the CellTiter-Blue Cell Viability Assay (Promega Biosciences, Inc., Madison, WI). Additional cell toxicity assays were conducted using the Neutral Red Assay (Sigma, St. Louis, MO).

    Statistical analysis.

    The arachidonic acid and prostaglandin data were analyzed by two-way or one-way analysis of variance (ANOVA), respectively, using Sigma Stat v3 software (Jandel Scientific, San Rafael, CA). Each data set passed the equal variance using a Levene Median test. Those data not normally distributed were transformed using the arcsine square root transformation prior to ANOVA, and the results were consistent with those obtained with the raw data. Post hoc pair-wise comparisons of means were performed by the Student-Newman-Keuls method. A p value <0.05 was considered statistically significant.

    RESULTS

    Immunoblot Detection of Phospholipase A2

    Lysates from unexposed cells were analyzed by Western blot to determine which PLA2 isoform(s) were expressed in rat myometrial cells isolated from late-gestation rat uteri and grown in cell culture. Analyses revealed the presence of the cytosolic calcium-dependent cPLA2 and the low-molecular-weight, secretory PLA2-IIA. Additionally, immunoblot analysis with an iPLA2 antibody detected both an 85 kDa protein as well as a related protein of 50 kDa in primary cultures of late-gestation rat myometrial cells (Fig. 1). The detection of the 50 kDa protein is not likely a result of unspecific binding, as the information from the antibody supplier (Upstate, Lake Placid, NY) reports detection of both bands (85 kDa and 50 kDa) can be competed out with peptide inhibition assay.

    PCB 50-Stimulated Arachidonic Acid Release

    PCB 50 stimulated the release of 3H-arachidonic acid from RMC in concentration- and time- dependent manners (ANOVA time, treatment, and time x treatment effects, p < 0.001; n = 3; Fig. 2). Following a 10-min exposure, there were no changes in the amounts of incorporated arachidonic acid released from PCB 50-treated (1–50 μM) or A23187 [GenBank] -treated (positive control) RMC compared to solvent controls (data not shown). After 20 min, exposure to 50 μM PCB 50 or 5 μM A23187 [GenBank] stimulated the release of incorporated arachidonic acid from RMC to 12.38 ± 0.91% and 11.14 ± 0.42%, respectively (Fig. 2A). These amounts were significantly greater than those observed in the nonexposed and solvent control cells (5.20 ± 0.23% and 4.75 ± 0.36%, respectively) (p < 0.05; Fig. 2A). A 20-min exposure to lower concentrations of PCB 50 (1–25 μM) did not stimulate arachidonic acid release from RMC compared with nonexposed and solvent controls (Fig. 2A).

    After 30 min, PCB 50 stimulated the release of incorporated arachidonic acid from RMC in a concentration-dependent manner to 11.51 ± 0.90%, 18.69 ± 3.35%, 30.88 ± 3.2%, and 48.70 ± 1.04% in cells exposed to 10, 15, 25, and 50 μM, respectively (p < 0.05; Fig. 2B). These values were significantly greater than those observed in the nonexposed cells (4.24 ± 0.16%) and solvent control cells (5.97 ± 0.22%) (p < 0.05). Moreover, the amount of incorporated arachidonic acid released by RMC exposed to 15, 25, and 50 μM PCB 50 was significantly greater at 30 min compared to 20 min (p < 0.05; Figs. 2A and 2B). The calcium ionophore A23187 [GenBank] (5 μM, positive control) also stimulated a significant release of arachidonic acid from RMC following a 30-min exposure (18.72 ± 1.14%) compared with nonexposed and solvent-exposed controls (p < 0.05). The Neutral Red and CellTiter-Blue Cell Viability Assays determined that the PCB (1–50 μM) and A23187 [GenBank] exposures were noncytotoxic to myometrial cells compared with DMSO solvent controls (data not shown).

    Effects of Calcium on PCB 50-Stimulated Arachidonic Acid Release

    To determine if the PCB 50-stimulated arachidonic acid release was dependent on the presence of extracellular calcium, the 30-min PCB exposures were repeated in the presence of the calcium chelator EGTA. When 1 mM EGTA was present in the culture media, the amount of incorporated arachidonic acid released by 15, 25, and 50 μM PCB 50 was significantly reduced to 6.80 ± 1.10%, 8.91 ± 1.07%, and 13.81 ± 2.3%, respectively, compared with amounts released under non-EGTA-containing conditions (p < 0.05; n = 3–6; Fig. 3A). These results suggest that the majority of PCB 50-stimulated release of arachidonic acid was dependent on the presence of calcium. However, even under the calcium-free conditions, the amount of arachidonic acid released from RMC following exposure to 50 μM PCB 50 was significantly, albeit modestly, greater than the amount released from solvent control RMC (13.81 ± 2.3% compared with 5.30 ± 0.60%, respectively) (p < 0.05; Fig. 3A), suggesting the involvement of a calcium-independent lipase at the higher PCB 50 concentration.

    In the presence of 10 μM of the L-type VOCC inhibitor nifedipine, arachidonic acid release was significantly reduced to 10.41 ± 0.30%, 9.32 ± 0.49%, and 15.46 ± 2.09% following exposure to 15, 25, or 50 μM PCB 50, respectively (p < 0.05; n = 3; Fig. 3B). There was still a significant amount of arachidonic acid released by 50 μM PCB 50 in the presence of nifedipine compared with solvent controls (15.46 ± 2.09% and 9.99 ± 0.23%, respectively) (p < 0.05; Fig. 3B), further supporting the role of a calcium-independent lipase activity at the highest PCB concentration tested.

    Effect of PLA2 Inhibition on PCB 50-Stimulated Arachidonic Acid Release

    There were no changes in myometrial cell viability following exposure to the PLA2 inhibitors MAFP (10 μM), BEL (10 μM) or manoalide (3 μM) compared with DMSO solvent controls (CellTiter-Blue assay, data not shown). Pretreatment with 10 μM of the cPLA2/iPLA2 inhibitor MAFP significantly attenuated PCB 50-stimulated release of arachidonic acid from late-gestation RMC. The amount of incorporated arachidonic acid released from RMC exposed to 15, 25, or 50 μM PCB was 11.36 ± 0.90%, 8.09 ± 0.80%, and 7.72 ± 0.05%, respectively (Fig. 4). These amounts were significantly less compared with PCB 50 alone (p < 0.05).

    To elucidate the contribution of iPLA2 to PCB 50-stimulated release of arachidonic acid from late-gestation RMC, cells were pretreated for 30 min with 10 μM of the iPLA2-selective inhibitor bromoenol lactone (BEL). BEL attenuated the 25 and 50 μM PCB 50-stimulated arachidonic acid release from RMC to 9.96 ± 0.80% and 23.72 ± 2.45%, respectively. These values were significantly less compared with PCB 50 alone (p < 0.05; Fig. 4). BEL had no effect on 15 μM PCB 50, indicating that PCB 50 only stimulates iPLA2 at the higher concentrations. Because MAFP inhibits both cPLA2 and iPLA2, whereas BEL inhibits iPLA2, the effects of MAFP on 15 μM PCB 50-stimulated arachidonic acid release are due to MAFPs inhibitory actions on cPLA2.

    The amount of arachidonic acid released by RMC exposed to 15 μM PCB 50 in the presence of the sPLA2-IIA inhibitor manoalide was not significantly different compared with amounts released by PCB 50 alone (Fig. 4). In contrast, manoalide significantly attenuated arachidonic acid released by 25 and 50 μM PCB 50 (to 9.08 ± 1.02% and 20.51 ± 5.31%, respectively) compared with amounts released by PCB 50 alone (p < 0.05; Fig. 4). Although 50 μM PCB 50-induced release of arachidonic acid from RMC was significantly attenuated in the presence of manoalide, it was still significantly greater compared with solvent controls (p < 0.05; Fig. 4).

    Effect of p38 MAPK Inhibition on PCB 50-Stimulated Release of Arachidonic Acid

    The p38 MAPK inhibitor SB 202190 significantly reduced the amounts of incorporated arachidonic acid released from RMC following exposure to PCB 50 (Fig. 5). In the presence of SB 202190, only 8.46 ± 0.28%, 10.31 ± 0.33%, and 15.80 ± 1.03% incorporated arachidonic acid was released from RMC following exposure to 15, 25, or 50 μM PCB 50, respectively, significantly reduced compared with cells exposed to PCB 50 without inhibitor (p < 0.05; Fig. 5). Nonetheless, a significant increase in the amount of arachidonic acid released by 50 μM PCB 50 was observed in the presence of SB 202190 compared with solvent controls (15.80 ± 1.03% compared to 7.67 ± 0.40%, respectively; p < 0.05), suggesting that a p38 MAPK-independent mechanism contributes to arachidonic acid release stimulated by 50 μM PCB 50 but not lower concentrations of PCB 50.

    Prostaglandin Production

    PCB 50 stimulated a significant release of prostaglandins from late-gestation RMC compared with solvent controls as measured using a Prostaglandin Screening EIA kit (p < 0.05; Fig. 6). The amount of prostaglandins released into the cell culture media following exposure to 15, 25, and 50 μM PCB 50 were 2675.03 ± 357.24, 3116.05 ± 274.52, and 3638.81 ± 81.55 pg/ml, respectively. These values were significantly greater compared with solvent controls (0 μM PCB 50, 1574.10 ± 307.15 pg/ml; p < 0.05).

    DISCUSSION

    Stimulus-induced activation of sPLA2-IIA (Cho, 2000), cPLA2 (Balsinde et al., 2000; Ghosh et al., 2004; Liberty et al., 2004), and iPLA2 (Gross et al., 1995; Lehman et al., 1993) are associated with arachidonic acid release and eicosanoid production. Previous reports have indicated that rat myometrial cells cultured from mid-gestation rat uteri possess calcium-dependent and calcium-independent PLA2 activity (Bae et al., 1999; Wang et al., 2001). In the present study, the depressed ability of PCB 50 to induce arachidonic acid release from RMC in the presence of PLA2 inhibitors indicates that late-gestation RMC also possess both calcium-dependent and calcium-independent PLA2 activity. To our knowledge, this is the first report that cultures of late-gestation rat myometrial cells express cPLA2, sPLA2-IIA, and iPLA2 in vitro.

    PCB 50 stimulated the release of arachidonic acid from late-gestation rat myometrial cells, in agreement with a previous report that PCB 50 releases arachidonic acid from a rat adrenal gland pheochromocytoma cell line (PC12) (Shin et al., 2002). Most, but not all, of the arachidonic acid release from RMC in response to PCB 50 was dependent on the presence of extracellular calcium, although a slight, yet statistically significant, calcium-independent release of arachidonic acid followed exposure to 50 μM PCB 50. In contrast to the current findings in myometrial cells, PCB 50-induced release of arachidonic acid from PC12 cells is independent of extracellular calcium (Shin et al., 2002). Possible explanation for the difference between the current findings and those of Shin et al. (2002) may be cell-specific differences in the calcium-dependency of the different phospholipase A2 isoforms or calcium-dependent cell signaling pathways targeted by PCB 50 in PC12 versus RMC cells.

    Group VI iPLA2 has a molecular weight of 85 kDa, although splice variants have been identified (Ma and Turk, 2001; Tang et al., 1997). The antibody against Group VI iPLA2 used in the current study recognizes the 85 kDa iPLA2 as well as a 50 kDa iPLA2-related protein, in agreement with the report of the supplier (Upstate, Lake Placid, NY). Moreover, the 50 kDa iPLA2-related protein showed a strong immunoreactivity against the iPLA2 antibody, whereas the 85 kDa isoform showed only a very faint immunoreactivity. The iPLA2 isoform that mediates the calcium-independent release of arachidonic acid from RMC following exposure to 50 μM PCB 50 is currently unknown and warrants further investigation. Because arachidonic acid can stimulate uterine contractions (Bae et al., 1999), this calcium-independent release of arachidonic acid in response to 50 μM PCB 50 is consistent with our finding that 50 μM PCB 50 stimulates late-gestation rat uterine contractions through an iPLA2-mediated pathway (Brant and Caruso, 2003).

    Inglefield and Shafer (2000) have identified nifedipine-sensitive, Aroclor 1254-induced Ca2+ oscillations in rat neocortical cells. Consistent with the findings that the commercial PCB mixture Aroclor 1242 stimulates L-type VOCC, our results demonstrated that PCB 50-induced release of arachidonic acid from cultures of RMC was sensitive to the L-type VOCC inhibitor nifedipine. These data, in combination with a requirement for extracellular calcium, suggest that PCB 50 stimulated arachidonic acid release from cultures of late-gestation RMC by a mechanism involving the influx of extracellular calcium through L-type VOCC.

    Depending on the concentration tested, approximately 19% to 49% of the incorporated 3H-arachidonic acid was released from RMC in response to PCB 50 exposure. These values represent a three- to eight-fold increase in stimulus-induced arachidonic acid release over solvent controls, comparable to levels of stimulus-induced arachidonic acid release observed with other smooth muscle cells. For example, Upmacis et al. (2004) report that a 30-min exposure to 100 or 500 μM peroxynitrite stimulates an approximate 4.5- and 7.5-fold increase, respectively, in arachidonic acid release above control values from rat vascular smooth muscle cells. Additionally, our findings in RMC are consistent with the reports that Aroclor 1242 (Olivero and Ganey, 2000) stimulate an approximate five-fold increase in arachidonic acid release from neutrophils compared with controls.

    Phosphorylation by p38 MAPK has been implicated in regulating the catalytic activity of both cPLA2 (Kramer et al., 1996) and iPLA2 (Yellaturu and Rao, 2003). The p38 MAPK inhibitor SB 202190 and the cPLA2/iPLA2 inhibitor MAFP significantly attenuated the arachidonic acid released from RMC in response to PCB 50, suggesting that phosphorylation by p38 MAPK may be involved in the activation of cPLA2 and iPLA2 in rat myometrial cells. Moreover, these data suggest a sensitivity of cytosolic PLA2 to PCB stimulation by a mechanism that involves p38 MAPK activation. The slight, yet significant release of arachidonic acid by 50 μM PCB in the presence of SB 202190 suggests that additional pathways contribute to PCB 50-induced activation of cPLA2. One possible pathway could involve activation of Ca2+/calmodulin kinase II, as this pathway does not require p38 MAPK activation and would therefore not be subject to SB 202190 inhibition (Kudo and Murakami, 2002).

    The current study demonstrated that the PCB 50-stimulated release of arachidonic acid from RMC occurred in a concentration-dependent, isoform-specific manner. Arachidonic acid released from RMC in response to 15 μM PCB 50 was inhibited by pretreatment with MAFP, but not BEL or manoalide. These results indicate the cPLA2 isoform was sensitive to 15 μM PCB 50-induced activation, but that iPLA2 and sPLA2 isoforms did not significantly contribute to arachidonic acid release by PCB 50 at this concentration. Arachidonic acid releases following exposure to 25 and 50 μM PCB 50 were approximately 1.6- and 2.5-fold greater compared with 15 μM PCB 50. Moreover, arachidonic acid releases in response to 25 and 50 μM PCB 50 were sensitive to BEL and manoalide inhibition. These findings suggest that, at higher concentrations of PCB 50, additional PLA2 isoforms are activated to release arachidonic acid from late-gestation rat myometrial cells.

    In addition to activation of PLA2, arachidonic acid can also be released from membrane phospholipids via sequential actions of phospholipase D (PLD), phosphatidic acid phosphohydrolase, and diacylglycerol lipase or through the sequential actions of phospholipase C (PLC) and diacylglycerol lipase. Rat myometrium expresses PLC (Wen et al., 1992) and PLD (Le Stunff et al., 2000). However, given the extent of inhibition in the presence of BEL, manoalide, and MAFP, activation of PLA2 is more likely the predominant pathway through which PCB 50 induces arachidonic acid release from myometrial cells.

    The ability of PCB 50 to stimulate the release of arachidonic acid from rat myometrial cells is consistent with results from a previous report indicating that the commercial PCB mixture Aroclor 1242 stimulates arachidonic acid release from rat myometrial cells (Bae et al., 1999). However, in contrast to the findings by Bae et al. (1999) that the majority (99%) of the arachidonic acid released by Aroclor 1242 remains as free arachidonate, the current study found that PCB 50 stimulates rat myometrial cells to release prostaglandins. The difference in the ability of Aroclor 1242 and PCB 50 to stimulate prostaglandin production may be due to the gestational age of the rat uteri from which myometrial cells were cultured. The Aroclor 1242 studies utilized myometrial cells isolated from mid-gestation (gd10) rat uterus whereas the current PCB 50 studies used cells isolated from late-gestation (gd20) rat uterus. Because COX-2 expression increases in the rat myometrium with advancing gestational age (Arslan and Zingg, 1996), the stimulation of prostaglandin release by PCB 50 and not Aroclor 1242 may be a result of the increased availability of COX-2 in the late-gestation myometrial cells. Alternatively, PCB50 and Aroclor 1250 may act on distinct pools of cellular phospholipids to promote different fates of the released arachidonic acid, as proposed by Tithof et al. (1998) for neutrophils. Whether PCB 50 targets a distinct phospholipid pool and whether the prostaglandins released by PCB 50 in the current study reflect metabolism of arachidonic acid by COX-1 or COX-2 remain for further study.

    It is possible that, in addition to serving as a substrate for prostaglandin production, the arachidonic acid released by PCB 50 may serve as a substrate for synthesis of other eicosanoids that can modulate on uterine contractions. For example, arachidonic acid can be metabolized to 5-hydroxyeicosatetraenoic acid (5-HETE) and 14,15-epoxyeicostrienoic acid (14,15-EET) products via the 5-lipoxygenase and CYP450 epoxygenase pathways, respectively. It would be of interest in future studies to examine whether arachidonic acid released by PCB 50 is metabolized to into 5-HETE or 14,15-EET in myometrial cells, as these eicosanoid products can stimulate uterine contractions (Bennett et al., 1987; Edwin et al., 1996; Gonzalez et al., 1997).

    It is important to acknowledge that the levels of PCB 50 shown to stimulate PLA2 in vitro (10 to 50 μM) are greater than total PCBs reported in human serum levels associated with decreased gestation length (Taylor et al., 1989) and PCB levels found in the general adult population (ATSDR, 2000). Direct comparisons between PCB levels used in vitro and those reported in vivo are problematic, as tissue concentrations of PCBs in vivo reflect an accumulation resulting from low-dose exposures over an extended time period. How PCB accumulation in tissues following an in vivo exposure relates to PCB accumulation in myometrial cells following an acute exposure in vitro is uncertain. However, PCBs have been reported to accumulate to a greater extent in uterine tissue compared to adipose tissue and blood during pregnancy (Polishuk et al., 1977), which could indicate that the uterus is a target for PCB toxicity.

    In conclusion, the current study indicates that PCB 50 stimulated the release of arachidonic acid from RMC through activation of multiple PLA2 isoforms by a mechanism involving the influx of extracellular calcium through L-type VOCC and activation of p38 MAPK. Additionally, a small, though significant, calcium-independent release of arachidonic acid from RMC occurred at a higher concentration (50 μM) of PCB 50, consistent with expression of both the 85 kDa iPLA2 and 50 kDa iPLA2-related protein. This calcium-independent release of arachidonic acid may reflect a sensitivity of iPLA2 to PCB 50-induced activation of p38 MAPK. Moreover, the current study provides evidence that the arachidonic acid released in response to PCB 50 exposure serves as a substrate for cycloxygenase, with subsequent production of prostaglandins. As such, PLA2 enzymes in late-gestation RMC can serve a target for PCB-induced release of arachidonic acid with the potential to elicit a functional response on uterine activity through subsequent prostaglandin production.

    NOTES

    Portions of this research were presented at the 43rd Annual Meeting of the Society of Toxicology, March 2004, Baltimore, MD.

    ACKNOWLEDGMENTS

    This study was supported by grants from the National Institute of Environmental Health Sciences (NIEHS), NIH to R. Loch Caruso (P42 ES04911) with additional support for K. Brant provided by a NIEHS Institutional Training Grant (T32-ES07062) and a University of Michigan Rackham Predoctoral Award. Conflict of interest: none declared.

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