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Research Article |
Department of Environmental Health Sciences,3 University of Michigan, Ann Arbor, Michigan 48109
Department of Pathobiology,4 University of Tennessee, Knoxville, Tennessee 37996
ABSTRACT
Phospholipase A2 (PLA2) enzymes catalyze the rate-limiting step in eicosanoid production by liberating arachidonic acid from membrane phospholipids. There is limited information regarding the expression pattern and activity of uterine PLA2 enzymes during pregnancy. Polychlorinated biphenyls (PCBs) are a group of persistent environmental toxicants previously associated with decreased gestation length that are capable of activating PLA2. The purpose of the present study was to determine whether uterine sensitivity to PCB stimulation is dependent on PLA2 expression, comparing rat uterine PLA2 expression in Gestational Day (gd) 10 versus gd20. Western blot analysis revealed a significant increase in the expression of calcium-dependent PLA2G2A and a 50-kDa protein immunoreactive to calcium-independent PLA2G6 antibody in gd20 compared to gd10 rat uterine tissue. The increased expression of the 50-kDa PLA2G6 was associated with a gestational age-related increase in endometrial calcium-independent PLA2 activity that was sensitive to inhibition by bromoenol lactone (P < 0.05). Longitudinal uterine strips isolated from gd10 or gd20 rat were suspended in muscle baths to evaluate uterine contractions following exposure to the ortho substituted congener PCB 50. Exposure to 50 and 100 µM PCB 50 significantly increased the frequency of gd20, but not gd10, uteri compared to solvent (dimethyl sulfoxide) controls (P < 0.05). Pharmacologic inhibition of PLA2G6, but not PLA2G2A, attenuated PCB-induced stimulation of gd20 uterine contractions (P < 0.05). These data suggest that PCB 50 stimulates uterine contractions by activating endometrial PLA2G6. Furthermore, gestation age-related sensitivity to PCB is associated with an increase in the expression of a previously unidentified 50-kDa PLA2G6 in rat uterus.
pregnancy, toxicology, uterus
INTRODUCTION
During pregnancy, the uterus must remain in a relatively quiescent state. At term, coordinated series of biochemical and physiological changes facilitate the development of contractions required for labor. The regulatory factors involved in maintaining the gestational uterine environment and the events leading to parturition are complex, interdependent, and species-specific. However, common components to the onset of parturition in both humans and rodents involve the mobilization of arachidonic acid from glycerophospholipids [1, 2]. Arachidonic acid and several of its eicosanoid metabolites are capable of stimulating uterine function [2–4]. Phospholipase A2 (PLA2) enzymes catalyze this rate-limiting step in eicosanoid formation.
PLA2 isoforms can be characterized based on differences in structure and catalytic activity, and include the calcium-dependent cytosolic PLA2G4A, the low-molecular-weight cysteine-rich secretory PLA2G2A, and the calcium-independent PLA2G6. The calcium-dependent PLA2 isoforms PLA2G4A and PLA2G2A have been localized in pregnant human myometrial tissue [5–7]. Although we previously reported evidence of a calcium-independent PLA2 activity in pregnant rat uterus [3, 8], specific information regarding calcium-independent PLA2 expression and activity in gestational tissues is limited.
Polychlorinated biphenyls (PCBs) are persistent environmental toxicants that have been associated with decreased gestation length [9, 10]. Previous reports have shown that PCBs, particularly the ortho-substituted congeners, activate PLA2 to release arachidonic acid from membrane phospholipids in uterine [3] and nonuterine cell types [3, 11–13]. A previous study by Shin et al. [13] showed that the ability of PCBs to stimulate arachidonic acid release from rat pheochromocytoma (PC12) cell membrane phospholipids related to the pattern of chlorine substitution on the biphenyl ring. In particular, ortho-substituted congers were more likely than non-ortho-substituted congeners to stimulate arachidonic acid release. The ortho-substituted congener PCB 50 (2,2',4,6-tetrachlorobiphenyl) was found to be the most potent of all congeners studied, inducing an approximate 3.5-fold greater release compared to controls [13]. This response was mediated through activation of PLA2 enzymes because the PCB 50-induced release of arachidonic acid from PC12 cells was attenuated in the presence of the PLA2G4A/PLA2G6 inhibitor methyl arachidonyl fluorophosphonate (MAFP) and the PLA2G6 inhibitor bromoenol lactone (BEL) [13].
The present study evaluated PLA2 expression patterns in the pregnant rat uterus. Additionally, PCB 50 was used as a model ortho-substituted PCB congener to examine whether PCBs stimulate uterine contraction frequency via activation of PLA2 and if sensitivity to stimulation is influenced by changes in PLA2 expression.
MATERIALS AND METHODS
Chemicals
PCB 50 (2,2',4,6-tetrachlorobiphenyl) was obtained from Ultra Scientific. Dimethyl sulfoxide (DMSO) was purchased from Sigma. The phospholipase inhibitor BEL and the PLA2G2A polyclonal antibody were from Cayman Chemical. Manoalide was from Biomol. The PLA2G6 and PLA2G4A antibodies were purchased from Upstate and Cell Signaling Technology, respectively. The glyceraldehyde-3-phosphate dehydrogenase (GAPD) antibody was obtained from Abcam, Inc. The ECF Western blotting reagent pack was from Amersham Biosciences. The nonradiolabeled phospholipids 1-palmitoyl-2-[arachidonoyl] phosphatidylcholine and 1-palmitoyl-2-[linoleoyl] phosphatidylcholine were from Avanti Polar Lipids. Radiolabeled 1-palmitoyl-2-[14C]arachidonoyl-phosphatidylcholine (14C-AA-PC) and 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine (14C-LA-PC) were obtained from American-Radiolabeled.
Animals
Timed-pregnant Sprague-Dawley rats were obtained from Harlan Laboratories and housed at 24 ± 1°C under a 12L:12D schedule. Animal housing and handling procedures complied with institutional and NIH guidelines for care and use of laboratory animals in research. Investigations were conducted in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science).
Preparation of Rat Uterine Tissue Homogenates
Uterine tissue (myometrium with attached endometrium) was excised from Gestation Day (gd) 10 and gd20 animals and pups and placenta were removed. Uteri were then rinsed in ice-cold calcium- and magnesium-free PBS (CMF-PBS) and snap-frozen in liquid nitrogen. The tissue was pulverized to powder under liquid nitrogen and homogenized in RIPA buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% SDS, 0.5% sodium deoxycholate, 1 mM DTT, 100 µM PMSF, 1% NP-40, and 1x protease inhibitor cocktail tablet (Roche Applied Science) using three 20-sec bursts of a polytron homogenizer (PT 2100, Kinematica) at setting 6. Homogenates were then filtered through cheesecloth to remove any unhomogenized tissue. Protein concentration was determined using the bicinchoninic acid protein assay kit from Pierce. BSA was used as the protein standard.
SDS-PAGE and Western Blotting
Uterine tissue homogenates (20 µg protein/lane) were subjected to electrophoresis using 10% (PLA2G6 and PLA2G4A) and 15% (PLA2G2A) SDS gels under denaturing conditions. Proteins were transferred to a polyvinyl fluoride membrane (Millipore Corporation). Nonspecific protein binding sites in membranes were blocked with 5% nonfat dry milk in 0.1% Tween-20 in TBS (19.98 mM TrisHCl, pH 7.6, 13.69 mM NaCl) (TBS-T) at room temperature for 2 h. Membranes were incubated with antibodies to PLA2G6, PLA2G4A, or PLA2G2A in 1:500, 1:800, and 1:1000 dilutions, respectively, in TBS-T containing milk overnight at 4°C. After three 15-min washes with fresh exchanges of TBS-T containing milk, membranes were incubated with antifluorescein alkaline phosphatase-conjugated secondary antibody (1:3300 dilution) for 1 h at room temperature. Membranes were then rinsed 3 times for 20 min in TBS-T. Protein bands were visualized using an enhanced fluorescent reagent (ECF Western blotting kit; Amersham Biosciences) on a FLA 5000 imaging system (Fujifilm Medical Systems U.S.A., Inc.). After visualization of protein bands, membranes were rinsed briefly in methanol to remove the fluorescent substrate and then reprobed for the housekeeping protein GAPD to control for variations in protein loading. Densitometry analysis was conducted using Multi Gauge version 2.2 software (Fujifilm Medical Systems U.S.A., Inc.). Changes in protein expression between gestational days were determined by calculating the changes in the ratio of PLA2 to the housekeeping protein GAPD.
Measurement of Cell-Free Calcium-Independent PLA2 Activity
Uterine tissue was excised from gd10 and gd20 rats, pups were removed, and tissue was rinsed in ice-cold CMF-PBS and snap-frozen in liquid nitrogen. In separate experiments, uterine strips were scraped of endometrium using the edge of a glass slide to assay myometrial PLA2 activity. Frozen uterine tissue (myometrium alone or myometrium with attached endometrium) was washed with Ca2+-free PBS containing 5 mM EDTA and 1 mM PMSF, resuspended in cold homogenizing buffer (50 mM Tris HCl, pH 7.4, 2 mM EGTA, 0.5 mM dithiothreitol, 20% glycerol, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonylchloride), placed on ice, and sonicated two times for 10 sec. The substrates 1-palmitoyl-2-[arachidonoyl] phosphatidylcholine (cold AA-PC), 1-palmitoyl-2-[arachidonoyl-1-14C] phosphatidylcholine (14C-AA-PC), 1-palmitoyl-2-[linoleoyl] phosphatidylcholine (cold LA-PC), and 1-palmitoyl-2-[linoleoyl-1-14C] phosphatidylcholine (14C-LA-PC) were used to assay for calcium-independent PLA2 activity. The substrates were dried under nitrogen and resuspended by sonication in assay buffer (400 µM Triton-X, 5 mM EDTA, 100 mM Hepes at pH 7.5, and 0.8 mM ATP) to a final optimum concentration of cold and radiolabeled substrate as determined in preliminary experiments. Experiments were performed in the presence and absence of the PLA2G4A/PLA2G6 inhibitor MAFP and the PLA2G6 inhibitor BEL. The experiments were terminated by addition of chloroform-methanol, 2:1 (v/v). The chloroform layer was extracted and lipids separated by thin-layer chromatography in a neutral lipid solvent (hexane:diethyl ether:glacial acetic acid, 7:3:0.2). The lipids were visualized by I2 vapor. The zones corresponding to fatty acid and phospholipid were cut out and radioactivity determined by scintillation counting.
Contractility Assessment
Mid- (gd10) and late-gestation (gd20) Sprague-Dawley rats were killed by CO2 asphyxiation. Uteri were excised, and embryos and fat were removed from both uterine horns. Longitudinal uterine strips (myometrium with or without attached endometrium; 2 mm x 20 mm) were excised from the midsection of each uterine horn. The strips were suspended in 50-ml muscle baths (custom-made by the University of Michigan Glass Shop), which contained prewarmed (37°C) physiologic saline solution (PSS; 116 mM NaCl, 4.6 mM KCl, 1.16 mM NaH2PO4 ·H2O, 1.16 mM MgSO4·7H2O, 21.9 mM NaHCO3, 1.8 mM CaCl2·2H2O, 11.6 mM dextrose, and 2.6 mM EDTA, pH 7.4). The muscle baths and buffer reservoirs were continuously bubbled with 95% O2 and 5% CO2. To record isometric contractions, one end of each uterine strip was tied with surgical silk to a stationary post and the other end was tied to a force transducer (Grass FT-03). Changes in contractile activity were monitored by polygraph. All strips were subjected to a 2.0-g preload tension and allowed to equilibrate at 37°C for 45 min. Strips were exposed to 0 (0.1% DMSO, solvent control), 10, 50, or 100 µM PCB 50. The 15-min interval after 1 h of exposure was taken for analysis. All contractility data were normalized with respect to basal activity (15 min before PCB exposure) and expressed as percentage of basal frequency or percentage of average basal peak force. A contraction was operationally defined as an increase in force that initiated at baseline, exceeded 25% of average basal force, and returned to baseline.
Evaluation of PLA2 Inhibition on PCB 50-Induced Uterine Contractions
To examine the role of PLA2 activation in PCB 50-induced stimulation of gd20 uterine contractions, strips were pretreated for 30 min with 3 or 6 µM of the PLA2G2A inhibitor manoalide or 10 µM of the PLA2G6 inhibitor BEL before PCB 50 treatment. These inhibitor concentrations are within the range of the reported IC50 for manoalide inhibition of PLA2G2A (4.7 µM) [14] and BEL inhibition of PLA2G6 [15–17].
Statistical Analysis
Gestation age-related differences in PLA2 gel band intensities were analyzed using a Student t-test. The PLA2 activity data were analyzed by two-way ANOVA. The contractility data were analyzed by one or two-way ANOVA, as appropriate. Statistical analyses were conducted using Sigma Stat v3 software (Jandel Scientific). As needed, the contractility data were transformed by the arcsine transformation to correct for the nonnormal distribution of percentage data before one-way ANOVA analysis. Post hoc pair-wise comparisons of means were performed by the Student-Newman-Keuls method. P < 0.05 was considered statistically significant.
RESULTS
Uterine Phospholipase A2 Expression
Western blot analysis was conducted to determine the expression patterns of phospholipase A2 enzymes in gd10 and gd20 rat uterus. Immunoblot analysis with anti-PLA2G4A revealed that PLA2G4A isoform is present in both gd10 and gd20 rat uterus, with no change in expression with advancing gestational age (Fig. 1). In contrast, expression of PLA2G2A was approximately 2.5-fold greater in gd20 uterine tissue compared to gd10 uterine tissue (P < 0.05; Fig. 2). Immunoblot analysis with the PLA2G6 antibody detected both an 85- and a 50-kDa protein (Fig. 3A), consistent with information from the antibody supplier (Upstate, Lake Placid, NY). Immunoblot analysis revealed a gestation age-related difference in expression of the 50-kDa and the 85-kDa immunoreactive bands (ANOVA, gestation age, molecular weight, gestation age x molecular weight interaction effects, P < 0.001; Fig. 3B). The higher molecular weight band (85-kDa) showed no change in expression between gd10 and gd20 (Fig. 3). In contrast, the expression of the lower molecular weight band (50-kDa) was significantly greater than that of the 85-kDa band at gd10 and gd20. Moreover, the expression of the 50-kDa protein detected with the PLA2G6 antibody was approximately 7.5-fold greater in tissue homogenates taken from gd20 rat uteri compared to gd10 rat uteri (P < 0.05; Fig. 3).
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Calcium-Independent PLA2 Activity in Mid- and Late-gestation Uteri
Because gestation age-related differences in PLA2G6 expression were observed, PLA2 activity experiments were conducted under calcium-free conditions in the presence and absence of the PLA2G6 inhibitor BEL and the PLA2G4A/PLA2G6 inhibitor MAFP. Activity of PLA2 was significantly increased under calcium-free conditions in uterus (myometrium with attached endometrium) at gd20 compared to gd10 when arachidonyl phospholipid substrate was used (Fig. 4A). In the absence of inhibitors, PLA2 activity with 14C-AA-PC increased from 32.69 ± 5.12 pmol·mg protein–1·h–1 at gd10 to 59.41 ± 4.97 pmol·mg protein–1·h–1 at gd20 (P < 0.05; Fig. 4A, open bars). Activity of PLA2 in gd10 uterus was significantly attenuated to 9.83 ± 7.00 and 15.79 ± 2.66 pmol·mg protein–1·h–1 in the presence of MAFP and BEL, respectively, compared with activity in the absence of inhibitors (P < 0.05). Additionally, both MAFP and BEL significantly inhibited PLA2 activity in gd20 uterus to 17.54 ± 5.88 and 17.54 ± 3.2 pmol·mg protein–1·h–1, respectively, compared with activity in the absence of inhibitors (P < 0.05). A similar pattern emerged when uterine PLA2 activity was assayed using 14C-LA-PC as a substrate. In the absence of inhibitors, PLA2 activity was significantly greater in gd20 uterus compared to gd10 uterus (22.13 ± 1.76 compared to 13.87 ± 1.20 pmol·mg protein–1·h–1, respectively; P < 0.05; Fig. 4B, open bars). There was a slight, though not statistically significant, decrease in gd10 PLA2 activity with MAFP and BEL treatment. In gd20 uterus, both MAFP and BEL significantly reduced PLA2 activity (from 22.13 ± 1.76 pmol·mg protein–1·h–1in the absence of inhibitors to 6.98 ± 3.56 and 6.34 ± 1.24 pmol·mg protein–1·h–1, respectively; P < 0.05; Fig. 4B). These results demonstrate a calcium-independent PLA2 activity in gd10 and gd20 rat uterus that is sensitive to MAFP and BEL.
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Because the previous experiments were conducted with uterine tissue that included attached endometrium, the experiments were repeated using myometrial tissue with endometrium removed to investigate gestational tissue expression of the calcium-independent PLA2 activity. Under calcium-free conditions, gd10 and gd20 myometrial tissue exhibited PLA2 activity with 14C-AA-PC as substrate, although no significant difference in PLA2 activity was observed between gd10 and gd20 uterus (Fig. 5). In gd10 myometrium, the inhibitors MAFP and BEL significantly attenuated arachidonyl phospholipid-stimulated activity from 38.75 ± 7.00 pmol·mg protein–1·h–1 (without inhibitor) to 7.62 ± 5.02 and 16.07 ± 7.01 pmol·mg protein–1·h–1, respectively (Fig. 5). Similarly, MAFP and BEL significantly attenuated activity in gd20 myometrium from 44.58 ± 3.64 pmol·mg protein–1·h–1 (no inhibitor) to 14.57 ± 5.27 and 9.13 ± 8.37 pmol·mg protein–1·h–1, respectively (P < 0.05; Fig. 5). These results demonstrate a calcium-independent PLA2 activity in gd10 and gd20 rat myometrium that is sensitive to MAFP and BEL. These findings also indicate that the gestation age-related increases in rat uterine calcium-independent PLA2 activity (Fig. 4) are caused by increases in endometrial as opposed to myometrial enzyme activity.
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Effects of PCB 50 on the Contraction Patterns of Mid- and Late-Gestation Uterine Strips
Contractility analysis was conducted on mid- and late-gestation uterine strips to determine whether the increases in PLA2G2A and 50-kDa PLA2G6 expression with advancing gestational age influence sensitivity to PCB stimulation. PCB 50 stimulated the frequency (ANOVA gestation day and treatment effects, P < 0.05; Fig. 6A) and force (ANOVA gestation day, treatment and gestation day x treatment interaction effects, P < 0.05; Fig. 6B) of uterine contractions in a gestation age-related manner. After 1 h of exposure, 50 and 100 µM PCB 50 induced significant increases in the frequency of contractions in gd20 uterine strips to 370.8 ± 85.6% and 385.2 ± 101.5%, respectively, compared to solvent control treated strips (87.0 ± 8.2%; P < 0.05; Fig. 6A). Furthermore, the contraction frequency was greater in gd20 uterine strips compared with gd10 uterine strips after exposure to 50 or 100 µM PCB 50 (P < 0.05; Fig. 6A). However, the contraction frequency of gd20 uterine strips exposed to 10 µM PCB 50 was not statistically significantly different from that of solvent controls. In contrast to gd20 uterus, there were no significant differences in the percentage basal frequency of gd10 uterine strips at any of the PCB 50 concentrations tested (10, 50 or 100 µM) compared to solvent controls.
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The average peak force of gd20 uterine contractions increased after 1 h of exposure to 100 µM (258.2 ± 104.3%) compared to solvent controls (142.5 ± 20.7%; P < 0.05; Fig. 6B). As with frequency, the 100 µM PCB stimulation of average peak force in gd20 uterine strips was significantly greater than that of gd10 uterine strips exposed under similar conditions (P < 0.05). No changes in the average peak force of gd10 contractions were observed following 10, 50, or 100 µM PCB exposure compared to solvent control.
Effects of PLA2 Inhibitors on PCB 50-Induced Stimulation of Contraction Frequency in gd20 Uterine Strips
To determine if PCB 50 stimulates the contraction frequency in gd20 uterine strips through PLA2 activation, uterine strips (with endometrium attached) were pretreated with isoform specific inhibitors for 30 min before 0 (0.1% DMSO, solvent control) or 50 µM PCB 50 exposure. Because the average peak force of uterine contractions was not affected by 50 µM PCB 50 (Fig. 6B), only changes in contraction frequency were evaluated in the PLA2 inhibitor experiments.
Uterine strips pretreated with the PLA2G2A inhibitor manoalide showed no statistically significant changes in contraction frequency compared with uterine strips exposed to PCB 50 alone (data not shown). In contrast, pretreatment of uterine strips with 10 µM of the PLA2G6 inhibitor BEL significantly reduced the 50 µM PCB 50-induced stimulation of basal contraction frequency from 370.8 ± 85.6% without BEL to 183.6 ± 60.6% in strips pretreated with BEL (P < 0.05; Fig. 7), a value not statistically significantly different from that of the solvent control strips.
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Effects of PCB 50 on the Contraction Patterns of Late-Gestation Uterine Strips in the Absence of Endometrium
Contractility analysis was conducted on late-gestation uterine strips for which the endometrium had been removed to determine whether PCB 50 activates endometrial PLA2G6 to stimulate contractions. In contrast to the results obtained with intact uterine strips (myometrium with attached endometrium), 50 µM PCB 50 did not stimulate contraction frequency in gd20 uterine strips that lacked attached endometrium, compared with solvent control-treated strips (Fig. 8).
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DISCUSSION
The arachidonic acid signaling cascade plays an important role in the onset of parturition in both humans and rodents. Phospholipase A2 enzymes regulate arachidonic signaling by liberating arachidonic acid from membrane phospholipids. Several studies have examined how PLA2 enzymes and activity change with the onset of labor [5, 18, 19], yet little is known regarding how expression and activity of uterine PLA2 enzymes may change during pregnancy.
To our knowledge, the present study is the first to identify the presence of a gestation age-related increase in the expression of a 50-kDa PLA2G6 in the rat uterus. The 50-kDa PLA2G6 immunoreactive band is reported by the supplier (Upstate) to be an isoenzyme of PLA2G6 because both the 85-kDa and 50-kDa bands can be competed by the immunizing peptide. The present study is also the first to show a significant gestation age-related increase in rat uterine calcium-independent PLA2 activity. The increased calcium-independent PLA2 activity was observed in uterine tissue with attached endometrium but not in uterine strips with endometrium removed. These results suggest that it is endometrial calcium-independent PLA2 activity that increases with advancing gestation in the rat. Given the lack of change in 85-kDa PLA2G6 expression, increased expression of the 50-kDa PLA2G6 may account for the gestation age-related increase in calcium-independent PLA2 activity observed in uterine strips with attached endometrium.
The functions of 85-kDa and 50-kDa PLA2G6 in the pregnant rat uterus remain unclear. Because PLA2G6 activity plays a role in lipid remodeling and the size of the rat uterus increases approximately 13-fold during gestation [20], increases in calcium-independent PLA2 activity may be a compensatory mechanism to ensure that lipid homeostasis is maintained as the uterus increases in size with advancing gestation. Alternatively, PLA2G6 may contribute to the uterine hypertrophy that occurs during pregnancy, because calcium-independent PLA2 activity mediates agonist-induced arachidonic acid release and growth of vascular smooth muscle cells [21]. Studies to determine if PLA2G6 contributes to uterine lipid homeostasis and/or hypertrophy during pregnancy are beyond the scope of the present investigation, however.
The present study also identified an increase in PLA2G2A protein expression in gd20 compared with gd10 rat uterus, consistent with evidence of pregnancy-associated changes in uterine PLA2G2A expression in the human. Previously, Slater et al. [22] reported that PLA2G2A mRNA was expressed in pregnant, but not nonpregnant, human myometrial tissue. Furthermore, human myometrial PLA2G2A mRNA expression increases in association with labor [22].
PLA2G2A contributes to several physiological functions that may yield insight into its role in uterine activity during pregnancy and labor. Secretory PLA2 enzymes are potent mediators of inflammation. Cytokines such as interleukin-1 are increased in fetal membranes and amniotic fluid throughout gestation and in association with labor [23–25]. PLA2G2A is upregulated by cytokines in the rat [26–28]. As such, it is possible that increases in rat uterine PLA2G2A expression are secondary to increases in cytokine production. PLA2G2A is also known to stimulate prostaglandin production [29]. The observed increases in PLA2G2A expression could reflect preparation for prostaglandin production at the time of labor. This is of particular importance in the rat because uterine-derived prostaglandins are responsible for luteolysis, which initiates a series of biochemical events that culminate in labor. Further studies are needed to elucidate which of these roles PLA2G2A plays in the rat and human uterus and how they relate to pregnancy maintenance.
The PCB mixtures Aroclor 1242, Aroclor 1248, and Aroclor 1254 stimulate contractions in midgestation rat uteri [30]. Additionally, PCBs stimulate contraction frequency of midgestation rat uteri in a congener-related manner, such that the lower chlorinated, ortho-substituted congeners acutely stimulate uterine contraction frequency whereas non-ortho-substituted congeners have no significant acute effect on uterine contraction frequency [31]. In the current study, we used an ortho-substituted congener, PCB 50, to evaluate how the gestation age-related increases in secretory and calcium-independent PLA2 expression affect uterine sensitivity to PCB stimulation. Consistent with the acute uterotonic properties of ortho-substituted PCBs [31], PCB 50 increased the frequency and average peak force of uterine contractions. However, stimulation of contractions by PCB 50 occurred in gd20 uterine strips but not in gd10 uterine strips, indicating that uterine sensitivity to PCB 50 is influenced by gestation age.
Although calcium-independent PLA2 activity was observed in uterine strips with and without attached endometrium from both gd10 and gd20, it was only when endometrium remained attached to the myometrium that increases in calcium-independent PLA2 activity with advancing gestation and stimulation of gd20 uterine strips by PCB 50 were observed. These results raise the possibility that PCB 50 activates endometrial PLA2G6 to stimulate late-gestation rat uterine contractions and that gestation age-related increase in endometrial 50-kDa PLA2G6 influences uterine sensitivity to PCB stimulation.
The physiologic role of PLA2G6 in the pregnant uterus remains unknown. Because stimulation of PLA2G6 may lead to the synthesis of uterotonic eicosanoids, it is plausible that activation of uterine PLA2G6 by PCBs may have an effect on uterine contractility, and consequently on parturition. In the current study, pharmacologic inhibition of PLA2G6 activity using BEL significantly attenuated PCB 50-induced stimulation of late-gestation rat uterine contractions, suggesting that activation of PLA2G6 can stimulate uterine function. These results also demonstrate for the first time a contribution of calcium-independent phospholipase A2 to total rat uterine phospholipase activity. Further characterization of calcium-independent PLA2 in gestational tissues is warranted in humans and other species to better understand the potential contribution of this enzyme in eicosanoid synthesis during maintenance of pregnancy or the onset of labor.
FOOTNOTES
1 Supported by grants from the National Institute of Environmental Health Sciences (NIEHS), NIH, to R.L.C. (P42 ES04911) and by an Institutional Training Grant (T32-ES07062) to K.B. Portions of this research were presented at the 36th annual meeting of the Society for the Study of Reproduction, 19–22 July 2003, Cincinnati, Ohio. 
2 Correspondence: Kelly Brant, Department of Environmental Health Sciences, University of Michigan, 1420 Washington Heights, Ann Arbor, MI 48109-2029. FAX: 734 763 8095; kbrant{at}umich.edu
Received: 31 August 2005.
First decision: 23 September 2005.
Accepted: 25 January 2006.
REFERENCES
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