Stimulation of Pregnant Rat Uterine Contraction by the Polychlorinated Biphenyl (PCB) Mixture Aroclor 1242 May Be Mediated by Arachidonic Acid Release through Activation of Phospholipase A2 Enzymes

The polychlorinated biphenyl (PCB) mixture Aroclor 1242 (A1242) increases frequency of contractions of pregnant rat uteri, suggesting a possible mechanism for decreased gestational age and increased spontaneous abortion in women and animals exposed to PCBs. In the present study, we hypothesized that A1242-induced stimulation of uterine contraction is mediated by arachidonic acid released by phospholipase A2 (PLA2) enzymes. Isometric uterine contraction was measured in longitudinal uterine strips isolated from gestation day 10 rat. Pretreatment of uterine strips with the PLA2 inhibitor (E)-6(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2one (HELSS) or manoalide, or an inhibitor of the G protein of PLA2, isotetrandrine, completely prevented the increase of contractile frequency induced by 50 mM A1242. However, the phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,Ndiphenylcarbamate (NCDC) and neomycin were unable to block stimulation of uterine contraction by A1242. In accordance, A1242 (100 mM) did not release inositol phosphates from myo[H]inositol-labeled myometrial cells, whereas myometrial cells prelabeled with [H]arachidonic acid released arachidonic acid in a concentrationand time-dependent manner after exposure to A1242 (10–100 mM). A1242 significantly stimulated arachidonic acid release in the absence of extracellular calcium, although the release was attenuated. Analysis of the eicosanoids released by A1242 indicated that only 0.83% of released [H]arachidonic acid was metabolized to eicosanoids and 99.07% remained as free arachidonate. Uterine contraction increased in strips exposed to exogenous arachidonic acid (1–100 mM). This study suggests that A1242 stimulates contraction in pregnant rat uterus by a mechanism involving PLA2mediated arachidonic acid release, and that arachidonic acid, rather than eicosanoids, may mediate A1242 uterotonic action in the uterus. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants previously used widely in industry (Safe, 1992). Exposure to PCBs is associated with spontaneous abortion and decreased gestational length in women (Leoni et al., 1989; Taylor et al., 1989) and nonhuman primates (Arnold et al., 1990). Timely and effective uterine contraction is a critical component of parturition. Increased frequency of uterine contractions before term is associated with preterm birth in women (Bell, 1983). In previous studies, we showed that noncoplanar orthosubstituted PCB congeners (Tsai et al., 1996) and PCB mixtures (Bae and Loch-Caruso, 1996) stimulate spontaneous oscillatory contractions of pregnant rat uterine muscle, suggesting a possible mechanism for disturbing the maintenance of pregnancy to term. Uterine muscle activity may be stimulated by many factors, including prostaglandins (PGs) metabolized from liberated arachidonic acid (Huszar and Roberts, 1982) and calcium released from intracellular stores by inositol 1,4,5-trisphosphate (Carsten and Miller, 1985). Released arachidonic acid can be metabolized by cyclooxygenases, lipoxygenases, or cytochrome P-450 enzymes (for review, see Piomelli, 1996). Most studies of uterine contraction Received for publication June 17, 1998. 1 This research was conducted as partial fulfillment of the requirements of the doctoral dissertation of J. Bae, and was supported by an National Institutes of Health (NIH) Grant to R. Loch-Caruso (P42-ES04911) with additional support provided by the Laboratory Animal Core of the Center for the Study of Reproduction at the University of Michigan (NIH P30-HD18258). Portions of this work were presented at the 37th Annual Meeting of the Society of Toxicology, Seattle, WA, March 1–5, 1998 (Toxicol Sci 42:102, 1998). ABBREVIATIONS: A1242, Aroclor 1242; BCS, bovine calf serum; carbachol, carbamylcholine chloride; CMF-PBS, calcium/magnesium-free PBS; DMSO, dimethyl sulfoxide; HELSS, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one; HETE, hydroxyeicosatetraenoic acid; 12HHT, 12-hydroxyheptadecatrienoic acid; PG, prostaglandin; 6-k-PGF1a, 6-keto-PGF1a; LT, leukotriene; NCDC, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate; PCB, polychlorinated biphenyl; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; TXB2, thromboxane B2. 0022-3565/99/2892-1112$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 289, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 289:1112–1120, 1999 1112 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from have focused on eicosanoids rather than arachidonic acid per se. However, in many cell types arachidonic acid itself serves as a second messenger (Piomelli, 1996). Arachidonic acid can be released by phospholipase A2 (PLA2) directly, by sequential action of phospholipase C (PLC) and diacylglycerol lipase, or by phospholipase D (PLD) and subsequent actions of phosphatidic acid phosphohydrolase and diacylglycerol lipase (for review, see Dennis, 1983). In uterine tissues, both PLA2 and PLC are involved in releasing arachidonic acid (Flint et al., 1986; Schrey et al., 1987). The activity of PLD has been detected in myometrium (Dokhac et al., 1995), but its ability and significance in releasing arachidonic acid in uterus is unknown. Others have shown that PCBs increase arachidonic acid release in neutrophils (Tithof et al., 1996) and stimulate inositol phosphate production from neutrophils and cerebellar granule cells (Tithof et al., 1995; Shafer et al., 1996). Therefore, in this study we investigated whether A1242-induced stimulation of uterine contraction is through the PLA2or PLC-mediated arachidonic acid release. Furthermore, we explored whether arachidonic acid or its metabolites are responsible for the uterotonic response elicited by A1242. Materials and Methods Chemicals. Aroclor 1242 (A1242) was purchased from Ultra Scientific (North Kingstown, RI). Manoalide, isotetrandrine, arachidonic acid, A23187, and (E)-6-(bromomethylene)tetrahydro-3-(1naphthalenyl)-2H-pyran-2-one (HELSS) were obtained from Biomol (Plymouth Meeting, PA). BSA, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), neomycin, carbamylcholine chloride (carbachol), EDTA, EGTA, deoxyribonucleases I, and type II collagenase were purchased from Sigma Chemical Co. (St. Louis, MO). [H]Arachidonic acid (210 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA). Myo-[2-H]inositol (15 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). HPLC standards of 6-keto-PGF1a, PGF2a, thromboxane B2, PGE2, PGD2, leukotriene (LT) B4, LTC4, LTD4, 12-hydroxyheptadecatrienoic acid (12-HHT), 15-hydroxyeicosatetraenoic acid (15-HETE), 12HETE, 5-HETE, and free arachidonic acid were purchased from Cayman Chemical (Ann Arbor, MI). C18 Sep-Pak cartridges were purchased from Waters Associates (Milford, MA). Dowex AG1-X8 (100–200 mesh, formate form) was obtained from BioRad Labs (Rockville Center, NY). RPMI medium and crude trypsin were obtained from Gibco BRL (Grand Island, NY) and defined, iron-supplemented bovine calf serum (BCS) was from HyClone (Logan, UT). Arachidonic acid stock was divided into aliquots and stored at 220°C, and arachidonic acid solution was made fresh for each experiment while aerated with N2. All chemicals were dissolved in dimethyl sulfoxide (DMSO) except carbachol and neomycin, which were dissolved in deionized water (di H2O). Final exposure concentrations of DMSO did not exceed 0.1%. Preparation of Uterine Strips. Female Sprague-Dawley rats aged 60 to 90 days and weighing 180 to 220 g were mated with males. Pregnant (gestation day 10) rats were obtained from Harlan (Indianapolis, IN) or the colony of the Reproductive Science Program at the University of Michigan. The animals were housed at 24 6 1°C under a 12-h light/12-h dark schedule. Pregnant rats were anesthetized with ether followed by exsanguination, a protocol required by collaborators with whom we shared tissue. After isolating uteri, embryos were removed. Longitudinal smooth muscle strips 1 mm wide by 20 mm long were cut from the mid-portion of horns that contained four implantation sites. Measurement of Spontaneous Oscillatory Contractions. The uterine strips were suspended in standard muscle baths that contained physiological salt solution composed of 116 mM NaCl, 4.6 mM KCl, 1.16 mM NaH2PO4zH2O, 1.16 mM MgSO4z7H2O, 21.9 mM NaHCO3, 1.8 mM CaCl2z2H2O, 11.6 mM dextrose, and 0.03 mM CaNa2EDTA at pH 7.4. The water-jacketed bath was maintained at 36°C and aerated with a mixture of 95% O2 and 5% CO2. The uterine strip was tied with surgical silk to a stationary post at one end and to an isometric force transducer at the other end. Isometric contractions of strips were monitored under constant passive force of 1.0 g. After a 40-min equilibration period, strips were challenged with 60 mM KCl to determine viability and maximum KCl-induced contraction force. After rinsing out the KCl, strips were allowed to equilibrate for 2 to 5 h to establish regular spontaneous oscillatory contractions. Contractions were measured by frequency (number of contraction/relaxation cycles in a 10-min period), because this was the most prominent parameter affected by A1242. After equilibration, the 10-min interval before any treatment was termed the basal