Phospholipase-mediated inhibition of spontaneous oscillatory uterine contractions by lindane in vitro.

Coordinated and forceful uterine contractions are necessary for parturition, yet few studies have examined mechanisms by which toxicants modify uterine muscle contractions (Loch-Caruso, 1999). The present study utilizes lindane, the γ-isomer of hexachlorocyclohexane, to investigate mechanisms of inhibition of spontaneous uterine contractions. Our laboratory initially investigated lindane’s actions on uterine contraction because it was one of several organochlorine substances found in significantly elevated concentrations (15–136 ppb) in the blood lipids of women who had experienced spontaneous abortions or premature delivery compared with women who had undergone full-term pregnancy (7–39 ppb) (Saxena et al., 1981; Wassermann et al., 1982). However, in vitro exposure of rat uterine strips isolated from pregnant (Gestation Day 10) rats to cumulative concentrations of lindane at 10-min intervals was found to depress spontaneous contractions at 10 μM lindane and abolish contractions at 30 μM lindane (Criswell and Loch-Caruso, 1999). The inhibitory action of lindane was in contrast to stimulation of uterine contraction frequency that we observed with DDT (Juberg and Loch-Caruso, 1991; Juberg et al., 1991, 1995) and polychlorinated biphenyl isomers (Tsai et al., 1996; Bae et al., 1999a,b, 2001), which were also elevated in the blood of women who experienced premature delivery or spontaneous abortion (Saxena et al., 1981; Wassermann et al., 1982). We subsequently used lindane as an inhibitor for probing physiologic and pathologic mechanisms of regulation of uterine contraction, and identified a previously unrecognized role for reactive oxygen species (Krieger and Loch-Caruso, 2001) and a previously unidentified myometrial phospholipase A2 activity (Wang et al., 2001). Lindane is an insecticide used as a pharmacological agent in veterinary and human medicine to treat scabies and lice (Smith, 1991). Although lindane’s use as an insecticide in agricultural, forestry, and household products has been restricted in the United States since 1977, lindane is a contaminant of 163 of the 1467 current or former U.S. EPA National Priorities List hazardous waste sites and its use as an insecticide continues in other countries (ATSDR, 1997). Lindane’s insecticidal activity is ascribed to neuroexcitation subsequent to inhibition of GABAA channels (Narahashi, 1976; Joy, 1982; Ogata et al., 1988). Accordingly, exposure to excessive amounts of lindane through misuse or unintentional exposure causes seizures, convulsions, and other signs of intoxication in humans (Starr and Clifford, 1972; Munk and Nantel, 1977; Davies et al., 1983), although actual amounts of lindane ingested were not determined in these reports. Rats administered a single oral dose of 30 mg lindane/kg exhibit convulsions within 10–30 min (Barron et al., 1995), and less serious neuroexcitation is observed in rats administered a single oral dose of 10 or 20 mg lindane/kg (Llorens et al., 1989; Llorens et al., 1990). In addition to neurotoxicity, lindane alters female reproductive processes in adult female rats. A single oral dose of 25 mg lindane/kg lengthens estrous cycles, and higher doses of 33 or 50 mg lindane/kg decrease sexual receptivity (Uphouse, 1987; Uphouse and Williams, 1989). Similarly, daily administration of 5–40 mg/kg lindane from 20 days of age until 110–125 days of age delayed the appearance of regular estrous cycles (Cooper et al., 1989). Although lindane exhibits weak estrogenic (Raizada et al., 1980) and antiestrogenic effects (Chadwick et al., 1988; Cooper et al., 1989) in vivo, it has no significant affinity for the estrogen receptor (α subtype) (Laws et al., 1994). In contrast, rats exposed to 10 mg lindane/kg/day for four generations showed no adverse reproductive effects (Palmer et al., 1978). Although lindane induces neurological hyperexcitability through inhibition of GABAA channels (Narahashi, 1976; Joy, 1982; Ogata et al., 1988), in vitro exposure to the GABAA channel inhibitor picrotoxin stimulates contraction of uterine strips from Gestation Day 10 rats (Criswell and Loch-Caruso, 1999). Because the picrotoxin response is opposite to the inhibitory response observed with lindane, this finding suggests that lindane-induced inhibition of uterine contraction is not mediated by GABAA channel inhibition. The uterine contractions necessary for parturition require the increased presence of gap junctions between the muscle cells of the myometrium (Garfield and Hayashi, 1981). The myometrial gap junctions facilitate coordination and synchronization of uterine contractions by providing low resistance intercellular pathways for the propagation of electrical and metabolic signals (Garfield and Yallampalli, 1994). Brief (4-min) exposures of myometrial cells in culture to 10–100 μM lindane inhibit gap junction intercellular communication as measured by Lucifer yellow dye transfer (Criswell et al., 1995). It has been suggested that lindane inhibits uterine contractions by inhibiting myometrial gap junction permeability (Criswell and Loch-Caruso, 1999), perhaps involving an oxidative stress-dependent mechanism (Krieger and Loch-Caruso, 2001), although the cellular pathway linking lindane-induced inhibition of myometrial gap junctions and contractility remains to be resolved. Various studies have explored phospholipase-coupled pathways as mechanisms of regulation of gap junctions. Activation of the phosphatidylinositol (PI)-specific phospholipase C (PLC) pathway precedes uncoupling of gap junctions in striatal astrocytes (Giaume et al., 1991). Protein kinase C (PKC) activation occurs downstream in the PLC and phospholipase D (PLD) pathways, and is associated with inhibition of gap junctions in several cell types and tissues (Murray and Fitzgerald, 1979; Yotti et al., 1979; Menkes et al., 1986; Sakata and Karaki, 1990; Sasaguri and Watson, 1990; Morrison and Vanhoutte, 1991; Watson and Karmazyn, 1991; Bastide et al., 1994; Kenne et al., 1994; Leibold et al., 1994; Mikalsen and Sanner, 1994; Rivedal et al., 1994; Kwak et al., 1995; Kwak and Jongsma, 1996), including myometrium (Nnamani et al., 1994; Criswell et al., 1995). Arachidonic acid, which is generated as a consequence of phospholipase A2 (PLA2) activation, inhibits gap junctions, also (Cole et al., 1985; Aylsworth et al., 1986; Giaume et al., 1989; de Haan et al., 1994; Miyachi et al., 1994; Criswell et al., 1995; Hii et al., 1995; Hayashi et al., 1997; Lavado et al., 1997). Lindane activates phospholipase signaling pathways in myometrium, as evidenced by lindane-stimulated generation of inositol phosphates (Criswell et al., 1994), activation of PKC (Criswell et al., 1995), and release of arachidonic acid (Criswell et al., 1995; Wang et al., 2001). The current study investigates the hypothesis that lindane-induced activation of a specific phospholipase pathway inhibits spontaneous oscillatory uterine contractions through inhibition of myometrial gap junctions. Lucifer yellow dye transfer between myometrial cells in culture was used to monitor changes in lindane-induced inhibition of gap junction communication and uterine strips in muscle baths were used to monitor changes in lindane-induced inhibition of uterine contractions in response to treatment with inhibitors of phospholipase pathways.