Phospholipase-Mediated Inhibition of Spontaneous Oscillatory Uterine Contractions by Lindane in Vitro

Abstract

Although regulation of uterine contractility is fundamental for parturition, mechanisms by which toxicants modify uterine muscle contractions remain poorly understood. In a previous cumulative concentration-response study, 10 μM lindane (γ-hexachlorocyclohexane) reduced contraction force and 30 μM lindane abolished contractions in Gestation Day 10 rat uterine strips when lindane was added to muscle baths at 10-min intervals. Other studies showed that brief (<10 min) exposures to 10–100 μM lindane inhibit gap junctions and activate phospholipase pathways in rat myometrial cells in culture. Consequently, lindane was used as a prototype toxicant with known uterine activity to investigate the hypothesis that activation of a specific phospholipase pathway provides a mechanistic link between inhibition of uterine contraction and inhibition of myometrial gap junctions. Uterine tissue and cells were pretreated with phospholipase pathway inhibitors to evaluate the role of phospholipase pathways in lindane's actions in the uterus. Concentrations of inhibitors were selected based on previous reports of effective concentrations for the enzyme activity and on pilot toxicity studies of the inhibitors on uterine contraction and gap junction communication. To monitor uterine contractions, longitudinal uterine strips were excised from Gestation Day 10 rats and suspended in isometric muscle baths, consistent with previous experiments. Exposure in vitro for 60 min to 10–50 μM lindane, an effective concentration range for the uterine responses of interest, revealed that 30 μM lindane rapidly abolished contractions. Subsequently, uterine strips were pretreated with phospholipase pathway inhibitors and then challenged with 30 μM lindane, the lindane concentration that elicited maximal inhibition of uterine contraction. Pretreatment with 20–50 μM of the phosphatidylinositol-specific phospholipase C inhibitor 1- O-octadecyl-2- O-methyl- sn-glycerol-3-phosphorylcholine (ET-18-OCH 3) reversed lindane-induced inhibition of spontaneous uterine contractions. Gap junction intercellular communication was monitored by injecting the fluorescent dye Lucifer yellow into rat myometrial cells grown in culture and assessing dye transfer to adjacent cells using epifluorescence microscopy. Similar to uterine contraction, pretreatment of cell cultures with phospholipase C inhibitors (30 μM ET-18-OCH 3, 50 μM tricyclodecan- p-yl-xanthogenate · K [D609] or 50 μM tricyclodecan- p-yl-xanthogenate · K or 2-nitro-4-carboxyphenyl- N,N-dophenylcarbamate [NCDC]) partially reversed inhibition of dye transfer by 100 μM lindane, a lindane concentration previously shown to abolish myometrial Lucifer yellow dye transfer under similar culture conditions. In contrast, pretreatment with 20 μM of bromoenol lactone (BEL) to inhibit the calcium-independent phospholipase A 2 or 100 mM ethanol to interrupt the phospholipase D pathway failed to prevent inhibition of spontaneous uterine contractions and inhibition of Lucifer yellow dye transfer by lindane (100 μM). These data suggest that lindane inhibits myometrial gap junctions and spontaneous oscillatory contractions by a phospholipase C-mediated pathway.