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 microM lindane (gamma-hexachlorocyclohexane) reduced contraction force and 30 microM 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 microM 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 microM lindane, an effective concentration range for the uterine responses of interest, revealed that 30 microM lindane rapidly abolished contractions. Subsequently, uterine strips were pretreated with phospholipase pathway inhibitors and then challenged with 30 microM lindane, the lindane concentration that elicited maximal inhibition of uterine contraction. Pretreatment with 20-50 microM 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 microM ET-18-OCH(3), 50 microM tricyclodecan-p-yl-xanthogenate.K [D609] or 50 microM tricyclodecan-p-yl-xanthogenate.K or 2-nitro-4-carboxyphenyl-N,N-dophenylcarbamate [NCDC]) partially reversed inhibition of dye transfer by 100 microM lindane, a lindane concentration previously shown to abolish myometrial Lucifer yellow dye transfer under similar culture conditions. In contrast, pretreatment with 20 microM 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 microM). These data suggest that lindane inhibits myometrial gap junctions and spontaneous oscillatory contractions by a phospholipase C-mediated pathway.

FIG. 1

The concentration-dependent effects of lindane on the time required for complete inhibition of spontaneous oscillatory contractions of uterine strips exposed in vitro to lindane. The time from the addition of lindane to complete cessation of contraction of each strip was recorded up to 60 min. Strips that contracted more than 60 min were assigned a score of 60 min. Strips exposed to 0 μM lindane were exposed to the solvent only (DMSO). The data are presented as the means ± SEM of 12 uterine strips. Data points sharing the same letter are not statistically different from each other (P < 0.05). Error bars not visible are contained within the symbols.

FIG. 2

The time- and concentration-dependent effects of lindane on (A) force and (B) frequency of contraction of spontaneously oscillating uterine strips exposed to lindane in vitro. Bars not visible are data means equal to zero. Error bars not visible are too small to be displayed graphically. Groups sharing the same letter are not statistically different from each other (P < 0.05).

FIG. 3

The concentration-dependent effects of ET-18-OCH₃ pretreatment on lindane-induced inhibition of spontaneous oscillatory uterine contractions. The time to complete cessation of spontaneous uterine oscillatory contraction was determined in uterine strips pretreated with ET-18-OCH₃ for 1 h prior to addition of 30 μM lindane (final concentration) to the ET-18-OCH₃-containing medium. The 0 μM concentration represents solvent (DMSO) controls. The data are presented as the means ± SEM of 12 uterine strips. Data points sharing the same letter are not statistically different from each other (P < 0.05).

FIG. 4

The time- and concentration-dependent effects of ET-18-OCH₃ pretreatment on (A) force and (B) frequency of contraction of uterine strips exposed to lindane. Uterine muscle strips were pretreated with solvent (DMSO) alone (control) or ET-18-OCH₃ for 1 h prior to exposure to 30 μM lindane. Pretreatment with ET-18-OCH₃ alone (10, 30, or 50 μM) did not affect the force and frequency of contraction (data not shown). The data are expressed as the means ± SEM of 12 uterine strips. Bars not visible are data means equal to zero. Groups within each graph that share the same letter are not statistically different from each other (P < 0.05).

FIG. 5

Effects of phospholipase pathway modulators on lindane-induced inhibition of myometrial gap junction communication as measured by Lucifer yellow dye transfer. Myometrial cells were exposed to 100 μM lindane for 30 min with or without 1-h pretreatment with the phospholipase inhibitors BEL (20 μM), D609 (50 μM), ET-18-OCH₃ (30 μM), NCDC (50 μM), or ethanol (100 mM) (filled bars), or to inhibitors or DMSO (solvent controls) alone (open bars). The data are expressed as the means ± SEM for 10 dishes (100–120 injected cells). Different letters denote means statistically different from each other (P < 0.05).