Four experiments were conducted to determine whether phospholipase (PL) A2 mediates the stimulatory effect of oxytocin on the release of prostaglandin (PG) F2α from ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes on the day after a steroid replacement protocol had been completed. The replacement protocol consisted of progesterone for 10 days (12 mg/day) followed by oestradiol on days 10 and 11 (100 μg/day) and had been shown previously to provide endometrial tissue that would release PGF2α in response to oxytocin in vitro. In experiment 1, oxytocin (10−7 m) and melittin (1·76 × 10−6 m; a stimulator of PLA2) stimulated release of PGF2α from tissue explants (P<0·05). Aristolochic acid (10−4 m; an inhibitor of PLA2) decreased oxytocin-and melittin-induced release of PGF2α by 77% and 71% respectively (P<0·05). Experiment 2 was conducted to establish the minimum inhibitory dose of aristolochic acid. Basal release of PGF2α was inhibited at 10−5 m aristolochic acid, but 10−4 m was required to block the stimulatory effect of oxytocin. Experiment 3 was carried out to identify the precise intracellular locus at which aristolochic acid was exerting its effect. Oxytocin (10−7 m), AlF4− (5 × 10−2m NaF, 10−5m AlCl3), melittin (1·76 × 10−6 m) and arachidonic acid (AA; 20 μg/ml) stimulated release of PGF2α (P<0·05). Aristolochic acid (10−4 m) blocked the release of PGF2α stimulated by oxytocin, AlF4− or melittin by >80% (P<0·05). However, aristolochic acid reduced AA-induced release by only 22% (P=0·09). Thus, aristolochic acid blocked responsiveness to stimulatory agents that act on, or proximal to, PLA2 but not responsiveness to AA whose stimulatory effect is exerted distal to PLA2. Experiment 4 was intended to establish the specificity of the inhibitory effect of aristolochic acid. This was accomplished by examining the effect of aristolochic acid on the ability of oxytocin to stimulate activity of PLC. Aristolochic acid (10−4 m) increased basal PLC activity by 29% (P<0·05) but had no effect on the activity induced by oxytocin (P>0·1). Thus, aristolochic acid blocked the ability of oxytocin to stimulate PGF2α release (experiments 1–3) but not its ability to stimulate the activity of PLC (experiment 4). The effects of melittin and aristolochic acid are consistent with the hypothesis that the stimulatory effect of oxytocin on PGF2α release from ovine endometrial tissue is mediated through PLA2.
Journal of Endocrinology (1994) 141, 491–496
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