CELLULAR MECHANISMS MEDIATING THE STIMULATION OF OVINE ENDOMETRIAL SECRETION OF PROSTAGLANDIN-F2-ALPHA IN RESPONSE TO OXYTOCIN - ROLE OF PHOSPHOLIPASE A(2)

Four experiments were conducted to determine whether phospholipase (PL ) A(2) mediates the stimulatory effect of oxytocin on the release of p rostaglandin (PG) F-2 alpha from ovine endometrial tissue. Caruncular endometrial tissue was collected from ovariectomized ewes on the day a fter a steroid replacement protocol had been completed. The replacemen t protocol consisted of progesterone for 10 days (12 mg/day) followed by oestradiol on days 10 and 11 (100 mu g/day) and had been shown prev iously to provide endometrial tissue that would release PGF(2 alpha) i n response to oxytocin in vitro. In experiment 1, oxytocin (10(-7) M) and melittin (1.76 x 10(-6) M; a stimulator of PLA(2)) stimulated rele ase of PGF(2 alpha) from tissue explants (P<0.05). Arirrolochic acid ( 10(-4) M; an inhibitor of PLA(2)) decreased oxytocin-and melittin-indu ced release of PGF(2 alpha) by 77% and 71% respectively (P<0.05). Expe riment 2 was conducted to establish the minimum inhibitory dose of ari stolochic acid. Basal release of PGF(2 alpha) was inhibited at 10(-5) M aristolochic acid, but 10(-4) M was required to block the stimulator y effect of oxytocin. Experiment 3 was carried out to identify the pre cise intracellular locus at which aristolochic acid was exerting its e ffect. Oxytocin (10(-7) M), AlF4- (5 x 10(-2) M NaF, 10(-5) M AlCl3), melittin (1.76 x 10(-6) M) and arachidonic acid (AA; 20 mu g/ml) stimu lated release of PGF(2 alpha) (P<0.05). Aristolochic acid (10(-4) M) b locked the release of PGF(2 alpha) stimulated by oxytocin, AlF4- or me littin by >80% (P<0.05). However, aristolochic acid reduced AA-induced release by only 22% (P=0.09). Thus, aristolochic acid blocked respons iveness to stimulatory agents that act on, or proximal to, PLA(2) but not responsiveness to AA whose stimulatory effect is exerted distal to PLA(2). Experiment 4 was intended to establish the specificity of the inhibitory effect of aristolochic acid. This was accomplished by exam ining the effect of aristolochic acid on the ability of oxytocin to st imulate 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 o xytocin to stimulate PGF(2 alpha) release (experiments 1-3) but not it s 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 PGF(2 alpha) release from o vine endometrial tissue is mediated through PLA(2).