Inhibition of Oxytocin Binding by Progesterone in Ovine Uteri
Speaker: Kathrin Dunlap
Abstract: Effects of progesterone (p4) and the antagonist RU 486 on binding of oxytocin (OT) to its uterine receptor (OTR) were studied. In Exp. 1, 11 ovariectomized (OVX) ewes were assigned to 3 groups (n = 3 or 4/grp). All ewes received s.c. injections to simulate an abbreviated estrous cycle: 25 mg estradiol-17b (e2)/day (d) for 2 d followed by p4 (10 mg/d for 5 d). Specific daily treatments were initiated on Day 8 and continued for 3 d as follows: Grps E) 25 mg e2 + vehicle; EP) 25 mg e2 + 10 mg p4; EPR) 25 mg e2 + 10 mg p4 + 10 mg RU 486. Endometrium removed on Day 11 was stored at -80&Mac176;C until analyzed for OTR. For Exp. 2, an aliquot of endometrium from group E was not frozen but was homogenized and centrifuged (100,000 x g) to recover plasma membranes. Membranes (1 mg protein/ml) were treated as follows: C) controls, P) p4 (5 ng/ml), and PR) p4 (5 ng/ml) + RU 486 (10 ng/ml). Membranes were incubated for 1 h (25&Mac176;C) and then stored (-80&Mac176;C) until OTR analysis. Scatchard analysis revealed high affinity OT binding sites (Kd = 1.01 x 10-10 M). In vivo p4 treatment suppressed binding of OT to OTR (E, 121 ± 18 vs EP, 40 ± 21 fmol/mg protein; p<0.01). Treatment with RU 486 blocked the effect of p4 (E vs EPR, 140 ± 24 fmol/mg protein; p >0.05). In vitro effects of p4 and RU 486 on concentrations of OTR were similar to those after in vivo treatment (C, 69 ± 5; P, 38 ± 5; PR, 62 ± 9 fmol/mg protein; p<0.05). In vitro binding of OT to receptors was less than in vivo. Because receptors may be lost due to freezing and thawing the in vitro experiment was repeated using 5 OVX ewes treated identically as Exp. 1 group E. After incubation, membranes were immediately analyzed for OTR. Binding of OT was similar to that of Exp. 2 (C, 273 ± 42 vs P, 128 ± 33 fmol/mg protein; p<0.05) but concentrations were greater. Effect of p4 was blocked by RU 486 (C vs PR, 245 ± 36 fmol/mg protein; p>0.05). Data suggest that p4 may be acting, at least in part, via a non-nuclear mechanism to suppress binding of OT to its uterine receptor in the ewe.