Signaling and Signal Transduction in Endocrine Tissues
(M733) NONGENOMIC ACTION OF PROGESTERONE INHIBITS OXYTOCIN-INDUCED PHOSPHOINOSITIDE HYDROLYSIS IN OVINE ENDOMETRIUM.
Bishop, Cecily1, Reeve, Reed1, Stormshak, Fredrick1, 1 Oregon State University, Corvallis, OR
ABSTRACT- Progesterone (P4) has been shown to act nongenomically via a putative plasma membrane receptor to inhibit oxytocin (OT) binding in the ovine endometrium. Experiments were conducted to further characterize the nongenomic effects of P4 on binding of OT to its receptor (OTR) and signal transduction in the ovine endometrium. Exp 1 was conducted to examine the dose-response relationship of P4 to OT binding. Endometrium was removed from ovariectomized ewes (n = 5) treated with P4 and estradiol-17 (E2) in a sequence to mimic an abbreviated estrous cycle. Plasma membranes under dominance of E2 to ensure upregulation of OTR were preincubated in the presence of P4 (0 to 2.5 ng/ml) for 1 h followed by OTR analysis using 5 nM [3H]-OT. Progesterone interfered with the binding of OT in a dose-dependent manner as determined by regression analysis (P < 0.001) with the lowest binding of OT occurring in membranes exposed to 2.5 ng P4/ml. Having established an effective inhibitory concentration of P4, Exp 2 was conducted to determine whether P4 could interfere with OT-induced phosphoinositide hydrolysis in vitro. Endometrium was recovered from ewes (n = 9) on Day 15 of the estrous cycle and divided into four explants assigned to a 2 × 2 factorial arrangement of treatments consisting of two levels of P4 (0 and 2.5 ng/ml) and two levels of OT (0 and 100 nM). Explants were preincubated with 10 Ci myo-[2-3H] inositol in Krebs Ringer Bicarbonate (KRB) containing 10 M unlabeled myo-inositol and 10 mM glucose for 2 h at 37°C under an atmosphere of 95% O2 - 5% CO2. Explants were placed in fresh KRB and incubated for an additional 30 min at which time explants were exposed to 10 mM LiCl alone or LiCl and P4 for another 10 min. Vehicle or OT were then added to explants for 20 min. Incubation was terminated, medium removed and trichloroacetic acid added, which was then analyzed for total inositol trisphosphate (IP3). Content was expressed as DPM [3H] IP3 / g tissue dry wt. Data were analyzed statistically by use of two-way ANOVA. Progesterone alone failed to affect phosphoinositide hydrolysis, but significantly interfered with OT stimulation of IP3 (P4 × OT interaction; P < 0.05). These data are interpreted to suggest that the observed reduction in OT-induced IP3 production is due to P4 inhibition of OT binding to its receptor.
KEY WORDS: Nongenomic, Progesterone, Oxytocin, Ovine