Second messenger systems in the action of LH and oxytocin on porcine endometrial cells in vitro

Abstract

LH/hCG as well as oxytocin receptors are present in the porcine endometrium. Oxytocin increases phosphatidylinositol hydrolysis in this tissue, but its action on adenylate cyclase activity is disputed. The second messenger system responding to LH/hCG in endometrial cells has not been established. In this study, we investigated the involvement of protein kinase A and C signaling mechanisms in the action of LH on porcine endometrial cells in vitro. The possibility of cAMP accumulation after treatment of endometrial cells with oxytocin was also investigated. Endometrial tissue was obtained from gilts during Days 12–15 of the estrous cycle. To study the adenylate cyclase system, endometrial cells were cultured for 48 h and then incubated with different doses of LH or oxytocin for 15, 30, 60, and 180 min. To study the phospholipase C system, dispersed cells were first labeled with myo-[ 3 H ]inositol and then treated with increasing doses of LH or 100 nM of oxytocin for 30 min. Time- and dose-dependent effect of LH and oxytocin on cAMP concentration was observed. After 30 min of incubation only the highest dose of LH (100 ng/ml) was able to increase cAMP concentration in medium ( P<0.05). Longer periods (1 and 3 h) caused increased cAMP accumulation after treatment with 10 and 100 ng/ml of LH ( P<0.001). Oxytocin-stimulated cAMP concentration was observed after 1 h when only the highest dose (1000 nM) of hormone was used ( P<0.01) and after 3 h of incubation with doses of 10–1000 nM ( P<0.01). LH (10 and 100 ng/ml) increased inositol phosphates (IPs) accumulation in endometrial cells after 30 min of incubation ( P<0.01). Oxytocin involvement in IPs synthesis was more apparent than was LH ( P<0.001 versus P<0.01). This is the first demonstration that LH receptor signaling leads to increased cAMP generation as well as IPs turnover in porcine endometrium. Oxytocin-dependent cAMP production in endometrial cells of swine was found after longer periods (3 h) of incubation. Our observations lead to the conclusion that both protein kinase A and C second messenger systems are involved in LH action and that oxytocin is able to stimulate adenylate cyclase activity in porcine endometrial cells.