Effect of Exogenous Sex Steroid Hormones on Distinct PG Synthesis Pathways in Bovine Myometrium
The effects of steroid hormone E2 alone or in combination with P4 on PGHS-2 gene expression were assessed (Fig. 2). Myocytes from the circular and the longitudinal layers of bovine myometrium were treated with 1 nM E2 (72 h) and/or 10 nM P4 (24 h). The level of PGHS-2 mRNA was determined after incubation of the cells for 3 h in the presence of 100 nM PMA (Fig. 2A). Messenger RNA levels were higher in the longitudinal layer (0.57 ± 0.08; PGHS-2:18S relative optical density [ROD] units, n = 3) than in the circular layer (0.29 ± 0.07; PGHS-2:18S ROD units, n = 3) and were dramatically inhibited by 10 nM P4 + E2, but not by E2 alone, in both layers (p < 0.05, n = 3). No effect of steroid hormone on PGHS-2 mRNA level was detected in the absence of PMA treatment (data not shown). buy flovent inhaler
The effect of steroid hormones on PGHS-2, PGHS-1, and PGI synthase protein levels was also assessed (Fig. 2B). Protein expression was analyzed after incubation of the cells for 6 h in the presence of 100 nM PMA. DEX, which is recognized as inhibiting PG production and PGHS-2 induction, was used as an inhibitory control. PGHS-2 protein level was higher in the longitudinal layer than in the circular layer and was dramatically inhibited by 10 nM P4 with or without E2 or 100 nM DEX but not by E2 alone. By contrast, PGHS-1 and PGI synthase protein levels were not affected by steroid hormone treatments.
FIG. 2. Expression of PGHS-2 mRNA and protein in PMA-treated bovine myometrial cells; effect of steroid hormones. Confluent myometrial cells, from the circular (C) and the longitudinal (L) layers, were pretreated with 1 nM E2 (72 h), 1 nM E2 (72 h) with 10 nM P4 (for the last 24 h), or 100 nM DeX (24 h); and 100 nM PMA was added for the last 3 h (mRNA) or 6 h (protein) of incubation. A) Total RNA was isolated and hybridized with PGHS-2 cDNA probe. The 18S rRNA was used to normalize data. Results represent the mean ± SEM of 3 separate experiments, *p < 0.05. B) Western blotting was carried out as described in Materials and Methods. Analysis with PGHS-1, PGHS-2, and PGI synthase antibodies was performed successively on the same membranes.