It is interesting to note that the effect of steroid hormones on PGHS-2 protein or mRNA level correlates well with PG accumulation in both myometrial layers (Table 1). Indeed, compared to those in the control group (6-h treatment with PMA in the absence of steroid), PG levels were not affected by E2 treatment whereas P4 or P4 + E2 significantly decreased the PG level in both layers. ventolin 100 mcg
Effect of Indomethacin and CGP 28238 on PGI2 Accumulation
The accumulation of PGI2 in myocytes cultured in the presence of PMA and increasing concentrations (0, 0.01, 0.1, 1, 10, 100 nM) of the PGHS inhibitors, indomethacin and CGP 28238, is illustrated in Figure 3. Both inhibitors induced a dose-dependent inhibition of PGI2 accumulation. The inhibitory effect was observed at low concentrations of indomethacin (1 nM, p < 0.05) in comparison to the specific PGHS-2 inhibitor, CGP 28238, which was effective only at concentrations above 10 nM (p < 0.05). When the two inhibitors were combined, their effect was additive for concentrations lower than 1 nM, after which it was equal to the effect with indomethacin alone.
Effect of CGP 28238 and Indomethacin on PGHS-2 mRNA Levels
The pattern of PGHS-2 mRNA expression in bovine myometrium was evaluated after treatment with 10 nM CGP 28238 or indomethacin in the presence of PMA (Fig. 4). By analogy with previous Western blot analysis, the PGHS-2 transcript (4.1 kilobases) was more abundant in the longitudinal layer, confirming preferential expression of this enzyme in this smooth muscle layer. The level of PGHS-2 mRNA in PMA-stimulated myocytes was decreased by 44.80 ± 7.67% (p < 0.01, n = 5) in the longitudinal layer as compared to 27.83 ± 7.62% (p < 0.05, n = 6) in the circular layer after treatment with 10 nM CGP 28238. Treatment with 10 nM indomethacin affected the PGHS-2 mRNA level slightly but not significantly (p > 0.05, n =3).
FIG. 3. Effect of PGHS inhibitors on PGI2 production in cultured myocytes. Myocytes from the longitudinal layers of bovine myometrium were incubated with CGP 28238 (solid circles), indomethacin (open circles), or CGP 28238 plus indomethacin (open squares) at the indicated concentrations, in the presence of 100 nM PMA. PGI2 production was measured after 24 h. Results represent the mean ± SEM of 3 separate experiments run in quadruplicate. Similar results were obtained in the circular layer. *p < 0.05 and **p < 0.01 when compared to untreated cells.
FIG. 4. Effect of CGP 28238 and indomethacin on PGHS-2 mRNA accumulation in bovine myometrium. Confluent myocytes from the circular (C) and longitudinal (L) myometrial layers were incubated for 24 h in the absence or presence of 10 nM CGP 28238 (open columns) or indomethacin (filled columns). During the last 3 h of incubation, 100 nM PMA was added. RNA extraction and Northern blot analysis were performed as described in Materials and Methods. Ten micrograms of total RNA was loaded in each lane. Each Northern blot was hybridized successively with PGHS-2 and 18S rRNA. Data are expressed as mean ± SEM from 5 or 6 separate experiments. *p < 0.05 and **p < 0.01 when compared to untreated cells.