PGHS and cPLA2 Protein Expression in Response to PMA in Cultured Myocytes
Figure 1A shows the effect of PMA on the pattern of PGHS-1, PGHS-2, and cPLA2 proteins in the two smooth muscle layers of the bovine myometrium. Under basal conditions, without PMA stimulation, two immunoreactive bands were detected after immunoblotting with antiserum directed against cPLA2 (approximately 85 kDa). After 6 h of PMA stimulation, only a single band with reduced mobility on SDS-PAGE was detected. After 24-h stimulation with the phorbol ester, corresponding to the maximal production of PGs, the signals were more intense, and two cPLA2 immunoreactive bands, probably corresponding to the phosphorylated and unphosphorylated enzyme, were observed.
A marked induction of immunoreactive PGHS-2 could be detected within 3 h after PMA treatment (data not shown), but it reached maximal levels after 6 h of incubation in the presence of phorbol ester. At that time point, the 69-kDa PGHS-2 protein appeared more abundant in the longitudinal than in the circular layer. antibiotics levaquin
The polyclonal antibody used against PGHS-1 recognized a band of approximately 72 kDa corresponding to the migration point of the platelet PGHS-1 preparation that we used as a control. No significant induction of immunoreactive PGHS-1 was observed in response to PMA.
PMA (100 nM) induced a time-dependent stimulation of PGE2 and PGI2 (6-keto-PGF1a) synthesis (Fig. 1B). The maximal phorbol ester effect was obtained after 24-h stimulation, with a predominance of PGI2 accumulation in the longitudinal layer (7200 ± 1800 pg/ml) in comparison to the circular layer (1600 ± 150 pg/ml), as previously described.
FIG. 1. Effect of PMA on steady state levels of cPLA2, PGHS-1, and PGHS-2 proteins in myocytes from the circular (C) and longitudinal (L) layers of bovine myometrium. When myocytes reached confluen-cy, the culture medium was replaced by RPMI without serum, and incubation was performed for 24 h in the absence or presence of 100 nM PMA for the indicated time periods. Cells were lysed in homoge-nization buffer, and immunoblot analysis was performed in the presence of the respective antibodies as described in Materials and Methods. PLA2 and PGHS Western blots were run successively on the same membranes. Extracts from human platelet or interleukin-1-treated osteoblasts were used as PGHS-1 or PGHS-2 positive control, respectively (M). PG accumulation in the incubation media was measured by ELISA as described in Materials and Methods. Results are representative of one of 3 experiments.
TABLE 1. Effect of 17^-estradiol and/or progesterone on PG accumulation in PMA-treated myocytes from the circular and longitudinal layers of bovine myometrium.3
|E2||5||86.25 ± 16.24NS||102.20 ± 41.56NS||86.00 ± 18.75NS||92.75 ± 9.67NS|
|P4||3||51.00 ± 8.71*||33.33 ± 6.65**||37.67 ± 16.17**||37.67 ± 13.42**|
|E2 + P4||5||45.00 ± 8.54**||33.33 ± 2.51**||34.67 ± 11.86**||33.67 ± 9.40**|
|DEX||3||18.00 ± 4.24**||19.00 ± 5.65**||19.50 ± 1.50**||3.00 ± 2.00**|
a Data are expressed as the percent of control (without treatment); values are mean ± SEM, n = 3 or 5 for each group. Treatment effect: * p < 0.05; ** p < 0.01; NS, not significant.