The modulation of placental lactogen release by opioids: a role for extracellular calcium

Abstract

We previously reported that kappa opioids stimulated the release of human placental lactogen (hPL) from trophoblastic cells and that this effect was prevented by co-incubation with naloxone. We also reported that adenylate cyclase was not directly involved in this process. In order to understand the post-receptor events mediating hPL release by opioids in the human placenta, we studied the role of extracellular calcium. Human trophoblastic cells obtained by trypsin digestion were cultured for 48 h in Ham's F-10 medium supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 μg/ml streptomycin. 45Ca 2+ influx was then measured by filtration on glass-fiber filters. We observed a time- and dose-dependent stimulation of 45Ca 2+ influx by ethylketocyclazocine (EKC) with an EC 50 of 0.5 nM and a maximal stimulation of 196% over control. This effect was completely blocked by naloxone, a non-specific opioid antagonist, and by nor-binaltorphimine, a specific kappa antagonist. We also demonstrated that U-50, 488 (kappa agonist) had the same stimulatory effect as EKC (221 ± 25% of control). D-Ala 2,NMe-Phe 4,Gly-ol 5)-enkephalin (DAGO) (mu agonist) slightly stimulated Ca 2+ influx (128 ± 5% of control, p > 0.05) whereas D-Ser 2,Leu, Thr 6)-enkephaIin (DSLET) (delta agonist) had no effect. Pre-incubation of trophoblastic cells with pertussis toxin (PTX) did not affect the EKC-induced 45Ca 2+ influx, suggesting that this placental opiate effect is not coupled with PTX-sensitive G proteins. The role of extracellular calcium in the regulation of hPL release by kappa opiates was confirmed by experiments in which ionomycin (ionophore) and Bay K 8644 (calcium-channel agonist) mimicked the stimulatory effect of EKC on hPL release. Also, without extracellular Ca 2+, the basal (control) and EKC-stimulated hPL release were significantly lower than with 1.8 mM Ca 2+. Moreover, nifedipine (calcium-channel antagonist) inhibited both basal and EKC-stimulated hPL release. These results suggest that calcium influx is one mechanism mediating opioid-stimulated hPL release by human term trophoblastic cells.