Neuropeptide Y (NPY) is a CRF secretagogue for human placental cells in culture. We have studied the involvement of intracellular calcium and calcium-dependent signaling in the NPY-induced CRF release in trophoblastic cells. The incubation of trophoblasts with NPY for 3 and 8 h led to a dose-dependent increase in CRF secretion. Also, NPY stimulated synthesis of this peptide hormone upon an 8-h incubation period. BIBP3226, a selective Y1 receptor antagonist, and pertussis toxin (PTX) eliminated these effects. NPY-stimulated CRF secretion was mostly prevented by loading cells with BAPTA-AM, suggesting that elevation of intracellular calcium is responsible for the increase of CRF secretion. However, this calcium chelator had no effect on CRF synthesis. Furthermore, U-73122, a phospholipase C-betas (PLC) inhibitor or xestospongin C, an inositol triphosphate receptor (InsP3-R) blocker, have partially prevented the effect of NPY on CRF synthesis and secretion. Therefore, the increase in CRF synthesis and secretion rely in part on the release of calcium from intracellular store. Interestingly, SKF 96365, an inhibitor of store operated calcium (SOC) influx, also partially blocked the NPY stimulatory effect on CRF release but not its synthesis, suggesting that calcium influx is also involved in this stimulation. In the syncytiotrophoblast, known to possess a NPY-activated protein kinase C (PKCs) activity, NPY also stimulated calcium calmodulin kinase II (CaMKII) and extracellular regulated kinase (ERK1/2) activities. In the present study, we observed that bisindolylmaleimide (BIM), a nonspecific PKCs inhibitor partially prevented the NPY-induced CRF release. On the other hand, autocamtide-2 related inhibitory peptide (AIP), a CaMKII inhibitor, prevented most of the stimulatory effect of NPY on both CRF synthesis and release. Go6976, an inhibitor of the conventional and mu PKCs and PD 098059, an inhibitor of the ERK cascade, had no effect on neither CRF synthesis nor release. Altogether, these results support a Y1 receptor-mediated PTX-sensitive induction on CRF synthesis and release by NPY from human placental trophoblasts. The stimulation of CRF synthesis by NPY seems to depend mainly on a PLC-beta to InsP3-R axis and on CaMKII activity. Also, the release of CRF depends on the PLC-beta to InsP3-R axis and CaMKII activity but also entails the participation of a calcium-independent PKCs.