Characterization of neuropeptide Y-mediated corticotropin-releasing factorsynthesis and release from human placental trophoblasts

Neuropeptide Y (NPY) is a CRF secretagogue for human placental cells in cul ture. 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-depende nt increase in CRF secretion. Also, NPY stimulated synthesis of this peptid e hormone upon an 8-h incubation period. BIBP3226, a selective Y, receptor antagonist, and pertussis toxin (PTX) eliminated these effects. NPY-stimula ted CRF secretion was mostly prevented by loading cells with BAPTA-AM, sugg esting that elevation of intracellular calcium is responsible for the incre ase of CRF secretion. However, this calcium chelator had no effect on CRF s ynthesis. Furthermore, U-73122, a phospholipase C-beta s (PLC) inhibitor or xestospongin C, an inositol triphosphate receptor (InsP(3)-R) blocker, hav e partially prevented the effect of NPY on CRF synthesis and secretion. The refore, the increase in CRF synthesis and secretion rely in part on the rel ease of calcium from intracellular store. Interestingly, SKF 96365, an inhi bitor of store operated calcium (SOC) influx, also partially blocked the NP Y stimulatory effect on CRF release but not its synthesis, suggesting that calcium influx is also involved in this stimulation. In the syncytiotrophob last, known to possess a NPY-activated protein kinase C (PKCs) activity, NP Y also stimulated calcium calmodulin kinase II (CaMKII) and extracellular r egulated kinase (ERK1/2) activities. In the present study, we observed that bisindolylmaleimide (BIM), a nonspecific PKCs inhibitor partially prevente d the NPY-induced CRF release. On the other hand, autocamtide-2 related inh ibitory peptide (AIP), a CaMKII inhibitor, prevented most of the stimulator y 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 res ults support a Y, receptor-mediated PTX-sensitive induction on CRF synthesi s and release by NPY from human placental trophoblasts. The stimulation of CRF synthesis by NPY seems to depend mainly on a PLC-beta to InsP(3)-R axis and on CaMKII activity. Also, the release of CRF depends on the PLC-beta t o InsP(3)-R axis and CaMKII activity but also entails the participation of a calcium-independent PKCs.