Function of Ca²⁺ in phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblast-like cells

We investigated the function of Ca²⁺ in the activation of phosphatidylcholine (PC)-hydrolyzing phospholipase D (PLD) in osteoblast-like MC3T3-E1 cells. Fetal calf serum (FCS) stimulated the formation of choline in a dose-dependent manner in the range between 0.6% and 10%. The effect of a combination of FCS and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C (PKC) activator, on the formation of choline was additive. Staurosporine, an inhibitor of protein kinases, enhanced the formation of choline induced by FCS, BAPTA/AM, a chelator of intracellular Ca²⁺, inhibited the formation of choline induced by FCS. The depletion of extracellular Ca²⁺ by EGTA markedly reduced the FCS-induced formation of choline, SK and F 96365, an inhibitor of receptor-operated Ca2+ entry, significantly inhibited the choline formation induced by FCS. On the other hand, nifedipine, an inhibitor of L-type voltage-dependent Ca²⁺ channels, had little effect on the choline formation, TMB-8, an inhibitor of Ca²⁺ mobilization from intracellular Ca²⁺ store, significantly inhibited FCS-induced choline formation, These results strongly suggest that Ca²⁺ mobilization, through both the influx via receptor-operated Ca²⁺ channel and the release from intracellular Ca²⁺ store, plays an important role in the activation of PLD in osteoblast-like cells.