Mechanism of phospholipase D activation induced by extracellular ATP in osteoblast-like cells

We have previously reported that extracellular ATP stimulates Ca²⁺ influx from extracellular space, resulting in the production of prostaglandin E₂ which mediates, at least in part, its proliferative effect on osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) stimulates phospholipase D in these cells. In the present study, we examined the effect of extracellular ATP on phosphatidylcholine-hydrolysing phospholipase D activity in MC3T3-E1 cells. ATP stimulated the formation of both choline and inositol phosphates dose-dependently in the range between 0.1 and 1 mM. The formation of choline by a combination of ATP and NaF, an activator of GTP-binding protein, was synergistic, whereas that of inositol phosphates was not. A combination of ATP and 12-O-tetradecanoylphorbol-13-acetate, a PKC activating phorbol ester, additively stimulated the formation of choline. Staurosporine, an inhibitor of PKC, had little effect on ATP-stimulated formation of choline. Choline formation was significantly reduced by chelating extracellular Ca²⁺ with EGTA, while being inhibited by W-7, an antagonist of calmodulin. These results suggest that extracellular ATP stimulates phospholipase D in a Ca²⁺/calmodulin-dependent manner in osteoblast-like cells, and that neither PKC activation nor GTP-binding protein is involved in this mechanism.