MECHANISM OF PHOSPHOLIPASE-D ACTIVATION-INDUCED BY EXTRACELLULAR ATP IN OSTEOBLAST-LIKE CELLS

We have previously reported that extracellular ATP stimulates Ca2+ inf lux from extracellular space, resulting in the production of prostagla ndin E(2) which mediates, at least in part, its proliferative effect o n osteoblast-like MC3T3-E1 cells, and that the activation of protein k inase C (PKC) stimulates phospholipase D in these cells. In the presen t study, we examined the effect of extracellular ATP on phosphatidylch oline-hydrolysing phospholipase D activity in MC3T3-E1 cells. ATP stim ulated the formation of both choline and inositol phosphates dose-depe ndently 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, wa s synergistic, whereas that of inositol phosphates was not. A combinat ion of ATP and 12-O-tetradecanoylphorbol-13-acetate, a PKC activating phorbol ester, additively stimulated the formation of choline. Stauros porine, an inhibitor of PKC, had little effect on ATP-stimulated forma tion of choline. Choline formation was significantly reduced by chelat ing extracellular Ca2+ with EGTA, while being inhibited by W-7, an ant agonist of calmodulin. These results suggest that extracellular ATP st imulates phospholipase D in a Ca2+/calmodulin-dependent manner in oste oblast-like cells, and that neither PKC activation nor GTP-binding pro tein is involved in this mechanism.