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.
Suzuki
A
A
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Shinoda
J
J
Oiso
Y
Y
Kozawa
O
O
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism*; D013268:Stimulation, Chemical; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002147:Calmodulin; D013449:Sulfonamides; D004533:Egtazic Acid; C017967:W 7; D000255:Adenosine Triphosphate; D010739:Phospholipase D; D002794:Choline; D013755:Tetradecanoylphorbol Acetate