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&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.
Department of Internal Medicine, Chubu National Hospital, National Institute for Longevity Sciences, Aichi, Japan.
H
H
Tokuda
A
A
Suzuki
Y
Y
Watanabe-Tomita
J
J
Shinoda
Y
Y
Imamura
Y
Y
Oiso
A
A
Igata
O
O
Kozawa
D016475:3T3 Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002794:Choline / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006868:Hydrolysis; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007093:Imidazoles; D010713:Phosphatidylcholines; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium