Thromboxane A2-stimulated phospholipase D in osteoblast-like cells: possible involvement of PKC.
We examined the effect of thromboxane A2 (TxA2) on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of TxA2, stimulated the formations of both choline and inositol phosphates in a dose-dependent manner in the range between 10 nM and 10 microM. The formation of choline stimulated by a combination of STA2 and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C-activating phorbol ester, was not additive. 1-(5-Isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinases, suppressed the formation of choline induced by STA2 as well as that by TPA, but 20 microM N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a control for H-7 as a protein kinase C inhibitor, had little effect. Calphostin C, a potent and specific inhibitor of protein kinase C, also suppressed the formation of choline induced by STA2. The STA2-induced formation of choline was significantly reduced by chelating extracellular Ca2+ with ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid. STA2 dose dependently stimulated 45Ca2+ influx from extracellular space. STA2 stimulated DNA synthesis of MC3T3-E1 cells and increased the number of these cells. These results suggest that TxA2 stimulates phospholipase D in osteoblast-like cells, resulting in the direction of their proliferation, and that the activation of protein kinase C is involved in the stimulation of phospholipase D.
Shinoda
J
J
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Suzuki
A
A
Oiso
Y
Y
Kozawa
O
O
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002794:Choline / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000494:pharmacology*
D007546:Isoquinolines; D009281:Naphthalenes; D010879:Piperazines; D013928:Thromboxane A2; C037866:STA 2; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004247:DNA; D011493:Protein Kinase C; D010739:Phospholipase D; C058819:calphostin C; D002794:Choline; D013755:Tetradecanoylphorbol Acetate