AJP - Endocrinology and Metabolism, Vol 269, Issue 3 E524-E529, Copyright © 1995 by American Physiological Society

ARTICLES

Thromboxane A2-stimulated phospholipase D in osteoblast-like cells: possible involvement of PKC

J. Shinoda, A. Suzuki, Y. Oiso and O. Kozawa
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.

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.

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