THROMBOXANE A(2)-STIMULATED PHOSPHOLIPASE-D IN OSTEOBLAST-LIKE CELLS - POSSIBLE INVOLVEMENT OF PKC

We examined the effect of thromboxane A(2) (TxA(2)) on phosphatidyl-ch oline-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. 9,11-Epithio-11,12-methanothromboxane A(2) (STA(2)), a stable analogue of TxA(2), stimulated the formations of both choline and inos itol phosphates in a dose-dependent manner in the range between 10 nM and 10 mu M. The formation of choline stimulated by a combination of S TA(2) and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C-activating phorbol ester, was not additive. 1-(5-Isoquinolinyl-sulf onyl)-2-methylpiperazine (H-7), an inhibitor of protein kinases, suppr essed the formation of choline induced by STA(2) as well as that by TP A, but 20 mu M N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004 ), a control for H-7 as a protein kinase C inhibitor, had little effec t. Calphostin C, a potent and specific inhibitor of protein kinase C, also suppressed the formation of choline induced by STA(2). The STA(2) -induced formation of choline was significantly reduced by chelating e xtracellular Ca2+ with ethylene glycol-bis(beta-amino-ethyl ether)-N,N ,N',N'-tetraacetic acid. STA(2) dose dependently stimulated Ca-45(2+) influx from extracellular space. STA(2) stimulated DNA synthesis of MC 3T3-E1 cells and increased the number of these cells. These results su ggest that TxA(2) stimulates phospholipase D in osteoblast-like cells, resulting in the direction of their proliferation, and that the activ ation of protein kinase C is involved in the stimulation of phospholip ase D.