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 μM. 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-Isoquinolinylsulfonyl)-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 μM 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(β-aminoethyl 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.