PROSTAGLANDIN F-2-ALPHA-STIMULATED PHOSPHOLIPASE-D ACTIVATION IN OSTEOBLAST-LIKE MC3T3-E1 CELLS - INVOLVEMENT IN SUSTAINED 1,2-DIACYLGLYCEROL PRODUCTION

In [H-3]myristic acid-labelled osteoblast-like MC3T3-E1 cells, prostaglandin F-2 alpha (PGF(2 alpha))-induced PLD activity was assessed by measuring the [(3)Hlphosphatidylethanol (PEt) formation in the presence of ethanol. Inhibition of the increase in intracellular Ca2+ concentration ([Ca2+](i)) by U73122, an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), or chelation of extracellular Ca2+ with EGTA or of intracellular Ca2+ with BAPTA, suppressed PGF(2 alpha)-induced phospholipase D (PLD) activation. Neither protein kinase C (PKC) inhibitors nor PKC down-regulation with phorbol 12-myristate 13-acetate affected PGF(2 alpha)-induced [H-3]PEt formation. In permeabilized cells, guanosine 5'-[gamma-thio]triphosphate enhanced PGF(2 alpha)'s potency in [H-3]PEt formation in the presence of Ca2+. The pretreatment of intact cells with pertussis toxin failed to inhibit PGF(2 alpha)- induced [H-3]PEt formation. PGF(2 alpha) caused a biphasic production of [H-3]1,2-diacylglycerol ([H-3]1,2-DAG) in [H-3]glycerol-labelled cells. The initial transient phase was decreased by U73122, whereas the late sustained phase was decreased by ethanol and the phosphatidic acid phosphohydrolase inhibitor, propranolol. From these results, it was suggested that PGF(2 alpha)-induced PLD activation was mediated by the dual control of the [Ca2+](i) increase due to PI-PLC activation and activation of pertussistoxin-insensitive G-protein, but not mediated by PKC, and also that PLD activation was involved in the late sustained 1,2-DAG generation in MC3T3-E1 cells.