Prostacyclin production after treatment of rat liver cells with tosyl-L-phenylalanine chloromethyl ketone and 12-O-tetradecanoylphorbol-13-acetate is mediated by a phorbol-ester-stimulated intermediate

The prostacyclin (PGI₂) production in rat liver cells by treatment by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is not dependent upon the simultaneous presence of both ligands. Preincubation of the rat liver cells with TPA followed by addition of TPCK, as well as preincubation of the cells with TPCK followed by addition of TPA, results in PGI₂ production. Maximum production is found after a 10 min incubation with TPA or after a 120 min incubation with TPCK. Preincubation with TPA for longer than 10 min or preincubation with TPCK for longer than 2 h results in reduced stimulation of PGI₂ production. Dexamethasone does not eliminate the effects of either preincubation or simultaneous addition of TPCK and TPA. EGTA does not affect either preincubation reaction but does completely inhibit PGI₂ production after simultaneous addition of the agents. Preincubation of the cells for 30 min with aspirin completely inhibits the TPCK-TPA-stimulated PGI₂ synthesis. The PGI₂ production following exogenous addition of arachidonic acid to the cells is unaffected by prior treatment of the cells with TPA, TPCK, or TPA plus TPCK. Taken together the data suggest that TPA stimulates the production of an intermediate which activates a Ca²⁺-dependent phospholipase activity. The intermediate is inactivated by a protease which is inhibited by the SH-reactive agent TPCK. The released arachidonic acid is oxygenated by the constitutively expressed cyclooxygenase (prostaglandin H synthase-1).