Proteolysis negatively regulates agonist-stimulated arachidonic acid metabolism

Phenylmethylsulphonyl fluoride, lactacystin (a selective inhibitor of the proteasome) and the peptide aldehydes carbobenzoxyleucylleucylnorvalinal and carbobenzoxyleucylleucylleucinal amplify the production of prostacyclin in rat liver cells incubated for 6 h with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the TPA-type tumour promoters teleocidin and aplysiatoxin. Such stimulation is not dependent upon the simultaneous presence of the inhibitor and TPA. Preincubation of the cells with TPA followed by addition of the inhibitor or preincubation with the inhibitor followed by addition of TPA results in amplified prostacyclin production. Phenylmethylsulphonyl fluoride, lactacystin, and carbobenzoxyleucylleucylnorvaline also enhance prostacyclin production after incubation with interleukin-1beta and transforming growth factor-alpha. The Ca2+ chelator ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid inhibits the phenylmethylsulphonyl fluoride-TPA or lactacystin-TPA amplifications. Cells, treated with phenylmethylsulphonyl fluoride, TPA, interleukin-1beta, lactacystin or the peptide aldehydes exhibit increased prostaglandin endoperoxide G/H synthase activity. The increased activities as well as the constitutive prostaglandin endoperoxide G/H synthase activity are inhibited by a selective prostaglandin endoperoxide G/H synthase-2 inhibitor, 1-[2-(4-fluorophenyl)-cyclopenten-1-yl]-4-(methysulphonyl)-b enzene, with an IC50 of approximately 0.5 microM. These results demonstrate that the C-9 rat liver cells express prostaglandin endoperoxide G/H synthase-2 constitutively and express induced prostaglandin endoperoxide G/H synthase-2. Inhibition of proteolytic activity amplifies agonist-stimulated arachidonic acid metabolism in these cells.