Regulation of eicosanoid biosynthesis by phorbol ester in Madin Darby canine kidney cells

We assessed the effects of the peptide agonist, bradykinin (BK), and phorbol myristate acetate (PMA) on prostaglandin E₂ (PGE₂) production, cyclooxygenase (COX) activity and mass, and arachidonic acid (AA) release in Madin Darby canine kidney (MDCK) cells. PMA stimulated PGE₂ production by increasing both AA release and the activity of COX. Using [³⁵S]methionine labeling and immunoprecipitation, we demonstrated that the increased COX activity is due to new COX synthesis. Actinomycin D and cycloheximide blocked the PMA-stimulated COX activity but not AA release. Both PMA-stimulated AA release and COX activity were reduced by the protein kinase C inhibitor staurosporine (STP). Glucocorticoids failed to alter PMA- or BK-stimulated PGE₂ production or COX synthesis. BK-stimulated PGE₂ production was reduced by STP, indicating BK acts in part through protein kinase C activation. BK increased PGE₂ production in PMA-treated cells, suggesting a protein kinase C-independent mechanism of action as well. BK did not stimulate any change in COX activity. We conclude that in MDCK cells PMA, but not BK, can stimulate both AA release and COX synthesis. Stimulation of COX synthesis requires either prolonged activation of protein kinase C and/or an additional nonprotein kinase C-mediated effect of PMA.