Inhibition by protein kinase-C inhibitor and cycloheximide of phorbol ester- and epidermal growth factor-induced arachidonic acid metabolism in cultured porcine thyroid cells

In an attempt to elucidate possible mechanism(s) for stimulated arachidonic acid metabolism by phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF) in porcine thyroid cells, we examined the effects of protein kinase inhibitors, isoquinolinesulfonamide derivatives (H-7 and HA-1004), and cycloheximide. The production of PGE2 stimulated by either PMA or EGF was strongly inhibited by H-7, with an ID50 value of approximately 20 to 25 mumol/L in each case, as well as by cycloheximide, with an ID50 value of less than 0.5 micrograms/mL in each case. In contrast, 100 mumol/L of HA-1004 showed less inhibition of PGE2 production provocated by either PMA or EGF. On the other hand, PGE2 production in basal or stimulated condition by exogenously added arachidonic acid, was inhibited to an even lesser extent by both H-7 and cycloheximide. The EGF- and PMA-stimulated release of 3H-arachidonic acid from the cells was also strongly inhibited by H-7 and cycloheximide. These results suggest an induction of synthesis of some proteins responsible for the release of arachidonic acid, which might be attributed to protein kinase-C activation in arachidonic acid metabolism stimulated by PMA or EGF. Moreover, PGE2 production was potently induced by PMA and slightly by EGF in the cyclooxygenase-inactivated cells by acetyl salicylate pretreatment, which also suggests that both agents might induce the synthesis of cyclooxygenase in cultured porcine thyroid cells, although we did not measure its activity.