Inhibition of prostaglandin E 2-responsive adenylyl cyclase in embryonal human kidney 293 cells by phorbol esters

Abstract

A clonal primary embryonal human kidney cell line, 293, increased cAMP production in response to prostaglandin E 2 (PGE 2) (0.02–2 μM). The purpose of this study was to show the effects of tumor-promoting phorbol esters (e.g., 4β-phorbol 12-myristate 13-acetate, PMA) on PGE 2-stimulated cAMP production. Pretreatment with PMA (0.2–200 nM) for 30 min markedly reduced PGE 2-Stimulated CAMP production in the presence of 0.5 mM isobutylmethylxanthine. The reduction by PMA was dose- and time-dependent. PMA seems to attenuate the increase in cAMP accumulation elicited by PGE 2 primarily, if not entirely, by inhibiting adenylyl cyclase activity, since we were unable to demonstrate an effect of PMA on the degradation half-life of cAMP in intact 293 cells. The action of PMA had some specificity for the agonist used; thus, PMA inhibited PGE 2 activated adenylyl cyclase but had no effect on the forskolin-activated enzyme. Co-pretreatment with PMA and H-7, an inhibitor of protein kinase C (PKC), partially prevented the PMA-induced attenuation of the PGE 2-stimulated cAMP accumulation, and 1-oleoyl-2-acetylglycerol, a synthetic diacylglycerol analog, partially mimicked the PMA action. Thus, PMA appeared to decrease cAMP production by a PKC-mediated mechanism, inhibiting adenylyl cyclase activity at a point other than the catalytic subunit of the enzyme in the kidney 293 cell line.