Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Abstract

Incubation of intact frog erythrocytes with 12- O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested β-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the V max of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity ( K D ) or the binding capacity ( B max) for the β-adrenergic antagonist [ 125I]cyanopindolol. In addition, agonist/[ 125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E 1-, forskolin-, NaF-, and MnCl 2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly ( t 1 2 ≅ 5 min ) and with an EC 50 of about 10 −7 to 10 −6 m. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4β-phorbol 12,13-dibutyrate, 4β-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4β-phorbol, 4α-phorbol 12,13-didecanoate, and 4- O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.