Differential abilities of phorbol esters in inducing protein kinase C (PKC) down-regulation in noradrenergic neurones

1 The ability of several phorbol ester protein kinase C (PKC) activators (p horbol 12, 13-dibutyrate, PDB, phorbol 12, 13-diacetate, PDA, and 12-deoxyp horbol 13-acetate, dPA) to downregulate PKC was studied by assessing their effects on electrical stimulation-induced (S-T) noradrenaline release from rat brain cortical slices and phosphorylation of the PKC neural substrate B -50 in rat cortical synaptosomal membranes. 2 In cortical slices which were incubated for 20 h with vehicle, acute appl ication of PDB, PDA and dPA (0.1-3.0 muM) enhanced the S-I noradrenaline re lease in a concentration-dependent manner to between 200-250% of control in each case. In slices incubated with PDB (1 muM for 20 h), subsequent acute application of PDB (0.1-3.0 muM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 muM) for 20 h, there was no reduction in the facilitatory effect of their respe ctive phorbol esters or PDB (0.1-3.0 muM) when acutely applied, indicating that PKC was not down-regulated. This was confirmed using Western blot anal ysis which showed that PDB (1 muM for 20 h) but not PDA (3 muM for 20 h) ca used a significant reduction in PKC alpha. 3 Incubation with PDB for 20 h, followed by acute application of PDB (3 muM ) failed to increase phosphorylation of B-50 in synaptosomal membranes, ind icating down-regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 muM) Or PDB (3 muM) induced a significant increase in B-50 phosphorylation. 4 Acutely all three phorbol esters elevate noradrenaline release to about t he same extent, yet PDA and dPA have lower affinities for PKC compared to P DB, suggesting unique neural effects for these agents. This inability to ca use functional down-regulation of PKC extends their unusual neural properti es. Their neural potency and lack of down-regulation may be related to thei r decreased lipophilicity compared to other phorbol esters. 5 We suggest that PKC down-regulation appears to be related to binding affi nity, where agents with high affinity, irreversibly insert PKC into artific ial membrane lipid and generate Ca2+-independent kinase activity which degr ades and deplete PKC. We suggest that this mechanism may also underlie the ability of PDB to down-regulate PKC in nerve terminals, in contrast to PDA and dPA.