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

Abstract

1 The ability of several phorbol ester protein kinase C (PKC) activators (phorbol 12, 13-dibutyrate, PDB; phorbol 12, 13-diacetate, PDA; and 12-deoxyphorbol 13-acetate, dPA) to downregulate PKC was studied by assessing their e ects on electrical stimulation-induced (S-I) 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 application of PDB, PDA and dPA (0.1 ± 3.0 mM) enhanced the S-I noradrenaline release in a concentration-dependent manner to between 200 ± 250% of control in each case. In slices incubated with PDB (1 mM for 20 h), subsequent acute application of PDB (0.1 ± 3.0 mM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 mM) for 20 h, there was no reduction in the facilitatory e ect of their respective phorbol esters or PDB (0.1 ± 3.0 mM) when acutely applied, indicating that PKC was not down-regulated. This was con®rmed using Western blot analysis which showed that PDB (1 mM for 20 h) but not PDA (3 mM for 20 h) caused a signi®cant reduction in PKCa. 3 Incubation with PDB for 20 h, followed by acute application of PDB (3 mM) failed to increase phosphorylation of B-50 in synaptosomal membranes, indicating down-regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 mM) or PDB (3 mM) induced a signi®cant increase in B-50 phosphorylation. 4 Acutely all three phorbol esters elevate noradrenaline release to about the same extent, yet PDA and dPA have lower a nities for PKC compared to PDB, suggesting unique neural e ects for these agents. This inability to cause functional down-regulation of PKC extends their unusual neural properties. Their neural potency and lack of down-regulation may be related to their decreased lipophilicity compared to other phorbol esters. 5 We suggest that PKC down-regulation appears to be related to binding a nity, where agents with high a nity, irreversibly insert PKC into arti®cial membrane lipid and generate Ca 2+ -independent kinase activity which degrades 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.

Involvement Of B‐50 (GAP‐43) Phosphorylation In The Modulation Of Transmitter Release By Protein Kinase C

“…Indeed, this is what was observed in intact synaptosomes 22 . However, in disrupted synaptosomes, the rank order of potency of the phorbol esters in causing B‐50 phosphorylation agreed with their potency on PKC and is highly correlated with phorbol ester effects on transmitter release ( r 2 = 0.99) 22 . The differences shown by the B‐50 phosphorylation studies suggest that B‐50 has an intraneuronal site of action that is difficult to access by lipophilic phorbol esters when the neural membrane is intact.…”

“…If B‐50 is involved in transmitter release, the order of potency of phorbol esters in causing its phosphorylation should correlate with their actions on transmitter release, including the observation that lipophilic phorbol esters (dPT and PMA) have anomalously low potency. Indeed, this is what was observed in intact synaptosomes 22 . However, in disrupted synaptosomes, the rank order of potency of the phorbol esters in causing B‐50 phosphorylation agreed with their potency on PKC and is highly correlated with phorbol ester effects on transmitter release ( r 2 = 0.99) 22 .…”

Distinct Protein Kinase C Isoforms Mediate Regulation of Vascular Endothelial Growth Factor Expression by A2A Adenosine Receptor Activation and Phorbol Esters in Pheochromocytoma PC12 Cells

“…PDA has been reported to activate but not promote the degradation of PKC␣ in rat brain cortical slices (62). As demonstrated in Fig.…”

The role of lipids and protein kinase Cs in the pathogenesis of diabetic retinopathy

“…There have been other reports of variation in phorbol ester potency; for example, cells from the cloned subline, KG‐1a, unlike their parental line (KG‐1 cells), are resistant to the differentiating effect of 12‐O‐tetradecanoylphorbol‐13‐acetate 28. Phorbol diacetate generally has a lower potency than other diesters but in isolated cases it has a greater potency due to changes in downregulation 29. More interesting, is that with PKCδ, a series of compounds all produced activation, but for each, the subcellular distribution was activator‐specific 30.…”