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 (phorbol 12, 13-dibutyrate, PDB; phorbol 12, 13-diacetate, PDA; and 12-deoxyphorbol 13-acetate, dPA) to down-regulate PKC was studied by assessing their effects 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 microM) 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 microM for 20 h), subsequent acute application of PDB (0.1 - 3.0 microM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 microM) for 20 h, there was no reduction in the facilitatory effect of their respective phorbol esters or PDB (0.1 - 3.0 microM) when acutely applied, indicating that PKC was not down-regulated. This was confirmed using Western blot analysis which showed that PDB (1 microM for 20 h) but not PDA (3 microM for 20 h) caused a significant reduction in PKCalpha. 3. Incubation with PDB for 20 h, followed by acute application of PDB (3 microM) 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 microM) or PDB (3 microM) induced a significant 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 affinities for PKC compared to PDB, suggesting unique neural effects 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 affinity, where agents with high affinity, irreversibly insert PKC into artificial 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.
Kotsonis
P
P
Novartis Institute for Medical Sciences, London WC1E 6BN. Abteilung Innere Medizin IV, Universitätsklinikum Freiburg, Freiburg 79106, Germany.
Funk
L
L
Prountzos
C
C
Iannazzo
L
L
Majewski
H
H
D000818:Animals; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects*; D066298:In Vitro Techniques; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D009638:Norepinephrine / Q000378:metabolism / Q000502:physiology*; D010703:Phorbol Esters / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013564:Sympathetic Nervous System / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D010703:Phorbol Esters; D011493:Protein Kinase C; D009638:Norepinephrine