P-0 PHOSPHORYLATION IN NERVES FROM NORMAL AND DIABETIC RATS - ROLE OFPROTEIN-KINASE-C AND TURNOVER OF PHOSPHATE GROUPS

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P-0 phosphate groups was studied in normal and experim entally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [P-32]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P-0, was increased 2-5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). Thi s increase was blocked by the protein kinase inhibitors, H-7 and staur osporine. Both the basal and PDB-stimulated phosphorylation of P-0 wer e significantly greater in segments of sciatic nerve from streptozotoc in-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P-0 and immunoblots of ner ve proteins revealed a decrease in the content of the protein kinase C alpha-isoform. The adenylate cyclase activator, forskolin, had no aff ect on the PDB-stimulated phosphorylation of P-0 in normal nerve but d ecreased phosphorylation in diabetic nerve. To measure turnover of P-0 phosphate groups, nerves were incubated with P-32 and incorporated la bel was then chased in radioactivity-free medium for up to 4 hours. P- 0 from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additio nal phosphate groups prelabeled in the presence of PDB disappeared aft er 2 hours of chase. P-0 phosphate groups from diabetic nerve displaye d similar turnover kinetics. When forskolin was added to the chase med ium, the turnover of P-0 phosphate moieties was accelerated in normal, but not in diabetic nerve. These findings clearly establish a promine nt role for protein kinase C in P-0 phosphorylation, provide evidence for heterogeneous turnover of P-0 phosphate groups and suggest that cy clic AMP-mediated processes may modulate P-0 phosphorylation. Further, these results indicate that the metabolism of P-0 phosphate moieties is perturbed in nerve from diabetic animals.