IN-VIVO PHOSPHORYLATION OF THE NA,K-ATPASE ALPHA-SUBUNIT IN SCIATIC-NERVES OF CONTROL AND DIABETIC RATS - EFFECTS OF PROTEIN-KINASE MODULATORS

The phosphorylation state of the Na,K-ATPase alpha subunit has been ex amined in P-32-labeled sciatic nerves of control and streptozotocin-tr eated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit P-32 labeling was analyzed after im munoprecipitation. In control nerves, the PKC activator phorbol 12-myr istate 13-acetate (PMA) had little effect on alpha-subunit P-32 labeli ng. In contrast, staurosporine, a PKC inhibitor, and extracellular cal cium omission decreased it. In Ca2+-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the P-32 labeling of the alpha subunit. The results suggest that nerve Na ,K-ATPase is tonically phosphorylated by PKC in a Ca2+-dependent manne r and that PKA modulates the phosphorylation process. In nerves of dia betic rats, PIMA increased P-32 labeling of the alpha subunit. In cont rast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significan t in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such ne rves. In conclusion, the altered effect of PK activators on Na,K-ATPas e phosphorylation state is consistent with the view that a defect in P KC activation exists in diabetic nerves.