Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: Phosphoprotein targets for nitric oxide action

Production of nitric oxide and the activation of protein kinases are requir ed for long-term potentiation of synaptic transmission at the giant synapse s in chicken ciliary ganglion. In the present study, we investigated the ab ility of nitric oxide to regulate the phosphorylation of endogenous protein s under conditions that induced long-term potentiation in intact ciliary ga nglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that t he nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of P-32(i)-labelled intact ciliary ganglion showed that the sodium nitropr usside (300 mu M) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts wh ich we have called nitric oxide-responsive phosphoproteins. A similar stimu lation of phosphorylation was achieved by 8-bromocyclic AMP (100 mu M), whi ch also induced long-term potentiation, but not by phorbol dibutyrate (2 mu M) that does not induce long-term potentiation in ciliary ganglion. When s ubcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-P-32]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-de pendent protein kinase substrates that gave rise to phosphopeptides co-migr ating with P50a, P50b and P41 from P-32(i)-labelled intact ciliary ganglion . P50a and P41 were derived from soluble proteins while P50b was derived fr om a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyc lic AMP-dependent protein kinase, but not for calcium and phospholipid-depe ndent protein kinase in vitro, suggesting that nitric oxide-responsive phos phoproteins are convergence points in information processing in vivo and th eir phosphorylation might represent an important mechanism in nitric oxide- mediated synaptic plasticity in ciliary ganglion. (C) 1999 IBRO. Published by Elsevier Science Ltd.