PROTEIN-KINASE-A AND NICOTINIC ACTIVATION OF BOVINE ADRENAL TYROSINE-HYDROXYLASE

1 Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated. 2 TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring (CO2)-C-14 evolved following the hydroxylation and rapid decarboxylation of [C-14]-tyrosine offered to the cells. 3 Nicotine (5 mu M), forskolin (1 mu M) and 8-bromo-cyclic AMP (8-Br-cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+. 4 TOH activation by ah three drugs was blocked by H89 (3-20 mu M), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3':5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8-Br-cyclic AMP. Both H89 and Rp-cAMPS inhibited basal TOH activity by 50-80%. 5 A structural analogue of H89, H85 (3-20 mu M), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 mu M) or basal TOH activity. Neither sodium nitroprusside (0.3-10 mu M) nor 8-Br-cyclic GMP (1 mM) increased TOH activity. 6 In digitonin-permeabilized chromaffin cells, forskolin (3 mu M), cyclic AMP (10 mu M) and Ca2+ (approx. 2 mu M free Ca2+ each increased TOH activity. The response to all three drugs was blocked by H89 (10 mu M)) which also reduced basal TOH activity in the permeabilized cells. 7 Maximal activation of TOH by forskolin was achieved with 10 mu M forskolin. This concentration was less than the EC(50) for forskolin-induced cyclic AMP accumulation in these cells. The activations of TOH by forskolin (10 mu M) and nicotine (5 mu M) were additive. 8 The results indicate that both basal TOH activity and nicotinic activation of TOH in bovine chromaffin cells require protein kinase A activity. However, it is unlikely that nicotinic activation of TOH is directly mediated by an activation of protein kinase A in response to elevated cyclic AMP levels. It is possible that protein kinase A plays a permissive role in allowing nicotinic cholinoceptors to activate TOH by another signalling pathway.