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Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase
Author(s) -
Marley Philip D.,
Thomson Kerrie A.,
Bralow Roger A.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb14958.x
Subject(s) - protein kinase a , forskolin , chemistry , chromaffin cell , nicotine , phosphorylation , protein kinase c , biochemistry , endocrinology , medicine , biology , adrenal medulla , catecholamine , in vitro
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 14 C0 2 evolved following the hydroxylation and rapid decarboxylation of [ 14 C]‐tyrosine offered to the cells. 3 Nicotine (5 μm), forskolin (1 μm) 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 Ca 2+ . 4 TOH activation by all three drugs was blocked by H89 (3–20 4mUm), 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 μm), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 μ m ) or basal TOH activity. Neither sodium nitroprusside (0.3–10 μ m ) nor 8‐Br‐cyclic GMP (1 mM) increased TOH activity. 6 In digitonin‐permeabilized chromaffin cells, forskolin (3 μm), cyclic AMP (10 μ m ) and Ca 2+ (approx. 2 μ m free Ca 2+ ) each increased TOH activity. The response to all three drugs was blocked by H89 (10 μm), which also reduced basal TOH activity in the permeabilized cells. 7 Maximal activation of TOH by forskolin was achieved with 10 μm 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 μm) and nicotine (5 μm) 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.