TYROSINE-HYDROXYLASE PHOSPHORYLATION IN BOVINE ADRENAL CHROMAFFIN CELLS - THE ROLE OF INTRACELLULAR CA2-1 RECEPTOR-STIMULATED PHOSPHORYLATION OF SER(8), SER(19), SER(31), AND SER(40)( IN THE HISTAMINE H)

Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histamine rgic H-1 receptors on cultured bovine adrenal chromaffin cells stimula ted a rapid increase in TOH phosphorylation (within 5 s) that was sust ained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellula r Ca2+. In contrast, the H-1-mediated response was abolished by preloa ding the cells with BAPTA acetoxymethyl ester (50 mu M) and significan tly reduced by prior exposure to caffeine (10 mM for 10 min) to deplet e intracellular Ca2+. Trypticphosphopeptide analysis by HPLC revealed that the H-1 response in the presence or absence of extracellular Ca2 resulted in a major increase in the phosphorylation of Ser(19) with s maller increases in that of Ser(40) and Ser(31). In contrast, although a brief stimulation with nicotine (30 mu M for 60 s) also resulted in a major increase in Ser(19) phosphorylation, this response was abolis hed in the absence of extracellular Ca2+. These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H-1-mediated TOH phosphorylation and may thus have a potentially impo rtant role in regulating catecholamine synthesis.