Studies on the regulatory mechanism of the tyrosine hydroxylase system in adrenal slices by using high-performance liquid chromatography with electrochemical detection

Abstract

A new method was developed to study the tyrosine hydroxylase (TH) system in rat adrenal slices by high-performance liquid chromatography (HPLC) with electrochemical detection (ED). TH activity was measured by determining DOPA, formed in adrenal slices containing all of the components of the TH system in the presence of an inhibitor of aromatic l-amino acid decarboxylase (NSD-1055), using a new and highly sensitive HPLC-ED method. The properties of the TH system in the slices were also examined. High K + (52 mM) stimulated the formation of DOPA in the slices; this stimulation was not observed in Ca 2+-free medium. Furthermore, the addition of ethyleneglycol bis ( β-aminoethylether)- N, N′-tetraacetic acid (EGTA) to a Ca 2+-free medium not only abolished the stimulation by high K + but also significantly inhibited the conversion of l-tyrosine to DOPA. This suggests that intracellular Ca 2+ may regulate TH activity in the adrenal medulla. The enzyme in homogenates of normal adrenal slices had two different K m values for the tetrahydropterin cofactor. Alteration of enzyme kinetics using homogenates of adrenal slices that had been treated in various ways suggests that changes in the proportions of the two forms of TH produced by intracellular Ca 2+ may regulate the activity of this enzyme in the adrenal medulla.