Dihydroxyphenylalanine production in rat brain striatal synaptosomes: stimulation by a calcium chelator

By inhibiting aromatic L-amino-acid decarboxylase (EC 4.1.1.28) in rat brain striatal synaptosomes, we have been able to measure dihydroxyphenylalanine production via high performance liquid chromatography-electrochemical oxidation. This dihydroxyphenylalanine assay was compared to a standard radioisotopic assay of catecholamine synthesis (14CO2 production from L-[1-14C]tyrosine) in terms of (1) units of activity, (2) effects of known inhibitory and stimulatory agents, and (3) effects of the calcium chelator, EGTA. The units of activity in the dihydroxyphenylalanine assay were 40% greater than the units in the radioisotopic assay, indicating a mixing of labeled and endogenous tyrosine pools before conversion of the labeled tyrosine to labeled dihydroxyphenylalanine. The inhibition of synthesis produced by either 3-iodotyrosine or 3,4-dihydroxyphenylethylamine was similar in the two assays, as was the stimulation produced by 8-bromo cyclic AMP. The calcium chelator, EGTA, also activated synthesis to the same extent in the two assays, indicating that the increase observed in the radioisotopic assay is not an artifact of altered precursor specific activity. These data thus indicate the general utility of the synaptosomal dihydroxyphenylalanine synthesis assay, and also demonstrate the specific advantages of this assay for analyzing the effects of agents such as EGTA, which can alter tissue catecholamine precursor levels.