Tyrosine hydroxylase regulation in rat striatal and olfactory tubercle slices—I: Activation induced by exposure to a calcium-free and high-magnesium medium

Abstract

Slices from rat corpus striatum and olfactory tubercle were incubated for 15 min at 37° in Krebs-Ringer phosphate (KRP) medium or Ca 2+-free KRP medium both in the presence and absence of high Mg 2+ (12 mM). Tyrosine hydroxylase activity and kinetic parameters were determined in the 20,000 g supernatant fraction prepared from slices. The omission of Ca 2+ from the incubation medium resulted in a moderate increase in the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, and a significant increase in the activity of tyrosine hydroxylase isolated from olfactory tubercle slices. The presence of Mg 2+ (12 mM) in the Ca 2+-free KRP medium produced a further increase in the activity of the enzyme isolated both from striatal and olfactory tubercle slices. The per cent of stimulation of enzyme activity induced by incubating the slices in a Ca 2+-free, high-Mg 2+ KRP medium was maximal when assayed at pH 7.0. The Mg 2+-induced activation of tyrosine hydroxylase was antagonized by increasing the Ca 2+ concentration (3.75 to 15.0 mM) in the medium in which the slices were incubated. The direct addition of Mg 2+ (5–20 mM) to striatal and olfactory tubercle homogenates also resulted in an increase in the activity of tyrosine hydroxylase. The addition of high concentrations of Ca 2+ (10 mM) to the homogenates also resulted in an increase in enzyme activity but this effect was additive to that produced by Mg 2+ (10 mM). Incubation of striatal slices in a Ca 2+-free, high-Mg 2+ KRP medium produced changes in the kinetic properties of tyrosine hydroxylase. The apparent m of the enzyme for 6-methyl-5,6,7,8-tetrahydropterine HCl (6-MPH 4) was decreased significantly from 0.85 to 0.40 mM, with no significant change in the V max. However, the K i of the enzyme for dopamine (DA) was unchanged. Magnesium ions (12 mM) added to KRP-high K + (55 mM) medium blocked the activation of tyrosine hydroxylase induced by K +-depolarization of striatal slices. However, Mg 2+ (12 mM) addition to the incubation medium did not block but actually further increased the tyrosine hydroxylase activation that results after incubating striatal slices in a KRP medium enriched with dibutyryl cAMP (1 mM). Moreover, the stimulating effect on tyrosine hydroxylase observed when assays were conducted in the presence of Mg 2+, ATP and cAMP remained unchanged in homogenates prepared from slices previously incubated in a Ca 2+-free, high-Mg 2+ KRP medium. The results reported in this work clearly demonstrate that a kinetic activation of tyrosine hydroxylase in dopaminergic nerve terminals can occur under conditions of diminished extracellular Ca 2+ and blockade of transmitter release. Cyclic AMP does not seem to play a role in the tyrosine hydroxylase activation induced by incubation of slices in Ca 2+-free and high-Mg 2+ medium. The results support the hypothesis that the increase in DA biosynthesis observed in dopaminergic neurons after inhibition of impulse flow may result primarily as a consequence of a diminished entrance of Ca 2+ into the nerve terminal.