TYROSINE-HYDROXYLASE REGULATION IN RAT STRIATAL AND OLFACTORY TUBERCLE SLICES .1. ACTIVATION INDUCED BY EXPOSURE TO A CALCIUM-FREE AND HIGH-MAGNESIUM MEDIUM

Slices from rat corpus striatum and olfactory tubercle were incubated for 15 min at 37° in Krebs-Ringer phosphate (KRP) medium or Ca2+-free KRP medium both in the presence and absence of high Mg2+ (12 mM). Tyrosine hydroxylase activity and kinetic parameters were determined in the 20,000 g supernatant fraction prepared from slices. The omission of Ca2+ 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 Mg2+ (12 mM) in the Ca2+-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 Ca2+-free, high-Mg2+ KRP medium was maximal when assayed at pH 7.0. The Mg2+-induced activation of tyrosine hydroxylase was antagonized by increasing the Ca2+ concentration (3.75 to 15.0 mM) in the medium in which the slices were incubated. The direct addition of Mg2+ (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 Ca2+ (10 mM) to the homogenates also resulted in an increase in enzyme activity but this effect was additive to that produced by Mg2+ (10 mM). Incubation of striatal slices in a Ca2+-free, high-Mg2+ 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-MPH4) was decreased significantly from 0.85 to 0.40 mM, with no significant change in the Vmax. However, the Ki 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, Mg2+ (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 Mg2+, ATP and cAMP remained unchanged in homogenates prepared from slices previously incubated in a Ca2+-free, high-Mg2+ 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 Ca2+ 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 Ca2+-free and high-Mg2+ 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 Ca2+ into the nerve terminal. © 1979.