Activation of tyrosine hydroxylase in rat striatal slices by K+-depolarization— Effect of ethanol

Abstract

Slices from rat corpus striatuum were incubated for 10 min at 37° in freshlyy oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K+ (55mM) media both in the presence and absence of ethanol (0.2 to 0.8%, w/v). Thereafter, the slices were homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 105,000 g supernatant fraction. The presence of K+ (55 mM) in the incubation media increased about 3-fold the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, when compared to that found in striatal slices incubated in normal KRP media. Incubation of striatal slices in a KRP-high K+ media also produced changes in the kinetic properties of tyrosine hydroxylase. The Km of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH4) was decreased from 0.82 to 0.09 mM and the Ki of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2 to 0.8%, w/v) added directly to the KRP-high K+ media markedly blocked the K+-induced activation of tyrosine hydroxylase as well as the kinetic alterations in the enzyme observed after K+-depolarization of the striatal slices. In contrast, the presence of ethanol did not modify the activity and kinetic characteristics of tyrosine hydroxylase isolated from slices incubated in normal KRP media. The results reported in this work suggest that the increase in DA synthesis observed in striatal slices after K+-depolarization might be mediated in part via an allosteric activation of tyrosine hydroxylase. This activation appears to be mediated by an increase in the affinity of the enzyme for the pterin cofactor and a decreased affinity for the end-product inhibitor DA. Also, the blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K+-depolarization seems to offer a likely explanation for the inhibitory effect of ethanol on K+-induced increase in DA synthesis reported recently by Gysling et al. (Biochem. Pharmac.25, 157 (1976)).