Activation of tryptophan hydroxylase from slices of rat brain stem incubated with agents which promote calcium uptake or intraneuronal release

Abstract

Incubation of slices of rat brain stem under conditions that promote calcium accumulation by nerve tissue (Na-free medium, ouabain. A23187) or intraneuronal release of calcium from mitochondrial stores (metabolic inhibitors, cyanide, azide, rotenone, guanidine and dicoumarol) has been found to result in an increase in the activity of tryptophan hydroxylase, prepared from the slice preparations in a low speed supernatant fraction and assayed in the presence of 200 μM L-tryptophan and 50 μM DL-6-methyl-5,6,7,8-tetrahydropterin (6-MPH 4). The increase in enzyme activity following treatment of the slices with Na +-free medium, ouabain, ionophore A23187, guanidine, cyanide or azide was reflected in altered kinetic properties of the enzyme, namely a decrease in the K m of the enzyme for both substrate and artificial reduced pterin cofactor. In addition, a modest increase in V max was observed, but was not always statistically significant. The alterations in kinetic properties obtained following the different treatments to the slice preparations were similar to those obtained with enzyme prepared from brain stem slices depolarized in a potassium-enriched incubation medium. This depolarization-induced activation of tryptophan hydroxylase is a calcium-dependent phenomenon. In agreement with this, no increase in enzyme activity was observed when calcium ions were omitted from the Na +-free medium and media containing ouabain or A23187. Removal of external calcium did not abolish the increase in enzyme activity obtained with metabolic inhibitors, presumably because these substances release calcium from mitochondria. The data are consistent with the view that a rise in free intraneuronal calcium triggers certain biochemical events which activate tryptophan hydroxylase.