Tryptophan hydroxylase activity in brain slices.

Numerous experimental data have shown that the rate of 5-hydroxytryptamine (5-HT) synthesis is enhanced by neuronal activity in mammalian central 5-HT neurons (Hamon and Glowinski, 1974; Knapp et al., 1975; Neckers et al., 1977; Boadle-Biber et al., 1983). The change of 5-HT synthesis may be due to rapid and reversible modifications of the activity of tryptophan hydroxylase (EC 1.14.16.4), which is the rate-limiting enzyme in the formation of 5-HT (Lovenberg et al., 1967; Ichiyama et al., 1970; Friedman et al., 1972). According to the recent in vitro studies, factors which affect the activity of this enzyme may control 5-HT synthesis in vivo. Tryptophan hydroxylase system in vivo is composed of tryptophan hydroxylase itself, the tetrahydrobiopterin cofactor, and dihydropteridine reductase which regenerates the enzymatically formed quinonoid dihydropterin to tetrahydropterin (Friedman et al., 1972). In this system the cofactor and substrates, tryptophan and molecular oxygen, are thought to be in subsaturating conditions (FernStrom and Wurtman. 1971; Carlsson, 1974). The regulatory mechanism of tryptophan hydroxylase, however, has been explored in vitro by using the enzyme extract from animals under various conditions and in the presence of sufficient amounts of a tetrahydropterin cofactor and artificial tetrahydropterin-regenerating system. A direct comparison has shown that there appears to be nearly a 350-fold difference between in vivo and in vitro activity of tryptophan hydroxylase in rat raphe nuclei (Meek and Lofstrandh, 1976), suggesting the experiments using cell-free assay system are not physiological and not suitable for the studies of physiological regulatory mechanisms of tryptophan hydroxylase. In order to study the physiological regulation of tryptophan hydroxylase, it would be necessary to determine the rate of conversion of tryptophan to 5-hydroxytryptophan (5-HTP) in a relatively intact tissue preparation such as tissue slices, in which all of the components of the enzyme system may be present at physiological levels. The usefulness of such slice systems for studying the regulatory mechanism of pteridine-requiring monooxygenases was proved on phenylalanine hydroxylase in rat liver slices (Milstien and Kaufman, 1975) and on tyrosine hydroxylase in rat adrenal slices (Togari et al., 1982) and in rat brain slices (Hirata et al., 1983). Using such a slice system of tryptophan hydroxylase, the effects of various pharmacological and physiological interventions on the hydroxylating system could be assessed.