Role of calmodulin in the activation of tryptophan hydroxylase

Tryptophan hydroxylase can be activated 2.0- to 2.5-fold in vitro by ATPa dn Mg2+. This apparent phosphorylation effect is not dependent on cyclic nucleotides but is dependent on the presence of calcium. The activation of tryptophan hydroxylase by ATP-Mg2+ reduces the apparent Km of the enzyme for its cofactor, 6-methyltetrahydropterin, from 0.21 to 0.09 mM. The addition of certain antipsychotic drugs known to bind to calmodulin in a phosphorylation reaction mixture prevents the activation to tryptophan hydroxylase by ATP-Mg2+ in the concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Preparation of calmodulin-free tryptophan hydroxylase by affinity chromatography on fluphenazine-Sepharose 4B yields an enzyme that is no longer activated by ATP-Mg2+, whereas the readdition of calmodulin to a calmodulin-free enzyme restores the responsiveness of tryptophan hydroxylase to ATP-Mg2+. This restoration is dependent on Ca2+. Taken together, these results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.     

Calcium-dependent activation of tryptophan hydroxylase by ATP and magnesium

Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg⁺⁺ in the presence of subsaturating concentrations of the cofactor 6-methyltetrahydropterin (6MPH₄). The activation of tryptophan hydroxylase under these conditions results from a reduction in the apparent K_m for 6MPH₄ from 0.21 mM to 0.09 mM. The activation requires Mg⁺⁺ and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg⁺⁺ on enzyme activity was enhanced by μM concentrations of Ca⁺⁺ and totally blocked by EGTA. These data suggest that tryptophan hydroxylase can be activated by a cyclic nucleotide independent protein kinase which requires low calcium concentrations for the expression of its activity.     

Partial purification of rabbit hind brain tryptophan hydroxylase by affinity chromatography.

Chromatography of crude homogenates of rabbit hind brains on ε-amino caproyl-D-tryptophan methyl ester-agarose gels provide enzyme fractions with specific activity 7–10 times higher than the starting material. The activity was found to be associated with two distinct components. While nearly forty-fold increase in specific activity can be achieved by purification of the homogenate on calcium phosphate gels prior to affinity chromatography, only a single active component was noted in such prepurified extracts.     

Characteristics of the activation by dithiothreitol and Fe2+ of tryptophan hydroxylase from the rat brain

The preincubation of tryptophan hydroxylase extracted from various areas of the central nervous system of the rat with 30 mM dithiothreitol and 50 M ferrous ammonium sulfate under nitrogen atmosphere resulted in a persistent increase of its activity. Studies on the enzyme characteristics indicated that this activation was associated with a doubling in itsV _max and a shift (from 7.6 to 7.2) of the optimal pH for its activity. In contrast, the molecular weight and the apparent affinities of tryptophan hydroxylase for its pterin cofactor and for tryptophan were not significantly altered by the preincubation with dithiothreitol and ferrous ammonium sulfate. Since this treatment did not prevent the stimulatory effects of various compounds (phosphatidylserine, ATP and Mg²⁺, Ca²⁺) on tryptophan hydroxylase activity, this might be a good procedure to activate this enzyme with only minor changes in its regulatory properties.     

Genetic control of tryptophan hydroxylase activity in the mouse brain

Interstrain differences in tryptophan hydroxylase activity have been studied in the brain stem of 12 inbred strains of mice. The Mendelian analysis has been performed using BALB/c and C57BL/6J mice. The activity of tryptophan hydroxylase was shown to be controlled by a single gene having two alleles with additive effect. It was suggested that this gene controls either the structure of enzyme's molecule, or its amount in a nervous tissue.     

Chronic amphetamine administration decreases brain tryptophan hydroxylase activity in cats

Chronic administration of d-amphetamine sulfate (7.5 mg/kg, i.p. every 12 hrs. for 6 days) to cats produced significant decreases in the Vmax of brain-stem and forebrain tryptophan hydroxylase when measured 1 day (?34 and ?46%) and 10 days (?17 and ?30%) after the final amphetamine injection. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) levels were decreased by a similar magnitude. A single injection of amphetamine (7.5 mg/kg) produced no significant changes in tryptophan hydroxylase activity, serotonin, or 5HIAA when measured 1 day after the injection. Neither acute nor chronic amphetamine treatment produced any significant changes in the Km of tryptophan hydroxylase for either tryptophan or the natural co-factor, tetrahydrobiopterin. These data suggest that chronic amphetamine treatment decreases central serotonergic neurotransmission by an action on the rate-limiting enzyme in serotonin biosynthesis.     

Depletion of tryptophan hydroxylase by 5,6-dihydroxytryptamine in rat brain. Time course and regional differences

—Wistar rats were injected intraventricularly with 75 μg 5,6-dihydroxytryptamine. Tryptophan hydroxylase was assayed in seven regions of the brain as well as spinal cord at intervals following injection. Spinal cord was depleted to 1 per cent of control by 12 days. Tectum was depleted to 32 per cent of control by 9 days. The time course of depletion in most regions was biphasic, with an early reduction of activity 1 h after injection with continued reduction of activity 1-2 days following injection, and a recovery of activity at 4-6 days. The activity again drops at 9-12 days, and this reduction of activity persists to varying degrees to 60 days, with slight recovery at this time in certain regions.     

Involvement of activator protein in the activation of tryptophan hydroxylase by cAMP-dependent protein kinase

The tryptophan hydroxylase activity of the crude extract from rat brain stem was stimulated approximately 2-fold by incubation with cAMP analogues under protein phosphorylating conditions. The cAMP-dependent activation process of the enzyme needed not only cAMP-dependent protein kinase but also activator protein. The kinetic properties of the enzyme activated by cAMP-dependent protein kinase were very similar to those of the enzyme activated by calmodulin-dependent protein kinase II.     

Dietary isoleucine inhibition of liver tryptophan hydroxylase

Dietary isoleucine administered to weanling rats for 25–32 days reduces 5-hydroxylation of tryptophan by liver homogenates. This inhibition occurs without alteration of central amines and appears unrelated to dietary stress.     

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.