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.     

Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylase in vitro.

Using low concentrations of substrates and cofactors, a comparison was made of the relative rates by which aminotransferases catalysed transaminations between aromatic amino acids and aromatic or aliphatic keto acids. Tryptophan aminotransferase in homogenates of rat midbrain and liver transaminated phenylpyruvate at a rate 70 to 150-fold greater than the rate with α-ketoglutarate at low concentrations of substrates. Phenylalanine aminotransferase in liver and midbrain also was more active with aromatic keto acids than with aliphatic keto acids. However, tyrosine aminotransferase in dialysed homogenates of midbrain transaminated α-ketoglutarate and phenylpyruvate at approximately equal rates. Fresh homogenates of midbrain contained an inhibitor which markedly decreased tyrosine aminotransferase activity with α-ketoglutarate but not with phenylpyruvate. Tyrosine aminotransferase in homogenates of rat liver transaminated α-ketoglutarate and phenylpyruvate at equal rates below 10 μM keto acid, but above 10 μM, transamination of α-ketoglutarate was favoured. With homogenates of liver, transamination of α-ketoglutarate, but not phenylpyruvate, by tyrosine was increased 650% by exogenous pyridoxal phosphate. Since tryptophan aminotransferase in the brain may compete with tryptophan hydroxylase for available tryptophan, a comparison was made of the relative activities of tryptophan hydroxylase and tryptophan aminotransferase. At concentrations above 7.5 μM phenylpyruvate, transamination was 8 to 17-fold greater than the rate of hydroxylation of 50 μM tryptophan.     

EARLY RESPONSE OF RAT BRAIN TRYPTOPHAN HYDROXYLASE ACTIVITY TO CYCLOHEXIMIDE, PUROMYCIN AND CORTICOSTERONE

Abstract— The activity of tryptophan hydroxylase was measured in whole homogenates of midbrain and forebrain areas of the rat brain. A significant elevation of tryptophan hydroxylase in midbrain and forebrain was found within 1 h after injection of corticosterone hemisuccinate Na salt (10mg/kg) into normal rats. A further elevation of tryptophan hydroxylase at 4 h after injection occurred only in the midbrain region. A rapid alteration of tryptophan hydroxylase was also observed following intracistemal injection of a protein synthesis inhibitor, cydoheximide. A significant depression of 50% of normal levels occurred both in midbrain and forebrain regions within 1 h. However. 4 h after injection only the midbrain tryptophan hydroxylase level was depressed, and this depression was 16% of normal levels. This temporal and spatial pattern following cydoheximide injection was not the result of changes in the ability of cydoheximide to inhibit in vivo protein synthesis since [³H]valine incorporation into protein was shown to be equally depressed at both 1 and 5 h in both the midbrain and forebrain. Puromycin blocked [³H]valine incorporation into proteins in the midbrain and forebrain. but only caused a depression of 16% of tryptophan hydroxylase in the midbrain at 4 h. The aminonucleoside derivative of puromycin has no effect on protein synthesis or on tryptophan hydroxylase. Cydoheximide had no effect on tryptophan hydroxylase in vitro. The data suggest that cydoheximide and corticosterone produce an early (1 h) effect on tryptophan hydroxylase unrelated to de novo protein synthesis in regions known to contain perikaryon (midbrain) and axon terminals (forebrain) of 5-HT-containing neurons. The later (4h) effects of these two compounds and puromycin on tryptophan hydroxylase in the perikaryon (midbrain) region of 5-HT-containing neurons probably result from alteration in de novo protein synthesis. The half time of tryptophan hydroxylase in midbrain region is calculated to be 12 h.     

GLUCOCORTICOIDS AS A REGULATORY FACTOR FOR BRAIN TRYPTOPHAN HYDROXYLASE

—The normal developmental rise of tryptophan hydroxylase levels in neonatal rat brain was blocked by adrenalectomy. Similarly, adrenalectomy prevented the rescrpine-induced elevation of tryptophan hydroxylase activity in brain stem of adult mice. In both cases, the effects of adrenalectomy could be reversed by replacement injections of corticosterone. Repeated injections of corticosterone (5 mg/kg daily) in fact induced a rise of brain tryptophan hydroxylase levels in neonatal brain. However, neither adrenalectomy nor repeated injections of large doses of the hormone (20 mg/kg, daily) was found to be effective in affecting the normal enzyme levels in adult brain. Apparent K_m of the enzyme for substrate was unchanged by corticosterone in vivo or in vitro. These results indicate that glucocorticoids have a significant role in the regulation of brain tryptophan hydroxylase: possibly as an inducing signal during neonatal development and as a permissive factor at adult age.     

Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland

The characterization and cellular localization of tryptophan hydroxylase mRNA in the human brainstem and pineal gland were investigated by using northern blot analysis and in situ hybridization histochemistry. Northern analysis of human pineal gland revealed the presence of two mRNA species that were absent in RNA isolated from human raphe. In situ hybridization experiments revealed very dense hybridization signal corresponding to tryptophan hydroxylase mRNA in cells throughout the pineal gland. In contrast, tryptophan hydroxylase mRNA was heterogeneously distributed in neurons in the dorsal and median raphe nuclei. Within the dorsal raphe, the ventrolateral and interfascicular subnuclei contained the greatest number of tryptophan hydroxylase mRNA-positive neurons. Also, the cellular concentration of tryptophan hydroxylase mRNA varied widely within the dorsal and median raphe. Comparison of the cellular concentration of tryptophan hydroxylase mRNA between the pineal gland and the raphe nuclei revealed an 11- and 46-fold greater average grain density of tryptophan hydroxylase mRNA positive cells in the pineal gland compared with the dorsal and median raphe, respectively. These findings are the first to demonstrate the cellular localization of tryptophan hydroxylase mRNA in the human brain and pineal gland as well as heterogeneity in the cellular concentration within and between these tissues.     

THE ROLE OF INTRANEURONAL 5-HT AND OF TRYPTOPHAN HYDROXYLASE ACTIVATION IN THE CONTROL OF 5-HT SYNTHESIS IN RAT BRAIN SLICES INCUBATED IN K+-ENRICHED MEDIUM

Abstract— The incubation of brain stem slices from adult rats in a K⁺-enriched medium containing a 5-HT uptake inhibitor (fluoxetine) significantly increased their capacity to synthesize 5-HT from tryptophan. The K+-induced stimulation of 5-HT synthesis was at least partly dependent on the depletion of the indoleamine in tissues since: (1) a good correlation was found between the respective changes in 5-HT release and synthesis evoked by high K⁺ concentrations in the presence of various 5-HT uptake inhibitors; (2) the modifications in endogenous 5-HT levels produced by in vim treatments with drugs (reserpine, pargyline) or by incubating slices with 5-HT altered the stimulating effect of high K⁺ concentrations and fluoxetine on 5-HT synthesis; (3) the replacement of Ca²⁺ by Co²⁺ (4 mM) or EGTA (0.1 mM) in the incubating medium completely prevented the increased 5-HT release and synthesis evoked by high K⁺ concentrations and fluoxetine. The extraction of tryptophan hydroxylase from incubated tissues revealed that the increased 5-HT synthesis occurring in K⁺-enriched medium was associated with an activation of this enzyme. Kinetic analyses indicated that this activation resulted from an increase in the V_max of tryptophan hydroxylase, its apparent affinities for both tryptophan and 6-MPH₄ being not significantly affected. In contrast to the tryptophan hydroxylase from tissues incubated in normal physiological medium, the activated enzyme from tissues depolarized by K⁺ was hardly stimulated by Ca²⁺-mediated phosphorylating conditions. This led to the proposition of a hypothetical model by which the Ca²⁺ influx produced by the neuronal depolarization would trigger the activity of a Ca²⁺-dependent protein kinase capable of activating tryptophan hydroxylase. Although this sequence is still largely speculative it must be emphasized that, as expected from such a model, the regional differences in the K⁺-evoked activation of tryptophan hydroxylase in slices (cerebral cortex > brain stem > spinal cord) were parallel to those of the Ca²⁺-dependent protein phosphorylation (r= 0.92) and those of the activating effect of phosphorylating conditions on soluble tryptophan hydroxylase (r= 0.96).     

Interaction of genetic and environmental components in the determination of tryptophan hydroxylase activity in the mouse brain in stress

The interaction between genetic and environmental components of phenotype variety in the response to cold and emotional stress of the brain serotonin system was studied in 11 inbred strains of mice. It was shown that the variety in the degree of tryptophan hydroxylase activity's changes under stress are mainly due to the genotypic differences. The presence of different genotypic systems controlling the activity of tryptophan hydroxylase under basal conditions and under stress was revealed. Differences in hereditary mechanisms determining the tryptophan hydroxylase reactions to different kinds of stress were noted.     

Effect of single and repeated methamphetamine treatment on neurotransmitter release in substantia nigra and neostriatum of the rat

There are tissue specific discrepancies in expression of tryptophan hydroxylase (TPH) between the pineal gland and brainstem. TPH mRNA levels in the pineal are much higher than in the brainstem, however, the two tissues contain comparable protein levels. This discrepancy could result from different translation efficiency of two of the TPH mRNA isoforms. Using PCR-based methods, we analyzed the relative expression, in pineal and brainstem, of two TPH mRNA isoforms differing in the length of their untranslated region (5'UTR). The levels of the TPHalpha were found to be 960-fold more abundant than the 51-nucleotide longer TPHbeta, in the pineal. TPHbeta was also detected for the first time in the brainstem, where TPHbeta/TPHalpha was about five-fold higher than in the pineal. To study the role of the different 5'UTRs, each was cloned in-frame upstream of luciferase, and transfected into PC12 cells. Both 5'UTRs enhanced luciferase activity, with TPHbeta 5'UTR being more effective than TPHalpha 5'UTR, indicating selective regulation of translation efficiency. We also examined whether physiological manipulations alter the distribution of the TPH mRNA isoforms. Repeated stress had no effect in pineal, but led to a marked preferential induction of TPHbeta in brainstem. Modulation of TPH gene expression in serotonergic neurons could result from selective and tissue specific regulation of its mRNA isoforms.     

Phosphorylation and Activation of Tryptophan Hydroxylase by Exogenous Protein Kinase A

Abstract: The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in V_max without alterations in the K_m for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.     

Evidence for the role of brain biogenic amines in depressed motor activity seen in chemically thyroidectomized rats.

Abstract— The effects of exposure to an antithyroid drug, methimazole, on brain tyrosine hydroxylase and tryptophan hydroxylase activity, as well as the levels of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid have been investigated in maturing brain. Daily treatment of neonatal rats with methimazole for 30 days induced chemical thyroidectomy as evidenced by significant impairment of body and brain growth. The activities or brain tyrosine hydroxylase and tryptophan hydroxylase and the levels of norepinephrine, dopamine and 5-hydroxytryptamine were markedly altered in a dose- and time-dependent manner in methimazole-treated rats. Conversely, the concentration of brain 5-hydroxyindoleacetic acid was elevated (46%) by methimazole administration. Treatment with the antithyroid drug failed to exert any significant effect on the endogenous levels of brain tryptophan, as well as on the activity of the deaminating enzyme, monoamine oxidase. Administration of triiodothyronine (25 or 100 μg/100 g) to hypothyroid rats for 30 days did not produce any appreciable effect upon the neurochemical parameters related to either norepinephrine or 5-hydroxytryptamine mctabolism. However, increasing the dose of triiodothyronine to 250 μg/100 g significantly elevated the levels of norepinephrine and 5-hydroxytryplamine as well as the activities of the two synthesizing enzymes, tyrosine hydroxylase and tryptophan hydroxylase. Brain 5-hydroxyindoleacetic acid levels were restored to normal values in thyroid hormone-deficient rats treated with this higher dose of triiodothyronine. Evidencc also was obtained to show that chemical thyroidectomy suppressed the spontancous locomotor activity in neonatal rats; the changes being apparent at 15 days of age. Our data support the view that thyroid hormone in neonatal life displays an important regulatory effect on the metabolism of norepinephrine, dopamine and 5-hydroxytryptamine. Since certain amines have been known to be implicated as the neurochemical substrates for behavioural arousal, it is conceivable that the observed hypoactivity in methimazolc-treated rats may, at least in part, be related to impaired maturation of norepinephrine and dopamine-synthesizing systems in brains of cretinous rats.     

Modulation of Tryptophan Hydroxylase Activity by Phospholipids: Stimulation Followed by Inactivation

The activity of tryptophan hydroxylase from the rat brainstem was stimulated rapidly three- to fourfold by the addition of phosphatidylinositol or phosphatidylserine. However, the activity of the enzyme once stimulated was decreased gradually by subsequent incubation with the phospholipid at 37 degrees C, reaching a level below the original activity after 1 h of incubation. The presence of ferrous ion almost perfectly protected the enzyme against this phospholipid inactivation. The activity of the enzyme inactivated by incubation with the phospholipid was not only restored, but also increased further by incubation at 37 degrees C with ferrous ion and dithiothreitol. Gel filtration analysis revealed that the enzyme stimulated by phosphatidylinositol was eluted in a void volume together with the phospholipid vesicles, but the enzyme inactivated by incubation with phosphatidylinositol was eluted at a later region apart from the vesicles. These results, taken together, suggest the possible involvement of cellular membranes in the regulation of tryptophan hydroxylase in the central nervous system.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

Tryptophan hydroxylase system in brain tissue slices assayed by high-performance liquid chromatography

A new method was developed to study the unsupplemented tryptophan hydroxylase system in brain tissue slices from the raphe nuclei of the rat by high-performance liquid chromatography (HPLC) with fluorescence detection. Tryptophan hydroxylase activity was measured by determining 5-hydroxytryptophan (5-HTP) accumulation in raphe nuclei slices containing all of the enzyme system (the hydroxylase, tetrahydrobiopterin, and dihydropteridine reductase) in the presence of NSD-1055 (an inhibitor of aromatic l-amino acid decarboxylase). An optimum temperature was observed at 25°C and the reaction progressed linearly for 60 min. The hydroxylation of tryptophan was maximal by the addition of 0.2 mM tryptophan in the medium. A maximum 1.5-fold activation was shown at 0.2 mM 6-methyltetrahydropterin in the presence of 10 mM dithiothreitol. Dithiothreitol alone did not affect the activity. A 1.5-fold activation was observed when incubation was carried out under gas phase of 95% oxygen and 5% CO₂ instead of air. The activity was inhibited by 75% at 10^?4 M p-chlorophenylalanine. Both A-23187, a calcium ionophore, and dibutyryl cyclic AMP (DBc-AMP) stimulated the hydroxylation of tryptophan. The activation by A-23187 plus DBc-AMP was more than additive, suggesting the two activating mechanisms by Ca²⁺ and cyclic AMP may be operating synergistically.     

Relationship between alternate routes of tryptophan metabolism following administration of tryptophan peroxidase inducers or stressors

Abstract— Administration of glucocorticoids to rats increased the activity of hepatic tryptophan peroxidase (EC 1.11.1.4) and lowered brain serotonin. Pretreatment with glucose diminished both of these effects. Administration of allylisopropylacetamide to adrenalecto-mized rats increased both the activity of tryptophan peroxidase and the level of brain serotonin but had no effect on tryptophan hydroxylase (EC 1.99.1.4) activity in the brain stem. The activity of tryptophan peroxidase was increased by the acute stress of laparotomy and by the chronic stress of a 72-h fast. Neither stressor affected brain serotonin levels appreciably. These results argue against the proposal that the activity of tryptophan peroxidase activity directly affects synthesis of brain serotonin by diverting tryptophan from the biosynthesis of this monoamine.     

Effect of food restriction on serotonin metabolism in rat brain

The brain concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased in rats maintained on restricted volume of low-protein or normal-protein diet, whereas these two agents decreased in rats fed low-protein diet ad libitum. In these two food-restricted groups brain 5-HT and 5-HIAA concentrations were not correlated with brain tryptophan hydroxylase activity, but the concentrations correlated closely with cerebral tryptophan concentrations. The cerebral tryptophan concentration in the two food-restricted groups was not consistent with the total or free tryptophan concentration in plasma. In these restricted rats cerebral tryptophan concentration was elevated, and, unlike the plasma tryptophan, it showed no diurnal variation. These results suggested that tryptophan uptake into the brain from plasma was enhanced by limiting food volume intake. Tryptophan uptake was increased by glucagon injection without changing the plasma tryptophan level, but injection of hydrocortisone or insulin had little or no effect on tryptophan concentration in either the plasma or brain.d-Glucose injection elevated plasma tryptophan concentration but decreased brain tryptophan concentration.     

Calcium activation of brain tryptophan hydroxylase.

Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba⁺⁺, Mg⁺⁺, and Mn⁺⁺, have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca⁺⁺ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.     

Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α -methyl- p -tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.     

Knockout mouse points to second form of tryptophan hydroxylase

A second form of tryptophan hydroxylase (TPH) is expressed in the brain by the gene Tph2. The presence of the gene was discovered when Tph 1(-/-)mice were found to express normal amounts of serotonin in brain, but not in the periphery. Additionally, Tph1(-/-) mice showed no observed behavioral differences from wild-type littermates. Veenstra-Vanderweele and Cook discuss the ramifications of these findings and what they might mean for designing drugs that target the expression and activity of TPH in differing tissues.     

CHARACTERIZATION OF THE Ca2+-INDUCED PROTEOLYTIC ACTIVATION OF TRYPTOPHAN HYDROXYLASE FROM THE RAT BRAIN STEM

The incubation of the 35,000 g supernatant of a rat brain stem homogenate in the presence of 7.5 mM-CaC1₂ for 10 min at 25°C resulted in a more than 2-fold increase in its tryptophan hydroxylase activity. This activation was irreversible and involved a reduction in the molecular weight of the enzyme, from 220,000 to 160,000. The partially proteolysed tryptophan hydroxylase, in contrast to the native enzyme, could not be activated by trypsin, sodium dodecyl sulphate, phosphatidylserine or phosphorylating conditions; dithiothreitol and Fe²⁺ were the only compounds whose stimulating effect on the enzymatic activity was not prevented by the Ca²⁺ -induced proteolysis of tryptophan hydroxylase. These findings suggest that the mol. wt. 60,000 fragment removed by the Ca²⁺ dependent neutral proteinase plays a critical role in the regulatory properties of tryptophan hydroxylase.