Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

Effects of thyrotropin releasing hormone and its analogue, DN 1417, on biopterin concentration and tryptophan hydroxylation in rat raphe slices

The effects of subchronic administration of thyrotropin releasing hormone (TRH) and its analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN 1417), on serotonin biosynthesis in situ were investigated in tissue slices of the midbrain raphe of rats. TRH or DN 1417 (10 mg/kg per day intraperitoneally) were administered to male Wistar rats for ten days. At twenty four hr after the last injection, tissue slices of the midbrain raphe were prepared and the rate of serotonin biosynthesis was estimated by measuring formation of 5-hydroxytryptophan (5-HTP) from tryptophan during inhibition of aromatic L-amino acid decarboxylase using high-performance liquid chromatography with fluorescence detection. Total biopterin content was determined by a specific radioimmunoassay. 5-HTP formation was decreased 22% and 29%, and total biopterin content 69% and 72%, in TRH- and DN 1417-treated rats, respectively. However, tryptophan concentration in raphe slices did not change. In contrast, the Vmax of tryptophan hydroxylase in the homogenate of the raphe nucleus in the presence of a saturating concentration of (6R)-L-erythro-tetrahydrobiopterin, the naturally occurring pterin cofactor, was significantly increased after repeated administration of TRH or DN 1417. These results indicate that reduction of in situ serotonin biosynthesis in tissue slices from the rats treated with TRH or DN 1417 subchronically contray to the increase in in vitro tryptophan hydroxylase may result from the decrease of the biopterin cofactor, and that changes in concentrations of the biopterin cofactor may play a regulatory role in serotonin biosynthesis in vivo under certain conditions.     

A monoclonal antibody against tyrosine hydroxylase: application in light and electron microscopy

The catecholaminergic neurons of the nervous system have been studied histochemically with fluorescent derivatives of catecholamines and immunocytochemically using antibodies against their biosynthetic enzymes. The immunocytochemical techniques yield permanent preparations and make possible ultrastructural studies and combined applications with other procedures. In this report, we describe the production and application of a high-affinity mouse monoclonal antibody against the rate-limiting enzyme in the biosynthetic pathway of the catecholamines, tyrosine hydroxylase. This antibody, coded TOHA1.1, has been used successfully to stain tyrosine hydroxylase immunoreactive sites in the known catecholaminergic neurons and fiber systems of rat brain in both light and electron microscopy. It has also been demonstrated that TOH A1.1 will immunoprecipitate phosphorylated tyrosine hydroxylase.     

Reduction in brain tyrosine hydroxylase activity following acetylcholinesterase blockade in rats.

Activation of cholinergic neurons in the brain is produced by administration of the acetylcholinesterase inhibitors physostigmine and diisopropylfluorophosphate (DFP). This activation has a biphasic effect on tyrosine hydroxylase (EC 4.14.3-) activity. The acute effect of DFP, 1 mg/kg, intraperitoneally, or physostigmine, 0.2 mg/kg, intravenously, or 10 mug, intraventricularly, was a rapid reduction in tyrosine hydroxylase activity in the hypothalamus. The activities of DOPA decarboxylase (EC 4.1.1.28) and dopamine-beta-hydroxylase (EC 1.14.17.1) were not changed. In contrast to the acute effect, chronic administration of physostigmine, 0.2 mg/kg, intravenously, twice daily for 7 days produced an increase in tyrosine hydroxylase activity in the hypothalamus. The rapid acute effects may be due to an allosteric inactivation of tyrosine hydroxylase, while the chronic effects may reflect enzyme induction.     

Subcellular distribution of biopterin in rat brain: the biochemical basis of its stability

Rat brain biopterin, the hydroxylase cofactor, was observed to distribute equally across regional subcellular fractions, rather than to codistribute neuronally with tyrosine and tryptophan hydroxylases for which it functions. Over a 24 h period with light/dark phasing, which some groups have shown to result in cycling of biopterin levels in striate and certain other regions, only the biopterin associated with the crude nuclear fraction of the striate (not associated with neurotransmitter synthesis) demonstrated a diurnal cycle. The selectivity of this perturbation response to the striate nuclear fraction suggests that (1) multiple subcellular loci of biopterin might exist independently in rat brain neurons and (2) the pterin's availability for neurotransmitter biosynthesis is limited beyond its apparent regional concentration. The demonstration of multiple independent sources of neuronal biopterin may be relevant to understanding why regional levels have been so resistant to efforts at pharmacological manipulation (only amphetamine and CRF have changed striate biopterin levels). It also shows that changes in regional hydroxylase cofactor levels may not be related to neurotransmitter synthesis, but instead may result from another presently unknown demand for the cofactor at a disparate neuronal site.     

Tyrosine Hydroxylase, Tryptophan Hydroxylase, Biopterin, and Neopterin in the Brains of Normal Controls and Patients with Senile Dementia of Alzheimer Type

The activities of tyrosine hydroxylase and tryptophan hydroxylase, and the concentrations of the biopterin cofactor and the precursor neopterin were measured in 14 regions of postmortem brains from four histologically verified patients of senile dementia of the Alzheimer type (SDAT) and eight histologically normal controls. Neopterin concentrations were measured in the human brain for the first time. The activities of tyrosine hydroxylase and tryptophan hydroxylase in the brains of patients with SDAT were significantly reduced in the substantia nigra and in the lateral segment of the globus pallidus, locus ceruleus, and substantia nigra, respectively. The concentrations of total biopterin in the brains of patients with SDAT were significantly reduced in the putamen and substantia nigra, but the total neopterin concentrations did not change significantly. These results suggest that the reduction in biogenic amines in SDAT might be related to reductions in biosynthetic enzymes associated with biogenic amines, due to destruction of monoaminergic neurons.     

Regulation of tryptophan and tyrosine hydroxylase

The synthesis of the neurotransmitters serotonin, norepinephrine, and the dopamine is regulated by the initial amino acid hydroxylases. Little is known about the factors that regulate the level of tryptophan hydroxylase in tissue. However, the level of tyrosine hydroxylase is regulated by transsynaptic induction. Acute regulation of in vivo hydroxylase activity appears to be by substrate availability in the case of tryptophan hydroxylase and possibly by feedback inhibition with tyrosine hydroxylase. A newly described phenomenon which has been termed “receptor mediated feedback inhibition” involving neuronal feedback regulation of the activity of both tyrosine and tryptophan hydroxylase may also have an important role.     

Inhibition of Tyrosine Hydroxylase by R and S Enantiomers of Salsolinol, 1-Methyl-6,7-Dihydroxy-1,2,3,4- Tetrahydroisoquinoline

Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.     

Similar Time Course Changes in Striatal Levels of Glutamic Acid Decarboxylase and Proenkephalin mRNA Following Dopaminergic Deafferentation in the Rat

An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long-lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle-treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.     

Immunocytochemical localization of neuronal antigens: tyrosine hydroxylase, substance P, [Met5]-enkephalin.

Neuronal antigens can be demonstrated histologically by numerous direct and indirect immunocytochemical techniques in which a specific antibody is identified by a marker compound such as fluorescein isothiocyanate, ferritin, or horseradish peroxidase. One of the more sensitive methods for the light and electron microscopic localizations of antigens in sections of tissue is the peroxidase-antiperoxidase (PAP) technique. The experimental procedures and the results obtained using this technique for the localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase, are described. The cellular and ultrastructural localization of the enzyme is demonstrated in perikarya, processes, and terminals of catecholaminergic neurons in rat brain. The immunocytochemical localization of tyrosine hydroxylase is compared to the localization of two peptides, substance P and [Met5]-enkephalin, in the A2 region of the medulla. These studies suggest that a synaptic interaction exists between the catecholaminergic neurons and neurons showing positive immunoreactivity for the peptides. The limitations of the PAP immunocytochemical technique are also discussed in relation to the immunocytochemical localization of tyrosine hydroxylase and other antigens.     

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.     

Tyrosine hydroxylase and cholecystokinin mRNA levels in the substantia nigra,ventral tegmental area,and locus ceruleus are unaffected by acute and chronic haloperidol administration

1. The studies described herein were designed to test the hypothesis that a neuroleptic, haloperidol, may alter the level of expression of the tyrosine hydroxylase and cholecystokinin genes in discrete brain regions. 2. In situ hybridization was employed to quantitate changes in concentration of mRNA for tyrosine hydroxylase and cholecystokinin in the ventral tegmental area, substantia nigra, and locus ceruleus after acute or chronic treatment with haloperidol or vehicle. 3. Haloperidol had no effect on the level of tyrosine hydroxylase or cholecystokinin mRNAs, in the ventral tegmentum, substantia nigra, or locus ceruleus, at either 3 or 19 days of drug administration. 4. These data suggest that haloperidol administration does not alter the level of tyrosine hydroxylase or cholecystokinin mRNAs in midbrain dopamine neurons of the rat.     

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.     

Comparative studies on the distribution of protein-o-carboxylmethyltransferase and tyrosine hydroxylase in rat brain by immunocytochemistry

The distribution of protein-O-carboxylmethyltransferase and tyrosine hydroxylase immunoreactivity in brain was compared with the use of highly specific polyclonal antibodies prepared against the native form of each enzyme. Protein-O-carboxylmethyltransferase was found in brain areas rich in catecholamine neurons as identified by tyrosine hydroxylase immunoreactivity. Rabbit anti-protein-O-carboxylmethyltransferase labeled cell bodies in the locus coeruleus, substantia nigra, and paraventricular nucleus whereas rabbit anti-tyrosine hydroxylase prepared against highly purified, native tyrosine hydroxylase from cultured PC12 cells labelled cell bodies in the same brain regions. In addition, the antibody to tyrosine hydroxylase made possible the visualization of very fine cortical processes containing tyrosine hydroxylase and very dense neuronal networks throughout the nigrostriatal pathway. The coincidence of protein-O-carboxylmethyltransferase and tyrosine hydroxylase in catecholamine rich brain areas provide an anatomical basis for the possibility that protein-O-carboxylmethyltransferase could modulate catecholaminergic neurotransmission.     

The role of reductants in the tyrosine hydroxylase reaction.

The role of several reducing systems in the tyrosine hydroxylase reaction has been studied. A significant dependence upon the reducing systems beyond that required to regenerate the oxidized cofactor has been observed. 2-Mercaptoethanol, NADPH, and ascorbate are each effective at reducing the cofactor, but their abilities to stimulate tyrosine hydroxylase vary over a threefold range. NADPH is a suitable reductant for the tyrosine hydroxylase reaction, even in the absence of pteridine reductase. A reducing system containing ascorbate, ferrous ion, and catalase gives unusually high enzyme activity and low blanks. This ascorbate system, in addition to being useful for in vitro enzyme assays, may serve as a model for the in vivo reaction. Ascorbate may play an important role in the hydroxylation of tyrosine in catecholaminergic tissues. This study demonstrates that an efficient reductant for the tyrosine hydroxylase reaction must, in addition to reducing the pterin cofactor, also interact effectively with the enzyme itself.     

Role of tryptophan hydroxylase phe313 in determining substrate specificity

The active site residue phenylalanine 313 is conserved in the sequences of all known tryptophan hydroxylases. The tryptophan hydroxylase F313W mutant protein no longer shows a preference for tryptophan over phenylalanine as a substrate, consistent with a role of this residue in substrate specificity. A tryptophan residue occupies the homologous position in tyrosine hydroxylase. The tyrosine hydroxylase W372F mutant enzyme does not show an increased preference for tryptophan over tyrosine or phenylalanine, so that this residue cannot be considered the dominant factor in substrate specificity in this family of enzymes.     

Effect of low doses of gamma-hydroxybutyric acid on serotonin,noradrenaline, and dopamine concentrations in rat brain areas

The effects of intraperitoneal administration of gamma-hydroxybutyric acid (GHB) on biogenic amine levels in hemispheres, hypothalamus, midbrain, and medulla-pons, and on tryptophan in serum and brain, were studied. One hour after GHB administration (50 and 100 mg/kg) significant increases of dopamine concentration were observed in the hemispheres with both doses and in the hypothalamus with the higher dose, but a significant decrease of noradrenaline in the hypothalamus. No significant changes of serotonin metabolism were observed. These results indicate that low doses of GHB selectively affect the catecholaminergic neuronal activity.     

Relationship Between Phenolamines and Catecholamines During Rat Brain Embryonic Development In Vivo and In Vitro

p-Octopamine and phenylethanolamine are present in the embryonic rat brain earlier than catecholamines. These phenolamines are localized mainly in the hypothalamus, where the level of p-octopamine is very high. The parallel developmental study of the activities of dopamine beta-hydroxylase, 3,4-dihydroxyphenylalanine decarboxylase, tyrosine hydroxylase, and monoamine oxidase shows that phenolamines are present in significant amounts in the hypothalamus until tyrosine hydroxylase and monoamine oxidase become catalytically active. The culture of embryonic hypothalamus at different ages shows that no tyrosine hydroxylase and monoamine oxidase activities can be detected if the tissue is cultured before 15 days. This clearly indicates that all the enzymes related to catecholamine biosynthesis are not triggered at the same time during the development of the rat brain. These results are discussed on the basis of the physiological importance of phenolamines in mammals and of the use of the developing rat brain as a model for the study of the onset of the catecholaminergic system and the decline of the octopamine.