Radiometric assay of tyrosine hydroxylase and tryptophan hydroxylase by Kalignost extraction procedures.
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Results for “Tryptophan Hydroxylase”
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Modulation of brain tryptophan hydroxylase activity by brain tryptophan content.
Although hydroxylation of tryptophan (TP) is considered to be the rate-limiting step in serotonin synthesis, the mechanism whereby tryptophan hydroxylase (TPH) participates in the regulation of serotonin synthesis is still in question. Since the brain TP concentration is probably near the Km for tryptophan hydroxylase, changes in brain TP content could affect the rate of its hydroxylation. However, it has not been established whether in vivo the activity of TPH and TP in several brain nuclei and other regions of rat brain after treatments known to lower brain TP levels. Chlorimipramine, loading with neutral amino acids and a TP-deficient diet decreased the TP content of some, but not all, brain regions studied. Whenever TP decrease withe TP injections. Several brain nuclei accumulate TP at different rates after a TP load. These data suggest possible mechanisms for regulation of serotonin synthesis in the face of fluctuating plasma levels of TP.
Amino Acids↗
Some properties of bovine pineal tryptophan hydroxylase.
Bovine pineal tryptophan hydroxylase is a pterin-dependent aromatic amino acid monooxygenase with a broad substrate specificity and with low Km values for the amino acid substrates, L-tryptophan and L-phenylalanine. p-Chlorophenylalanine, an inhibitor of the tryptophan hydroxylation in the brain, also serves as a good substrate of the bovine pineal enzyme. The full activity of this enzyme is detected in vitro only after preincubation with dithiothreitol under reductive conditions. The enzyme is profoundly and more or less specifically inhibited by L-5HTP, the product of tryptophan hydroxylation, suggesting a possible regulatory role of this hydroxylated amino acid. The Km of the enzyme for tetrahydrobiopterin, a presumed natural cofactor, is significantly higher than the expected tissue concentration if the cofactor is assumed to be uniformly distributed in the tissue. These properties of bovine pineal tryptophan hydroxylase are distinguishable from those of the hydroxylase in the brain indicating that the hydroxylation of tryptophan in the brain leading to the synthesis of serotonin and the reaction in the pineal gland leading to the formation of melatonin are catalyzed by different tryptophan hydroxylases and are probably under separate regulatory mechanisms.
Animals↗
Further studies on tryptophan hydroxylase from neoplastic murine mast cells.
Tryptophan hydroxylase (tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4) purified from the neoplastic murine mast cells by hydroxylapatite chromatography following ammonium sulfate fractionation showed maximum activity at pH 6.0 in the presence of 2-mercaptoethanol, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine and Fe2+, and pH 7.6 to 8.0 in the absence of addED Fe2+. The Km values were 38.5 muM and 22.2 muM for tryptophan, 298 muM and 204 muM for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetra-hydropteridine, and 6.45% for oxygen in either presence or absence of added Fe-2+, respectively. From kinetic data the reaction mechanism of tryptophan hydroxylation appears to be of the sequential, rather than the ping-pong, type. Tryptophan hydroxylase from mast cells was considerably inhibited by o-phenanthroline like phenylalanine hydroxylase as well as tyrosine hydroxylase from other sources, and its Ki was between 1.2 muM and 4.53 muM. It was found that the inhibition by o-phenanthroline was competitive with respect to both tryptophan and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not molecular oxygen under the assay conditions employed.
Animals↗
A serotonergic innervation of noradrenergic neurons in nucleus locus coeruleus: demonstration by immunocytochemical localization of the transmitter specific enzymes tyrosine and tryptophan hydroxylase.
Immunocytochemical localization of the neurotransmitter synthesizing enzymes, tyrosine and tryptophan hydroxylase, was used to determine whether the noradrenergic neurons in the nucleus locus coeruleus of the rat are innervated by serotonergic (5-HT) neurons. Specific antibodies were prepared to tyrosine hydroxylase, purified from the bovine adrenal medulla, and tryptophan hydroxylase, purified from rat midbrain. These were localized by both light and electron microscopy by the use of the peroxidase-antiperoxidase method. In the nucleus locus coeruleus, tyrosine hydroxylase was contained in the cytoplasm, proximal axons, and dendrites of intrinsic neurons. Tryptophan hydroxylase, on the other hand, was only contained within processes surrounding the perikarya and dendrites of the catecholaminergic neurons. The processes labeled with tryptophan hydroxylase were unmyelinated, ranged in size from 0.1 to 1.4 micron, and consisted of terminal varicosities separated by intervaricose segments. Although in close approximation to noradrenergic neurons, these processes, presumably axons, rarely formed synatic contacts with thickened membrane specializations. In processes, tryptophan hydroxylase was associated with subcellular organelles which had size and distribution of microtubules, and small and large synaptic vesicles. These observations provide a morphological basis to support the hypothesis that the activity of noradrenergic neurons may be modulated by a direct action of 5-HT neurons.
Animals↗
Serotonergic neurons in the peripheral nervous system: identification in gut by immunohistochemical localization of tryptophan hydroxylase.
A specific antibody to tryptophan hydroxylase [L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] has been used to localize the enzyme immunohistochemically in neurons of the mammalian gut. The enzyme was found in perikarya of intestinal neurons of mice, rats, and guinea pigs. Neurons containing the enzyme survived for up to 3 weeks in organotypic tissue culture and were intrinsic to the gut. These neurons are probably serotonergic and are the first such neurons to be found in the peripheral nervous system.
Animals↗
Characteristics of the activation by dithiothreitol and Fe(2+) 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 muM 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 its Vmax 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 MG(2+), Ca(2+)) on tryptophan hydroxylase activity, this might be a good procedure to activate this enzyme with only minor changes in its regulatory properties.
Animals↗
Serotonergic innervation of the forebrain: effect of lesions on serotonin and tryptophan hydroxylase levels.
Concentrations of serotonin and tryptophan hydroxylase activity in individual hypothalamic and limbic nuclei, as well as cortical regions, were measured by radioenzymatic microassays after dorsal raphe nucleus lesions and various surgical transections of the brain stem. Fourteen days after lesioning of the dorsal raphe nucleus there were 43-65% decreases in 5-HT concentrations of certain hypothalamic and limbic nuclei. More pronounced decreases were found in the parietal cortex and in the locus coeruleus and substantia nigra. Degenerations of nerve terminals in various regions of the hypothalamus and the limbic system were detected by electron microscopic studies 1-8 days after dorsal raphe nucleus lesions. There was no change in the 5-HT concentration of the spinal cord following dorsal raphe nucleus lesions. Surgical transections at the level of mesencephalic-pontine junction caused no significant changes in 5-HT concentrations or tryptophan hydroxylase levels in the hypothalamus, limbic system, cerebral cortex or spinal cord. Serotonergic fibers ascending from the mesencephalic raphe nuclei constitute a well circumscribed bundle dorsal to the interpeduncular nucleus. The axons enter the hypothalamus between the mammillary body and crus cerebri just ventral to the medial forebrain bundle. The ascending pathway contains all the ascending 5-HT fibers innervating the hypothalamus and the rostral limbic and cortical areas.
Animals↗
Conformational influences on brain tryptophan hydroxylase by submicromolar calcium: opposite effects of equimolar lithium.
Tryptophan hydroxylase from rat midbrain, EGTA-pretreated and dialyzed, manifested allosteric properties with respect to its substrate tryptophan, cofactor tetrahydrobiopterin, and the calcium ion. Kinetic studies suggest two preferred enzyme conformations in the presence of low concentrations of the cosubstrates: a higher affinity form manifesting hyperbolic substrate kinetics, induced by submicromolar (0.4--0.8 microM) calcium in vitro and cocaine in vivo, and a lower affinity form exaggerating cooperativity with respect to substrate, induced by submicromolar (0.4 to 0.8 microM) lithium in vitro and lithium in vivo. Lithium's effect on serotonin biosynthesis may be due to its antagonism of the positive effector influence of calcium on tryptophan hydroxylase, either as a negative effector or by blocking the calcium site.
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Activation of brain tryptophan hydroxylase by a phosphorylating system.
A significant activation of tryptophan hydroxylase (TPH) was achieved by the addition of 1 mM ATP and 10 mM MgCl2 to 100,000g supernatant prepared from mouse midbrain. The activation produced an increase of enzyme activity by 50-70% above control. Cyclic AMP was not a necessary component in the activation as initially suspected; neither was the involvement of a cyclic AMP-dependent protein kinase detected. The enzyme activation by Mg+2-ATP was totally retained after dialysis, thus excluding the possibility of an allosteric effect. In contrast to the activation, TPH was found to be sensitive to inactivation by acid phosphatase, a dephosphorylating enzyme. These findings are consistent with the proposal that the enzyme activation may involve a phosphorylation process. Furthermore, the activation by Mg+2-ATP in the brain preparations was only minimally observed from neonatal mice and could be abolished from adult mice by administration of hydrocortisone. It appears that the phosphorylation process, as postulated here, may be under developmental and hormonal influences.
Acid Phosphatase↗
Coincidence of blockade of synaptosomal 5-hydroxytryptamine uptake and decrease in tryptophan hydroxylase activity: effects of fenfluramine.
A single injection of fenfluramine hydrochloride resulted in a short-term increase in striate synaptosomal conversion of tryptophan to serotonin (5-HT) in rat brain. In contrast, D- and L-amphetamine sulfate resulted in a short-term decrease of this index of 5-HT biosynthesis. None of the amphetamines studied altered the kinetics of synaptosomal uptake of L-[3-14C]-tryptophan measured in the same striate preparation. Within 4 hours after fenfluramine administration, 3H-5-HT uptake into synaptosomes was markedly decreased; it returned to control levels in 10 to 14 days. Intrasynaptosomal tryptophan hydroxylase activity dropped markedly within 4 hours of drug administration and remained depressed for 10 to 14 days, its return to control levels coinciding with that of 3H-5-HT uptake. Only 5-HT cell body enzyme prepared from the lateral midbrain (B9) demonstrated a reduction in activity comparable to that seen in the synaptosomes; very small decreases occurred in cell body enzyme prepared from whole midbrain (B7, B8, B9) or medial midbrain (B7, B8). Lateral midbrain tryptophan hydroxylase activity returned to control levels by 8 days. In vitro, fenfluramine (100 muM) affected none of these indices of central 5-HT synthesis except 3H-5-HT uptake, which it reduced, and synaptosomal 3H-5-HT release, which it facilitated. The effects of fenfluramine on 5-HT biosynthesis persisted longer than those of D-amphetamine, which lasted less than 24 hours. However, the fenfluramine effects were much shorter than those reported for p-chloroamphetamine, which persist for up to 3 months. These three amphetamines apparently affect the lateral midbrain raphe nuclei selectively.
Animals↗
Abnormalities in grooming behavior and tryptophan hydroxylase activity in the superior colliculi in cats with pontile and frontal neocortical lesions.
Cats with pontile or frontal neocortical lesions display a dissociation of the appetitive and consummatory components of grooming behavior when their body surface is tactually stimulated, an abnormal behavior that waxes and wanes with the seasons of the year. Tryptophan hydroxylase activity is significantly decreased in the superior colliculi of cats with pontile lesions and of cats with frontal neocortical lesions. The results suggest that the change in tryptophan hydroxylase activity is mediated neuronally and is a transneuronal effect on the serotonergic input to the superior colliculi. Pharmacological manipulations of the serotonergic system in normal cats failed to induce the abnormal behavior, indicating that other factors are involved in the genesis of the abnormal behavior.
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Effect of short- and long-term treatment with cocaine on rat brain tryptophan hydroxylase.
Short-term (5 days) treatment with cocaine (10 mg/kg, i.p.) inhibited the soluble tryptophan hydroxylase while the particulate enzyme was essentially unchanged. In contrast, long-term (45 days) treatment resulted in an increased activity of the particulate enzyme and a return to normal of the soluble enzyme activity.
Animals↗
Calcium dependence of rat brain tryptophan hydroxylase.
The effects of various concentrations of ionized Ca were examined on the activity of rat brain tryptophan hydroxylase previously treated with EGTA. Within the range of ionized Ca-concentrations though to be physiologically important (10(-8) to 10(-5) M), no significant activation of the enzyme occurred, although activation was observed at higher concentrations of the metal.
Animals↗
Effect of stress on serotonin concentration and tryptophan hydroxylase activity of brain nuclei.
The effects of a single stressful stimulus on serotonin (5-HT) concentration and of repeated stressful stimuli on tryptophan hydroxylase (THy) activity were measured in individual hypothalamic nuclei and other rat brain regions using specific sensitive radioisotopic enzymatic microassays. Two h after formalin injection, 5-HT levels were increased in the dorsal raphe (RDN). Acute immobilization stress, however, reduced the concentrations of 5-HT in several of the hypothalamic nuclei: the medium eminence (ME), lateral amygdaloid nucleus, hippocampus, cingulate cortex (CCX) and RDN. Five consecutive daily immobilization periods failed to alter THy activity in any region examined. These results suggest a rapid release of 5-HT both from the cells and the axon terminals in the central nervous system, and failure, even with repeated stresses, to elicit induction of increased levels of the biosynthetic enzyme regulating the synthesis of the neurotransmitter.
Animals↗
Temporal effects of fornix transection on brain tryptophan hydroxylase activity and plasma corticosterone levels.
The effect of fornix transection on plasma corticosterone and on midbrain (MID), septum/preoptic (S/POA) or hippocampal (HIP) tryptophan hydroxylase activity (THA) was studied in adult male rats. A mild stress resulted in higher levels of plasma corticosterone in operated as compared to sham-operated rats 6 and 40 h post-transection; however, at 30 days post-operation no difference was found. The response of THA to fornix transection was region-specific. No significant change in the S/POA region was found at any time. THA in the HIP, a terminal area of 5-HT fibers, showed a progressive fall over time to values 80% below normal levels. This result suggests that most of the 5-HT fibers to the HIP had been severed. A 28 h half-life for HIP THA was calculated. THA in the MID, an area known to contain the majority of 5-HT cell bodies with ascending fibers, was significantly reduced compared to sham controls at 6 h, 40 h, and 8 days post-transection. However, at 30 days post-operation no difference was found. The depression in MID THA by its rapid onset, the distance from the fornix transection site, and its return to normal after 30 days, is thought to be due to a transneuronal effect on the serotonin-containing neurons in MID raphe. The fall in MID THA at a time when plasma corticosterone levels are increased in fornix-transected rats may be compared with the situation in normal, stressed and adrenalectomized rats where MID THA andplasma corticosterone levels change in the same direction. The data suggest that the glucocorticoid effects on MID 5-HT containing neurons are mediated transneuronally through the hormone concentrating cells in the HIP.
Animals↗
Redundant mechanisms regulating brain tyrosine and tryptophan hydroxylases.
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Amino Acids↗
Diurnal changes in the kinetic properties of tryptophan hydroxylase from rat brain.
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