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.     

PROPERTIES OF PARTIALLY PURIFIED PIG BRAIN STEM TRYPTOPHAN HYDROXYLASE

—Tryptophan hydroxylase form pig brain has been purified using a method which involved sonic disintegration of a whole homogenate, ammonium sulphate fractionation, hydroxylapatite fractionation, column chromatography on Sephadex G-100 or G-200 and finally electrophoresis on poly-acrylamide gel. The enzyme was stabilized during purification by tryptophan and dithiothreitol. The partially purified enzyme has a molecular weight of 55,000-60,000 as measured by gel-filtration. The K_m of the soluble partially purified enzyme was 0-4 mm , which differed significantly from that of the particulate enzyme (0·02mm ). Enzyme activity was not stimulated by ferrous ion. However, it was inhibited by the chelating agents 8-hydroxyquinoline, O-phenanthroline and EDTA. In contrast to dopamine, high concentration of tryptophan (10 mm ), 5-hydroxytryptamine, tryptamine and tyramine at 0-5 mm concentration did not inhibit the enzyme in the presence of dimethyltetrahydropterin (DMPH4). A number of monoamine oxidase inhibitors, phenelzine, pheniprazine and chlorgyline at 1 mm strongly inhibit the formation of 5-hydroxytryptamine. Evidence is presented for the presence of an endogenous inhibitor of tryptophan hydroxylase.     

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.     

THE DISTRIBUTION OF TRYPTOPHAN HYDROXYLASE IN CAT BRAIN

Abstract— The distribution of tryptophan hydroxylase in the cat brain was investigated and found to parallel roughly the distribution of serotonin. The most active areas are the caudate nucleus, septal area, anterior perforating substance, hypothalamus, amygdala and various areas of the midbrain.     

THE ACTIVATION OF THIAMINE DIPHOSPHATASE BY ATP IN RAT BRAIN

• 1 Thiamine diphosphatase (TDPase) of brain was activated by a low concentration of ATP.
• 2 In thiamine-deficient rats TDPase activity in the brain increased significantly relative to that in pair-fed animals, while in liver it decreased by the same amount as in the pair-fed controls.
• 3 Liver TDPase was localized almost entirely in the soluble fraction, but in the brain it was bound to insoluble protein. On treatment with Triton X-100 brain TDPase activity increased.
• 4 The ATP content of the brain of deficient rats, but not the ADP or AMP content, was significantly higher than in the control group. The level of inorganic phosphate in the brain and spinal cord of deficient animals was elevated markedly, while that of P-creatine was unchanged.
• 5 The possible roles of brain TDPase in relation to nerve conduction and the blood-brain barrier are discussed.

     

REGIONAL TRANSPORT OF TRYPTOPHAN IN RAT BRAIN

Abstract— Tryptophan uptake was studied in brain slices and synaptosomes prepared from regions known to vary in the numbers of serotoninergic cell bodies and nerve endings that they contain. The rate of tryptophan uptake was highest in hypothalamus for both types of preparation. Differences among the regions were much more pronounced in isolated nerve endings (synaptosomes). Loading with tryptophan did not affect the uptake into tissue slices. Tryptophan accumulation in hypothalamus synaptosomes was reduced after intraventricular injection of 5,7–dihydroxytryptamine whereas no change was observed in synaptosomes prepared from cerebellum under the same conditions; accumulation by synaptosomes prepared from the hypothalamic and hippocampal regions was reduced after raphe lesions.     

A SENSITIVE MICROASSAY FOR TRYPTOPHAN HYDROXYLASE IN BRAIN

—A specific and sensitive, radioisotopic microassay for tryptophan hydroxylase (EC 1.14.36) is described, which is capable of determining enzymatic activity in as little as 5 μg of crude brainstem homogenate. 5-Hydroxytryptophan, the immediate product of hydroxylation of tryptophan is enzymatically converted to N-acetylserotonin. A radioisotopic label is then introduced by the enzymatic methylation of N-acetylserotonin in the presence of [³H]methyl-S-adenosyl-methionine. The [³H]-melatonin thus formed is isolated by extraction and counted. With this assay, the activity in individual hypothalamic nuclei (arcuate nucleus, median eminence, suprachiasmatic nucleus, and medial forebrain bundle) has been measured.     

PROPERTIES AND REGIONAL DISTRIBUTION OF TRYPTOPHAN HYDROXYLASE IN THE CHICKEN BRAIN

Tryptophan hydroxylase in young chicken brain had a pH optimum of 7.5–8, depending on the buffer used. It had apparent K_m values for tryptophan and tetrahydrobiopterin of 49 μM and 32 μM respectively. The enzyme in chicken brain, but not rat brain, was cold-shock labile but was stable for up to 4 days at — 20°C. Lability was observed both in tissues and homogenates of these tissues subjected to cold shock, but the extent of loss of activity varied between brain regions. Supernatant fractions did not lose activity after cold shock. The highest level of tryptophan hydroxylase was found in the rostral region of the chicken brainstem. High levels were also found in the caudal region of the brainstem, the midbrain, thalamus, caudate and cerebral cortex. The cerebellum and optic chiasma contained only traces of activity.     

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.     

TYROSINE HYDROXYLASE IN RAT BRAIN: DEVELOPMENTAL CHARACTERISTICS

Abstract— The development of tyrosine hydroxylase (tyrosine 3-hydroxylase, EC 1.14.3.a) activity has been examined in whole rat brain and in various regions and subcellular fractions thereof. The specific activity of tyrosine hydroxylase increased almost 15-fold from 15 days of gestation to adulthood. With maturation, those regions of the brain that contain only terminals of the catecholaminergic neurons showed the greatest increases in enzyme activity. There was a shift in the subcellular distribution of tyrosine hydroxylase from the soluble fraction in the fetal brain to the synaptosomal fraction in the adult brain. Tyrosine hydroxylase, dopamine hydroxylase (EC 1.14.2.1) and the specific uptake mechanism for norepinephrine appear to develop in a coordinated fashion.     

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).     

DIURNAL RHYTHM AND TURNOVER OF TRYPTOPHAN HYDROXYLASE IN THE PINEAL GLAND OF THE RAT

Tryptophan hydroxylase in the pineal gland of the rat was found to undergo a diurnal rhythm in activity with an elevated activity at night. The rhythm was abolished in constant light. Cycloheximide (15 mg/kg, i.p.), administered both at night and during the day, caused a rapid decay in activity suggesting that tryptophan hydroxylase was subject to a rapid turnover in vivo. The primary site of control appeared to be at the level of translation since actinomycin D had no effect. Some relevant properties of the enzyme were studied. Thiol-containing compounds were shown to substantially protect pineal tryptophan hydroxylase from inactivation at 0°C but provided little protection at higher temperatures. The inactivation process appeared to be independent of oxygen. The activity of the enzyme, lost after ageing at 0°C. could be recovered by incubation with dithiothreitol under anaerobic conditions. Fresh enzyme, or enzyme inactivated at 37°C could not be activated by this process. A re-examination of the action of p-chlorophenylalanine (PCPA) on pineal tryptophan hydroxylase revealed that an irreversible inactivation occurred within 6h (25% of initial activity) followed by a recovery within 24 h. The rapid turnover of the enzyme is the probable reason for the failure of previous studies to observe an irreversible inhibition of this enzyme by PCPA.     

DETERMINATION OF SOME MOLECULAR PARAMETERS OF TRYPTOPHAN HYDROXYLASE FROM RAT MIDBRAIN AND MURINE MAST CELL

Amphetamine, a potent sympathomimetic amine, has powerful stimulant actions in the central nervous system. These actions are believed to be related to the influence of amphetamine on release and uptake of catecholamine neurotransmitters. The [¹⁴C]deoxyglucose method makes it possible to study changes in cerebral metabolic rate in different areas of gray and white matter. Because of the close relationship between metabolic rate and functional activity, this method may be used to identify specific structures in the brain in which functional activity is altered. The [¹⁴C]deoxyglucose method was used to explore for changes in metabolic rate produced by d-and l-amphetamine (5 mg/kg) in forty gray and four white matter structures in normal conscious rats. d-Amphetamine produced increases in local cerebral glucose utilization in a number of components of the extrapyramidal motor system, as well as in some other structures known to contain dopamine-producing and/or dopaminoceptive cells. The largest increases after d-amphetamine administration occurred in the subthalamic nucleus and the zona reticulata of the substantia nigra. l-Amphetamine produced increases in some but not all of these same structures, and these were generally smaller than those observed with d-amphetamine. Decreases in local cerebral glucose utilization after either d- or l-amphetamine administration were found in the habenula and the suprachiasmatic nuclei of the hypothalamus. The effects in the suprachiasmatic nuclei may reflect their normal diurnal rhythm in metabolic rate. These results indicate that amphetamines may influence behavior through effects on specific regions of the brain. Only some of these regions have previously been studied as possible sites of action of amphetamine.     

EFFECTS OF IMMOBILIZATION AND FOOD DEPRIVATION ON RAT BRAIN TRYPTOPHAN METABOLISM

Abstract— Withdrawal of food or immobilization both led to changes in rat brain tryptophan metabolism. Brain tryptophan and 5-hydroxyindolylacetic acid concentrations both increased while changes in 5-hydroxytryptamine were much smaller. Changes were greater upon withdrawal of food. The brain tryptophan change did not appear merely to reflect an overall increase of brain amino acid concentrations, brain tyrosine concentration being only slightly increased by food withdrawal and significantly decreased upon immobilization. Plasma tryptophan did not increase. The changes in brain indole metabolism were not abolished by adrenalectomy. Results are discussed in relation to the regulation of brain serotonin metabolism.     

INHIBITION OF GUINEA-PIG BRAIN TYROSINE HYDROXYLASE BY CATECHOLS AND BIOPTERIN

—The inhibition by catechols and biopterin of tyrosine hydroxylase from guineapig caudate nuclei has been examined. Inhibitory constants of 10–20 μm were obtained for dopamine and noradrena-line and 150–250 μm for l -DOPA and dihydroxyphenylacetic acid. When examined under similar conditions homovanillic acid was found not to be inhibitory. Using an acetone dried powder as the source of tyrosine hydroxylase no change in K_m or V_max was observed when cyclic AMP or Ca²⁺ were added to the medium. Enzyme mechanisms and a possible explanation of the mechanisms controlling catechol synthesis are discussed.     

HISTOCHEMISTRY OF RAT BRAIN STEM MONOAMINE OXIDASE DURING MATURATION

—Monoamine oxidase (MAO) activity in the nuclei and tracts of the medulla and pons of the rat from birth to 90 days is reported. Prominent MAO activity was present in the locus coeruleus and nucleus ambiguus at birth. At 5 days a weak reaction localized mainly within the neuropil and glia cells was detected in several other nuclei. By 10 days all nuclei were identified with MAO activity varying from weak to intense, the activity showing further increases at 15 and 20 days. Staining in nerve fibres was negligible at 5 days but increased rapidly to 15 days in some tracts when the characteristic beading pattern was distinct. At 30 days differentiation in intensity of MAO activity between the nuclei diminished and no increase was apparent after 55 days. The results are compared with the distribution of brain stem acetylcholinesterase during maturation and also with regions specific in catecholamine or serotonin content in the adult rat brain stem. This and an earlier study on the cerebrum suggest that MAO is another component of the brain that falls into the caudal-rostral concept of biochemical maturation and that it fits into a group of enzymes exhibiting a similar pattern of increase in activity during development.     

ALLOSTERIC ACTIVATION OF HYPOTHALAMIC TYROSINE HYDROXYLASE BY IONS AND SULPHATED MUCOPOLYSACCHARIDES

Abstract— The kinetics of canine hypothalamic tyrosine hydroxylase were studied in the presence of various ions and sulphated mucopolysaccharides. Enzymic activity was dependent on ionic strength, a specific sulphate effect and the presence of the highly sulphated mucopolysaccharide, heparin. Whereas both sulphate and heparin activated tyrosine hydroxylase by increasing V_max heparin, but not sulphate, also increased the affinity of the enzyme for the synthetic cofactor, 2-amino-4-hydroxy-6,7-dirnethyl-5,6,7,8-tetrahydropteridine, by nearly an order of magnitude. Other rnucopolysaccharides, such as chondroitin sulphate and hyaluronic acid, were not effective as activators of tyrosine hydroxylase. The allosteric activation of tyrosine hydroxylase by heparin may serve to 'sensitize' the enzyme to low levels of its end product, norepinephrine.     

INCREASE OF BRAIN TRYPTOPHAN BY ELECTROCONVULSIVE SHOCK IN RATS

—ECS markedly increased tryptophan and 5-hydroxyindoleacetic acid levels in brain. Brain serotonin and plasma tryptophan levels were unaffected.     

EFFECTS OF HYPOPHYSECTOMY ON RNA METABOLISM IN RAT BRAIN STEM

Abstract— Ribosomal aggregates were isolated from rat brain stem and characterized as polysomes by sedimentation analysis and by their sensitivity to RNase and EDTA treatment.
Three weeks following hypophysectomy there was a significant decrease in the content of large polysomes in the rat brain stem. The incorporation of radioactive uridine into RNA was studied using a double-labelling technique with [³H]- and [¹⁴C]uridine and labelling periods of 70 and 180 min. It was found that after hypophysectomy the incorporation of radioactive uridine into total, nuclear and cytoplasmic RNA and in polysomes was decreased after 70 and 180 min. Information on the nature of the rapidly-labelled RNA in the various subcellular fractions was obtained by sucrose gradient sedimentation analysis.
After 70 min of labelling the nucleus contained heterogeneous RNA with a considerable fraction of RNA sedimenting faster than 28 S. In the cytoplasmic fraction heterogeneous 4 to 30 S RNA was found, presumably associated with RNP particles, whereas after 180 min the polyribosomal aggregates were also labelled.
The present results indicate a profound effect of hypophysectomy on the metabolism of all species of brain RNA investigated.