DOPAMINE-β-HYDROXYLASE IN THE RAT BRAIN: DEVELOPMENTAL CHARACTERISTICS

Abstract— A sensitive and specific assay for dopamine-8-hydroxylase (DBH) in the rat brain has been developed. The enzyme in the brain has requirements for cofactors and affinity for substrate similar to DBH in the adrenal medulla. DBH activity was demonstrable in the brain of the fetal rat at 15 days of gestation; there was an increase in DBH activity with maturation that preceded and paralleled the rise in levels of endogenous norepinephrine until 3 weeks after birth. There was a shift in the distribution of total DBH activity from the caudal to the rostral regions of the brain during development. In the adult brain, DBH was highly localized in the nerve terminals. Between 17 days of gestation and adult-hood, there was 2300-fold increase in the DBH activity that sedimented with sheared-off nerve terminals.     

DEVELOPMENTAL CHARACTERISTICS OF PHENYLETHANOLAMINE AND OCTOPAMINE IN THE RAT BRAIN

Abstract— Phenylethanolamine and octopamine have been detected in the developing rat brain. Maximum concentration of these amines occurs early in development (16-17 days of gestation). At this developmental stage, the brain concentration of these amines is higher than that of norepinephrine. There is a sharp decline in the phenylethanolamine and octopamine concentrations on day 18 of gestation to approximately those of the adult. This decrease coincides with an increase in-monoamine oxidase activity of fetal brain, with an increase in the activities of tyrosine hydroxylase and dopamine-β-hydroxylase, and with the appearance of a saturable active uptake mechanism for norepinephrine. The administration of iproniazid, a monoamine oxidase inhibitor, to pregnant rats produced an increase in phenylethanolamine, octopamine and norepinephrine concentrations in the fetal rat brain at 16 days of gestation. p -Chlorophenylalanine, an inhibitor of phenylalanine hydroxylase, decreased fetal brain norepinephrine; this drug increased brain levels of phenylethanolamine and octopamine. The combined administration of iproniazid, p -chlorophenylalanine and phenylalanine to pregnant rats resulted in increased concentrations of octopamine and in a several-fold increase of phenylethanolamine levels; norepinephrine concentrations were sharply reduced. The possible significance of these findings in relation to pathological conditions such as phenylketonuria is discussed.     

LOCALIZATION OF TYROSINE TRANSAMINASE IN RAT BRAIN

The distribution of rat brain tyrosine transaminase was determined in brain regions and in subcellular fractions. The results indicate that brain tyrosine transaminase is predominantly a mitochondrial enzyme. Its localization on the inner mitochondrial membrane is suggested. The results are discussed in relation to the function of tyrosine as precursor of catecholamines in the central nervous system.     

URIDINE PHOSPHORYLASE FROM RAT BRAIN: CHARACTERISTICS AND DEVELOPMENTAL COURSE

—Uridine phosphorylase (uridine: orthophosphate ribosyltransferase; EC 2.4.2.3) from rat brain was purified and its properties were studied. The enzyme resembled preparations made from other mammalian sources. Its pH optimum was between 7·6 and 8·0. An examination of its action on various substrates showed rates of reaction in the order: uridine > deoxyuridine > thymidine > cytidine. The enzyme showed a requirement for phosphate which could also be satisfied by arsenate. The activity of the enzyme was protected from heat inactivation by uridine and by phosphate. In brain and liver the activity of the enzyme increased five- to ten-fold between 10 and 20 days of life. Injections of cortisol or of uridine did not increase the enzymic activity.     

EFFECTS OF HYPOXIA ON TYROSINE HYDROXYLASE ACTIVITY IN RAT CAROTID BODY

Abstract— Tyrosine hydroxylase (TH) activity was measured in the carotid body. superior cervical ganglion and adrenal glands of the rat under normal conditions and at 48 h following exposure of the animals for 1-3 h in a low O₂ atmosphere. Basal TH levels were 5-6 nmol/h/mg tissue for both the carotid body and the ganglion. Forty-eight hours after hypoxia, there was an increase in enzyme activity in both tissues which paralleled the severity of the hypoxia but was greater in the carotid body than the superior cervical ganglion. Thus, following exposure to 5% O₂ in N₂ for two 30-min periods (20-min interim), TH activity had increased by 50% in the carotid body and 33% in the ganglion; after exposure to 10% O₂ in N₂ for 3 h (continuous), TH levels were increased by 37% in the carotid body and 12% in the ganglion. In the adrenal gland, basal TH activity was 3.42 ± 1.87 nmol/h/mg tissue, and this value was unchanged following either level of hypoxia.     

THE EFFECTS OF REPEATED-LYSERGIC ACID DIETHYLAMIDE INJECTIONS ON CATECHOLAMINE LEVELS AND TYROSINE HYDROXYLASE ACTIVITY IN RAT BRAIN REGIONS

Daily injections of 100 μg/kg of d -lysergic acid diethylamide (LSD) for 14 days produced a significant decrease in the dopamine level in rat brain corpus striatum which was still apparent 15 days after the last LSD treatment. Further LSD injections did not change the amount of dopamine depletion. In cerebral cortex, 14 days of LSD injections produced a significant decrease in the norepinephrine level and a significant increase in tyrosine hydroxylase activity. The elevated tyrosine hydroxylase activity was still present 15 days after the final LSD injection but only in those animals receiving daily vehicle injections during this period. Pre-treatment of rats with daily saline injections for 2 weeks before the 2 week period of LSD treatment prevented both the reduced norepinephrine content and elevated tyrosine hydroxylase activity usually found 24 h after the last LSD injection.     

酪氨酸羟化酶基因载体－pCMVTH 质粒的构建及在大鼠骨骼肌内的表达

为用转基因方法治疗巴金森氏病大鼠模型,本研究采用分子克隆技术,将合成多巴胺的关键酶－酪氨酸羟化酶（ＴＨ）的基因,克隆进入以巨细胞病毒ＣＭＶ为启动子的载体质粒内,经限制性内切酶定位分析证实该重组的ＤＮＡ质粒的可靠性。携带ＴＨ基因的ＰＣＭＶＴＨ质粒以ＬＩＰＯ－ＦＥＣＴＩＮ介导,在培养的原代骨骼肌细胞中高效表达。本研究为进一步用转基因的细胞植入脑内以治疗巴金森氏病打下一定基础。     

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.     

TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

外源性TH基因在帕金森氏病鼠脑内的表达——行为和TH免疫组化的研究

用成年大鼠７５只,给右侧黑质区注射６－羟基多巴胺（６－ＯＨＤＡ）,损毁黑质多巴胺能神经元,制备偏侧帕金森氏病（ＰＤ）鼠模型。四周后,注射阿朴吗啡（ＡＰＯ）诱发大鼠向左侧旋转。旋转数为每分钟７次以上的３５只ＰＤ鼠作实验用。其中实验组１５只,对照组２０只。向实验组ＰＤ鼠右侧纹状体多点植入含大鼠酪氨酸羟化酶ｃＤＮＡ（ＴＨｃＤＮＡ）的真核表达载体ｐＳＶＫ３－ＴＨ和脂质体Ｌｉｐｏｆｅｃｔｉｎ混合的基因转染复     

DEVELOPMENTAL PROFILES OF GANGLIOSIDES IN HUMAN AND RAT BRAIN

Abstract— The developmental profiles of individual gangliosides of human brain were compared with those of rat brain. Interest was focused mainly on the pre- and early postnatal development. Human frontal lobe cortex covering the period from 10 foetal weeks to adult age and the cerebrum of rat from birth to 21 days were analysed. Lipid-NANA and lipid-P were followed; in the rat, also protein and brain weight. A limited number of samples of human cerebral white matter and cerebellar cortex were also studied. The following major results were obtained:

• 1 The ganglioside concentration increased approximately three-fold within a short period: in rat cerebrum, from birth to the 17th day; in human cerebral cortex, from the 15th foetal week to the age of about 6 months. The largest increase in the rat brain occurred by the 11th to the 13th day; in human brain by term. The relative increase of gangliosides during this period was more rapid than that of phospholipids.
• 2 A hitherto unknown distinct early period of ganglioside and phospholipid formation in rat occurred by the second to fourth day.
• 3 The changes in brain ganglioside pattern, characteristic of the developmental stages of the rat, were found to be equally pronounced in the human brain.
• 4 Regional developmental differences in the ganglioside pattern were demonstrated in human brain. A characteristic white matter pattern, rich in monosialogangliosides, had developed by the age of 1 year. The increase in ganglioside concentration and the formation of the definitive ganglioside pattern of cerebellar cortex occurred later than in cerebral cortex. This cerebellar pattern was characterized by a very large trisialoganglioside fraction.
• 5 The two periods of rapid ganglioside metabolism in rat brain preceded the two periods of rapid protein biosynthesis.

     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

SEMIAUTOMATED FLUORIMETRIC DETERMINATION OF TYROSINE IN RAT BRAIN AND HUMAN PLASMA

Abstract— The evidence is presented to show that the fiuorimetric method of W aalkes & U denfriend (1957) cannot be used directly for the determination of free tyrosine in acid deproteinized extracts of rat brain, owing to the reaction of peptide bound tyrosine in some proteins present in the acid extracts. A method is given to obtain a fraction containing free tyrosine in the acid extracts of rat brain and to determine its tyrosine content fluorimetrically by autoanalysis. An autoanalysis method is also given for the fiuorimetric determination of free tyrosine directly in 5₀ trichloroacetic acid extracts of the plasma.     

RAT BRAIN STEM TRYPTOPHAN HYDROXYLASE: MECHANISM OF ACTIVATION BY CALCIUM

Abstract— The activity of soluble tryptophan hydroxylase from rat brain stem was increased in presence of mm concentrations of calcium. Similarly to that observed by treating the enzyme with sodium dodecyl sulphate or trypsin, this activation resulted mainly from an increased affinity of tryptophan hydroxylase for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). In addition, the optimal pH for the enzymic activity was shifted from 7.6 to 7.9 following activation by calcium, sodium dodecyl sulphate or trypsin.
Under the assay conditions used for measuring tryptophan hydroxylase activity, calcium also stimulated a neutral proteinase. This latter enzyme could be eliminated from the solution of tryptophan hydroxylase by filtration through Sephadex G 200. The resulting partially purified tryptophan hydroxylase could be activated by calcium only when the neutral proteinase was included in the assay mixture. In support of this conclusion, the effect of calcium on tryptophan hydroxylase was very small in the new born rat when the activity of the neutral proteinase was low. In addition, the activating effect of Ca²⁺ could be antagonized not only by a chelating agent like EGTA but also (partially) by specific inhibitors of proteinases such as benzethonium and PMSF.
These results strongly suggest that the activation of tryptophan hydroxylase by calcium is the consequence of a partial proteolysis of the enzyme by the calcium-dependent neutral proteinase. Therefore, the physiological significance of this irreversible effect is doubtful.     

NERVE GROWTH FACTOR AND THE ACTIVITY OF TYROSINE HYDROXYLASE IN ORGAN CULTURES OF RAT SUPERIOR CERVICAL GANGLIA

Abstract— Superior cervical ganglia from young rats were cultured in the absence of serum. The effect of nerve growth factor on the level of tyrosine hydroxylase was studied. In the absence of nerve growth factor the specific activity of tyrosine hydroxylase fell by more than 50% within 48 h. In the presence of nerve growth factor the total and specific activities were maintained and even increased in the same period. Both the 2.5 S and the 7 S forms of nerve growth factor were effective. Oxidized nerve growth factor had no effect except when present in very high concentration. Purified antibody to nerve growth factor was inhibitory. Insulin had only a slight effect in this system, but dibutyryl CAMP elevated tyrosine hydroxylase activity substantially. Propranolol inhibited the action of nerve growth factor but its action appeared to be nonspecific and unrelated to its action on the β-adrenergic receptor. Changes in the activity of dihydropteridine reductase paralleled those seen in tyrosine hydroxylase.     

BRAIN TYROSINE HYDROXYLATION: ALTERATION OF OXYGEN AFFINITY IN VIVO BY IMMOBILIZATION OR ELECTROSHOCK IN THE RAT

Abstract— The rates of brain tyrosine and tryptophan hydroxylation, estimated in vivo from the accumulation of DOPA and 5-hydroxytryptophan after the administration of a decarboxylase inhibitor, appear dependent on the availability of oxygen as a substrate. During two types of physical stress, electroshock and curare-immobilization, the rate of brain tyrosine hydroxylation was greater than in unstressed controls and was not significantly decreased when the stresssed animals were made hypoxic. The loss of oxygen dependence by brain tyrosine hydroxylation during stress was observed in several brain regions and was not associated with alterations in the concentrations of brain tyrosine. tryptophan, serotonin, dopamine or norepinephrine. The rate of brain tryptophan hydroxylation was not affected by stress and remained oxygen dependent. The increase in catecholamine synthesis during stress appears to be the result of increased catecholaminergic nerve impulse flow. These experiments are consistent with the hypothesis that during neuronal stimulation an allosteric change in tyrosine hydroxylase increases the affinity of the enzyme for oxygen allowing greater catecholamine synthesis despite limiting concentrations of this substrate.     

THE DE NOVO SYNTHESIS OF TYROSINE HYDROXYLASE IN RAT SUPERIOR CERVICAL GANGLIA IN VITRO: THE EFFECT OF NERVE GROWTH FACTOR

Abstract— An immunoprecipitation technique has been employed to measure the rate of synthesis of tyrosine hydroxylase in organ cultures of rat superior cervical ganglia and the effect of nerve growth factor on that rate. Ganglia which have been maintained in culture for 16 h without nerve growth factor synthesize tyrosine hydroxylase; the hydroxylase comprises approx 0.2% of the newly synthesized soluble protein. While the total amount of tyrosine hydroxylase synthesized de novo increases in the presence of physiological levels of nerve growth factor, the differential rate of tyrosine hydroxylase synthesis is essentially unchanged. At higher levels of nerve growth factor (3–10 μg/ml) there is a small increase in the differential rate of tyrosine hydroxylase synthesis. The major action of nerve growth factor appears to be on the survival of the tissue, but a small effect on the induction of tyrosine hydroxylase is evident at high levels of nerve growth factor.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.     

DEVELOPMENTAL AND REGIONAL VARIATIONS IN NEUROTRANSMITTER-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN CELL-FREE PREPARATIONS FROM RAT BRAIN

Investigations have been carried out on regional and developmental variations in the properties of adenylate cyclase systems in participate preparations from rat brain. EGTA was routinely included in the assay medium to minimize differences in the state of activation of these systems resulting from variations in their exposure to endogenous Ca²⁺. At birth, adenylate cyclase activity was much higher in the hindbrain-medullary preparations than in comparable fractions from cerebellum, cerebral cortex or subcortex (including midbrain, corpus striatum, hypothalamus and hippocampus). Adenylate cyclase activity increased during early development in preparations from all areas of the brain. Maximal levels were reached at 14 days of age or later. These levels were not greatly altered in the young adult animal, except in the hindbrain-medullary area, where a decrease in activity was observed. Adenylate cyclase systems in cerebral cortical and subcortical preparations were activated by norepinephrine and dopamine throughout development. Serotonin also stimulated adenylate cyclase activity in these preparations from young animals but was much less effective in comparable fractions from adult rats. The response to dopamine was diminished with age in cerebral cortical preparations, but not in subcortical fractions. The responses to norepinephrine increased in both brain regions during early development. Adenylate cyclase systems in particulate preparations from the cerebellum and hindbrain-medullary areas exhibited relatively poor responses to the biogenic amines. Detailed studies of the properties of the cerebral cortical adenylate cyclase systems revealed enhancement of activity by Ca²⁺ and F^? at all stages of development with the maximal activation at 2–3 weeks of age. The results suggest that developmental differences in hormonal sensitivity of adenylate cyclase systems from diverse areas of the brain are related to changes in the proportions of the receptor-enzyme complexes responsive to the different biogenic amines.     

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.