THE METABOLISM OF TRITIATED DOPAMINE IN REGIONS OF THE RAT BRAIN IN VIVO—I

Abstract— A simple combination of acid- and base-exchange resin columns enabled a more complete separation of amine, acid and neutral metabolites of catecholamines from individual small brain samples to be made. Fractions containing individual catecholamines or metabolites were obtained in aqueous eluates suitable for fluorimetric or radioisotopic analysis. With consistent intraventricular injection and brain dissection techniques, this separation method enables a study of the metabolism of catecholamines in regions of the rat brain and the effects of drugs on this metabolism.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

大鼠脑皮层中多巴胺受体的检定

本研究使用放射配基结合分析法,建立了对大鼠脑皮层多巴胺Ｄ＿１和Ｄ＿２型受体的检测方法。测得正常大鼠脑皮层中Ｄ＿１受体的Ｂ＿ｍａｘ,为１０３±５３ｐｍｏｌ／ｇ蛋白,Ｋ＿ｄ值为３．２±１．２ｎｍｏｌ／Ｌ;Ｄ＿２受体的Ｂ＿ｍａｘ,为２２７±７９ｐｍｏｌ／ｇ蛋白,Ｋ＿ｄ值为２．６±ｌ．３ｎｍｏｌ／Ｌ。     

METABOLISM OF THE ASPARTYL MOIETY OF N-ACETYL-l-ASPARTIC ACID IN THE RAT BRAIN

The metabolism of N-acetyl-l -aspartic acid (NAA) was studied in rat brain. [Aspartyl-U-¹⁴C]NAA was metabolized predominantly by deacylation. Studies of NAA biosynthesis from l -[U-¹⁴C]aspartic acid have confirmed previous reports that NAA turns over slowly in rat brain. However, intracerebrally-injected N-acetyl-l -[U-¹⁴C]asparticacid was rapidly metabolized. Exogenous NAA appears to be taken up rapidly into a small, metabolically-active pool. This pool serves as substrate for a tricarboxylic acid cycle associated with the production of glutamate for the biosynthesis of glutamine. The bulk of the NAA content in brain appears to be relatively inactive metabolically.     

DOI诱导的大鼠头部抽动模型脑内多巴胺转运体的研究

目的研究DOI诱导的大鼠头部抽动模型脑内多巴胺转运体改变,探讨Tourette综合征发病与突触前多巴胺神经纤维支配的关系。方法将30只Wistar大鼠随机分为模型组和对照组,应用DOI诱导大鼠头部抽动作为Tourette综合征动物模型。两组以99mTc-TRODAT-l作为配体,采用放射自显影和γ测量方法观察大鼠脑内多巴胺转运体分布及其密度。结果放射自显影结果表明模型组大鼠脑内银颗粒密度显著高于对照组,尤其是纹状体。γ测量结果显示,模型组纹状体每克脑组织含放射性占注射剂量的百分比(%ID/g)与对照组相比显著增高(P<0.01),模型组海马和大脑皮层与对照组相比有统计学差异(P<0.05)。结论DOI诱导的大鼠头部抽动模型脑内多巴胺转运体密度较正常大鼠显著增加,Tourette综合征发病与突触前多巴胺神经纤维支配增加密切相关。     

CHOLINE ACETYLTRANSFERASE CONTENT IN DISCRETE REGIONS OF THE RAT BRAIN STEM

—Choline acetyltransferase (ChAc) content of 50 separate rat brain stem nuclei and cerebellum removed by microdissection was determined using a sensitive radiometric assay. The distribution of ChAc activity is uneven, with extremely high levels in the cranial motor nuclei and the nucleus salivatorius. Low ChAc concentrations were observed in the cranial sensory nuclei, the nuclei of the reticular formation, the raphe nuclei and the nuclei of the acoustic system. The lowest ChAc levels were measured in the cerebellum. Comparison of the distribution of ChAc with histochemical localization of acetylcholinesterase revealed generally good agreement, and notable exceptions are discussed.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

THE SUBCELLULAR LOCALIZATION OF HISTIDINE DECARBOXYLASE IN VARIOUS REGIONS OF RAT BRAIN

Abstract— The subcellular distribution of histidine decarboxylase (assayed by two different isotopic methods) and several biochemical markers (lactate dehydrogenase, DOPA decarboxylase and protein) was determined in rat cerebral cortex. After differential centrifugation, the enzyme activity was found mainly in the crude mitochondrial and soluble fractions. Further separation of the former on discontinuous sucrose gradients showed that the particulate histidine decarboxylase (HD) was found in the synaptosomal fraction. After osmotic shock, HD activity appeared in the supernatant fraction suggesting that a major portion of the enzyme is localized in the cytoplasm of cortical nerve endings. By analogy with other brain amines, this finding, together with the presence of histamine in synaptic vesicles (K ataoka and de R obertis , 1967), can be taken as further support for the hypothesis of a role as neurotransmitter for histamine.
Various brain regions were homogenized under conditions leading to synaptosome formation. The distribution of HD between 'particulate' and soluble fractions differed from one region to the other, but did not give any clear-cut indication of regions rich in cell bodies or nerve terminals.     

THE EFFECT OF FASTING ON THE METABOLISM OF 5-HYDROXYTRYPTAMINE AND DOPAMINE IN THE BRAIN OF THE MOUSE

Abstract— The turnover of 5-hydroxytryptamine in the forebrain and of dopamine in the striatum was studied in mice fasted for 20 h. Such mice showed an increased tissue concentration of 5-hydroxyindoleacetic acid in the forebrain and an increased accumulation of this acid after probenecid. Fasted mice also showed a higher concentration of homovanillic acid in the striatum than fed mice. However, the administration of probenecid produced a smaller increase in homovanillic acid concentration in fasted than in fed mice. The decay of dopamine following α-methyl- p -tyrosine was reduced in fasted mice at 2 h, but not at 1 h or 6 h after administration of the inhibitor. The possibility that fasting increases the activity of some dopaminergic neurones while decreasing the activity of others is considered. The existence of a pool of homovanillic acid at a site within the striatum where the probenecid-sensitive transport is not effective is postulated.     

SYNTHESIS AND METABOLISM OF l-KYNURENINE IN RAT BRAIN

Abstract— A method for the quantitative analysis of femtomole amounts of kynurenine (along with tryptophan, 3-hydroxykynurenine and kynuramine) in rat brain using high pressure liquid chroma-tography and electron-capture GLC is described. Endogenous concentrations of these substances in rat brain regions were measured, and their formation after the injection of radioactive tryptophan or kynurenine was determined. Kynurenine was formed from tryptophan in brain and was also taken up from the periphery. Extracerebral kynurenine was calculated to account for 60% of the cerebral pool of kynurenine. The cerebral rates of synthesis of kynurenine and 3-hydroxykynurenine were 0.29 and 0.17nmol/g/h. The turnover rate of kynurenine in the brain was 1.02 nmol/g/h measured from [¹⁴C]tryptophan or 1.14 nmol/g/h from [³H]kynurenine injected intraperitoneally. Kynuramine levels in different areas of the brain were similar to those of tryptamine. Following intraperitoneal injection of [¹⁴C]tryptophan, the presence of anthranilic, 3-hydroxyanthranilic, xanthurenic, kynurenic and quinaldic acids was demonstrated in the brain.     

METABOLISM OF A TRYPTOPHAN LOAD IN THE HYPOTHALAMUS AND OTHER BRAIN REGIONS

Abstract— Results confirm previous findings that after injecting rats with 50mg/kg tryptophan the percentage increase of 5-hydroxytryptamine metabolism (as shown by 5-hydroxyindolylacetic acid changes) is particularly small in the hypothalamus. However, 15–30 min after tryptophan injection (when brain 5-hydroxytryptamine changes were maximal) percentage 5-hydroxytryptamine increases in the hypothalamus and in the rest of the brain were comparable. The small 5-hydroxyindolylacetic acid changes in the hypothalamus are consistent with a long 5-hydroxytryptamine turnover time therein as indicated by experiments using pargyline or probenecid and by the relatively small increases of 5-hydroxytryptamine after injecting tryptophan into tranylcypromine treated rats. When 5-hydroxytryptamine synthesis was partially inhibited by p -chlorophenylalanine and tryptophan was injected, there was a large percentage rise of hypothalamic 5-hydroxytryptamine but the concentration found in rats given neither drug was not attained and 5-hydroxyindolylacetic acid showed little change. Elsewhere in the brain 5-hydroxytryptamine attained concentrations comparable to those in rats given neither drug and 5-hydroxyindolylacetic acid rose considerably. Results are discussed in relation to the contributions made to brain 5-hydroxytryptamine turnover by functional and non-functional metabolism.     

DEVELOPMENT OF MITOCHONDRIAL PYRUVATE METABOLISM IN RAT BRAIN

The activities of a number of mitochondrial enzymes involved in the metabolism of pyruvate during development of the rat brain were investigated. The rates of decarboxylation of [1-¹⁴C]pyruvate to ¹⁴CO₂ via pyruvate dehydrogenase and the fixation of H¹⁴CO₃^? in the presence of pyruvate via pyruvate carboxylase by brain homogenates were very low in newborn rats. These rates increased markedly by about four-fold and 15-fold respectively during 10–35 postnatal days. The rates of the fixation of H¹⁴CO₃^? by cerebral homogenates were supported by the development of the activity of pyruvate carboxylase in rat brain. The activities of citrate synthase, aconitase, NAD-malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and phosphoenol-pyruvate carboxykinase were very low in the particulate fraction of the newborn rat brain. The activities of all these enzymes increased makedly by about three- to 10-fold during 10–35 days after birth. The activity of mitochondrial phosphoenolpyruvate carboxykinase from rat brain was not precipitated by an antibody prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase suggesting that cerebral mitochondrial enzyme is immunologically different from that of the cytosolic form in hepatocytes. The significance of the development of the cerebral mitochondrial metabolism is discussed in relation to biochemical maturation of the brain.     

REGIONAL BRAIN STUDY OF INDOLEAMINE METABOLISM IN THE RAT IN ACUTE HEPATIC FAILURE

Abstract— Tryptophan, 5-hydroxytryptamine and 5-hydroindoleacetic acid were found to be greatly increased in various parts of the brains of rats in acute hepatic failure following two stage hepatic devascularization. However, the increases in 5-hydroxytryptamine and 5-hydroxyindoleacetic acid varied by region and are not explicable solely in terms of increased concentrations of tryptophan. The results are discussed in terms of differences in the regional metabolism of 5-hydroxyindoleamines. Plasma free fatty acids, albumin, total tryptophan and free tryptophan were measured in plasma in hopes of elucidating the mechanism responsible for the cerebral elevation of tryptophan. Increased plasma free tryptophan appears sufficient to explain the rapid increase in brain tryptophan. The relationship between these results and recent observations in hepatic encephalopathy is discussed.     

EFFECTS OF DI-ISOPROPYLFLUOROPHOSPHATE ON THE METABOLISM OF CHOLINE AND PHOSPHATIDYLCHOLINE IN RAT BRAIN

—The subcutaneous administration of 2·0 mg DFP per kg to rats causes a diminution in the lysophosphatidylcholine content in the brain, which is followed by a decrease of glycerylphosphorylcholine concentration and by a reduced post mortem choline increase. This supports the hypothesis that a post mortem increase in choline is due to phosphatidylcholine breakdown. Since the amount of phosphatidylcholine in brains of di-isopropylfluorophosphate-treated rats increases, it is concluded that phospholipase A is inhibited by di-isopropylfluorophosphate, which corresponds to findings of other authors in vitro. The activity of glycerylphosphorylcholine diesterase (EC 3.1.4.2) is not altered.     

THE EFFECTS OF INTOXICATING DOSES OF ETHANOL UPON INTERMEDIARY METABOLISM IN RAT BRAIN

Abstract— The effect of acute (8-min) and prolonged (13-h) exposures to high doses of ethanol upon the intermediary metabolites of rat brain has been studied, with the use of a new freezing technique which minimizes post-mortem changes. Injection of ethanol (80 mmol/kg body wt) produced general anaesthesia within 8 min after administration. At this time there were increases in the brain contents of glucose, glucose-6-phosphate and citrate; there was no change in arterial pCO₂. Rats under ethanol anaesthesia for 13 h showed increases in brain contents of glycogen, glucose and glucose 6-phosphate; and decreases in lactate, pyruvate, α-oxoglutarate and malate. Under similar experimental conditions, arterial pCO₂, increased from 37 to 51 Torr. The changes in levels of metabolites after injection of ethanol were similar to those after administration of many volatile anaesthetic agents or elevation of brain CO₂ by other means. Although brain levels of malate and α-oxoglutarate decreased after prolonged exposure to ethanol, the mitochondrial redox state was maintained. Accordingly, the levels of glutamate and aspartate fell in accordance with the law of mass action. The maintenance of the cytoplasmic and mitochondrial redox states in the brain during ethanol intoxication was in marked contrast to the effects on the liver. We suggest that the different effects observed in brain and liver result from the action of ethanol upon the nerve cell membrane in brain, whereas the primary target in liver is alcohol dehydrogenase.     

EFFECTS OF ELECTRICAL STIMULATION OF THE NIGROSTRIATAL PATHWAY OF THE RAT ON DOPAMINE METABOLISM

Abstract— Electrical stimulation of the nigrostriatal dopaminergic pathway in rat brain elicited a frequency and current intensity-dependent increased in the formation of homovanillic acid in the basal ganglia. The accumulation of the acid in probenecid-treated animals was constant over 1 h, when maximally stimulated at 25 Hz and 300 μA. Dopamine levels remained unchanged during stimulation. When prior to stimulation the inhibitor of catecholamine synthesis α-methyl- p -tyrosine methyl ester was administered, dopamine levels declined biphasically. Tyrosine and nomifensine, a dopamine uptake inhibitor, and apomorphine had no major effect on the formation of homovanillic acid, whilst α-methyl- p -tyrosine prevented its formation. Our data suggest that dopamine in the striatum is compartmentalized and that the newly-synthesized amine is released and converted to homovanillic acid. Apomorphine decreases dopamine flux only when dopaminergic neurons are at rest. When depolarized neither access of the precursor nor reuptake seem to influence the conversion of dopamine to homovanillic acid.