IN VITRO STUDIES ON THE INTERACTION OF BRAIN MONOAMINE OXIDASE WITH 5,6- AND 5,7-DIHYDROXYTRYPTAMINE12

[¹⁴C]5,6-Dihydroxytryptamine ([¹⁴C] 5,6-DHT) and [¹⁴C]5,7-dihydroxytryptamine ([¹⁴C]5,7-DHT) were deaminated to toluene-isoamylalcohol extractable products when incubated with homogenates of rat hypothalamus or pons-medulla oblongata. [¹⁴C]5,6-Dihydroxyindole acetic acid ([¹⁴C]5.6-DHIAA) and [¹⁴C]5,7-dihydroxyindole acetic acid ([¹⁴C]5,7-DHIAA) were detected as MAO metabolites by TLC besides non-identified components. The conversion of [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT obeyed, at least initially, Michaelis-Menten kinetics (K_m 5,7-DHT: 0.5 × 10^?3M; K_m 5,6-DHT: 1.25 × 10^?3M). Inhibition of the reaction by the MAO A inhibitor, clorgyline, resulted in a typical double sigmoidal inhibition curve indicating that both amines are metabolized by both types of MAO (A and B). In deprenyl inhibition studies, however, 5,7- and 5,6-DHT seemed to be preferred substrates of MAO A. Incubation of rat brain homogenates with [¹⁴C]5,6-DHT and [¹⁴C]5,7-DHT or with the MAO metabolites [¹⁴C]5,6-DHIAA and [¹⁴C]5,7-DHIAA caused a time-dependent break-down of the dihydroxylated indole compounds with subsequent binding of radioactivity to perchloric acid insoluble tissue components. 5,6-DHT inactivated MAO in rat brain homogenates parallel to its decomposition and extensive protein binding. The inactivation of MAO by 5,6-DHT and the extensive binding of radioactivity to protein were antagonized by dithiothreitol (DTT), glutathione (GSH) and L-ascorbic acid. Reduction of [O₂] in the incubation medium slightly attenuated the inactivation of MAO by 5,6-DHT. Catalase or superoxide dismutase failed to prevent MAO from being inactivated by 5,6-DHT. The results suggest that oxidation products of 5,6-DHT, e.g. its corresponding o-quinone, are involved in the inactivation of MAO in vitro and mainly responsible for the binding of radioactivity to brain proteins in vitro. Similar mechanisms may also be operative in the in vivo neurotoxicity of 5,6-DHT. The lack of inactivation of MAO by 5,7-DHT in vitro correlated with a low degree of radioactivity binding (from [¹⁴C]5,7-DHT) to homogenate protein pellets; the binding to proteins was barely influenced by GSH, cysteine, DTT and l -ascorbic acid. These latter findings do not provide a plausible explanation for the mechanism(s) involved in the well known in vivo neurotoxicity of 5,7-DHT.     

EFFECT OF OESTRADIOL ON TURNOVER OF TYPE A MONOAMINE OXIDASE IN BRAIN

Abstract— Administration of oestrogen (oestradiol-17β or oestradiol-17β-benzoate) to ovariectomized (OVX) rats for 1–4 weeks results in an approx 30% decrease in the activity of monoamine oxidase (MAO) in the basomedial-hypothalamus (BM-Hyp) and corticomedial-amygdala (CM-Amy) but not in cerebral cortex. Further investigation shows that (1) decreased MAO activity in the BM-Hyp and CM-Amy occurs only in Type A MAO (serotonin as substrate) and does not occur in Type B MAO (phenylethylamine as substrate); (2) decreased MAO activity does not occur when a single large dose of oestrogen is given i. v. or when homogenates from oestrogen treated rats are mixed with homogenates from OVX rats suggesting that direct enzyme inhibition is not responsible for the change in activity; (3) oestrogen administration to OVX rats increases the rate constant of degradation for MAO in BM-Hyp and CM-Amy but not in cerebral cortex as determined in turnover studies using pargyline, an irreversible inhibitor of MAO. The increased rate of degradation results in shorter half lives ( t 1/2) for MAO in the BM-Hyp and CM-Amy of oestrogen treated rats. In OVX rats the t 1/2 is 9.8 days in BM-Hyp and 12.7 days in CM-Amy. Oestrogen administration results in a t 1/2 of 7.6 days in BM-Hyp and 7.8 days in CM-Amy. The possible relationship between oestrogen dependent decreased MAO activity and estrogen dependent lordosis behavior is 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.     

NONEXISTENCE OF A TYPE C MONOAMINE OXIDASE IN RAT BRAIN

Abstract— The possible existence of type C MAO, distinct from type A and type B, in circumventricular structures of rat brain was examined by histological studies on the inhibitory effects of clorgyline. a preferential type A MAO inhibitor and deprenyl, a preferential type B inhibitor, on enzyme. Brain slices were preincubated with the inhibitors and then incubated with 5-HT, the substrate for type A MAO, and stained for MAO activity. Deposits of the product formazan were detected in circumventricular structures of slices of brain preincubated with clorgyline and deprenyl at concentrations of 10^-7–10^-4m at room temperature for 5 min. When the slices were preincubated with either of these inhibitors at room temperature for 60 min, strong activity was observed in this region, whereas when they were preincubated with either 10^-5m -clorgyline or 10^-5m -deprenyl for 20 and 30 min at 37°C, no MAO activity was seen in any region of the brain. Thus, at the higher preincubation temperature, lower concentrations of each inhibitor and a shorter preincubation period were required for inhibition of the enzyme. Preincubation for 60 min at 37°C with a combination of 10^-7m -clorgyline and 10^-8m -deprenyl did not inhibit the enzyme in the circumventricular region completely, but at the same temperature, concentrations of 10^-7m of both inhibitors inhibited the enzyme completely in 10min, Thus the effects of the inhibitors are synergistic. These results indicate that the inhibitory effects of the two inhibitors on the enzyme in circumventricular structures of the brain is time- and temperature-dependent. Moreover, the activity seems to be sensitive to deprenyl even when 5-HT is used as substrate. The results do not support the idea of the existence of type C MAO, distinct from type A and type B MAO.     

SOME EFFECTS OF MONOAMINE OXIDASE INHIBITORS ON THE METABOLISM OF γ-AMINOBUTYRIC ACID IN RAT BRAIN

(1) The inhibitor of γ-aminobutyrate transaminase (GABA-T), amino-oxyacetic acid (AOAA), drastically reduced the activity of GABA-T to 30 per cent of the control value, with a corresponding increase of brain GABA, but had no effect on the activity of glutamate decarboxylase (GAD). (2) The monoamine oxidase (MAO) inhibitors phenelzine, phenylpropylhydrazine and phenylvalerylhydrazine, lowered GABA-T activity to 58, 49 and 48 per cent, respectively; this was associated with a marked elevation of brain GABA. (3) The action of phenelzine and phenylpropylhydrazine in vivo and in vitro could be abolished by pre-treatment of the tissue with the structurally related MAO inhibitors phenylisopropylhydrazine and trans-2-phenylcyclopropylamine. These had no action on the GABA system in vivo, either on the GABA content or on the GABA-T activity. These latter drugs, however, were unable to influence the effects of AOAA either on GABA or on GABA-T. (4) The possible mechanism of action on GABA and the enzyme activities of the GABA system is discussed.     

PURIFICATION AND PROPERTIES OF MITOCHONDRIAL MONOAMINE OXIDASE IN BEEF BRAIN

Abstract— Monoamine oxidase was purified approximately 40-fold from beef brain mitochondria. The purification procedure involved extraction with a non-ionic detergent (Nonion NS-210) after heat treatment, ammonium sulphate fractionation, chromatographies on DEAE-cellulose and Sepharose 6B, and a continuous flow electrophoresis. A major component (enzyme 1) with a higher specific activity and a minor component (enzyme 2) with a lower specific activity were separated. Properties of both enzymes towards kynuramine including pH-optimum and K_m values were similar, but the enzyme 1 had the higher specific activity towards tyramine whereas that of enzyme 2 was towards normetane-phrine. Fluorescence spectra indicated that the enzyme 1 is a flavoprotein. Copper was not detected, and copper chelating agents did not inhibit the enzyme. p -Chloromercuribenzoate and JV-ethylmaleimide inhibited the enzyme, indicating the presence of the essential SH-groups.     

THE EFFECTS OF METOPIRONE AND ADRENALECTOMY ON THE REGULATION OF THE ENZYMES MONOAMINE OXIDASE AND CATECHOL-O-METHYL TRANSFERASE IN DIFFERENT BRAIN REGIONS

Abstract— The role of glucocorticoids in the regulation of the enzymes monoamine oxidase (MAO) and catechol- O -methyltransferase (COMT) in brain regions has been studied. Glucocorticoids were blocked by Metopirone. The activities of MAO and COMT were determined in the hypophysis, hypothalamus, pineal gland and in the rest of brain. All the cerebral tissues except the pineal gland demonstrated highest MAO activity 8 h after Metopirone administration, when glucocorticoids were at the lowest level. Prolonged treatment for 10 days significantly augmented MAO activity in brain, hypophysis and hypothalamus, and COMT in the hypophysis increased by 56 per cent. The COMT activity in the rest of the brain did not change significantly with either short or prolonged administration. Complete ablation of the adrenal cortex resulted in a 167 per cent rise in MAO activity of the hypophysis. Metopirone and hydrocortisone inhibit MAO and COMT in vitro. The results suggest that glucocorticoids in the circulation of normal animals inhibit the activities of MAO and COMT. The inhibition or ablation of these hormones removes this rate-limiting control of catecholamine degradation resulting in higher activities of MAO and COMT. Metopirone, an inhibitor of MAO and COMT in vitro , acts in the opposite direction in vivo due to its inhibitory effects on corticoid biosynthesis.     

EFFECT OF THE ADMINISTRATION OF L-5-HYDROXYTRYPTOPHAN AND A MONOAMINE OXIDASE INHIBITOR ON GLUCOSE METABOLISM IN RAT BRAIN

The effects of the presence of large amounts of 5-HT and of its precursor 5-HTP in brain on cerebral utilization of glucose were studied. [U-¹⁴C]Glucose was injected to fed rats that had previously been treated with L-5-HTP, L-5-HTP and an inhibitor—N-[β-(2-chlorophenoxy)-ethyl]-cyclopropylamine hydrochloride (Lilly-51641)-of MAO, or Lilly-51641 alone. Such treatment increased the concentrations of 5-HTP and 5-HT in the brain. After treatment with 5-HTP and Lilly-51641, and to a lesser extent with Lilly-51641 alone, the concentration of glucose in plasma was increased. However, the uptake of glucose by the brain did not appear to be proportionately increased, and this suggested an impairment in this mechanism. After the administration of Lilly-51641 alone and more especially of Lilly-51641 plus 5-HTP, the concentration of glucose in the brain was increased. This increase was thought to be due to an impairment of glucose utilization, because the flux of ¹⁴C from glucose to amino acids in the brain was reduced. The concentrations of most major amino acids in the brain were not greatly affected by these treatments. GABA and alanine concentrations in the brain were modestly increased after treatment with 5-HTP alone or in combination with Lilly-51641. The present results suggest that the metabolism of glucose to amino acids in the brain is altered when the concentration of 5-HTP, or more especially that of 5-HT, in the brain is increased.     

大豆磷脂对老龄大鼠单胺氧化酶,血小板,动脉内膜的影响

本文观察了饲料添加剂大豆磷脂对老龄大鼠脑组织单胺氧化酶(MAO)活力、血小板聚集功能及主动脉内膜的影响。结果表示喂大豆磷脂24月龄大鼠组的大脑皮层、小脑、海马的MAO活力较对照组高,接近3月龄组;二磷酸腺苷(ADP)诱导血小板释放三磷酸腺苷(ATP)的量高于3月龄组,低于对照组;主动脉内膜扫描电镜观察内皮细胞轮廓清晰可见,有少数桥样结构,表面未见斑块样附着物,对照组则呈粥样硬化形态特征。上述结果提示,大豆磷脂作为饲料添加剂对某些衰老现象有明显的改善。     

单胺氧化酶的抑制作为杀虫脒对澳氰菊酯的增效作用

杀虫脒对拟除虫菊酯具有增效作用, 其与溴氰菊酯的共毒系数高达890.3, 增效显著.当用溴氰菊酯单独处理美洲蜚蠊时, 血淋巴内酪胺增加125%, 章鱼胺增加108%;当杀虫脒与溴氰菊酯(1:1)混用处理美洲蜚蠊时, 引起酪胺及章鱼胺增加的量更多, 其分别增加925%及500%, 杀虫脒对蜚蠊体内的单胺氧化酶(MAO)有显著的抑制作用.由此推断, 杀虫脒抑制单胺氧化酶后, 引起酪胺及章鱼胺的积累(不能进行氧化脱氨所致), 造成不正常的生理效应, 这应是杀虫脒对溴氰菊酯的增效机制.     

DETERMINATION OF MONOAMINE OXIDASE IN RAT BRAIN BY ELECTRON SPIN RESONANCE TECHNIQUES

—The recent development of spin-labeling techniques has permitted its application to the measurement of the concentration of rat brain mitochondrial monoamine oxidase (MAO) and detection of possible multiple forms of this enzyme. Spin-labeled p-hydroxyamphetamine (SHA) was used as a probe of the active site of the enzyme. The binding of SHA to MAO was monitored by measuring the intensity of the × band ESR spectra obtained with a Varian V-4502 ESR spectrometer. The measured volume of each sample was about 0·02 ml and time required for each measurement was 5 min. In the presence of MAO, a linear relationship was observed between spectral intensity and the concentration of the spin-label in a range of 4 × 10^?6m to 10^?3m . A plot of the amount of enzyme-inhibitor complex formed against the concentration of SHA suggested the existence of at least three different affinities of SHA to the possible multiple forms of MAO. Measurements made using a constant concentration of the spin-label (3·21 × 10^?5m ) resulted in a calibration curve with two plateaus when the concentration of the enzyme-inhibitor complex was plotted against the apparent concentration of MAO. These data also suggested that spin-labeled hydroxyamphetamine had distinctly different affinities toward the multiple forms of MAO that were present in the partially purified preparation employed. Measurements made with constant concentrations of SHA and MAO but with varying temperatures (4-56°C) resulted in a curve with two plateaus which also suggested the existence of at least three different binding affinities of the enzyme preparation for the inhibitor. This ESR technique is simple, rapid, accurate and fairly sensitive. It was also applied to the measurement of MAO in human platelets.     

STUDIES IN VIVO ON THE RELATIONSHIP BETWEEN BRAIN TRYPTOPHAN, BRAIN 5-HT SYNTHESIS AND HYPERACTIVITY IN RATS TREATED WITH A MONOAMINE OXIDASE INHIBITOR AND L-TRYPTOPHAN

Abstract— The effect of l -tryptophan loading upon the amount of 5-HT accumulating in the brains of rats pretreated with a monoamine oxidase inhibitor was studied. The amount of brain 5-HT accumulated increased with increasing tryptophan dosages and brain tryptophan concentrations up to a tryptophan dose of 120 mg/kg body wt. and a brain tryptophan of about 70 μg/g brain. Above this dose and concentration no further increase in brain 5-HT accumulation occurred. After monoamine oxidase inhibition and tryptophan loading gross hyperactivity and hyperpyrexia occurred. Monoamine oxidase inhibition, tryptophan administration and intact aromatic amino acid decarboxylase activity were all collectively essential for the production of hyperactivity and hyperpyrexia. DL-Parachlorophenyl-alanine prevented both the occurrence of hyperactivity and the increased accumulation of, brain 5-HT. Indices of hyperactivity correlated with the amount of brain 5-HT accumulating in 1 h after tryptophan loading but not with the overall concentration of brain 5-HT, suggesting that hyperactivity was dependent upon the rate of 5-HT synthesis. Reserpine and tetra-benazine pretreatment speeded the onset and rate of development of the hyperactive state without altering the synthesis of brain 5-HT. It is suggested that when monoamine oxidase is inhibited and the rate of 5-HT synthesis is increased, granular uptake and storage of 5-HT and other rate-limiting mechanisms for 5-HT inactivation are unable to prevent 5-HT 'spilling over’to produce hyperactivity. The crucial dependence of 5-HT synthesis upon brain tryptophan concentration and the ability of intraneuronal metabolism, when monoamine oxidase activity is intact, to cope with increased 5-HT synthesis and prevent ‘spillover’, raise the possibility that brain 5-HT synthesis is normally in excess of functional needs, and suggest that intraneuronal metabolism and the intraneuronal organization of 5-HT pools are of more importance than synthesis in regulating the amount of 5-HT available for functional activity.     

MONOAMINE OXIDASE (EC 1.4.3.4): ISOLATION AND CHARACTERIZATION OF MULTIPLE FORMS OF THE BRAIN ENZYME

Monoamine oxidase (MAO) in crude mitochondrial preparations from rat brain was solubilized, and different MAO-active fractions were separated by agarose columns and by Sephadex electrophoresis. Any combination of these techniques yielded at least three fractions possessing MAO activity as measured by assays using radioactive serotonin and benzylamine as substrates. The molecular weight of one of the MAO forms was found to be approximately 400,000 daltons while another was at least 1.5 × 10⁶ daltons. The crude mitochondria1 MAO was inhibited by [¹⁴C]-labelled pargyline and then solubilized and the radioactivity of the soluble and particulate MAO was compared to the enzyme activity found in the soluble and particulate fractions. Our studies suggest that appreciable MAO activity is lost upon solubilization and that the conformation of MAO may be altered.     

THE EFFECT OF MONOAMINE OXIDASE INHIBITORS ON SOME ARYLALKYLAMINES IN RAT STRIATUM

The trace amines phenylethylamine, tryptamine, p-tyramine and m-tyramine have been measured in the striatum of both control and MAO-treated rats. Dose-response and time-response studies have been carried out with clorgyline and deprenyl, inhibitors which preferentially inhibit the A and B forms of MAO, respectively, and with tranylcypromine and phenylethylhydrazine, which are used clinically in the treatment of depression. Phenylethylamine was increased by 1 mg/kg of deprenyl, but was unaffected by clorgyline at doses up to 50 mg/kg, while the tyramines and tryptamine were increased by low doses of clorgyline, but were increased only by much greater doses of deprenyl than those required to affect phenylethylamine. Phenylethylamine is oxidized by the B form of MAO, but tryptamine and the tyramines appear to be oxidized by both A and B MAO. The observed proportional increases in trace amine levels are much greater than those observed for the classical neurotransmitters, noradrenaline, dopamine and 5-hydroxytryptamine. As these increases are differential, selective manipulation of trace amine concentrations is possible.     

HISTOCHEMICAL MAPPING OF MONOAMINE OXIDASE AND LACTIC DEHYDROGENASE IN THE PONS AND MESENCEPHALON OF SQUIRREL MONKEY (SAIMIRI SCIUREUS)

The detailed distribution of lactate dehydrogenase and monoamine oxide has been described at various levels through the pons and mesencephalon of the brain of the squirrel monkey. A comparison of the two enzymes has given an interesting picture of their selective localization in the different nuclei. Particular attention has been paid to select sections at comparative levels. Marked monoamine oxidase activity has been observed in the nucleus interpeduncularis, the nucleus centralis superior, the nucleus annularis, and in the neuropil and in the cytoplasm of neurons of some other nuclei. Particularly strong lactate dehydrogenase activity has, however, been observed in the corpus geniculatum laterale and mediale, the nucleus pulvinaris, the superior and inferior colliculi, the substantia grisea centralis and some of the nuclei of cranial nerves.     

恒河猴红细胞免疫功能的研究

本文应用花环法测定了健康恒河猴红细胞上C_(3b)受体和免疫复合物的数量。恒河猴红细胞上C_(3b)受体花环率的数量为12.83±1.95％,免疫复合物花环率为6.37±1.25％。研究结果表明恒河猴的红细胞,除了具有携氧、运输气体等功能外,还具有重要的免疫功能。     

MULTIPLE FORMS OF MONOAMINE OXIDASE IN DEVELOPING BRAIN: TISSUE AND SUBSTRATE SPECIFICITIES

Brains, hearts and livers from newborn and adult rats were assayed for monoamine oxidase activity using gel electrophoretic techniques. The results suggest that each of the tissues possesses multiple forms (isoenzymes) of monoamine oxidase and that these forms are different for the various tissues. Further, the forms of monoamine oxidase in the neonatal tissues differ from those in the corresponding adult tissue. These different forms of monoamine oxidase have different substrate specificities. Using 5-hydroxy[¹⁴C]tryptamine as substrate, we have demonstrated that the monoamine oxidase patterns appearing on the gel do indeed possess monoamine oxidase activity.     

MULTIPLE BINDING SITES OF HUMAN BRAIN MONOAMINE OXIDASE AS INDICATED BY SUBSTRATE COMPETITION

Abstract— Six endogenous substrates of monoamine oxidase (EC 1.4.3.4) (serotonin, l -norepinephrine, dopamine, tyramine, tryptamine and β -phenethylamine) were used separately and in pairs with human brain mitochondrial extracts. Apparent K ₁ values were obtained from experiments in which only 1 of 2 substrates was isotopically labelled, and these values were compared with experimental K _m values. β -Phenethylamine appears to be metabolized at enzyme active sites independent from those which bind serotonin. The substrate l -norepinephrine competes with serotonin for an enzyme site, but also may be catalysed at an additional site which is independent of serotonin binding. Experiments in which [¹⁴C]tryptamine was combined with [³E]serotonin indicated that tryptamine is a much more potent inhibitor of serotonin oxidation than was predicted from K _m values. It is suggested that the competition among substrates of MA0 which is observed in uitro may have relevance to in uiuo mechanisms for control of biogenic amine concentrations.