Comparison of the Properties of Semipurified Mitochondrial and Cytosolic Monoamine Oxidases from Rat Brain

Mitochondrial and cytosolic monoamine oxidases were purified 220- and 129-fold, respectively, from rat brain. The purification procedure involved extraction (without the use of detergents for mitochondrial monoamine oxidase), ammonium sulfate precipitation, and chromatography on Sephadex G-25 and a DEAE-cellulose column. The properties of both enzymes with kynuramine as substrate, including Km values and pH optima at different kynuramine concentrations; the Rf values on polyacrylamide gel electrophoresis; and the thermal inactivation patterns were different. 2-Mercaptoethanol, together with heat treatment, released the flavin and decreased the enzyme activity differentially for the two enzymes. The absorption spectrum showed a "Red shift" in the absorption maxima when the spectra of the non-Triton-treated purified preparations were compared with those of the Triton-treated ones, thus possibly revealing that the mitochondrial and the cytosolic monoamine oxidases may be two different enzyme entities.     

Alterations of Tubulin Function Caused by Chronic Antidepressant Treatment in Rat Brain

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, G_s, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity.2. Male Sprague–Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what.3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin–GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats.4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between G_s and the catalytic subunit of adenylyl cyclase but also tubulin interaction with G_s in the cerebral cortex of the rat.     

Influence of Convulsants on Rat Brain Activities of Alanine Aminotransferase and Aspartate Aminotransferase

There exist differences between 12-day-old and adult rats in the onset of seizures induced by some inhibitors of glutamate decarboxylase (GAD). The aim of study was to investigate if there are differences between both groups in activities of rat brain alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the enzymes involved in glutamate metabolism, after the administration of 3-mercaptopropionic acid as specific GAD inhibitor or isoniazid as less specific general inhibitor of pyridoxal enzymes. Activities of both aminotransferases in a supernatant 20,000 g of the whole brain (containing predominantly cytosolic isoforms of enzymes) were increased at the beginning of 3-mercaptopropionic acid-induced generalized tonic-clonic seizures. At isoniazid-induced generalized tonic-clonic seizures, a significant increase in both enzyme activities was observed in adult rat brain. In the 12-day-old rat brain, ALT and AST activities reached about 40% and about 50–60% of adult control levels, respectively. In in vitro experiments, no influence of 3-mercaptopropionic acid on transaminase activities was found and an inhibitory effect of isoniazid on the enzymes was confirmed. Increased aminotransferase activities might participate in the enhanced synthesis of excitatory amino acid neurotransmitters in the nervous system, which may take a part in the initiation of epileptic seizures. Alternatively, the increased AST activity may be connected with an increased transport of NADH from the cytosol to mitochondria, while the increased ALT activity would represent the transformation of pyruvate to alanine as a consequence of increased glycolysis.     

Semicarbazide-Sensitive Amine Oxidase (SSAO) in the Brain

Semicarbazide-sensitive amine oxidase (SSAO) is widely distributed in almost tissues. However, its presence in brain microvessels is still controversial. The affinity of SSAO towards benzylamine (Bz) is considerably higher than that of monoamine oxidase (MAO). SSAO plays a role in the toxicity of several environmental and endogenous amines. SSAO-mediated production of toxic aldehydes has been proposed to be related to pathophysiological conditions. The most potent of inhibition of SSAO in monkey brain was observed by tricyclic antidepressant drug imipramine, as compared to tetracyclic drug maprotiline or non-cyclic drug nomifensine. An endogenous SSAO modulator in rat brain cytosol after immobilization stress (IMMO) was found and that this inhibitor could be induced by IMMO. SSAO activity in rat brain might be regulated by the level of this inhibitor. Semicarbazide, a SSAO inhibitor, enhances the formation of OH products of efflux/oxidation due to 1-methyl-4-phenylpyridinium ion (MPP⁺). The precise physiological functions of SSAO could play an important role in the control of energy balance in adipose tissue. SSAO could play an important role in the regulation of adipocyte homeostasis.     

Effects of Antidepressant Drug Imipramine on Gene Expression in Rat Prefrontal Cortex

We have investigated gene expression changes produced by acute and chronic daily treatment with a prototypical antidepressant, imipramine, using DNA microarrays. The analysis of similarities in gene expression patterns among functionally related genes revealed four expression profile cluster areas that showed a highly significant overrepresentation of several functional classes. Genes encoding for proteins involved in cAMP metabolism, postsynaptic membrane proteins, and proto-oncogenes were overrepresented in different cluster areas. Furthermore, we found that serine proteases as a group were similarly regulated by chronic antidepressant treatment. Our data suggest that cAMP metabolism, synaptic function, and protein processing by serine proteases may be important targets of antidepressant treatment and potential objects for antidepressant drug development.     

木豆素对慢性不可预见温和应激小鼠的抗抑郁作用

目的观察木豆素于抗抑郁方面的作用,并初步探讨其可能的作用机制。方法采用小鼠慢性不可预见温和应激模型(chronic unpredictable mild stress,CUMS),进行糖水偏好实验评价经木豆素处理的小鼠的快感缺失情况,采用酶联免疫吸附测定法测定小鼠血清皮质酮的含量,采用LC-MS/MS法检测小鼠皮层和海马中多种神经递质的含量。结果木豆素可以逆转CUMS引起的糖水偏爱指数降低和血清皮质酮水平升高。并且与正常组相比,模型组小鼠皮层和海马中相关神经递质的含量发生了显著的改变,而木豆素给药对于CUMS小鼠体内相关神经递质具有明显的调节作用。结论木豆素可能通过降低血清皮质酮水平和调节脑内神经递质来实现抗抑郁作用。     

Selective Effects of Neonatal Hypothyroidism on Monoamine Oxidase Activities in the Rat Brain

Hypothyroidism of mild intensity was obtained with prenatal and neonatal submission of Long-Evans rats to an iodide-rich diet. Chronic daily administration of methimazole to iodide-supplemented Long-Evans pups or to iodine-deprived Charles-River rats through the first 29–30 days of age provoked severe hypothyroidism. Monoamine oxidase type A (MAO-A) and not type B (MAO-B) activity was consistently, although slightly (by approximately 20%), increased in the hypothyroid brain. Triiodothyronine (T₃)-induced hyperthyroidism did not affect MAO activity. Replacement therapy with T₃ did not normalize MAO-A activity in hypothyroidism. Methimazole displayed a competitive and reversible in vitro inhibition of MAO-A but not MAO-B activity. Although this effect was obtained at concentrations far higher than those estimated to reach the brain after a single injection of the goiterogen, the occurrence of accumulation processes in the metabolism-deficient hypothyroid neonate rs cannot be excluded. Thus, MAO-A activity might be either directly depressed during the goiterogenic treatment, or increased as the result of some kind of rebound effect after interruption of methimazole administration.     

Effects of Chronic Ethanol Administration on Serotonin Metabolism in the Various Regions of the Rat Brain

Changes in serotonin (5-HT) and 5-hydroxy indole acetic acid (5-HIAA), its major metabolite, in cerebral cortex, corpus striatum and hippocampus were investigated at 10^th and 21^st days of chronic ethanol ingestion in Wistar rats. Ethanol (7.2% v/v) was given to rats in a modified liquid diet. Biochemical analysis was performed in two groups of ethanol-treated and control rats (n = 6 for each group). Rats in each group were decapitated at the 10^th and 21^st days of ethanol consumption. Brains were removed and cerebral cortex, corpus striatum and hippocampus were dissected. 5-HT and 5-HIAA levels were measured in respective brain regions by using high performance liquid chromatography. In cerebral cortex and corpus striatum, 5-HT levels were significantly lower than control at the 10^th day of ethanol consumption. At the 21^st day, the levels tended to remain low, but not significantly different statistically. In hippocampus, 5-HIAA levels were significantly higher than control at 10^th day of ethanol consumption. Increased 5-HIAA level returned to control values at the 21^st day of ethanol consumption. Our results suggest that, 5-HT clearly seems to play a critical role in the brain at the 10^th day of chronic ethanol consumption.     

Differential Effects of Angiotensin II and Eledoisin on Monoamine Oxidase A and B Activities in Rat Brain

Intracerebroventricular injections of angiotensin II caused 108, 62, and 54% increases in monoamine oxidase A activities in rat hippocampus, hypothalamus, and striatum, respectively. These activatory effects were abolished by simultaneous injections of eledoisin. No significant changes of monoamine oxidase B activities were found under the same experimental conditions. Neither angiotensin II nor elodoisin changed substrate/inhibitor affinities of both isoenzymes. These data indicate that angiotensin II and tachykinin transmitter systems may exert opposite, long-term regulatory effects on monoaminergic neurons in rat brain.     

Diabetic Modulation of the Temperature Kinetics Properties of Cytochrome Oxidase Activity in Rat Brain Mitochondria

The effects of alloxan-diabetes and subsequent treatment with insulin on temperature kinetics properties of cytochrome oxidase activity from rat brain mitochondria were examined. The enzyme activity decreased only at the late stage of diabetes which was not normalized by insulin treatment; however at early stage of diabetes hyper-stimulation occurred. In the control animals the Arrhenius plot was chair shaped with three energies of (E₁, E₂ and E₃) and two phase transition temperatures (T_t1 and T_t2). At early diabetic stage the Arrhenius plot became biphasic and E₁ and E₂ decreased_; insulin treatment reversed chair-shaped pattern with increase in E₂. These changes correlated with transient changes in the phospholipids profiles especially decreased acidic phospholipids. The temperature kinetics parameters were minimally affected at the late stage of diabetes or by insulin treatment. Thus at the late stage the brain tissue seems to have readjusted to its insulin homeostasis.     

Stimulation of the Na+/K+ Pump Activity During Electrogenic Uptake of Acidic Amino Acid Transmitters by Rat Brain Synaptosomes

Addition of D-aspartate, a substrate for the high-affinity transport of acidic amino acid transmitters, to suspensions of rat brain synaptosomes increased the rate of O2 consumption, uptake of 86Rb, and transport of 2-[3H]deoxyglucose. Stimulation of all three processes was abolished in the presence of ouabain. D-Aspartate had no effect on respiration in the medium in which NaCl was replaced by choline chloride. The ratio of the ouabain-sensitive increase in 86Rb uptake to that in O2 consumption was 12 to 1, which gives a calculated 86Rb(K+)/ATP of 2. It is concluded that electrogenic, high-affinity transport of sodium-D-aspartate into synaptosomes stimulates the activity of the Na+/K+ pump through an increase in [Na+]i.     

Zinc and Calcium Reduce Lead Induced Perturbations in the Aminergic System of Developing Brain

Since alterations in monoamines and monoamine oxidase (MAO) have been postulated to play a role in toxic effects of lead (Pb) on the central nervous system, we have examined the protective effects of calcium (Ca²⁺) and zinc (Zn²⁺) supplementation on Pb-induced perturbations in the levels of monoamines and the activity of MAO. Swiss albino mice were lactationally exposed to low (0.2%) and high (1%) levels of Pb-acetate via drinking water of the mother. Pb-exposure commenced on postnatal day (PND) 1, continued up to PND 21 and stopped at weaning. Ca²⁺ or Zn²⁺ (0.02% in 0.2% Pb–water or 0.1% in 1% Pb–water) was supplemented separately to the mother up to PND 21. The levels of monoamines (epinephrine, norepinephrine, dopamine and serotonin) and the activity of MAO in the brain regions such as hippocampus, cortex, cerebellum and medulla of young (1 month old) and adult (3 month old) mice were determined in the synaptosomal fractions. The synaptosomal monoamines though increased with low level (0.2%) Pb-exposure, significantly decreased with high level (1%) Pb-exposure in all the brain regions in both the age groups. In general, the young mice seem to be more vulnerable to Pb-neurotoxicity. Ca²⁺ or Zn²⁺ supplementation significantly reversed the Pb-induced perturbations both in the levels of monoamines and in the activity of MAO. However, the recovery in monoamine levels and MAO activity was more pronounced with Ca²⁺ supplementation as compared to Zn²⁺. These results provide evidence that dietary Ca²⁺ and/or Zn²⁺ provide protection against Pb-induced neurotoxic effects.     

Studies on Hormonal Effects on Hepatic Histidase and Alanine Transaminase of Rats Fed on Either Histidine Controlled or Imbalanced Diet

Effects of several hormones on liver histidase activity were investigated to study the mechanism of elevation of the activity of histidase when rats were fed on a histidine imbalanced diet.

Continuous injection of thyroxine (5~10 µg/rat/day) diminished histidase activity, and hypophysectomy increased histidase activity as compared with that of non treated animals. No change was observed on alanine transaminase (GPT) by above treatments.

Neither histamine, insulin, triamcinolone, adrenaline, nor adrenalectomy affected on the activity of histidase. Injection of histidine to rats fed on a histidine imbalanced diet made the activities of histidase and GPT tend to decrease.     

Effects of Undernutrition on Glutamatergic Parameters in Rat Brain

Early restriction of nutrients during the perinatal period has marked repercussions on CNS ontogeny, Leading to impaired functions. This study investigated the effects of pre- and postnatal (up to 75 days) undernutrition (diet: 8% protein; normonourished group: 25% protein) on some glutamatergic and behavioral parameters of rats. Undernutrition reduced: (i) seizures caused by ICV quinolinic acid (QA) administration; (ii) Na-independent [³H]glutamate binding in cell plasma membranes of cerebral cortex, and (ii) basal [³H]glutamate release from synaptosomal preparation. Behavioral parameters related to locomotion, anxiety, or memory were not affected. These results indicate that our model of undernutrition decreased the sensitivity to QA as convulsing agent and point to some putative glutamatergic parameters involved in this effect.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

Effects of NMDA on Carbachol-Stimulated Phosphatidylinositol Resynthesis in Rat Brain Cortical Slices

N-methyl-D-aspartate (NMDA) inhibits carbachol-stimulated phosphoinositide breakdown in rat brain cortical slices but not in isolated membranes (1). To gain insight into the mechanisms, we examined the effects of NMDA on carbachol-stimulated [³H]inositol phosphate and intermediates of phosphatidylinositol cycle accumulation in rat cortical slices. The inhibition is primarily on the synthesis of inositol phospholipids subsequent to activation of muscarinic cholinergic receptors. In the absence of lithium, NMDA inhibited carbachol-stimulated [³²P]PtdIns but not [³²P]PtdOH synthesis. Carbachol-stimulated CDP-DAG formation required trace amount of Ca²⁺ and the response was inhibited by NMDA at low but not high extracellular Ca²⁺ concentrations. The inhibition due to NMDA was only seen at millimolar extracellular Mg²⁺. The inhibition of carbachol-stimulated CDP-DAG formation was not affected by adding tetrodotoxin or cobalt chloride suggesting the inhibitory effect was not due to releasing of neurotransmitters. The inhibitory effects of NMDA could be abolished by MK-801, the specific NMDA receptor associated channel antagonist. When cortical slices were preincubated with ligands and lithium to allow the build up of CDP-DAG, carbachol stimulated the incorporation of [³H]Ins into [³H]PtdIns. However, this response was not inhibited by NMDA. These results suggest that CDP-DAG synthesis is the primary site of regulation by NMDA. Because CDP-DAG cytidyltransferase requires Mg²⁺ as cofactor and is sensitive to Ca²⁺ it is possible that NMDA inhibits ligand-stimulated PtdIns breakdown by blocking the replenish of agonist-sensitive PtdIns pool through changes of divalent cation homeostasis.     

IDPN-Induced Monoamine and Hydroxyl Radical Changes in the Rat Brain

--iminodipropionitrile (IDPN)-induced monoamine and hydroxyl radical changes in the rat brains were studied. IDPN caused decreases in 5-HT and 5-HIAA levels in all brain regions, strongly indicating that IDPN's neurotoxicity primarily affects 5-HT containing neurons. Dopamine and its metabolites' levels decreased in the some regions, most likely due to depression of dopamine metabolic turnover. Our results more clearly demonstrate IDPN-induced monoamine alterations in the rat brain more than previous reports. To clarify one of the pathogenesis of IDPN-induced neurological disorders, we measured hydroxyl radical levels. 2,3-DHBA increased at 1st day, and decreased in some regions at 7th days after discontinuing IDPN. We conclude, hydroxyl radical formation causes neuronal damage, and monoamine changes contribute to IDPN-induced neurological disorder.     

Effects of intrastriatal kainic acid injection on [3H]dopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the type A and B forms of monoamine oxidase

Abstract: Intrastriatal injections of kainic acid (KA) were utilized to investigate the cellular localization of postsynaptic dopamine (DA) metabolism by type A and B monoamine oxidase (MAO) in rat striatum. At 2 days postinjection, maximal degeneration of cholinergic and γ-aminobutyric acid (GABA)ergic neurons was observed and found to be associated with a significant decrease in both type A and B MAO activity. However, over the next 8-day period, when only the process of gliosis appeared to be occurring, a selective return to control of type B MAO activity was seen. When the metabolism of [³H]DA (10^?7 M) was examined in 8-day KA-lesioned rat striatal slices, an increase in [³H]dihydroxyphenylacetic acid (DOPAC) and [³H]homovanillic acid (HVA) formation was observed. The KA-induced elevation of [³H]DOPAC formation (but not [³H]HVA) was abolished by the DA neuronal uptake inhibitor nomifensine. This is consistent with earlier findings suggesting that HVA is formed exclusively within sites external to DA neurons. Experiments with clorgyline and/or deprenyl revealed that the relative roles of type A and B MAO in striatal DA deamination remained unchanged following KA (90% deamination by type A MAO) even though total deamination was substantially enhanced. At high concentrations of [³H]DA (10^?5 M), deamination by type B MAO could be increased to 30% of the total MAO activity; however, this was observed in both control and KA-lesioned striata. These results suggest that KA-sensitive neurons contain type A and/or type B MAO. Moreover, whereas these neurons may metabolize DA, a major portion of postsynaptic DA deamination appears to occur within glial sites of rat striatal tissue. Furthermore, glial cells would appear to contain functionally important quantities of both type A and B MAO.