The Novel Neuropsychotropic Agent Milacemide Is a Specific Enzyme-Activated Inhibitor of Brain Monoamine Oxidase B

The novel neuropsychotropic agent milacemide hydrochloride (2-n-pentylaminoacetamide HCl) is a highly selective substrate of the B form of monoamine oxidase (EC 1.4.3.4; MAO). Under the in vitro conditions used in the present study, milacemide acts as an enzyme-activated, partially reversible inhibitor of MAO-B. A reversible inhibition of MAO-A activity is also observed at high concentrations. The inhibitory activity of milacemide is significantly greater for MAO-B. In vivo, after single or repeated oral administration, a specific inhibition of MAO-B is apparent in brain and liver, with a lack of inhibition of the MAO-A activity. In contrast to the irreversible inhibitory action of L-deprenyl, the recovery of MAO-B activity in vivo after milacemide administration is significantly faster, a result suggesting that it is a partially reversible inhibitor. The selective inhibitory effect of milacemide for MAO-B in vivo is confirmed by its potentiation of phenylethylamine-induced stereotyped behavior, whereas vasopressor responses to tyramine were not affected. These observations suggest that milacemide could enhance dopaminergic activity in the brain and could be used as therapy for Parkinson's disease in association with L-3,4-dihydroxyphenylalanine.     

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.     

Expression of Functional Human Monoamine Oxidase A and B cDNAs in Mammalian Cells

Monoamine oxidase (MAO) A and B are important enzymes that metabolize biogenic amines throughout the body. Previous studies had suggested that both MAO A and B consist of two subunits of molecular masses of 63 and 60 kilodaltons, respectively. The cDNAs encoding one subunit of human liver MAO A and B have been expressed in mammalian cells by transfection of the individual clones. The proteins expressed from these cDNAs are shown to be catalytically active. Similar to the endogenous enzymes, the expressed MAO A prefers serotonin as a substrate and is sensitive to the inhibitor clorgyline. In contrast, the expressed MAO B prefers phenylethylamine as a substrate and is sensitive to the inhibitor deprenyl. These results suggest that a single polypeptide of MAO A (or B), existing as either a monomer or homodimer, is enzymatically active. The ability to obtain functional MAO A and B from their respective cDNA clones allows us to study further the structure and function relationships of these important enzymes.     

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.     

Relationship Between Platelet Monoamine Oxidase B Activity and Alleles at the MAOB Locus

Genetic variations in monoamine oxidase (MAO)-B activity have been proposed to have a contributory role in several neurologic and psychiatric diseases. Variations in activity could affect rates of degradation of exogenous amines, including toxins, precursors of toxins (like 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), or false transmitters, and of endogenous amines, such as neurotransmitters. In this study a highly polymorphic (GT)n repeat element was used to mark alleles at the MAOB locus. The MAOB allele status and levels of platelet MAO-B activity were determined for 41 control males. No correlation was noted between specific alleles and levels of MAO-B activity in this sample set. This suggests that the structural gene for MAOB is not usually the primary determinant of activity levels in platelets.     

Cimoxatone Is a Reversible Tight-Binding Inhibitor of the A Form of Rat Brain Monoamine Oxidase

Abstract: Cimoxatone is a fully reversible inhibitor selective for the A form of monoamine oxidase. The inhibition is so potent against this enzyme form that it acts as a tight-binding inhibitor. Use of this inhibitor indicates that in rat brain homogenates the concentration of monoamine oxidase A is approximately 8–11 pmol-mg protein⁻¹. Values similar to this were obtained by clor-gyline titration and both methods gave values similar to those found with a [³H]harmaline binding assay.     

Inhibition of Brain Type A Monoamine Oxidase and 5-Hydroxytryptamine Uptake by Two Amphetamine Metabolites, p-Hydroxyamphetamine and p-Hydroxynorephedrine

Two amphetamine metabolites, p-hydroxyamphetamine (p-OHA) and p-hydroxynorephedrine (p-OHN), selectively inhibited the A form of monoamine oxidase (MAO) in rat and mouse forebrain homogenates. Of these two metabolites, p-OHA inhibited MAO-A more strongly than p-OHN. This MAO-A-selective inhibition by p-OHA or p-OHN was found to be competitive with respect to deamination of its substrate, 5-hydroxytryptamine (5-HT). The degree of MAO-A inhibition was not changed by 90 min of preincubation of the enzyme preparations with either metabolite, and the activity inhibited by p-OHA after the preincubation recovered completely to the control level after repeated washing. Uptake of 5-HT or dopamine into mouse forebrain synaptosomes was highly reduced by both p-OHA and p-OHN. Both metabolites were more potent in reducing dopamine uptake than in reducing 5-HT uptake. In reduction of 5-HT and of dopamine uptake, p-OHA was more potent than p-OHN. These results indicate that p-OHA is a more selective inhibitor of brain MAO-A activity and 5-HT uptake than its subsequent metabolite, p-OHN. These two actions of p-OHA might, together with possible 5-HT efflux into the synaptic cleft, greatly contribute to head twitch, a brain 5-HT-mediated animal behavior induced by p-OHA.     

Deamination of 5-Hydroxytryptamine by Both Forms of Monoamine Oxidase in the Rat Brain

Abstract: K _m and V _max values of monoamine oxidase (MAO) A and B towards 5-hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l -deprenyl. K _m values of 178 and 1170μ m , and V _max values of 0.73 and 0.09 nmol·mg protein⁻¹·min⁻¹ towards 5-hydroxytryptamine were found for MAO-A and -B, respectively. The K ₁ for 5-hydroxytryptamine as a competitive inhibitor of β-phenethylamine oxidation by MAO-B was found to be 1400 μm. The significance of these findings is discussed.     

A and B Forms of Monoamine Oxidase Within the Monoaminergic Neurons of the Rat Brain

The inhibition of the A and B forms of monoamine oxidase (MAO) inside and outside serotonergic, noradrenergic, and dopaminergic synaptosomes in homogenates of rat hypothalamus or striatum by clorgyline, a selective and irreversible MAO-A inhibitor, and selegiline, a selective and irreversible MAO-B inhibitor, was examined. Intrasynaptosomal deamination at low concentrations of the substrates [14C]5-hydroxytryptamine ([14C]5-HT; 0.1 microM), [14C]noradrenaline (0.25 microM), [14C]3,4-dihydroxyphenylethylamine ([14C]dopamine; 0.25 microM), and [14C]tyramine (0.25 microM) was hindered by selective uptake inhibitors (citalopram, maprotiline, and amfonelic acid) in the incubation media. Thus, the difference between the deamination of 14C-amine in the absence and presence of the appropriate selective uptake inhibitor provided a measure of deamination in the specific aminergic synaptosomes. This was verified by determining the loss of MAO activity within noradrenergic and serotonergic systems after degeneration of the nerve terminals by the neurotoxins N-chloroethyl-N-ethyl-2-bromobenzylamine and p-chloroamphetamine. Results with the two inhibitors revealed that the A and B forms were responsible for 80 and 20%, respectively, of the deamination of [14C]5-HT within serotonergic synaptosomes from the hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes from the hypothalamus and that of [14C]dopamine and [14C]tyramine within the striatal dopaminergic synaptosomes were due to MAO-A. About 10% of the deamination of [14C]noradrenaline, [14C]dopamine, and [14C]tyramine outside the noradrenergic or dopaminergic synaptosomes was brought about by the B form, with the remainder being deaminated by MAO-A.     

Inhibition of Monoamine Oxidase by N-Methylisoquinolinium Ion

N-Methylisoquinolinium ion (N-MIQ) has been found to inhibit the biosynthesis of catecholamines; it inhibited tyrosine hydroxylase activity in striatal tissue slices. In this article, the effects of N-MIQ and an analogue, N-methylquinolinium ion, on monoamine oxidase (MAO) activity were examined to see their effects on the catabolism of catecholamines. MAO-A in human placental mitochondria was strongly inhibited by N-MIQ in competition with the substrate. The apparent Ki value of N-MIQ was found to be 20.4 +/- 1.1 microM, whereas that of N-methylquinolinium ion was 54.6 +/- 4.5 microM. MAO-B in human brain synaptosomes and liver mitochondria was found to be inhibited by N-MIQ, but the inhibition proved to be noncompetitive. The inhibition of MAO-B by N-MIQ was completely reversible by dialysis of the incubation mixture. MAO-A in human brain and liver mitochondria was more sensitive to the inhibitor than MAO-B. By quantitative analysis of N-MIQ, using HPLC, it was found not to be catabolized by the incubation with mitochondria, suggesting that the inhibition was due to N-MIQ itself and not due to any metabolic product. The inhibition of MAO by N-MIQ is discussed in terms of its possible involvement of the etiology of parkinsonism.     

Dopamine Sulfoconjugation in the Rat Brain: Regulation by Monoamine Oxidase

An increase of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain such as is found following 3,4-dihydroxyphenylalanine (L-DOPA) administration or an intraventricular injection of free dopamine did not result in DA sulfate formation, despite the presence of phenolsulfotransferase activity in various regions of the brain and the high affinity of DA for this enzyme. However, when rats were pretreated with pargyline, a monoamine oxidase inhibitor, the same treatment with L-DOPA or free DA led to active synthesis of DA sulfate. The increase in DA sulfate was significantly correlated with the degree of monoamine oxidase inhibition and directly proportional to free DA concentrations in the hypothalamus (r = 0.86), striatum (r = 0.54), and brainstem (r = 0.89). The highest ratio of DA sulfate to free DA was found in the hypothalamus, suggesting that sulfoconjugation is most active in this region. Prior treatment of rats with 6-hydroxydopamine did not decrease DA sulfate concentrations, indicating that sulfoconjugation occurs most likely in extraneuronal tissues not destroyed by the neurotoxin. The results are compatible with the notion that phenolsulfotransferase may be highly compartmentalized and that inhibition of monoamine oxidase allows the newly generated free DA to become accessible to the sulfoconjugating enzyme, resulting in increase in DA sulfation.     

Gonadal Influences on the Sexual Differentiation of Monoamine Oxidase Type A and B Activities in the Rat Brain

Abstract: The sex-dependent differentiation of monoamine oxidase (MAO) in the hypothalamus of 60-day-old, Charles River rats was found to involve only type A (MAO-A), and not type B (MAO-B) enzyme. In vivo inhibition of type A by clorgyline, and type B by (−)deprenyl, however, tended to decrease the specific activity of both types of MAO to a smaller extent in the female than in the male hypothalamus. When masculinization was prevented by neonatal administration of estradiol (E) to males, hypothalamic MAO-A and MAO-B activities increased in both control and MAO-inhibited rats. Androgenization of females, however, had little effect on the MAO activity. Whereas the effects of neonatal estrogenization were attributable neither to a direct influence of E nor to a sexual difference in the peripheral clearance of the MAO-inhibitor used, single, high doses of steroids to adult, but not to newborn rats, did acutely affect the kinetics of MAO-A. The activity of MAO-A was also decreased by high concentrations of E or TS in vitro. The imprinting for patterns of hypothalamic MAO-A and MAO-B in the two sexes results, probably, from genetic predetermination. Neonatal changes in the homeostasis of gonadal hormones may result in type-MAO nonspecific effects in adulthood, whereas the short-term effects of high concentrations of steroids may be selective for the A form.     

Inhibition of Monoamine Oxidase by 7-Chloro-4-Nitrobenzofurazan

7-Chloro-4-nitrobenzofurazan (NBD-Cl) is a potent inhibitor of both types of monoamine oxidase (MAO). NBD-Cl competitively inhibited the oxidative deamination of kynuramine catalyzed by human placenta MAO-A, the oxidative deamination of benzylamine catalyzed by bovine liver MAO-B, the oxidative deamination of serotonin catalyzed by rat brain MAO-A, and the oxidative deamination of phenylethylamine catalyzed by rat brain MAO-B. In addition, a time-dependent inactivation of MAOs by NBD-Cl has been demonstrated upon incubation of the enzyme preparations with NBD-Cl at pH 9, but not at pH 7.5. The time-dependent inhibition of MAO by NBD-Cl could be prevented by the addition of 4-nitrophenyl azide, an active site-directed label of MAO, during incubation of the enzyme with NBD-Cl. On the basis of these findings, it is suggested that at pH 9, NBD-Cl modifies one (or more) essential lysine residue(s) in the active sites of the two types of MAO.     

The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO-A toward dopamine (120 microM) is lower than the Km of MAO-B toward this substrate (340 microM). The activity of MAO-A was lower in old rats than in young rats, and the same degree of decrease was found for 5-hydroxytryptamine as for dopamine as substrates for this enzyme form. The activity of MAO-B was higher in the old rats, the degree of increase being the same for dopamine as for beta-phenethylamine as substrates for this enzyme form. The Ki values of the inhibition of MAO-A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the Ki values for the inhibition of MAO-B by these compounds.     

A Caution in the Use of Tritiated Substrates for Monoamine Oxidase Assays

Abstract: A monoamine oxidase assay utilizing generally labeled [³H]-serotonin as substrate became nonlinear after only ～5% conversion of initial c.p.m. to product. Subsequent analysis showed that a significant proportion of the tritium label was readily exchangeable into water and that monoamine oxidase activity increased release of label as water. The use of generally labeled substrates for oxidase activities is not recommended.     

Inhibition of Monoamine Oxidase by 3,4-Dihydroxyphenylserine

The effects of diastereomers of 3,4-dihydroxyphenylserine (DOPS) on the enzyme activity of monoamine oxidase (MAO) in human placenta and liver mitochondria were examined. Both L- and D-threo-DOPS were found to inhibit MAO-A in human placental mitochondria in competition with the substrate, and the Ki values for L- and D-threo-DOPS obtained were 68.3 and 125 microM, respectively. The inhibitory effect of L-threo-DOPS on both MAO-A and -B activity was confirmed in human liver mitochondria, and MAO-A was found to be more sensitive to the inhibitor. Other isomers of DOPS, L- and D-erythro-DOPS, were found to inhibit MAO activity, but the inhibition was noncompetitive with the substrate. The inhibitory effects of DOPS isomers were not affected by the presence of NSD-1055, an inhibitor of aromatic L-amino acid decarboxylase, suggesting that the inhibition is the direct effect of DOPS, and not of norepinephrine produced by the decarboxylase.     

Phenelzine Causes an Increase in Brain Ornithine that is Prevented by Prior Monoamine Oxidase Inhibition

Phenelzine (PLZ), a nonselective irreversible inhibitor of monoamine oxidase (MAO), also inhibits GABA-transaminase (GABA-T), markedly increasing brain GABA levels. PLZ is also a substrate for MAO, and studies suggest that a metabolite formed by the action of this enzyme on PLZ may be responsible for the increase in GABA observed. We have recently found that PLZ also elevates brain ornithine (ORN), an amino acid precursor to both glutamate (and GABA) and the polyamines, and have conducted dose- and time-response studies on this effect. Rats were treated with vehicle or PLZ doses (7.5, 15 or 30 mg/kg i.p.), and brains were collected 3 h later. In the time-response study, animals were treated with vehicle or PLZ (15 mg/kg i.p.) and brains were collected 1–24 h later. To determine whether a metabolite formed by the action of MAO on PLZ may be responsible for the elevation in brain ORN observed, animals were pretreated with vehicle or the MAO inhibitor tranylcypromine (TCP) before vehicle or PLZ (15 mg/kg), and brains collected 3 h later. ORN levels (measured by an HPLC procedure) were dose- and time-dependently increased in PLZ-treated animals, with levels reaching approximately 650% of control at 6 and 12 h. Pretreatment with TCP completely abolished the PLZ-induced increase in brain ORN, suggesting, as with GABA, that a metabolite of PLZ formed by the action of MAO is responsible for the elevation of brain ORN observed. The possible contribution of increased ORN to therapeutic and/or neuroprotective properties of PLZ is discussed.