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.     

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.     

Oxidation of N-Methyl-1,2,3,4-Tetrahydroisoquinoline into the N-Methyl-Isoquinolinium Ion by Monoamine Oxidase

N-Methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) was found to be oxidized by monoamine oxidase (MAO) into N-methylisoquinolinium ion, which was proved to inhibit enzymes related to the metabolism of catecholamines, such as tyrosine hydroxylase, aromatic-L-amino acid decarboxylase, and MAO. NMTIQ was oxidized by both types A and B MAO in human brain synaptosomal mitochondria. Oxidation was dependent on the amount of MAO sample and the reaction time. Enzyme activity with respect to NMTIQ reached optimum at a pH of approximately 7.25, as was the case with other substrates. Type A MAO had higher activity for this substrate than type B. The Km and Vmax values of the oxidation by types A and B MAO were 571 +/- 25 microM and 0.29 +/- 0.06 pmol/min/mg protein, and 463 +/- 43 microM and 0.16 +/- 0.03 pmol/min/mg protein, respectively. The Vmax values of types A and B MAO for NMTIQ were much smaller than those for other substrates such as kynuramine. NMTIQ was the first tetrahydroisoquinoline shown to be oxidized into the isoquinolinium ion by MAO in the brain.     

Monoamine Oxidase-Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro

In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50 = 3-5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1-100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1-10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 microM). At this time, the SR 95191-induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki = 68 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Ganglioside GM1 Causes Expression of Type B Monoamine Oxidase in a Rat Clonal Pheochromocytoma Cell Line, PC12h

The effects of ganglioside supplementation of culture medium on monoamine oxidase (MAO) type A and B activities in a rat clonal pheochromocytoma cell line, PC12h, were examined. The MAO activity in PC12h cells proved to be mainly due to type A MAO, and type B MAO activity was negligible. After supplementation of the culture medium with ganglioside GM1, the PC12 cells were found to express type B MAO activity after 4 days of culture, and the amount of type B activity increased with the number of days of culture. After 3 weeks of culture in the presence of GM1, type B activity was about 10% of the total, whereas in control cells type B MAO activity was only about 0.6% of the total. By kinetic analyses of type A and B MAO in PC12h cells after 3 weeks of culture, the increase of type B MAO activity was found to be due to the increase in amount of type B MAO; the Km values were almost the same and only the Vmax values were increased in the cells supplemented with GM1. Among gangliosides tested GM1 was the most effective in causing expression of type B MAO activity, whereas nerve growth factor was not effective. These results suggest that GM1 and other gangliosides may be involved in the expression of type B MAO in nerve cells and in the regulation of levels of the biogenic amines in the brain.     

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.     

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.     

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.     

Inhibition of Monoamine Oxidase by Phenyl Azides

We had previously shown that 4-fluoro-3-nitrophenyl azide (FNPA) is a competitive inhibitor of both types of monoamine oxidase (MAO) in the dark, but it is a preferential photoaffinity label for only the type B MAO (MAO-B). Recently we synthesized a number of arylazido compounds with structures related to FNPA and determined the effects of these compounds on the two types of MAO in rat brain cortex. We found that the fluoro group of FNPA was not required for the inhibition of MAO activities because neither the presence nor the position of the fluoro group affected its inhibition of MAO. On the other hand, both the nitro and the azido groups of FNPA were shown to be important for FNPA inactivation of two types of MAO. The inhibitory potency was significantly lower for compounds without either group. Furthermore, we found that all nitrophenyl azide isomers except 2-nitrophenyl azide were photodependent inhibitors of MAO-B. Under the same experimental conditions none of the compounds photoinactivated MAO-A. On the basis of these findings, mechanisms for FNPA inhibition of the two types of MAO are discussed.     

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.     

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.     

Structural Features of Human Monoamine Oxidase A Elucidated from cDNA and Peptide Sequences

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.     

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 Aromatic L-Amino Acid Decarboxylase and Tyrosine Aminotransferase by the Monoamine Oxidase Inhibitor Phenelzine

The concentration of p-tyramine in the rat striatum was increased significantly by intraperitoneal injection of phenelzine (5 or 100 mg/kg). Unlike other monoamine oxidase (MAO) inhibitors, phenelzine had no effect on p-tyramine levels in the first 1-2 h following injection. The high dose of phenelzine increased the p-tyramine levels much more than the low dose. In addition, the high dose of phenelzine increased striatal p-tyrosine levels significantly 12 h after injection. Further studies showed that phenelzine inhibited the tyrosine aminotransferase activity of rat liver homogenates; the IC50 was 50 microM. Phenelzine also inhibited the aromatic L-amino acid decarboxylase activity of rat brain homogenate with an IC50 of 25 microM. Following intraperitoneal injection of 100 mg/kg phenelzine, the initial concentration of phenelzine in the striatum appears to be high enough to inhibit aromatic L-amino acid decarboxylase. It is suggested that the multiple enzyme inhibition caused by administration of high doses of phenelzine accounts for its unusual effects on striatal p-tyramine levels compared with other MAO inhibitors, i.e., its initial lack of effect on p-tyramine levels followed later by very large increases in p-tyramine levels.     

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.     

Monoamine Oxidase Activity of Mitochondria Prepared from Rat Liver and Rat Heart

Abstract: The effect of agents that change the respiratory state of the mitochondrion on tyramine oxidation was investigated. Neither uncoupler nor ADP and P_t in the presence of substrate produced any change in the rate of tyramine oxidation, as judged by direct measurement of tyramine oxidation or by H₂O₂ production. We conclude that previously reported depression of monoamine oxidase activity by stimulated respiration was due to oxygen depletion.     

MDL 72145, an Enzyme-Activated Irreversible Inhibitor with Selectivity for Monoamine Oxidase Type B

Abstract: MDL 72145, ( E )-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine hydrochloride, was designed and synthesised as a potential enzyme-activated irreversible inhibitor of monoamine oxidase (MAO). In vitro , the compound displayed time-dependent pseudo-first-order irreversible inhibitory characteristics with high selectivity for the B form of rat brain mitochondrial MAO At 10°C the K_t and T₅₀ values for the B enzyme were 40 μ M and 1.7 min, respectively, while these same kinetic constants for the A enzyme were 131 μ M and 14.5 mm, respectively. Selective protection against inactivation of the two forms of MAO by MDL 72145 was obtained by preincu-bating the enzyme with suitable concentrations of the selective A and B substrates, 5-hydroxytryptamine and benzylamine.