Brain region differences and some characteristics of monoamine oxidase type a and b activities in the vervet monkey

Monoamine oxidase in the vervet monkey showed greater variations in activity in six brain regions when tyramine or phenylethylamine was used as the substrate (3.8- to 4.1-fold differences) than when serotonin was the substrate (1.8-fold differences). With phenylethylamine and tyramine as substrates, the highest MAO specific activities were found in the hypothalamus and the lowest in the cerebellum and cortex. With serotonin as the substrate, the highest specific activities were in the mesencephalon and cortex. The inhibition of tyramine deamination by clorgyline and deprenyl yielded biphasic plots indicative of the presence of MAO-A and MAO-B enzyme forms in the vervet brain. On the basis of these inhibitor curves, the vervet brain could be estimated to contain approximately 85% MAO-B and 15% MAO-A, in contrast to rat brain which contains 45% MAO-B and 55% MAO-A. The inhibition of serotonin deamination by deprenyl in vervet brain yielded a biphasic plot, suggesting that some serotonin deamination in the vervet is accomplished by the MAO-B enzyme form. Estimations of the relative amounts of MAO-A and MAO-B based on inhibitor curves or based on substrate ratios yielded proportionate results which were in close agreement across the different brain regions, supporting the validity of these approaches to estimating MAO-A and MAO-B activities.     

Molecular characteristics of a single and novel form of carp (Cyprinus carpio) monoamine oxidase

Two mammalian monoamine oxidases (MAO), MAO-A and MAO-B, are similar in primary structures but have unique substrate/inhibitor selectivities. Carp (Cyprinus carpio) contains a MAO enzyme (C-MAO) with properties different from MAO-A and MAO-B. To determine the molecular characteristics of C-MAO and its phylogenetic relationship with other fish and mammalian MAOs, the primary structure of C-MAO was estimated. The putative C-MAO cDNA encodes 526 amino acids with 59.001 Da, and the deduced amino acid sequence showed as much as 68.9% homology with some mammalian MAO-A proteins, 69.8% homology with some mammalian MAO-B proteins, and as much as 92.4% homology with some fish MAOs. Comparison of two regions in the polypeptide sequence of C-MAO determining possible substrate/inhibitor preferences of MAO-A and MAO-B showed both 79.5% homologies.     

Absence of tissue-bound semicarbazide-sensitive amine oxidase activity in carp tissues

We have previously reported that carp (Cyprinus carpio) tissue mitochondria contain a novel form of monoamine oxidase (MAO), which belongs neither to MAO-A nor to MAO-B of the mammalian enzyme. This conclusion results from the findings that the carp MAO was equally sensitive to a selective MAO-A inhibitor clorgyline and to the MAO-B selective inhibitor l-deprenyl, when tyramine, a substrate for both forms, serotonin or beta-phenylethylamine, a substrate for either A or B-form of mammalian MAO, was used. In the present study, we tried to detect another amine oxidase, termed tissue-bound semicarbazide-sensitive amine oxidase (SSAO), activity in carp tissues. As definition of SSAO was used, such as insensitivity to inhibition of the kynuramine oxidizing activity by an MAO inhibitor pargyline and high sensitivity to the SSAO inhibitor semicarbazide. The results indicated that the oxidizing activity was selectively and almost completely inhibited by 0.1 mM pargyline alone or a combination of 0.1 mM pargyline plus 0.1 mM semicarbazide, but not by 0.1 mM semicarbazide alone. We also tried to detect any SSAO activity by changing experimental conditions, such as lower incubation temperature, higher enzyme protein concentration, a lower substrate concentration and different pH's in the reaction, as the enzyme source. However, still no SSAO activity could be detected in the tissues. These results conclusively indicate that carp tissues so far examined do not contain SSAO activity.     

Modification of substrate-inhibitor affinities of human platelet monoamine oxidase B in vitro

The rate of benzylamine utilization by monoamine oxidase (MAO)-B from human blood platelets was 2-4 times higher than that for octopamine. Both activities were inhibited 100% by 10(-7) M deprenyl (a specific MAO-B inhibitor) and were not affected by clorgyline (a specific MAO-A inhibitor) or by polyclonal antibodies to MAO-A. The preincubation of platelet MAO-B with purified MAO-A from mitochondrial membranes of human placenta resulted in appearance of excess octopamine activity. This additional activity was not precipitated by antibodies to MAO-A or inhibited by deprenyl but was inhibited by clorgyline. Incubation of the MAO-A preparation from placenta at 45 degrees C for 15 min before its preincubation with MAO-B caused 50% loss of both activities. Protease inhibitors had no effect on the modification of MAO. These data indicate that MAO-A or a factor tightly bound to it can modify MAO-B yielding a form of the enzyme with both MAO-A and MAO-B substrate and inhibitor affinities and MAO-B immunospecificity.     

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.     

Type A monoamine oxidase is the target of an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, leading to apoptosis in SH-SY5Y cells

Mitochondrial monoamine oxidase (MAO) has been considered to be involved in neuronal degeneration either by increased oxidative stress or protection with the inhibitors of type B MAO (MAO-B). In this paper, the role of type A MAO (MAO-A) in apoptosis was studied using human neuroblastoma SH-SY5Y cells, where only MAO-A is expressed. An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, an MAO-A inhibitor, reduced membrane potential, DeltaPsim, in isolated mitochondria, and induced apoptosis in the cells, which 5-hydroxytryptamine, an MAO-A substrate, prevented. In contrast, beta-phenylethylamine, an MAO-B substrate, did not suppress the DeltaPsim decline by N-methyl(R)salsolinol. The binding of N-methyl(R)salsolinol to mitochondria was inhibited by clorgyline, a MOA-A inhibitor, but not by (-)deprenyl, an MAO-B inhibitor. RNA interference targeting MAO-A significantly reduced the binding of N-methyl(R)salsolinol with simultaneous reduction in the MAO activity. To examine the intervention of MAO-B in the apoptotic process, human MAO-B was transfected to SH-SY5Y cells, but the sensitivity to N-methyl(R)salsolinol was not affected, even although the activity and protein of MAO increased markedly. These results demonstrate a novel function of MAO-A in the binding of neurotoxins and the induction of apoptosis, which may account for neuronal cell death in neurodegenerative disorders, including Parkinson's disease.     

Synthesis and characterization of immobilized dopamine beta-hydroxylase in membrane-bound and solubilized formats

Dopamine beta-hydroxylase (DBH) catalyzes the beta-hydroxylation of dopamine to norepinephrine. The enzyme in chromaffin granules occurs in a soluble form and a form confined to the surrounding membrane. DBH was noncovalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase and the resulting DBH-IAM stationary phase was enzymatically active and was shown to mimic the membrane-bound form of the enzyme. DBH was also covalently immobilized onto a silica-based support containing, glutaraldehyde-P (Glut-P). The resulting DBH-Glut-P interphase was also enzymatically active, reproducible and shown to display characteristics of the solubilized enzyme. The results demonstrate that the different immobilization methods utilized for the enzyme can be used to quantitatively and qualitatively determine the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions for the two distinct forms of the enzyme. These new entities can be used in basic biochemical studies as well as in high throughput screening of substances for DBH substrate/inhibitor properties.     

4-(O-Benzylphenoxy)-N-Methylbutylamine (Bifemelane) and Other 4-(O-Benzylphenoxy)-N-Methylalkylamines as New Inhibitors of Type A and B Monoamine Oxidase

4-(O-Benzylphenoxy)-N-methylbutylamine (Bifemelane, BP-N-methylbutylamine), a new psychotropic drug, was found to inhibit monoamine oxidase (MAO) in human brain synaptosomes. It inhibited type A MAO (MAO-A) competitively and type B (MAO-B) noncompetitively. BP-N-methylbutylamine had a much higher affinity to MAO-A than an amine substrate, kynuramine, and it was a more potent inhibitor of MAO-A than of MAO-B. The Ki values of MAO-A and -B were determined to be 4.20 and 46.0 microM, respectively, while the Km values of MAO-A and -B with kynuramine were 44.1 and 90.0 microM, respectively. The inhibition of MAO-A and -B by BP-N-methylbutylamine was found to be reversible by dialysis of the incubation mixture. MAO-A in human placental and liver mitochondria and in a rat clonal pheochromocytoma cell line, PC12h, was inhibited competitively by BP-N-methylbutylamine, while MAO-B in human liver mitochondria was inhibited noncompetitively, as in human brain synaptosomes. BP-N-methylbutylamine was not oxidized by MAO-A and -B. The effects of other BP-N-methylalkylamines, such as BP-N-methylethylamine, -propylamine, and -pentanylamine, on MAO activity were examined. BP-N-methylbutylamine was the most potent inhibitor of MAO-A, and BP-N-methylethylamine and -propylamine inhibited MAO-B competitively, whereas BP-N-methylbutylamine and -pentanylamine inhibited it noncompetitively. Inhibition of these BP-N-methylalkylamines on MAO-A and -B is discussed in relation to their chemical structure.     

Deamination of Aliphatic Amines by Monoamine Oxidase A and B Studied Using a Bioluminescence Technique

Deamination of n-octylamine and n-decylamine has been studied in various tissues using a new bioluminescence technique. Selectivity of n-octylamine and n-decylamine as substrates for monoamine oxidase (MAO) A or B has been determined using both clorgyline and (-)-deprenyl inhibition curves and kinetic parameters. Homogenates of rat brain, liver and heart containing predominantly MAO-A or -B were prepared by preincubation for 60 min with (-)-deprenyl or clorgyline (30 nM), respectively. Human placenta (MAO-A) and platelet (MAO-B) were used as reference tissues containing only one MAO form. In tissues (rat liver, brain) containing both MAO forms in equal proportion, inhibition curve studies showed a preference of both substrates for the B form of the enzyme; however, where MAO-A was the major form (rat heart, human placenta), clorgyline was the more effective inhibitor. In the beef brain cortex n-octylamine showed marked preference for MAO-B, whereas n-decylamine was selective toward-MAO-A. Kinetic studies in general supported the picture of greater selectivity of the aliphatic amine substrates for deamination by MAO-B, as reflected by lower Km values for this enzyme type. However, n-octylamine was more selective for MAO-B than n-decylamine in both kinetic and inhibition curve studies. The deamination of these aliphatic amine substrates cannot be explained only by reference to the binary classification of MAO into types A and B.     

Inhibition of Type A Monoamine Oxidase by Methylquinolines and Structurally Related Compounds

Abstract: A series of methylquinolines (MQ) were found to inhibit markedly type A monoamine oxidase (MAO) in human brain synaptosomal mitochondria. 4-MQ and 6-MQ inhibited type A MAO (MAO-A) competitively and 7- and 8-MQ inhibited MAO-A noncompetitively. Among these four isomers of MQ, 6-MQ was the most potent inhibitor; the K _i value toward MAO-A was 23.4 ± 1.8 μ M , which was smaller than the K _m value toward kynuramine, ± amine substrate, 46.2 ± 2.8 μ M . On the other hand, MQ were very weak inhibitors of type B MAO (MAO-B) and 8-MQ did not inhibit MAO-B in brain synaptosomal mitochondria. The inhibition of MAO-A proved to be reversible; by dialysis the inhibition of MQ was completely reversible. The affinity of these isomers of MQ toward MAO-A or -B was confirmed further with human liver mitochondria as sources of MAO-A and -B and with human placental mitochondria and rat pheochromocytoma PC12h cell line as sources of MAO-A. The relationship of the chemical structure of structurally related quinoline and isoquinoline derivatives to inhibition of the activity of type A or B MAO was examined.     

Multiple forms of monoamine oxidase in rabbit platelets.

Rabbit platelets were found to contain both types A and B MAO activities. The specific enzymatic activity of rabbit platelet MAO was higher for the substrate serotonin than for phenylethylamine. The K_m's for rabbit platelet MAO indicated that the MAO-B enzyme was similar to human platelet MAO and that both MAO-A and MAO-B enzymes in the rabbit platelet are similar to the corresponding forms in the rabbit brain. The drugs clorgyline and deprenyl confirmed the existence of types A and B MAO in the platelet and furthermore indicated that the type A form accounted for approximately 90% of the total enzymatic activity. Amitriptyline at low (micromolar) concentrations selectively inhibited MAO-B activity in both rabbit platelets and brain.     

Molecular cloning and characterization of monoamine oxidase A and B genes in pig.

Monoamine oxidase (MAO) is a key enzyme in the degradation of monoamine neurotransmitters, which are physiologically and pathologically important for animals and humans. MAO exists in two forms, coded by separate genes. Here we cloned and sequenced full length cDNAs of MAO-A and MAO-B in pig. The putative proteins include 527 and 520 amino acids, respectively. The sequences alignment and homology analysis showed that MAO-A and MAO-B are conserved among mammals. The amino acid residues and domains, which have been demonstrated to be important for MAO-A and MAO-B binding of ligands, substrates and inhibitors, were conserved in the pig.     

Differential Postnatal Development of Monoamine Oxidases A and B in the Blood-Brain Barrier of the Rat

Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [³H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [³H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO-A and -60,000 for MAO-B. SDS-PAGE resuits of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.     

Inhibition of rat brainstem monoamine oxidase activity by CGP 6085 A

CGP 6085 A [4-(5,6-dimethyl-2-benzofuranyl)piperidine] HCl, a known serotonin inhibitor, also inhibits rat brainstem monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in both in vivo and in vitro experiments. Serotonin (5-HT) deamination by MAO-A is inhibited 35% at a dose of 100 mg/kg i.p. in vivo. Similar experiments show a maximal 20% decrease in phenylethylamine (PEA) deamination by MAO-B at a dosage of 30 mg/kg i.p. Over the range of 0.1 to 10 mg/kg i.p., CGP 6085 A decreases 5-HIAA levels in the brainstem. This in vivo inhibition of MAO activity is confirmed by in vitro experiments. In vitro studies in rat brainstem mitochondrial preparations show a dose-dependent, reversible, inhibition of MAO using tyramine as the substrate for the enzyme reaction. With an in vitro IC50 of 2-3 microM, the potency of CGP 6085 A is comparable to pargyline.     

Docking studies on monoamine oxidase-B inhibitors: estimation of inhibition constants (K(i)) of a series of experimentally tested compounds

Monoamine oxidase (EC1.4.3.4; MAO) is a mitochondrial outer membrane flavoenzyme that catalyzes the oxidation of biogenic amines. It has two distinct isozymic forms designated MAO-A and MAO-B, each displaying different substrate and inhibitor specificities. They are the well-known targets for antidepressant and neuroprotective drugs. Elucidation of the X-ray crystallographic structure of MAO-B has opened the way for molecular modeling studies. A series of experimentally tested (1-10) model compounds has been docked computationally to the active site of the MAO-B enzyme. The AutoDock 3.0.5 program was employed to perform automated molecular docking. The free energies of binding (DeltaG) and inhibition constants (K(i)) of the docked compounds were calculated by the Lamarckian Genetic Algorithm (LGA) of AutoDock 3.0.5. Excellent to good correlations between the calculated and experimental K(i) values were obtained.     

Effect of selective monoamine oxidase A and B inhibitors on footshock induced aggression in paired rats

Effects of a selective monoamine oxidase (MAO)--A inhibitor, clorgyline, a selective MAO-B inhibitor, deprenyl, and a non-selective MAO inhibitor, nialamide, were investigated on footshock-induced aggression (FIA) in paired rats. The doses and pretreatment times of the inhibitors used were based on an earlier reported in vivo dose-response and time-course study. In addition, apomorphine, a dopaminergic receptor agonist, and beta-phenylethylamine, a preferred substrate for MAO-B, were also used to garner corroborative evidence. The results of the study indicate that selective MAO-A inhibitors are likely to attenuate FIA by augmenting central serotonergic activity, while selective MAO-B inhibitors accentuate the behaviour by facilitating dopaminergic activity. A permissive role for noradrenaline could not be delineated by the available data.     

Monoamine oxidase and semicarbazide-sensitive amine oxidase in spontaneously hypertensive and in normotensive control rats

The aim of the present work was to compare monoamine oxidase (MAO) and semicarbazide sensitive amine oxidase (SSAO) activity in several tissues from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Contribution of MAO-A, -B and SSAO to the metabolism of each substrate in each tissue was defined from experiments where the decrease of oxidative deamination of each substrate at a given concentration was measured as a function of increasing concentrations of a selective MAO-A, -B or SSAO inhibitor. In the heart, aorta and, to a lesser extent, the femoral arteries MAO-A activity was higher in SHR than in WKY. Similarly in the liver the enzyme activity was higher in SHR than in WKY but was due to the -B form of MAO. In all the other tissues studied (duodenum, brain, lungs, adrenals and kidneys) no difference in MAO-A, MAO-B or SSAO activity was found between SHR and WKY, except for the kidneys and brain, if the differences in the weights of these organs in SHR are taken into account.     

The oxidation of adrenaline and noradrenaline by the two forms of monoamine oxidase from human and rat brain

The selective monoamine oxidase inhibitors clorgyline and (−)-deprenyl were used to study the distribution of monoamine oxidase-A and -B (MAO-A, MAO-B) activities towards (−)-noradrenaline and (+),(−)-adrenaline in homogenates from seven different regions of human brain. The activities towards 5-hydroxytryptamine and 2-phenethylamine, which are essentially specific substrates for the A- and B-forms, respectively, under the conditions used in this work, were also determined. Noradreanline and adrenaline were substrates for both forms of the enzyme in all regions studied. The total MAO activity was found to be highest in the hypothalamus and lowest in the cerebellar cortex. Use of the selective MAO inhibitors clorgyline and (−)-deprenyl also showed adrenaline and noradrenaline to be substrates for both forms of the enzyme in rat brain. In human cerebral cortex and rat brain the two forms were found to have similar K_m-values and maximum velocities towards adrenaline. These values for the two forms were also found to be similar in human cerebral cortex when noradrenaline was used as the substrate. In contrast MAO-A showed a significantly lower K_m and a higher maximum velocity towards noradrenaline in rat brain. These results suggest that the rat may not provide a close model of the human for studies on the effects of MAO inhibitors on brain noradrenaline metabolism.     

Monoamine Oxidase in Rat and Bovine Endocrine

Monoamine oxidase (MAO) was characterized in tissue homogenates from rat pancreatic islets, rat neurohypophysis and adenohypophysis, and rat and bovine adrenal medulla and adrenal cortex. Phenylethylamine was preferentially deaminated by rat pancreatic islet and bovine adrenal medulla MAO and with slight preference by rat neurohypophysis MAO, whereas 5-hydroxytryptamine was preferentially deaminated by MAO from all other endocrine tissues. Tyramine was a good substrate for all tissues. Clorgyline, a selective inhibitor of MAO-A, preferentially inhibited deamination of 5-hydroxytryptamine by all tissue homogenates, whereas deprenyl, a selective inhibitor of MAO-B, preferentially inhibited deamination of phenylethylamine. Km values for 5-hydroxytryptamine and tyramine were higher by one to two decimal powers than for phenylethylamine in homogenates from all endocrine tissues. Km values were significantly lower for 5-hydroxytryptamine and significantly higher for phenylethylamine in rat and bovine adrenal cortex than in adrenal medulla. According to these results, the contributions of MAO-B to total enzyme activity were 70% for rat pancreatic islets, 45% for rat neurohypophysis, 15% for rat adenohypophysis, 20% for rat adrenal medulla, 10% for rat adrenal cortex, 60% for bovine adrenal medulla, and 20% for bovine adrenal cortex. PC 12 cells also contained predominantly MAO-A (90%); however, an increased Km for phenylethylamine and a sensitivity of deamination of this MAO-B substrate to inhibition by clorgyline are indicators of abnormal behavior of MAO in this clonal rat pheochromocytoma cell line.     

Different effects of monoamine oxidase inhibition on MPTP depletion of heart and brain catecholamines in mice

Pargyline, an inhibitor of monoamine oxidase type B (MAO-B), did not prevent the depletion of heart norepinephrine 24 hr after a single dose of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in mice. In mice killed 24 hr after the last of 4 daily doses of MPTP, the depletion of dopamine in the striatum and of norepinephrine in the frontal cortex was completely prevented by pargyline, but the depletion of heart norepinephrine was not prevented. These results with pargyline are the same as results obtained earlier with deprenyl, another selective inhibitor of MAO-B. The doses of pargyline and of deprenyl that were used resulted in almost complete inhibition of MAO-B activity (phenylethylamine as substrate) in brain, heart and liver of mice. Deprenyl did not inhibit MAO-A activity (serotonin as substrate) in brain, but pargyline caused some inhibition of MAO-A in brain. In heart and liver, serotonin was oxidized only at about 1/10 the rate of phenylethylamine oxidation, suggesting that MAO-B predominates in these tissues. Both pargyline and deprenyl caused some inhibition of serotonin deamination in heart and liver, suggesting that the oxidation may have been due partly to MAO-B. Experiments with selective MAO inhibitors in vitro showed that only about 20% of the oxidation of serotonin was occurring via MAO-B in heart and liver. The in vitro oxidation of MPTP by MAO in mouse brain, heart and liver was almost completely inhibited by pretreatment with either pargyline or deprenyl. Neither pargyline nor deprenyl had any significant effect on the concentrations of MPTP in brain or heart one-half hr after injection of MPTP into mice. The concentrations of the metabolite, MPP+ (1-methyl-4-phenyl-pyridinium), were markedly reduced in brain and in heart by pretreatment with either pargyline or deprenyl. The data suggest that MPP+ formation, which is necessary for the depletion of brain catecholamines after MPTP injection, may not be necessary for depletion of norepinephrine in heart. Since the oxidation of MPTP in vitro was inhibited more by pargyline or deprenyl pretreatment than was the appearance of MPP+ in vivo, the possibility exists that some MPP+ formation might occur by an enzyme other than MAO.