Species differences in the deamination of dopamine and other substrates for monoamine oxidase in brain

Cortex and caudate specimens from human, non-human primate and rodent brains were examined for their ability to deaminate dopamine and for their sensitivity to irreversible monoamine oxidase (MAO) inhibitors. Using inhibition curves obtained with clorgyline, deprenyl and pargyline to estimate the relative proportions of MAO-A and MAO-B activity, dopamine was found to be deaminated predominantly by MAO-A in rat cortex and caudate. In contrast, dopamine was primarily an MAO-B substrate in human and vervet cortex and caudate. When clorgyline inhibition curves with tyramine or dopamine as substrate were compared in human, vervet and rat cortex, more pronounced species differences were found with dopamine than with tyramine. In all three species caudate tended to be more sensitive to inhibition by low concentrations of clorgyline than was cortex, suggesting a higher proportion of MAO-A activity in caudate. Similar species differences were also found when MAO-A activities were estimated using serotonin (5-HT): -phenylethylamine (PEA) ratios (5-HT/5-HT + PEA). These ratios with selective substrates were highly correlated with clorgyline inhibition curves obtained with tyramine as substrate across 29 brain regions and tissues from different rodent and primate species (r=0.85, P<0.001). Data from both the substrate ratios and the clorgyline inhibition curves confirmed the relative predominance of MAO-B activity in primate brain regions (70–85%) as compared to rat brain regions (45%). Smaller species differences were observed in liver. Species differences in the proportion of brain MAO-A and B activities and in the deamination of dopamine and other substrates for MAO may have important implications in regard to the widespread use of rodent rather than primate models in the study of biogenic amine metabolism and of drugs affecting amine function.     

Styrene inhibits monoamine oxidase A, but not monoamine oxidase B in monkey brain mitochondria

The effects of styrene on mitochondrial monoamine oxidase (MAO) activity in rat and monkey brains were compared in vitro. After preincubation at 25 degrees C for 20 min with 1 mM styrene monomer MAO-A activity in monkey brain was inhibited potently using 5-HT (for MAO-A substrate), but MAO-B activity in monkey brain and platelets were slightly inhibited using beta-PEA (for MAO-B substrate). Styrene monomer also competitively inhibited MAO-A activity in a dose-dependent manner. MAO-A in monkey brain was inhibited by styrene in ascending order of potency: styrene trimer>styrene dimer>styrene monomer. In contrast styrene monomer slightly inhibited both MAO-A and MAO-B activities in rat brain mitochondria. In the present study styrene monomer potently inhibits MAO-A activity, but not MAO-B activity, in monkey brain mitochondria in vitro. These results indicate the inhibiting action of styrene differs depending on animal species and MAO isoforms.     

Inhibition by Zn(2+) of A-form monoamine oxidase in monkey brain mitochondria

The effects of ZnSO(4) on mitochondrial monoamine oxidase (MAO) activity in monkey brain were compared with those in rat and rabbit, in vitro. After preincubation at 25 degrees C for 20 min with 1 microM ZnSO(4), MAO-A activity in monkey brain was about 50% using serotonin (5-HT) as a substrate, and the inhibition was proportional to the concentration of ZnSO(4). However, ZnSO(4) had no effect on MAO-B activity in monkey brain using beta-phenylethylamine (beta-PEA) as a substrate. The inhibition by ZnSO(4) of MAO-A activity was competitive and reversible. CdSO(4) also inhibits MAO-A, but not MAO-B in monkey brain mitochondria. ZnSO(4) did not inhibit either MAO-A or MAO-B activity in rat and rabbit brain mitochondria. These results indicate that the inhibiting action of Zn(2+) differs depending on animal species. In monkey brain mitochondria, MAO-A was highly sensitive to Zn(2+) and MAO-B was less sensitive. These results also suggest that Zn(2+) may regulate the level of catecholamine content in monkey brain.     

Substrate selectivity of monoamine oxidase A, monoamine oxidase B, diamine oxidase, and semicarbazide-sensitive amine oxidase in COS-1 expression systems

The substrate selectivity of monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), diamine oxidase (DAO), and semicarbazide-sensitive amine oxidase (SSAO) was investigated in the absence of chemical inhibitors using the COS-1 cells expressed with respective amine oxidase. Serotonin (5-hydroxytryptamine), 1-methylhistamine, and histamine were preferentially oxidized by MAO-A, SSAO, and DAO, respectively, at a low substrate concentration. In contrast, benzylamine, tyramine, and beta-phenylethylamine served as substrates for all of MAO-A, MAO-B, and SSAO. Each amine oxidase showed broad substrate selectivity at a high substrate concentration. The cross-inhibition was remarkable in MAO-A and MAO-B, especially in MAO-A, but not in SSAO and DAO. A study of the substrate selectivity of amine oxidases should include consideration of the effects of substrate concentration and specific chemical inhibitors.     

Relation between brain monoamine oxidase (MAO) activity and the firing rate of locus coeruleus neurons

Summary Utilizing a specific low substrate concentration technique, intrasynaptosomal MAO-A and MAO-B activities within the rat brain noradrenaline system were studied. It was found that mainly MAO-A was localized intrasynaptosomally, whereas MAO-B contributed with less than 15% of the total intrasynaptosomal MAO activity, a phenomenon that was also observed within the central dopamine system. It is suggested that the intrasynaptosomal pool of MAO in the noradrenaline and the dopamine systems may reflect the density of innervation of the respective system throughout the brain. In addition, the effects of various selective monoamine oxidase (MAO) inhibitors on the noradrenergic intrasynaptosomal MAO activity as well as on the neuronal firing rate of noradrenaline containing cells in the locus coeruleus (LC) were investigated. Pretreatment with the MAO-A selective inhibitors clorgyline (10 mg/kg, i.p., 1 h) or (+)-FLA 336 (1 mg/kg, i.p., 1 h) caused a significant depression (40%) of mean spontaneous firing rate of LC neurones, randomly encountered throughout the LC. The MAO-B selective inhibitor pargyline (10 mg/kg, i.p., 1 h) was found to lack effect in this regard. However, pretreatment with (–)-deprenyl (10 mg/kg, i.p., 1 h), equally a selective MAO-B inhibitor, markedly suppressed the spontaneous firing rate of LC units. This inhibition by (–)-deprenyl was blocked by pretreatment with SK&F 525 A (50 mg/kg, i.p., 30 min), an inhibitor of microsomal drug metabolizing enzymes. Thus, the depression of LC units by (–)-deprenyl seems to be executed by a metabolite, e. g. l-amphetamine. Taken together, the present electrophysiological and biochemical results show that the neuronal depression of noradrenaline neurones in the LC by MAO-inhibitors is specifically related to the inhibition of MAO-A. Furthermore, the data indicate a relationship between the activity of intrasynaptosomally located MAO-A and the neuronal activity of central noradrenaline pathways.     

The inhibition of monoamine oxidase activity by various antidepressants: differences found in various mammalian species

The effects of the antidepressant drugs zimeldine, imipramine, maprotiline or nomifensine on mitochondrial monoamine oxidase (MAO) activity in mouse, rat, dog and monkey brains were compared in vitro. Mouse, rat, dog and monkey brain MAO-B activities were inhibited by zimeldine more potently than MAO-A activity. Imipramine inhibited MAO-B more potently than MAO-A activity in mouse and rat brains. When dog and monkey brains were investigated, MAO-A activity was inhibited more potently than MAO-B activity at high concentrations of imipramine, while at low concentrations, MAO-B activity was more potently inhibited. Maprotiline and nomifensine inhibited mouse and rat brain MAO-B activity more potently than MAO-A activity, while the inverse was true for dog and monkey brains. All four drugs are competitive inhibitors of MAO-A, but noncompetitive inhibitors of MAO-B in all animal brains. The respective Ki values of these reagents for monkey brain MAO-A and MAO-B were low compared to those of mouse, rat and dog. These results indicate that monkey brain MAOs are more sensitive to antidepressant drugs than those in rodent brain.     

Stereoselectivity and isoenzyme selectivity of monoamine oxidase inhibitors. Enantiomers of amphetamine, N-methylamphetamine and deprenyl

The enantiomers of amphetamine, N-methylamphetamine and deprenyl were studied, using a solubilised rat liver mitochondrial monoamine oxidase (MAO) preparation, as competitive inhibitors of MAO-A and MAO-B (5-hydroxytryptamine and beta-phenylethylamine as substrate respectively). Only in the case of deprenyl enantiomers inhibiting MAO-B was a preference shown towards the [R]-configuration enantiomer justifying the use of [R]-(-)-deprenyl (as compared to the racemate) for the specific inhibition of MAO-B. Recalculation of the observed Ki values in terms of the base form of the inhibitor indicated that the activity of all enantiomers fell within a narrow, approximately 25-fold range when inhibiting MAO-B. The selectivity of inhibition of MAO-B by [R]-(-)-deprenyl cannot therefore be attributed to any specific structural features of the MAO-B isoenzyme form but rather to a lack of affinity of this enantiomer towards MAO-A.     

Effect of low-dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine-induced dopamine release in vivo

1. Chronic treatment with low doses of the selective monoamine oxidase (MAO) type B inhibitors selegiline [(-)-deprenyl] and rasagiline, causes elevation in extracellular level of 3,4-dihydroxyphenylethylamine (dopamine) in the rat striatum in vivo (Lamensdorf et al., 1996). The present study was carried out to determine whether this effect of selegiline could be the result of an inhibition of the high-affinity dopamine neuronal transport process. 2. Changes in activity of the dopamine transporter (DAT) in vivo following selegiline treatment were evaluated indirectly by microdialysis technique in the rat, from the change in striatal dopamine extracellular concentration following systemic amphetamine administration (4 mg kg(-1), i.p.). Striatal levels of the DAT molecule were determined by immunoblotting. Uptake of [3H]-dopamine was determined in synaptosomes from selegiline-treated animals. 3. Amphetamine-induced increase in striatal extracellular dopamine level was attenuated by one day and by chronic (21 days) treatment with selegiline (0.25 mg kg(-1), s.c.). 4. Striatal levels of DAT were elevated after 1 and 21 days treatment with selegiline, but were not affected by clorgyline, rasagiline, nomifensine or amphetamine. 5. The increase in DAT expression, and attenuation of amphetamine-induced dopamine release, were not accompanied by a change in [3H]-dopamine uptake in synaptosomes of selegiline-treated animals. 6. The results suggest that a reversible inhibition of dopamine uptake occurs following chronic low dose selegiline treatment in vivo which may be mediated by an increase in endogenous MAO-B substrates such as 2-phenylethylamine, rather than by the inhibitor molecule or its metabolites. Increased DAT expression appears to be a special property of the selegiline molecule, since it occurs after one low dose of selegiline, and is not seen with other inhibitors of MAO-A or MAO-B. The new DAT molecules formed following selegiline treatment appear not to be functionally active.     

Inhibition of rat brain monoamine oxidase activity by cerebral anti-ischemic agent, ifenprodil

Ifenprodil, which is clinically used as a cerebral vasodilator, inhibited rat brain type A (MAO-A) and type B (MAO-B) monoamine oxidase activity. It did not, however, affect rat lung semicarbazide-sensitive amine oxidase. The degree of inhibition of either form of MAO was not changed by 30 min preincubation of the enzyme preparations at 37 degrees C with ifenprodil. Modes of inhibition of MAO-A and MAO-B by ifenprodil were competitive towards oxidation of their respective substrates, 5-hydroxytryptamine and benzylamine, with Ki values of 75 microM for inhibition of MAO-A and 110 microM for inhibition of MAO-B.     

Selectivity of clorgyline and pargyline as inhibitors of monoamine oxidases A and B in vivo in man

During 4 weeks of treatment with clorgyline, a selective MAO-A inhibitor, platelet monoamine oxidase (MAO) activity was unchanged. During a similar 4-week crossover treatment period with pargyline, a selective MAO-B inhibitor, platelet MAO activity was essentially completely inhibited in the same individuals. The differential effects of the two drugs on platelet MAO, which consists exclusively of the MAO-B form, suggests that the in vitro selectivity of clorgyline, and possibly of pargyline, on MAO-A and MAO-B may be maintained in vivo during long-term administration in man. Reductions in blood pressure, heart rate, and plasma amine oxidase activity were generally similar in magnitude during treatment with both drugs, however, suggesting that either these effects are nonspecific consequences of both MAO-A and MAO-B inhibition, or that pargyline also inhibited MAO-A activity.     

Enzymic and molecular characteristics of a new form of monoamine oxidase, distinct from form-A and form-B

The present study was undertaken to clarify the enzymic and molecular properties of monoamine oxidase (MAO) in carp brain. In particular, its sensitivities to selective MAO inhibitors, kinetic properties and molecular weight were compared with those of the enzyme in carp liver. The selective and potent MAO-A and MAO-B inhibitors FLA 788(+), FLA 336(+), MD 780236 and benzylcyanide caused dose-dependent inhibitions of MAO activity in both carp brain and liver; the inhibition curves were all single-sigmoidal, and the degrees of inhibition of the activities towards 5-hydroxytryptamine (5-HT, selective MAO-A substrate), tyramine (substrate for both forms of MAO) and beta-phenylethylamine (PEA, selective MAO-B substrate) were similar. This was also the case for inhibition of activity in carp brain by the irreversible and selective MAO-A and MAO-B inhibitors clorgyline and I-deprenyl, indicating the presence in both preparations of a single MAO which differs from either form of MAO. Studies on the substrate specificities and Km values for these three substrates and the inhibitory effects of some compounds suggested that the enzymic characters of MAO in carp preparations were similar and that these enzymes might be FAD-containing enzymes, like MAO in various mammals. By labelling the preparations with radioactive pargyline and then subjecting them to sodium dodecyl sulfate electrophoresis, the apparent molecular weights of carp brain and liver MAO were estimated as 60,000 daltons. The same value was also obtained for rat brain and liver mitochondrial MAO-B. These results indicate that by the present definitions of MAO-A and MAO-B, MAO in carp brain and liver is similar to, but distinct from, both these forms of MAO.     

Evidence for an extraneuronal location of monoamine oxidase in renal tissues

Summary (1) Homogenates of renal cortex and renal medulla of control and 6-hydroxydopamine-denervated cat kidneys were prepared. (2) Monoamine oxidase (MAO) activity was determined with [³H]-5-hydroxytryptamine ([³H]-5HT) and [¹⁴C]--phenylethylamine ([¹⁴C]--PEA) as preferential substrates for MAO-A and MAO-B, respectively. (3) The endogenous dopamine and noradrenaline tissue contents of control and chemicallydenervated kidneys were compared with the MAO activities. (4) The results show that a 70% depletion of monoamine content by chemical denervation resulted only in a 23% reduction of MAO-A activity in the renal cortex, whereas MAO-13 was unaffected either in the cortical or the medullary zones; in the renal medulla MAO-A activity was not changed by denervation. Most of the MAO activity in the cat kidney is of the B type (74%) and is located in the renal cortex.On leave from Faculdade de Farmácia, Universidade de Coimbra, Portugal Send offprint requests to P. Soares-da-Silva at the above address     

Interaction of psychotropic drugs with monoamine oxidase in rat brain

Literature observations indicate that some psychotropic drugs may have inhibitory activity towards monoamine oxidase (MAO). This study was undertaken to assess the potency, isozyme selectivity and mechanism of inhibition of representative first- and second-generation antidepressant drugs towards rat brain MAO-A and MAO-B. Five tricyclic antidepressants (imipramine, trimipramine, clomipramine, amitriptyline and doxepine) and three selective serotonin reuptake inhibitors (fluoxetine, fluvoxamine and citalopram) were examined. They showed inhibitory activity towards MAO-A and MAO-B, with clear selectivity for MAO-B (Ki in the micromolar range). Their mechanism of inhibition was competitive towards MAO-B and of a mixed competitive type towards MAO-A. The results suggest that some of the drugs examined might also contribute an MAO inhibitory effect in chronically treated patients.     

Inhibition of rodent brain monoamine oxidase and tyrosine hydroxylase by endogenous compounds - 1,2,3,4-tetrahydro-isoquinoline alkaloids

Four different noncatecholic and one catecholic tetrahydroisoquinolines (TIQs), cyclic condensation derivatives of beta-phenylethylamine and dopamine with aldehydes or keto acids, were examined for the inhibition of rat and mouse brain monoamine oxidase (MAO) and rat striatum tyrosine hydroxylase (TH) activity. Simple noncatecholic TIQs were found to act as moderate (TIQ, N-methyl-TIQ, 1-methyl-TIQ) or weak (1-benzyl-TIQ), MAO B and MAO A inhibitors. 1-Methyl-TIQ inhibited more potently MAO-A than MAO-B; the similar but more modest effect was exerted by salsolinol. Only salsolinol markedly inhibited TH activity, being competitive with the enzyme biopterin cofactor. The inhibition of MAO and TH by TIQs is discussed in relation to their ability to regulate monoamine metabolism.     

Studies of monoamine oxidase and semicarbazide-sensitive amine oxidase. I. Inhibition by a selective monoamine oxidase-B inhibitor, MD 780236

In vitro studies of the effect of MD 780236, a selective monoamine oxidase (MAO)-B inhibitor, on a semicarbazide-sensitive amine oxidase (SSAO) in rat testis and lung showed that this compound dose-dependently inhibited SSAO activity. The extents of inhibition of MAO-A, -B and SSAO in these two rat tissues by this compound after 30 min of preincubation were found to be MAO-B greater than MAO-A greater than SSAO. This selectivity was also evident in preparations without preincubation. Degree of inhibition of SSAO was not significantly influenced by pretreatment with either 10(-3) M clorgyline, I-deprenyl or 10(-4) M SKF 525A. Inhibition of SSAO was not enhanced by varying the time of preincubation of the enzyme and the compound, indicating direct action on and reversible inhibition of SSAO. The inhibition of SSAO by MD 780236 was non-competitive with or without preincubation, with a K1 value of 110 muM. Although MD 780236 is a selective and "suicide substrate" inhibitor of MAO-B, these present results indicate that this compound may also inhibit SSAO activity, but by a mechanism different from that for MAO-B. These findings confirm an earlier hypothesis that compounds that inhibit both MAO and SSAO have totally different modes of action on these two different amine oxidases.     

The deamination of dopamine by human brain monoamine oxidase

The deamination of dopamine has been studied in seven regions of human brain. Both A and B forms of the enzyme were found to be active towards this substrate. The ratio of activities of MAO-A: MAO-B was found to vary considerably from brain region to brain region, from about 1:1 for the cerebral and cerebellar cortex to about 1:2 for the pons and medulla oblongata. Enzyme titration studies and comparisons of the substrate specificities of MAO-A and MAO-B across the brain indicated that dopamine was metabolised by the same MAO active centres as other monoamines. In the cerebral cortex, the Km values of MAO-A and -B towards dopamine were found to be 210 and 230 microM, respectively, indicating that the relative contributions of these two forms towards the oxidation of this substrate will not be significantly affected by changes in its concentration.     

Substrate and inhibitor selectivity of human heart monoamine oxidase.

The subcellular distribution, inhibitor sensitivity, thermostability and pH profiles of monoamine oxidase (MAO) from samples of human heart obtained at post mortem have been investigated with several substrates. A simple subcellular fractionation showed that, with either tyramine or benzylamine as substrate, about 50 per cent of the MAO activity was found in the mitochondrial fraction, with negligible quantities in the high speed supernatant. From the use of clorgyline, it appears that 5-HT is a substrate for MAO-A, benzylamine and β-phenethylamine are substrates for MAO-B, while tyramine and dopamine are substrates for both forms of the enzyme, d-Amphetamine was shown to be a selective competitive inhibitor of MAO-A, of similar potency to that observed with MAO from rat liver. No significant difference between the thermostability at 50° of the MAO activity towards 5-HT and benzylamine was observed. Preliminary results for the effect of pH on human heart MAO are presented. The results are discussed with respect to similar data obtained for MAO from other human and animal tissues.     

Comparison of intra- and extrasynaptosomal monoamine oxidase-A and -B activities in the striatum and frontal cortex of two mice strains with different sensitivities to the neurotoxic actions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

Conditions for the assay of the intra- and extrasynaptosomal rates of deamination of dopamine and noradrenaline by the two forms of monoamine oxidase (MAO) have been determined in striatal and frontal cortical homogenates, respectively, from C57 BL/6 mice. The activities obtained were compared with the corresponding activities found for NMRI mice, a strain less sensitive to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) than the C57 BL/6 strain. In both strains, the intra- and extrasynaptosomal deamination of dopamine in the striatal homogenates was brought about predominantly by MAO-A. No significant differences between the two strains were found for the intra- or extrasynaptosomal MAO-A or -B activities towards dopamine in striatal homogenates. On the other hand, the striatal dopamine concentrations were higher in the C57 BL/6 mice than in the NMRI mice. The concentrations of the dopamine metabolites DOPAC and HVA were similarly higher, suggesting that the rate of turnover of dopamine is the same for the two strains. In frontal cortical homogenates, MAO-A predominated in the deamination of noradrenaline both intra- and extrasynaptosomally. The extrasynaptosomal rates of deamination of noradrenaline were similar in the two mice strains, whereas the intrasynaptosomal MAO-A activity was significantly higher for the C57 BL/6 mice. These results concur with and extend to the noradrenergic system the conclusion previously made by Jossan et al. (1987) for the dopaminergic system that although MAO-B activity is necessary for expression of MPTP neurotoxicity, it is not the rate-limiting step for the development of the neurotoxic effects.     

Zinc benzoate, a contaminating environmental compound derived from polystyrene resin inhibits A-type monoamine oxidase

The contaminants in deionized and distilled water (DDI water) boiled with polystyrene resin inhibited A-type monoamine oxidase (MAO, MAO-A preferentially deaminates serotonin and norepinephrine and regulates these amines concentration) activity in monkey brain mitochondria. To identify these contaminants, we attempted measurements by HPLC, FT-IR and NMR. The compound inhibiting MAO-A activity was zinc benzoate. Although it potently inhibited MAO-A activity, zinc benzoate did not effect MAO-B in monkey brain mitochondria. It also reversibly and competitively inhibited MAO-A activity in a dose-dependent manner. Zinc benzoate, however, did not inhibit either MAO-A or -B activities in rat brain mitochondria. These results indicate that zinc benzoate, which inhibits MAO-A activity, is easily incorporated in DDI water by boiling polystyrene and also may be a contaminating environmental chemical compound that alters the levels of serotonin and norepinephrine in the central nervous system.     

Effect of lamotrigine on the activities of monoamine oxidases A and B in vitro and on monoamine disposition in vivo

Recent clinical evidence indicates that the broad spectrum anticonvulsant drug lamotrigine is effective against the depressive phase of bipolar illness and the difficult to treat rapid cycling form of the disorder. However, the molecular mechanism underlying this therapeutic action remains uncertain. Given that inhibition of the A-type of monoamine oxidase (MAO) is a proven antidepressant mechanism, we investigated the effects of lamotrigine on MAO activities in vitro and on monoamine disposition in vivo. In vitro, lamotrigine inhibited rat brain MAO activities with Ki values (MAO-A, 15 microM; MAO-B, 18 microM) potentially within the therapeutic range for this drug. The effects of lamotrigine on the MAO-A activities of rat brain and human liver preparations were almost identical suggesting minimal species or tissue variation. In contrast, there was no (MAO-A) or minimal (MAO-B) reduction in brain MAO activities when assayed ex vivo following the administration of lamotrigine to rats. In vivo brain microdialysis failed to detect meaningful alterations in extracellular hippocampal or frontal cortex monoamine concentrations. Furthermore, lamotrigine did not modulate oral tyramine-induced hypertension in rats or 5-hydroxytryptophan-induced head shaking in mice, providing strong evidence that the drug does not perturb monoamine metabolism in vivo. The absence of observable effects of lamotrigine on monoamine disposition in vivo may be explained by the competitive and highly reversible nature of the interaction of lamotrigine with MAO isoforms. Thus, altered monoamine metabolism in vivo is unlikely to account for the antidepressant action of the drug in bipolar depression.