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.     

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.     

Intra- and extra-dopamine-synaptosomal localization of monoamine oxidase in striatal homogenates from four species

MAO-A and MAO-B activities within and outside dopaminergic synaptosomes in homogenates of striatal tissue from pig, cat, rat and human brains have been studied by using a specific "low substrate concentration technique" with dopamine. It was found that within the synaptosomes, MAO-A activity predominated in all species. Outside the synaptosomes there were more pronounced differences and only in the rat did MAO-A predominate, while in the other species MAO-B predominated. When estimating MAO-A and -B activities with a conventional method the activity of MAO-B predominated in man, cat and pig. Thus, also in species where the MAO-B activity (as estimated in a conventional way) was dominating, the intrasynaptosomal deamination of dopamine was brought about mainly by MAO-A. The "low substrate concentration technique", more adequately reflects physiological conditions by taking into account the higher concentration of monoamine transmitter substrates within the monoamine neurons. With this technique it was found that in all species (with the possible exception of man) the oxidation rate was higher within than that outside the DA-synaptosomes. In man the unavoidable longer time between death and estimation of the enzyme activity may be the cause of the deviating result.     

The effects of peptide and nonpeptide angiotensin II-receptor ligands on the activity of brain monoamine oxidase in water-repleted rats

The effects of i.c.v. administered peptide and nonpeptide ANG II-receptor ligands (losartan, EXP 3174, saralasin and sarmesin) on monoamine oxidase A (MAO-A) and MAO-B activities in the frontal cortex, striatum, hypothalamus and hippocampus of water-repleted rats were investigated. Alterations in MAO-A and MAO-B activities were found in different rat brain regions after ANG II which depended on the isoenzyme type and brain structure. MAO-A activity significantly increased in the frontal cortex and hypothalamus, brain regions containing AT1 receptors, mainly. MAO-A and MAO-B activities were affected differently by all studied ANG II-receptor ligands, which in most cases antagonized the effect of ANG II (losartan, an AT1-nonpeptide receptor antagonist being the most effective). There was no clearcut relationship between the inhibition of ANG II-induced water intake and the changes of MAO-A and MAO-B activities under the effect of the ANG II-receptor antagonists studied.     

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.     

Computer visualisation of the active site of monoamine oxidase-A by means of selective inhibitors

Computer visualisation of the active site of monoamine oxidase (MAO) is based on an assumption that the specific and reversible interaction of a ligand (substrate or inhibitor) with the substrate-binding region of the active site requires shape complementarity. The size of the ligand must allow its accommodation at the substrate-binding region. Analysis of the MAO-inhibitory activity of rigid analogues of isatin and pirlindole revealed a dependence between three-dimensional linear sizes of these molecules and the efficacy of inhibition of both MAO-A and MAO-B. However, flexible molecules did not exhibit any dependence between linear sizes and MAO-B inhibitory potency, possibly because they folded into compact structures could fit into the substrate-binding pocket of MAO-B. 'Moulding' of the substrate/inhibitor binding region by superposition of effective MAO-A inhibitors from various groups of chemicals allowed the shape of substrate/inhibitor binding region to be visualised. 'Removal of contents' from this mould yielded a cavity, which corresponded to the shape of substrate/inhibitor binding region. Such cavity can be used to evaluate the most probable positions known inhibitors take in binding to it. The docking procedure can also be used for searching molecular databases for new inhibitors. Pilot experiments revealed that relatively rigid compounds, which did not fit to this cavity, were poor inhibitors of MAO-A.     

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.     

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.     

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.     

Intestinal metabolism of tyramine by both forms of monoamine oxidase in the rat

The two forms of monoamine oxidase (MAO) in rat intestine and brain homogenates were found to have different K_m and V_max values towards tyramine. The K_m values for the A-form of the enzyme towards this substrate were around 120 μM in both cases, whereas the values for the B-form were about 240 μM. As a consequence, the ratio of activities (MAO-A: MAO-B) towards tyramine are dependent upon the substrate concentration. The MAO-A-selective inhibitors, toloxatone and cimoxatone, were found to be competitive inhibitors of the oxidation of tyramine by the A-form of this enzyme in the rat intestine, with K_i values of 3.4 μM and 3.7 nM respectively. The significance of these results in relation to the “cheese effect”, a pressor response to tyramine after monoamine oxidase inhibition, are discussed.     

Effect of milacemide on extracellular and tissue concentrations of dopamine and 5-hydroxytryptamine in rat frontal cortex.

1. Milacemide is a glycine prodrug which is both an inhibitor and a substrate for monoamine oxidase-type B (MAO-B) and also an inhibitor of MAO-type A (MAO-A). Its effects on dopamine and 5-hydroxytryptamine (5-HT) metabolism in rat frontal cortex tissue and dialysate were evaluated. 2. Dialysate dopamine concentrations increased linearly and dose-dependently after milacemide administration (100, 200, 400 mg kg-1, i.p.), peaking at 1 h. A concomitant dose-dependent decrease in dialysate 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations was observed but these changes were smaller (27% and 40% respectively) than the change in dopamine (125% after 400 mg kg-1 milacemide). 3. Dialysate 5-HT was significantly increased only at 1.5 h after giving milacemide 400 mg kg-1. Dialysate 5-hydroxyindoleacetic acid (5-HIAA) concentration was not affected. 4. Milacemide (400 mg kg-1) at 1.5 h post-administration significantly increased frontal cortex tissue concentrations of dopamine and 5-HT; the percentage increase in dopamine being about four times that of 5-HT. Metabolite concentrations, including 5-HIAA, decreased. Changes in tissue and dialysate dopamine, DOPAC and HVA were approximately proportionate to each other. 5. The results are explicable in terms of an inhibition by milacemide of MAO-A.     

Relevance of benzyloxy group in 2-indolyl methylamines in the selective MAO-B inhibition

1. Previous studies with indolyl derivatives as monoamine oxidase (MAO) inhibitors have shown the relevance of the indole structure for recognition by the active site of this enzyme. We now report a new series of molecules with structural features which determine the selectivity of MAO inhibition. 2. A benzyloxy group attached at position 5 of the indole ring is critical for this selective behaviour. Amongst all of these benzyloxy-indolyl methylamines, N-(2-propynyl)-2-(5-benzyloxyindol)methylamine FA-73 was the most potent MAO-B 'suicide' inhibitor studied. 3. The Ki values for MAO-A and MAO-B were 800+/-60 and 0.75+/-0.15 nM, respectively. These data represent a selectivity value of 1066 for MAO-B, being 48 times more selective than L-deprenyl (Ki values of 376+/-0.032 and 16.8+/-0.1 nM for MAO A and MAO-B, respectively). The IC50 values for dopamine uptake in striatal synaptosomal fractions from rats were 150+/-8 microM for FA-73 and 68 +/- 10 microM for L-deprenyl whereas in human caudate tissue the IC50 values were 0.36+/-0.015 microM for FA-73 and 0.10+/-0.007 microM for L-deprenyl. Moreover, mouse brain MAO-B activity was 90% ex vivo inhibited by both compounds 1 h after 4 mg kg(-1) administration, MAO-A activity was not affected. 4. These novel molecules should provide a better understanding of the active site of monoamine oxidase and could be the starting point for the design of further selective, non-amphetamine-like MAO-B inhibitors with therapeutic potential for the treatment of neurological disorders.     

The Anticonvulsant FCE 26743 is a Selective and Short-acting MAO-B Inhibitor Devoid of Inducing Properties towards Cytochrome P450-dependent Testosterone Hydroxylation in Mice and Rats

Abstract— The effects of the potent anticonvulsant FCE 26743 ((S)-2-(4-(3-fluorobenzyloxy)benzylamino)propionamide) on monoamine oxidase (MAO) activity were measured in-vitro and ex-vivo using rat tissue homogenates. In-vitro, FCE 26743 showed potent and selective inhibitory properties towards liver MAO-B, with IC50 values about 10^?7 m for MAO-B and higher than 10^?5 m for MAO-A. When determined ex-vivo in brain, the ED50 value for the inhibition of MAO-B was 1·1 mg kg^?1 (p.o.) 1 h post-dosing, whereas MAO-A remained virtually unaffected after administration of 60 mg kg^?1. Similar effects were seen in liver. Following oral administration of 5 mg kg^?1 FCE 26743 to rats, brain MAO-B inhibition was 79% after 1 h and 13% after 24 h, indicating that FCE 26743 behaves as a short-acting MAO-B inhibitor. The ability of FCE 26743 to act as a MAO substrate was assessed in mice by measuring the urinary excretion of alaninamide, a potential metabolite of FCE 26743 which would result from the action of MAO. No alaninamide was detectable in the 0–8 h urines after administration of a 119 mg kg^?1 dose, suggesting that FCE 26743 is not, or only to a small degree, a substrate of MAO. The effects of FCE 26743 on cytochrome P450 enzymes involved in testosterone hydroxylation were determined in rats after repeated administration. No induction of the cytochrome P450 system was noted.     

Effect of diethylnitrosamine on monoamine oxidase in rat liver

The effect of diethylnitrosamine (DEN), a well-known experimental carcinogen, toward MAO-A and MAO-B activity of rat liver was investigated. The oxidations of both beta-PEA (MAO-B) and 5-HT (MAO-A) were inhibited by DEN. The K1 values of DEN in the inhibition of rat liver MAO-A and MAO-B activity were determined. The kinetic data show that DEN is a competitive, MAO-B selective inhibitor and its inhibitory effect on MAO-B is about 4-fold more potent than that on MAO-A. DEN might change the proportions of the multiple forms of MAO activity in tumor cells.     

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.     

Concentration dependence of the oxidation of tyramine by the two forms of rat liver mitochondrial monoamine oxidase

The two forms of monoamine oxidase in rat liver mitochondria were shown to have different K_m and maximum velocity values with tyramine as the substrate. K_m values of 107±15 μM and 579±45μM were determined for the A- and B-form respectively at pH 7.2 in air-saturated buffer. The maximum velocity of the A-form was found to be approximately half of that of the B-form under these conditions. A consequence of these differences is that the ratio of activities of MAO-A:MAO-B determined from clorgyline inhibition curves will be dependent upon the concentration of tyramine used to assay for enzyme activity. As a fixed concentration of tyramine, the height of the plateau in a clorgyline inhibition curve will also be affected by the presence of a selective competitive inhibitor. Procaine, an MAO-A selective competitive inhibitor, was found to increase the K_m value of the MAO-A towards tyramine to 505 ± 172 μM and under these conditions the plateau-height of the clorgyline inhibition curve was not significantly affected by variations of the tyramine concentration over a 20-fold range.     

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.     

Cardiovascular responses to combined treatment with selective monoamine oxidase type B inhibitors and L-DOPA in the rat

BACKGROUND AND PURPOSE: Postural hypotension is a common side-effect of L-DOPA treatment of Parkinson's disease, and may be potentiated when L-DOPA is combined with selegiline, a selective inhibitor of monoamine oxidase B (MAO-B). Rasagiline is a new, potent and selective MAO-B inhibitor, which does not possess the sympathomimetic effects of selegiline. We have studied the effects of these selective MAO inhibitors, L-DOPA and dopamine on the cardiovascular system of the rat. EXPERIMENTAL APPROACH: Blood pressure and heart rate was measured in conscious rats following acute or chronic administration of rasagiline, selegiline and L-DOPA, by comparison with the selective MAO-A inhibitor clorgyline, or the MAO-A/B inhibitor tranylcypromine. Cardiovascular responses, catecholamine release, and modification of pressor response to dopamine were studied in pithed rats. KEY RESULTS: In conscious rats neither rasagiline nor selegiline caused significant potentiation of the effects of L-DOPA (50, 100, 150 mg.kg(-1)) on blood pressure or heart rate at doses which selectively inhibited MAO-B, but L-DOPA responses were potentiated by clorgyline and tranylcypromine. In rats treated twice daily for 8 days with L-DOPA and carbidopa, selegiline (5 mg.kg(-1)) but not rasagiline (0.2 mg.kg(-1)) caused a significant hypotensive response to L-DOPA and carbidopa, although both drugs caused similar inhibition of MAO-A and MAO-B. In pithed rats, selegiline but not rasagiline increased catecholamine release and heart rate, and potentiated dopamine pressor response at MAO-B selective dose. CONCLUSIONS AND IMPLICATIONS: The different responses to the two MAO-B inhibitors may be explained by the amine releasing effect of amphetamine metabolites formed from selegiline.     

Drug-induced changes in motor activity after selective MAO inhibition

The increase in motor activity produced in mice by phenylethylamine (PEA), L-DOPA and amphetamine was evaluated after selective inhibition of MAO Type A (by clorgyline) or Type B (by low doses of pargyline). PEA-induced motor stimulation was intensified in the presence of MAO-B inhibition, but not when MAO-A was inhibited. This was paralleled by higher concentrations of brain and plasma PEA (after injection) in mice in which there was inhibition of MAO-B compared with control or MAO-A inhibition. Conversely, L-DOPA produced significant stimulation only when MAO-A was inhibited. The clorgyline pretreatment resulted in larger increases in brain dopamine concentrations (in the striatum, olfactory tubercles and in the area containing the substantia nigra) than did MAO-B inhibition. This effect occurred both in mice receiving L-DOPA + inhibitor and in mice receiving the inhibitor alone. Amphetamine-induced stimulation was increased following the inhibition of either form of MAO, and this was not the result of changes in the distribution or metabolism of amphetamine. These results support the concept that MAO-A and MAO-B deaminate different substrates in the rodent CNS and that amphetamine may utilize either dopamine or PEA in producing its stimulant effects.