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.     

Studies of monoamine oxidases: Effect of Triton X-100 and bile salts on monoamine oxidase in brain mitochondria

Triton X-100 and the bile salts, cholate and deoxycholate, detergents often used in the solubilization of monoamine oxidase (MAO) from mitochondria, have been found to cause an inhibition of the enzyme activity. With beef brain mitochondria, it was found that there was a differential effect of Triton X-100 on the putative MAO types A and B, with MAO-A being more susceptible to inhibition by Triton X-100. This was indicated by the greater loss of serotonin-deaminating than of phenyl ethylamine-deaminating activity in the presence of Triton X-100. Although the bile salts also caused substantial inactivation at concentrations above 0.1%, no differentiation between MAO types could be made. Kinetic studies of the inhibition by Triton X-100 indicated two different mechanisms were occurring with the two MAO types. The inhibition was competitive for MAO-A, but uncompetitive for MAO-B. Removal of Triton X-100 by co-polymer beads restored some, but not all of the activity for both MAO-A and MAO-B types. This suggests that the activity loss may have been due in part to inactivation when the enzyme was separated from the mitochondrial membrane.     

Some effects of non-ionic detergent triton X-100 on rat liver monoamine oxidase

The non-ionic detergent Triton X-100, an agent used to solubilize mitochondrial membrane monoamine oxidase (EC 1.4.3.4, MAO), has been shown to inhibit markedly MAO activity. The inhibition was non-competitive in nature. Triton X-100 changed the susceptibility of MAO toward clorgyline, a specific type A MAO inhibitor, and deprenyl, a type B inhibitor. Its effect on the temperature dependence of the initial velocity revealed that the transition temperatures for p-tyramine and serotonin (22°) and β-phenylethylamine (16° and 27°) were not changed. The stability of the MAO decreased considerably, however, in the presence of Triton X-100, and its inactivation was particularly pronounced somewhat higher temperatures.     

Comparative lipophilicities of substrates of monoamine oxidase

Oil/water partition coefficients of various substrates of monoamine oxidase (MAO) and kinetic parameters of MAO-A and -B of rat liver at two pH values, pH 7 and pH 9, were investigated. Octanol, heptane or benzene were chosen for the oil phases. The deamination of the biogenic amines 5-hydroxytryptamine (5-HT), tyramine, 2-phenethylamine (PEA) and benzylamine was studied at pH 7 and pH 9. Results indicated all four substrates were very hydrophilic, and the oil/water partition coefficients of benzylamine and PEA were higher than those of 5-HT and tyramine. The changes in Km and Vmax values at pH 7 and pH 9 indicated that the affinities of MAO-A towards 5-HT and tyramine slightly increased at pH 9 and those of MAO-B towards tyramine and benzylamine also increased at pH 9, while uncharged amines at pH 9 amounted to about a hundred times of those at pH 7. It is concluded that the mitochondrial MAO bound to the membrane may metabolize charged molecules as well as uncharged counterparts.     

Biochemical and pharmacologic properties of 2614W94, a reversible,competitive inhibitor of monoamine oxidase-A

2614W94 [3-(1-trifluoromethyl)ethoxyphenoxathiin 10,10-dioxide] is a selective, reversible inhibitor of monoamine oxidase-A with a competitive mechanism of inhibition and a K_i value of 1.6 nM with serotonin as substrate. In pretreated rats, the ED₅₀ value after single oral dosing was 1.7 mg/kg, similar to an ED₅₀ value of 1.1 mg/kg estimated in the 5-hydroxytryptophan potentiation test. Maximal inhibition of monoamine oxidase-A (MAO-A) was observed by 0.5 h after dosing, suggesting rapid transport to brain. Inhibition in brain was maintained for several hours, followed by a gradual reversal with a half-time of 7.2 h. Brain levels of parent compound were higher than plasma levels at all times after dosing. No significant inhibition of MAO-B was observed. After preincubation of MAO with 2614W94 at 37°C, the inhibition was reversed by dialysis. Concentrations of serotonin, norepinephrine, and dopamine were clearly elevated in brains of rats after single oral doses, whereas levels of MAO metabolites were decreased. In a rat model designed to show blood pressure elevations in response to a threshold dose of orally administered tyramine, 2614W94 compared well with moclobemide, an MAO-A selective inhibitor that has not been associated with problems relating to dietary tyramine. The two stereoisomers of 2614W94 were both potent MAO-A inhibitors. In vitro and in vivo properties of 2614W94 suggest that this compound and its close analogs are among the most potent MAO-A inhibitors known and that they may have therapeutic potential as safe new antidepressant/anxiolytic agents. Drug Dev. Res. 45:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Mechanism of monoamine oxidase-A inhibition by BW 1370U87

BW 1370U87 is a potent, selective inhibitor of rat and human MAO-A with a competitive mechanism of action. K_i = 0.01 μM with either serotonin or tyramine as substrate. After preincubation of BW 1370U87 with mitochondrial MAO, full enzyme activity was restored by dialysis. Following oral administration to rats, BW 1370U87 inhibited brain MAO-A in a dose-dependent manner, with a duration greater than 6 hr, but less than 24 hr. No significant inhibition of MAO-B by BW 1370U87 was observed either in vitro or ex vivo. The selectivity, reversibility, and competitive kinetics of the inhibition by BW 1370U87 may contribute to an improved safety profile with this novel MAO-A inhibitor.     

Substrate-selective activation of rat liver mitochondrial mono amine oxidase by oxygen

The activation by oxygen of the activity of monoamine oxidase (MAO) in preparations of rat liver mitochondrial membrane vesicles has been studied. The increase of activity with the substrates 5-hydroxytryptamine, tyramine, β-phenethylamine and benzylamine were in all cases uncompetitive. However, the degree of activation depended upon the particular substrate used. An attempt has been made to explain these results by use of an assumption that MAO activity can be divided into two forms unrelated to MAO-A and MAO-B. These two forms, tentatively called MAO-1 and MAO-2, differ in their Michaelis constants for oxygen.     

Possible heterogeneity of type B monoamine oxidase in pig heart mitochondria

The metabolism in vitro of 5-hydroxytryptamine (5-HT), tyramine and benzylamine by pig heart mitochondrial monoamine oxidase (MAO) has been studied. Linear Lineweaver-Burk plots yielded estimated K_m values (at pH 7.8) of 475 μM (5-HT) and 292 μM (tyramine). In contrast, linear regions of a downward-curving reciprocal plot revealed the presence of a high- and low-affinity metabolizing site (estimated K_m of 39 and 853 μm respectively) for benzylamine. Studies with the irreversible MAO inhibitor clorgyline indicated that metabolism of the three substrates in this tissue was brought about by type B MAO alone. However, the apparent sensitivity toward clorgyline of each substrate-metabolizing activity was not identical. This was due to different degrees of rapid or possibly instantaneous inhibition of enzyme activity toward each substrate. This rapid inhibition appeared to be both partially reversible and irreversible to a relative degree depending upon the substrate-metabolizing activity studied; additional time-dependent inhibition developing with prolonged preincubation was a first-order process, with a similar half-life, whichever substrate was used to assay MAO activity. Ackermann-Potter and Lineweaver-Burk plots also demonstrated differences in the inhibitory effects of clorgyline upon metabolism of each substrate. The ability of 5-HT, tyramine and benzylamine to inhibit each other's deamination in vitro was also investigated. Enzyme activity was measured by radiochemical assay with each labeled substrate in the presence and absence of the other non-labeled amines. Lineweaver-Burk analysis revealed a competitive interaction between tyramine and benzylamine, whereas mixed-type inhibition patterns were obtained for mixtures containing 5-HT/tyramine or 5-HT/benzylamine. In this latter case, the present inhibition data could only be assessed accurately with the low-affinity catalytic site for benzylamine. The kinetics of heat denaturation indicated both a thermolabile and thermostable component of each substrate-metabolizing activity. Some substrate-dependent differences in the relative proportions of these components were found. These experiments are discussed in relation to similar studies by other workers and suggest that pig heart MAO may, in fact, be heterogeneous.     

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.     

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.     

Time-dependent inhibition of monoamine oxidase by β-phenethylamine

Several reports have suggested that monoamine oxidase activity towards β-phenethylamine is inhibited by high concentrations of that substrate. This inhibition is not found if initial velocities are measured, but there is a slower time-dependent inhibition at higher β-phenethylamine concentrations. Such time-dependent inhibition is not found with tyramine as substrate or upon incubation of the enzyme with the reversible inhibitor amphetamine. The inhibition is not due to the accumulation of phenacetaldehyde, phenylethanol or phenacetic acid, or to a reaction of any of these three products either with each other or with β-phenethylamine. Although the inhibition is time-dependent, the inactivated enzyme slowly regains activity upon removal of the β-phenethylamine. A model is proposed to explain the observed inhibition.     

The effect of tris buffers on rat liver mitochondrial monoamine oxidase

The effect of tris buffers upon the monoamine oxidase (MAO) activity in rat liver mitochondria has been investigated. Tris buffer was shown to inhibit MAO in a non-competitive manner with a Ki of 15–25 mm. Tyramine, 5-hydroxytryptamine and β-phenethylamine but not benzylamine oxidations were all inhibited by tris buffer. All inhibitions, except that of 5-HT, were completely reversible. It is suggested that these effects are produced by conformational changes in the structure of the MAO. The significance of these results is discussed with respect to the use of tris buffers in the extraction and estimation of the activity of MAO.     

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 comparative influence of pyrazidol, inkazan and other antidepressant monoamine oxidase inhibitors on the pressor effect of tyramine

In experiments on conscious normotensive male Wistar rats the new antidepressants, reversible MAO-A inhibitors, pyrazidole and incazane, as well as moclobemid increased the pressor effect of orally administered tyramine. The drugs potentiated also the pressor effect of intravenous tyramine. More prolonged potentiation of tyramine action was produced by moclobemid, less prolonged by incazane. The potentiation by the studied MAO-A inhibitors of the pressor effect of tyramine reflects the inhibition of the activity of MAO-A and the first-pass metabolism of tyramine in the gut and liver, as well as the inhibition of intraneuronal MAO activity in noradrenergic nerve endings and the potentiation of sympathetic 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.     

Amethocaine-induced inhibition of mitochondrial monoamine oxidase activity

Amethocaine (tetracaine) (1-10 microM) produces a concentration-dependent in-vitro inhibition of mitochondrial membrane-bound MAO activity towards tyramine (18-84% in brain and 19-84% in liver) and 5-hydroxytryptamine (5-HT) (23-94% in brain and 20-100% in liver). At relatively higher concentrations (25-300 microM) of amethocaine, benzylamine oxidation is inhibited in brain (24-91%) and liver (29-100%). The extent of MAO inhibition is appreciably reduced when preincubation time of the enzyme with a low concentration (7.5 microM) of amethocaine is increased from zero to 45 min. This inhibition is reversible. The Km of MAO for tyramine is increased in brain (106-473%) and liver (121-352%) in the presence of amethocaine (2-7.5 microM) accompanied by a decrease in Vmax (21-51% in brain and 18-57% in liver). Similarly the Km of MAO for 5-HT is increased to the extent of 79-336% in brain and 51-225% in liver and the corresponding Vmax is decreased by 35-55% and 39-74%, respectively, in the presence of 2-5 microM amethocaine. At relatively higher concentrations (25-100 microM), amethocaine increases the Km of MAO for benzylamine in brain (25-101%) and liver (26-85%) and decreases the Vmax by 28-64% and 32-63% in the respective tissues. Thus these results suggest that amethocaine preferentially inhibits MAO-A and the nature of inhibition is reversible and of mixed type.     

Tyramine infusions and selective monoamine oxidase inhibitor treatment

The relationship between changes in IV tyramine pressor sensitivity accompanying selective monoamine oxidase (MAO) inhibitor treatment and estimates of MAO-A and MAO-B inhibition in vivo were studied. Reductions in platelet MAO activity provided an index of MAO-B inhibition, while changes in plasma 3-methoxy-4-hydroxyphenethylene glycol (MHPG) were used as an hypothesized reflection of MAO-A inhibition. Chronic treatment with the MAO-A inhibitor clorgyline and the MAO-B inhibitor pargyline showed significant inhibition of the alternate MAO enzyme as well, although this crossover effect was greater for pargyline than clorgyline. The MAO-B inhibitor deprenyl appeared to maintain the greatest degree of MAO inhibition selectivity in vivo. Tyramine pressor sensitivity changes accompanying administration of the MAO inhibitors were highly correlated with decreases in plasma MHPG (r=0.92), supporting our previous data indicating the rank order of clorgyline > pargyline > deprenyl for enhancement of tyramine pressor sensitivity and, thus, suggesting that tyramin potentiation is primarily a function of MAO-A rather than MAO-B inhibition. Changes in plasma MHPG are suggested to provide a potentially useful clinical index of in vivo MAO-A inhibition.Presently with the Biological Psychiatry Branch, NIMH     

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.     

次黄嘌呤对单胺氧化酶的抑制作用

实验证明给小鼠po次黄嘌呤25～500 mg/kg时,对肝和脑中单胺氧化酶B(MAOB)活性的抑制作用与剂量成明显的量—效关系,对MAO-A活性的抑制较弱,且无明显的量—效关系。给小鼠一次po次黄嘌呤500 mg/kg,于给药后16h,对MAO抑制作用最明显。sc时,对肝中MAO活性抑制也以给药后16 h最明显,但对脑中MAO活性抑制不明显。离体实验证明,次黄嘌呤对MAO-B的抑制为竞争性,对MAO-A则为混合型抑制。