Studies of monoamine oxidases: Inhibition of bovine brain MAO in intact mitochondria by selective inhibitors

The inhibition of mitochondrial monoamine oxidase (MAO) from beef brain cortex by the selective inhibitors, clorgyline, harmaline, Deprenyl and pargyline, was compared using five substrates: serotonin (5-HT), β-phenylethylamine (PEA), tyramine, tryptamine and dopamine. Dose-response studies, consistent with the classification of MAO, types A and B, indicated that serotonin deamination was more sensitive to clorgyline and harmaline inhibition than was phenylethylamine. However, the curves for all substrates were double-sigmoidal, rather than being a single sigmoid curve for 5-HT and PEA. Deprenyl and pargyline did not exhibit any marked selectivity for inhibiting PEA deamination without prior preincubation of enzyme and inhibitor. The rate of inhibition was variable and was dependent upon the substrate, the nature of the inhibitor and the inhibitor concentration. Dual inhibitor studies, using the “type A” inhibitor, clorgyline, and the “type B” inhibitor, Deprenyl, together, resulted in almost complete MAO inhibition, regardless of substrate. Combining the two type A inhibitors, clorgyline and harmaline, or the two type B inhibitors, deprenyl and pargyline, resulted in inhibitions that were equal to or only slightly greater than the inhibition produced by a single inhibitor. These results suggested that there are at least two distinct sites in beef brain MAO from cortical mitochondria which may be interacting. The deamination of all substrates occurs at both sites.     

Studies on monoamine oxidase. XVIII. Enzymic properties of placental monoamine oxidase.

Enzymic properties of partially purified monoamine oxidase (MAO) from human placenta were studied with tyramine, serotonin and benzylamine as substrates. The highest activity was obtained with serotonin and almost no activity was observed with benzylamine. These results are similar to those obtained with rat placental MAO, but different from those with rabbit placental MAO. The Km values for serotonin and tyramine were found to be 0.21 mM and 0.23 mM, respectively and the pH optimum was 8.1 with either substrate. The thermal inactivation curves of this enzyme with the two substrates were identical. The pI curves for inhibition of MAO activity by harmine, pargyline and iproniazid were similar and almost the same pI 50 values for the respective inhibitors were obtained with the two substrates. MAO in human placenta differs from that in other organs, such as liver, brain and plasma from the standpoint of the substrate specificity and the inhibitor sensitivity. The possibility that human placenta contains a single form of MAO is discussed on the basis of the present results.     

Effect of lignocaine on monoamine oxidase activity of brain and liver

In vivo administration of a single dose (100-150 mg/kg, i.p.) of lignocaine produces no change in MAO activity, while long-term treatment (50 mg/kg/day for 15 and 30 consecutive days, i.p.) produces a slight but appreciable inhibition of MAO activity with tyramine or serotonin but not with benzylamine as substrate in both rat brain and liver mitochondria. Lignocaine (2-20 mM) inhibits (in vitro) both brain and liver mitochondrial MAO activity, using tyramine, serotonin and benzylamine as substrates, in a concentration-dependent manner. Furthermore, lignocaine produces a marked in vitro inhibition of serotonin and tyramine oxidation in MAO-A and not in MAO-B preparation of rat brain. Ackermann-Potter plots of MAO indicate that lignocaine-induced inhibition of MAO activity is reversible in nature. Lineweaver-Burk plots show that lignocaine (2-10 mM) produces a significant increase in Km and decrease in Vmax of MAO for tyramine and serotonin in both brain and liver. Similarly Km and Vmax values are changed using benzylamine as substrate in the presence of relatively higher concentrations of lignocaine (5-20 mM). These results suggest that lignocaine-induced inhibition of mitochondrial membrane-bound MAO activity of both neuronal and non-neuronal tissues is associated with its conformational change.     

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.     

Characterization of rat skeletal muscle monoamine oxidase.

Optimal conditions for deamination of 5-hydroxytryptamine in rat skeletal muscle were determined. The presence of monoamine oxidase (MAO) A and MAO B isozymes was demonstrated by the use of tyramine (a substrate of both forms), specific substrates (serotonin and benzylamine), and specific inhibitors (clorgyline and deprenyl) of MAO A and B respectively. A 6.5:3.5 ratio of MAO A to B was found using a whole muscle homogenate, while a 7.5:2.5 ratio was found with isolated mitochondria. Thermal inactivation studies demonstrated that skeletal muscle MAO A is more susceptible to heat inactivation than MAO B. The approximate proportion of muscle homogenate MAO which is present in sympathetic nerves was found to be 18 per cent, as determined by treating rats with 6-hydroxydopamine and quantifying the decrease in activity. Significant inhibition of MAO activity was observed after administration in vivo of the MAO inhibitors pargyline, tranylcypromine and harmaline.     

Characteristics of mitochondrial and synaptosomal monoamine oxidase in monkey brain

Enzymic properties of monoamine oxidase (MAO) from monkey brain were studied. High MAO activity was observed in the mesencephalon and dienecephalon of the brain. Highest activity in every region of the brain was found with tyramine as a substrate. Monkey brain mitochondrial MAO showed a different substrate specificity and different Km and Vmax values than the enzyme from mice, rats, guinea pigs and rabbits. The pH activity curves were all bell-shaped, but the pH optima were remarkably different with the various substrates used. The activities of various substrates at pH 7.2 were compared with those at the pH optimum. At the pH optima, the activity was about 1.2-fold higher with tyramine and dopamine, 2-fold higher with beta-phenylethylamine (beta-PEA) and 3-fold higher with serotonin (5-HT) and benzylamine. These results were almost similar when synaptosomes from monkey brain were used. MAO activities with 5-HT and beta-PEA were strongly inhibited by much lower concentrations of clorgyline and deprenyl, respectively. Plateau-shaped inhibition curves by these inhibitors were obtained with tyramine as the substrate. These results indicate that both the A- and B-form of MAO appear to be uniformly distributed in monkey brain, and the A-form of MAO represents approximately 35% and 50% of the total MAO activity in mitochondria and synaptosomes, respectively.     

MAO types in guinea pig liver mitochondria

MAO of guinea pig liver mitochondria actively deaminated dopamine, tyramine, serotonin and 5-methoxy-tryptamine, while tryptamine, 5-methyl-tryptamine and 7-methyl-tryptamine were moderately deaminated. Very little deamination occurred when benzylamine. noradrenaline and β-phenylethylamine were used as substrates. The in vitro inhibition patterns of MAO of guinea pig liver mitochondria by some selective inhibitors were investigated in the presence of tyramine, tryptamine and serotonin. Tryptamine oxidation showed biphasic inhibition pattern with harmaline, clorgyline and Lilly 51641, while the inhibition curves in the presence of pargyline and deprenyl were sigmoidal. The inhibition curves for tyramine oxidation were biphasic with all the inhibitors except pargyline. Serotonin-MAO inhibition curves, on the other hand, were sigmoidal with all the inhibitors except Lilly 51641. Thermal treatment of guinea pig liver mitochondria produced rapid inactivation of serotonin and tryptamine oxidizing activity, while benzylamine oxidizing activity was found to be most thermostable.     

Monoamine oxidase (XXXVI). Characteristics of benzylamine oxidase in the dog serum]

Enzymic properties of monoamine oxidase (MAO) in dog serum were studied and the following results were obtained. Some of enzymic properties of MAO in dog serum differed from that of mitochondrial MAO. When dog serum was fractionated by ammonium sulfate, proteins were concentrated in two fractions, such as 25 approximately 33% and 67 approximately 80% of saturated ammonium sulfate fraction, while MAO activity was concentrated in 40 approximately 50% of saturated ammonium sulfate fraction. The reaction rate of MAO in dog serum was found to be proportional to enzyme concentration. The optimum pH of MAO in dog serum was 7.0 which differed from that of MAO in rabbit serum (pH 8.0). Tris-HCl buffer strongly inhibited MAO activity in dog serum. When benzylamine was used as substrate, the highest activity was obtained compared with the other substrate used. The activities with butylamine, amylamine, beta-phenylethylamine and tyramine showed about 30% while tryptamine and serotonin showed 3 approximately 10% compared to that with benzymlamine as substrate. The value of pI50 of catron was about 3 X 10(-6) M and that of harmaline was about 3 X 10(-5) M, but pargyline did not inhibit MAO activity in dog serum at the concentration of 1 X 10(-4) M.     

Inhibition patterns of monoamine oxidase in sub-fractions of rat brain mitochondria in presence of some selective inhibitors.

In vitro inhibition patterns of MAO activity in different mitochondrial sub-fractions of rat brain in presence of harmine, harmaline and deprenyl with tyramine and serotonin as substrates were investigated. The results indicate that type A and type B MAO are distributed in different ratios among the mitochondrial sub-fractions.     

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.     

Effect of ether,chloroform and carbon dioxide on monoamine inactivation

To determine if anaesthetic agents alter monoamine inactivation, we exposed tissue homogenates (liver, kidney and brain) from mice and rabbits to ether and chloroform vapors and carbon dioxide gas. These anaesthetic agents inhibited monoamine oxidase (MAO) activity against tryptamine and serotonin. Concentrations of anaesthetic agents that are achieved in the plasma of man during general anaesthesia caused a 27% (ether) and 49% (chloroform) reduction in mouse liver MAO; higher concentrations caused a 95% inhibition mouse or rabbit liver MAO. Kinetic analysis with tryptamine as substrate indicate that ether and chloroform are noncompetitive, reversible MAO inhibitors that preferentially inhibit Type B MAO. Ether and chloroform cause noncompetitive inhibition of serotonin oxidation by mouse liver MAO and competitive inhibition of serotonin oxidation by mouse brain and kidney MAO. Ether or chloroform did not alter catechol-O-methyltransferase activity from tissues of mice. Isolated blood platelets (rabbit and human) were used as a model system for neuronal uptake. Ether caused an irreversible inhibition of serotonin uptake by platelets.     

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.     

The effects of thyroid hormones on monoamine oxidase in the rat heart

The administration of thyroxine to young male rats produced an increase in the specific activity of their cardiac monoamine oxidase (MAO). A reduction in the circulating concentrations of thyroid hormones, brought about by 2-thiouracil, led to a decrease. The relative change in activity produced was greater with tyramine than with benzylamine as substrate. By following the time-course of the return of enzyme activity, with tyramine as substrate, after a single injection of pargyline in vivo, it was concluded that both excess and lack of thyroid hormones cause their effects on MAO activity by changing the rate of synthesis of the enzyme and not its degradation rate constant. The degradation rate constant did change with the age of the animal. The MAO activity, which increased towards tyramine as substrate in hyperthyroid rat hearts, behaved in the same way as that of controls to heat treatment, irreversible inhibition by pargyline or by clorgyline and also in K_m determinations. The pattern for benzylamine oxidation was similar, except for the effect of the inhibitor clorgyline which shifted the plateau region of the double sigmoid inhibition curve significantly using enzyme from hyperthyroid rat hearts. The plateau region was also shown to be affected by the age of the animal. The possibility is discussed that the increased cardiac MAO activity produced by thyroid hormones and by the growth of the animal is mediated by that form of the enzyme primarily responsible for the oxidation of tyramine. Mixed substrate experiments suggested that tyramine oxidation could be inhibited competitively by benzylamine.     

Studies on the nature of the increased monoamine oxidase activity in the rat heart after adrenalectomy

The increased activity, induced by adrenalectomy, of the enzyme monoamine oxidase (MAO) in the rat heart was found to resemble closely that present in the hearts of control animals. No significant differences were observed in the nature of the response to heat denaturation, changes in pH or to inhibition by pargyline or clorgyline. The relative activities using the substrates tyramine, 5-hydroxytryptamine, dopamine or benzylamine were the same. No evidence was found to suggest the presence of a heat-stable or dialysable inhibitor of enzyme activity. In young rats there was an increase in the relative enzyme activity using benzylamine as substrate, compared with the activity using tyramine, in the first few days after adrenalectomy. No effect of adrenalectomy could be detected upon the MAO activity in the rat brain or liver. It is concluded that the increase in rat heart MAO following adrenalectomy cannot be due to the synthesis of an enzyme with different catalytic properties, nor to the transformation of the existing enzyme into one of increased catalytic ability but with different properties.     

The turnover of the A- AND B-forms of monoamine oxidase in rat liver

The inhibition of rat liver monoamine oxidase was determined following the intraperitoneal injection of the inhibitors clorgyline and pargyline in order to establish the concentration ranges in which substrate-selective inhibition occurred. The rate of recovery of the activity of the A-form of the enzyme after inhibition by clorgyline was determined using tyramine and serotonin as substrates, and the rate of recovery of the activity of the B-form after inhibition by pargyline was determined using tyramine and 2-phenylethylamine as substrates. No significant differences could be detected between the rates of recoveries of the two forms which corresponded to a rate constant for degradation of the enzyme of about 0.27 day ^-1.     

Selective inhibition by amiflamine of monoamine oxidase type A in rat brain,liver and duodenum

Summary Amiflamine (FLA 336(+)), N-desmethylamiflamine (FLA 788(+)) and N,N-didesmethylamiflamine (FLA 668(+)) were examined for their monoamine oxidase (MAO) inhibitory effects in rat brain, liver and duodenum and were compared with the irreversible inhibitors clorgyline and (-)-deprenyl. The potency of each FLA compound was the same in each tissue both in vitro and after oral administration with either serotonin or tyramine as substrate. The in vitro effect of FLA 788(+) was 2–6 times stronger than that of amiflamine although the compounds were equipotent after oral administration. FLA 668(+) was 2–3 times less potent than amiflamine in vitro and had very poor activity after oral administration. The deamination of phenethylamine was weakly afected by the three FLA compounds. Clorgyline inhibited strongly the deamination of serotonin and tyramine in the duodenum after oral administration, being 1,000 times more potent than in the brain and the liver. Similar results were obtained for (-)-deprenyl which, however, was more potent in inhibiting the deamination of phenethylamine than that of serotonin and tyramine. Amiflamine was a reversible MAO inhibitor with no MAO inhibitory capacity 24 h after a single oral dose. On the other hand the irreversible inhibitor clorgyline had a maximal effect on brain MAO 48 h after a single dose while the inhibitory effect in the duodenum had almost disappeared. The influence of amiflamine on the excretion of acid and basic metabolites of orally administered ¹⁴C-tyramine (58 mol/kg) in rat was examined. Amiflamine, at doses that strongly inhibited MAO-A in rat brain, only slightly affected the excretion of ¹⁴C-labelled acid in urine during 6 and 24 h after the tyramine administration. The results in this study suggest that other factors than a low interaction with intestinal MAO may be of importance for the low tyramine potentiating effect obtained after oral administration of amiflamine.     

Substrate- and inhibitor-related characteristics of human platelet monoamine oxidase

Human platelet monoamine oxidase (MAO) preferentially deaminated benzylamine and phenylethylamine, two substrates relatively specific for type B MAO, in comparison to 5-hydroxytryptamine, a substrate specific for type A MAO. In studies comparing human platelet and rat brain MAO specific activities, benzylamine and 5-hydroxytryptamine deamination by platelets was approximately 90 and 2 per cent, respectively, that of brain, while platelet deamination of dopamine, tryptamine and tyramine was 20 per cent or less than that of brain. Among sixteen drugs studied, platelet MAO activity was selectively inhibited by low concentrations of the MAO-B inhibitors, deprenyl and pargyline, and was relatively insensitive to the MAO-A inhibitors, clorgyline and Lilly 51641. These observations, in addition to the simple sigmoid inhibition curves obtained with increasing concentrations of either clorgyline or deprenyl, suggest that platelet MAO consists of essentially one distinguishable form of MAO which most closely resembles the MAO type B found in other tissues.     

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     

Effects of zinc ion on type A monoamine oxidase in monkey brain mitochondria

The effects of ZnSO(4) on types A and B monoamine oxidase (MAO) isozymes in monkey brain mitochondria were investigated, in vitro. Type A MAO activity in monkey brain decreased to about 50% with 1 microM ZnSO(4) using serotonin as a substrate, and this inhibition was proportional to the concentration of ZnSO(4). ZnSO(4) had no effect, however, on type B MAO activity in monkey brain using beta-phenylethylamine as a substrate. The inhibition by ZnSO(4) of type A MAO activity was competitive and reversible. ZnSO(4) did not inhibit either type A or type B MAO activity in rat brain mitochondria. Almost similar results were also obtained when ZnCl(2) was used, in vitro. These results indicate that the inhibiting action of zinc ion differs depending on animal species and organ. Type A MAO in monkey brain mitochondria was highly sensitive to zinc ion, while type B activity was less sensitive.     

Harmaline and its sulfur analogue: Increase of cerebellar cyclic GMP and inhibition of monoamine oxidase

Harmaline and its benzo[b]thiophene analogue $\text{(“S-}\text{harmaline}\text{”)}$ are shown to competitively inhibit monoamine oxidase (MAO). The inhibition was more marked with 5-hydroxytryptamine as a substrate than with tyramine or β-phenylethylamine. After i.p. administration, S-harmaline in contrast to harmaline inhibited the MAO in the brain more potently than in the liver. Harmaline produced a greater and longer lasting increase of cGMP in the cerebellum than S-harmaline. The S-harmaline was also less tremorogenic, thus indicating a connection between cGMP increase and tremor, but not between MAO inhibition and cGMP increase.