Blood-Brain Barrier Monoamine Oxidase: Enzyme Characterization in Cerebral Microvessels and Other Tissues from Six Mammalian Species, Including Human

We studied the enzyme monoamine oxidase (MAO) in isolated cerebral microvessels, and in mitochondria-enriched brain and liver preparations from six mammalian species, including human. We also studied MAO distribution in various tissues and in discrete brain regions of the rat. MAO was assessed by measuring the specific binding of [3H]pargyline, an irreversible MAO inhibitor, and the rates of oxidation of known MAO substrates: benzylamine, tyramine, tryptamine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Molecular forms of MAO were examined by using specific MAO inhibitors, and by polyacrylamide gel electrophoresis after [3H]pargyline binding. In general, the liver from all species had higher MAO levels than the brain, with minor variation among species in their brain and liver MAO content. However, there were remarkable species differences in brain microvessel MAO, with rat microvessels having one of the highest MAO activity among all tissues, whereas MAO activities in brain microvessels from humans, mice, and guinea pigs were very low. In most rat tissues, including the brain, there was a preponderance of MAO-B over MAO-A. The only exceptions were the heart and skeletal muscle. Estimates of MAO half-life in rat brain microvessels, rat brain, and rat liver indicated that microvessel MAO had a higher turnover rate. The reasons underlying the remarkable enrichment of rat cerebral microvessels with MAO-B are unknown, but it is evident that there are marked species differences in brain capillary endothelium MAO activity. The biological significance of these findings vis a vis the role of MAO as a "biochemical blood-brain barrier" that protects the brain from circulating neurotoxins and biogenic amines should be investigated.     

Differences in the Structure of A and B Forms of Human Monoamine Oxidase

Abstract: [³H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in human tissues were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [³H]Pargyline was bound to MAO A in a crude mitochondrial fraction from the placental trophoblast of a male newborn and to MAO B in blood platelets from the umbilical vein of the same newborn. [³H]Pargyline was also bound to MAO A and B in a crude mitochondrial fraction from cultured skin fibroblasts of a male adult and to MAO B in blood platelets from the same individual. Specific labeling of proteins associated with type A or type B activity in fibroblast cells was achieved by preincubation with selective B or A inhibitors, respectively. For all tissues, SDS-PAGE of [³H]pargyline-bound samples revealed a labeled protein band of apparent molecular weight 63,000 for MAO A and 60,000 for MAO B. When SDS-solubilized, [³H]pargyline-labeled MAO A and B proteins from the same male newborn were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [³H]pargyline-labeled peptides were obtained. These findings indicate that distinct enzyme molecules are associated with the A and B types of human MAO activity.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

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

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

MELATONIN: DEACETYLATION TO 5-METHOXYTRYPTAMINE BY LIVER BUT NOT BRAIN ARYL ACYLAMIDASE

Abstract— Rat liver and brain slices were incubated in vitro with [³H]melatonin. Liver slices synthesized small amounts of [³H]5-methoxyindoleacetic acid ([³H]5-MIAA) along with other melatonin metabolites including 6-hydroxymelatonin. Pretreatment of animals prior to killing with the irreversible monoamine oxidase inhibitor pargyline allowed [³H]5-methoxytryptamine ([³H]5-MT) to be recovered from the incubation. No [³H]5-MIAA or [³H]5-MT could be detected in incubations with hypothalamic slices or following intraventrieular injection of [³H]melatonin. The possibility that the deacetylase aryl acylamidase was in part responsible for the deacetylation occurring in liver slices was examined. Liver aryl acylamidase was able to utilize [³H]melatonin as substrate to produce [³H]5-MT. Furthermore, the liver enzyme was inhibited by melatonin ( K_i. 1 m m ) when tested with the alternate substrate o -nitroacetanalide. Brain aryl acylamidase did not generate any detectable [³H]5-MT nor was it inhibited by melatonin. These results suggest that 5-MT is not formed in brain from melatonin although trace amounts of 5-MT in the periphery could be derived from this precursor.     

MD 240928 and harmaline: opposite selectivity in antagonism of the inactivation of types A and B monoamine oxidase by pargyline in mice

In mice treated 24 hrs earlier with pargyline (20 mg/kg i.p), both type A and type B monoamine oxidase (MAO-A and MAO-B) were partially inactivated in brain, heart and liver. The abilities of two short-acting, reversible inhibitors of MAO to antagonize that inactivation were compared. Pretreatment with MD 240928 at doses of 10, 20 or 30 mg/kg i.p. antagonized the inactivation of type B MAO but did not alter the inactivation of type A MAO in all three tissues. In contrast, pretreatment with harmaline at a dose of 10 mg/kg i.p. antagonized the inactivation of type A MAO but did not alter the inactivation of type B MAO. Antagonism of the pargyline-induced inactivation is interpreted as being due to the transient selective inhibition of MAO-A by harmaline and of MAO-B by MD 240928, preventing the mechanism-based inactivation of those enzymes by pargyline. The selective protection by harmaline is in agreement with earlier results with that compound in rats; the selective protection by MD 240928 is the first report of selective protection against MAO-B inactivation.     

Deamination of 5-Hydroxytryptamine by Both Forms of Monoamine Oxidase in the Rat Brain

Abstract: K _m and V _max values of monoamine oxidase (MAO) A and B towards 5-hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l -deprenyl. K _m values of 178 and 1170μ m , and V _max values of 0.73 and 0.09 nmol·mg protein⁻¹·min⁻¹ towards 5-hydroxytryptamine were found for MAO-A and -B, respectively. The K ₁ for 5-hydroxytryptamine as a competitive inhibitor of β-phenethylamine oxidation by MAO-B was found to be 1400 μm. The significance of these findings is discussed.     

EVIDENCE FOR THE SYNTHESIS AND STORAGE OF 5-HYDROXYTRYPTAMINE IN TWO SEPARATE POOLS IN THE BRAIN

Abstract— Evidence is presented to support the hypothesis that 5-hydroxytryptamine (5-HT) in the rat brain is synthesized by two separate pathways and stored in two or more compartments. Lysergic acid diethylamide in doses down to 50 μg/kg was shown to reduce the formation of 5-[³H]HT from [³H]tryptophan in the presence of a monoamine oxidase inhibitor, although the total rate of accumulation of 5-HT was unchanged. Conversely, adrenalectomy was found to increase the total synthesis of 5-HT measured in the same way, although the amount of 5-[³H]HT formed suggested that there was no increase in the synthesis of the amine. In a third experiment it was found that electrical stimulation of 5-HT-containing nerves following labelling of 5-HT stores with [³H]tryptophan led to a biphasic disappearance of 5-[³H]HT. It is suggested that the method of measuring 5-HT synthesis by measuring 5-[³H]HT formed from[³H]tryptophan in the presence of a monoamine oxidase inhibitor may be a way of selectively measuring the turnover of the functional pool of 5-HT.     

Inhibition of rat brainstem monoamine oxidase activity by CGP 6085 A

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

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

Localization and Mechanism of Thymidine Transport in the Central Nervous System

Abstract: The localization and mechanism of thymidine and deoxyuridine transport in the central nervous system were studied in vivo and in vitro . Previous studies have shown that thymidine enters brain from blood in part via the CSF. In vitro , isolated adult bovine cerebral microvessels, which readily concentrated and phosphorylated deoxyglucose, were unable to concentrate thymidine and deoxyuridine. In vivo , [³H]thymidine (0.2 μ M ) and [³H]deoxyuridine(0.4 μ M ) were not extracted more readily than [¹⁴C]sucrose in a single pass through the cerebral circulation of rats. In vivo , [³H]thyrnidine retention in CSF and brain after entry from blood was increased when the efflux of [³H]thymidine from CSF and the phosphorylation of [³H]thymidine in brain were depressed by the intraventricular injection of unlabeled thymidine. These studies and previous work suggest that the transfer of thymidine (and deoxyuridine) through the blood-brain barrier in either direction must be extremely low. The present studies are consistent with the postulate that thymidine is transported by an active transport system in the choroid plexus that transfers thymidine from blood into the CSF; from the CSF, the thymidine enters brain cells and is phosphorylated.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Inhibition of brain monoamine oxidase activity by the generation of hydroxyl radicals: potential implications in relation to oxidative stress

Monoamine oxidase (MAO) is an enzyme involved in brain catabolism of monoamine neurotransmitters whose oxidative deamination results in the production of hydrogen peroxide. It has been documented that hydrogen peroxide derived from MAO activity represents a special source of oxidative stress in the brain. In this study we investigated the potential effects of the production of hydroxyl radicals (*OH) on MAO-A and MAO-B activities using mitochondrial preparations obtained from rat brain. Ascorbic acid (100 microM) and Fe2+ (0.2, 0.4, 0.8, and 1.6 microM) were used to induce the production of *OH. Results showed that the generation of *OH significantly reduced both MAO-A (85-53%) and MAO-B (77-39%) activities, exhibiting a linear correlation between both MAO-A and MAO-B activities and the amount of *OH produced. The reported inhibition was found to be irreversible for both MAO-A and MAO-B. Assuming the proven contribution of MAO activity to brain oxidative stress, this inhibition appears to reduce this contribution when an overproduction of *OH occurs.     

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

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

Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels

Abstract: The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [³H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [³H]diazepam binding in vitro (IC₅₀= 2.3 ± 0.6 nmol/liter). [³H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant ( K _D) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [³H]Ro 15-1788 from its binding site was of the same rank order as found previously in [³H]diazepam binding. Autoradiograms of [³H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [³H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Development and Localization of the Thymidine Phosphorylating Systems in Brain

Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[³H]thymidine at 37°C under 95% O₂-5% CO₂. When slices from all brain regions of 2-day-old rabbits were incubated in [³H]thymidine for 30 min, tissue-to-medium ratios of ³H were between 2 and 4 and declined with age, and the percentages of the total ³H in perchloric acid homogenates of brain slices as [³H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the ³H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [³H]thymidine for 30 min, the highest percentage of [³H]thymidine phosphates plus [³H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [³H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [³H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span.     

Brain-to-blood elimination of 24S-hydroxycholesterol from rat brain is mediated by organic anion transporting polypeptide 2 (oatp2) at the blood-brain barrier

24S-Hydroxycholesterol (24S-OH-chol), a major cerebral cholesterol metabolite, is an endogenous ligand for the liver X receptor and is a potential stimulant of cholesterol release from glial cells. The elimination mechanism of 24S-OH-chol from the brain is one of the key issues for understanding cerebral cholesterol homeostasis. The purpose of the present study was to clarify the molecular mechanism of the elimination process of 24S-OH-chol across the blood–brain barrier (BBB). After an intracerebral injection in rats, [³H]24S-OH-chol was eliminated from the brain and the radioactivity derived from [³H]24S-OH-chol was detected in the plasma, while [³H]cholesterol was not significantly eliminated from the brain. Co-administration of unlabeled 24S-OH-chol significantly inhibited the [³H]24S-OH-chol elimination, while no inhibitory effect was seen at the same concentration of cholesterol. The [³H]24S-OH-chol elimination was inhibited by co-administration of probenecid, but not by benzylpenicillin. Pre-administration of digoxin completely inhibited the elimination. Xenopus laevis oocytes expressing rat oatp2 exhibited significant transport of [³H]24S-OH-chol, and this was inhibited by unlabeled 24S-OH-chol and digoxin, indicating that rat oatp2 transports 24S-OH-chol. These results are the first direct demonstration that 24S-OH-chol undergoes elimination from the brain to blood across the BBB via a carrier-mediated process, which involves oatp2 expressed at the BBB in rats.     

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

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

Formation of the Neurotransmitter Glycine from the Anticonvulsant Milacemide Is Mediated by Brain Monoamine Oxidase B

Milacemide (2-n-pentylaminoacetamide) is a secondary monoamine that in the brain is converted to glycinamide and glycine. This oxidative reaction was suspected to involve the reaction of monoamine oxidase (MAO). Using mitochondrial preparations from tissues that contain MAO-A and -B (rat brain and liver), MAO-A (human placenta), and MAO-B (human platelet and bovine adrenal chromaffin cell), it has been established that mitochondria containing MAO-B rather than MAO-A oxidize (H2O2 production and glycinamide formation) milacemide. The apparent Km (30-90 microM) for milacemide oxidation by mitochondrial MAO-B preparations is significantly lower than that for milacemide oxidation by mitochondrial MAO-A (approximately 1,300 microM). In vitro MAO-B (l-deprenyl and AGN 1135) rather than MAO-A (clorgyline) selectively inhibited the oxidation of milacemide. These in vitro data are matched by ex vivo experiments where milacemide oxidation was compared to oxidation of serotonin (MAO-A) and beta-phenylethylamine (MAO-B) by brain mitochondria prepared from rats pretreated with clorgyline (0.5-10 mg/kg) and l-deprenyl (0.5-10 mg/kg). Furthermore, in vivo experiment demonstrated that l-deprenyl selectively increased the urinary excretion of [14C]milacemide and the total radioactivity with a concomitant decrease of [14C]glycinamide. Such changes were not observed after clorgyline treatment, but were evident only at doses beyond clorgyline selectivity. The present data therefore demonstrate that milacemide is a substrate for brain MAO-B, and its conversion to glycinamide, further transformed to the inhibitory neurotransmitter, glycine, mediated by this enzyme may contribute to its pharmacological activities.