NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO.4 231 MDMA (Ecstasy) Inhibition of MAO Type A and Type B: Comparisons with Fenfluramine and Fluoxetine (Prozac) Efthimia T. Kokotos Leonardi, M.S. and Efrain C. Azmitia, Ph.D. 3,4-Methylenedioxymethamphetamine (MDMA), a fenfiuramine (FEN) and fiuoxetine (FLUOX) were serotonin (5-HT) neurotoxin, has been shown to promote compared to those of MDMA. The rank order potency of the release of serotonin (5-HT) and block its reuptake. these drugs for MAO-A inhibition was The increased buildup of extracellular 5-HT should MDMA>FLUOX>FEN, whereas for MAO-B inhibition, normally be degraded by monoamine oxidase (MAO). The FLUOX>MDMA>FEN. A combination of FLUOX and effects of both enantiomers of MDMA were examined on MDMA at their respective lC50 did not inhibit MAO MAO-A and monoamine oxidase-B (MAO-B) activity in activity more than either drug alone at equivalent rat brain homogenates. Both enantiomers competitively concentrations. These results indicate that the actions of inhibited 5-HT catabolism by rat brain MAO-A. The Ki FEN do not appear to involve MAO inhibition. MDMA of MDMA for MAO-A was 22 J.lmollL. A mixed type of (ecstasy) produced a preferential inhibition of MAO-A inhibition by MDMA was observed for phenethylamine (1C50 = 44 J.lmoIlL), which should increase extracellular catabolism by MAO-B for both optical antipodes. 5-HT. This may explain its high toxicity potential. Logistical analysis of concentration response curves for Finally, FLUOX (Prozac) showed an inhibition of MDMA inhibition of MAO-A and MAO-B show an lC50 MAO-B (1C50 = 80 J.lmoIlL, which may increase the of 44 J.lmollL for inhibition of MAO-A by MDMA. The intracellular content of 5-HT. This may contribute to its lC50 value of MDMA inhibition of MAO-B was 370 therapeutic potential. In contrast, FEN appears to be a J.lmollL, showing a selective potency for MAO-A poor inhibitor of both MAO-A and MAO-B. inhibition. The MAO inhibitory properties of [Neuropsychopharmacology 10:231-238, 1994] KEY WORDS: Serotonin; Phenethylamine; Enantiomer for serotonin is 1170 IlmollL (Fowler and Tipton 1982; Garrick and Murphy 1982). Monoamine oxidase A is Monoamine oxidase (MAO) is an enzyme with two sub­ inhibited by nanomolar concentrations of clorgyline types (E. C.1.4.3.4) characterized by their differentialre­ (Johnston 1968). Monoamine oxidase B (MAO-B) has sponses to the irreversible inhibitors clorgyline and a higher affinityfor phenethylamine than MAO-A and depreny1. Monoamine oxidase A (MAO-A) has a higher is inhibited by nanomolar concentrations of deprenyl affinity for serotonin (5-HT) than MAO-B; the KM of (Garrick and Murphy 1982; Yang and Neff1974). Dopa­ MAO-A for serotonin is 99 IlmollL, the KM of MAO-B mine (DA) is metabolized with equal affinity by both subtypes (Yang and Neff 1974). Further evidence for two molecular forms of MAO has been provided by the From the Department of Biology, New York University, New York, New York. cloning of two distinct MAO genes (Bach et a1. 1988) Address correspondence to Dr. Efrain C. Azmitia, Department of and their subsequent functional expression inCOS cells Biology, New York University, 100 Washington Square East, New (Lan et a1. 1989). York, NY 10003. Received November 7, 1993; revised February 16, 1994; accepted Both neurons and glia contain monoamine oxidases February 17, 1994. that catabolize the classical monoamine neurotransmit- © 1994 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0893-133X/94/$7.00 232 E.T. Kokotos Leonardi and E.C. Azmitia NEUROPSYCHOPHARMACOLOGY 1994-VOL.10, NO. 4 ters. Monoamine oxidase A is the predominant form to the transporter for these agents is FLUOX>FEN = in catecholaminergic neurons, whereas MAO-B is the MDMA (Poblete et al. 1989). prevalent form in glia (Levitt et al. 1982; Westlund et The similar actions of FEN, FLUOX and MDMA on al. 1985). Although MAO-B has a higher affinity than serotonin transporter binding suggest they may pos­ MAO-B for serotonin, MAO-B is the major molecular sess common effects upon other serotonergic param­ form found within serotonergic neurons (Westlund et eters, such as monoamine oxidase activity, in vitro. The al. 1985). present report examines the effectsof both enantiomers 3,4-Methylenedioxymethamphetamine (MDMA) of MDMA on catabolism of [3H]-serotonin and p4C]­ binds with high affInity to the 5-HT transporter pro­ phenethylamine by rat brain monoamine oxidase in tein (Poblete et al. 1989) and has been shown to be a vitro. In addition, we compared the effects of fluoxe­ potent releaser of serotonin by a Ca2+-independent tine (FLUOX) and fenfluramine (FEN) to MDMA on rat mechanism (Berger et al. 1992; Gu and Azmitia 1989; brain MAO activity. Our results suggest that inhibition Johnson et al. 1986; Schmidt 1987; Schmidt et al. 1987). of MAO-A by MDMA may contribute to an accumula­ MDMA has been demonstrated to produce a depletion tion of extracellular 5-HT. The inhibition of MAO-B ac­ of serotonin that may be reversed in acute stages by tivity by FLUOX may increase intracellular 5-HT, agents that bind to the serotonin transporter and block whereas the actions of FEN do not appear to involve serotonin reuptake into presynaptic terminals (Azmitia MAO inhibition. et al. 1990; Schmidt 1987). It also inhibits the 5-HT re­ uptake system (Steele et al. 1987), resulting in an in­ creased amount of extracellular 5-HT. However, little MATERIALS AND METHODS attention has been paid to the fate of extracellular 5-HT. Parachloroamphetamine (PCA) is another sub­ Male Sprague-Dawley rats weighing 200 to 250 g (Ta­ stituted amphetamine that is a potent releaser of sero­ conic Farms, Germantown, NY) were housed two per tonin and has a biphasic depletion of serotonin similar cage and given free access to food and water. The to that observed with MDMA (Ask and Ross 1987; animals were maintained on a 12-hour light/dark cy­ Berger et al. 1992; Fuller et al. 1975; Gu and Azmitia cle. Animals were euthanized with C02 and decapi­ 1989; Gu 1993; Hwang and van Woert 1980; Mamounas tated according to a protocol approved by the NYU An­ and Molliver 1988; Poblete et al. 1989; Ross and Froden imal Welfare Committee. Brains were rapidly removed 1977). As with MDMA, the depletion of serotonin resul­ and placed on ice in 0.32 mollL sucrose. After removal tant from PCA may be reversed in its acute phase by of the cerebellum, brains were homogenized in 10-fold serotonin uptake blockers (Fuller et al. 1975; Ross et al. volume/weight in 0.32 mollL sucrose using 10 strokes 1977). Parachloroamphetamine has been shown to in­ with a Teflon/glass homogenizer. P1 pellets were pre­ hibit MAO-A activity in rat brain homogenates with a pared by sedimentation of homogenates at 800 x g for Ki value of 1.31 IlmollL (Fuller 1966). The toxicity of 10 minutes at 2°C in a Sorvall RC5C centrifuge (Sorvall PCA is also affected by the amount of releasable sero­ Instruments, DuPont, Chadds Ford, PA). Supernatants tonin into the extracellular space. For instance, ifsero­ were resedimented at 14,000 xg at 2°C for 15 minutes tonin release is decreased by parachlorophenylalanine to obtain a crude mitochondrial P2 pellet. Resultant (pCP A) and reserpine, the level of toxicity is reduced pellets were resuspended in 500 ilL 0.32 mollL sucrose (Berger et al. 1989). and stored at -70°C until use. The anorectic compound, fenfluramine (FEN), is Prior to the MAO assay, homogenates were thawed a halogenated amphetamine that has actions in and brought up to lOx volume/original weight 0.01 serotonergic axon terminals similar to those of MDMA mollL sodium phosphate buffer (PB), pH 7.4 and and PCA (Mamounas and Molliver 1988; Molliver and dialyzed to remove endogenous monoamines by a Molliver 1990; O'Hearn et al. 1988). Like MDMA and modiflcationof the method described by Patterson, et PCA, FEN causes the release of 5-HT from presynaptic al. (1973). Briefly, homogenates were dialyzed in 2 mL terminals (Borroni et al. 1983) and inhibits the reuptake aliquots using 3500 mw cutoff dialysis tubing (Spec­ of serotonin into its terminals (Belin et al. 1976; Kan­ trap or) against 0.01 mollL PB, pH 7.4 at 4°C for 2 hours nengiesser et al. 1976). The effects of FEN are blocked with three successive changes of 1L buffer. MAO as­ by 5-HT uptake inhibitors, as in MDMA and PCA (Hek­ says were performed immediately following this di­ matpanah and Peroutka 1990). These observations sug­ alysis. gest that the carrier-mediated release of serotonin and Monamine oxidase activity was assayed using the inhibition of its reuptake are critical components [3H]-5-HT (25Ci/mmol, 1IlCi/mL, New England Nu­ in the mechanism of these drugs. FEN, FLUOX, and clear, Boston, MA) as a substrate for MAO-A at fmal MDMA bind to the serotonin transporter protein with concentrations ranging from 12.5IlmollL to 400IlmollL. high affinity; the rank order potencies for binding p4C]-phenethylamine at a fmal concentration of 5 NEUROPSYCHOPHARMACOLOGY 1994-VOL. 10, NO. 4 MDMA Inhibition of MAO-A 233 15000 tillation counting in Beckman lS 1801 scintillation 14000 ... counter (Fullerton, CA) with a counting efficiency 13000 • � 0 uM !.IOMA � o '" 5 uU UOUA " 12000 " = 10 uU UOMA of 40%. c " - 50 uU I.IDUA 11000 E • = 100 uU UO!.l I 10000 � D = 500 u!.l UD e Comparison of FEN, FLUOX, and MDMA � ... 9000 A = 1 m!.l !.IDMA � '" 8000 < E Fenfluramine, FLUOX, and each enantiomer of MDMA I .....