Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of l -DOPA-treated monkeys

BRAIN RESEARCH Brain Research738(1996)53-59

Research report

oxidase-dependent metabolism of dopamine in the striatum and substantialnigra of L-DOPA-treated monkeys Donato A. Di Monte *, Louis E. DeLanney, Ian Irwin, Joyce E. Royland, Piu Chan, Michael W. Jakowec, J. William Langston The Parkinson’s Institute, 1170 Morse Avenue, Sunnyvale, CA 94089, USA

Accepted18June 1996

Abstract The effects of monoamine oxidase (MAO) inhibitors on the metabolism of dopamine synthesized from exogenous L-DOPA were investigated in the striatum and substantial nigra of squirrel monkeys. Administration of a single dose of L-DOPA (methyl ester, 40 mg/kg, i.p.) caused a significant increase in the levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)and in the DOPAC/dopamineratio in the putamen,caudateand substantialnigra. These changeswere more pronouncedin the substantialnigra than in the striatumand withinthe striatum of L-DOPA-treated monkeys, levels of doparnine and its metabolizes were higher in the putamen than in the caudate nucleus. When L-DOPA treatment was preceded by the injection of clorgyline or deprenyl at a concentration (1 mg\kg) which selectively inhibited MAO A or MAO B, respectively, striatrd dopamine was increased while the striatal DOPAC and HVA levels and DOPAC/dopamine ratio were significantly reduced as compared to the values obtained with L-DOPA alone. The two MAO inhibitors also counteracted the increase in the DOPAC and HVA levels and DOPAC/dopamine ratio induced by L-DOPA in the substantial nigra. Thus, both MAO A and MAO B contribute to the metabolism of dopamine when higher levels of this neurotransmitter are generated from L-DOPA in the squirrel monkey. The extent of reduction of dopamine catabolism (as assessed by the decrease in DOPAC and HVA levels) in the striatum and substantialnigra was similar with clorgyline and deprenyl even if the ratio MAO A/MAO B was approximately 1 to 10. This indicates that, though catalyzed by both MAO A and MAO B, dopamine delamination following treatment with L-DOPA preferentially involves MAO A. Keywords: Monoamineoxidase;Dopamine;L-DOPA;Substantialnigra; Striatum;Parkinsonism;Deprenyl;Monkey

1. Introduction The respective roles of monoamineoxidase(MAO)A and MAO B in striatal dopamine turnover have been extensively investigated in the rodent model, and experimental evidence suggests that, under normal conditions (i.e., intact tissue of untreated animals), MAO A is primarily responsible for doparnine dearnination. Studies in rats have revealed that striatal levels of 3,4-dihydroxyphenylacetic acid (DOPAC), the product of doparnine metabolism by MAO, are significantly reduced after administration of the selective MAO A inhibitor clorgyline while they are not affected by the MAO B inhibitor deprenyl at selective concentrations [8,18]. Similarly, the efflux of DOPAC

* Correspondingauthor.Fax: + 1 (40S)734-8522.

measured using microdialysis in the rat striatum is decreased by clorgyline, but remains unchanged after deprenyl administration [3,12]. Furthermore, dopamine delaminationis inhibited in striatal synaptosomes from clorgyline-treated, but not deprenyl-treated rats [3]. In this latter study, deprenyl became effective only at a concentration (10 mg/kg) which is likely to be non-selective and thus to inhibit MAO A as well as MAO B activity [18]. While MAO A activity appears to account for most, if not all, of the dopamine metabolism under normal conditions, it is possible that a contribution of MAO B may become evident during drug-induced increased doparnine turnover. This may occur as a consequence of the administration of the dopamine precursor L-DOPA. R has been shown that clorgyline significantly counteracts the increase in DOPAC level induced by L-DOPA in the rat striatum; in contrast, DOPAC concentration was not different in animals injected with both L-DOPA and deprenyl as com-

0006-8993/96/$15.00Copyright0 1996ElsevierScienceB.V. AUrightsreserved. PZZS0006-8993(96)00761

-5

54

D.A. Di Monte et aL/Brain Research 738 (1996) 53-59

pared to L-DOPA alone [4]. More recently, L-DOPA has been reported to increase markedly the extracellular level of DOPAC in dialysate fractions from rat striatum [24]. This increase was completely reversed by the combined administration of clorgyline and L-DOPA while neither deprenyl nor Ro 19-6327, another MAO B inhibitor, exhibited any effect. Taken together, these findings suggest that, in the rodent model, delamination by MAO A represents the primary mechanism for dopamine turnover not only under basal conditions but also after L-DOPA administration. Studies on the effects of L-DOPA on dopamine metabolism bear important therapeutic implications since (i) L-DOPA is the primary drug for the treatment of Parkinson’s disease, and (ii) it has been hypothesized that dopamine-induced oxidative stress may affect the function and survival of dopaminergic nigrostriatal neurons in patients undergoing chronic L-DOPA treatment [21]. It is noteworthy therefore that rats may not be the most reliable model for obtaining insight into the roles of MAO A and MAO B in dopamine catabolism after the administration of L-DOPA to humans. This is because brain levels of MAO A and MAO B are significantly different in humans as compared to rodents. In particular, the ratio MAO A/MAO B is much lower in the human brain than in the rat brain [7,26], thus raising the possibility that MAO B may contribute more significantly to dopamine deamination in the human nigrostriatal system. Previous studies have reported a low MAO A/MAO B ratio in the brain of other non-human primates [19,26], thus suggesting that monkeys are a more suitable model than rodents for evaluating the MAO-dependent metabolism of dopamine synthesized from L-DOPA in the human brain. In this study, dopamine metabolism was investigated in squirrel monkeys treated with L-DOPA alone or in combination with the MAO A inhibitor clergy line or the MAO B inhibitor deprenyl. The effects of these treatments were compared not only in the putamen and caudate nucleus which are sites of the dopaminergic terminals, but also in the substantial nigra where dopaminergic cell bodies are located. This is particularly important since the metabolism of dopamine synthesized from exogenous L-DOPA remains virtually unexplored in the substantial nigra.

2. Materials and methods 2.1. Animals and drug treatment All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Measurements of animal specimens reported in this study were conducted as part of a detailed investigation of the neurochemical, behavioral

and pathological effects of L-DOPA. Monkeys were housed individually in standard stainless steel primate cages (1 m3) with free access to water and with a daily diet of monkey chow and fresh fruit. A total of 12 squirrel monkeys (Saimiri boliviensis, > 10 years old) were used. Animals were divided into four treatment groups and each animal received a total of three injections. On day one, 3 monkeys (2 males and 1 female) were injected with saline, 3 animals were given the MAO A inhibitor clorgyline (1 mg/kg) and 3 were injected with the MAO B inhibitor deprenyl (1 mg/kg) via the intraperitoneal route. Deprenyl- and clorgyline-treated animals were all males. Twenty-four hours later, all 9 animals received a single subcutaneous injection of benserazide (12.5 mg/kg) followed 30 min later by an injection of L-DOPA (methyl ester, 40 mg/kg, i.p.). The fourth group of 2 males and 1 female served as control and was given an identical series of injections of saline. The mg/kg dose of L-DOPA chosen for this study is similar to that used by previous investigations in rodents (e.g., [24]). Three hours after the last injection, all animals were sacrificed by an overdose of sodium pentobarbital and their brains were rapidly removed and dissected on ice. Coronal slices (3 mm thick) were obtained using a brain dissection block. The left putamen and the left caudate nucleus were dissected from the slice at the level of the anterior commissure and immersed into 1 ml of ice-cold 5% (v/v) concentrated (60%) perchloric acid for assays of dopamine, DOPAC and homovanillic acid (HVA). Right putamen and right caudate obtained from the same slice were immersed in ice-cold 50 mM phosphate buffer (pH 7.4) and processed for MAO A and MAO B assays. The entire left substantialnigra (both pars compacta and reticulate) was also dissected and placed in perchloric acid for dopamine, DOPAC and HVA measurements. 2.2. Sample preparation and assays Acid-extracted samples were sonicated, then centrifuged (10 000 X g, 15 rein), and the supematants were used for determinations of dopamine, DOPAC and HVA by HPLC with electrochemical detection [13]. Samples were analyzed within one week of the preparation procedures. Preliminary work in our laboratories has established that levels of dopamine, DOPAC and HVA remain stable after acid extraction, sonication and immediate freeze of the samples at –70”C for at least one week. Values of dopamine and dopamine metabolizes were calculated on the basis of dopamine, DOPAC and HVA standards injected at different concentrations and at regular intervals between samples (i.e., every 10 samples). Samples in buffer were sonicated and frozen at –70”C overnight. MAO A and MAO B activities were measured as previously described [27] using [14C]5-hydroxytryptamine (1 X 10-4 M, final concentration) and [14C]phenylethylamine (0.25 X 10–4 M, final concentration) as selec-

D.A. Di Monte et al. /Brain Research 738 (1996) 53-59

tive substrates for MAO A and MAO B, respectively. Preliminary experiments were performed to ensure that enzyme activity was linear with respect to time and enzyme concentration, and that no more than 10–20% of the substrate was consumed during the incubation period (30 min at 37”C). Reactions were carried out with 30 p,g protein, and protein content was measured using the method of Lowry et al. [16].

55

1 +

.31M

**

*

2.3. Chemicals L-DOPA (methyl ester), benserazide, dopamine and DOPAC and HVA were obtained from Sigma Chemical Co. (St. Louis, MO). Clorgyline (hydrochloride) and deprenyl (hydrochloride) were purchased from Research Biochemical International (Natick, MA). [14C]5-hydroxytryptamine (binoxalate) and [14C]phenylethykimine (hydrochloride) were obtained from New England Nuclear (Boston, MA). All other chemicals and solvents were reagent grade. 2.4. Statistics Statistical analysis was performed using the StudentNewman-Keuls test after a two-way analysis of variance (ANOVA).

* T

dopamine

DOPAC

HVA

Fig. 1. Effects of L-DOPAadministrationon the levels of dopamine, DOPACandHVAin the putamen.Monkeys(n = 3/group) wereinjected with:(i) saline(emptybars);(ii) benserazide/L-DOPA(12.5/40 mg/kg, striped bas), (iii) deprenyl (1 mg/kg) prior to benserazide/L-DOPA (light shadedbars), or (iv) clorgyline(1 mg/kg) prior to benserazide/& DOPA(dark bars). Animalswere killed 3 h after saline or L-DOPA,and dopamineand its metabolizesweremeasuredin a section of the putarnen. Vatues are means ~S.E.M. “ Statistically different (P < 0.05) from the satine control group. + Statistically different(P < 0.05) from the L-DOPA alone group.

3. Results 3.1. Effects of L-DOPA on the level and metabolism of striatal dopamine

250

Three hours after the administration of L-DOPA to squirrel monkeys a marked increase in doparnine levels was measured in both the putamen (Fig. 1) and the caudate nucleus (Fig. 2). This increase was greater in the putamen (+ 81%) than in the caudate (+60%) and, indeed, dopamine levels following L-DOPA treatment became statistically different between the two striatal areas (P < 0.05). The rise of striatal dopamine was paralleled by an increase in concentrations of both doparnine metabolizes, DOPAC and HVA (Figs. 1 and 2). While HVA levels doubled after L-DOPA administration, changes in DOPAC were even more pronounced. Furthermore, similar to the effect of L-DOPA on dopamine, f=DOPA-induced enhancement of striatal DOPAC was greater in the putamen (+14 times) than in the caudate (+11 times). The difference between the DOPAC levels in the putamen and caudate after L-DOPA treatment did not reach statistical significance, however. Changes in the ratio DOPAC/dopamine can be used to estimate changes in the rate of intracellular dopamine metabolism via MAO. As reported in Table 1, DOPAC\dopamine was approximately seven times higher

2oo-

150-

1oo-

* s! -1-

50

01

dopamine

DOPAC

HVA

Fig. 2. Effects of L-DOPA administration on the levels of dopamine, DOPAC and HVA in the caudate nucleus. Monkeys (n= 3/group) were injected with: (i) satine (empty bars); (ii) benserazide\L-DOpA (12.5/40 mg/kg, striped bars), (iii) deprenyl (1 mg/kg) prior to benserazide/LDOPA (light shaded bars), or (iv) clorgyline (1 mg/kg) prior to benserazide/L-DOPA (dark bars). Animals were killed 3 h after saline or L-DOPA, and doparnine and its metabolizes were measured in a section of the caudate. Values are means ~S.E.M. “ Statistically different (P<

0.05) from the saline controlgroup. + Statisticallydifferent(P< 0.05) from the L-DOPAalone group.

56

D.A. Di Monte et al. /Brain Research 738 (1996) 53-59

Table I DOPAC/dopamine ratio in the striatum and substantialnigra of monkeys treated with L-DOPA alone or in combination with MAO inhibitors Treatment

Saline L-DOPA L-DOPA + clorgyline L-DOPA + deprenyl

DOPAC/dopamine ratio Putamen

Caudate

Substantialnigra

0.029 + 0.221 * 0.062 ~ 0.067 +

0.033 + 0,006 0.213 ~ 0.051 * 0.069 & 0.010 ‘ * 0.075 f 0.014 * *

0.065 ~ 0.682 + 0.243 + 0.187 +

0.004 0.053 “ 0.007 “ ‘ 0.010 “ *

0.007 0.109 * 0,035 * * 0,029 “ “

“ Statistically different (P< 0.05) from the saline control group. * “ Statistically different (P< 0.05) from the L-DOPA alone group. Monkeys were injected with saline (n= 3), L-DOPA alone (40 mg/kg, n = 3) or L-DOPA with clorgyline (1 mg/kg, n = 3) or deprenyl (1 mg/kg, n = 3). MAO ifiibitors were administered 24 h prior to L-DCJPA, md L.DOPA treatment was always preceded by an injection of benserazide (12.5 mg/kg). Animals were killed 3 h after tbe L-DOPA injection.

in the striatum of L-DOPA-treated monkeys as compared to saline controls.

values measured in saline controls, when L-DOPA administration was preceded by injections of MAO inhibitors (Figs. 1 and 2). Data in Table 1 show that the DOPAC\dopamine ratio was also reduced by the co-administration of L-DOPA and MAO inhibitors. The extent of reductions of DOPAC and HVA levels and DOPAC/dopamine ratio was similar regardless of whether MAO A was inhibited with clorgyline or MAO B was blocked by deprenyl.

3.2. Effects of MAO inhibitors on L-DOPA-induced changes in the level and metabolism of striatal dopamine Measurements of MAO A and MAO B revealed no difference in individual enzyme activity between the putamen and caudate of squirrel monkeys (Table 2). In both areas, however, MAO B activity was approximately ten times higher than MAO A. Administration of L-DOPA did not significantly change MAO levels. In contrast, when clorgyline was injected before L-DOPA, MAO A was reduced to approximately 109ZO of the basal level with no change in MAO B. Deprenyl decreased MAO B to less than 5% of the basal level while it did not affect MAO A activity. Thus, both clorgyline and deprenyl at the concentration used in this study (1 mg/kg) acted as selective inhibitors of MAO A and MAO B, respectively. A trend toward higher striatal dopamine was observed in monkeys treated with either clorgyline or deprenyl prior to L-DOPA (Figs. 1 and 2) as compared to animals injected only with L-DOPA. These differences reached statistical significance (f’ = 0.04) when dopamine levels were compared in the putamen of animals treated with deprenyl/LDOPA vs. L-DOPA alone. Both DOPAC and HVA levels were significantly decreased, although not completely reduced to the basal

3.3. Effects of L-DOPA and MAO inhibitors on the level and metabolism of dopamine in the substantial nigra The dopamine level in the substantial nigra was greatly enhanced after L-DOPA administration (Fig. 3). Indeed, the extent of nigral dopamine increase was much greater than that caused by L-DOPA in the striatum (soo~o vs. 60-80%). Similarly, the extent of increase in DOPAC and HVA caused by L-DOPA in the substantial nigra was more pronounced than that measured in the striatum (60 times vs. 11 to 14 times for DOPAC, 8 times vs. 2 times for HVA) (Fig. 3). The DOPAC/dopamine ratio was approximately ten times higher in the substantial nigra of LDOPA-treated monkeys as compared to saline controls (Table 1). Co-administration of MAO inhibitors and L-DOPA did not seem to cause any additional increase in nigral dopamine concentration as compared to L-DOPA alone

Table 2 MAO-A and MAO-B activities in the striatum of monkeys treated with L-DOPA alone or in combination with MAO inhibitors MAO-B

Treatment

MAO-A Putamen

Caudate

Putamen

Caudate

Saline L-DOPA L-DOPA + clorgyline L-DOPA + deprenyl

6.03 ~ 5.91 * 0.52 t 5,73 +

6.03 t 4,90 * 0.45 + 4.75 *

65.1 + 63.0 + 66.6 + 2.20 +

51.9 + 48.1 + 51.1 + 1.75 f

1.3 1.2 0.05 ‘ 0.9

1,4 0.3 0.08 ‘ I.0

1,5 11.0 8.1 0.4 ‘

5.0 2.6 3.2 0.4 *

* Statistically different (P < 0.05) from other groups. Monkeys were injected with saline (n = 3), L-DOPA alone (40 mg/kg, n = 3) or L-DOPA with clorgyline (1 mg/kg, n =2) or deprenyl (1 mg/kg, mdL-DOpA treatment was always preceded by an injection of benserazide (12.5 n = 3), MAO i~ibitors were administered 24 h priortoL-f)opA, mg/kg). Animals were killed 3 h after the L-DOPA injection,

D.A. Di Monte et al. /Brain Research 738 (1996) 53-59

*

*

++

dopamine

DOPAC

HVA

Fi.q. 3. Effects of L-DOPA administration on the levels of domrnine, DOPAC and HVA in the substantialnigra. Monkeys (n= 3/group) were

injectedwith:(i) saline(emptybars);(ii) benserazide/L-DOPA(12.5/40 mg/kg, s~ped b~s), (iii) deprenylO mg/kg) pfior to ~nser=ide/Ldopa (Iight shadedbars), or (iv) clorgyline(1 mg/kg) prior to benserazide/L-DOPA (dark bars). Animals were killed 3 h after saline or L-DOPA, and dopamine and its metabolizes were measured in a section of the substantial nigra. Vafues are means +S.E.M. * Statistically different (P< 0.05) from the saline controlgroup. + Statisticallydifferent(P<

().05)from the L-DOPAalone group.

(Fig. 3). However, these combined treatments significantly counteracted the L-DOPA-induced increase in nigral DOPAC and HVA concentrations and DOPAC/dopamine ratio (Fig. 3 and Table 1).

4. Discussion The findings of this study address two major experimental questions concerning the effects of a single dose of L-DOPA on the level and metabolism of nigrostriatal doparnine and the involvement of MAO A and/or MAO B in the metabolism of L-DOPA-generated dopamine. When the effects of L-DOPA are compared in the putarnen vs. the caudate and in the striatum vs. the substantial nigra of squirrel monkeys, significant differences can be found. Within the striatum, the extent of L-DOPA-induced increase in both dopamine and DOPAC levels was greater in the putamen than in the caudate nucleus. The reason(s) for these unequal effects is unclear although anatomical (e.g., density and distribution of terminals) and/or pharmacological (e.g., doparnine storage capability) differences within the striatal doparninergic system may be involved. Results from previous studies further support this concept. For example, differences between the putamen and caudate have been reported in age-related changes in dopamine levels and in relation to sensitivity to dopaminergic damage [10,11,17]. In both cases and similar to results of the

57

present study, the putamen appeared to be more significantly affected. Elucidation of the anatomical and pharmacologic differences within the striatum has important implications, as it may provide insight into the mechanism(s) responsible for the uneven pattern of dopamine depletion in the striatum of patients with Parkinson’s disease [14]. Interestingly, dopamine depletion in these patients was more complete and severe in the putamen than in the caudate nucleus. Previous data in rodents have indicated that the pharmacological consequences of L-DOPA administration can be different in the striatum as compared to the substantial nigra [5]. In the present investigation, a comparison of the effects of L-DOPA in the striatum vs. the substantial nigra of monkeys reveals that the enhancement of doparnine, DOPAC and HVA levels was of a much greater magnitude in the substantial nigra. Following L-DOPA treatment, nigral dopamine levels, although increased fivefold, were still approximately half of the striatal dopamine concentrations. It is noteworthy, however, that the levels of DOPAC increased 60-fold in the substantialnigra and became higher than in the striatum. DOPAC is the product of the intracellular metabolism of dopamine via MAO which also generates hydrogen peroxide. Although DOPAC may be further metabolized to HVA in the extracellulm compartment, tissue levels of DOPAC as well as the DOPAC/dopamine ratio can be used to estimate the rate of intracellular dopamine metabolism and H202 formation. Thus, higher levels of DOPAC in the substantialnigra as compared to the striatum suggest that a greater amount of the newly synthesized dopamine undergoes intracellular metabolism via MAO. They may also reflect higher HZ02 production raising the possibility that nigral cell bodies may be at greater risk than striatal terminals for oxidative stress induced by L-DOPA treatment. The toxic consequences of H202 generation may also be more pronounced in the substantial nigra due to the interaction of oxygen radicals with neuromelanin and transition metals [6]. The second major aim of this investigation was to determine the respective roles of MAO A and MAO B in the metabolism of doparnine generated from L-DOPA. Inhibition of MAO activity prior to L-DOPA administration resulted in a significant reduction of doparnine metabolism as indicated by the greatly decreased levels of DOPAC and HVA and DOPAC/dopamine ratio in both the striatum and the substantialnigra. The effects of clorgyline and deprenyl, although assessed in relatively small groups of monkeys (n = 3/treatment), were consistent and statistically significant in the putarnen, caudate and substantial nigra. Interestingly, DOPAC and HVA reductions were achieved with either clorgyline or deprenyl, indicating that both MAO A and MAO B contribute to the dearnination of dopamine synthesized from exogenous LDOPA. It is noteworthy, however, that the extent of inhibition of dopamine metabolism was similar with clor-

58

D.A. Di Monte et al./Brain Research 738 (1996) 53–59

gyline or deprenyl despite the fact that MAO A activity is much lower than MAO B in monkey striatum. This suggests that dopamine delamination after L-DOPA treatment, though catalyzed by both MAO A and MAO B, preferentially involves MAO A. These results are apparently at odds with those of other investigations in which little or no evidence for a role of MAO B in the metabolism of dopamine generated from L-DOPA was found [4,24]. The most likely explanation for these differences is the fact that previous work has been performed in the rat model. To the best of our knowledge, this is the first investigation in which the respective roles of MAO A and MAO B in L-DOPA-generated dopamine turnover are determined in non-human primates. The ratio MAO A/MAO B is approximately 1/10 in the striatum of squirrel monkeys vs. 2/1 in the rat brain [26]. Thus, it is likely that, under conditions of elevated tissue levels of dopamine, MAO B would contribute to dopamine turnover in the monkey striatum. On the other hand, MAO B involvement may be less important in the presence of relatively high MAO A activity as in the case of the rodent striatum. That the relative proportion of the two MAO isozymes plays a role in determining their contribution to dopamine metabolism is emphasized by data suggesting that, even in rats, MAO B may catalyze striatal dopamine delamination when MAO A is inhibited [1,3]. The involvement of MAO B in dopamine turnover under our experimental conditions raises an important issue concerning the site for this metabolic process. Immunocytochemical studies indicate that dopaminergic terminals contain MAO A but not MAO B [15,25], suggesting that the delamination of dopamine generated in the monkey striatum after L-DOPA administration occurs outside these terminals. Possible sites for the MAO B-mediated conversion of dopamine include other neuronal cells (e.g., serotonergic neurons) and/or non-neuronal glial elements which exhibit MAO B [15,25]. The latter possibility is particularly intriguing in view of the fact that uptake sites for dopamine have been demonstrated on astrocyte plasma membranes [20]. Another implication of the present results relates to the interpretation of findings on doparrtine metabolism in different animal models. Changes in the levels of MAO A and MAO B are apparently capable of affecting the routes of dopamine delamination as well as the sites where this reaction may occur. Thus, the inter-species variability of MAO A/MAO B ratio, such as that between rodents and human and non-human primates, should always be taken into careful consideration during comparative analyses of dopamine metabolism. The involvement of MAO B in dopamine metabolism and the ability of deprenyl to counteract the increase in dopamine turnover after L-DOPA administration to monkeys supports the therapeutic use of MAO B inhibitors in Parkinson’s disease. The efficacy of deprenyl in the treatment of parkinsonism has been suggested by several clini-

cal studies [2,22,23]. As a MAO B inhibitor, deprenyl could decrease the rate of dopamine metabolism, thus (i) enhancing the level of dopamine available for neurotransmission, and (ii) possibly preventing the damaging effects of high dopamine turnover. The action of MAO B inhibitors toward dopamine metabolism in humans may result not only from the high level of MAO B in the primate brain, as shown in this study, but also from a greater dopamine affinity of MAO B than MAO A in human brain [9]. Two caveats need to be emphasized, however, when drawing conclusions from the present results obtained by injecting a single dose of L-DOPA to monkeys. First, chronic treatment with L-DOPA in Parkinson’s disease may affect the respective roles of MAO A and MAO B in dopamine metabolism. Second, the biodisposition of dopamine may be changed in the nigrostriatal pathway of patients with Parkinson’s disease due to the loss of dopaminergic neurons. Therefore, the role of MAO B in the delamination of dopamine synthesized from L-DOPA needs to be confirmed by future studies in which L-DOPA is administered chronically to animals with a lesioned nigrostriatal pathway.

Acknowledgements This work was supported by The Parkinson’s Institute. J.E.R. was supported by a fellowship from the National Parkinson Foundation. Authors wish to thank Dr. Giselle Petzinger for her comments on this manuscript. The technical assistance of T. Hall, M. Thiruchelvam and M. Verma and the help of D. Rosner in the preparation of the manuscript are greatly appreciated.

References [I] Azzaro, A.J., King, J., Kotzuk, J., Schoepp, D.D., Frost, J. and Schochet, S., Guinea pig striatum as a model of human dopamine delamination:the role of monoamine oxidase isozyme ratio, localization and affinity for substrate in synaptic dopamine metabolism, J. Neurochem., 45 (1985) 949-956. [2] Birkmayer, W., Knoll, J., Riederer, P. and Youdim, M.B., (–)-Deprenyl leads to prolongation of L-DOPA efficacy in Parkinson’s disease, Mod. Probl. Pharmacopsych., 19 (1983) 170-176. [3 Butcher, S.P., Fairbrother, 1.S., Kelly, J,S. and Arbuthnott, G.W., Effects of selective monoamine oxidase inhibitors on the in vivo release and metabolism of dopamine in the rat striatum, J. Neur-ochem,,55 (1990) 981–988. [4 Buu, N.T. and Angers, M., Effects of different monoamine oxidase inhibitors on the metabolism of L-DOPA in the rat brain, Biockem. Pharrnacol., 36 (1987) 1731–1735. [5] DeBoer, P., Wachtel, S.R. and Abercrombie, E.D., Biochemistry and pharmacology of L-DOPA in relation to basal ganglia circuitry. In G. Percheron, J.S. McKenzie and J. Feger (Eds.), The Basal Ganglia IV, Plenum Press, New York, 1994, pp. 395–401. [6] Fahn, S. and Cohen, G., The oxidant stress hypothesis in Parkinson’s disease: evidence supporting it, Ann. Neurol., 32 (1992) 804-812.

D.A. Di Monte et al. /Brain Research 738 (1996) 53-59 [7] Fowler, C.J., Wiberg, A., Oreland, L., Marcusson, J. and Winblad, B., The effect of age on the activity and molecuku properties of human brain monoamine oxidase, J. Neural Trans., 49 (1980) 1-20. [8] Garrett, M.C. and Soares-da-Silva, P., Role of type A and B monoamine oxidase on the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) in tissue from the brain of the rat, Neuropharmacology, 29 (1990) 875–879. [9] Glover,V., Sandier,M., Owen, F. and Riley, G.J., Dopamineis a monoamineoxidaseB substratein man, Nature, (1977) 265 80–81. [10] Irwin, I., DeLanney, L.E., Forno, L.S., Finnegan, K.T., Di Monte, D.A. and Langston, J.W., The evolution of nigrostriatal neurochemical changes in the MPTP-treated squirrel monkey, Brain Res., 531 (1990) 242-252. [11] Irwin, I., DeLanney, L.E., McNeill, T., Chan, P., Fomo, L.S., Murphy, G.R., Di Monte, D.A., Sandy, M.S. and Langston, J.W., Aging and the nigrostriatal dopamine system: a non-human primate study, Neurodegeneration, 3 (1994) 251–265. [12] Kate, T., Dong, B., Ishii, K. and Kinemuchi, H., Brain dialysis: in vivo metabolism of dopatnine and serotonin by monoamine oxidase A but not B in the striatum of unrestrained rats, 1 Neurochem., 46 (1986) 1277-1282. [13] Kilpatrick, L, Jones, M. and Phillipson, O., A semiautomated analysis method for catecholamines, indoleamines and some prominent metabolizes in microdissected regions of the nervous system: an isocratic HPLC technique employing coulometric detection and minimal sample preparation, J. Neurochern., 46 (1986) 1865–1876. [14] Kish, S.J., Shannak, K. and Homykiewicz, O., Uneven pattern of dopamine loss in the striatum of patients with idiopathic Parkinson’s disease, N. Engl. 1 Med., 318 (1988) 876-880. [15] Levitt, P., Pintar, J.E. and Breakefieid, X.O., Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons, Proc. NatL Acad. Sci. USA, 79 (1982) 6385– 6389. [16] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [17] Moratalla, R., Quinn, B., DeLrmney, L.E., Irwin, I., Langston, J.W. and Graybiel, A.M., Differential vulnerability of primate caudate-

59

putamen and striosome-matrix dopamine systems to the neurotoxic effects of l-methyl-4-phenyl- l,2,3,6-tetrahydropyndine, Proc. Natl. Acad. Sci, USA, 89 (1992) 3859-3863. [18] Paterson, I.A., Juorio, A.V., Berry, M.D. and Zhu, M.Y., Inhibition of mono~ine oxidase-B by ( – )-deprenyl potentates neuronal responses to dopamine agonists but does not inhibit dopamine catabolism in the rat striatum, J. PlrarmacoL Exp. Ther., 258 (1991) 1019-1026. [19] Riachi, N.J. and Harik, S.1., Monoamine oxidases of the brains and livers of macaque and cercopithecus monkeys, Exp. Neurol., 115 (1992) 212-217. [20] Semenoff, D. and Kimelberg, H.K., Autoradiography of high affinity uptake of catecholamine by primary astrocyte cultures, Brain Res., 348 (1985) 125-136. [21] Spina, M.B. and Cohen, G., Exposure of striatal synaptosomes to L-DOPA increases levels of oxidized ghrtathione, J. Pharmacol. Exp. Ther., 247 (1988) 502-507. [22] Tetmd, J.W. and Langston, J.W., The effect of deprenyl (selegiline) on the natural history of Parkinson’s disease, Science, 245 (1989) 519-522. [23] The Parkinson Study Group, Effect of deprenyl on the progression of disability in early Parkinson’s disease, N. Engl. J Med., 321 (1989) 1364-1371. [24] Wachtel, S.R. and Abercrombie, E.D., L-3,4-dihydroxyphenylalanine-induced dopamine release in the striatum of intact and 6-hydroxydopamine-treated rats: differential effects of monoamine oxidase A and B inhibitors, J. Neurochenz., 63 (1994) 108-117. [25] Westlund, K.N., Denney, R.M., Kochersperger, L.M., Rose, R.M. and Abell, C.W., Distinct monoamine oxidase A and B populations in primate brain, Science, 230 (1985) 181–183. [26] Willoughby, J., Glover, V., Sarrdler, M., Albanese, A., Jenner, P. and Marsden, C.D., Monoamine oxidase activity and distribution in marmoset brain: implications for MPTP toxicity, Neurosci. L@., 90 (1988) 100-106. [27] Yu, P.H. and Hertz, L., Differential expression of type A and type B monoamine oxidase of mouse astrocytes in primary cultures, J. Neurochem., 39 (1982) 1492-1495.