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Dopamine metabolism in the guinea pig striatum: role of monoamine oxidase A and B
ELSEVIER
European Journal of Pharmacology 254 (1994) 213-220
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Dopamine metabolism in the guinea pig striatum: role of monoamine oxidase A and B Augusto V. Juorio *, I. Alick Paterson, Meng-Yang Zhu Neuropsychiatric Research Unit, Medical Research Building, University of Saskatchewan, Saskatoon, Saskatchewan S7N OW£ Canada (Received 7 October 1993; revised MS received 9 December 1993; accepted 15 December 1993)
Abstract
These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig affects the actions of (-)-deprenyl on dopamine metabolism in whole tissue or in extracellular fluid. The administration of (-)-deprenyl in doses that do not affect monoamine oxidase A activity (1-4 mg kg-1, 2 h) increases striatal 2-phenylethylamine and dopamine concentrations and reduces 3,4-dihydroxyphenylacetic acid. No effects were observed on striatal homovanillic acid, 5-HT and 5-hydroxyindole acetic acid. Inhibition of monoamine oxidase A with clorgyline with doses up to 8 mg kg- 1 (2 h) does not affect striatal 2-phenylethylamine but increases dopamine and 5-HT concentrations and reduces 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid. (-)-Deprenyl (2-4 mg kg -1) did not change the extracellular concentrations of dopamine but the higher dose produced a limited reduction in extracellular 3,4-dihydroxyphenylacetic acid. Inhibition of monoamine oxidase A and monoamine oxidase B with pargyline (75 mg kg -1, 2 h) significantly increased the levels of extracellular dopamine and reduced those of their acid metabolites. These results show that in the guinea pig striatum inhibition of monoamine oxidase B by (-)-deprenyl impairs the metabolism of dopamine in the whole tissue but does not produce a marked increase in extracellular dopamine. Key words: Monoamine oxidase type B; (-)-Deprenyl; Dopamine; Microdialysis; 2-Phenylethylamine; (Guinea pig)
1. Introduction
Dopamine is a substrate for both monoamine oxidase A and monoamine oxidase B in vitro (Fowler and Tipton, 1984) and ( - ) - d e p r e n y l is a potent irreversible inhibitor for the oxidative deamination of dopamine in human brain homogenates (Garrick and Murphy, 1980) and it has been assumed that ( - ) - d e p r e n y l acts by inhibiting dopamine catabolism in vivo (Marsden, 1990). The acute administration of ( - ) - d e p r e n y l , however, does not alter the rat brain levels of dopamine or its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid (Azzaro et al., 1985; Hovevey-Sion et al., 1989; Knoll, 1983; Waldmeier et al., 1976, Paterson
* Corresponding author. Neuropsychiatric Research Unit, A l l 4 MR Building, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7NOW0. Tel. (306) 966-8818, fax (306) 966-8830, e-mail [email protected]. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 3 ) E 0 9 1 2 - C
et al., 1991) nor does it alter extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid or 3-methoxytyramine (Butcher et aI., 1990; Kato et al., 1986) but it significantly inhibits monoamine oxidase B (Knoll and Magyar, 1972). Monoamine oxidase B preferentially metabolizes 2-phenylethylamine (Yang and Neff, 1973) which is also present in the brain but in low concentrations (Durden et al., 1973; Philips et al., 1978; Juorio, 1988). The turnover of 2-phenylethylamine is quite rapid (Durden and Philips, 1980) and its brain concentrations are markedly increased by acute treatment with ( - ) - d e p r e n y l (Philips and Boulton, 1979; Paterson et al., 1991). Electrophysiological studies have demonstrated that 2-phenylethylamine and ( - ) - d e p r e n y l potentiate striatal neuron responses to dopamine agonists suggesting that 2-phenylethylamine may mediate some of the actions of ( - ) deprenyl (Paterson et al., 1990, 1991). The finding that monoamine oxidase A / m o n o a m i n e oxidase B ratio in the guinea pig striatum is similar to that of the human striatum (Azzaro et al., 1985) sug-
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A. IC Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
gested that the guinea pig striatum is a better model of human dopamine metabolism. These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig striatum affects the actions of ( - ) - d e p r e n y l on dopamine metabolism in the whole tissue as well as its extracellular levels. For comparison, the effects of ( - ) - d e p r e n y l and clorgyline on the striatal concentrations of 5-HT and 5-hydroxyindole acetic acid were also determined.
2. Materials and methods
2.1. Animals Male English Short-Hair guinea pigs (300-400 g) were obtained from Charles River (Montreal). They were housed in a 12 h light/dark cycle (lights on at 7.00 a.m.) with free access to food and water for at least 5 days before the start of the experiment. The experimental protocol was approved by the University Committee of Animal Care and Supply.
2.2. Measurement of 2-phenylethylamine, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-HT and 5-hydroxyindole acetic acid Guinea-pigs were stunned and decapitated, the brain was removed and chilled in ice-cold saline and the striatum, consisting mainly of the head of the caudate nucleus and including some of the underlying putamen (approx. between 130 and 170 mg) was dissected out on ice. Tissues were immediately frozen in dry ice and weighed. Two striata from one guinea pig were homogenized in 0.1 N perchloric acid containing E D T A (0.25 mM), sodium metabisulfite (0.1 mM). Isoproterenol (100 n g / m l ) and [2H4] 2-phenylethylamine were added as internal standards. After centrifugation of the tissue homogenate, 250/zl of the supernatant were removed for assay by high pressure liquid chromatography with electrochemical detection of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-HT and 5-hydroxyindole acetic acid (Kwok and Juorio, 1986). The remaining supernatant was used to measure 2-phenylethylamine; for this, the tissue homogenates were derivatized with 5-dimethylamino-L-naphthalene sulphonyl (dansyl) chloride. The resultant derivatives extracted into an organic phase, evaporated to a small volume and separated chromatographically in two different unidimensional systems (Juorio and Kazakoff, 1984). 2-Phenylethylamine estimations were carried out by a high resolution mass spectrometric selected ion monitoring technique. Complete details concerning this procedure have been described (Durden et al., 1973).
2.3. Measurement of monoamine oxidase actit,ity The tissue was homogenised in ice-cold potassium phosphate buffer and centrifuged at 40000 x g for 30 min. The pellet was resuspended and centrifuged again. The activity of monoamine oxidase A was determined using [14C]5-HT bioxalate and the activity of monoamine oxidase B was determined using [~4C]2-phenylethylamine hydrochloride (Wurtman and Axelrod, 1963; Yu, 1985). The radiolabelled substrate was mixed with unlabelled substrate to yield a final concentration of 1 X 10 4 M for 5-HT and 0.25 X 10 4 M for 2-phenylethylamine. The reaction mixture contained potassium phosphate buffer, striatal membranes (40-50 mg protein) and 0.05 /zCi of the substrate. The reaction occurred for 30 min at 37°C and was terminated by the addition of 250 /zl 2 M citric acid. The acid and aldehyde products were extracted into 1 ml of t o l u e n e / e t h y l acetate (1 : 1, v/v); 500/zl of the organic extract was used for liquid scintillation counting to determine acid metabolite production. The protein concentration in the membrane preparation was determined by Lowry's method.
2.4. Intracerebral microdialysis Guinea pigs were anaesthetised with a mixture of urethane and sodium pentobarbital (1 g / k g and 10 m g / k g respectively, i.p.), placed in a stereotaxic frame and body temperature (rectal) was maintained at 37 + 0.2°C. Concentric microdialysis probes (Carnegie Medicin C M A / 1 0 , 3 mm tip) were placed in the striaturn at AP + 2.0 mm, L + 3.7 ram, V - 9 . 0 mm (with respect to bregma, flat skull orientation). The probes were perfused with an artificial cerebrospinal fluid (composition in mM: Na + 150.0; K + 3.5; Ca 2+ 1.2; Mg 2+ 0.8, C1- 128.9; HCO 3 26.0; SO 2- 0.8; PO~ 1.0; glucose 10.0; ascorbate 0.01; pH 7.35 at 25°C) at a rate of 2 / z l / m i n . The probes gave 16-19% recovery of dopamine in vitro. Samples were collected into 40 tzl of 0.1 M perchloric acid containing 3.6 ng of isoproterenol (internal standard for high performance chromatography with electrochemical detection) over a 20 min period. 50/zl of the sample were injected onto the HPLC column and dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were detected at a glassy carbon electrode at - 0 . 7 5 mV. The limits of detection for this system are: dopamine, 2 pg; 3,4-dihydroxyphenylacetic acid, 2 pg; homovanillic acid, 50 pg. The levels in 50 /~1 of a baseline sample were: dopamine, 4-16 pg; 3,4-dihydroxyphenylacetic acid, 3-4 ng; homovanillic acid, 7 - 9 ng. Dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were stable within 1 h of probe insertion. After 1 h, 3 samples were collected to determine basal levels of
A. 1~. Juorio et aL / European Journal of Pharmacology 254 (1994) 213-220
these substances, drug or vehicle was injected and sampling continued for 3-4 h (Ungerstedt, 1984).
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Fig. 1. Effects of (-)-deprenyl administration on the activity of striatal m o n o a m i n e oxidase A (MAO-A) (open circles) and monoamine oxidase B (MAO-B) (closed circles), expressed as percentage of the control (saline) values. (-)-Deprenyl or saline was administered by i.p. injection 2 h before death. The values indicate means ( + S . E . M . ) obtained from 9-15 experiments. * * P < 0 . 0 1 , Newman-Keuls test with respect to saline.
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The effects of different doses of (-)-deprenyl and clorgyline on monoamine oxidase activity and the striatal concentrations of amines and acid metabolites were analyzed by one-way analysis (ANOVA) of variance and the statistical differences between groups determined by the Newman-Keuls test. The effects of pargyline on the monoamine oxidase activity and the concentrations of monoamines and metabolites were analysed by the Student's t-test. The effects of ( - ) - d e prenyl on urethane-pentobarbital treated animals were analyzed by ANOVA. Statistical analysis of the dialysis experiments were performed by two-way ANOVA ( D r u g - between subjects factor, T i m e - w i t h i n sub-
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Fig. 2. Effects of different doses of (-)-deprenyl on the striatal concentrations of (A) 2-phenylethylamine (PE), (B) dopamine (DA), (C) 3,4-dihydroxyphenylacetic acid (DOPAC), (D) homovanillic acid (HVA), (E) 5-HT and (F) 5-hydroxyindole acetic acid (5-HIAA). ( - )-Deprenyl or saline was administered by i.p. injection 2 h before death. Each point represents the mean + S.E.M. of 11-21 determinations. ** P < 0.01, Newman-Keuls test with respect to saline.
A. I/. Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
216
jects factor). The microdialysis results are shown as a percentage of the mean of the three baseline samples.
2.6. Drugs ( - ) - D e p r e n y l hydrochloride was obtained from Research Biochemicals (Natick, MA, USA), pargyline hydrochloride from Sigma Chemical Company (St. Louis, MO, USA) and clorgyline hydrochloride generously supplied by May & Baker, Essex, UK.
0.033 nmol of [14C]2-phenylethylamine min ~ mg of protein -] (n = 12). The administration of ( - ) - d e p r e nyl only reduced the activity of monoamine oxidase A at a dose of 8 mg k g - ' (F(6,68)= 5.08, P < 0.001). Monoamine oxidase B activity was reduced (F(6,80) = 17.77, P < 0.001) with all doses of ( - )-deprenyl (0.25-8 mg k g - ' , 2 h i.p.) by 67 to 90% (Fig. 1). The administration of pargyline (75 mg kg-1, 2 h i.p.) produced complete inhibition of the activity of monoamine oxidase A and monoamine oxidase B (to 6 and 1%, respectively) (results not shown).
3.2. Effects of monoamine oxidase inhibition on striatal amines and metabolites
3. Results
3.1. The effects of (-)-deprenyl and pargyline on monoamine oxidase activity The striatal activity of monoamine oxidase A was 0.026 + 0.001 nmol of [14C]5-HT rain- 1 mg of protein- a (n = 12) and that of monoamine oxidase B was 0.226 +
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Fig. 3. Effects of different doses of clorgyline on the striatal concentrations of (A) 2-phenylethylamine (PE), (B) dopamine (DA), (C) 3,4-dihydroxyphenylacetic acid (DOPAC), (D) homovanillic acid (HVA), (E) 5-HT and (F)5-hydroxyindole acetic acid (5-HIAA). Clorgyline or saline was administered by i.p. injection 2 h before death. Each point represents the m e a n _+ S.E.M. of 9 - 1 7 determinations. ** P < 0.01, Newman-Keuls test with respect to saline.
A.V. Juorio et aL / European Journal of Pharmacology 254 (1994) 213-220
(-)-Deprenyl had no effect on homovanillic acid (F(5,81) = 0.76), 5-HT (F(5,81) = 1.04) and 5-hydroxyindole acetic acid (F(5,80)= 0.33). Comparisons using the Newman-Keuls test showed that 2-phenylethylamine concentrations were significantly increased by 1-4 mg kg -1 of (-)-deprenyl and dopamine levels were increased by 4 mg kg-1 while 3,4-dihydroxyphenylacetic acid was decreased after treatment with 2 and 4 mg kg-1 (Fig. 2). The administration of clorgyline (0.5-8 mg kg-1, 2 h i.p.) did not produce significant changes in striatal 2-phenylethylamine concentrations (F(5,48)= 1.09). In contrast, it increased the levels of dopamine (F(5,70) = 7.21, P < 0.001), reduced the concentrations of 3,4dihydroxyphenylacetic acid (F(5,70) = 4.80, P < 0.001) and did not affect homovanillic acid (F(5,70)= 1.81). Comparisons using the Newman-Keuls test showed that dopamine concentrations were significantly increased by 4 and 8 mg kg-t of clorgyline and 3,4-dihydroxyphenylacetic acid levels were reduced by 2-8 mg kg-1 while 5-HT was increased by 8 mg kg- 1 and 5-hydroxyindole acetic acid was decreased after treatment with 4 and 8 mg kg-1 (Fig. 3). In order to evaluate the effect of urethane-pentobarbital anaesthetic used for the microdialysis experiments, rats were treated with saline, (-)-deprenyl, urethane-pentobarbital and urethane-pentobarbital plus (-)-deprenyl (Table 1). Two-way analysis of variance indicated that (-)-deprenyl significantly changed the 2-phenylethylamine levels (F(1,44)= 14.35, P < 0.001) but no interactions between the treatments were observed. (-)-Deprenyl produced no changes in dopamine concentrations (F(1,50) = 2.07), but reduced the concentrations of 3,4-dihydroxyphenylacetic acid (F(1,50) = 27.11, P < 0.001) and homovanillic acid (F(1,50) = 4.98, P < 0.05) (Table 1). Treatment with urethane-pentobarbital had no effect on the levels of 2-phenylethylamine (F(1,44)= 0.15) or dopamine (F(1,50)= 1.96) but changed the concentrations of 3,4-dihydroxyphenylacetic acid (F(1,50) = 7.75, P < 0.01) and homovanillic acid (F(1,50) = 13.49, P < 0.001) (Table 1).
Table 1 Effects of saline, urethane-pentobarbital (1.75 g kg - I and 5 mg kg 1 respectively) and ( - )-deprenyl (4 mg kg- ]) on the striatal concentrations of 2-phenylethylamine (PE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) Treatment
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Fig. 4. Effects of saline (open squares) and (-)-deprenyl (4 mg k g - i ) (closed squares) on the striatal extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The results are expressed as percentages of basal release. (-)-Deprenyl or saline was administered by i.p. injection at 0 time (indicated by arrow). Each point represents the mean+S.E.M, of 6 determinations. There was no effect of ( - ) - d e prenyl on dopamine levels but there were decreases in 3,4-dihydroxyphenylacetic acid (all samples following injection, P < 0.05-P < 0.01) and homovanillic acid levels (40-60 min sample and subsequent samples, P < 0.05-P < 0.01).
Treatment with pargyline (75 mg kg -1, 2 h i.p.) produced significant increases in the striatal concentrations of 2-phenylethylamine, dopamine and 5-HT while the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole acetic acid were markedly reduced (results not shown).
3.3. Effects of (-)-deprenyl on the extracellular concentration of dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid Intraperitoneal administration of saline or (-)-deprenyl (2-4 mg kg -1) had no effect on extracellular dopamine levels (F(2,16) = 2.35) but the 4 mg/kg dose of (-)-deprenyl did alter 3,4-dihydroxyphenylacetic acid and homovanillic acid levels (Fig. 4). There was an overall effect of drug on 3,4-dihydroxyphenylaeetic acid levels (F(2,16)= 4.76, P < 0 . 0 5 ) and a significant
218
A.V. Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
drug-time interaction (F(22,176) = 1.968, P < 0.01). Further analysis showed that there was a significant decrease in 3,4-dihydroxyphenylacetic acid levels in all samples after injection of the 4 mg/kg dose of (-)-deprenyl. There was no overall effect of drug on homovanillic acid levels (F(2,16)= 2.34) but there was a significant drug-time interaction (F(22,176)= 3.38, P < 0.001). Further analysis showed that there was a significant decrease in homovanillic acid levels in the third and subsequent samples (40-60 min onwards) after injection of the 4 mg/kg dose of (-)-deprenyl. Treatment with pargyline (75 mg kg -~) produced significant increases in the extracellular levels of dopamine and significant reductions in the extracellular levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (drug-time interactions: dopamine, F(11,110)= 4.21, P < 0.001; 3,4-dihydroxyphenylacetic acid, F(11,110) = 15.78, P < 0.001; homovanillic acid, F ( l l , l l 0 ) = 19.51, P < 0.001) (Fig. 5). Analysis of simple effects showed that following injection of pargyline dopamine was increased and 3,4-dihydroxyphenylacetic acid was decreased in the second (20-40 min) and subsequent samples and homovanillic acid was decreased in the third (40-60 min) sample onwards.
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4. Discussion
Specific inhibition of monoamine oxidase B by ( - ) deprenyl (Fig. 1) increased levels of 2-phenylethylamine in the guinea pig striatum, and reduced striatal 3,4-dihydroxyphenylacetic acid levels (Fig. 2). Although the levels of dopamine and homovanillic acid were not altered, these results demonstrate that specific inhibition of monoamine oxidase B does alter dopamine metabolism. The administration of clorgyline (2-8 mg kg-~) produced a dose dependent reduction in striatal 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid, increases in dopamine and 5-HT and no changes in 2-phenylethylamine (Fig. 3) suggesting that these effects are the consequence of inhibition of monoamine oxidase A. Combined inhibition of monoamine oxidase A and monoamine oxidase B with pargyline produced significant increases in the striatal concentrations of 2-phenylethylamine, dopamine and 5-HT while the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole acetic acid were markedly reduced. These results demonstrate that in the guinea pig striatum both type A and type B monoamine oxidase are able to metabolize dopamine. This is in agreement with earlier work (Azzaro et al., 1985) that has shown that in the guinea pig striatum, substantial changes in dopamine metabolism are only observed after inhibition of both type A and B monoamine oxidase. These findings further support the contention put forward earlier that in contrast to
Fig. 5. Effects of saline (open squares) and pargyline (75 mg kg i) saline (open squares) on the striatal extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The results are expressed as percentages of basal release. Pargyline or saline was administered by i.p. injection at 0 time (indicated by arrow). Each point represents the mean _+S.E.M. of 6 determinations. Following injection of pargyline, dopamine was increased and 3,4-dihydroxyphenylacetic acid was decreased in the second (20-40 min) and subsequent samples and homovanillic acid was decreased in the third (40-60 min) sample onwards.
the rat where dopamine is mainly metabolized by monoamine oxidase A (Azzaro et al., 1985; Paterson et al., 1991), in the guinea pig dopamine is metabolized by both type A and type B monoamine oxidase. The question that follows is: what is the reason of this difference? It has been reported that in the guinea pig the ratio of monoamine oxidase B/monoamine oxidase A is 3 : 1 and monoamine oxidase B is a significant factor in dopamine metabolism (Azzaro et al., 1985). This is different from the rat striatum where it is reported that the ratio monoamine oxidase B/monoamine oxidase A is 1:3 (Azzaro et al., 1985) and specific inhibition of monoamine oxidase B does not alter dopamine metabolism (Paterson et al., 1991). The determination of the relative abundances of monoamine oxidase A and B, however, is not simple and may be subject to many uncontrolled variables, making the accuracy of these reported ratios suspect (Berry et al., 1994). Im-
A.V. Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
munohistochemical studies in the rat and primate brain have demonstrated that nigrostriatal neurons contain monoamine oxidase A and that both monoamine oxidase A and monoamine oxidase B are present in extraneuronal locations (Levitt et al., 1982; Westlund et al., 1988a,b; see Berry et al., 1993 for a review). So far, there seem to be no histochemical investigations of the guinea pig brain. In a series of experiments carried out with guinea pig striatal synaptosomes, it has been demonstrated that [14C]dopamine was deaminated by type A monoamine oxidase to the extent of 65% and the remaining 35% by monoamine oxidase B (Ross, 1987). The present findings agree with the importance of monoamine oxidase A for the metabolism of striatal dopamine. The extent to which monoamine oxidase B is acting, however, will have to wait demonstration by a immunohistochemical technique because in these experiments, the differentiation between dopamine, noradrenaline, and 5-HT synaptosomes is dependent on the specificities of the uptake inhibitors (Ross, 1987). In addition, it is not known whether these inhibitors will interfere with the uptake of dopamine into the glial component of the synaptosome preparation. At present, the simplest assumption is that the distribution of monoamine oxidase A and monoamine oxidase B in the guinea pig striatum is not fundamentally different from that in the rat and primate brain. In such a case, irrespective of the ratio of monoamine oxidase A and B activities, the increased dependence on monoamine oxidase B for dopamine metabolism in the guinea pig would suggest that there is more extraneuronal metabolism of dopamine in the guinea pig striatum than in the rat. This hypothesis is supported by the higher levels of homovanillic acid in the guinea pig striatum, which are indicative of more extraneuronal metabolism of dopamine by catechol-O-methyl transferase. Similarly high homovanillic acid levels have been kown to occur in the human striatum (Hornykiewicz, 1973; Rinne and Sonninen, 1973). Interestingly, the levels of homovanillic acid in the macaque caudate are very high (25/zg g-1) and inhibition of monoamine oxidase B results in strong inhibition of dopamine metabolism and marked increases in dopamine levels (Boulton et al., 1992). The extracellular concentration of dopamine in the guinea pig striatum was not changed by the acute administration of ( - ) - d e p r e n y l (Fig. 4) even though the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the guinea pig extracellular fluid were moderately reduced following administration of the higher dose of ( - ) - d e p r e n y l (4 mg kg-1) (Fig. 4). These observations are in agreement with the changes in dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in the striata of anaesthetised animals. In contrast, combined inhibition of both monoamine oxidase A and monoamine oxidase B
219
by pargyline produced a significant increase in the extracellular concentration of dopamine, as well as decreasing metabolite concentrations (Fig. 5). These results demonstrate that although monoamine oxidase B inhibition is sufficient to alter dopamine metabolism, it is not sufficient to produce a marked overflow of dopamine into the extracellular compartment. These findings are similar to those in the rat brain (Butcher et al., 1990). A recent biochemical and electrophysiological study in the rat striatum has shown that specific inhibition of monoamine oxidase B by ( - ) - d e p r e n y l potentiates striatal neuron responses to dopamine receptor agonists without affecting dopamine turnover; these findings are in agreement with the explanation that the potentiation is mediated by increases in 2phenylethylamine (Paterson et al., 1991). In the guinea pig striatum, inhibition of monoamine oxidase B by ( - ) - d e p r e n y l increases 2-phenylethylamine striatal concentration at the same dose range (2-4 mg kg -~) that reduces the concentration of 3,4-dihydroxyphenylacetic acid (Fig. 2). These results suggest that similarly to the primates (Garrick and Murphy, 1980), monoamine oxidase B in the guinea pig striatum plays a role in dopamine metabolism. The reduction in dopamine metabolism which occurred is not sufficient, however, to raise the levels of extracellular dopamine (Fig. 4). If ( - ) - d e p r e n y l potentiates dopamine neuronal responses in the guinea pig, the mechanism could be through the increases that it produces in 2-phenylethylamine concentrations. It is concluded that in the guinea pig striatum, monoamine oxidase B has a significant role in the metabolism of dopamine but that inhibition of monoamine oxidase B does not impair dopamine metabolism sufficiently to increase dopamine overflow from the synapse. The results further agree with the suggestion that the rat brain represents a poor model for the study of human dopamine deamination (Garrick and Murphy, 1980; Fowler and Strolin Benedetti, 1983; Azzaro et al., 1985).
5. Acknowledgements The authors wish to thank Dr. B.A. Davis for the synthesis of deuterated 2-phenylethylamine,Dr. D.A. Durden for supervision of the mass spectrometric analyses, E.P. Zarycki, M. Beszterda and S. Ambrose for technical assistance and Dr. A.A. Boulton for helpful discussion. Saskatchewan Health, Deprenyl Research Ltd., Canada and the Schizophrenia Society of Saskatchewan provided financial support.
6. References Azzaro, A.J., J. King, J. Kotzuk, D.D. Schoepp, J. Frost and S. Schochet, 1985, Guinea pig striatum as a model of human
220
A. V. Juorio et al. / European Journal of Pharmacology 254 (1994) 213 220
dopamine deamination: the role of monoamine oxidase isozyme ratio, localization, and affinity for substrate in synaptic dopamine metabolism, J. Neurochem. 45, 949. Berry, M.D., A.V. Juorio and I.A. Paterson, 1994, Functional role of monoamine oxidases A and B in the mammalian central nervous system, Prog. Neurobiol. (in press). Boulton, A.A., G. Ivy, B. Davis, D. Durden, A.V. Juorio, P. Yu, A. Medonca, W. Milgram, P.H. Wu and I.A.A. Paterson, 1992, Inhibition of monoamine oxidase-B alters dopamine metabolism in primate caudate, Proc. Am. Soc. Neurochem. 23, 225. Butcher, S.P., I.S. Fairbrother, J.S. Kelly and G.W. Arbuthnott, 1990, Effects of selective monoamine oxidase inhibitors on the in vivo release and metabolism of dopamine in the rat striatum, J. Neurochem. 55, 981. Durden, D.A. and S.R. Philips, 1980, Kinetic measurement of the turnover rates of phenylethylamine and tryptamine in vivo in the rat brain, J. Neurochem. 34, 1725. Durden, D.A., S.R. Philips and A.A. Boulton, 1973, Identification and distribution of /3-phenylethylamine in the rat, Can. J. Biochem. 51, 995. Fowler, C.J. and M. Strolin Benedetti, 1983, The metabolism of dopamine by both forms of monoamine oxidase in the rat brain and its inhibition by cimoxatone, J. Neurochem. 40, 1534. Fowler, C.J. and K.F. Tipton, 1984, On the substrate specificities of the two forms of monoamine oxidase, J. Pharm. Pharmacol. 36, Ill. Garrick, N.A. and D.L. Murphy, 1980, Species differences in the deamination of dopamine and other substrates for monoamine oxidase in brain, Psychopharmacology 72, 27. Hornykiewicz, O., 1973, Dopamine in the basal ganglia. Its role and therapeutic implications (including the clinical use of L-DOPA), Br. Med. Bull. 29, 172. Hovevey-Sion, D., I.J. Kopin, R.W. Stull and D.S. Goldstein, 1989, Effects of monoamine oxidase inhibitors on levels of catechols and homovanillic acid in striatum and plasma, Neuropharmacology 28, 791. Juorio, A.V., 1988, Brain /3-phenylethylamine: localization, pathways, and interrelation with catecholamines, in: Progress in Catecholamine Research, Part B: Central Aspects. Neurology and Neurobiology, Vol. 42B, eds. M. Sandler, A. Dahlstr6m and R.H. Belmaker (Alan Liss, New York) p. 433. Juorio, A.V. and C.W. Kazakoff, 1984, The presence of fl-phenylethylamine, p-tyramine, m-tyramine and tryptamine in ganglia and foot muscle of the garden snail (Helix pomatia), Experientia 40, 549. Kato, T., B. Dong, K. Ishii and H. Kinemuchi, 1986, Brain dialysis: in vivo metabolism of dopamine and serotonin by monoamine oxidase A but not B in the striatum of unrestrained rats, J. Neurochem. 46, 1277. Knoll, J., 1983, Deprenyl (selegiline): the history of its development and pharmacological action, Acta Neurol. Scand. (Suppl.) 95, 57. Knoll, J. and K. Magyar, 1972, Some puzzling effects of some
monoamine oxidase inhibitors, Adv. Biochem. Psychopharmacol. 5, 393. Kwok, R.P.S. and A.V. Juorio, 1986, The concentration of striatal tyramine and dopamine metabolism in diabetic rats and the effect of insulin administration, Neuroendocrinology 43, 590. Levitt, P., J.E. Pintar and X.O. Breakefield, 1982, lmmunocytochemical demonstration of monoamineoxidase B in brain astrocytes and serotonergic neurons, Proc. Natl. Acad. Sci. USA 79, 6385. Marsden, C.D., 1990, Parkinson's disease, Lancet 335, 948. Paterson, I.A., A.V. Juorio and A.A. Boulton, 1990, 2-Phenylethylamine: a modulator of catecholamine transmission in the mammalian central nervous system?, J. Neurochem. 55, 1827. Paterson, I.A., A.V. Juorio, M.D. Berry and M.-Y. Zhu, 1991, Inhibition of monoamine oxidase-B by (-)-deprenyl potentiates neuronal responses to dopamine agonists but does not inhibit dopamine catabolism in the rat striatum, J. Pharmacol. Exp. Ther. 258, 1019. Philips, S.R. and A.A. Boulton, 1979, The effect of monoamine oxidase inhibitors on some arylalkylamines in rat striatum, J. Neurochem. 33, 159. Philips, S.R., A.A. Boulton and B. Rozdilsky, 1978, Evidence for the presence of m-tyramine, p-tyramine, tryptamine and phenylethylamine in the rat brain and several areas of human brain, Biol. Psychiatry 13, 51. Rinne, U.K. and V. Sonninen, 1973, Brain catecholamines and their metabolites in parkinsonian patients, Arch. Neurol. 28, 107. Ross, S.B., 1987, Distribution of the two forms of monoamine oxidase within monoaminergic neurons of the guinea pig brain, J. Neurochem. 48, 609. Ungerstedt, U., 1984, Measurement of neurotransmitter release by intracranial dialysis, in: Measurement of Neurotransmitter Release "in Vivo, ed. C.A. Marsden (John Wiley and Sons, New York) p. 81. Waldmeier, P.C., A. Delini-Stula and L. Maitre, 1976, Preferential deamination of dopamine by an A type monoamine oxidase in rat brain, Naunyn-Schmied. Arch. Pharmacol. 292, 9. Westlund, K.N., R.M. Denney, L.M. Kochersperger, L.M. Rose and C.W. Abell, 1988a, Distinct monoamine oxidase populations in primate brain, Science 230, 181. Westlund, K.N., R.M. Denney, R.M. Rose and C.W. Abell, 1988b, Localization of distinct monoamine oxidase A and monoamine oxidase B cell populations in human brain stem, Neuroscience 25, 439. Wurtman, R.J. and J. Axelrod, 1963, A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol. 12, 2353. Yang, H.-Y.T. and N.H. Neff, 1973, /3-Phenylethylamine: a specific substrate for type B monoamine oxidase of brain, J. Pharmacol. Exp. Ther. 187, 365. Yu, P.H., 1985, Monoamine oxidase, in: Neuromethods. 5. Neurotransmitter Enzymes, eds. A.A. Boulton, G.B. Baker and P.H. Yu (Humana Press, Clifton, NJ) p. 235.
European Journal of Pharmacology 254 (1994) 213-220
0P
Dopamine metabolism in the guinea pig striatum: role of monoamine oxidase A and B Augusto V. Juorio *, I. Alick Paterson, Meng-Yang Zhu Neuropsychiatric Research Unit, Medical Research Building, University of Saskatchewan, Saskatoon, Saskatchewan S7N OW£ Canada (Received 7 October 1993; revised MS received 9 December 1993; accepted 15 December 1993)
Abstract
These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig affects the actions of (-)-deprenyl on dopamine metabolism in whole tissue or in extracellular fluid. The administration of (-)-deprenyl in doses that do not affect monoamine oxidase A activity (1-4 mg kg-1, 2 h) increases striatal 2-phenylethylamine and dopamine concentrations and reduces 3,4-dihydroxyphenylacetic acid. No effects were observed on striatal homovanillic acid, 5-HT and 5-hydroxyindole acetic acid. Inhibition of monoamine oxidase A with clorgyline with doses up to 8 mg kg- 1 (2 h) does not affect striatal 2-phenylethylamine but increases dopamine and 5-HT concentrations and reduces 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid. (-)-Deprenyl (2-4 mg kg -1) did not change the extracellular concentrations of dopamine but the higher dose produced a limited reduction in extracellular 3,4-dihydroxyphenylacetic acid. Inhibition of monoamine oxidase A and monoamine oxidase B with pargyline (75 mg kg -1, 2 h) significantly increased the levels of extracellular dopamine and reduced those of their acid metabolites. These results show that in the guinea pig striatum inhibition of monoamine oxidase B by (-)-deprenyl impairs the metabolism of dopamine in the whole tissue but does not produce a marked increase in extracellular dopamine. Key words: Monoamine oxidase type B; (-)-Deprenyl; Dopamine; Microdialysis; 2-Phenylethylamine; (Guinea pig)
1. Introduction
Dopamine is a substrate for both monoamine oxidase A and monoamine oxidase B in vitro (Fowler and Tipton, 1984) and ( - ) - d e p r e n y l is a potent irreversible inhibitor for the oxidative deamination of dopamine in human brain homogenates (Garrick and Murphy, 1980) and it has been assumed that ( - ) - d e p r e n y l acts by inhibiting dopamine catabolism in vivo (Marsden, 1990). The acute administration of ( - ) - d e p r e n y l , however, does not alter the rat brain levels of dopamine or its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid (Azzaro et al., 1985; Hovevey-Sion et al., 1989; Knoll, 1983; Waldmeier et al., 1976, Paterson
* Corresponding author. Neuropsychiatric Research Unit, A l l 4 MR Building, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7NOW0. Tel. (306) 966-8818, fax (306) 966-8830, e-mail [email protected]. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 3 ) E 0 9 1 2 - C
et al., 1991) nor does it alter extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid or 3-methoxytyramine (Butcher et aI., 1990; Kato et al., 1986) but it significantly inhibits monoamine oxidase B (Knoll and Magyar, 1972). Monoamine oxidase B preferentially metabolizes 2-phenylethylamine (Yang and Neff, 1973) which is also present in the brain but in low concentrations (Durden et al., 1973; Philips et al., 1978; Juorio, 1988). The turnover of 2-phenylethylamine is quite rapid (Durden and Philips, 1980) and its brain concentrations are markedly increased by acute treatment with ( - ) - d e p r e n y l (Philips and Boulton, 1979; Paterson et al., 1991). Electrophysiological studies have demonstrated that 2-phenylethylamine and ( - ) - d e p r e n y l potentiate striatal neuron responses to dopamine agonists suggesting that 2-phenylethylamine may mediate some of the actions of ( - ) deprenyl (Paterson et al., 1990, 1991). The finding that monoamine oxidase A / m o n o a m i n e oxidase B ratio in the guinea pig striatum is similar to that of the human striatum (Azzaro et al., 1985) sug-
214
A. IC Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
gested that the guinea pig striatum is a better model of human dopamine metabolism. These studies were carried out to determine whether the greater abundance of monoamine oxidase B in the guinea pig striatum affects the actions of ( - ) - d e p r e n y l on dopamine metabolism in the whole tissue as well as its extracellular levels. For comparison, the effects of ( - ) - d e p r e n y l and clorgyline on the striatal concentrations of 5-HT and 5-hydroxyindole acetic acid were also determined.
2. Materials and methods
2.1. Animals Male English Short-Hair guinea pigs (300-400 g) were obtained from Charles River (Montreal). They were housed in a 12 h light/dark cycle (lights on at 7.00 a.m.) with free access to food and water for at least 5 days before the start of the experiment. The experimental protocol was approved by the University Committee of Animal Care and Supply.
2.2. Measurement of 2-phenylethylamine, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-HT and 5-hydroxyindole acetic acid Guinea-pigs were stunned and decapitated, the brain was removed and chilled in ice-cold saline and the striatum, consisting mainly of the head of the caudate nucleus and including some of the underlying putamen (approx. between 130 and 170 mg) was dissected out on ice. Tissues were immediately frozen in dry ice and weighed. Two striata from one guinea pig were homogenized in 0.1 N perchloric acid containing E D T A (0.25 mM), sodium metabisulfite (0.1 mM). Isoproterenol (100 n g / m l ) and [2H4] 2-phenylethylamine were added as internal standards. After centrifugation of the tissue homogenate, 250/zl of the supernatant were removed for assay by high pressure liquid chromatography with electrochemical detection of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-HT and 5-hydroxyindole acetic acid (Kwok and Juorio, 1986). The remaining supernatant was used to measure 2-phenylethylamine; for this, the tissue homogenates were derivatized with 5-dimethylamino-L-naphthalene sulphonyl (dansyl) chloride. The resultant derivatives extracted into an organic phase, evaporated to a small volume and separated chromatographically in two different unidimensional systems (Juorio and Kazakoff, 1984). 2-Phenylethylamine estimations were carried out by a high resolution mass spectrometric selected ion monitoring technique. Complete details concerning this procedure have been described (Durden et al., 1973).
2.3. Measurement of monoamine oxidase actit,ity The tissue was homogenised in ice-cold potassium phosphate buffer and centrifuged at 40000 x g for 30 min. The pellet was resuspended and centrifuged again. The activity of monoamine oxidase A was determined using [14C]5-HT bioxalate and the activity of monoamine oxidase B was determined using [~4C]2-phenylethylamine hydrochloride (Wurtman and Axelrod, 1963; Yu, 1985). The radiolabelled substrate was mixed with unlabelled substrate to yield a final concentration of 1 X 10 4 M for 5-HT and 0.25 X 10 4 M for 2-phenylethylamine. The reaction mixture contained potassium phosphate buffer, striatal membranes (40-50 mg protein) and 0.05 /zCi of the substrate. The reaction occurred for 30 min at 37°C and was terminated by the addition of 250 /zl 2 M citric acid. The acid and aldehyde products were extracted into 1 ml of t o l u e n e / e t h y l acetate (1 : 1, v/v); 500/zl of the organic extract was used for liquid scintillation counting to determine acid metabolite production. The protein concentration in the membrane preparation was determined by Lowry's method.
2.4. Intracerebral microdialysis Guinea pigs were anaesthetised with a mixture of urethane and sodium pentobarbital (1 g / k g and 10 m g / k g respectively, i.p.), placed in a stereotaxic frame and body temperature (rectal) was maintained at 37 + 0.2°C. Concentric microdialysis probes (Carnegie Medicin C M A / 1 0 , 3 mm tip) were placed in the striaturn at AP + 2.0 mm, L + 3.7 ram, V - 9 . 0 mm (with respect to bregma, flat skull orientation). The probes were perfused with an artificial cerebrospinal fluid (composition in mM: Na + 150.0; K + 3.5; Ca 2+ 1.2; Mg 2+ 0.8, C1- 128.9; HCO 3 26.0; SO 2- 0.8; PO~ 1.0; glucose 10.0; ascorbate 0.01; pH 7.35 at 25°C) at a rate of 2 / z l / m i n . The probes gave 16-19% recovery of dopamine in vitro. Samples were collected into 40 tzl of 0.1 M perchloric acid containing 3.6 ng of isoproterenol (internal standard for high performance chromatography with electrochemical detection) over a 20 min period. 50/zl of the sample were injected onto the HPLC column and dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were detected at a glassy carbon electrode at - 0 . 7 5 mV. The limits of detection for this system are: dopamine, 2 pg; 3,4-dihydroxyphenylacetic acid, 2 pg; homovanillic acid, 50 pg. The levels in 50 /~1 of a baseline sample were: dopamine, 4-16 pg; 3,4-dihydroxyphenylacetic acid, 3-4 ng; homovanillic acid, 7 - 9 ng. Dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels were stable within 1 h of probe insertion. After 1 h, 3 samples were collected to determine basal levels of
A. 1~. Juorio et aL / European Journal of Pharmacology 254 (1994) 213-220
these substances, drug or vehicle was injected and sampling continued for 3-4 h (Ungerstedt, 1984).
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Fig. 1. Effects of (-)-deprenyl administration on the activity of striatal m o n o a m i n e oxidase A (MAO-A) (open circles) and monoamine oxidase B (MAO-B) (closed circles), expressed as percentage of the control (saline) values. (-)-Deprenyl or saline was administered by i.p. injection 2 h before death. The values indicate means ( + S . E . M . ) obtained from 9-15 experiments. * * P < 0 . 0 1 , Newman-Keuls test with respect to saline.
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The effects of different doses of (-)-deprenyl and clorgyline on monoamine oxidase activity and the striatal concentrations of amines and acid metabolites were analyzed by one-way analysis (ANOVA) of variance and the statistical differences between groups determined by the Newman-Keuls test. The effects of pargyline on the monoamine oxidase activity and the concentrations of monoamines and metabolites were analysed by the Student's t-test. The effects of ( - ) - d e prenyl on urethane-pentobarbital treated animals were analyzed by ANOVA. Statistical analysis of the dialysis experiments were performed by two-way ANOVA ( D r u g - between subjects factor, T i m e - w i t h i n sub-
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Fig. 2. Effects of different doses of (-)-deprenyl on the striatal concentrations of (A) 2-phenylethylamine (PE), (B) dopamine (DA), (C) 3,4-dihydroxyphenylacetic acid (DOPAC), (D) homovanillic acid (HVA), (E) 5-HT and (F) 5-hydroxyindole acetic acid (5-HIAA). ( - )-Deprenyl or saline was administered by i.p. injection 2 h before death. Each point represents the mean + S.E.M. of 11-21 determinations. ** P < 0.01, Newman-Keuls test with respect to saline.
A. I/. Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
216
jects factor). The microdialysis results are shown as a percentage of the mean of the three baseline samples.
2.6. Drugs ( - ) - D e p r e n y l hydrochloride was obtained from Research Biochemicals (Natick, MA, USA), pargyline hydrochloride from Sigma Chemical Company (St. Louis, MO, USA) and clorgyline hydrochloride generously supplied by May & Baker, Essex, UK.
0.033 nmol of [14C]2-phenylethylamine min ~ mg of protein -] (n = 12). The administration of ( - ) - d e p r e nyl only reduced the activity of monoamine oxidase A at a dose of 8 mg k g - ' (F(6,68)= 5.08, P < 0.001). Monoamine oxidase B activity was reduced (F(6,80) = 17.77, P < 0.001) with all doses of ( - )-deprenyl (0.25-8 mg k g - ' , 2 h i.p.) by 67 to 90% (Fig. 1). The administration of pargyline (75 mg kg-1, 2 h i.p.) produced complete inhibition of the activity of monoamine oxidase A and monoamine oxidase B (to 6 and 1%, respectively) (results not shown).
3.2. Effects of monoamine oxidase inhibition on striatal amines and metabolites
3. Results
3.1. The effects of (-)-deprenyl and pargyline on monoamine oxidase activity The striatal activity of monoamine oxidase A was 0.026 + 0.001 nmol of [14C]5-HT rain- 1 mg of protein- a (n = 12) and that of monoamine oxidase B was 0.226 +
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A.V. Juorio et aL / European Journal of Pharmacology 254 (1994) 213-220
(-)-Deprenyl had no effect on homovanillic acid (F(5,81) = 0.76), 5-HT (F(5,81) = 1.04) and 5-hydroxyindole acetic acid (F(5,80)= 0.33). Comparisons using the Newman-Keuls test showed that 2-phenylethylamine concentrations were significantly increased by 1-4 mg kg -1 of (-)-deprenyl and dopamine levels were increased by 4 mg kg-1 while 3,4-dihydroxyphenylacetic acid was decreased after treatment with 2 and 4 mg kg-1 (Fig. 2). The administration of clorgyline (0.5-8 mg kg-1, 2 h i.p.) did not produce significant changes in striatal 2-phenylethylamine concentrations (F(5,48)= 1.09). In contrast, it increased the levels of dopamine (F(5,70) = 7.21, P < 0.001), reduced the concentrations of 3,4dihydroxyphenylacetic acid (F(5,70) = 4.80, P < 0.001) and did not affect homovanillic acid (F(5,70)= 1.81). Comparisons using the Newman-Keuls test showed that dopamine concentrations were significantly increased by 4 and 8 mg kg-t of clorgyline and 3,4-dihydroxyphenylacetic acid levels were reduced by 2-8 mg kg-1 while 5-HT was increased by 8 mg kg- 1 and 5-hydroxyindole acetic acid was decreased after treatment with 4 and 8 mg kg-1 (Fig. 3). In order to evaluate the effect of urethane-pentobarbital anaesthetic used for the microdialysis experiments, rats were treated with saline, (-)-deprenyl, urethane-pentobarbital and urethane-pentobarbital plus (-)-deprenyl (Table 1). Two-way analysis of variance indicated that (-)-deprenyl significantly changed the 2-phenylethylamine levels (F(1,44)= 14.35, P < 0.001) but no interactions between the treatments were observed. (-)-Deprenyl produced no changes in dopamine concentrations (F(1,50) = 2.07), but reduced the concentrations of 3,4-dihydroxyphenylacetic acid (F(1,50) = 27.11, P < 0.001) and homovanillic acid (F(1,50) = 4.98, P < 0.05) (Table 1). Treatment with urethane-pentobarbital had no effect on the levels of 2-phenylethylamine (F(1,44)= 0.15) or dopamine (F(1,50)= 1.96) but changed the concentrations of 3,4-dihydroxyphenylacetic acid (F(1,50) = 7.75, P < 0.01) and homovanillic acid (F(1,50) = 13.49, P < 0.001) (Table 1).
Table 1 Effects of saline, urethane-pentobarbital (1.75 g kg - I and 5 mg kg 1 respectively) and ( - )-deprenyl (4 mg kg- ]) on the striatal concentrations of 2-phenylethylamine (PE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) Treatment
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Fig. 4. Effects of saline (open squares) and (-)-deprenyl (4 mg k g - i ) (closed squares) on the striatal extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The results are expressed as percentages of basal release. (-)-Deprenyl or saline was administered by i.p. injection at 0 time (indicated by arrow). Each point represents the mean+S.E.M, of 6 determinations. There was no effect of ( - ) - d e prenyl on dopamine levels but there were decreases in 3,4-dihydroxyphenylacetic acid (all samples following injection, P < 0.05-P < 0.01) and homovanillic acid levels (40-60 min sample and subsequent samples, P < 0.05-P < 0.01).
Treatment with pargyline (75 mg kg -1, 2 h i.p.) produced significant increases in the striatal concentrations of 2-phenylethylamine, dopamine and 5-HT while the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole acetic acid were markedly reduced (results not shown).
3.3. Effects of (-)-deprenyl on the extracellular concentration of dopamine, 3, 4-dihydroxyphenylacetic acid and homovanillic acid Intraperitoneal administration of saline or (-)-deprenyl (2-4 mg kg -1) had no effect on extracellular dopamine levels (F(2,16) = 2.35) but the 4 mg/kg dose of (-)-deprenyl did alter 3,4-dihydroxyphenylacetic acid and homovanillic acid levels (Fig. 4). There was an overall effect of drug on 3,4-dihydroxyphenylaeetic acid levels (F(2,16)= 4.76, P < 0 . 0 5 ) and a significant
218
A.V. Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
drug-time interaction (F(22,176) = 1.968, P < 0.01). Further analysis showed that there was a significant decrease in 3,4-dihydroxyphenylacetic acid levels in all samples after injection of the 4 mg/kg dose of (-)-deprenyl. There was no overall effect of drug on homovanillic acid levels (F(2,16)= 2.34) but there was a significant drug-time interaction (F(22,176)= 3.38, P < 0.001). Further analysis showed that there was a significant decrease in homovanillic acid levels in the third and subsequent samples (40-60 min onwards) after injection of the 4 mg/kg dose of (-)-deprenyl. Treatment with pargyline (75 mg kg -~) produced significant increases in the extracellular levels of dopamine and significant reductions in the extracellular levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (drug-time interactions: dopamine, F(11,110)= 4.21, P < 0.001; 3,4-dihydroxyphenylacetic acid, F(11,110) = 15.78, P < 0.001; homovanillic acid, F ( l l , l l 0 ) = 19.51, P < 0.001) (Fig. 5). Analysis of simple effects showed that following injection of pargyline dopamine was increased and 3,4-dihydroxyphenylacetic acid was decreased in the second (20-40 min) and subsequent samples and homovanillic acid was decreased in the third (40-60 min) sample onwards.
400
-
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~
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¢o 150. 100¢o 50.
:5 ._=
c
0 r l l J l r J ~ J T l l
HVA
E
150100500 t l l l l l J l J l l l Time (minutes)
4. Discussion
Specific inhibition of monoamine oxidase B by ( - ) deprenyl (Fig. 1) increased levels of 2-phenylethylamine in the guinea pig striatum, and reduced striatal 3,4-dihydroxyphenylacetic acid levels (Fig. 2). Although the levels of dopamine and homovanillic acid were not altered, these results demonstrate that specific inhibition of monoamine oxidase B does alter dopamine metabolism. The administration of clorgyline (2-8 mg kg-~) produced a dose dependent reduction in striatal 3,4-dihydroxyphenylacetic acid and 5-hydroxyindole acetic acid, increases in dopamine and 5-HT and no changes in 2-phenylethylamine (Fig. 3) suggesting that these effects are the consequence of inhibition of monoamine oxidase A. Combined inhibition of monoamine oxidase A and monoamine oxidase B with pargyline produced significant increases in the striatal concentrations of 2-phenylethylamine, dopamine and 5-HT while the levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole acetic acid were markedly reduced. These results demonstrate that in the guinea pig striatum both type A and type B monoamine oxidase are able to metabolize dopamine. This is in agreement with earlier work (Azzaro et al., 1985) that has shown that in the guinea pig striatum, substantial changes in dopamine metabolism are only observed after inhibition of both type A and B monoamine oxidase. These findings further support the contention put forward earlier that in contrast to
Fig. 5. Effects of saline (open squares) and pargyline (75 mg kg i) saline (open squares) on the striatal extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The results are expressed as percentages of basal release. Pargyline or saline was administered by i.p. injection at 0 time (indicated by arrow). Each point represents the mean _+S.E.M. of 6 determinations. Following injection of pargyline, dopamine was increased and 3,4-dihydroxyphenylacetic acid was decreased in the second (20-40 min) and subsequent samples and homovanillic acid was decreased in the third (40-60 min) sample onwards.
the rat where dopamine is mainly metabolized by monoamine oxidase A (Azzaro et al., 1985; Paterson et al., 1991), in the guinea pig dopamine is metabolized by both type A and type B monoamine oxidase. The question that follows is: what is the reason of this difference? It has been reported that in the guinea pig the ratio of monoamine oxidase B/monoamine oxidase A is 3 : 1 and monoamine oxidase B is a significant factor in dopamine metabolism (Azzaro et al., 1985). This is different from the rat striatum where it is reported that the ratio monoamine oxidase B/monoamine oxidase A is 1:3 (Azzaro et al., 1985) and specific inhibition of monoamine oxidase B does not alter dopamine metabolism (Paterson et al., 1991). The determination of the relative abundances of monoamine oxidase A and B, however, is not simple and may be subject to many uncontrolled variables, making the accuracy of these reported ratios suspect (Berry et al., 1994). Im-
A.V. Juorio et al. / European Journal of Pharmacology 254 (1994) 213-220
munohistochemical studies in the rat and primate brain have demonstrated that nigrostriatal neurons contain monoamine oxidase A and that both monoamine oxidase A and monoamine oxidase B are present in extraneuronal locations (Levitt et al., 1982; Westlund et al., 1988a,b; see Berry et al., 1993 for a review). So far, there seem to be no histochemical investigations of the guinea pig brain. In a series of experiments carried out with guinea pig striatal synaptosomes, it has been demonstrated that [14C]dopamine was deaminated by type A monoamine oxidase to the extent of 65% and the remaining 35% by monoamine oxidase B (Ross, 1987). The present findings agree with the importance of monoamine oxidase A for the metabolism of striatal dopamine. The extent to which monoamine oxidase B is acting, however, will have to wait demonstration by a immunohistochemical technique because in these experiments, the differentiation between dopamine, noradrenaline, and 5-HT synaptosomes is dependent on the specificities of the uptake inhibitors (Ross, 1987). In addition, it is not known whether these inhibitors will interfere with the uptake of dopamine into the glial component of the synaptosome preparation. At present, the simplest assumption is that the distribution of monoamine oxidase A and monoamine oxidase B in the guinea pig striatum is not fundamentally different from that in the rat and primate brain. In such a case, irrespective of the ratio of monoamine oxidase A and B activities, the increased dependence on monoamine oxidase B for dopamine metabolism in the guinea pig would suggest that there is more extraneuronal metabolism of dopamine in the guinea pig striatum than in the rat. This hypothesis is supported by the higher levels of homovanillic acid in the guinea pig striatum, which are indicative of more extraneuronal metabolism of dopamine by catechol-O-methyl transferase. Similarly high homovanillic acid levels have been kown to occur in the human striatum (Hornykiewicz, 1973; Rinne and Sonninen, 1973). Interestingly, the levels of homovanillic acid in the macaque caudate are very high (25/zg g-1) and inhibition of monoamine oxidase B results in strong inhibition of dopamine metabolism and marked increases in dopamine levels (Boulton et al., 1992). The extracellular concentration of dopamine in the guinea pig striatum was not changed by the acute administration of ( - ) - d e p r e n y l (Fig. 4) even though the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the guinea pig extracellular fluid were moderately reduced following administration of the higher dose of ( - ) - d e p r e n y l (4 mg kg-1) (Fig. 4). These observations are in agreement with the changes in dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in the striata of anaesthetised animals. In contrast, combined inhibition of both monoamine oxidase A and monoamine oxidase B
219
by pargyline produced a significant increase in the extracellular concentration of dopamine, as well as decreasing metabolite concentrations (Fig. 5). These results demonstrate that although monoamine oxidase B inhibition is sufficient to alter dopamine metabolism, it is not sufficient to produce a marked overflow of dopamine into the extracellular compartment. These findings are similar to those in the rat brain (Butcher et al., 1990). A recent biochemical and electrophysiological study in the rat striatum has shown that specific inhibition of monoamine oxidase B by ( - ) - d e p r e n y l potentiates striatal neuron responses to dopamine receptor agonists without affecting dopamine turnover; these findings are in agreement with the explanation that the potentiation is mediated by increases in 2phenylethylamine (Paterson et al., 1991). In the guinea pig striatum, inhibition of monoamine oxidase B by ( - ) - d e p r e n y l increases 2-phenylethylamine striatal concentration at the same dose range (2-4 mg kg -~) that reduces the concentration of 3,4-dihydroxyphenylacetic acid (Fig. 2). These results suggest that similarly to the primates (Garrick and Murphy, 1980), monoamine oxidase B in the guinea pig striatum plays a role in dopamine metabolism. The reduction in dopamine metabolism which occurred is not sufficient, however, to raise the levels of extracellular dopamine (Fig. 4). If ( - ) - d e p r e n y l potentiates dopamine neuronal responses in the guinea pig, the mechanism could be through the increases that it produces in 2-phenylethylamine concentrations. It is concluded that in the guinea pig striatum, monoamine oxidase B has a significant role in the metabolism of dopamine but that inhibition of monoamine oxidase B does not impair dopamine metabolism sufficiently to increase dopamine overflow from the synapse. The results further agree with the suggestion that the rat brain represents a poor model for the study of human dopamine deamination (Garrick and Murphy, 1980; Fowler and Strolin Benedetti, 1983; Azzaro et al., 1985).
5. Acknowledgements The authors wish to thank Dr. B.A. Davis for the synthesis of deuterated 2-phenylethylamine,Dr. D.A. Durden for supervision of the mass spectrometric analyses, E.P. Zarycki, M. Beszterda and S. Ambrose for technical assistance and Dr. A.A. Boulton for helpful discussion. Saskatchewan Health, Deprenyl Research Ltd., Canada and the Schizophrenia Society of Saskatchewan provided financial support.
6. References Azzaro, A.J., J. King, J. Kotzuk, D.D. Schoepp, J. Frost and S. Schochet, 1985, Guinea pig striatum as a model of human
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