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Raven Press, Ltd., New York 0 1990 lnternational Society for Neurochemistry
Effects of Selective Monoamine Oxidase Inhibitors on the In Vivo Release and Metabolism of Dopamine in the Rat Striatum Steven P. Butcher, *Iain S . Fairbrother, John S. Kelly, and *Gordon W. Arbuthnott Department of Pharmacology, University of Edinburgh Medical School; and *MRC Brain Metabolism Unit, University Department of Pharmacology, University of Edinburgh, Edinburgh, Scotland
Abstract: Brain microdialysis was used to examine the in vivo efflux and metabolism of dopamine (DA) in the rat striatum following monoamine oxidase (MAO) inhibition. Relevant catecholamines and indoleamines were quantified by HPLC coupled with a electrochemical detection system. The MAO-B inhibitor selegiline only affected DA deamination at a dose shown to inhibit partially type A MAO. Alterations in DA and metabolite efflux were not observed when using the MAO-B-selective dose of 1 mg/kg of selegiline. At 10 mg/kg, selegiline reduced the efflux of DA metabolites to -70% of basal values without affecting DA efflux. K+and veratrine-stimulated DA efflux was not affected by selegiline. Experiments using amphetamine and the DA uptake inhibitor nomifensine demonstrated that the effect of selegiline on DA metabolism was unlikely to be mediated either by inhibition of DA uptake or by an indirect effect of its metabolite amphetamine. The possibility that the effect of selegiline is mediated via a nonspecific inhibition of M A 0 is discussed. In contrast, the MAO-A inhibitor clorgyline inhibited basal DA metabolism and increased basal and depolarisation-induced DA efflux. A 1 mg/kg dose of clorgyline reduced basal DA metabolite efflux (40-60% of control val-
ues) without affecting DA efflux. At I0 mg/kg of clorgyline, DA efflux increased to 253 k 19% of basal values, whereas efflux of DA metabolites was reduced to between 15 and 26% of control values. The release of DA induced by K+ and veratrine was not affected by I mg/kg of clorgyline but was increased by -200% following pretreatment with 10 mg/kg of clorgyline. The nonselective M A 0 inhibitor pargyline caused similar but more pronounced alterations in these parameters. For example, using a 75 mg/kg dose of pargyline, efflux of DA increased maximally to 310 ? 20% of basal values, and the release of DA induced by K+ and veratrine was increased by -300% following pargyline pretreatment. These data suggest that DA metabolism is mediated principally by MAOA in the rat striatum. However, under conditions of MAOA inhibition, a component of metabolism mediated by the type B enzyme becomes apparent. Key Words: Monoamine oxidase - Isoenzymes - Clorgyline - Selegiline - Dopamine-Striatum-Brain microdialysis. Butcher S. P. et al. Effects of selective monoamine oxidase inhibitors on the in vivo release and metabolism of dopamine in the rat striatum. J. Neurochem. 55, 98 1-988 ( 1990).
The presence of two forms of the catecholamineand indoleamine-metabolising enzyme monoamine oxidase (MAO) has been demonstrated in rat brain (Johnston, 1968; Houslay et al., 1976). These isoenzymes, termed types A and B, have been classified according to their substrate and inhibitor specificity (Johnston, 1968; Christmas et al., 1972; Knoll and Magyar, 1972; Yang and Neff, 1973, 1974; Schoepp and Azzaro, 1981a). MAO-A preferentially deaminates serotonin [5-hydroxytryptamine (5-HT)I and is more sensitive to inhibition by clorgyline, whereas MAO-B
metabolises /?-phenylethylamine (/?-PEA)and is inhibited more potently by selegiline. The neurotransmitter dopamine (DA) is a substrate for both forms of the enzyme (Yang and Neff, 1974; Houslay et al., 1976; Green et al., 1977). Differences between the cellular localisation of the two isoenzymes have also been reported. In the rat striaturn, MAO-A appears to be localised predominantly within dopaminergic nigrostriatal terminals, whereas MAO-B is found exclusively within intrinsic neurones and dial cells (Demerest et al., 1980; Oreland
Received October 2, 1989; revised manuscript received January 10, 1990; accepted February 16, 1990. Address correspondence and reprint requests to Dr. S. P. Butcher at Department of Pharmacology, University of Edinburgh Medical School, I George Square, Edinburgh EH8 9JZ, Scotland, U.K.
Abbreviations used: DA, dopamine; WPAC, 3,4-dihydroxyphenylacetic acid; ECD, electrochemicaldetection; S-HIAA, 5-hydroxyindoleacetic acid; 5-HT, S-hydroxytryptamine; HVA, homovanillic acid; MAO, monoamine oxidase: 3-MT, 3-methoxytyramine;PEA, phenylethylamine.
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S. P. BUTCHER ET AL.
et al., 1980; Levitt et al., 1982: Francis et al., 1985). Evidence suggestive of a small component of MAO-A activity located within striatal neurones and glial cells has also been reported (Schoepp and Azzaro, 1983; Francis et al., 1985). Studies on the functional significance of the two isoenzymes in DA metabolism have in general monitored the effects of the selective M A 0 inhibitors either on in vitro DA deamination or on brain tissue levels of DA and its metabolites. A role for MAO-A in the metabolism of striatal DA has been clearly demonstrated, but the relevance of MAO-B remains unclear, because MAO-B-selective doses of selegiline did not affect DA metabolism (Braestrup et al., 1975; Waldmeier et al., 1976; Green et al., 1977; Demerest et al., 1980; Kato et al., 1986; but see Yang and Neff, 1974). &PEA metabolism is, however, inhibited under identical conditions (Green et al., 1977: Demerest et al., 1980), and selegiline is reported to potentiate PEA-induced behavioural responses at doses that did not affect catecholamine metabolism (Braestrup et al., 1975). These data suggest that MAO-B is indeed present in the striatum. The apparentally preferential deamination of striatal DA by MAO-A may therefore be due to (a) differences in the cellular localisation of the two MA0 isoenzymes related to DA compartmentalisation, (b) the lower affinity of DA for MAO-B compared with MAO-A (Schoepp and Azzaro, 1 9 8 1 ~ Harsing ; and Vizi, 1984; Azzaro et al., 1985), and (c) the relative proportions of the two isoenzymes within the striatum (Schoepp and Azzaro, I98 1a; Azzaro et al., 1985). In the present study, we have examined the role of the two isoenzymes in the regulations of striatal DA release and metabolism using in vivo brain microdialysis. This technique allows quantification of DA and its metabolites within the extracellular compartment of the brain (Ungerstedt, 1984). The effects of M A 0 inhibitors on the in vivo release of DA were of particular interest, because the therapeutic effects of these drugs may be mediated in part by their ability to enhance dopaminergic neurotransmission. Previous studies on the effect of M A 0 inhibitors on DA efflux have used in vitro preparations (Schoepp and Azzaro, 1982; Harsing and Vizi, I984), and data reporting the in vivo effects of these drugs on DA release have not been published. Thus, Kato et al. ( 1 986) have reported in vivo effects of M A 0 inhibitors on DA metabolites under resting conditions, but the chromatographic system used by these workers did not allow DA levels to be determined. We have in addition studied the effects of M A 0 inhibitors on chemically stimulated DA efflux, i.e., when extracellular concentrations of DA are elevated. Under these conditions, DA metabolism may be directed to other cellular compartments, because the presynaptic uptake mechanism is flooded, thereby allowing metabolism of DA by alternative catabolic routes. A preliminary account of these data has been published (Fairbrother et al., 1987).
MATERIALS AND METHODS Dialysis probe construction Dialysis probes were constructed as described previously (Sandberg et al., 1986). In brief, two lengths of vitreous silica tubing (VSI70/110; Scientific Glass Engineering) were inserted into a 5-6-mm length of dialysis tubing (Cuprophan B4AH; external diameter, 0.3 mm: molecular cutoff, 5,000). The distance between the silica tubes, which act as the fluid inlet and outlet, was adjusted to 3 mm, and a length of plasticcoated tungsten wire (WT3T; Clark Electromedical) was inserted into the dialysis probe to provide rigidity. The two ends of the dialysis tubing were sealed with cyanoacrylate glue. In vitro recovery of catecholamines from dialysis probes was tested by inserting individual probes into a stirred solution containing relevant catecholamines. Recovery of DA and related catecholamines by individual probes was typically 10%.
-
Surgical procedure The concentration of DA and its metabolites in the extracellular fluid was determined using the brain microdialysis method. Dialysis probes were positioned under stereotaxic guidance into the striata [AP 0.5; ML k2.5 (Paxinos and Watson, 1982)] of halothane-anaesthetised rats (Wistar albino; weighing 250-300 g) and were perfused with oxygenated Krebs-bicarbonate buffer (composition, in mM: 124 NaCI, 3.3 KCI, 1.25 K2HP04, 2.4 MgS04, 25 NaHC03, and 2.5 CaCI,, pH 7.4) at 1.25 pl/min for 60 min before collection of dialysates. Dialysates were then collected in 20-min fractions, and catecholamine levels were determined immediately.
M A 0 activity M A 0 activity was measured in striatal synaptosomes using a modification of the method described by Demerest et al. (1980). Rats were anaesthetised using halothane and were injected with M A 0 inhibitors as described in the text. After 120 min, rats were decapitated, and the striata were quickly dissected out. A crude mitochondria1 pellet was prepared following homogenisation of brain tissue in 0.32 Msucrose and 5 mM Tris-HC1 (pH 7.4) buffer. The homogenate was centrifuged at 800 g for 10 min, and the supernatant was recovered and centrifuged at 15,000g for 20 min. The mitochondrial/synaptosomal pellet was resuspended in 50 mM sodium/ potassium phosphate buffer (pH 7.4). One hundred microliters of the tissue preparation was added to 900 p1 of 50 m M sodium/potassium phosphate buffer that had been prewarmed to 37°C. Ten microliters of either DA (final assay concentration, 100 p M ) or 5-HT (final assay concentration, 100 p M ) was added to each assay tube, and incubations were continued at 37°C for 15 min. Assays were terminated by addition of 200 gl of 2 M citric acid, and following centrifugation at 15,000 g for 2 min, catecholamine or indoleamine content was determined using an HPLC with electrochemical detection (ECD) system by injection of 20 p1 of the resulting supernatant.
HPLC/ECD analysis of catecholamines Dialysate concentrations of DA and related metabolites were assayed using reverse-phase HPLC with ECD as described previously (Butcher et al., 1988). Twenty microliters of each dialysate was injected onto a 5-pm (particle size) U1trasphere C-18 column (250 X 4.6 mm). An isocratic mobile phase consisting of 100 mM citrate/sodium acetate buffer
M A 0 INHIBITORS AND DA RELEASE (pH 5.2) containing 5% methanol, 2% tetrahydrofuran, and 100 &ml of octanesulphonic acid (an ion pairing agent) was used to elute the catecholamines and indoleamines. A glassy carbon electrode set at 0.7 V was used to detect catecholamines. The system was calibrated using 2-ng standards injected in 20 pl of Krebs-bicarbonate buffer. Catecholamines were identified with reference to retention time and quantified by peak height analysis. The detection limit of the HPLC/ ECD system was -20 fmol, and the typical dialysate content ofbasal samples was -80 fmol, i.e., signal/noise ratio of 2 3 .
Drug administration Selegiline (0.1-10 mg/kg), pargyline (75 mg/kg), clorgyline (1-10 mg/kg), and amphetamine (4 mg/kg) were administered as single intraperitoneal injections. All other substances were perfused directly into the striatum via the dialysis probe (see figure and table legends for details).
Materials Selegiline was a generous gift from Brittania Pharmaceuticals Ltd. All other drugs were supplied by either Sigma Ltd. or Fisons.
RESULTS Effects of M A 0 inhibitors on deamination of DA and 5-HT In synaptosomal preparations from control animals, DA was deaminated to 3,4-dihydroxyphenylaceticacid (DOPAC) at a rate of 0.85 k 0.04 nmol/min/mg of protein. Pretreatment for 120 rnin with selegiline at 1 mg/kg did not affect DA deamination (102 .+ 10% of control values). However, at 10 mg/kg (120 rnin of pretreatment), selegiline inhibited DOPAC production to 73 f 9% of control values (Table 1). 5-HT was deaminated to 5-hydroxyindoleacetic acid (5-HIAA) TABLE 1. Effects o f M A 0 inhibitors on deamination of DA and 5-HT Deamination (nmol/min/mg of protein) DOPAC
5-HIAA
Control
0.85 ? 0.04
0.83 +_ 0.14
Selegiline 1 mg/kg 10 mg/kg
0.86 i 0.10 0.63fO.11”
0.85 t 0.13 0.60 t 0.10“
Clorgyline 1 mukg 10 rngjkg
0.21 ? 0.106 0.14 -t 0.08’
0.24 t 0. lob 0.03 O.Olb
Pargyline (75 mgjkg)
0.04 -t 0.02’
0.05 -t 0.03b
*
983
at a rate of 0.83 nmol/min/mg of protein in control animals. Selegiline at a dose of 1 mg/kg (120 rnin of pretreatment) did not inhibit 5-HIAA production (102 .+ 15% of control values), whereas at 10 mg/kg, 5-HIAA production was reduced to 72 f 18%of control values (Table 1). Clorgyline at a dose of 1 mg/kg (120 rnin of pretreatment) reduced DOPAC production to 25 2 9% of control values and 5-HIAA production to 29 2 13% ofcontrol values (Table 1). At 10 mg/kg, DOPAC production was reduced to 16 & 8% of control values, and that of 5-HIAA was reduced to 4 1: 2% of control vatues. Pargyline (75 mg/kg; 120 rnin of pretreatment) reduced DOPAC and 5-HIAA production to 4 k 3 and 6 2 3% of control values, respectively (Table 1).
Effects of selegiline on catecholamine and indoleamine efflux The effects of selegiline (0.1- 10 mg/kg, i.p.) on basal efflux of catecholamines and indoleamines are shown in Fig. 1. No significant effects on dialysate DA or 3methoxytyramine (3-MT) levels were observed at any of the selegiline doses tested. Lower doses of selegiline (0.1 and 1 mg/kg) did not affect efflux of either DA or 5-HT metabolites. However, at 10 mg/kg, a significant reduction in DOPAC (62 .+ 1 1 % of basal efflux; p < 0.05), homovanillic acid (HVA) (78 F 8% of basal efflux; p < 0.05), and 5-HIAA (88 f 5% of basal efflux; p < 0.05) levels was noted. Statistical analysis was performed by comparing basal efflux with efflux 120 min following drug injection using a Mann-Whitney U test. The possibility that the effect of selegiline is mediated by its metabolite, amphetamine, was examined by administration of the DA uptake inhibitor nomifensine, which is known to inhibit the reduction in dialysate DOPAC level induced by amphetamine (Butcher et al., 1988). Administration of nomifensine (20 mg/kg, i.p.) increased the DA efflux to 250 +- 18% of basal efflux but did not affect the selegiline-evoked reduction in DOPAC efflux (Fig. 2). The nomifensine-induced increase in DA efflux was similar in animals treated with nomifensine alone and in animals receiving both nomifensine and selegiline (Fig. 2). Effects of clorgyline on catecholamine and indoleamine efflux The effects of clorgyline (1- 10 mg/kg, i.p.) on basal efflux of catecholamines and indoleamines are shown in Fig. 3. At the 1 mg/kg dose, DA and 3-MT efflux tended to increase, although this effect proved to be inconsistent. In contrast, DOPAC, HVA, and 5-HIAA efflux was reduced (maximal decreases in basal efflux, 40 6, 59 +. 7, and 65 k 4%, respectively). The higher clorgyline dose (10 mg/kg) elevated DA efflux maximally to 253 +- 19% of basal efflux, and 3-MT levels were increased to 500 .+ 30% of basal efflux. DA and 5-HT metabolite levels were reduced dramatically under these conditions. The DOPAC content was decreased to 15 k 5 % of basal efflux, that of HVA to 26 _+
M A 0 activity was measured by investigating the deamination of DA and 5-HT to DOPAC and 5-HIAA, respectively, in synaptosomes prepared from animals pretreated with M A 0 inhibitors. Relevant compounds were quantified by HPLC/ECD. Data are mean f SEM values from six independent experiments. Statistical analysis was performed using a Mann-Whitney U test: “p < 0.05, ’p < 0.01 for comparison with the corresponding control.
J Neurochon , Ibl 55. N o 3, 1990
S. P. BUTCHER ET AL.
984 DOPAC
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,
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10 (m)mg/kg, on dialysate efflux of DA, DOPAC. HVA. 3-MT. and 5-HIAA. Results are given as percentages of basal efflux and are mean k SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Selegiline- was given (intraperitoneal injection) following fraction 2 (arrow). Mean i SEM basal efflux in dialysates was as follows: DA, 0.1 1 rt 0.02 pmol/20 min; DOPAC, 19.6 k 1.9 prnol/20 rnin; HVA, 10.9 i0.6 prnol/ 20 rnin; 5-HIAA, 5.6 k 0.6 pmol/20 min; and 3MT, 0.06 k 0.02 pmol/20 min.
8
Fraction Number
f 8% of basal efflux, and that of 5-HIAA to 55 f 4% of basal efflux. Effects of pargyline on catecholamine and indoleamine efflux The effects of pargyline (75 mg/kg, i.p.) on basal efflux of catecholamines and indoleamines are shown in Fig. 3. DA efflux was elevated maximally to 310 f 20% of basal efflux, and that of 3-MT was increased to 670 f 60% of basal efflux. DOPAC efflux was reduced progressively over a 120-min period to 8 f 1% of basal efflux, that of HVA to 14 t- 3% of basal efflux, and that of 5-HIAA to 52 f 3% of basal efflux. The reductions in dialysate DOPAC and HVA levels were significantly greater than that induced by 10 mg/kg of clorgyline ( p < 0.05),as were the increases in dialysate DA and 3-MT levels ( p < 0.05). Statistical analyses DOPAC
DA
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Fraction Number
FIG. 2. Effects of nomifensine (20 mg/kg, i.p.) on alterations in dialysate DA and DOPAC levels induced by selegiline (10 mg/kg, i.p.). Results are given as percentages of basal efflux and are mean SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Drugs were given after collection of fraction 2 (arrow): selegiline (O),nomifensine (A), and nomifensineplus selegiline (m).
*
J. Ncwrochem , Vu/ 55, No. 3, 1990
were performed by comparing the effects of drugs at 120 rnin using a Mann-Whitney U test.
Effects of M A 0 inhibitors on chemically evoked DA release Increasing the K+ concentration in the perfusion medium (the Na+ concentration was reduced accordingly) leads to a calcium-dependent efflux of DA from the striatum (Fairbrother et al., 1990b). The effects of pretreatment with M A 0 inhibitors on this outflow of DA was examined. Neither selegiline (10 mg/kg; 120 rnin of pretreatment) nor clorgyline ( 1 mg/kg; 120 rnin of pretreatment) significantly affected K+-induced DA release (Figs. 4 and 5). In contrast, both pargyline (75 mg/kg; 120 rnin of pretreatment; Fig. 4) and clorgyline (10 mg/kg; 120 rnin of pretreatment; Fig. 5) faciliated the release of DA. The release of DA induced by K+ was increased by 186% following clorgyline pretreatment and by 283% following pargyline pretreatment (Table 2). The reduction in dialysate DOPAC levels induced by K+ (Figs. 4 and 5) was qualitatively similar in animals treated with either M A 0 inhibitor. A similar pattern was observed when other chemical stimulants were used. Thus, the DA efflux produced by veratrine [which induces release of DA via a camerindependent mechanism (Fairbrother et al., 1990a)l was not affected by selegiline (10 mg/kg) or clorgyline (1 mg/kg), whereas both pargyline (75 mg/kg) and clorgyline (10 mg/kg) resulted in an increase in the release of DA induced by veratrine (all 120 rnin of pretreatment; Table 2). The facilitation was again greater in pargyline-treated animals compared with animals treated with clorgyline (10 mdkg): 175% facilitation following clorgyline versus 262% facilitation following pargyline (Table 2; p < 0.05). The release of
985
M A 0 INHIBITORS AND DA RELEASE lr
4 r
FIG. 3. Effects of clorgyline, 1 (0)and 10 (A) mg/kg, and pargyline. 75 mg/kg (m), on dialysate efflux of DA, DOPAC, HVA, 3-MT. and 5-HIAA. Results are given as percentages of basal efflux and are mean 2 SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Drugs (injected intraperitoneally) were given following fraction 2 (arrow). Mean t SEM basal efflux in dialysates was as follows: DA, 0.09 k 0.01 pmol/20 min; DOPAC, 15.5 t 0.83 pmol/20 min; HVA, 9.7 t 1.Ipmol/20 min; 5-HIAA. 4.8 t 0.6 pmol/20 min; and 3-MT, 0.06 t 0.01 pmol/20 min.
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DA induced by amphetamine was unaffected by prior selegiline pretreatment (10 mg/kg; 120 min of pretreatment; Table 2 ) but was increased by pretreatment with 10 mg/kg of clorgyline (Table 2; 193%facilitation following clorgyline; p < 0.0 1).
DISCUSSION The present data demonstrate that the MAO-B inhibitor selegiline at a dose of 10 mg/kg alters the basal overflow of both DA and 5-HT metabolites. These results contrast with those of Kato et al. (1986), although the use of unanaesthetised animals in the latter study may explain this discrepancy. Because this effect was insensitive to nomifensine, a DA uptake inhibitor that blocks the dialysate response to amphetamine (Butcher
-
DA
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x
-
30
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x
10
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ib
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w
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et al., 1988), a direct action of selegiline is suggested rather than an effect of its metabolite amphetamine (Karoum et al., 1982). The possibility that selegiline acts as a DA uptake inhibitor (Lai et al., 1980; Azzaro and Demerest, 1982) would also appear unlikely, because selegiline, unlike nomifensine, does not inhibit amphetamine-induced DA efflux (Table 2). It should not, however, be assumed that this effect is mediated solely by inhibition of MAO-B. High doses, e.g., 10 mg/kg, of selegiline are reported to inhibit partially MAO-A activity as assessed by an inhibition of the deamination of the preferred MAO-A substrate, 5-HT (Green et al., 1977; Demerest et al., 1980). A similar profile was obtained in the present study (see Table l), and the small but consistent fall in basal overflow of 5-HIAA noted in dialysis experiments using 10 mg/kg
0.2 O
1
3
5
7
9
11
1
3
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7
04 02
9 1 1
Fraction Number
1
3
5
7
9
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1
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FIG. 4. Effects of selegiline (10 mg/kg, i.p.) and pargyline (75mg/
FIG. 5. Effects of clorgyline (1 and 10 mg/kg, i.p.) on K+-induced
kg, i.p.) on K+evokedalterations in dialysate DA and DOPAC levels. Results are given as percentages of basal efflux and are mean f SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Selegiline and pargyline were given following collection of fraction 2 (arrow a), and K+ (90 mM) was infused via the dialysis probe for 20 min after collection of fraction 8 (arrow b): K+ (A), Kf plus selegiline (O),and Kf plus pargyline (m).
alterations in dialysate DA and DOPAC levels. Results are given as percentages of basal efflux and are mean ? SEM (bars) values from six independent experiments. Each fraction representsa 20min collection period. Clorgyline was injected after collection of fraction 2 (arrow a), and K+ (90 mnn) was infused via the dialysis probe for 20 min following collection of fraction 8 (arrow b): K+ (A), K+ plus 1 mg/kg of clorgyline (O), and K+ plus 10 mg/kg of clorgyline (m).
J. Neurochem.. Vol. 55, No. 3. 1990
S. P. BUTCHER ET AL.
986
TABLE 2. Effect o f M A 0 inhibitors on K'. and veratrine-evoked DA eflux Veratrine K+ (90 mM) (100 &nl) Control
1.13+0.13
2.65i0.36
Clorgyline 1 mg/kg 10 mg/kg
1.35 f 0.37 3.35 + 0.55 2.10 t 0.20" 4.80 f 0.70"
Amphetamine (4 mg/kg, i.p.) 1.5kO.2
2.9 f 0.60"
Pargyline (75 mg/kg) 3.20 f 0.68"." 6.93 f 1.2"," Selegiline (10 mdkg) 1.26
* 0.45
3.15 f 0.58
1.3 t 0.2
Endogenous DA levels in dialysates were measured by HPLC/ ECD. Results are mean f SEM values for the release of DA induced (in picomoles) above M A 0 pretreatment levels by K+ or veratrine during a 20-min period of stimulant perfusion (n = 6). Amphetamine data represent DA elllux over a 60-min postinjection period. Statistical analysis was performed using a Mann-Whitney U-test: "p < 0.02 for comparison of release in the presence and absence of the M A 0 inhibitor, ' p < 0.05 for comparison of clorgyline versus pargyline facilitation.
of selegiline suggests that the selectivity of this drug cannot be assumed. Lower, more selective doses of selegiline did not affect catecholamine or indoleamine metabolism. These data suggest that selegiline (1 0 mg/ kg) may exert its action via a nonselective inhibition of MAO-A. Data obtained using selegiline should be compared with results obtained when clorgyline was used to inhibit MAO-A or pargyline was used to impair both forms of MAO. Metabolite responses to these inhibitors were qualitatively similar to those reported by Kato et al. (1986). At an MAO-A-selective clorgyline dose of 1 mg/kg, dramatic reductions (40-65% of basal efflux) in the dialysate content of DOPAC, HVA, and 5-HIAA were observed. Increasing the clorgyline dose to 10 mg/ kg leads to even greater reductions in metabolite levels. Pargyline (75 mg/kg) inhibited basal overflow of DOPAC and HVA to a greater extent than 10 mg/kg of clorgyline, whereas the reduction in 5-HIAA content was similar in both cases. These data may be interpreted in two ways: (a) Under conditions of total MAO-A blockade, an MAO-B-mediated component of catecholamine metabolism becomes apparent, or (b) the 10 mg/kg dose of clorgyline does not fully inhibit MAO-A. The finding that 5-HT deamination was essentially abolished when using 10 mg/kg of clorgyline argues against the latter possibility (see Table 1). It has been suggested previously that MAO-B may play a role in catecholamine metabolism when MAO-A is inhibited (Green et al., 1977; Schoepp and Azzaro, 1982, 1983; Azzaro et al., 1985). The present study lends support to this proposal because DA, in contrast to 5HT, can still be metabolised to some extent following pretreatment with 10 mg/kg of clorgyline (Table 1). These data do, however, suggest that DA metabolism in striaturn is mediated predominately by MAO-A.
J Nrurochem , Vol $5,N o 3. 1990
Two further points merit consideration when comparing the effects of clorgyline and pargyline. First, M A 0 inhibitors were found to elevate extracellular levels of DA, and this may reflect the enhancement of dopaminergic neurotransmission mediating the therapeutic effects of these compounds. Basal efflux of DA was, however, not altered by the 1 mg/kg dose of clorgyline, even though metabolite levels are suppressed. Increasing the clorgyline dose to 10 mg/kg elevated the extracellular DA level but not to the same degree as that observed with pargyline. This suggests that although the dialysate DA content increases following MAO-A inhibition, total blockade of both forms of M A 0 is required to elevate maximally extracellular DA levels. These data are consistent with results reported by both Green et al. (1977), who demonstrated that behavioural effects of M A 0 inhibitors are only observed following complete inhibition of both forms of the enzyme, and Harsing and Vizi (1984), who showed using in vitro experiments that clorgyline reduces DOPAC efflux before DA release is affected. A second point of interest concerns extracellular levels of 3-MT. Neither 1 mg/kg of clorgyline nor selegiline (10 mg/kg) altered basal 3-MT overflow, whereas 10 mg/kg of clorgyline elevated 3-MT efflux. However, 3-MT levels are again elevated to a greater extent following pargyline pretreatment. Thus, the catechol-0-methyltransferase catabolic pathway becomes significant following blockade of MAO-A, presumably because 3-MT cannot be converted to HVA. The specificity of this effect suggests that MAO-A is preferentially involved in the metabolism of 3-MT, and this may reflect extraneuronal metabolism because catechol-0-methyltransferase activity is thought to be located outside dopaminergic terminals in the striaturn (Kaplan et al., 1979). The more pronounced effect of pargyline does, however, suggest a role for MAO-B in 3-MT deamination in the absence of MAO-A activity. These data, therefore, support the proposed functional role of M A 0 outside dopaminergic terminals under certain conditions (see Schoepp and Azzaro, 1982, 1983). One possibility not explored by Kato et al. (1986) was that an action of MAO-B is only apparent following release of DA. Thus, under resting conditions, extracellular DA is thought to be recaptured by uptake mechanisms. Following depolarisation, extracellular DA levels rise dramatically and may perhaps overload the neuronal uptake mechanism, thereby allowing access to nonneuronal sites of metabolism. GIial uptake sites for DA and 5-HT have been demonstrated (Pelton et al., 1981; Semeloff and Kimelberg, 1985), and MA0 activity is reported to reside in intrinsic striatal neurones and glial elements (Pelton et al., 1981; Levitt et al., 1982; Schoepp and Azzaro, 1983; Francis et ai., 1985). We have examined this possibility by elevating the extracellular DA level using several stimuli. Infusion of 90 mM Kf via the dialysis probe led to
M A 0 INHIBITORS AND DA RELEASE a marked increase in DA efflux and a reduction in dialysate DOPAC levels. Pretreatment with either selegiline (10 mg/kg) or a low clorgyline dose (1 mg/kg) did not affect the release of DA induced by K+. In contrast, pretreatment with 10 mg/kg of clorgyline or 75 mg/kg of pargyline markedly enhanced K+-induced DA release. A similar profile was obtained when DA effluxwas stimulated with veratrine, and these data are therefore consistent with the in vitro experiments of Harsing and Vizi ( 1984). Pargyline was again found to be a more effective enhancer of efflux than 10 mg/kg of clorgyline, a result suggesting that MAO-B may be involved in the deamination of DA in the absence of MAO-A activity. Thus, maximal facilitation of stimulated release is only observed following inhibition of both forms of MAO. These data suggest that under both resting and depolarising conditions DA metabolism principally involves MAO-A. However, under conditions of MAOA inhibition, an MAO-B component of catecholamine metabolism becomes apparent. Two mechanisms may underlie the effects of MAO-A inhibition on extracellular DA levels: (a) MAO-A inhibition may prevent DA catabolism following release, or (b) MAO-A inhibition may increase the pool of DA available for release. Because nomifensine does not affect extracellular DOPAC levels, the first possibility is unlikely unless metabolism of released DA is switched to extraneuronal sites when the neuronal uptake system is blocked. If this is the case, the finding that selegilinedoes not affect nomifensine-induced DA efflux suggests that MAO-B is not involved in DA deamination following blockade of the neuronal DA transporter. An alternative explanation has been suggested by Zetterstrom et al., (1988), who proposed that MA0 inhibition increases the synaptic pool of DA available for release. Although this hypothesis is also consistent with the present data, Schoepp and Azzaro (198 I b) have reported that DA synthesis is inhibited by clorgyline (perhapsby feedback inhibition of tyrosine hydroxylase). These workers suggested that the size of the releasable pool could be increased by preventing the deamination of DA once it had been taken up into dopaminergic terminals (Liccione and Azzaro, 1988). Acknowledgment: S. P. Butcher was a n MRC Training Fellow, and I. S. Fairbrother was supported by studentships from t h e MRC a n d Edinburgh University Faculty of Medicine. W e would like to t h a n k Avril Davidson for preparing synaptosomal preparations and Audrey Kerr for typing the manuscript.
REFERENCES Azzaro A. J. and Demerest K. T. (1982) Inhibitory effect of type A and type B monoamine oxidase inhibitory on synaptosomal accumulation of ['Hldopamine: a reflection of antidepressant potency. Biochem. Pharmacol. 31,2 195-2 197.
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Avaro A. J., King J., Kotzuk J., Schoepp D. D., Frost J., and Schochet S. (1985) Guinea-pig striatum as a model of human dopamine deamination: the role of monoamine oxidase isozyme ratio, localisation, and affinity for substrate in synaptic dopamine metabolism. J. Neurochem. 45,949-956. Braestrup C., Andersen H., and Randrup A. (1 975) The monoamine oxidase B inhibitor deprenyl potentiates phenylethylamine behavior. Eur. J. Pharmacol. 34, 18I - 187. Butcher S. P., Fairbrother I. S., Kelly J. S., and Arbuthnott G. W. (1988) Amphetamine-induced dopamine release in the rat striatum: an in vivo microdialysis study. J. Neurochem. 50, 346355. Christmas A. J., Coulson C. J., Maxwell D. P., and kddell D. (1972) A comparison of the pharmacological and biochemical properties of substrate-selective monoamine oxidase inhibitors. Br. J. Pharmacol. 45,490-503. Demerest K. T., Smith D. J., and Azzaro A. J. (1980) The presence of the type A form of monoamine oxidase within nigrostriatal dopamine containing neurons. J. Pharmacol. Exp. Ther. 215, 461-468. Fairbrother I. S., Butcher S. P., Kelly J. S., and Arbuthnott G. W. (1987) A physiological role for MAO-B in the metabolism of dopamine? (Abstr) 3r. J. Phannacol. 92,612P. Fairbrother I. S., Arbuthnott G. W., Kelly J. S., and Butcher S. P. (19904 In vivo mechanisms underlying dopamine release from rat nigrostriatal terminals: I. Studies using veratrine and ouabain. J. Neurochem. 54, 1834- 1843. Fairbrother I. S., Arbuthnott G. W., Kelly J. S., and Butcher S. P. (1990b) In vivo mechanisms underlying dopamine release from rat nigrostriatal terminals: 11. Studies using potassium and tyramine. J. Neurochem. 54, 1844-1 85 I . Francis A,, Pierce L. B., and Roth J. A. (1985) Cellular localisation of M A 0 A and B in brain. Evidence from kainic acid lesions in striatum. Brain Rex 334, 59-64. Green A. R., Mitchell B. D., Tordoff A. F. C., and Youdim M. B. H. (1977) Evidence for dopamine deamination by both type A and type B monoamine oxidase in rat brain in vivo and for the degree of inhibition of enzyme necessa.ry for increased functional activity of dopamine and 5-hydroxytryptamine. Br. J. Pharmacol. 60,343-349. Harsing L. A. and Vizi E. S. (1984) Release of endogenous dopamine from rat isolated striatum: effect of clorgyline and (-)-deprenyl. Br. J. Pharmacol. 83, 741-749. Houslay M. D., Tipton K. F., and Youdim M. B. H. (1976) Multiple forms of monoamine oxidase: fact and artefact. Lije Sci. 19, 467-478. Johnston J. P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem. Pharmacol. 17, 1285-1297. Kaplan G. P., Hartman B. K., and Creveling C. E. (1979) Immunohistochemical demonstration of catechol-O-methyl transferase in mammalian brain. Brain Res. 167, 241-250. Karoum F., Chuang L. W., Eisler T., Calne D. B., Liebovitz M. R., Quitkin F. M., Klein D. F., and Wyatt R. J. (1982) Metabolism of (-)-deprenyl to amphetamine and metamphetamine may be responsible for deprenyl's therapeutic benefit: a biochemical assessment. Neurology 32, 503-509. Kato T., Dong B., Ishii K., and Kinemuchi H. (1986) Brain dialysis: in vivo metabolism of dopamine and serotonin by monoamine oxidase A but not B in the striatum of unrestrained rats. J. Neilrochem. 46, 1277-1282. Knoll J. and Magyar K. (1972) Some puzzling pharmacologic effects of monoamine oxidase inhibitors. Adv. Biochem. Psychopharmacol. 5, 393-408. Lai J. C. K., Leung T. K. C., Guest J. F., and Davison A. N. (1980) The monoamine oxidase inhibitors clorgyline and deprenyl also affect uptake of dopamine, noradrenaline and serotonin by rat brain synaptosomal preparations. Biochem. Pharmacol. 29, 2763-2767. Levitt P., Pintar J., and Breakefield X. ( 1 982) Immunocy-tochemical
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demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurones. Proc. Natl. Acud. Sci. USA 79, 63858389. Liccione J. and Azzaro A. J. (1988) Different roles for type A and type B monoamine oxidase in regulating synaptic dopamine at D- 1 and D-2 receptors associated with adenosine-3'-5'-cyclic monophosphate (cyclic AMP) formation. Naunjw Schmiedebergs Arch. Pharmacol. 337, 151-158. Oreland L., Fowler C . J., Carlsson A,, and Magnusson T. (1980) Monoamine oxidase-A and -B activity in the rat brain after hemitransection. Life Sci. 26, 139-146. Paxinos G. and Watson C. ( 1 982) The Rut Bruin in Siereotaxic Coordinates. Academic Press, Sydney. Pelton E. W.. Ktmelberg H . K., Shepherd S. V., and Bourke R. S. ( 1 98 I ) Dopamine and noradrenaline uptake and metabolism by astroglial cells in culture. L@ Sci.28, 1655-1663. Sandberg M., Butcher S. P., and Hagberg H. (1986) Extracellular overflow of neuroactive amino acids during severe insulin induced hypoglycemia: in vivo microdialysis of the rat hippocampus. J. Neurochem. 47, 178-184. Schoepp D. D. and Azzaro A. J. (19810) Specificity of endogenous substrates for types A and B monoamine oxidase in rat striatum. J . Neurochem. 36,2025-203 1. Schoepp D. D. and Azzaro A. J. (19816) Alterations of dopamine synthesis subsequent to selective type A monoamine oxidase inhibition. J. Neurochem. 37, 527-530. Schoepp D. D. and Azzaro A. J. (1982) The role of type A and type B monoamine oxidase in the metabolism of released
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['Hldopamine from rat striatal slices. Biochem. Pharmacol. 31, 296 1-2968. Schoepp D. D. and Azzaro A. J. (1983) Effects of intrastriatal kainic acid injection on ['Hldopamine metabolism in rat striatal slices: evidence for postsynaptic glial cell metabolism by both the A and B forms of monoamine oxidase. J. Nezirochem. 40, 13401348. Semeloff D. and Kimelberg H. K. (1985) Autoradiography of high affinity uptake of catecholamine by primary astrocyte cultures. Brain Res. 348, 125-1 36. Ungerstedt U. (1984) Measurements of neurotransmitter release by intracranial dialysis, in Meastireincnt qfNeitrorransmitierReleuse In Vivo (Marsden C. A,, ed), pp. 81-105. John Wiley and Sons, Chichester. Waldmeier P. C., Delini-Stula A.. and Maitre L. (1976) Preferential deamination of dopamine by an A type monoamine oxidase in rat brain. Naunyn Schrniedebergs Arch. Pharmacol. 292,9- 14. Yang H. Y. T. and Neff N. H. (1973) (3-Phenylethylamine: a specific substrate for type B monoamine oxidase of brain. J. Pharmacol. Exp. ?'her. 187,365-37 1. Yang H. Y. T. and Neff N. H. (1974) The monoamine oxidase of brain: selective inhibition with drugs and the consequences for the metabolism of biogenic amine. J. Pharmacol. ESP. Ther. 189, 733-740. Zetterstrom T., Sharp T., Collin A. K., and Ungerstedt U. (1988) In vivo measurements of extracellular dopamine and DOPAC in the rat striatum after various dopamine releasing drugs; implications for the origin ofextracellular DOPAC. Ezir. J. Pharmacol. 148, 327-334.
Raven Press, Ltd., New York 0 1990 lnternational Society for Neurochemistry
Effects of Selective Monoamine Oxidase Inhibitors on the In Vivo Release and Metabolism of Dopamine in the Rat Striatum Steven P. Butcher, *Iain S . Fairbrother, John S. Kelly, and *Gordon W. Arbuthnott Department of Pharmacology, University of Edinburgh Medical School; and *MRC Brain Metabolism Unit, University Department of Pharmacology, University of Edinburgh, Edinburgh, Scotland
Abstract: Brain microdialysis was used to examine the in vivo efflux and metabolism of dopamine (DA) in the rat striatum following monoamine oxidase (MAO) inhibition. Relevant catecholamines and indoleamines were quantified by HPLC coupled with a electrochemical detection system. The MAO-B inhibitor selegiline only affected DA deamination at a dose shown to inhibit partially type A MAO. Alterations in DA and metabolite efflux were not observed when using the MAO-B-selective dose of 1 mg/kg of selegiline. At 10 mg/kg, selegiline reduced the efflux of DA metabolites to -70% of basal values without affecting DA efflux. K+and veratrine-stimulated DA efflux was not affected by selegiline. Experiments using amphetamine and the DA uptake inhibitor nomifensine demonstrated that the effect of selegiline on DA metabolism was unlikely to be mediated either by inhibition of DA uptake or by an indirect effect of its metabolite amphetamine. The possibility that the effect of selegiline is mediated via a nonspecific inhibition of M A 0 is discussed. In contrast, the MAO-A inhibitor clorgyline inhibited basal DA metabolism and increased basal and depolarisation-induced DA efflux. A 1 mg/kg dose of clorgyline reduced basal DA metabolite efflux (40-60% of control val-
ues) without affecting DA efflux. At I0 mg/kg of clorgyline, DA efflux increased to 253 k 19% of basal values, whereas efflux of DA metabolites was reduced to between 15 and 26% of control values. The release of DA induced by K+ and veratrine was not affected by I mg/kg of clorgyline but was increased by -200% following pretreatment with 10 mg/kg of clorgyline. The nonselective M A 0 inhibitor pargyline caused similar but more pronounced alterations in these parameters. For example, using a 75 mg/kg dose of pargyline, efflux of DA increased maximally to 310 ? 20% of basal values, and the release of DA induced by K+ and veratrine was increased by -300% following pargyline pretreatment. These data suggest that DA metabolism is mediated principally by MAOA in the rat striatum. However, under conditions of MAOA inhibition, a component of metabolism mediated by the type B enzyme becomes apparent. Key Words: Monoamine oxidase - Isoenzymes - Clorgyline - Selegiline - Dopamine-Striatum-Brain microdialysis. Butcher S. P. et al. Effects of selective monoamine oxidase inhibitors on the in vivo release and metabolism of dopamine in the rat striatum. J. Neurochem. 55, 98 1-988 ( 1990).
The presence of two forms of the catecholamineand indoleamine-metabolising enzyme monoamine oxidase (MAO) has been demonstrated in rat brain (Johnston, 1968; Houslay et al., 1976). These isoenzymes, termed types A and B, have been classified according to their substrate and inhibitor specificity (Johnston, 1968; Christmas et al., 1972; Knoll and Magyar, 1972; Yang and Neff, 1973, 1974; Schoepp and Azzaro, 1981a). MAO-A preferentially deaminates serotonin [5-hydroxytryptamine (5-HT)I and is more sensitive to inhibition by clorgyline, whereas MAO-B
metabolises /?-phenylethylamine (/?-PEA)and is inhibited more potently by selegiline. The neurotransmitter dopamine (DA) is a substrate for both forms of the enzyme (Yang and Neff, 1974; Houslay et al., 1976; Green et al., 1977). Differences between the cellular localisation of the two isoenzymes have also been reported. In the rat striaturn, MAO-A appears to be localised predominantly within dopaminergic nigrostriatal terminals, whereas MAO-B is found exclusively within intrinsic neurones and dial cells (Demerest et al., 1980; Oreland
Received October 2, 1989; revised manuscript received January 10, 1990; accepted February 16, 1990. Address correspondence and reprint requests to Dr. S. P. Butcher at Department of Pharmacology, University of Edinburgh Medical School, I George Square, Edinburgh EH8 9JZ, Scotland, U.K.
Abbreviations used: DA, dopamine; WPAC, 3,4-dihydroxyphenylacetic acid; ECD, electrochemicaldetection; S-HIAA, 5-hydroxyindoleacetic acid; 5-HT, S-hydroxytryptamine; HVA, homovanillic acid; MAO, monoamine oxidase: 3-MT, 3-methoxytyramine;PEA, phenylethylamine.
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et al., 1980; Levitt et al., 1982: Francis et al., 1985). Evidence suggestive of a small component of MAO-A activity located within striatal neurones and glial cells has also been reported (Schoepp and Azzaro, 1983; Francis et al., 1985). Studies on the functional significance of the two isoenzymes in DA metabolism have in general monitored the effects of the selective M A 0 inhibitors either on in vitro DA deamination or on brain tissue levels of DA and its metabolites. A role for MAO-A in the metabolism of striatal DA has been clearly demonstrated, but the relevance of MAO-B remains unclear, because MAO-B-selective doses of selegiline did not affect DA metabolism (Braestrup et al., 1975; Waldmeier et al., 1976; Green et al., 1977; Demerest et al., 1980; Kato et al., 1986; but see Yang and Neff, 1974). &PEA metabolism is, however, inhibited under identical conditions (Green et al., 1977: Demerest et al., 1980), and selegiline is reported to potentiate PEA-induced behavioural responses at doses that did not affect catecholamine metabolism (Braestrup et al., 1975). These data suggest that MAO-B is indeed present in the striatum. The apparentally preferential deamination of striatal DA by MAO-A may therefore be due to (a) differences in the cellular localisation of the two MA0 isoenzymes related to DA compartmentalisation, (b) the lower affinity of DA for MAO-B compared with MAO-A (Schoepp and Azzaro, 1 9 8 1 ~ Harsing ; and Vizi, 1984; Azzaro et al., 1985), and (c) the relative proportions of the two isoenzymes within the striatum (Schoepp and Azzaro, I98 1a; Azzaro et al., 1985). In the present study, we have examined the role of the two isoenzymes in the regulations of striatal DA release and metabolism using in vivo brain microdialysis. This technique allows quantification of DA and its metabolites within the extracellular compartment of the brain (Ungerstedt, 1984). The effects of M A 0 inhibitors on the in vivo release of DA were of particular interest, because the therapeutic effects of these drugs may be mediated in part by their ability to enhance dopaminergic neurotransmission. Previous studies on the effect of M A 0 inhibitors on DA efflux have used in vitro preparations (Schoepp and Azzaro, 1982; Harsing and Vizi, I984), and data reporting the in vivo effects of these drugs on DA release have not been published. Thus, Kato et al. ( 1 986) have reported in vivo effects of M A 0 inhibitors on DA metabolites under resting conditions, but the chromatographic system used by these workers did not allow DA levels to be determined. We have in addition studied the effects of M A 0 inhibitors on chemically stimulated DA efflux, i.e., when extracellular concentrations of DA are elevated. Under these conditions, DA metabolism may be directed to other cellular compartments, because the presynaptic uptake mechanism is flooded, thereby allowing metabolism of DA by alternative catabolic routes. A preliminary account of these data has been published (Fairbrother et al., 1987).
MATERIALS AND METHODS Dialysis probe construction Dialysis probes were constructed as described previously (Sandberg et al., 1986). In brief, two lengths of vitreous silica tubing (VSI70/110; Scientific Glass Engineering) were inserted into a 5-6-mm length of dialysis tubing (Cuprophan B4AH; external diameter, 0.3 mm: molecular cutoff, 5,000). The distance between the silica tubes, which act as the fluid inlet and outlet, was adjusted to 3 mm, and a length of plasticcoated tungsten wire (WT3T; Clark Electromedical) was inserted into the dialysis probe to provide rigidity. The two ends of the dialysis tubing were sealed with cyanoacrylate glue. In vitro recovery of catecholamines from dialysis probes was tested by inserting individual probes into a stirred solution containing relevant catecholamines. Recovery of DA and related catecholamines by individual probes was typically 10%.
-
Surgical procedure The concentration of DA and its metabolites in the extracellular fluid was determined using the brain microdialysis method. Dialysis probes were positioned under stereotaxic guidance into the striata [AP 0.5; ML k2.5 (Paxinos and Watson, 1982)] of halothane-anaesthetised rats (Wistar albino; weighing 250-300 g) and were perfused with oxygenated Krebs-bicarbonate buffer (composition, in mM: 124 NaCI, 3.3 KCI, 1.25 K2HP04, 2.4 MgS04, 25 NaHC03, and 2.5 CaCI,, pH 7.4) at 1.25 pl/min for 60 min before collection of dialysates. Dialysates were then collected in 20-min fractions, and catecholamine levels were determined immediately.
M A 0 activity M A 0 activity was measured in striatal synaptosomes using a modification of the method described by Demerest et al. (1980). Rats were anaesthetised using halothane and were injected with M A 0 inhibitors as described in the text. After 120 min, rats were decapitated, and the striata were quickly dissected out. A crude mitochondria1 pellet was prepared following homogenisation of brain tissue in 0.32 Msucrose and 5 mM Tris-HC1 (pH 7.4) buffer. The homogenate was centrifuged at 800 g for 10 min, and the supernatant was recovered and centrifuged at 15,000g for 20 min. The mitochondrial/synaptosomal pellet was resuspended in 50 mM sodium/ potassium phosphate buffer (pH 7.4). One hundred microliters of the tissue preparation was added to 900 p1 of 50 m M sodium/potassium phosphate buffer that had been prewarmed to 37°C. Ten microliters of either DA (final assay concentration, 100 p M ) or 5-HT (final assay concentration, 100 p M ) was added to each assay tube, and incubations were continued at 37°C for 15 min. Assays were terminated by addition of 200 gl of 2 M citric acid, and following centrifugation at 15,000 g for 2 min, catecholamine or indoleamine content was determined using an HPLC with electrochemical detection (ECD) system by injection of 20 p1 of the resulting supernatant.
HPLC/ECD analysis of catecholamines Dialysate concentrations of DA and related metabolites were assayed using reverse-phase HPLC with ECD as described previously (Butcher et al., 1988). Twenty microliters of each dialysate was injected onto a 5-pm (particle size) U1trasphere C-18 column (250 X 4.6 mm). An isocratic mobile phase consisting of 100 mM citrate/sodium acetate buffer
M A 0 INHIBITORS AND DA RELEASE (pH 5.2) containing 5% methanol, 2% tetrahydrofuran, and 100 &ml of octanesulphonic acid (an ion pairing agent) was used to elute the catecholamines and indoleamines. A glassy carbon electrode set at 0.7 V was used to detect catecholamines. The system was calibrated using 2-ng standards injected in 20 pl of Krebs-bicarbonate buffer. Catecholamines were identified with reference to retention time and quantified by peak height analysis. The detection limit of the HPLC/ ECD system was -20 fmol, and the typical dialysate content ofbasal samples was -80 fmol, i.e., signal/noise ratio of 2 3 .
Drug administration Selegiline (0.1-10 mg/kg), pargyline (75 mg/kg), clorgyline (1-10 mg/kg), and amphetamine (4 mg/kg) were administered as single intraperitoneal injections. All other substances were perfused directly into the striatum via the dialysis probe (see figure and table legends for details).
Materials Selegiline was a generous gift from Brittania Pharmaceuticals Ltd. All other drugs were supplied by either Sigma Ltd. or Fisons.
RESULTS Effects of M A 0 inhibitors on deamination of DA and 5-HT In synaptosomal preparations from control animals, DA was deaminated to 3,4-dihydroxyphenylaceticacid (DOPAC) at a rate of 0.85 k 0.04 nmol/min/mg of protein. Pretreatment for 120 rnin with selegiline at 1 mg/kg did not affect DA deamination (102 .+ 10% of control values). However, at 10 mg/kg (120 rnin of pretreatment), selegiline inhibited DOPAC production to 73 f 9% of control values (Table 1). 5-HT was deaminated to 5-hydroxyindoleacetic acid (5-HIAA) TABLE 1. Effects o f M A 0 inhibitors on deamination of DA and 5-HT Deamination (nmol/min/mg of protein) DOPAC
5-HIAA
Control
0.85 ? 0.04
0.83 +_ 0.14
Selegiline 1 mg/kg 10 mg/kg
0.86 i 0.10 0.63fO.11”
0.85 t 0.13 0.60 t 0.10“
Clorgyline 1 mukg 10 rngjkg
0.21 ? 0.106 0.14 -t 0.08’
0.24 t 0. lob 0.03 O.Olb
Pargyline (75 mgjkg)
0.04 -t 0.02’
0.05 -t 0.03b
*
983
at a rate of 0.83 nmol/min/mg of protein in control animals. Selegiline at a dose of 1 mg/kg (120 rnin of pretreatment) did not inhibit 5-HIAA production (102 .+ 15% of control values), whereas at 10 mg/kg, 5-HIAA production was reduced to 72 f 18%of control values (Table 1). Clorgyline at a dose of 1 mg/kg (120 rnin of pretreatment) reduced DOPAC production to 25 2 9% of control values and 5-HIAA production to 29 2 13% ofcontrol values (Table 1). At 10 mg/kg, DOPAC production was reduced to 16 & 8% of control values, and that of 5-HIAA was reduced to 4 1: 2% of control vatues. Pargyline (75 mg/kg; 120 rnin of pretreatment) reduced DOPAC and 5-HIAA production to 4 k 3 and 6 2 3% of control values, respectively (Table 1).
Effects of selegiline on catecholamine and indoleamine efflux The effects of selegiline (0.1- 10 mg/kg, i.p.) on basal efflux of catecholamines and indoleamines are shown in Fig. 1. No significant effects on dialysate DA or 3methoxytyramine (3-MT) levels were observed at any of the selegiline doses tested. Lower doses of selegiline (0.1 and 1 mg/kg) did not affect efflux of either DA or 5-HT metabolites. However, at 10 mg/kg, a significant reduction in DOPAC (62 .+ 1 1 % of basal efflux; p < 0.05), homovanillic acid (HVA) (78 F 8% of basal efflux; p < 0.05), and 5-HIAA (88 f 5% of basal efflux; p < 0.05) levels was noted. Statistical analysis was performed by comparing basal efflux with efflux 120 min following drug injection using a Mann-Whitney U test. The possibility that the effect of selegiline is mediated by its metabolite, amphetamine, was examined by administration of the DA uptake inhibitor nomifensine, which is known to inhibit the reduction in dialysate DOPAC level induced by amphetamine (Butcher et al., 1988). Administration of nomifensine (20 mg/kg, i.p.) increased the DA efflux to 250 +- 18% of basal efflux but did not affect the selegiline-evoked reduction in DOPAC efflux (Fig. 2). The nomifensine-induced increase in DA efflux was similar in animals treated with nomifensine alone and in animals receiving both nomifensine and selegiline (Fig. 2). Effects of clorgyline on catecholamine and indoleamine efflux The effects of clorgyline (1- 10 mg/kg, i.p.) on basal efflux of catecholamines and indoleamines are shown in Fig. 3. At the 1 mg/kg dose, DA and 3-MT efflux tended to increase, although this effect proved to be inconsistent. In contrast, DOPAC, HVA, and 5-HIAA efflux was reduced (maximal decreases in basal efflux, 40 6, 59 +. 7, and 65 k 4%, respectively). The higher clorgyline dose (10 mg/kg) elevated DA efflux maximally to 253 +- 19% of basal efflux, and 3-MT levels were increased to 500 .+ 30% of basal efflux. DA and 5-HT metabolite levels were reduced dramatically under these conditions. The DOPAC content was decreased to 15 k 5 % of basal efflux, that of HVA to 26 _+
M A 0 activity was measured by investigating the deamination of DA and 5-HT to DOPAC and 5-HIAA, respectively, in synaptosomes prepared from animals pretreated with M A 0 inhibitors. Relevant compounds were quantified by HPLC/ECD. Data are mean f SEM values from six independent experiments. Statistical analysis was performed using a Mann-Whitney U test: “p < 0.05, ’p < 0.01 for comparison with the corresponding control.
J Neurochon , Ibl 55. N o 3, 1990
S. P. BUTCHER ET AL.
984 DOPAC
"p&?# ;I,, ;I,,
! 2.4 3
OA
-: oe ^O
$
+ VA I
0
2
W
4
6
8
2
, ,
..
4
6
,
,
8
5HIAA
Mt
t
4.5 3t
0-4 2
4
6
2
FIG. 1. Effects of selegiline, 0.1 (O),1 (A), and '
,
4
.
,
6
,
, 8
10 (m)mg/kg, on dialysate efflux of DA, DOPAC. HVA. 3-MT. and 5-HIAA. Results are given as percentages of basal efflux and are mean k SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Selegiline- was given (intraperitoneal injection) following fraction 2 (arrow). Mean i SEM basal efflux in dialysates was as follows: DA, 0.1 1 rt 0.02 pmol/20 min; DOPAC, 19.6 k 1.9 prnol/20 rnin; HVA, 10.9 i0.6 prnol/ 20 rnin; 5-HIAA, 5.6 k 0.6 pmol/20 min; and 3MT, 0.06 k 0.02 pmol/20 min.
8
Fraction Number
f 8% of basal efflux, and that of 5-HIAA to 55 f 4% of basal efflux. Effects of pargyline on catecholamine and indoleamine efflux The effects of pargyline (75 mg/kg, i.p.) on basal efflux of catecholamines and indoleamines are shown in Fig. 3. DA efflux was elevated maximally to 310 f 20% of basal efflux, and that of 3-MT was increased to 670 f 60% of basal efflux. DOPAC efflux was reduced progressively over a 120-min period to 8 f 1% of basal efflux, that of HVA to 14 t- 3% of basal efflux, and that of 5-HIAA to 52 f 3% of basal efflux. The reductions in dialysate DOPAC and HVA levels were significantly greater than that induced by 10 mg/kg of clorgyline ( p < 0.05),as were the increases in dialysate DA and 3-MT levels ( p < 0.05). Statistical analyses DOPAC
DA
3r
I
021
Fraction Number
FIG. 2. Effects of nomifensine (20 mg/kg, i.p.) on alterations in dialysate DA and DOPAC levels induced by selegiline (10 mg/kg, i.p.). Results are given as percentages of basal efflux and are mean SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Drugs were given after collection of fraction 2 (arrow): selegiline (O),nomifensine (A), and nomifensineplus selegiline (m).
*
J. Ncwrochem , Vu/ 55, No. 3, 1990
were performed by comparing the effects of drugs at 120 rnin using a Mann-Whitney U test.
Effects of M A 0 inhibitors on chemically evoked DA release Increasing the K+ concentration in the perfusion medium (the Na+ concentration was reduced accordingly) leads to a calcium-dependent efflux of DA from the striatum (Fairbrother et al., 1990b). The effects of pretreatment with M A 0 inhibitors on this outflow of DA was examined. Neither selegiline (10 mg/kg; 120 rnin of pretreatment) nor clorgyline ( 1 mg/kg; 120 rnin of pretreatment) significantly affected K+-induced DA release (Figs. 4 and 5). In contrast, both pargyline (75 mg/kg; 120 rnin of pretreatment; Fig. 4) and clorgyline (10 mg/kg; 120 rnin of pretreatment; Fig. 5) faciliated the release of DA. The release of DA induced by K+ was increased by 186% following clorgyline pretreatment and by 283% following pargyline pretreatment (Table 2). The reduction in dialysate DOPAC levels induced by K+ (Figs. 4 and 5) was qualitatively similar in animals treated with either M A 0 inhibitor. A similar pattern was observed when other chemical stimulants were used. Thus, the DA efflux produced by veratrine [which induces release of DA via a camerindependent mechanism (Fairbrother et al., 1990a)l was not affected by selegiline (10 mg/kg) or clorgyline (1 mg/kg), whereas both pargyline (75 mg/kg) and clorgyline (10 mg/kg) resulted in an increase in the release of DA induced by veratrine (all 120 rnin of pretreatment; Table 2). The facilitation was again greater in pargyline-treated animals compared with animals treated with clorgyline (10 mdkg): 175% facilitation following clorgyline versus 262% facilitation following pargyline (Table 2; p < 0.05). The release of
985
M A 0 INHIBITORS AND DA RELEASE lr
4 r
FIG. 3. Effects of clorgyline, 1 (0)and 10 (A) mg/kg, and pargyline. 75 mg/kg (m), on dialysate efflux of DA, DOPAC, HVA, 3-MT. and 5-HIAA. Results are given as percentages of basal efflux and are mean 2 SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Drugs (injected intraperitoneally) were given following fraction 2 (arrow). Mean t SEM basal efflux in dialysates was as follows: DA, 0.09 k 0.01 pmol/20 min; DOPAC, 15.5 t 0.83 pmol/20 min; HVA, 9.7 t 1.Ipmol/20 min; 5-HIAA. 4.8 t 0.6 pmol/20 min; and 3-MT, 0.06 t 0.01 pmol/20 min.
c
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Fraction Number
DA induced by amphetamine was unaffected by prior selegiline pretreatment (10 mg/kg; 120 min of pretreatment; Table 2 ) but was increased by pretreatment with 10 mg/kg of clorgyline (Table 2; 193%facilitation following clorgyline; p < 0.0 1).
DISCUSSION The present data demonstrate that the MAO-B inhibitor selegiline at a dose of 10 mg/kg alters the basal overflow of both DA and 5-HT metabolites. These results contrast with those of Kato et al. (1986), although the use of unanaesthetised animals in the latter study may explain this discrepancy. Because this effect was insensitive to nomifensine, a DA uptake inhibitor that blocks the dialysate response to amphetamine (Butcher
-
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et al., 1988), a direct action of selegiline is suggested rather than an effect of its metabolite amphetamine (Karoum et al., 1982). The possibility that selegiline acts as a DA uptake inhibitor (Lai et al., 1980; Azzaro and Demerest, 1982) would also appear unlikely, because selegiline, unlike nomifensine, does not inhibit amphetamine-induced DA efflux (Table 2). It should not, however, be assumed that this effect is mediated solely by inhibition of MAO-B. High doses, e.g., 10 mg/kg, of selegiline are reported to inhibit partially MAO-A activity as assessed by an inhibition of the deamination of the preferred MAO-A substrate, 5-HT (Green et al., 1977; Demerest et al., 1980). A similar profile was obtained in the present study (see Table l), and the small but consistent fall in basal overflow of 5-HIAA noted in dialysis experiments using 10 mg/kg
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7
9
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FIG. 4. Effects of selegiline (10 mg/kg, i.p.) and pargyline (75mg/
FIG. 5. Effects of clorgyline (1 and 10 mg/kg, i.p.) on K+-induced
kg, i.p.) on K+evokedalterations in dialysate DA and DOPAC levels. Results are given as percentages of basal efflux and are mean f SEM (bars) values from six independent experiments. Each fraction represents a 20-min collection period. Selegiline and pargyline were given following collection of fraction 2 (arrow a), and K+ (90 mM) was infused via the dialysis probe for 20 min after collection of fraction 8 (arrow b): K+ (A), Kf plus selegiline (O),and Kf plus pargyline (m).
alterations in dialysate DA and DOPAC levels. Results are given as percentages of basal efflux and are mean ? SEM (bars) values from six independent experiments. Each fraction representsa 20min collection period. Clorgyline was injected after collection of fraction 2 (arrow a), and K+ (90 mnn) was infused via the dialysis probe for 20 min following collection of fraction 8 (arrow b): K+ (A), K+ plus 1 mg/kg of clorgyline (O), and K+ plus 10 mg/kg of clorgyline (m).
J. Neurochem.. Vol. 55, No. 3. 1990
S. P. BUTCHER ET AL.
986
TABLE 2. Effect o f M A 0 inhibitors on K'. and veratrine-evoked DA eflux Veratrine K+ (90 mM) (100 &nl) Control
1.13+0.13
2.65i0.36
Clorgyline 1 mg/kg 10 mg/kg
1.35 f 0.37 3.35 + 0.55 2.10 t 0.20" 4.80 f 0.70"
Amphetamine (4 mg/kg, i.p.) 1.5kO.2
2.9 f 0.60"
Pargyline (75 mg/kg) 3.20 f 0.68"." 6.93 f 1.2"," Selegiline (10 mdkg) 1.26
* 0.45
3.15 f 0.58
1.3 t 0.2
Endogenous DA levels in dialysates were measured by HPLC/ ECD. Results are mean f SEM values for the release of DA induced (in picomoles) above M A 0 pretreatment levels by K+ or veratrine during a 20-min period of stimulant perfusion (n = 6). Amphetamine data represent DA elllux over a 60-min postinjection period. Statistical analysis was performed using a Mann-Whitney U-test: "p < 0.02 for comparison of release in the presence and absence of the M A 0 inhibitor, ' p < 0.05 for comparison of clorgyline versus pargyline facilitation.
of selegiline suggests that the selectivity of this drug cannot be assumed. Lower, more selective doses of selegiline did not affect catecholamine or indoleamine metabolism. These data suggest that selegiline (1 0 mg/ kg) may exert its action via a nonselective inhibition of MAO-A. Data obtained using selegiline should be compared with results obtained when clorgyline was used to inhibit MAO-A or pargyline was used to impair both forms of MAO. Metabolite responses to these inhibitors were qualitatively similar to those reported by Kato et al. (1986). At an MAO-A-selective clorgyline dose of 1 mg/kg, dramatic reductions (40-65% of basal efflux) in the dialysate content of DOPAC, HVA, and 5-HIAA were observed. Increasing the clorgyline dose to 10 mg/ kg leads to even greater reductions in metabolite levels. Pargyline (75 mg/kg) inhibited basal overflow of DOPAC and HVA to a greater extent than 10 mg/kg of clorgyline, whereas the reduction in 5-HIAA content was similar in both cases. These data may be interpreted in two ways: (a) Under conditions of total MAO-A blockade, an MAO-B-mediated component of catecholamine metabolism becomes apparent, or (b) the 10 mg/kg dose of clorgyline does not fully inhibit MAO-A. The finding that 5-HT deamination was essentially abolished when using 10 mg/kg of clorgyline argues against the latter possibility (see Table 1). It has been suggested previously that MAO-B may play a role in catecholamine metabolism when MAO-A is inhibited (Green et al., 1977; Schoepp and Azzaro, 1982, 1983; Azzaro et al., 1985). The present study lends support to this proposal because DA, in contrast to 5HT, can still be metabolised to some extent following pretreatment with 10 mg/kg of clorgyline (Table 1). These data do, however, suggest that DA metabolism in striaturn is mediated predominately by MAO-A.
J Nrurochem , Vol $5,N o 3. 1990
Two further points merit consideration when comparing the effects of clorgyline and pargyline. First, M A 0 inhibitors were found to elevate extracellular levels of DA, and this may reflect the enhancement of dopaminergic neurotransmission mediating the therapeutic effects of these compounds. Basal efflux of DA was, however, not altered by the 1 mg/kg dose of clorgyline, even though metabolite levels are suppressed. Increasing the clorgyline dose to 10 mg/kg elevated the extracellular DA level but not to the same degree as that observed with pargyline. This suggests that although the dialysate DA content increases following MAO-A inhibition, total blockade of both forms of M A 0 is required to elevate maximally extracellular DA levels. These data are consistent with results reported by both Green et al. (1977), who demonstrated that behavioural effects of M A 0 inhibitors are only observed following complete inhibition of both forms of the enzyme, and Harsing and Vizi (1984), who showed using in vitro experiments that clorgyline reduces DOPAC efflux before DA release is affected. A second point of interest concerns extracellular levels of 3-MT. Neither 1 mg/kg of clorgyline nor selegiline (10 mg/kg) altered basal 3-MT overflow, whereas 10 mg/kg of clorgyline elevated 3-MT efflux. However, 3-MT levels are again elevated to a greater extent following pargyline pretreatment. Thus, the catechol-0-methyltransferase catabolic pathway becomes significant following blockade of MAO-A, presumably because 3-MT cannot be converted to HVA. The specificity of this effect suggests that MAO-A is preferentially involved in the metabolism of 3-MT, and this may reflect extraneuronal metabolism because catechol-0-methyltransferase activity is thought to be located outside dopaminergic terminals in the striaturn (Kaplan et al., 1979). The more pronounced effect of pargyline does, however, suggest a role for MAO-B in 3-MT deamination in the absence of MAO-A activity. These data, therefore, support the proposed functional role of M A 0 outside dopaminergic terminals under certain conditions (see Schoepp and Azzaro, 1982, 1983). One possibility not explored by Kato et al. (1986) was that an action of MAO-B is only apparent following release of DA. Thus, under resting conditions, extracellular DA is thought to be recaptured by uptake mechanisms. Following depolarisation, extracellular DA levels rise dramatically and may perhaps overload the neuronal uptake mechanism, thereby allowing access to nonneuronal sites of metabolism. GIial uptake sites for DA and 5-HT have been demonstrated (Pelton et al., 1981; Semeloff and Kimelberg, 1985), and MA0 activity is reported to reside in intrinsic striatal neurones and glial elements (Pelton et al., 1981; Levitt et al., 1982; Schoepp and Azzaro, 1983; Francis et ai., 1985). We have examined this possibility by elevating the extracellular DA level using several stimuli. Infusion of 90 mM Kf via the dialysis probe led to
M A 0 INHIBITORS AND DA RELEASE a marked increase in DA efflux and a reduction in dialysate DOPAC levels. Pretreatment with either selegiline (10 mg/kg) or a low clorgyline dose (1 mg/kg) did not affect the release of DA induced by K+. In contrast, pretreatment with 10 mg/kg of clorgyline or 75 mg/kg of pargyline markedly enhanced K+-induced DA release. A similar profile was obtained when DA effluxwas stimulated with veratrine, and these data are therefore consistent with the in vitro experiments of Harsing and Vizi ( 1984). Pargyline was again found to be a more effective enhancer of efflux than 10 mg/kg of clorgyline, a result suggesting that MAO-B may be involved in the deamination of DA in the absence of MAO-A activity. Thus, maximal facilitation of stimulated release is only observed following inhibition of both forms of MAO. These data suggest that under both resting and depolarising conditions DA metabolism principally involves MAO-A. However, under conditions of MAOA inhibition, an MAO-B component of catecholamine metabolism becomes apparent. Two mechanisms may underlie the effects of MAO-A inhibition on extracellular DA levels: (a) MAO-A inhibition may prevent DA catabolism following release, or (b) MAO-A inhibition may increase the pool of DA available for release. Because nomifensine does not affect extracellular DOPAC levels, the first possibility is unlikely unless metabolism of released DA is switched to extraneuronal sites when the neuronal uptake system is blocked. If this is the case, the finding that selegilinedoes not affect nomifensine-induced DA efflux suggests that MAO-B is not involved in DA deamination following blockade of the neuronal DA transporter. An alternative explanation has been suggested by Zetterstrom et al., (1988), who proposed that MA0 inhibition increases the synaptic pool of DA available for release. Although this hypothesis is also consistent with the present data, Schoepp and Azzaro (198 I b) have reported that DA synthesis is inhibited by clorgyline (perhapsby feedback inhibition of tyrosine hydroxylase). These workers suggested that the size of the releasable pool could be increased by preventing the deamination of DA once it had been taken up into dopaminergic terminals (Liccione and Azzaro, 1988). Acknowledgment: S. P. Butcher was a n MRC Training Fellow, and I. S. Fairbrother was supported by studentships from t h e MRC a n d Edinburgh University Faculty of Medicine. W e would like to t h a n k Avril Davidson for preparing synaptosomal preparations and Audrey Kerr for typing the manuscript.
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