Neuropharmacology Vol. 29, No. 10, pp. 815419,
1990
0028-3908/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc
Printed in Great Britain. All rights reserved
ROLE OF TYPE A AND B MONOAMINE OXIDASE ON THE FORMATION OF 3,4-DIHYDROXYPHENYLACETIC ACID (DOPAC) IN TISSUES FROM THE BRAIN OF THE RAT M. C. GARRETT* and P. SoAnt%+DA-Sn.vAt Laboratorio de Farmacologia, Faculdade de Medicina, 4200 Porto, Portugal (Accepted 3 May 1990) Summary-The role of monamine oxidase (MAO), type A and B, on the deamination of doparnine in the striatum, nucleus accumbens and frontal cortex of the rat was studied. Levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) tissue were quantified by means of high pressure liquid chromatography with electrochemical detection. Rats were given pargyline (75 mg/kg), selegyline (5 mg/kg) or clorgyline (2 mg/kg) by the intraperitoneal route, 60 min before sacrifice; in another set of experiments, clorgyline (2 mg/kg, i.p.) was given 15 or 30 min before sacrifice. Only clorgyline and pargyline were found to reduce significantly the formation of DOPAC and HVA in all the three areas of brain under study (83-97% reduction). The inhibition of deamination of dopamine by clorgyline and pargyline was accompanied by an increase in levels of dopamine in tissue. The increase of the levels of amine in tissue, as a result of inhibition of MAO, was more marked in the frontal cortex (52% increase) and the accumbens (39% increase), than in the striatum (25% increase). The results suggest that a substantial amount of DOPAC in brain derives from the deamination of dopamine by MAO,. Key
words-striatum,
accumbens, frontal cortex, dopamine, DOPAC, monoamine oxidase.
The two major metabolites of dopamine in brain are 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The DOPAC is formed by the action of monoamine oxidase (MAO) and is partially O-methylated to HVA by catechol-Omethyltransferase (COMT); an alternative pathway for the formation of HVA is that of the deamination by MAO of 3-methoxytryramine (3-MT), a metabolite of dopamine, resulting from a direct O-methylation of the amine (Kopin, 1985). The quantification of metabolites of dopamine in tissue, cerebrospinal fluid (CSF) or perfusates or dialysates of brain, has been widely used as an indirect measure of both metabolism or utilization of the amine. More recently, however, it has been demonstrated that only 3-MT can be accepted as an index of release of amine (for review see Wood and Altar, 1988). Evidence along this line has also been presented in studies
of MAO in the brain differs in that MAO, is nearly absent from neural elements and MAO, is distributed to both neuronal and non-neuronal tissues (Waldmeier, 1987). If, in fact, most of the MAO in nerve terminals is of type A and DOPAC largely derives from a newly-synthesized pool of dopamine which has never been released (Soares-da-Silva and Garrett, 1990; Zetterstrijm et al., 1988), then the inhibition of MAOa would be without effect upon levels of DOPAC, whereas inhibition of MAO, would produce a decrease in the formation of this metabolite of dopamine. The present report describes findings on the effects of a non-selective inhibitor and selective inhibitors of MAO,., and MAO, on the levels of dopamine and metabolites in tissue in the striatum, nucleus accumbens and frontal cortex of the rat.
showing that most of the DOPAC in brain derives from a pool of dopamine which has never been released (Commissiong, 1985; RackC, Bijhm and Muscholl, 1987; Soares-da-Silva, 1987; Zetterstrom, Sharp, Collin and Ungerstedt, 1988; Westerink, Damsma, Vries and Koning, 1987; Wood and Altar, 1988). Two subtypes of MAO have been described, MAOA and MAO,. The distribution of the two forms
Animals, drugs and dissection
*On leave from Service de Neurologia e Neurocirurgia, Faculdade de Medicina, 4200 Porto, Portugal. tTo whom all correspondence should be addressed.
METHODS
Male Wistar rats (Bioterio do Instituto Gulbenkian de Ciencia, Oeiras, Portugal), weighing 250-300 g were used. The animals were kept 5 per cage under controlled environmental conditions (12 hr light/dark cycle and room temperature 24°C). Food and tap water were allowed ad Zibitum.The experiments were all carried out during daytime. Pargyline (75 mg/kg), selegyline (5 mg/kg) and clorgyline (2 mg/kg) were injected intraperitoneally (i.p.) into groups of 5 rats, control animals receiving the same volume of saline by the same route; 60 min 875
876
M.C. GARRETT and
after the injection, the animals were sacrificed by decapitation. Two other groups of rats were given clorgyline (2 mg/kg, i.p.) and sacrificed 15 and 30 min after the injection. The brains were quickly removed and placed on an ice-cold glass-plate. A coronal cut was made at the anterior border of the pons and the posterior portion of the brain was discarded. The lateral parts of the anterior portion were removed by two saggital cuts, 4mm to the right and left of the median piane. The block of tissue obtained was divided symmetrically by a saggital cut in the midline, the corpus striatum and frontal cortex dissected free and slices, containing the nucleus accumhens selected, according to the atlas of Paxinos and Watson (1982). Assay procedure
The assay of dopamine, DOPAC and HVA was performed by means of HPLC-ECD, as described in Soares-da-Silva and Garrett (1990). In brief, after dissection of the striatum, nucleus accumbens and frontal cortex, the tissues were placed in 500~1 of 0.1 mol/l perchloric acid, with 1250 pg of dihydroxybenzylamine added (internal standard), immediately frozen and stored at -30°C; this procedure was found to completely remove dopamine, DOPAC and HVA from the tissues. After gentle thawing 100 ~1 of the supernatant was taken and filtered on Millipore (MFl) microfilters; 50 ~1 of the filtered supernatant was then directly injected into the HPLC column. The HPLC-ECD system consisted of a pump (Gihon model 302) connected to a manometric module (Gilson model 802 C) and a stainless steel 5 ilrn ODS column (Biophase) of 25cm length. Samples were injected into the column by means of an automatic sample injector (Gilson model 23l), connected to a Gilson dilutor (model 401). The detection was carried out electrochemically by means of a thin-layer cell. with a glassy carbon working electrode, an Ag/AgCl reference electrode and an amperometric detector (model 304 LC-4A, Bioanalytical Systems). The detector cell was operated at i-0.75 V. The current produced was monitored using a Spectra Physics integrator (model SP4270). The mobile phase was a degassed solution of citric acid (0.1 mmoh’l), sodium octylsulphate (0.5 mmol~l), sodium acetate (0.1 molil), ethylene diamine tetra acetic acid (EDTA, 0.17 mmol/l), dibutylamine (1 mmol’l) and methanol (10% v/v), pH 3.5 and pumped at a rate of I .Oml/min. Under these conditions, the lower limits of detection for dopamine, DOPAC and HVA were 10, 20 and 40 pg, respectively. Calculations were based on the recovery of the internal standard added to tissues; in the tissue samples, the recovery of dihydroxybenzylamine was about 95%. Sources qf drugs and chemicals
Drugs used were: clorgyline hydrochloride (May and Baker, Ltd. Dagenham, U.K.), dihydroxybenzylamine (Sigma Chemical Company. St Louis, Missouri. U.S.A.) 3,4-dihydroxyphenylacetic acid
P. SOARES-DA-SILVA (DOPAC; Sigma), dopamine hydrochloride (Sigma), homovanilic acid (Sigma), pargyline hydrochloride (Sigma) and selegyline (( -)-deprenyl) was a gift from Professor J. Knoll, Department of Pharmacology, Semmelweiss University of Medicine, Budapest, Hungary.
Results are mean values +SEM of 5 experiments per group; n is the number of animals used. Statistical significance was determined using the Tukey-Kramer method; a probability of less than 0.05 was assumed to denote a significant difference. RESULTS
Administration of the MAO, selective inhibitor, selegyline was found not to alter the concentrations of dopamine, DOPAC and HVA in the three areas of brain studied, the striatum, nucleus a~urn~ns and frontal cortex (Figs l-3). In contrast, administration of the non-selective MAO inhibitor, pargyline results in a marked reduction (92-97%) of levels of DOPAC and HVA in both the striatum and nucleus accumbens; this effect on the formation of metabolites of dopamine was accompanied by an increase (27-37%) of levels of dopamine in tissue (Figs 1 and 2). Sixty min after the MAO, selective inhibitor, clorgyline had been given, a decrease of DOPAC (83-90% reduction) and HVA (73-77% reduction) was also observed in the three areas of brain (Figs l-3). As found for pargyline, administration of clorgyline, 60 min before sacrifice resulted in an increase in levels of dopamine in tissue; this effect was more marked in the frontal cortex (52% increase) and the accumbens (39% increase), than in the striatum (25% increase). The effect of clorgyline on the deamination of dopamine in all the three areas of brain (striatum, accumbens and frontal cortex) was found to be time-dependent (Figs l-3). Also, in all the three areas of brain studied the decline of levels of DOPAC was faster than that of HVA, after clorgyline had been given (Figs l-3). DISCUSSION
The aim of present work was to study the role of MAO, type A and B, on the deamination of dopamine in the striatum, nucleus accumbens and frontal cortex of the rat. The results presented show that only the non-selective MAO inhibitor, pargyline and the MAO, selective inhibitor, clorgyline reduced the deamination of dopamine into DOPAC; the MAO inhibitor, selegyline did not affect the deamination of dopamine in either area. Therefore, it appears that most of the dopamine in all the three areas of brain is deaminated by MAO, and MAOB is unimportant in the metabolism of the amine. The results presented here agree with previous observations showing that inhibition of MAO,
MAO,
and formation
of DOPAC
* I
i
*.! II30
Cont
Seleq
Parg
.
r2
30 mln
Clorgyline
(2 mg/kg) and selegyline (Seleg.; 5 mg/kg) on levels of the rat. Control rats (Cont.) were injected intraperitoneally with saline (4 ml/kg) and tissues were taken 60 min after the injection of drugs or at 15, 30 or 60 min intervals, as indicated for clorgyline. Each column represents the mean of 5 experiments; vertical lines show SEM. *Significantly different from control values (P < 0.01) by the Tukey-Kramer method. Dopamine, 0; DOPAC, q; HVA, q .
Fig. 1. Effects of pargyline
of dopamine,
(Parg.; 75 mg/kg),
clorgyline
DOPAC and HVA in the striatum
results in an increase in levels of dopamine in the striatum, whereas inhibition of MAO, did not
alter levels of the amine and of its metabolites in tissue (Waldemeier, Delini-Stula and Maitre, 1976; Oreland, Arai and Stenstrom, 1983; Azzaro, King, Kotzuk, Schoepp, Frost and Schochet, 1985; Kato, Dong, Ishii and Kinemuchi, 1986; Offori, Breton, Hof and Schorderet, 1986; Nguyen and Angers, 1987; Hovevey-Sion, Kopin, Stull and Goldstein, 1989). It could be argued that the lack of effect of selegyline on the deamination of dopamine was a dose-related problem. However, Waldemeier et al. (1976) have shown in tissues from the brain of the rat that 0.3 and 3.0mg/kg selegyline were found to reduce by 65% and 75%, respectively, the deamination of /I-phenylethylamine, a specific substrate for MAO,. In addition, Kato et al. (1986) administered a dose of selegyline twice that used in the present
study and still could not find any changes in levels of dopamine, DOPAC and HVA in the striatum. It appears, therefore, that the lack of effect of selegyline on the deamination of dopamine derives from the fact that most of the dopamine is deaminated intraneuronally where the activity of MAO is of the A type. In agreement with this view are the results of Oreland et al., (1983) who clearly demonstrated in the striatum of the rat that 84% of the total deamination of dopamine was carried out by MAO, intrasynaptosomally, 11% was deaminated by MAO, extrasynaptosomally and the remaining 5% deaminated by MAO, (2% intra- and 3% extrasynaptosomally). Thus, the data reported here confirms the previous suggestion that dopamine in the striatum is mainly deaminated by MAO, and extends this conclusion to the nucleus accumbens and frontal cortex.
*
30
Seleg Fig. 2. Effects of dopamine, intraperitoneally 30 or 60 min vertical lines
Parg
CIorgyline
of pargyline (Parg.; 75 mg/kg), clorgyline (2 mg/kg) and selegyline (Seleg.; 5 mg/kg) on levels DOPAC and HVA in the nucleus accumbens of the rat. Control rats (Cont.) were injected with saline (4 ml/kg) and tissues were taken 60 min after the injection of drugs or at 15, intervals, as indicated for clorgyline. Each column represents the mean of 5 experiments; show SEM. *Significantly different from control values (P < 0.01) by the Tukey-Kramer ; HVA, a. method. Dopamine, 0; DOPAC,
M. C. GARRETTand P. SOARES-DA-SILVA
878 300
1
60 mln
30
Seleg
Cont
Clorgyline
Fig. 3. Effects of clorgyline (2 mg/kg) and selegyline (Seleg.; 5 mg/kg) on levels of dopamine, HVA in the (4 ml/kg) and indicated for *Significantly
The
suggestion
DOPAC and frontal cortex of the rat. Control rats (Cont.) were injected intraperitoneally with saline tissues were taken 60 min after the injection of drugs or at 15, 30 or 60 min intervals, as clorgyline. Each column represents the mean of 5 experiments; vertical lines show SEM. different from control values (P < 0.01) by the TukeyyKramer method. Dopamine, 0; DOPAC, F2I; HVA, q .
that
dopamine
is mainly
deami-
MAO, also agrees with the findings that a substantial amount of DOPAC derives from a newly-formed pool of dopamine (see Soares-da-Silva and Garrett, 1990). Evidence favouring the view that most of newly-formed dopamine is mainly deaminated by MAO, has also been provided in the studies of Offori et al. (986) and Nyuyen and Angers (1987). These authors have reported that administration of L-3,Cdihydroxyphenylalanine (L-DOPA) led to an increase in levels of dopamine in the striatum and this effect was potentiated by previous administration of the MAO, inhibitor clorgyline, but not by selegyline. Other types of evidence, suggesting that most of the dopamine is deaminated intraneuronally, has also been presented (see Rack& et al., 1987; Rack& and Muscholl, 1986; Sharp, ZetterstrGm and Ungerstedt, 1986). Another finding, further supporting the view that a substantial amount of recently-synthesized dopamine is deaminated before its release has occurred, is that the increase in levels of dopamine in tissue after administration of clorgyline (60 min) or pargyline, differed between the three areas of brain studied, the rank order being frontal cortex > nucleus accumbens > striatum. In fact, the increase in levels of dopamine in tissue, after inhibition of MAO, were greater in areas presenting the highest rates of synthesis of dopamine. This view is also consistent with biochemical studies showing that the rate of utilization of dopamine in the frontal cortex is greater than that in mesolimbic and nigrostriatal dopamine terminal areas (Sharp et al., 1986; Soares-da-Silva and Garrett, 1990). Moreover, electrophysiological studies have shown that the firing rate in dopaminergic neurones in the frontal cortex is greater than in mesolimbic and nigrostriatal pathways (for review see Bannon and Roth, 1983). Therefore, it might be hypothesized that differences in the formation nated
intraneuronally
by
of deaminated metabolites of dopamine, between different areas of the brain largely derive from differences in the rates of formation of dopamine and not to differences in MAO activity. As has been reported by other authors, the decline of DOPAC after inhibition of MAO was faster than that for HVA (Dedek, Baumes, Tien-Due, Gomeni and Korf, 1979; Westerink and Spaan, 1982; Westerink, Bosker and Wirix, 1984; Sharp et al., 1986). This would agree with the concept that a substantial amount of HVA derives from a pool of DOPAC formed before inhibition of MAO took place. In all these studies, inhibition of MAO has been performed with non-selective inhibitors, but similar results were found in the present study with selective inhibition of MAO,. In conclusion, the results presented here demonstrate that deamination of dopamine in the stiatum, nucleus accumbens and frontal cortex of the rat, is predominantly dependent on MAO,. If in fact most of MAO, is located intraneuronally (and MAO, extraneuronally), then the results reported would suggest that most of the dopamine is deaminated inside nerve endings. Acknowledgements-This work was supported by a grant from Instituto National de Investigacao Cientifica (FmPl). The HPLC-ECD system used in the present study was kindly donated by The Alexander von Humboldt Foundation (F.R.A.).
REFERENCES Azzaro A. J., King J., Kotzuk
J., Schoepp D. D., Frost J. and Schochet S. (1985) Guinea striatum as a model of human dopamine deamination: the role of monoamine oxidase isozyme ratio, localization and affinity for substrate in synaptic dopamine metabolism. J. Neurochem. 45: 949-956. Bannon M. J. and Roth R. H. (1983) Pharmacology of mesocortical dopamine neurons. Pharmac. Rev. 35: 53-68.
MAO,
and formation
Commissiong J. W. (1985) Monoamine metabolites: their relationship and lack of relationship to monoaminergic neuronal activity. Eiochem. Pharmac. 34: 1127-I 131. Dedek J., Baumes R., Tien-Due N., Gomeni R. and Korf J. (1979) Turnover of free and conjugated (sulphonxy) dihydroxyphenylacetic acid in rat striatum. J. Neurochem. 33: 687-695. Hovevey-Sion D., Kopin I. J., Stull R. W. and Goldstein D. S. (1989) Effects of monoamine oxidase inhibitors on levels of catechols and homovanilic acid in striatum and plasma. Neuropharmacology 28: 791-797. 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. Neurochem. 46: 1277-1282. Kopin I. J. (1985) Catecholamine metabolism: Basic aspects and clinical significance. Pharmac. Rev. 37: 333-364. Nguyen T. B. and Angers M. (1987) Effect of different monoamine oxidase inhibitor on the metabolism of L-DOPA in the rat brain. Biochem. Pharmac. 36: 1731-1735. Offori S., Breton C., Hof P. and Schorderet M. (1986) Investigation of dopamine content, synthesis and release in the rabbit retine in vitro: I. Effects of dopamine amphetamine and L-DOPA deprecursors, reserpine, carboxylase and monoamine oxidase inhibitors. J. Neurothem. 47: 119991206. Oreland L., Arai Y. and Stenstrom A. (1983). The effect of deprenyl (selegyline) on intra- and extraneuronal dopamine oxidation. Acta neural. stand. Suppl. 95: 81-85. Paxinos G. and Watson C. (1982) The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. RackC K., Biihm E. and Muscholl E. (1987) The role of cytoplasmic (newly synthesized) dopamine for the spontaneous and electrically evoked release of dopamine and its metabolites from the isolated neurointermediate lobe of the rat pituitary gland in vitro. Nuunyn-Schmiedeberg Arch. Pharmac. 335: 21-27. Rack& K. and Muscholl E. (1986) Release of endogenous 3,4-dihydroxyphenylethylamine and its metabolites from the isolated neurointermediate lobe of the rat pituitary gland. Effects of electrical stimulation and of inhibition of
of DOPAC
879
monoamine oxidase and reuptake. J. Neurochem. 46: 745-752. Sharp T., Zetterstrijm T. and Ungerstedt U. (1986) An in vivo study of dopamine release and metabolism in rat brain regions using intracerebral dialysis. J. Neurochem. 47: 113-122. Soares-da-Silva P. (1987) Does brain 3,4-dihydroxyphenylacetic acid (DOPAC) reflect dopmaine release? J. Pharm. Pharmac. 39: 127-129. Soares-da-Silva and Garrettt M. C. (1990) A kinetic study of the rate of formation of dopamine, 3,4-dihydroxynhenvlacetic acid (DOPAC) and homovanillic acid (HVA) in the rat brain: implications for the origin of DOPAC. Neuropharmacology 29: 869-874. Waldmeier P. C. (1987) Amine oxidases and their endogenous substrates (with special reference to monoamine oxidase and the brain). J. Neural Trans. Suppl. 23: 55-72. Waldmeier P. C., Delini-Stula and Maitre L. (1976) Preferential deamination of dopamine by an A type monoamine oxidase in rat brian. Naunyn-Schmiedebergs Arch. Pharmat. 292: 9-14. Westerink B. H. C., Bosker F. J. and Wirix E. (1984) Formation and metabolism of dopamine in nine areas of the rat brain: modifications by haloperidol. J. Neurochem. 42: 1321-1327. Westerink B. H. C., Damsma G., de Vries J. B. and Koning H. (1987) Dopamine re-uptake inhibitors show inconsistent effects on the in vivo release of dopamine as measured by intracerebral dialysis in the rat. Eur. J. Pharmac. 135: 123-128. Westerink B. H. C. and Spaan S. J. (1982) Simultaneous determination of the formation rate of dopamine and its metabolite 3,4-dihydroxyphenylacetic (DOPAC) in various rat brain areas. Brain Res. 252: 239-245. Wood P. L. and Altar A. (1988) Dopamine release in vivo from nigrostriatal, mesolimbic, and mesocortical neurons: Utility of 3-methoxytryramine measurements. Pharmac. Rev. 40: 163-187. Zetterstriim T., Sharp T., Collin A. K. and Ungerstedt U. (1988) In oivo measurement of extracellular dopamine and DOPAC in rat striatum after various dopamine-releasing drugs; implications for the origin of the extracellular DOPAC. Eur. J. Pharmac. 148: 327-334. I
I
1990
0028-3908/90$3.00+ 0.00 Copyright 0 1990Pergamon Press plc
Printed in Great Britain. All rights reserved
ROLE OF TYPE A AND B MONOAMINE OXIDASE ON THE FORMATION OF 3,4-DIHYDROXYPHENYLACETIC ACID (DOPAC) IN TISSUES FROM THE BRAIN OF THE RAT M. C. GARRETT* and P. SoAnt%+DA-Sn.vAt Laboratorio de Farmacologia, Faculdade de Medicina, 4200 Porto, Portugal (Accepted 3 May 1990) Summary-The role of monamine oxidase (MAO), type A and B, on the deamination of doparnine in the striatum, nucleus accumbens and frontal cortex of the rat was studied. Levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) tissue were quantified by means of high pressure liquid chromatography with electrochemical detection. Rats were given pargyline (75 mg/kg), selegyline (5 mg/kg) or clorgyline (2 mg/kg) by the intraperitoneal route, 60 min before sacrifice; in another set of experiments, clorgyline (2 mg/kg, i.p.) was given 15 or 30 min before sacrifice. Only clorgyline and pargyline were found to reduce significantly the formation of DOPAC and HVA in all the three areas of brain under study (83-97% reduction). The inhibition of deamination of dopamine by clorgyline and pargyline was accompanied by an increase in levels of dopamine in tissue. The increase of the levels of amine in tissue, as a result of inhibition of MAO, was more marked in the frontal cortex (52% increase) and the accumbens (39% increase), than in the striatum (25% increase). The results suggest that a substantial amount of DOPAC in brain derives from the deamination of dopamine by MAO,. Key
words-striatum,
accumbens, frontal cortex, dopamine, DOPAC, monoamine oxidase.
The two major metabolites of dopamine in brain are 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The DOPAC is formed by the action of monoamine oxidase (MAO) and is partially O-methylated to HVA by catechol-Omethyltransferase (COMT); an alternative pathway for the formation of HVA is that of the deamination by MAO of 3-methoxytryramine (3-MT), a metabolite of dopamine, resulting from a direct O-methylation of the amine (Kopin, 1985). The quantification of metabolites of dopamine in tissue, cerebrospinal fluid (CSF) or perfusates or dialysates of brain, has been widely used as an indirect measure of both metabolism or utilization of the amine. More recently, however, it has been demonstrated that only 3-MT can be accepted as an index of release of amine (for review see Wood and Altar, 1988). Evidence along this line has also been presented in studies
of MAO in the brain differs in that MAO, is nearly absent from neural elements and MAO, is distributed to both neuronal and non-neuronal tissues (Waldmeier, 1987). If, in fact, most of the MAO in nerve terminals is of type A and DOPAC largely derives from a newly-synthesized pool of dopamine which has never been released (Soares-da-Silva and Garrett, 1990; Zetterstrijm et al., 1988), then the inhibition of MAOa would be without effect upon levels of DOPAC, whereas inhibition of MAO, would produce a decrease in the formation of this metabolite of dopamine. The present report describes findings on the effects of a non-selective inhibitor and selective inhibitors of MAO,., and MAO, on the levels of dopamine and metabolites in tissue in the striatum, nucleus accumbens and frontal cortex of the rat.
showing that most of the DOPAC in brain derives from a pool of dopamine which has never been released (Commissiong, 1985; RackC, Bijhm and Muscholl, 1987; Soares-da-Silva, 1987; Zetterstrom, Sharp, Collin and Ungerstedt, 1988; Westerink, Damsma, Vries and Koning, 1987; Wood and Altar, 1988). Two subtypes of MAO have been described, MAOA and MAO,. The distribution of the two forms
Animals, drugs and dissection
*On leave from Service de Neurologia e Neurocirurgia, Faculdade de Medicina, 4200 Porto, Portugal. tTo whom all correspondence should be addressed.
METHODS
Male Wistar rats (Bioterio do Instituto Gulbenkian de Ciencia, Oeiras, Portugal), weighing 250-300 g were used. The animals were kept 5 per cage under controlled environmental conditions (12 hr light/dark cycle and room temperature 24°C). Food and tap water were allowed ad Zibitum.The experiments were all carried out during daytime. Pargyline (75 mg/kg), selegyline (5 mg/kg) and clorgyline (2 mg/kg) were injected intraperitoneally (i.p.) into groups of 5 rats, control animals receiving the same volume of saline by the same route; 60 min 875
876
M.C. GARRETT and
after the injection, the animals were sacrificed by decapitation. Two other groups of rats were given clorgyline (2 mg/kg, i.p.) and sacrificed 15 and 30 min after the injection. The brains were quickly removed and placed on an ice-cold glass-plate. A coronal cut was made at the anterior border of the pons and the posterior portion of the brain was discarded. The lateral parts of the anterior portion were removed by two saggital cuts, 4mm to the right and left of the median piane. The block of tissue obtained was divided symmetrically by a saggital cut in the midline, the corpus striatum and frontal cortex dissected free and slices, containing the nucleus accumhens selected, according to the atlas of Paxinos and Watson (1982). Assay procedure
The assay of dopamine, DOPAC and HVA was performed by means of HPLC-ECD, as described in Soares-da-Silva and Garrett (1990). In brief, after dissection of the striatum, nucleus accumbens and frontal cortex, the tissues were placed in 500~1 of 0.1 mol/l perchloric acid, with 1250 pg of dihydroxybenzylamine added (internal standard), immediately frozen and stored at -30°C; this procedure was found to completely remove dopamine, DOPAC and HVA from the tissues. After gentle thawing 100 ~1 of the supernatant was taken and filtered on Millipore (MFl) microfilters; 50 ~1 of the filtered supernatant was then directly injected into the HPLC column. The HPLC-ECD system consisted of a pump (Gihon model 302) connected to a manometric module (Gilson model 802 C) and a stainless steel 5 ilrn ODS column (Biophase) of 25cm length. Samples were injected into the column by means of an automatic sample injector (Gilson model 23l), connected to a Gilson dilutor (model 401). The detection was carried out electrochemically by means of a thin-layer cell. with a glassy carbon working electrode, an Ag/AgCl reference electrode and an amperometric detector (model 304 LC-4A, Bioanalytical Systems). The detector cell was operated at i-0.75 V. The current produced was monitored using a Spectra Physics integrator (model SP4270). The mobile phase was a degassed solution of citric acid (0.1 mmoh’l), sodium octylsulphate (0.5 mmol~l), sodium acetate (0.1 molil), ethylene diamine tetra acetic acid (EDTA, 0.17 mmol/l), dibutylamine (1 mmol’l) and methanol (10% v/v), pH 3.5 and pumped at a rate of I .Oml/min. Under these conditions, the lower limits of detection for dopamine, DOPAC and HVA were 10, 20 and 40 pg, respectively. Calculations were based on the recovery of the internal standard added to tissues; in the tissue samples, the recovery of dihydroxybenzylamine was about 95%. Sources qf drugs and chemicals
Drugs used were: clorgyline hydrochloride (May and Baker, Ltd. Dagenham, U.K.), dihydroxybenzylamine (Sigma Chemical Company. St Louis, Missouri. U.S.A.) 3,4-dihydroxyphenylacetic acid
P. SOARES-DA-SILVA (DOPAC; Sigma), dopamine hydrochloride (Sigma), homovanilic acid (Sigma), pargyline hydrochloride (Sigma) and selegyline (( -)-deprenyl) was a gift from Professor J. Knoll, Department of Pharmacology, Semmelweiss University of Medicine, Budapest, Hungary.
Results are mean values +SEM of 5 experiments per group; n is the number of animals used. Statistical significance was determined using the Tukey-Kramer method; a probability of less than 0.05 was assumed to denote a significant difference. RESULTS
Administration of the MAO, selective inhibitor, selegyline was found not to alter the concentrations of dopamine, DOPAC and HVA in the three areas of brain studied, the striatum, nucleus a~urn~ns and frontal cortex (Figs l-3). In contrast, administration of the non-selective MAO inhibitor, pargyline results in a marked reduction (92-97%) of levels of DOPAC and HVA in both the striatum and nucleus accumbens; this effect on the formation of metabolites of dopamine was accompanied by an increase (27-37%) of levels of dopamine in tissue (Figs 1 and 2). Sixty min after the MAO, selective inhibitor, clorgyline had been given, a decrease of DOPAC (83-90% reduction) and HVA (73-77% reduction) was also observed in the three areas of brain (Figs l-3). As found for pargyline, administration of clorgyline, 60 min before sacrifice resulted in an increase in levels of dopamine in tissue; this effect was more marked in the frontal cortex (52% increase) and the accumbens (39% increase), than in the striatum (25% increase). The effect of clorgyline on the deamination of dopamine in all the three areas of brain (striatum, accumbens and frontal cortex) was found to be time-dependent (Figs l-3). Also, in all the three areas of brain studied the decline of levels of DOPAC was faster than that of HVA, after clorgyline had been given (Figs l-3). DISCUSSION
The aim of present work was to study the role of MAO, type A and B, on the deamination of dopamine in the striatum, nucleus accumbens and frontal cortex of the rat. The results presented show that only the non-selective MAO inhibitor, pargyline and the MAO, selective inhibitor, clorgyline reduced the deamination of dopamine into DOPAC; the MAO inhibitor, selegyline did not affect the deamination of dopamine in either area. Therefore, it appears that most of the dopamine in all the three areas of brain is deaminated by MAO, and MAOB is unimportant in the metabolism of the amine. The results presented here agree with previous observations showing that inhibition of MAO,
MAO,
and formation
of DOPAC
* I
i
*.! II30
Cont
Seleq
Parg
.
r2
30 mln
Clorgyline
(2 mg/kg) and selegyline (Seleg.; 5 mg/kg) on levels of the rat. Control rats (Cont.) were injected intraperitoneally with saline (4 ml/kg) and tissues were taken 60 min after the injection of drugs or at 15, 30 or 60 min intervals, as indicated for clorgyline. Each column represents the mean of 5 experiments; vertical lines show SEM. *Significantly different from control values (P < 0.01) by the Tukey-Kramer method. Dopamine, 0; DOPAC, q; HVA, q .
Fig. 1. Effects of pargyline
of dopamine,
(Parg.; 75 mg/kg),
clorgyline
DOPAC and HVA in the striatum
results in an increase in levels of dopamine in the striatum, whereas inhibition of MAO, did not
alter levels of the amine and of its metabolites in tissue (Waldemeier, Delini-Stula and Maitre, 1976; Oreland, Arai and Stenstrom, 1983; Azzaro, King, Kotzuk, Schoepp, Frost and Schochet, 1985; Kato, Dong, Ishii and Kinemuchi, 1986; Offori, Breton, Hof and Schorderet, 1986; Nguyen and Angers, 1987; Hovevey-Sion, Kopin, Stull and Goldstein, 1989). It could be argued that the lack of effect of selegyline on the deamination of dopamine was a dose-related problem. However, Waldemeier et al. (1976) have shown in tissues from the brain of the rat that 0.3 and 3.0mg/kg selegyline were found to reduce by 65% and 75%, respectively, the deamination of /I-phenylethylamine, a specific substrate for MAO,. In addition, Kato et al. (1986) administered a dose of selegyline twice that used in the present
study and still could not find any changes in levels of dopamine, DOPAC and HVA in the striatum. It appears, therefore, that the lack of effect of selegyline on the deamination of dopamine derives from the fact that most of the dopamine is deaminated intraneuronally where the activity of MAO is of the A type. In agreement with this view are the results of Oreland et al., (1983) who clearly demonstrated in the striatum of the rat that 84% of the total deamination of dopamine was carried out by MAO, intrasynaptosomally, 11% was deaminated by MAO, extrasynaptosomally and the remaining 5% deaminated by MAO, (2% intra- and 3% extrasynaptosomally). Thus, the data reported here confirms the previous suggestion that dopamine in the striatum is mainly deaminated by MAO, and extends this conclusion to the nucleus accumbens and frontal cortex.
*
30
Seleg Fig. 2. Effects of dopamine, intraperitoneally 30 or 60 min vertical lines
Parg
CIorgyline
of pargyline (Parg.; 75 mg/kg), clorgyline (2 mg/kg) and selegyline (Seleg.; 5 mg/kg) on levels DOPAC and HVA in the nucleus accumbens of the rat. Control rats (Cont.) were injected with saline (4 ml/kg) and tissues were taken 60 min after the injection of drugs or at 15, intervals, as indicated for clorgyline. Each column represents the mean of 5 experiments; show SEM. *Significantly different from control values (P < 0.01) by the Tukey-Kramer ; HVA, a. method. Dopamine, 0; DOPAC,
M. C. GARRETTand P. SOARES-DA-SILVA
878 300
1
60 mln
30
Seleg
Cont
Clorgyline
Fig. 3. Effects of clorgyline (2 mg/kg) and selegyline (Seleg.; 5 mg/kg) on levels of dopamine, HVA in the (4 ml/kg) and indicated for *Significantly
The
suggestion
DOPAC and frontal cortex of the rat. Control rats (Cont.) were injected intraperitoneally with saline tissues were taken 60 min after the injection of drugs or at 15, 30 or 60 min intervals, as clorgyline. Each column represents the mean of 5 experiments; vertical lines show SEM. different from control values (P < 0.01) by the TukeyyKramer method. Dopamine, 0; DOPAC, F2I; HVA, q .
that
dopamine
is mainly
deami-
MAO, also agrees with the findings that a substantial amount of DOPAC derives from a newly-formed pool of dopamine (see Soares-da-Silva and Garrett, 1990). Evidence favouring the view that most of newly-formed dopamine is mainly deaminated by MAO, has also been provided in the studies of Offori et al. (986) and Nyuyen and Angers (1987). These authors have reported that administration of L-3,Cdihydroxyphenylalanine (L-DOPA) led to an increase in levels of dopamine in the striatum and this effect was potentiated by previous administration of the MAO, inhibitor clorgyline, but not by selegyline. Other types of evidence, suggesting that most of the dopamine is deaminated intraneuronally, has also been presented (see Rack& et al., 1987; Rack& and Muscholl, 1986; Sharp, ZetterstrGm and Ungerstedt, 1986). Another finding, further supporting the view that a substantial amount of recently-synthesized dopamine is deaminated before its release has occurred, is that the increase in levels of dopamine in tissue after administration of clorgyline (60 min) or pargyline, differed between the three areas of brain studied, the rank order being frontal cortex > nucleus accumbens > striatum. In fact, the increase in levels of dopamine in tissue, after inhibition of MAO, were greater in areas presenting the highest rates of synthesis of dopamine. This view is also consistent with biochemical studies showing that the rate of utilization of dopamine in the frontal cortex is greater than that in mesolimbic and nigrostriatal dopamine terminal areas (Sharp et al., 1986; Soares-da-Silva and Garrett, 1990). Moreover, electrophysiological studies have shown that the firing rate in dopaminergic neurones in the frontal cortex is greater than in mesolimbic and nigrostriatal pathways (for review see Bannon and Roth, 1983). Therefore, it might be hypothesized that differences in the formation nated
intraneuronally
by
of deaminated metabolites of dopamine, between different areas of the brain largely derive from differences in the rates of formation of dopamine and not to differences in MAO activity. As has been reported by other authors, the decline of DOPAC after inhibition of MAO was faster than that for HVA (Dedek, Baumes, Tien-Due, Gomeni and Korf, 1979; Westerink and Spaan, 1982; Westerink, Bosker and Wirix, 1984; Sharp et al., 1986). This would agree with the concept that a substantial amount of HVA derives from a pool of DOPAC formed before inhibition of MAO took place. In all these studies, inhibition of MAO has been performed with non-selective inhibitors, but similar results were found in the present study with selective inhibition of MAO,. In conclusion, the results presented here demonstrate that deamination of dopamine in the stiatum, nucleus accumbens and frontal cortex of the rat, is predominantly dependent on MAO,. If in fact most of MAO, is located intraneuronally (and MAO, extraneuronally), then the results reported would suggest that most of the dopamine is deaminated inside nerve endings. Acknowledgements-This work was supported by a grant from Instituto National de Investigacao Cientifica (FmPl). The HPLC-ECD system used in the present study was kindly donated by The Alexander von Humboldt Foundation (F.R.A.).
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