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Release of monoamines from striatal slices by phenelzine and β-phenylethylamine
Rag. Neum-Wychopharmacol6 Bill. l?sychiat. Printed in Great Britain. All rightn resmved.
1883. Vol. 7. pp. 797400 Copyright
RELEASE OF MONOAMUUES FROM STRIATAL SLICES BY PB p-P-AMINE
0
027848481n3 $0.00 + so 1983 l’ergamon FWSS Ltd.
ANJI
LILLIAN E. DYCK
Psychiatric Research Division, CMK Building, University of Saskatchewan Saskatoon, Saskatchewan, Canada
(Final form, July, 1983)
Abstract
Dyck, Lillian B.: Release of monoamines from striatal slices by phenelzine and B-phenylethylamine. Prog. Neuro-Psychopharmacol.6 Biol. Psychiat. 1983. 7(4-4):797-800. ^ 1. Slices of strlatum obtained from control rats were incubatedwEh 'll-serotonin('H-5HT) or '%dopamine (14C-DA) in the presence of pargyline; then, they were subjected to a rapid transfer technique during which they were washed either with a normal Krebs buffer or o.necontaining known quantities of phenelzine (PEH) or B-phenylethylamine(PE). 2. Both PEH and PE were able to stimulate releases of 3H-5HT and 14C-DA that were greater than control. More 14C-DA than 3H-5HT was released by both compounds. Much lower concentrationsof PE than of PEH were required to stimulate monoamine release. 3. Other rats were injected intraperitoneallywith behaviourally effective doses of either PEH or PE and were killed at various times. 4. The concentrations of PEH or of PE in the striata of these animals were very high 15 min after I.P. Injection, but declined rapidly thereafter. 5. These results may be interpreted to suggest that after I.P. injection, sufficient levels of PEH and PE were attained in the rat striatum to stimulate release of endogenous 5HT and DA.
Keywords: monoamine release, phenelsine, phenylethylamine
Abbreviations: dopamine (DA), phenelzine (PEH), B-phenylethylamine(PE), serotonin (5-HT)
Introduction
PEH is an antidepressant which is structurally similar to the trace amine, PE. Injection of PEH (Dourlsh et al 1982 and In press) or of PE (Sloviter et al 1980, Dourish 1981) Into rats rapidly elicits short periods of behavioural stimulation, the components of which are predominantly dopaminergic and serotonergic, respectively. The purpose of this study was to determine whether a monoamine releasing effect of PEH or of PE might contribute to the behavioural stimulation elicited by these compounds. In order to investigate this possibility, the -in vitro ability of PEH and PE to stimulate release of preloaded radiolabelled 5-HT and DA from rat striatal slices was examined. Additionally, the concentrations of PkH and PE in rat striatum at various times following the injection of behaviourally effective doses of these compounds was determined and compared to the -in vitro concentrationswhich caused monoamine release.
797
798
L.E. Dyck
Methods
Uptake and Release Experiments
Male Wistar rats were killed by cervical dislocation and the brain removed. The head of the striatum was dissected out rapidly, weighed (about 15 mg) and chopped in:! 0.2 mm slices. The ylices were placed in 4 ml Erebs-Henseleitbuffer containing 10 M pargyline and 1.1 x IO- M ascorbic acid and were preincubated (20 min at 37'C) in a transfer assembly with continual oxy enation. Following this, the slices were transferred to fresh buffer -9 containing 1 x 10 M 14C-DA, or 3H-5HT, and were incubated for 15 min at 37'C. After this Incubation, the slices were transferred at 2 min intervals through a series of ten tubes containing 4 ml of buffer. The last five tubes contained various concentrations of PE or PEH prepared in the Erebs buffer or just the Erebs buffer. The radioactivity released into the various tubes, or fractions, as well as the radioactivityleft in the slices was quantified by liquid scintillation counting. The amount of radioactivity in each sample was expressed as a percentage of total radioactivity. More than 95% of the radioactivity in the slices or in the various release fractions was unchanged amine.
Mass SpectrometricAnalysis of PE and PEH
The amounts of PE or PEH in rat striata obtained from PE-treated or PEH-treated rats were measured as described elsewhere (see Durden et al 1973, Philips and Boulton 1979). Briefly, the rats were killed, the strlata were dissected out quickly, and homogenized in 0.1 N HCl to which known amounts of db-PE or d4-PEH had been added. The homogenates were reacted with dansyl chloride (Sigma Chemical Co., St. Louis, MO.) to convert the compounds to their dansyl-derivativeswhich were extracted and separated unidimensionallysequentially on two different silica gel thin layers. The amounts of PE and PEH quantified from the relative signals due to the molecular ion of dansyl-PE or dansyl acetone phenylethylhydrazineand were their internal standards. Values from saline-treated rats were subtracted from PEtreated rats.
Results Effects of PEH and PE on the Release of 3H-5HT and 14C-DA
The addition of PEH to tubes 6 through 10 of the release experiments caused dosedependent releases of 3H-5HT and 14C-DA from the preloaded striatal slices (see Fig. 1). While the PEH-induced release of 3H-5HT was significantly elevated with only 1 mM PEH, 14C-DA release was significantly increased by 100 M and 1 mM PEH. 1 mM PEH released significantlymore 14C-DA than 3H-5HT.
The addition of PE (0.08, 0.8, 8.0 pM) to tubes p4through 10 of the release experiments also caused dose-dependent releases of H-5HT and C-DA (data not shown); 0.8 and 8.0 pM PE caused significant releases of 3H-5HT and "C-DA. More "C-DA than 3H-5HT was released by PE.
Amounts of PEH and PE in Bat Striatum
From Table 1, it can be seen that large concentrationsof PEH and PE (about 26 mg/g) were attained In the rat strlatum 15 min after I.P. injection of 100 mg/kg PEH or 50 mg/kg PE respectively. The levels of both PEH and PE were considerably smaller at later times.
799
Monoamine release by phenelzine and phenylethylamine
“C-DA
3H-5 HT
0
5
10
0
10
5 Fraction
Fraction
Fig. 1. The effect of PEH on the release of 3H-5HT and 14C-DA from striatal slices
Table 1
Concentrations of PEH and PE in Rat Striatum After I.P. Administration of PEH (100 mg/kg) or PE (50 mg/kg)
PE
PEH Time
pg/g
IaM
0.25 h
25.7k5.1
189k37 (4)
0.5 1.0 h 2.0 h
6.0f0.6 1.1kO.l O.SkO.2
44+ 8+ 5 1 (8) 3k 1 (4)
pglg 26.5 k5.4 0.02k0.01 1.5 kO.8
r&l 219 k44
(6)
13 0 .2+ f 0 7 .1 I!;
Values are mean + S.E., number of replicates in brackets. Assuming that the specific gravity of the tissue was 1, M values were calculated.
800
L.E. Dyck
Discussion
In an earlier study, which used a single concentration (10 pM) of PEH, PEH was found to release 5HT, but not DA, from rat striatal tissue (Baker et al 1980). Tbe present data are qualitatively similar. While a preferential release of DA rather than a release of both DA and 5HT would be more compatible with the dopaminergic nature of the initial effects of PEH, perhaps the endogenoue amines are less easily released; hence, insufficient PEH may be present to release endogenous 5HT. Interestinglythe time course of PEH accumulation paralleled the time course of the initial phase of behavioural activation (Dourish et al 1982 and in press).
The present data agree with previous findings regarding the effect of PE on the release of labelled DA and 5HT (Baker et al 1976, Raiteri et al 1977, Dyck 1981). It is evident that much lower concentrations of PE than of PEH were required to stimulate the release of DA and 5HT. From the time course of striatal PE accumulation following I.P. injection of 50 mg/kg, it appears that sufficient PE was present at 30 and 60 min to cause release of DA and/or 5HT. In addition, it is also possible, however, that PE exerted a direct post-synaptic effect (Antelman et al 1977, Hansen et al 1980, Sloviter et al 1980).
Acknowledgements
I thank Dr. A.A. Boulton for helpful comments and criticisms, Dr. D.A. Durden for supervising the mass spectrometric analyses, Dr. B.A. Davis for synthesizing the deuterated compounds, Mr. R. Mag-Atas for technical assistance and Saek. Health for financial support.
References
(1977) Phenylethylamine: evidence for a direct, post-synaptic dopamine-receptorstimulating action. Brain Bee. 127: 317-322. BAKER, G.B., BERTOLLINI, A., DEL CARMINE, R., KBANE, P.E. and MARTI.L. (1976) Interaction of B-phenylethylaminewith dopamine and noradrenaline in the central nervous system of the rat. J. Warm. Pharmac. 28: 456-457. BAKER, G.B., HIOB, L.E. and DEWHURE, W.G. (1980) Effects of monoamine oxidase inhibitors on release of dopamine and 5-hydroxytryptaminefrom rat striatum -in vitro. Cell Mol. Biol. 26: 182-186. DOURISH,~.T., BOULTON, A.A. and DYCK, L.E. (1982) Biphasic behavioural stimulation induced by a monoamine oxidase inhibiting antidepressant. Prog. Neuropsychopharmac.Biol. Psychiat. 2: 383-388. DOURISH, C.T., DEWAR, K., DYCK, L.E. and BOULTON, A.A. (in press) Potentiation of the behavioural effects of the antidepressant phenelzine by deuterium substitution. Psychopharmac. DYCK, L.E. (1981) Release of radiolabelled dopamine, E-tyramine and dopamine in rat striatal slices by some aminotetralins. Neurochem. Res. 6: 365-375. HANSEN, T.R., GREENBERG, J. and MOSNAIM, A.D. (1980) Mrez effect of phenylethylamine upon isolated rat aortic strip. Eur. J. Pharmacol. 63: 95-101. RAITERI, M., DEL CARMINE, R., BERTOLLINI, A. and LEVI,G. (1977) Effect of sympathomimetic amines on the synaptosomal transport of noradrenaline, dopamine and 5-hydroxytryptamine. Eur. J. Pharmac. 41: 133-143. SLOVITER, R.S., COER, J.D. and DRUST, E.G. (1980) Serotonergic properties of B-phenylethylaminein rats. Neuropharmac.-19: 1071-1074.
ANTELMAN, S.M., EDWARDS, D.J. and LIN, M.
Inquiries and reprint requests should be addressed to: Dr. L.E. Dyck Psychiatric Research Division CMR Building, University of Saskatchewan Saskatoon S7N OWO
1883. Vol. 7. pp. 797400 Copyright
RELEASE OF MONOAMUUES FROM STRIATAL SLICES BY PB p-P-AMINE
0
027848481n3 $0.00 + so 1983 l’ergamon FWSS Ltd.
ANJI
LILLIAN E. DYCK
Psychiatric Research Division, CMK Building, University of Saskatchewan Saskatoon, Saskatchewan, Canada
(Final form, July, 1983)
Abstract
Dyck, Lillian B.: Release of monoamines from striatal slices by phenelzine and B-phenylethylamine. Prog. Neuro-Psychopharmacol.6 Biol. Psychiat. 1983. 7(4-4):797-800. ^ 1. Slices of strlatum obtained from control rats were incubatedwEh 'll-serotonin('H-5HT) or '%dopamine (14C-DA) in the presence of pargyline; then, they were subjected to a rapid transfer technique during which they were washed either with a normal Krebs buffer or o.necontaining known quantities of phenelzine (PEH) or B-phenylethylamine(PE). 2. Both PEH and PE were able to stimulate releases of 3H-5HT and 14C-DA that were greater than control. More 14C-DA than 3H-5HT was released by both compounds. Much lower concentrationsof PE than of PEH were required to stimulate monoamine release. 3. Other rats were injected intraperitoneallywith behaviourally effective doses of either PEH or PE and were killed at various times. 4. The concentrations of PEH or of PE in the striata of these animals were very high 15 min after I.P. Injection, but declined rapidly thereafter. 5. These results may be interpreted to suggest that after I.P. injection, sufficient levels of PEH and PE were attained in the rat striatum to stimulate release of endogenous 5HT and DA.
Keywords: monoamine release, phenelsine, phenylethylamine
Abbreviations: dopamine (DA), phenelzine (PEH), B-phenylethylamine(PE), serotonin (5-HT)
Introduction
PEH is an antidepressant which is structurally similar to the trace amine, PE. Injection of PEH (Dourlsh et al 1982 and In press) or of PE (Sloviter et al 1980, Dourish 1981) Into rats rapidly elicits short periods of behavioural stimulation, the components of which are predominantly dopaminergic and serotonergic, respectively. The purpose of this study was to determine whether a monoamine releasing effect of PEH or of PE might contribute to the behavioural stimulation elicited by these compounds. In order to investigate this possibility, the -in vitro ability of PEH and PE to stimulate release of preloaded radiolabelled 5-HT and DA from rat striatal slices was examined. Additionally, the concentrations of PkH and PE in rat striatum at various times following the injection of behaviourally effective doses of these compounds was determined and compared to the -in vitro concentrationswhich caused monoamine release.
797
798
L.E. Dyck
Methods
Uptake and Release Experiments
Male Wistar rats were killed by cervical dislocation and the brain removed. The head of the striatum was dissected out rapidly, weighed (about 15 mg) and chopped in:! 0.2 mm slices. The ylices were placed in 4 ml Erebs-Henseleitbuffer containing 10 M pargyline and 1.1 x IO- M ascorbic acid and were preincubated (20 min at 37'C) in a transfer assembly with continual oxy enation. Following this, the slices were transferred to fresh buffer -9 containing 1 x 10 M 14C-DA, or 3H-5HT, and were incubated for 15 min at 37'C. After this Incubation, the slices were transferred at 2 min intervals through a series of ten tubes containing 4 ml of buffer. The last five tubes contained various concentrations of PE or PEH prepared in the Erebs buffer or just the Erebs buffer. The radioactivity released into the various tubes, or fractions, as well as the radioactivityleft in the slices was quantified by liquid scintillation counting. The amount of radioactivity in each sample was expressed as a percentage of total radioactivity. More than 95% of the radioactivity in the slices or in the various release fractions was unchanged amine.
Mass SpectrometricAnalysis of PE and PEH
The amounts of PE or PEH in rat striata obtained from PE-treated or PEH-treated rats were measured as described elsewhere (see Durden et al 1973, Philips and Boulton 1979). Briefly, the rats were killed, the strlata were dissected out quickly, and homogenized in 0.1 N HCl to which known amounts of db-PE or d4-PEH had been added. The homogenates were reacted with dansyl chloride (Sigma Chemical Co., St. Louis, MO.) to convert the compounds to their dansyl-derivativeswhich were extracted and separated unidimensionallysequentially on two different silica gel thin layers. The amounts of PE and PEH quantified from the relative signals due to the molecular ion of dansyl-PE or dansyl acetone phenylethylhydrazineand were their internal standards. Values from saline-treated rats were subtracted from PEtreated rats.
Results Effects of PEH and PE on the Release of 3H-5HT and 14C-DA
The addition of PEH to tubes 6 through 10 of the release experiments caused dosedependent releases of 3H-5HT and 14C-DA from the preloaded striatal slices (see Fig. 1). While the PEH-induced release of 3H-5HT was significantly elevated with only 1 mM PEH, 14C-DA release was significantly increased by 100 M and 1 mM PEH. 1 mM PEH released significantlymore 14C-DA than 3H-5HT.
The addition of PE (0.08, 0.8, 8.0 pM) to tubes p4through 10 of the release experiments also caused dose-dependent releases of H-5HT and C-DA (data not shown); 0.8 and 8.0 pM PE caused significant releases of 3H-5HT and "C-DA. More "C-DA than 3H-5HT was released by PE.
Amounts of PEH and PE in Bat Striatum
From Table 1, it can be seen that large concentrationsof PEH and PE (about 26 mg/g) were attained In the rat strlatum 15 min after I.P. injection of 100 mg/kg PEH or 50 mg/kg PE respectively. The levels of both PEH and PE were considerably smaller at later times.
799
Monoamine release by phenelzine and phenylethylamine
“C-DA
3H-5 HT
0
5
10
0
10
5 Fraction
Fraction
Fig. 1. The effect of PEH on the release of 3H-5HT and 14C-DA from striatal slices
Table 1
Concentrations of PEH and PE in Rat Striatum After I.P. Administration of PEH (100 mg/kg) or PE (50 mg/kg)
PE
PEH Time
pg/g
IaM
0.25 h
25.7k5.1
189k37 (4)
0.5 1.0 h 2.0 h
6.0f0.6 1.1kO.l O.SkO.2
44+ 8+ 5 1 (8) 3k 1 (4)
pglg 26.5 k5.4 0.02k0.01 1.5 kO.8
r&l 219 k44
(6)
13 0 .2+ f 0 7 .1 I!;
Values are mean + S.E., number of replicates in brackets. Assuming that the specific gravity of the tissue was 1, M values were calculated.
800
L.E. Dyck
Discussion
In an earlier study, which used a single concentration (10 pM) of PEH, PEH was found to release 5HT, but not DA, from rat striatal tissue (Baker et al 1980). Tbe present data are qualitatively similar. While a preferential release of DA rather than a release of both DA and 5HT would be more compatible with the dopaminergic nature of the initial effects of PEH, perhaps the endogenoue amines are less easily released; hence, insufficient PEH may be present to release endogenous 5HT. Interestinglythe time course of PEH accumulation paralleled the time course of the initial phase of behavioural activation (Dourish et al 1982 and in press).
The present data agree with previous findings regarding the effect of PE on the release of labelled DA and 5HT (Baker et al 1976, Raiteri et al 1977, Dyck 1981). It is evident that much lower concentrations of PE than of PEH were required to stimulate the release of DA and 5HT. From the time course of striatal PE accumulation following I.P. injection of 50 mg/kg, it appears that sufficient PE was present at 30 and 60 min to cause release of DA and/or 5HT. In addition, it is also possible, however, that PE exerted a direct post-synaptic effect (Antelman et al 1977, Hansen et al 1980, Sloviter et al 1980).
Acknowledgements
I thank Dr. A.A. Boulton for helpful comments and criticisms, Dr. D.A. Durden for supervising the mass spectrometric analyses, Dr. B.A. Davis for synthesizing the deuterated compounds, Mr. R. Mag-Atas for technical assistance and Saek. Health for financial support.
References
(1977) Phenylethylamine: evidence for a direct, post-synaptic dopamine-receptorstimulating action. Brain Bee. 127: 317-322. BAKER, G.B., BERTOLLINI, A., DEL CARMINE, R., KBANE, P.E. and MARTI.L. (1976) Interaction of B-phenylethylaminewith dopamine and noradrenaline in the central nervous system of the rat. J. Warm. Pharmac. 28: 456-457. BAKER, G.B., HIOB, L.E. and DEWHURE, W.G. (1980) Effects of monoamine oxidase inhibitors on release of dopamine and 5-hydroxytryptaminefrom rat striatum -in vitro. Cell Mol. Biol. 26: 182-186. DOURISH,~.T., BOULTON, A.A. and DYCK, L.E. (1982) Biphasic behavioural stimulation induced by a monoamine oxidase inhibiting antidepressant. Prog. Neuropsychopharmac.Biol. Psychiat. 2: 383-388. DOURISH, C.T., DEWAR, K., DYCK, L.E. and BOULTON, A.A. (in press) Potentiation of the behavioural effects of the antidepressant phenelzine by deuterium substitution. Psychopharmac. DYCK, L.E. (1981) Release of radiolabelled dopamine, E-tyramine and dopamine in rat striatal slices by some aminotetralins. Neurochem. Res. 6: 365-375. HANSEN, T.R., GREENBERG, J. and MOSNAIM, A.D. (1980) Mrez effect of phenylethylamine upon isolated rat aortic strip. Eur. J. Pharmacol. 63: 95-101. RAITERI, M., DEL CARMINE, R., BERTOLLINI, A. and LEVI,G. (1977) Effect of sympathomimetic amines on the synaptosomal transport of noradrenaline, dopamine and 5-hydroxytryptamine. Eur. J. Pharmac. 41: 133-143. SLOVITER, R.S., COER, J.D. and DRUST, E.G. (1980) Serotonergic properties of B-phenylethylaminein rats. Neuropharmac.-19: 1071-1074.
ANTELMAN, S.M., EDWARDS, D.J. and LIN, M.
Inquiries and reprint requests should be addressed to: Dr. L.E. Dyck Psychiatric Research Division CMR Building, University of Saskatchewan Saskatoon S7N OWO