Absence of 2-phenylethylamine binding after monoamine oxidase inhibition in rat brain

European Journal of Pharmacology, 210 (1992) 189-193 •

189

~ 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52226

Absence of 2-phenylethylamine binding after monoamine oxidase inhibition in rat brain X i n - M i n Li, A u g u s t o V. J u o r i o , I. A l i c k P a t e r s o n a n d A l a n A. B o u l t o n Neuropsychiatric Research Unit, Medical Research Building, Unit'ersity of Saskatchewan, Saskatoon. Saskatchewan, Canada S7N OWO

Received 18 July 1991, revised MS received 4 October 1991, accepted 22 October 1991

Earlier work has suggested the existence of saturable and highly specific binding sites for [3H]2-phenylethylaminc in rat forebrain membranes. Since monoamine oxidase (MAO) was not inhibited during the assay, the [3H]2-phenylethylamine binding may have been affected by an interaction between 2-phenylethylamine and the enzyme. This is an investigation of [3H]2-phenylethylamine binding to rat forebrain membranes in the presence of two MAO inhibitors, ( -)-deprenyl and pargyline. The results show that the high affinity specific binding of [3H]2-phenylethylamine to rat forebrain membranes is inhibited by pretreatment of the membrane with the MAO inhibitors and in vivo injection of the MAO inhibitors in a concentration-dependent manner. In the presence of higher concentrations of MAO inhibitors, the specific binding of [3H]2-phenylethylaminc is completely blocked, suggesting that the binding sites reported earlier represent binding to MAO-B. 2-Phenylethylamine; 2-Phenylethylamine binding sites; MAO (monoamine oxidase); MAO (monoamine oxidase) inhibitor; Pargyline; ( )-Deprenyl

I. Introduction 2-Phenylethylamine has a heterogeneous distribution in invertebrate, rat and human neural tissues where it occurs in low endogenous concentrations (Durden et al., 1973; Philips et al., 1978; Juorio and Philips, 1976; Saavedra, 1974). Electrolytic or 6-hydroxydopamine lesion of the substantia nigra resulted in a decrease of striatal 2-phenylethylamine in rats pretreated with a monoamine oxidase ( M A O ) inhibitor (Greenshaw et al., 1986). In addition, electrical stimulation of the nigrostriatal pathway produced a reduction in the M A O inhibitor-induced accumulation of striatal 2-phenylethylamine (Juorio et al., 1991), suggesting that 2-phenylethylamine co-exists with dopamine in the nigrostriatal system. In electrophysiological investigations on single neurones in rat brain, small doses of 2-phenylethylamine possess a modulatory action on neuronal responses to the catecholamines noradrenaline (NA) and dopamine (DA) (Jones and

Correspondence to: X.-M. Li, Neuropsychiatric Research Unit, Medical Research Building, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0. Tel. 1.306.966 8815, fax 1.306.966 8830.

Boulton, 1980; Paterson and Boulton, 1988; Paterson et al., 1991). As a result of these experiments, it has b e e n proposed that 2-phenylethylamine acts as neuromodulator of dopaminergic transmission (see review by Paterson et al., 1990). The occurrence of saturable and highly specific binding sites for [3H]2-phenylethylamine in rat forebrain m e m b r a n e s has been described (Hauger et al., 1982). The binding of 2-phenylethylamine was not displaced by neuroleptics, tricyclic antidepressants, benzodiazepines, a m p h e t a m i n e or compounds which bind to noradrenergic, dopaminergic and cholinergic binding sites. In these studies, however, M A O was not inhibited and since 2-phenylethylamine is a very good substrate of M A O - B with high affinity and Vm~×, [3H]2-phenylethylamine binding may have been affected by an interaction between 2-phenylethylamine and the enzyme (Nguyen and Juorio, 1989). In the absence of M A O - B inhibition and even at the low t e m p e r a t u r e that the assay was carried out (0°C) (Hauger et al., 1982), it is likely that some of the tritiated and cold 2-phenylethylamine in the assay was metabolized by the enzyme. The present experiments have d e t e r m i n e d whether [3H]2-phenylethylamine binding in rat neuronal m e m b r a n e s is observed after M A O inhibition produced by parenteral administration of two irreversible M A O inhibitors ( - ) - d e p r e n y l and pargyline.

190

2. Materials and methods

2.1. Animals Male Wistar rats (Charles River Canada, Montreal, Quebec) weighing 230-250 g were used. The animals, housed in groups of six to eight in hanging wire cages, had free access to food and water and were kept on a 12 h light/dark cycle (light on at 6 a.m.) at a temperature of 19-21°C.

2.2. Tissue preparation The rats were stunned and decapitated, their brains immediately removed and cooled with ice-cold saline. The forebrain areas rostrat to the colliculi was dissected out on ice. The tissue was homogenized in 100 volumes ( w / v ) of ice-cold buffer (50 mM Tris HCI, pH 7.5) using a Brinkmann Polytron (setting 5 for 15 s), centrifuged at 40 000 × g for 15 min at 4°C. The resulting pellet was re-suspended in an equal volume of buffer, centrifuged again and then diluted with Tris HCI buffer for binding assay and with H 2 0 for MAO assay.

2.3. [-~H]2-Phenylethylamine binding The binding assays were performed basically follow the procedure of Hauger et al. (1982). The various mixtures in 50 mM Tris HC1, pH 7.5, including 2-phenylethylamine and a membrane preparation (20 mg wet tissue weight per ml) were incubated for 1 h at 0°C and terminated by rapid filtration through Whatman G F / B glass fiber filters. Liquid scintilation counting was performed in 10 ml ACS cocktail using Beckman LS 5000 TD counter at efficiency about 37% for 3H. Specific binding is defined as the difference of radioactivity bound in the presence and absence of the unlabeled 2-phenylethylamine (100/xM). In the saturation studies the concentrations of [3H]2-phenylethylamine ranged from 12.5 to 400 nM. To study the effects of different MAO inhibitors on 2-phenylethylamine binding 100 nM [3H]2-phenylethylamine was used. All the binding assays were performed in triplicates and the binding data from the saturation studies were analyzed by the equilibrium binding data analysis program (EBDA) (McPherson, 1983).

2. 4. Inhibition of MA 0 To inhibit MAO activity, we performed two experiments: (1) Before the incubation for binding assay, treatment of membrane preparations with 100 ~ M

pargyline for 30 min at 37°C, following by one wash with Tris HCI buffer (50 raM, pH 7.5) before the incubation of binding assay; (2) Administration of single doses of ( - ) - d e p r e n y l and pargyline (1, 5, 10, 5(1, 100 mg kg ~) given by intraperitoneal (i.p) injection 4 h before decapitation and dissection of brain tissue.

2. 5. MA 0 actit,ity assay The MAO assay was adopted from Yu and Boulton (1979). The reaction mixture contained 0.05 M potassium phosphate buffer (pH 7.5 at 25°C), an appropriate [14C]-labelled substrate (0.05/xCi) mixed with the nonradioactive substrate to yield final concentrations 10 - 4 M 5-HT (MAO-A) or 0.25 × 10 - 4 M 2-phenylethylamine (MAO-B) and rat forebrain membranes in final volume of 200 /xl. The enzyme reactions were carried out at 37°C for 30 rain, then terminated by adding 250 /xl 2 mol citric acid. The formed acid products were extracted into 1 ml of toluene: ethylacetate (1 : 1, v/v). The organic extracts (500 ~1) were transferred to counting vials for liquid scintilation counting in 10 ml ACS cocktail using Beckman LS 5000 TD counter at efficiency about 90% for J4C. Blank values were obtained from reaction mixtures containing no enzyme. The protein concentration was determined by the Lowry method (Lowry et al., 1951), using bovine serum albumin as a standard and MAO activity (nmol rain-l mg protein- l ) was calculated.

2.6. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) and the statistical differences between groups were determined by the Newman-Keuls test (Winer, 1971). Linear regression was analyzed by the least-squares method and correlation by Pearson's product-moment method. Analysis of variance was performed on a Macintosh microcomputer using the CLR A N O V A program (Clearlake Research, Houston, Texas, U.S.A.).

2. 7. Drugs and chemicals Pargyline hydrochloride, 5-HT, 2-phenylethylamine and bovine serum albumin were obtained from Sigma Chemicals, St. Louis, MO, ( - ) - d e p r e n y l hydrochloride from Research Biochemicals Natick, MA., [3H]2-phenylethylamine hydrochloride from Amersham, England, [14C]2-phenylethylamine hydrochloride and [14C]5-hydroxytryptamine bioxalate from NEN, Boston, MA. All other reagents and solvents were of analytical grade.

191 MAO-A

3. Results

125] ~ O pZ O u.. O ~

3.1. Effect of addition of pargyline to neuronal membranes In the absence of MAO inhibition, the specific binding of [3H]2-phenylethylamine to rat forebrain membranes was saturable. Transformation by Scatchard analysis revealed that Ko = 155 nM and the Bin,~ = 1268 fmol mg protein ~ (fig. 1A). These values are quite close to those reported earlier (Hauger et al., 1982). The pretreatment of rat forebrain membranes with pargyline (100 /zM) before the binding assay was carried out, however, prevented the [3H]2-phenylethylamine binding to rat forebrain membranes and the remaining binding was not saturable (fig. 1B).

100 75 50 25 0 0 MAO-B 125

O nIZ

1

5

10

50

100

10

50

100

100 75

0 ,, 0

,'~,"

50 25

~

0 0

3.2. Effects of ( - )-deprenyl and pargyline injections

1

5

PE B I N D I N G

Four hours after administration (i.p.) of different doses of MAO inhibitors (1, 5, 10, 50, 100 mg/kg), it was shown that both ( - ) - d e p r e n y l and pargyline could block the specific binding of[ 3H]2-phenylethylamine to forebrain membranes and inhibit MAO activities in a dose-dependent manner (figs. 2, 3). Following ( - ) - d e prenyl administration, 2-phenylethylamine binding was reduced to the levels of 73, 35, 25, 13 and 8% of the control values at the doses of 1, 5, 10, 50 and 100 mg kg =, respectively (fig. 2, F(5,66)=502, P < 0.001). ( - ) - D e p r e n y l produced a selective inhibition on MAO-B and a dose of 1 mg kg J inhibited MAO-B to 48% ef the controls (fig. 2, F(5,30) = 2121, P < 0.001). MAO-A was inhibited with doses that were higher than 5 mg kg I (fig. 2, F(5,30)= 971, P < 0.001). For pargyline-injected rat forebrain tissues, the binding was lowered to 65, 47, 29, 21 and 16% of the control values at the doses of 1, 5, 10, 50 and 100 mg kg ], respectively (fig. 3, F(5,661 = 68, P < 0.001). MAO-A activity

2,0•

D.

100

=J c¢

75

X
50

o~

0

25

1

5

10 mg/kg,

50

100

i.p. 4 h

Fig. 2. Dose course of the effect of ( )-deprenyl (i.p. 4 h) on specific [~H]2-phenylethylamine (100 nM) binding to rat forebrain membranes, MAO type A and type B activities. * P < 0.01 vs. control (maximal binding of control, 596_+53 fmol mg protein t activity of control, 0.041_+0.001 and 0.138_+0.008 nmol min ] mg protein ] for MAO-A and MAO-B, respectively).

was reduced significantly after the doses of pargyline were 5 mg kg i or higher (F(5,30)= 420, P < 0.001). As expected, MAO-B activity was reduced with all

B

0.5

1.6-.

LLI 0,. u'J

Z

DEPRENYL,

-13

(3 zi z m c~ m u.

125

0

0.6

A

(3 z

0.4 1.2-

0.3 0.8

KD = 155nM Bmax = 1268 frnoles/mg protein

0.4

0.0

.

.

1;o

.

.

.

0.1 0.0

,oo

[3H]-PHENYLETHYLAMINE

0.2

0 (nM)

100

200

300

400

[3H]-PHENYLETHYLAMINE

500 (nM)

Fig. 1. Saturation curves of specific [3H]2-phenylethylamine binding to rat forebrain membranes without MAO inhibition (A) and to rat forebrain membranes which were treated with 100/xM pargyline at 37°C for 30 min before the incubation (B). After transformation of equilibrium binding data by Scatchard analysis, the value of K a was 155 nM and the Bin, Xwas 1268 fmol mg protein i in the absence of pargyline. The concentration of [3H]2-phenylethylamine ranged between 16 and 480 nM.

192 MAO-A

forebrain neuronal membranes and the inhibition of MAO-B activity (r = 0.996, P < 0.01 for pargyline, r = 0.986, P < 0.01 for deprenyl) (fig. 4). There was no correlation between the inhibition of the [3H]2-phenylethylamine binding and inhibition of MAO-A.

125 ~) n,IZ O ,O u. O o~

100 75 50 25 0 0

8~

I.-Z 0 0 ~. 0 ~

1

5

10

50

100

MAO-B 125

4. Discussion

100 75 50 25 0 0

1

5

10

1

5

10

50

100

PE BINDING 125 Z Z

100

._1

75

X =E

50

" O

25 0 0

PARGYLINE,

mg/kg,

50

100

i.p. 4 h

Fig. 3. Dose course of the effect of pargyline (i.p. 4 h) on specific [3HJ2-phenylethylamine (100 nM) binding to rat forebrain membranes, MAO type A and type B activities. * P < 0.01 vs. control (maximal binding of control, 541 + 24 fmol mg protein t, activity of control, 0.043_+0.001 and 0.129_+0.005 nmol min I mg protein i for MAO-A and MAO-B, respectively).

doses used (fig. 3, 1-100 mg kg - j , F(5,30)=211, P < 0.001). There was a significant correlation between the inhibition of [3H]2-phenylethylamine binding by rat

PARGYLINE >i.m > I0 <

100-

R=

Using the same binding conditions that were used by Hauger et al. (1982), 50 mM Tris HCI, p H 7.5, 1 h at 0°C and without inhibition of MAO, we find similar experimental binding kinetic parameters (fig. 1A). The specific binding of [3H]2-phenylethylamine was saturable and Scatchard analysis revealed a dissociation constant ( K j ) of 155 nM and a maximum number of binding sites (B ..... ) of 1268 fmol mg p r o t e i n - ] comparing with a dissociation constant (K d) of 55 nM and a maximum number of binding sites (B .... ) of 1078 fmol mg protein l described by Hauger et al. (1982). However, by inhibiting M A O either in vitro and in vivo, the specific binding of 2-phenylethylamine was significantly reduced to an extent dependent on the degree of inhibition of MAO-B. With the lower doses of ( - )-deprenyl or pargyline which inhibit MAO-B but not M A O - A the 2-phenylethylamine specific binding was already significantly reduced (figs. 2, 3). With higher doses or concentrations of ( - ) - d e p r e n y l or pargyline, both M A O - B and M A O - A were inhibited (MAO-B was more sensitive), the 2-phenylethylamine binding was totally blocked. Pargyline and ( - ) - d e p r e n y l at low doses, are irreversible MAO-B-selective inhibitors. There was a very good correlation between the inhibition of the 2-phenylethylamine binding and the inhibition of MAO-B activity (fig. 4) and with no significant correlation with M A O - A activity. In both pretreatment

DEPRENYL

.

100 "

80.

80"

60.

60"

40.

40

20

20"

R=0.986

6 =E x <

P, o~

0

0 0

2

4

%OF MAXIMAL

20 BINDING

40

60

% OF MAXIMAL

80

100

BINDING

Fig. 4. Correlation beween [3H]2-phenylethylamine binding and MAO-B activity after MAO inhibitor treatments. The regression line was fitted by least-squares linear regression•

193

of membrane and in vivo administration of the MAO inhibitors, MAO was inhibited irreversibly, and it is unlikely that the MAO inhibitors were present in binding medium in significant concentrations. This minimizes the possibility that the MAO inhibitors are acting as antagonists of a putative 2-phenylethylamine receptor. Under these conditions therefore there is no binding of 2-phenylethylamine to membrane receptor site and we conclude that the specific binding of [3H]2-phenylethylamine to rat forebrain membranes reported earlier (Hauger et al., 1982) is most likely the consequence of binding to MAO-B. Previous studies from this laboratory have shown that there may be a functional relationship between 2-phenylethylamine and DA. IontophoreticaIly applied 2-phenylethylamine potentiates neuronal responses to iontophoretically applied DA without affecting the spontaneous firing rate (Jones and Boulton, 1980). lontophoretically applied 2-phenylethylamine also potentiates caudate neurone responses to iontophoretically applied D A and electrical stimulation of the substantia nigra (Paterson et al., 1990). With the spontaneously active cells, injection of 2-phenylethylamine resulted in a decrease in the ITs0 (which is calculated by multiplying the iontophoretic current by the time to 50% inhibition of the firing rate) of the responses to both apomorphine and (+_)-2-(N-phenethy[-N-propyl) amino-5-hydroxytetralin (PPHT), indicating an increase in the neuronal sensitivity to these agonists (Paterson et al., 1991). These electrophysiological observations are supported by studies on the neuronal membrane fluidity using electron spin resonance, that is, the change in membrane fluidity induced by D A was potentiated by the addition of 0.5 /xM 2-phenylethylamine even though this concentration of 2-phenylethylamine had no effect on membrane fluidity by itself (Harris et al., 1988). On the basis of these findings, it has been proposed that 2-phenylethylamine may have a neuromodulatory role in D A neurotransmission. 2Phenylethylamine somehow can potentiate the effect of D A and it is likely that a postsynaptic mechanism is involved in the modulation of D A transmission (Paterson et al., 1990). These findings suggest that there must be a receptor site through which 2-phenylethylamine mediates its actions. The present findings do not exclude the existence of such a site because we only performed the binding studies in one buffer system. It remains to be demonstrated whether 2-phenylethylamine regulates some D A or NE postsynaptic receptor types or some post-receptor events of D A transmission.

Acknowledgements We wish to thank Saskatchewan Health and the Canadian MRC for continuing financial support. X.-M.L. is a Saskatchewan Health Research Board Fellow.

References Durden, D.A., S.R. Philips and A.A. Boulton, 1973, Identification and distribution of /3-phenylethylamine in the rat, Can. J. Biochem. 51,995. Greenshaw, A.J., A.V. Juorio and T.-V. Nguyen, 1986, Depletion of striatal /3-phenylethylamine following dopamine but not 5-ttT denervation, Brain Res, Bull. 15, 477. Harris, J., S. Trivedi and B.L. Ramakrishna, 1988, A contribution to the neuromodulatory/neurotransminer role of trace amines, in: Trace Amines. Comparative and Clinical Neurobiology, eds. A.A. Boulton, A.V. Juorio and R.G.FI. Downer (Humana Press, Clifton, NJ) p. 213. Hauger, R.I., P. Skolnick and S.M. Paul, 1982, Specific [3Hl/3-phenylethylamine binding sites in rat brain, Eur. J. Pharmacol. 83, 147. Jones, R.S.G. and A.A. Boulton, 1980, Interactions between p-tyramine, m-tyramine or/3-phenylethylamine and dopamine on single neurons in the cortex and caudate nucleus of the rat, Can. J. Physiol. Pharmacol. 58, 222. Juorio, A.V. and S.R. Philips, 1976, Arylalkylamines in Octopus tissues, Neurochem. Res. 1,501. Juorio, A.V., I.A. Paterson, M.Y. Zhu and G. Matte, 199l, Electrical stimulation of the substantia nigra and changes of 2-phenylethylamine in the rat striatum, J. Neurochem. 56, 213. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. McPherson, G.A., 1983, A practical computer-base approach to the analysis of radioligand binding experiments, Comput. Programs Biomed. 17, 107. Nguyen, T.V. and A.V. Juorio, 1989, Binding sites for brain trace amines, Cell. Mol. Neurobiol. 9, 297. Paterson, I.A. and A.A. Boulton, 1988, ~-Phenylethylamine enhances single cortical neurone responses to noradrenaline in the rat, Brain Res. Bull. 20, 173. Paterson, I.A., A.V. Juorio and A.A. Boulton, 1990, 2-Phenylethylamine: a modulator of catecholamine transmission in the mammalian central nervous system?, J. Neurochem. 55, 1297. Paterson, I.A., A.V. Juorio, M.D. Berry and M.Y. Zhu, 1991, Inhibition of monoamine oxidase-B by ( - ) - d e p r e n y l potentiates neuronal responses to dopamine agonists but does not inhibit dopamine catabolism in the rat striatum, J. Pharmacol. Exp. Ther. 258 (in press). Philips, S.R., B. Rozdilsky and A.A. Boulton, 1978, Evidence for the presence of m-tyramine, p-tyramine, tryptamine and phenylethylamine in the rat brain and several areas of the human brain, Biol. Psychiat. 13, 51. Saavedra, J.M., 1974, Enzymatic isotopic assay for the presence of /3-phenylethylamine in brain, J, Neurochem. 22, 211. Winer, B.J., 1971, Statistical Principles in Experimental Design (McGraw-Hill Inc., New York). Yu, P.H. and A.A. Boulton, 1979, Activation of platelet monoamine oxidase by plasma in the human, Life Sci. 25.31.