Psycho
Psychopharmacology 48, 295-301 (1976)
pharmacology 9 by Springer-Verlag 1976
Mepiprazole, a New Psychotropic Drug" Effects on Uptake and Retention of Monoamines in Rat Brain Synaptosomes P. PLACHETA*, E. SINGER, W. KRIWANEK, and G. HERTTING Institute of Pharmacology, Vienna, Austria and Institute of Pharmacology, Freiburg i. Br., Federal Republic of Germany
Abstract. The influence of mepiprazole (EMD 16,923), a new pyrazol-ylalkyl-piperazine derivative, on the uptake of 3H-norepinephrine (NE), 3H-dopamine (DA), and 3H-serotonin (5-HT) into rat brain synaptosomes from cerebral cortex, corpus striatum, and hypothalamus was investigated in comparison with several psychotropic drugs, including oxypertine, d-amphetamine, imipramine, desipramine, chlorimipramine, amitriptyline, and chlorpromazine in vitro. Mepiprazole was a relatively weak inhibitor of monoamine uptake and exhibited its strongest action on the hypothalamic 5-HT uptake, being almost equipotent with desipramine (ICso = 0.9 gM). Furthermore, the influence of the drugs on the retention of 3H-amines previously taken up by whole rat brain synaptosomes was studied. Unlike the tricyclic antidepressants, mepiprazole as well as oxypertine and d-amphetamine markedly increased the efflux of' radioactivity during a 20-min incubation at 37~ at low concentrations (10 -6 to 10 -5 M), whereas at 10 -.4 M all drugs greatly enhanced the efflux. The ability of mepiprazole to increase 5-HT concentration at the receptor level by a combination of neuronal uptake ;inhibition and release is discussed in relationship to the central actions of the drug.
INTRODUCTION Mepiprazole (EMD 16,923) is a representative compound of a new class of psychotropic drugs, the pyrazolyl-alkyl-piperazine derivatives (Fig. 1). It has been found to have a strong tranquillizing action in several laboratory animals, including rats and rhesus monkeys (Mfiller-Calgan, 1971 ; Miiller-Calgan, 1974). Mepiprazole has a low neuroleptic potency and only weak if any adrenolytic or anticholinergic properties (Goncalves, 1972). Clinically, mepiprazole has been shown to be effective as a minor tranquillizer in various emotional and psychosomatic conditions (Henkel and Bastine, 1972; Goncalves and Fieguth, 1973; Cuasnicu, 1974; Dotevall and Groll, 1974). Among the target symptoms tested the relief of anxiety and depression has been observed frequently (Foguet, 1974; P61dinger, 1975). In view of the possible involvement of biogenic amines in the central actions of mepiprazole the present study was designed to investigate its effects on some neurotransmitter mechanisms in the rat brain in vitro. The effects of several centrally active drugs have been attributed to their inhibitory activity on the neuronal reuptake system or to their potency
Key words: Synaptosomes - Monoamine uptake Monoamine release - Mepiprazole - Oxypertine d-Amphetamine - Imipramine - Desipramine Chlorimipramine - Amitriptyline - Chlorpromazine.
92 H C /
MEPIPRAZOLE t- (m - chlorop henyt) - 4 - [2- (5 - me thyl - 3-pyrezoty/) piperezine
*
Address for offprints: Dr. Peter Placheta, Institute of Pharma-
cology, University of Vienna, W~hringer Strage 13a, A- 1090Vienna, Austria.
dihydrochloride
EMD
16923
Fig. 1
- ethyl] -
296
Psychopharmacology 48 (1976)
to e v o k e o r a t t e n u a t e the release o f n e u r o t r a n s m i t t e r s . W e h a v e c o m p a r e d the effects o f m e p i p r a z o l e w i t h t h e effects o f s o m e o t h e r c e n t r a l l y active d r u g s , i n c l u d i n g oxypertine, d-amphetamine, imipramine, desipramine, chlorimipramine, amitriptyline, and chlorpromazine o n the u p t a k e o f 3 H - l a b e l e d n o r e p i n e p h r i n e ( N E ) , dopamine (DA), and serotonin (5-HT) into synaptos o m e s p r e p a r e d f r o m rat b r a i n c e r e b r a l cortex, c o r p u s s t r i a t u m , a n d h y p o t h a l a m u s , respectively. F u r t h e r m o r e , the i n f l u e n c e o f these d r u g s o n t h e release o f 3 H - N E , 3 H - D A , a n d 3 H - 5 - H T f r o m w h o l e rat b r a i n s y n a p t o s o m e s was studied. S y n a p t o s o m e s ( p i n c h e d - o f f n e r v e t e r m i n a l s ) h a v e p r o v e d to be a v a l u a b l e t o o l for the i n v e s t i g a t i o n o f d r u g effects o n n e u r o n a l u p t a k e a n d release m e c h a n i s m s in vitro.
MATERIALS
AND
METHODS
Chemicals and Drugs. All reagents used were of analytical grade. l-[73H]-norepinephrine (10.9 Ci/mmol), aH-dopamine (3,4 dihydroxy[ring-G-3H]phenylethylamine hydrochloride; 2.9 Ci/mmol), and 3H-serotonin (5-hydroxy[G-3H]tryptamine creatinine sulfate; 17.3 Ci/mmol) were purchased from the Radiochemical Centre, Amersham. The drugs were: mepiprazole (E. Merck AG); oxypertine (Winthrop); imipramine, desipramine, and chlorimipramine (Ciba-Geigy AG); amitriptyline (Merck, Sharp & Dohme); damphetamine (Smith, Kline & French); chlorpromazine (Bayer AG); and nialamid (Pfizer). All concentrations indicated are for the free base.
Animals. Male adult Sprague-Dawley rats (240--300 g) were sacrificed by cervical dislocation, their brains rapidly removed, placed on a chilled glass plate and the brain areas dissected as described by Glowinski and Iversen (1966). For the uptake studies three brain areas were used: cerebral cortex, corpus striatum, and hypothalamus. When necessary, tissues from 2 to 6 rats were pooled, weighed, a n d homogenized for preparation of synaptosomes. In the release studies whole brains (without cerebellum) were used.
Tissue Preparation. A crude synaptosomal fraction was prepared by differential centrifngation using the method of Whittaker (1969). Tissues were homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose in a Thomas B 10001 glass homogenizer with a motor driven loosely fitting teflon pestle using six complete up and down strokes within approximately 1 min. Homogenates were centrifuged at 1000 x g for 10 rain at 4 ~C. The pellet (P1) was discarded and the supernatant ($1) was centrifuged at 14000 xg for 15 min at 4 ~C. The remaining pellet (P2) containing synaptosomes and other cellular components was washed once with ice-cold sucrose, centrifuged again, and carefully resuspended in ice-cold incubation medium. The incubation medium contained: 100mM NaCI; 5 mM KC1; 15 mM NazHPO~, 15 mM NaHaPO4, pH 7.0; 10 mM d-glucose; 40mM sucrose; 12.SpM nialamide and 25gg/ml ascorbic acid. Uptake Studies. For estimation of the kinetic constants of 3Hamine uptake portions of the resupended P2-pellet, equivalent to approximately 20 mg (cerebral cortex), 2 mg (corpus striatum), or 12.5 mg (hypothatamus) original tissue wet wt, respectively, were preincubated for 4 min at 37~ in a shaking water bath under air. Then, the 3H-labeled amines were added in 20 ~tl of medium to the samples (final volume of 1.0 ml) and the incubations were continued for another 5 min. Incubations were stopped by cooling the samples on ice followed by immediate ultrafiltration with cellulose nitrate filters (Schleicher & Schiill, BA 90, 0.6 gm
pore size, 26 mm in diameter) by suction. The filters were rinsed 3 times with 4 ml of ice-cold medium, blotted and placed into scintillation vials containing 2ml of ethyleneglycolemonoethylether (E. Merck AG) and total 3H-radioactivity was determined by liquid scintillation spectrometry. The quenching was corrected by internal standardization. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. The synaptosomal protein yield ranged from 25 to 40 mg/g of tissue wet wt. Uptake was expressed as the initial rate of accumulation during 5 min/mg of synaptosomal protein. Values were corrected for 'uptake' at 0~C which accounted for less than 5 of the radioactivity taken up in control samples incubated at 37~C. Kinetic analysis was performed by plotting the reciprocal of the amine concentration against the reciprocal of the initial uptake velocity according to Lineweaver and Burk (1934). When the effects of drugs on the amine uptake was studied, drugs at various concentrations were included during the 4-min preincubation period. Then, the 3H-amines were added to give a final concentration of 0.05 JaM. ICs0-values (drug concentration required to inhibit the uptake during 5 min by 50 ~) were determined graphically within the range of at least four concentrations of inhibitor by plotting inhibitor concentration against percent inhibition of control uptake using a logarithmic probability paper.
Release Studies. The Pz-pellet was prepared essentially as described for the uptake studies from a whole rat brain (without cerebellum) and preloaded with 0.05 gM 3H-labeled amine during 15 rain at 37~ in an incubation volume of 10 ml/g original tissue wet wt. The particles were then centrifuged at 14000 x g for 15 min at 4~C, washed, centrifuged again, and resuspended in ice-cold incubation medium. The radioactivity taken up by the synaptosomes was fully retained at 0~C over a period of at least 20 min. When incubated at 37~C for 20 rain there was a spontaneous efflux of radioactivity that amounted to about 25 7o of the initial value. Portions of precharged synaptosomes (0.2 ml) were incubated for 20 min at 37~C without or with varying concentrations of drugs in a final volume of 1.0 ml. The incubations were stopped by cooling the samples on ice and the radioactivity retained in the synaptosomes was determined as described in the uptake studies. The ability of drugs to release amines during 20 min at 37~C was assessed by comparing the radioactivity retained in drug-treated samples with control samples as percent of radioactivity initially accumulated.
RESULTS
Kinetic Constants of 3H-Amine Uptake into Synaptosomes. I n t h e r a n g e o f c o n c e n t r a t i o n s o f a m i n e s u s e d i n o u r e x p e r i m e n t s the i n c o r p o r a t i o n o f r a d i o a c t i v i t y was a l i n e a r f u n c t i o n o f t i m e for at least 5 m i n i n all b r a i n areas. T h e r e f o r e , t h e u p t a k e rate d u r i n g 5 - m i n i n c u b a t i o n was c h o s e n for the e s t i m a t i o n o f k i n e t i c c o n s t a n t s . K i n e t i c a n a l y s i s was p e r f o r m e d a c c o r d i n g to L i n e w e a v e r a n d B u r k (1934) u s i n g at least five c o n c e n t r a t i o n s o f a m i n e r a n g i n g f r o m 0.04 to 0.5 g M . T h i s was r e p e a t e d at least 3 times to c o n f i r m h o m o g e n e i t y o f the p r e p a r a t i o n s . T h e a p p a r e n t Kinv a l u e s for the u p t a k e o f 3 H - N E , 3 H - D A , a n d 3 H - 5 - H T are s h o w n i n T a b l e 1.
Inhibition by Drugs of 3H-Amine Uptake into Synaptosomes Prepared from Cerebral Cortex, Corpus Striatum, and Hypothalamus. T h e effects o f d r u g s o n the u p t a k e o f 3 H - a m i n e s i n t o the s y n a p t o s o m a l p r e p a r a -
P. Placheta et al. : Mepiprazole on Synaptosomes
297
Table 1. Apparent Kin-values of 3H-amine uptake into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus
Table 3. Effect of drugs on uptake of 3H-DA into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus
3H-amine
Drug
Km (p.M) Cortex cerebri
Corpus striatum
Hypothalamus
Norepinephrine
0.27 • 0.02" (3) b
1.05 +_ 0.12 (3)
0.25 __+0.03 (3)
Dopamine
0.08 • 0.01 (5)
0.07 • 0.01 (9)
0.11 _ 0.02 (3)
Serotonin
0.05 • 0.01 (3)
0.13 • 0.03 (3)
0.05 • 0.02 (3)
a Mean +_ S.E.M. b Number of experiments. Synaptosomes (P2-pellet) were preincubated for 4 m i n at 37~ and incubated for another 5 min with at least five different concentrations of 3H-amine ranging from 0.04 to 0.5 p-M in duplicate. Km-values were estimated from double reciprocal plots according to Lineweaver and Burk. Table 2. Effect of drugs on uptake of 3H-NE into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
ICso (p-M)
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
IC5o (p-M) Cortex cerebri
Corpus striatum
Hypothalamus
11.0 1.8 0.21 19.0 17.0 12.0 11.5 15.0
~2.0 1.7 0.24 19.0 14.0 9.5 8.5 9.5
3.0 1.1 0.15 3.4 1.8 5.0 3.1 8.2
Experimental conditions and estimation of ICs0-values were identical with that in Table 2 except that 3H-DA 0.05 p-M was used in place of 3H-NE. Control uptake rates of 3H-DA during 5 rain were (mean _+ S.E.M.) 1.96 • 0.16 pmol/mg protein/rain, in cortical; 21.00 + 1.73 pmol/mg protein/rain, in striatal; and 2.25 + 0.21 pmol/mg protein/rain, in hypothalamic tissue preparations, respectively.
ICso values should be in the order of the inhibitor constants (Ki values) (Cheng and Prusoff,/973).
Inhibition of aH-NE Uptake. IC50-values of 3H-NE
Cortex cerebri
Corpus striatum
HypothaIamus
5.2 0.8 0.06 3.3 1.6 1.5 1.0 4.5
11.5 1.4 0.11 27.0 12.5 4.2 5.0 4.9
5.1 0.4 0.12 0.09 0.003 0.07 0.03 0.07
Synaptosomes were preincubated for 4 rain at 37 ~C with or without drugs and incubated for another 5 min with 3H-NE 0.05 p-M. Uptake in the presence of drugs was determined with at least four drug concentrations in triplicate. Control uptake rates of 3H-NE during 5 rain were (mean _+ S.E.M.) 1.40 • 0.22 pmol/ mg protein/rain, in cortical; 7.93 _+ 1.08 pmol/mg protein/min, in striatal; and 1.15 • 0.18 pmol/mg protein/rain, in hypothalamic tissue preparations, respectively.
tion was evaluated by determining the drug concentration that lowers amine uptake in a 5-min incubation by 50 ~ (IC~o). For these studies an amine concentration of 0.05 gM was chosen. Using a substrate concentration below or equal to the Kin-value of the high affinity uptake should avoid the possible interference by nonspecific or low affinity uptake as well as by random binding or passive diffusion with higher concentrations (Snyder and Coyle, 1969; Harris and Baldessarini, /973; Wong et al., ~973); furthermore,
uptake are shown in Table 2. Among the drugs tested two different patterns of uptake inhibition were observed. The antidepressants imipramine, desipramine, chlorimipramine, and amitriptyline as well as the neuroleptic chlorpromazine exhibited a particular strong inhibition of NE uptake in the hypothalamus region. In contrast, their inhibitory potencies in cerebral cortex or striatal tissue were much less pronounced. On the other hand, mepiprazole, oxypertine, and d-amphetamine, though different in their relative potencies, exhibited a quite uniform inhibitory effect on the NE uptake in the three brain areas. In this group of drugs, d-amphetamine was more effective than oxypertine; mepiprazole was a relatively weak inhibitor of NE uptake.
Inhibition of 3H-DA Uptake. Table 3 shows the ICs0values for the inhibition of 3H-DA uptake by drugs. Mepiprazole, the tricyclic antidepressants, and chlorpromazine were only weak inhibitors of DA accumulation with a slightly more pronounced inhibition of uptake into hypothalamic than into cortical or striatal tissues. In contrast, oxypertine had a strong inhibitory potency and inhibited DA uptake about equally in all three brain areas, d-Amphetamine exhibited the strongest inhibition of DA uptake showing ICsovalues in the order of 0.2 pM.
Inhibition of 3H-5-HT Uptake. As shown in Table 4 the tricyclic antidepressants were the most potent
298
Psychopharmacology 48 (1976)
Table 4. Effect of drugs on uptake of 3H-5-HT into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus
Table 5. Influence of drugs on the retention of radioactivity in whole rat brain synaptosomes preloaded with 3H-NE Drug
Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptytine Chlorpromazine
ICso (gM) Cortex cerebri
Corpus striatum
Hypothalamus
1.4 1.0 5.2 0.14 1.3 0.026 0.29 4.0
2.1 1.1 2.9 0.5 3.0 0.1 0.9 4.7
0.9 0.9 8.1 0.09 0.75 0.026 0.2 2.3
Experimental conditions and estimation of ICs0-values were identical with that in Table 2 except that 3H-5-HT 0.05 gM was used in place of 3H-NE. Control uptake rates of 3H-5-HT during 5 rain were (mean 4-_ S.E.M.) 2.44 • 0.12 pmol/mg protein/min, in cortical; 4.03 • 0.35 pmol/mg protein/min, in striatal; and 3.77 + 0.23 pmol/mg protein/rain, in hypothalamic tissue preparations, respectively.
drugs. The order of potency was: chlorimipramine > imipramine > amitriptyline > desipraminein each brain area. Mepiprazole and oxypertine were less effective in their abilities to inhibit 5-HT uptake. In comparison, d-amphetamine and chlorpromazine were the weakest inhibitors of 5-HT uptake.
Influence of Drugs on the Retention of 3H-Amines Previously Taken up into Synaptosomes. When synaptosomes prepared from whole rat brain were preloaded with 3H-amines (NE, DA, or 5-HT) and then incubated at 37~ C a time-dependent efflux of radioactivity was observed. After 20 rain incubation control samples retained (mean _+S.E.M.) 73.6 + 0.9 % (NE); 73.4_+0.7~ (DA); and 75.4• (5-HT) of total radioactivity at zero time.
Retention of 3H-NE. Table 5 shows the influence of drugs at concentrations of 10 -~, 10 -5, and 10 .4 M on the retention of radioactivity in synaptosomes preloaded with 3H-NE. At 10 .6 M only oxypertine and d-amphetamine significantly increased the efflux of radioactivity. At 10 .5 M mepiprazole also caused a significant release. At the highest concentration tested (10 .4 M) all drugs significantly enhanced the release.
Retention of 3H-DA. The data are shown in Table 6. At 10 -6 M mepiprazole, oxypertine, and d-amphetamine significantly increased the efflux of radioactivity. At 10 -5 M release was further enhanced by these drugs. At this concentration chlorimipramine, amitriptyline, and chlorpromazine caused a smaller but
Drug concentration (M) (percent of total radioactivity)
(lo-~) Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
74.2 64.8 59.5 69.9 69.6 72.6 72.2 70.4
• • 4• • • • •
1.9 0.9 b 1.5 b 2.2 2.0 1.3 1.3 2.2
(lO-~)
(lO-+)
66.4 61.2 55.1 70.2 70.4 70.4 71.5 69.7
48,3 44.6 52.0 40.3 36,0 13.7 37.8 20.4
4- 0.7 a 4- 0.6 b 4- 1.2 b _+ 3.1 4- 1.4 • 1.5 • 1.4 -!-_2.6
• 1.6 b 4- 1.0 b • 1.7 b • 3.46 -t- 2.9 b __. 1.3 b 4- 1.46 +_ 1.4 b
a P < 0:01 b P < 0.001 Synaptosomes prepared from whole rat brain (without cerebellum) were first incubated with 0.05 gM 3H-NE for 15 rain at 37~ After centrifugation synaptosomes were washed and resuspended in 3H-NE free medium and incubated for another 20 rain at 37~ without (control samples) or with drugs at three different concentrations. Total radioactivity at zero time of the second incubation period was (mean • S.E.M.) 222.7 • 20.2 x 103 dprn/ nag protein. The radioactivity retained in synaptosomes at the end of the 20 rain incubation at 37~C was expressed as percent of total radioactivity and represents the mean 4- S.E.M. of at least four determinations at each drug concentration. Retention of radioactivity in control samples was 73.6 + 0.9 %. Significance of differences between control and drug treated samples was calculated using Student's t-test.
Table 6. Influence of drugs on retention of radioactivity in whole rat brain synaptosomes preloaded with 3H-DA Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
Drug concentration (M) (percent of total radioactivity) (10 -6 )
(10 -5 )
(10 -4 )
67.6 55.9 39.2 71.2 72.9 74.5 72.1 73.0
51.0 32.0 31.0 70.9 74.3 59.6 66.3 66.0
17.0 12.0 28:1 12.6 13.2 14.1 17.9 14.4
4- 2.0 a + 1.6 b 4-_ 1.2 b • 2.9 • 0.8 • 0.4 • 0.6 • 1.4
+ 2.3 b 4- 1.6 b +_ 0.8 b +_ 2.7 4- 1.6 • 1-6 b • 0A b + 0.9 b
4-_ 1.5 b + 1.0 b + 1A b ___ 1.2 b +_ 0.2 b _+ 1.66 • 1,1 b + 1.9 b
P < 0.01 b P < 0,001 Experimental conditions and expression of data were identical with that in Table 5 except that synaptosomes were preloaded with 3H-DA 0.05 ~tM in place of 3H-NE. Total radioactivity was 216.4 • 10,1 x 103 dpm/mg protein. Radioactivity retained in control samples was 73,4 • 0.7%.
significant increment of release, too. At I 0 - 4 M drugs had a very strong effect.
all
Retention of 3H-5-HT. Table 7 shows the effect of drugs on the retention of 3H-5-HT. At 10 - 6 M only oxypertine and d-amphetamine significantly increased
299
P. Placheta et al. : Mepiprazole on Synaptosomes Table 7. Influence of drugs on the retention of radioactivity in whole rat brain synaptosomes preloaded with-3H-5-HT Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
Drug concentration (M) (percent of total radioactivity)
Oo -+)
(io-~)
71.1 _+ 2.3 61.8 • 1.8 a 61.3 __+ 1.4 a 73,0 • 2.3 71.8 • 3.4 71.1 • 0.5 72,2 • 0.5 71.7 • 0.3
53.9 39.7 33.7 72.1 70.8 67.0 67.6 67.8
• • • + + • • •
(io-~) 2.0" 0.4 a 0.9" 3.4 3.0 0.4 a 0.5 a 0.4 a
23.0 15.2 19.1 27.6 25.4 16.9 24.4 15:7
• 0.7" • 0.7 ~ _+ 0.8" • 2.1" • 1.9 a • 1.4 a +_ 1.1" • 1.4 a
P < 0.001 Experimental conditions and expression of data were identical with that in Table 5 except that synaptosomes were preloaded with 3H-5-HT 0.05 t~M in place of 3H-NE. Total radioactivity was 160.9 4- 9.3 x 103 dpm/mg protein. Radioactivity retained in control samples was 75.4 + 1.1 ~.
the efflux. At 10 -s M chlorimipramine, amitriptyline, and chlorpromazine reduced radioactivity only to approximately 67 ~ (significant) whereas mepiprazole (54 ~), oxypertine (40 ~), and d-amphetamine (34 ~) had a stronger effect. When present at 10-4M all drugs greatly enhanced the release of radioactivity. DISCUSSION Increasing evidence has been presented for the involvement of biogenic amines in the central action of psychotropic drugs. For example, imipraminelike drugs owe their antidepressant activity at least in part to their ability to block effectively reuptake of either NE or 5-HT in central neurons (Ross and Renyi, 1967; Carlsson et al., 1969; Horn et al., 1971). Centrally stimulating drugs like amphetamine apparently act through a strong releasing effect on neuronally stored catecholamines (Carlsson et al., 1966; Glowinski et al., 1966). On the other hand, neuroleptic drugs like chlorpromazine are believed to exert their ant• effect predominantly on a postsynaptic site by blocking dopamine receptors (van Rossum, 1966; Kebab• et al., 1972). This paper was restricted to studying the effects of mepiprazole on some presynaptic neurotransmitter mechanisms in comparison to several other psychotropic drugs whose actions on brain biogenic amine uptake and release are comparatively well documented. In the present study the tricyclic antidepressants were found to be very strong inhibitors of hypothalamic NE uptake, whereas they had a low inhibitory action in striatal tissue. This is in accordance with the
data of Horn et al. (1971) and Shaskan and Snyder (1970). It is assumed that desipramine exerts its beneficial effect on psychomotor retardation and drive through its particular inhibitory action on neuronal NE uptake (Carlsson et aI., 1969). Considering the weak inhibitory potency on NE uptake of mepiprazole this drug cannot be expected to act by this mechanism. In addition to the generally low affinity ofmepiprazole to NE uptake sites, no preference of hypothalamic sites was observed. Mepiprazole is also a very weak inhibitor of DA uptake into rat brain synaptosomes. However, no pharmacologic or clinical effects of mepiprazole have been established so far from which the involvement of DA uptake blockade in the CNS might be expected. It is well documented that 5-HT uptake is more sensitive to inhibition by tricyclic antidepressants with a tertiary than with a secondary configuration, the most effective being chlorimipramine (Carlsson et al., 1969; Shaskan and Snyder, 1970; Ross and Renyi, 1975). This is confirmed by our data showing the order of potency: chlorimipramine > imipramine > amitriptyline > desipramine in each brain area. The action of mepiprazole on amine uptake was generally weak and was not markedly different in the three brain areas. However, its inhibitory potency on 5-HT uptake was somewhat more pronounced than its effect on catecholamine uptake. Thus, the ICs0-values of mepiprazole for 5-HT uptake were very similar to that of desipramine. We have observed in previous experiments (Placheta et al., 1975) an inhibition of 5-HT uptake blockade in vitro using whole brain synaptosomes prepared from rats given 20 mg/ kg mepiprazole 30, 60, or 90 rain before sacrifice. This effect was comparable with that of desipramine 25 mg/kg in the same experimental set but was weaker than after 25 mg/kg imipramine. In separate studies (Hertting et al., in preparation) mepiprazole like imipramine reduced the release of 5-HT in the rat brain in vivo elicited by 4-methyl-c~-ethyl-metatyramine (H 75/12). In addition, mepiprazole significantly reduced the 5-HT turnover in the rat brain. From these observations it might be concluded that mepiprazole reduces 5-HT turnover by a neuronal feedback mechanism by blocking 5-HT uptake, thus increasing the concentration at receptor sites as discussed by Corrodi and Fuxe (1968). Mepiprazole increased the spontaneous efflux of radioactivity when it was included in a 20-rain incubation of 3H-amine precharged synaptosomes. At 10 .6 M only the retention of 3H-DA was significantly reduced, whereas at 10 -5 or 10-4M efflux of all amines was significantly enhanced. This effect of mepiprazole was somewhat less pronounced than the
300 effect o f d-amphetamine which was included in this study because it is an accepted releasing agent and o f oxypertine which has been reported to lower biogenic amine levels in the rat brain in vivo by a depleting action on the storage vesicles (Hassler, 1970). A t 10 -6 M the tricyclic antidepressants and c h l o r p r o m a zine generally did not enhance the release. At 10 -5 M, however, a small but significant D A and 5-HT release was elicited by chlorimipramine, amitriptyline, and c h l o r p r o m a z i n e ( W o n g et al., 1973; Heikkila et al.,
1975). A t a concentration as high as 1 0 - 4 M all drugs greatly enhanced the efflux o f radioactivity f r o m the synaptosomes. Several investigators have observed the release o f biogenic amines by tricyclic antidepressants at high concentrations using various tissue preparations (Hamberger, 1967; Leitz et al., 1970; N g et al., 1970; W o n g et al., 1973; A z z a r o et al., 1974; Heikkila et al., 1975). O u r in vitro experiments provide additional evidence that tricyclic antidepressants at low concentrations which can be expected to block effectively the neuronal u p t a k e do n o t enhance the efflux o f biogenic amines, whereas at the highest concentrations tested (10 -4 M) the drugs induce release. H o w ever, this releasing effect p r o b a b l y is not involved in their antidepressant action. I n contrast, mepiprazole like d - a m p h e t a m i n e and oxypertine caused a significant increase o f the efflux o f amines even at low concentrations. This observation suggests that the release o f biogenic amines m a y at least in part contribute to the action o f mepiprazole in therapeutic doses. In conclusion, mepiprazole was f o u n d to be only a weak inhibitor o f active amine transport in the cortex cerebri, corpus striatum, and h y p o t h a l a m u s o f the rat brain in vitro. This contrasts with the particular inhibition pattern o f the standard antidepressant drugs included in this study. Unlike the tricyclic c o m p o u n d s tested, mepiprazole elicited release o f previously accumulated amines in vitro even in the low concentration range. Considering the fact that m e p o p r a z o l e inhibits p r e d o m i n a n t l y 5-HT uptake and has some releasing action u p o n this amine, too, one might speculate that 5-HT could be involved in the drug's action. This is supported by some in vivo observations indicative o f increased 5-HT concentrations at the receptor level. However, this report deals only with a presynaptic site o f action. F u r t h e r studies are needed which m a y clarify whether or n o t other mechanisms such as receptor stimulation are involved.
REFERENCES Azzaro, A. J., Ziance, R. J., Rutledge, C. O.: The importance of neuronal uptake of amines for amphetamine-induced release
Psychopharmacology 48 (1976) of 3H-norepinephrine from isolated brain tissue. J. Pharmacol. exp. Ther. 189, 110-118 (1974) Carlsson, A., Corrodi, H., Fuxe, K., H6kfelt, T.: Effect of antidepressant drugs on the depletion of intraneuronal brain 5hydroxytryptamine stores caused by 4-methyl-c~-ethyl-metatyramine. Europ. J. Pharmacol. 5, 357-366 (1969) Carlsson, A., Fuxe, K., Hamberger, B., Lindquist, M. : Biochemical and histochemical studies on the effects of imipramine-like drugs and (+)-amphetamine on central and peripheral catecholamine neurons. Acta physiol, scan& 67, 481-497 (1966) Cheng, Y., Prusoff, W. H. : Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 ~o inhibition (I5o) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099-3108 (1973) Corrodi, H., Fuxe, K. : The effect of imipramine on central monoamine neurons. J. Pharm. Pharmacol. 20, 230- 231 (1968) Cuasnicfi, A.: Comparaci6n doble-ciego del nuevo tranquilizante Mepiprazol con diazepan. Therapia (Revista Mensual de Terapeutica) 49, 34-- 40 (1974) Dotevall, G., Groll, E. : Controlled clinical trial of mepiprazole in irritable bowel syndrome. Brit. med. J. 1974IV, I6-18 Foguet, G. : Evaluaci6n terapdutica de un nuevo tranquilizante menor. E1 Dia Medico 46, 698- 704 (1974) Glowinski, J., Axelrod, J., Iversen, L.L.: Regional studies of catecholamines in the rat brain. IV. Effects of drugs on the disposition and metabolism of 3H-norepinephrine and 3H-dopamine. J. Pharmacot. exp. Ther. 153, 30-41 (1966) Glowinski, J., Iversen, L. L. : Regional studies of catecholamines in the rat brain. I. The disposition of 3H-norepinephrine, 3Hdopamine and 3H-dopa in various regions of the brain. J. Neurochem. 13, 655- 669 (1966) Goncalves, N.: Das klinische Wirkungsbild des Tranquilizers Mepiprazol bei hospitalisierten Schizophrenen. Psychopharmacologia (Berl.) 25, 281-290 (1972) Goncalves, N., Fieguth, G. : Ver/inderung der Aufmerksamkeitsund Konzentrationsleistung chronisch Schizophrener unter zus/itzlicher Behandlung mit Mepiprazol und ihre Bedeutung f/Jr die Resozialisierung. Pharmakopsychiat. 6, 167-177 (1973) Harris, J. E., Baldessarini, R. J. : Uptake of 3H-catecholamines by homogenates of rat corpus striatum and cerebral cortex: Effects of amphetamine analogues. Neuropharmacology 12, 669- 679 (1973) Hassler, R., Bak, I. J., Kim, J. S. : Unterschiedliche Entleerung der Speicherorte fiir Noradrenaiin, Dopamin und Serotonin als Wirkungsprinzip des Oxypertins. Nervenarzt 41, 105-118 (1970) Heikkila, R. E., Orlansky, H., Cohen, G. : Studies on the distinction between uptake inhibition and release of 3H-dopamine in rat brain tissue slices. Biochem. Pharmacol. 24, 847- 852 (1975) Hamberger, B. : Reserpine-resistant uptake of catecholamines in isolated tissues of the rat. Acta physiol, scand. 71, Suppl. 295, 1 - 56 (1967) Henkel, D., Bastine, R.: Vergleichende Untersuchungen zur systematischen Desensibilisierung in Gruppen mit verbal-muskul~irer und pharmakologischer Entspannungsinduktion. Z. klin. Psychol. 1, 150-163 (1972) Horn, A. S., Coyle, J. D., Snyder, S. H.: Catecholamine uptake by synaptosomes from rat brain. Structure-activity relationships of drugs with differential effects on dopamine and norepinephrine neurons. Molee. Pharmacol. 7, 66-80 (1971) Kebabian, J. W., Petzold, G.L., Greengard, P. : Dopamine sensitive adenylate cyclase in the caudate nucleus of the rat brain and its similarity to the "dopamine receptor". Proc. nat. Acad. Sci. (Wash.) 69, 2145-2149 (1972)
P. Placheta et al. : Mepiprazole on Synaptosomes Leitz, F. H., Stefano, F. J. E. : Desipramine-induced release of'norepinephrine from heart. Biochem. Pharmacol. 19, 1797-180~l (1970) Lineweaver, H., Burk, D. : The determination of enzyme dissociation constants. J. Amer. chem. Soc. $6, 658-666 (1934) Lowry, O., Rosebrough, N., Farr, A., Randall, R. : Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) Mfiller-Calgan, H. : Psychopharmacological characteristics of certain new tranquilizing piperazine derivatives in rats and rhesus monkeys. In: Proceedings of 5th World Congress on Psychiatry, Mexico City (197I) Mtiller-Calgan, H. : A pharmacological model with rhesus monkeys for the prediction of selective depression of psychic functions. Activ. nerv. sup. (Praha) 16, 1 - 3 (1974) Ng, K. Y., Chase, T. N., Kopin, I. J.: Drug-induced release of 3H-norepinephrine and aH-serotonin from brain slices. Nature (Lond.) 228, 468- 469 (1970) Placheta, P., Singer, E., Kriwanek, W., Hertting, G.: The effect of mepiprazoIe on the uptake of norepinephrine, dopamine, and serotonin into rat brain synaptosomes. Naunyn-Schmiedeberg's Arch. Pharmacol., Suppl. 287, R4 (1975) P61dinger, W. : Clinical trial of 3-methyl-5-(/LN'-[N-m-chlorophenylpiperazinoJethyl)-pyrazole dihydrochloride (mepiprazole) in the therapy of psychovegetative disorders. Int. Pharmacopsychiat. 10, 1 - 8 (1975)
301 Ross, S. B., Renyi, A. L.: Inhibition of the uptake of tritiated catecholamines by antidepressant and related agents. Europ. J. Pharmacol. 2, 181 - 186 (1967) Ross, S. B., Renyi, A. L. : Tricyclic antidepressant agents. I. Comparison of the inhibition of the uptake of 3H-noradrenaline and 14G5-hydroxytryptamine in slices and crude synaptosome preparations of the midbrain-hypotbalamus region of the rat brain. Acta pharmacol. (Kbh.) 36, 382-394 (1975) Shaskan, E. G., Snyder, S. H. : Kinetics of serotonin accumulation into slices from rat brain : Relationship to catecholamine uptake. J. PharmacoL exp. Ther. 175, 404-418 (1970) Snyder, S. H., CoyIe, J. T. : Regional differences in 3H-norepinephrine and 3H-dopamine uptake into rat brain homogenates. J. Pharmacol. exp. Ther. 165, 78-86 (1969) Van Rossum, J. M. : The significance of dopamine-receptor blockade for the mechanism of action of neuroleptic drugs. Arch. int. Pharmacodyn. 160, 492-494 (1966) Whittaker, V. P. : The synaptosome. In: Handbook of neurochemistry, Vol. 2, A.A. Lajtha, ed., pp. 327-364. New York: Plenum Press 1969 Wong, D. T., Horng, J., Fuller, R. W. : Kinetics of serotonin accumulation into synaptosomes of rat brain-Effects of amphetamine and chloroamphetamines. Biochem. Pharmacol. 22, 311 322 (1973) Received December 2, 1975
Psychopharmacology 48, 295-301 (1976)
pharmacology 9 by Springer-Verlag 1976
Mepiprazole, a New Psychotropic Drug" Effects on Uptake and Retention of Monoamines in Rat Brain Synaptosomes P. PLACHETA*, E. SINGER, W. KRIWANEK, and G. HERTTING Institute of Pharmacology, Vienna, Austria and Institute of Pharmacology, Freiburg i. Br., Federal Republic of Germany
Abstract. The influence of mepiprazole (EMD 16,923), a new pyrazol-ylalkyl-piperazine derivative, on the uptake of 3H-norepinephrine (NE), 3H-dopamine (DA), and 3H-serotonin (5-HT) into rat brain synaptosomes from cerebral cortex, corpus striatum, and hypothalamus was investigated in comparison with several psychotropic drugs, including oxypertine, d-amphetamine, imipramine, desipramine, chlorimipramine, amitriptyline, and chlorpromazine in vitro. Mepiprazole was a relatively weak inhibitor of monoamine uptake and exhibited its strongest action on the hypothalamic 5-HT uptake, being almost equipotent with desipramine (ICso = 0.9 gM). Furthermore, the influence of the drugs on the retention of 3H-amines previously taken up by whole rat brain synaptosomes was studied. Unlike the tricyclic antidepressants, mepiprazole as well as oxypertine and d-amphetamine markedly increased the efflux of' radioactivity during a 20-min incubation at 37~ at low concentrations (10 -6 to 10 -5 M), whereas at 10 -.4 M all drugs greatly enhanced the efflux. The ability of mepiprazole to increase 5-HT concentration at the receptor level by a combination of neuronal uptake ;inhibition and release is discussed in relationship to the central actions of the drug.
INTRODUCTION Mepiprazole (EMD 16,923) is a representative compound of a new class of psychotropic drugs, the pyrazolyl-alkyl-piperazine derivatives (Fig. 1). It has been found to have a strong tranquillizing action in several laboratory animals, including rats and rhesus monkeys (Mfiller-Calgan, 1971 ; Miiller-Calgan, 1974). Mepiprazole has a low neuroleptic potency and only weak if any adrenolytic or anticholinergic properties (Goncalves, 1972). Clinically, mepiprazole has been shown to be effective as a minor tranquillizer in various emotional and psychosomatic conditions (Henkel and Bastine, 1972; Goncalves and Fieguth, 1973; Cuasnicu, 1974; Dotevall and Groll, 1974). Among the target symptoms tested the relief of anxiety and depression has been observed frequently (Foguet, 1974; P61dinger, 1975). In view of the possible involvement of biogenic amines in the central actions of mepiprazole the present study was designed to investigate its effects on some neurotransmitter mechanisms in the rat brain in vitro. The effects of several centrally active drugs have been attributed to their inhibitory activity on the neuronal reuptake system or to their potency
Key words: Synaptosomes - Monoamine uptake Monoamine release - Mepiprazole - Oxypertine d-Amphetamine - Imipramine - Desipramine Chlorimipramine - Amitriptyline - Chlorpromazine.
92 H C /
MEPIPRAZOLE t- (m - chlorop henyt) - 4 - [2- (5 - me thyl - 3-pyrezoty/) piperezine
*
Address for offprints: Dr. Peter Placheta, Institute of Pharma-
cology, University of Vienna, W~hringer Strage 13a, A- 1090Vienna, Austria.
dihydrochloride
EMD
16923
Fig. 1
- ethyl] -
296
Psychopharmacology 48 (1976)
to e v o k e o r a t t e n u a t e the release o f n e u r o t r a n s m i t t e r s . W e h a v e c o m p a r e d the effects o f m e p i p r a z o l e w i t h t h e effects o f s o m e o t h e r c e n t r a l l y active d r u g s , i n c l u d i n g oxypertine, d-amphetamine, imipramine, desipramine, chlorimipramine, amitriptyline, and chlorpromazine o n the u p t a k e o f 3 H - l a b e l e d n o r e p i n e p h r i n e ( N E ) , dopamine (DA), and serotonin (5-HT) into synaptos o m e s p r e p a r e d f r o m rat b r a i n c e r e b r a l cortex, c o r p u s s t r i a t u m , a n d h y p o t h a l a m u s , respectively. F u r t h e r m o r e , the i n f l u e n c e o f these d r u g s o n t h e release o f 3 H - N E , 3 H - D A , a n d 3 H - 5 - H T f r o m w h o l e rat b r a i n s y n a p t o s o m e s was studied. S y n a p t o s o m e s ( p i n c h e d - o f f n e r v e t e r m i n a l s ) h a v e p r o v e d to be a v a l u a b l e t o o l for the i n v e s t i g a t i o n o f d r u g effects o n n e u r o n a l u p t a k e a n d release m e c h a n i s m s in vitro.
MATERIALS
AND
METHODS
Chemicals and Drugs. All reagents used were of analytical grade. l-[73H]-norepinephrine (10.9 Ci/mmol), aH-dopamine (3,4 dihydroxy[ring-G-3H]phenylethylamine hydrochloride; 2.9 Ci/mmol), and 3H-serotonin (5-hydroxy[G-3H]tryptamine creatinine sulfate; 17.3 Ci/mmol) were purchased from the Radiochemical Centre, Amersham. The drugs were: mepiprazole (E. Merck AG); oxypertine (Winthrop); imipramine, desipramine, and chlorimipramine (Ciba-Geigy AG); amitriptyline (Merck, Sharp & Dohme); damphetamine (Smith, Kline & French); chlorpromazine (Bayer AG); and nialamid (Pfizer). All concentrations indicated are for the free base.
Animals. Male adult Sprague-Dawley rats (240--300 g) were sacrificed by cervical dislocation, their brains rapidly removed, placed on a chilled glass plate and the brain areas dissected as described by Glowinski and Iversen (1966). For the uptake studies three brain areas were used: cerebral cortex, corpus striatum, and hypothalamus. When necessary, tissues from 2 to 6 rats were pooled, weighed, a n d homogenized for preparation of synaptosomes. In the release studies whole brains (without cerebellum) were used.
Tissue Preparation. A crude synaptosomal fraction was prepared by differential centrifngation using the method of Whittaker (1969). Tissues were homogenized in 10 volumes (w/v) of ice-cold 0.32 M sucrose in a Thomas B 10001 glass homogenizer with a motor driven loosely fitting teflon pestle using six complete up and down strokes within approximately 1 min. Homogenates were centrifuged at 1000 x g for 10 rain at 4 ~C. The pellet (P1) was discarded and the supernatant ($1) was centrifuged at 14000 xg for 15 min at 4 ~C. The remaining pellet (P2) containing synaptosomes and other cellular components was washed once with ice-cold sucrose, centrifuged again, and carefully resuspended in ice-cold incubation medium. The incubation medium contained: 100mM NaCI; 5 mM KC1; 15 mM NazHPO~, 15 mM NaHaPO4, pH 7.0; 10 mM d-glucose; 40mM sucrose; 12.SpM nialamide and 25gg/ml ascorbic acid. Uptake Studies. For estimation of the kinetic constants of 3Hamine uptake portions of the resupended P2-pellet, equivalent to approximately 20 mg (cerebral cortex), 2 mg (corpus striatum), or 12.5 mg (hypothatamus) original tissue wet wt, respectively, were preincubated for 4 min at 37~ in a shaking water bath under air. Then, the 3H-labeled amines were added in 20 ~tl of medium to the samples (final volume of 1.0 ml) and the incubations were continued for another 5 min. Incubations were stopped by cooling the samples on ice followed by immediate ultrafiltration with cellulose nitrate filters (Schleicher & Schiill, BA 90, 0.6 gm
pore size, 26 mm in diameter) by suction. The filters were rinsed 3 times with 4 ml of ice-cold medium, blotted and placed into scintillation vials containing 2ml of ethyleneglycolemonoethylether (E. Merck AG) and total 3H-radioactivity was determined by liquid scintillation spectrometry. The quenching was corrected by internal standardization. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. The synaptosomal protein yield ranged from 25 to 40 mg/g of tissue wet wt. Uptake was expressed as the initial rate of accumulation during 5 min/mg of synaptosomal protein. Values were corrected for 'uptake' at 0~C which accounted for less than 5 of the radioactivity taken up in control samples incubated at 37~C. Kinetic analysis was performed by plotting the reciprocal of the amine concentration against the reciprocal of the initial uptake velocity according to Lineweaver and Burk (1934). When the effects of drugs on the amine uptake was studied, drugs at various concentrations were included during the 4-min preincubation period. Then, the 3H-amines were added to give a final concentration of 0.05 JaM. ICs0-values (drug concentration required to inhibit the uptake during 5 min by 50 ~) were determined graphically within the range of at least four concentrations of inhibitor by plotting inhibitor concentration against percent inhibition of control uptake using a logarithmic probability paper.
Release Studies. The Pz-pellet was prepared essentially as described for the uptake studies from a whole rat brain (without cerebellum) and preloaded with 0.05 gM 3H-labeled amine during 15 rain at 37~ in an incubation volume of 10 ml/g original tissue wet wt. The particles were then centrifuged at 14000 x g for 15 min at 4~C, washed, centrifuged again, and resuspended in ice-cold incubation medium. The radioactivity taken up by the synaptosomes was fully retained at 0~C over a period of at least 20 min. When incubated at 37~C for 20 rain there was a spontaneous efflux of radioactivity that amounted to about 25 7o of the initial value. Portions of precharged synaptosomes (0.2 ml) were incubated for 20 min at 37~C without or with varying concentrations of drugs in a final volume of 1.0 ml. The incubations were stopped by cooling the samples on ice and the radioactivity retained in the synaptosomes was determined as described in the uptake studies. The ability of drugs to release amines during 20 min at 37~C was assessed by comparing the radioactivity retained in drug-treated samples with control samples as percent of radioactivity initially accumulated.
RESULTS
Kinetic Constants of 3H-Amine Uptake into Synaptosomes. I n t h e r a n g e o f c o n c e n t r a t i o n s o f a m i n e s u s e d i n o u r e x p e r i m e n t s the i n c o r p o r a t i o n o f r a d i o a c t i v i t y was a l i n e a r f u n c t i o n o f t i m e for at least 5 m i n i n all b r a i n areas. T h e r e f o r e , t h e u p t a k e rate d u r i n g 5 - m i n i n c u b a t i o n was c h o s e n for the e s t i m a t i o n o f k i n e t i c c o n s t a n t s . K i n e t i c a n a l y s i s was p e r f o r m e d a c c o r d i n g to L i n e w e a v e r a n d B u r k (1934) u s i n g at least five c o n c e n t r a t i o n s o f a m i n e r a n g i n g f r o m 0.04 to 0.5 g M . T h i s was r e p e a t e d at least 3 times to c o n f i r m h o m o g e n e i t y o f the p r e p a r a t i o n s . T h e a p p a r e n t Kinv a l u e s for the u p t a k e o f 3 H - N E , 3 H - D A , a n d 3 H - 5 - H T are s h o w n i n T a b l e 1.
Inhibition by Drugs of 3H-Amine Uptake into Synaptosomes Prepared from Cerebral Cortex, Corpus Striatum, and Hypothalamus. T h e effects o f d r u g s o n the u p t a k e o f 3 H - a m i n e s i n t o the s y n a p t o s o m a l p r e p a r a -
P. Placheta et al. : Mepiprazole on Synaptosomes
297
Table 1. Apparent Kin-values of 3H-amine uptake into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus
Table 3. Effect of drugs on uptake of 3H-DA into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus
3H-amine
Drug
Km (p.M) Cortex cerebri
Corpus striatum
Hypothalamus
Norepinephrine
0.27 • 0.02" (3) b
1.05 +_ 0.12 (3)
0.25 __+0.03 (3)
Dopamine
0.08 • 0.01 (5)
0.07 • 0.01 (9)
0.11 _ 0.02 (3)
Serotonin
0.05 • 0.01 (3)
0.13 • 0.03 (3)
0.05 • 0.02 (3)
a Mean +_ S.E.M. b Number of experiments. Synaptosomes (P2-pellet) were preincubated for 4 m i n at 37~ and incubated for another 5 min with at least five different concentrations of 3H-amine ranging from 0.04 to 0.5 p-M in duplicate. Km-values were estimated from double reciprocal plots according to Lineweaver and Burk. Table 2. Effect of drugs on uptake of 3H-NE into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
ICso (p-M)
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
IC5o (p-M) Cortex cerebri
Corpus striatum
Hypothalamus
11.0 1.8 0.21 19.0 17.0 12.0 11.5 15.0
~2.0 1.7 0.24 19.0 14.0 9.5 8.5 9.5
3.0 1.1 0.15 3.4 1.8 5.0 3.1 8.2
Experimental conditions and estimation of ICs0-values were identical with that in Table 2 except that 3H-DA 0.05 p-M was used in place of 3H-NE. Control uptake rates of 3H-DA during 5 rain were (mean _+ S.E.M.) 1.96 • 0.16 pmol/mg protein/rain, in cortical; 21.00 + 1.73 pmol/mg protein/rain, in striatal; and 2.25 + 0.21 pmol/mg protein/rain, in hypothalamic tissue preparations, respectively.
ICso values should be in the order of the inhibitor constants (Ki values) (Cheng and Prusoff,/973).
Inhibition of aH-NE Uptake. IC50-values of 3H-NE
Cortex cerebri
Corpus striatum
HypothaIamus
5.2 0.8 0.06 3.3 1.6 1.5 1.0 4.5
11.5 1.4 0.11 27.0 12.5 4.2 5.0 4.9
5.1 0.4 0.12 0.09 0.003 0.07 0.03 0.07
Synaptosomes were preincubated for 4 rain at 37 ~C with or without drugs and incubated for another 5 min with 3H-NE 0.05 p-M. Uptake in the presence of drugs was determined with at least four drug concentrations in triplicate. Control uptake rates of 3H-NE during 5 rain were (mean _+ S.E.M.) 1.40 • 0.22 pmol/ mg protein/rain, in cortical; 7.93 _+ 1.08 pmol/mg protein/min, in striatal; and 1.15 • 0.18 pmol/mg protein/rain, in hypothalamic tissue preparations, respectively.
tion was evaluated by determining the drug concentration that lowers amine uptake in a 5-min incubation by 50 ~ (IC~o). For these studies an amine concentration of 0.05 gM was chosen. Using a substrate concentration below or equal to the Kin-value of the high affinity uptake should avoid the possible interference by nonspecific or low affinity uptake as well as by random binding or passive diffusion with higher concentrations (Snyder and Coyle, 1969; Harris and Baldessarini, /973; Wong et al., ~973); furthermore,
uptake are shown in Table 2. Among the drugs tested two different patterns of uptake inhibition were observed. The antidepressants imipramine, desipramine, chlorimipramine, and amitriptyline as well as the neuroleptic chlorpromazine exhibited a particular strong inhibition of NE uptake in the hypothalamus region. In contrast, their inhibitory potencies in cerebral cortex or striatal tissue were much less pronounced. On the other hand, mepiprazole, oxypertine, and d-amphetamine, though different in their relative potencies, exhibited a quite uniform inhibitory effect on the NE uptake in the three brain areas. In this group of drugs, d-amphetamine was more effective than oxypertine; mepiprazole was a relatively weak inhibitor of NE uptake.
Inhibition of 3H-DA Uptake. Table 3 shows the ICs0values for the inhibition of 3H-DA uptake by drugs. Mepiprazole, the tricyclic antidepressants, and chlorpromazine were only weak inhibitors of DA accumulation with a slightly more pronounced inhibition of uptake into hypothalamic than into cortical or striatal tissues. In contrast, oxypertine had a strong inhibitory potency and inhibited DA uptake about equally in all three brain areas, d-Amphetamine exhibited the strongest inhibition of DA uptake showing ICsovalues in the order of 0.2 pM.
Inhibition of 3H-5-HT Uptake. As shown in Table 4 the tricyclic antidepressants were the most potent
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Psychopharmacology 48 (1976)
Table 4. Effect of drugs on uptake of 3H-5-HT into rat brain synaptosomes prepared from cerebral cortex, corpus striatum, and hypothalamus
Table 5. Influence of drugs on the retention of radioactivity in whole rat brain synaptosomes preloaded with 3H-NE Drug
Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptytine Chlorpromazine
ICso (gM) Cortex cerebri
Corpus striatum
Hypothalamus
1.4 1.0 5.2 0.14 1.3 0.026 0.29 4.0
2.1 1.1 2.9 0.5 3.0 0.1 0.9 4.7
0.9 0.9 8.1 0.09 0.75 0.026 0.2 2.3
Experimental conditions and estimation of ICs0-values were identical with that in Table 2 except that 3H-5-HT 0.05 gM was used in place of 3H-NE. Control uptake rates of 3H-5-HT during 5 rain were (mean 4-_ S.E.M.) 2.44 • 0.12 pmol/mg protein/min, in cortical; 4.03 • 0.35 pmol/mg protein/min, in striatal; and 3.77 + 0.23 pmol/mg protein/rain, in hypothalamic tissue preparations, respectively.
drugs. The order of potency was: chlorimipramine > imipramine > amitriptyline > desipraminein each brain area. Mepiprazole and oxypertine were less effective in their abilities to inhibit 5-HT uptake. In comparison, d-amphetamine and chlorpromazine were the weakest inhibitors of 5-HT uptake.
Influence of Drugs on the Retention of 3H-Amines Previously Taken up into Synaptosomes. When synaptosomes prepared from whole rat brain were preloaded with 3H-amines (NE, DA, or 5-HT) and then incubated at 37~ C a time-dependent efflux of radioactivity was observed. After 20 rain incubation control samples retained (mean _+S.E.M.) 73.6 + 0.9 % (NE); 73.4_+0.7~ (DA); and 75.4• (5-HT) of total radioactivity at zero time.
Retention of 3H-NE. Table 5 shows the influence of drugs at concentrations of 10 -~, 10 -5, and 10 .4 M on the retention of radioactivity in synaptosomes preloaded with 3H-NE. At 10 .6 M only oxypertine and d-amphetamine significantly increased the efflux of radioactivity. At 10 .5 M mepiprazole also caused a significant release. At the highest concentration tested (10 .4 M) all drugs significantly enhanced the release.
Retention of 3H-DA. The data are shown in Table 6. At 10 -6 M mepiprazole, oxypertine, and d-amphetamine significantly increased the efflux of radioactivity. At 10 -5 M release was further enhanced by these drugs. At this concentration chlorimipramine, amitriptyline, and chlorpromazine caused a smaller but
Drug concentration (M) (percent of total radioactivity)
(lo-~) Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
74.2 64.8 59.5 69.9 69.6 72.6 72.2 70.4
• • 4• • • • •
1.9 0.9 b 1.5 b 2.2 2.0 1.3 1.3 2.2
(lO-~)
(lO-+)
66.4 61.2 55.1 70.2 70.4 70.4 71.5 69.7
48,3 44.6 52.0 40.3 36,0 13.7 37.8 20.4
4- 0.7 a 4- 0.6 b 4- 1.2 b _+ 3.1 4- 1.4 • 1.5 • 1.4 -!-_2.6
• 1.6 b 4- 1.0 b • 1.7 b • 3.46 -t- 2.9 b __. 1.3 b 4- 1.46 +_ 1.4 b
a P < 0:01 b P < 0.001 Synaptosomes prepared from whole rat brain (without cerebellum) were first incubated with 0.05 gM 3H-NE for 15 rain at 37~ After centrifugation synaptosomes were washed and resuspended in 3H-NE free medium and incubated for another 20 rain at 37~ without (control samples) or with drugs at three different concentrations. Total radioactivity at zero time of the second incubation period was (mean • S.E.M.) 222.7 • 20.2 x 103 dprn/ nag protein. The radioactivity retained in synaptosomes at the end of the 20 rain incubation at 37~C was expressed as percent of total radioactivity and represents the mean 4- S.E.M. of at least four determinations at each drug concentration. Retention of radioactivity in control samples was 73.6 + 0.9 %. Significance of differences between control and drug treated samples was calculated using Student's t-test.
Table 6. Influence of drugs on retention of radioactivity in whole rat brain synaptosomes preloaded with 3H-DA Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
Drug concentration (M) (percent of total radioactivity) (10 -6 )
(10 -5 )
(10 -4 )
67.6 55.9 39.2 71.2 72.9 74.5 72.1 73.0
51.0 32.0 31.0 70.9 74.3 59.6 66.3 66.0
17.0 12.0 28:1 12.6 13.2 14.1 17.9 14.4
4- 2.0 a + 1.6 b 4-_ 1.2 b • 2.9 • 0.8 • 0.4 • 0.6 • 1.4
+ 2.3 b 4- 1.6 b +_ 0.8 b +_ 2.7 4- 1.6 • 1-6 b • 0A b + 0.9 b
4-_ 1.5 b + 1.0 b + 1A b ___ 1.2 b +_ 0.2 b _+ 1.66 • 1,1 b + 1.9 b
P < 0.01 b P < 0,001 Experimental conditions and expression of data were identical with that in Table 5 except that synaptosomes were preloaded with 3H-DA 0.05 ~tM in place of 3H-NE. Total radioactivity was 216.4 • 10,1 x 103 dpm/mg protein. Radioactivity retained in control samples was 73,4 • 0.7%.
significant increment of release, too. At I 0 - 4 M drugs had a very strong effect.
all
Retention of 3H-5-HT. Table 7 shows the effect of drugs on the retention of 3H-5-HT. At 10 - 6 M only oxypertine and d-amphetamine significantly increased
299
P. Placheta et al. : Mepiprazole on Synaptosomes Table 7. Influence of drugs on the retention of radioactivity in whole rat brain synaptosomes preloaded with-3H-5-HT Drug
Mepiprazole Oxypertine d-Amphetamine Imipramine Desipramine Chlorimipramine Amitriptyline Chlorpromazine
Drug concentration (M) (percent of total radioactivity)
Oo -+)
(io-~)
71.1 _+ 2.3 61.8 • 1.8 a 61.3 __+ 1.4 a 73,0 • 2.3 71.8 • 3.4 71.1 • 0.5 72,2 • 0.5 71.7 • 0.3
53.9 39.7 33.7 72.1 70.8 67.0 67.6 67.8
• • • + + • • •
(io-~) 2.0" 0.4 a 0.9" 3.4 3.0 0.4 a 0.5 a 0.4 a
23.0 15.2 19.1 27.6 25.4 16.9 24.4 15:7
• 0.7" • 0.7 ~ _+ 0.8" • 2.1" • 1.9 a • 1.4 a +_ 1.1" • 1.4 a
P < 0.001 Experimental conditions and expression of data were identical with that in Table 5 except that synaptosomes were preloaded with 3H-5-HT 0.05 t~M in place of 3H-NE. Total radioactivity was 160.9 4- 9.3 x 103 dpm/mg protein. Radioactivity retained in control samples was 75.4 + 1.1 ~.
the efflux. At 10 -s M chlorimipramine, amitriptyline, and chlorpromazine reduced radioactivity only to approximately 67 ~ (significant) whereas mepiprazole (54 ~), oxypertine (40 ~), and d-amphetamine (34 ~) had a stronger effect. When present at 10-4M all drugs greatly enhanced the release of radioactivity. DISCUSSION Increasing evidence has been presented for the involvement of biogenic amines in the central action of psychotropic drugs. For example, imipraminelike drugs owe their antidepressant activity at least in part to their ability to block effectively reuptake of either NE or 5-HT in central neurons (Ross and Renyi, 1967; Carlsson et al., 1969; Horn et al., 1971). Centrally stimulating drugs like amphetamine apparently act through a strong releasing effect on neuronally stored catecholamines (Carlsson et al., 1966; Glowinski et al., 1966). On the other hand, neuroleptic drugs like chlorpromazine are believed to exert their ant• effect predominantly on a postsynaptic site by blocking dopamine receptors (van Rossum, 1966; Kebab• et al., 1972). This paper was restricted to studying the effects of mepiprazole on some presynaptic neurotransmitter mechanisms in comparison to several other psychotropic drugs whose actions on brain biogenic amine uptake and release are comparatively well documented. In the present study the tricyclic antidepressants were found to be very strong inhibitors of hypothalamic NE uptake, whereas they had a low inhibitory action in striatal tissue. This is in accordance with the
data of Horn et al. (1971) and Shaskan and Snyder (1970). It is assumed that desipramine exerts its beneficial effect on psychomotor retardation and drive through its particular inhibitory action on neuronal NE uptake (Carlsson et aI., 1969). Considering the weak inhibitory potency on NE uptake of mepiprazole this drug cannot be expected to act by this mechanism. In addition to the generally low affinity ofmepiprazole to NE uptake sites, no preference of hypothalamic sites was observed. Mepiprazole is also a very weak inhibitor of DA uptake into rat brain synaptosomes. However, no pharmacologic or clinical effects of mepiprazole have been established so far from which the involvement of DA uptake blockade in the CNS might be expected. It is well documented that 5-HT uptake is more sensitive to inhibition by tricyclic antidepressants with a tertiary than with a secondary configuration, the most effective being chlorimipramine (Carlsson et al., 1969; Shaskan and Snyder, 1970; Ross and Renyi, 1975). This is confirmed by our data showing the order of potency: chlorimipramine > imipramine > amitriptyline > desipramine in each brain area. The action of mepiprazole on amine uptake was generally weak and was not markedly different in the three brain areas. However, its inhibitory potency on 5-HT uptake was somewhat more pronounced than its effect on catecholamine uptake. Thus, the ICs0-values of mepiprazole for 5-HT uptake were very similar to that of desipramine. We have observed in previous experiments (Placheta et al., 1975) an inhibition of 5-HT uptake blockade in vitro using whole brain synaptosomes prepared from rats given 20 mg/ kg mepiprazole 30, 60, or 90 rain before sacrifice. This effect was comparable with that of desipramine 25 mg/kg in the same experimental set but was weaker than after 25 mg/kg imipramine. In separate studies (Hertting et al., in preparation) mepiprazole like imipramine reduced the release of 5-HT in the rat brain in vivo elicited by 4-methyl-c~-ethyl-metatyramine (H 75/12). In addition, mepiprazole significantly reduced the 5-HT turnover in the rat brain. From these observations it might be concluded that mepiprazole reduces 5-HT turnover by a neuronal feedback mechanism by blocking 5-HT uptake, thus increasing the concentration at receptor sites as discussed by Corrodi and Fuxe (1968). Mepiprazole increased the spontaneous efflux of radioactivity when it was included in a 20-rain incubation of 3H-amine precharged synaptosomes. At 10 .6 M only the retention of 3H-DA was significantly reduced, whereas at 10 -5 or 10-4M efflux of all amines was significantly enhanced. This effect of mepiprazole was somewhat less pronounced than the
300 effect o f d-amphetamine which was included in this study because it is an accepted releasing agent and o f oxypertine which has been reported to lower biogenic amine levels in the rat brain in vivo by a depleting action on the storage vesicles (Hassler, 1970). A t 10 -6 M the tricyclic antidepressants and c h l o r p r o m a zine generally did not enhance the release. At 10 -5 M, however, a small but significant D A and 5-HT release was elicited by chlorimipramine, amitriptyline, and c h l o r p r o m a z i n e ( W o n g et al., 1973; Heikkila et al.,
1975). A t a concentration as high as 1 0 - 4 M all drugs greatly enhanced the efflux o f radioactivity f r o m the synaptosomes. Several investigators have observed the release o f biogenic amines by tricyclic antidepressants at high concentrations using various tissue preparations (Hamberger, 1967; Leitz et al., 1970; N g et al., 1970; W o n g et al., 1973; A z z a r o et al., 1974; Heikkila et al., 1975). O u r in vitro experiments provide additional evidence that tricyclic antidepressants at low concentrations which can be expected to block effectively the neuronal u p t a k e do n o t enhance the efflux o f biogenic amines, whereas at the highest concentrations tested (10 -4 M) the drugs induce release. H o w ever, this releasing effect p r o b a b l y is not involved in their antidepressant action. I n contrast, mepiprazole like d - a m p h e t a m i n e and oxypertine caused a significant increase o f the efflux o f amines even at low concentrations. This observation suggests that the release o f biogenic amines m a y at least in part contribute to the action o f mepiprazole in therapeutic doses. In conclusion, mepiprazole was f o u n d to be only a weak inhibitor o f active amine transport in the cortex cerebri, corpus striatum, and h y p o t h a l a m u s o f the rat brain in vitro. This contrasts with the particular inhibition pattern o f the standard antidepressant drugs included in this study. Unlike the tricyclic c o m p o u n d s tested, mepiprazole elicited release o f previously accumulated amines in vitro even in the low concentration range. Considering the fact that m e p o p r a z o l e inhibits p r e d o m i n a n t l y 5-HT uptake and has some releasing action u p o n this amine, too, one might speculate that 5-HT could be involved in the drug's action. This is supported by some in vivo observations indicative o f increased 5-HT concentrations at the receptor level. However, this report deals only with a presynaptic site o f action. F u r t h e r studies are needed which m a y clarify whether or n o t other mechanisms such as receptor stimulation are involved.
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