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Differential effects of several dopamine uptake inhibitors and releasing agents on locomotor activity in normal and in reserpinized mice
Life Sciences, Vol. 28, pp. 1867-1873 Printed in the U.S.A.
Pergamon Press
DIFFERENTIAL EFFECTS OF SEVERAL DOPAMINE UPTAKE INHIBITORS AND RELEASING AGENTS ON LOCOMOTOR ACTIVITY IN NORMALAND IN RESERPINIZED ~ICE
Richard E. Heikkila Department of Neurology College of Medicine and Dentistry of New Jersey Rutgers Medical School University Heights Piscataway, New Jersey 08854 (Received in final form February 2, 1981)
SUMMARY Several drugs that were potent dopamine uptake i n h i b i t o r s in v i t r o (including GBR 13,069 and GBR 13,098, amfonelic acid, mazindol, Win 35,065 and Win 35,428) caused large increases in locomotor a c t i v i t y in normal mice but not in reserpinized mice. A d d i t i o n a l l y , these drugs given to reserpinized mice p r i o r to the dopamine releasinm a~ent d-amphetamine, were able to prevent the normal increase in a c t i v i t y caused by d-amphetamine. The data demonstrate markedly d i f f e r e n t in vivo effects of dopamine uptake i n h i ~ i t o r s and dopamine releasing agents. I t is well-known that drugs which increase dopaminergic a c t i v i t y d i r e c t l y , for example by dopamine receptor stimulation, as well as drugs which increase dopaminergic a c t i v i t y i n d i r e c t l y , for example by blockade of dopamine uptake or by f a c i l i t a t i o n of dopamine release, increase locomotor a c t i v i t y or stereotyped behavior in rodents. For example, i t was recently shown ( I ) that several rather potent dopamine uptake i n h i b i t o r s including methylphenidate mazindol, nomifensine and amfonelic acid increased locomotor a c t i v i t y in normal mice. However, these same agents did not increase a c t i v i t y in reserpinized mice. In contrast amphetamine, a drug thought by some to be an uptake i n h i b i t o r but by most to be a dopamine releasing agent (2,3), increased locomotor a c t i v i t y in both normal and reserpinized mice ( I ) . Furthermore, the four above mentioned dopamine uptake i n h i b i t o r s , given to reserpinized mice p r i o r to amphetamine, were able to prevent the increased a c t i v i t y normally brought about by amphetamine. I t was concluded that the amphetamine-like drugs owed t h e i r behavioral effect to dopamine release and the methylphenidatel i k e owed t h e i r behavioral effects to the blockade of uptake of synaptically released dopamine. In the present study we have given several drugs classified as dopamine uptake inhibitors from in vitro studies (including mazindol and amfonelic acid, which were done for comparative purposes) to both normal and reserpinized mice, and measured their effects on locomotor activity. We have also tested the effects of several of the drugs on amphetamine-induced increases in activity in reserpinized mice. The data will confirm and extend the concepts set forth by Ross, (1). 0024-3205/81/171867-07$02.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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MATERIALS AND METHODS The accumulation of 3H-do,amine into r a t neostriatal tissue slices was done with previously published standard methods (2,3). B r i e f l y , the neostriatum was dissected from male Sprague-Dawley rats (150 to 200 q) after decapitation. The tissue was sliced with a razor blade and then cross-chopped at 0.2 mm with a Mcllwain-Mickle tissue chopper. The slices ( I . 0 x 0.2 x 0.2 mm) were dispersed into 200 volumes of a modified Kreb's Ringer phosphate buffer at pH 7.a containing E.6 mM glucose, 1.3 mM EDTA, 1.7 mM ascorhic acid and 0.08 mM pargyline hydrochloride (3). Ten milligrams of tissue contained in 2 ml of the buffer was added to 8 ml of the buffer, and the samples equilibrated at 37oc for 5-10 min. Then 3H-dopamine (3H-DA, 12 Ci/mmole, New England Nuclear, Boston, Mass.) and the appropriate concent r a t i o n of the drug to be studied were added simultaneously. There were 4 concentrations used for each drug; these concentrations were chosen from preliminary experiments to qive a response ranging from 20% to 80% effect. The maximum concentration used was IO-~M. The samples ( t r i p l i c a t ~ t o quadruplicates) were mixed with shaking at 37oc for 15 min., the contents rapidly f i l t e r e d through 2.1 cm Whatman f i l t e r paper discs and rinsed with ice-cold saline. Radioactivity was measured by l i q u i d s c i n t i l l a t i o n spectrometry. In release studies, the 3H-DA was added and uptake was done as above. Then after the f i l t r a t i o n and saline rinse, the f i l t e r paper discs with the adhering tissue slices were placed in 30 ml beakers containing I0 ml of the buffer. The drugs at appropriate concentrations were then added, the samples equilibrated for 15 min., and then r a d i o a c t i v i t y remaining in the slices was measured after f i l t r a t i o n . GBR 13,0~9 { l - { 2 - { B i s (~-fluorophenyl) methoxy} ethyl} -~- (3-phenyl -2propenyl) piperazine dimethane sulfonate} and GBR 13,098 { I - { 2 {Bis (~fluorophenyl) methoxy} e t h y l } - 4 - {3- (4-fluorophenyl) propyl} piperazine dimethane sulfonate} were kindly supplied by Dr. W. Hespe (Gist-Brocades, Haarlem, The Netherlands); amfonelic acid ( 7 - b e n z y l - l - e t h y l - l , 4 - d i h y d r o - 4 - o x o 1,8- naphthyridine -3- carboxylic acid), Win 35,065-2 (Methyl(-)-3B-phenylImH, 5m H-tropane - 2B-carboxylate 1,5-naphthalene disulfonate), and Win 35,428 (Methyl (-) - 3B - (p-fluorophenyl) -ImH, 5m H-tropane-2~ -carboxylate 1,5 naphthalene disulfonate) were kindly supplied by Dr. Robert Clarke (Sterling-Winthrop Research I n s t i t u t e , Renssalaer, New York); mazindol (5Hydroxy-5-(41-chlorophenyl)-2,3-dihydro - 5H- imidazo (2, l-a) isoindole) was kindly supplied by Dr. W. Houlihan (Sandoz Pharmaceutics, E. Hanover, New Jersey); CDCI (N-cyclo-propylmethyl- I 0 , I I - dihydro - 5H-dibenzo- {a,d} cyclohepten -5-imine) was kindly supplied by Dr. W.F. Herblin (Dupont, Wilmington, Delaware.) The d-amphetamine sulfate was obtained from Smith, Kline and French (Philadelphia, Pa.), desmethylimipra~ine hydrochloride (DMI) was obtained from U.S.V. (Tuckahoe, New York), cocaine hydrochloride from Squibb (Princeton, New Jersey), phenmetrazine hydrochloride from the National Formulary (Washington, D.C.) and reserpine (Serpasil) from Ciba-Geigy (Summit, New Jersey). The mazindol and CDCI were dissolved in I00 ul of INHCI and 9.9 cc of d i s t i l l e d water, the amfonelic acid was dissolved in I00 ul of IN NaOH and 9.9 cc of d i s t i l l e d water, and all other drugs were dissolved in d i s t i l l e d water alone. For a c t i v i t y measurements, 4 male Swiss-Webster mice were put in each cage of a Varimex A c t i v i t y Chamber (Columbus I n s t . , Columbus, Ohio). The mice were allowed to acclimate to t h e i r surroundings for at least 2 hours. Some mice received reserpine at 5 mq/kg i . p . ; others received vehicle or no i n j e c t i o n . The mice then received i . p . injections of the drugs (0.I ml/lO g body weight) to be studied at appropriate times. A c t i v i t y was measured for successive 15 min. periods for I-2 hours p o s t - i n j e c t i o n . Each experiment was repeated
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several times. RESULTS ED50 values f o r several drugs as i n h i b i t o r s of 3H-DA uptake in v i t r o in n e o s t r i a t a l tissue s l i c e s are given in Table I. The f i r s t six drugs, with ED50 values of less than 1 uV a l l may be c l a s s i f i e d as r e l a t i v e l y potent inh i b i t o r s of 3H-DA uptake. CDCI and cocaine were less potent. All eight of these druQs were quite weak releasing aqents, with ED50 values f o r release of 3H-DA considerably higher than t h e i r respective EDSO values for blockade of 3H-DA uptake (data not shown; ED50 values enerally well above I0 uM). Drugs 1 to 8 thus caused a potent i n h i b i t i o n of ~H-DA uptake at concentrations at which they were quite weak in causing release. The drugs may thus be c l a s s i f i e d as true i n h i b i t o r s of uptake from this in v i t r o data. (For d i s cussion of these points, see references 2,3). Except CDCI and DMI all of the drugs listed in Table l ( i . e . numbers l through 6, and 8) caused large increases in locomotor a c t i v i t y in normal mice (Table l ) . The doses used ranged from 2 mg/kg to 20 mg/kg for the active drugs. These doses were chosen from preliminary screenin~ experiments to cause close to maximal a c t i v i t y . In separate experiments all drugs showed clear dose-response relationships. DMI and CDCI also caused no significant increases in a c t i v i t y at doses as high as 50 mg/kg or doses as low as l mg/kg. TABLE I
A comparison between the effects of drugs as uptake inhibitors for 3H-DA and their stimulatory effects on locomotor a c t i v i t y . Uptake data are given as mean ED50 values ± S.D. in uM (point of 50% inhibition) for at least three experiments. Locomotor a c t i v i t y data represent mean one hour a c t i v i t y ! S.D. for four mice for three to five experiments. Locomotor a c t i v i t y in vehicle treated controls (H20) was 560 ! 151. Similar values were obtained when the vehicle was dilute acid or base. NO.
DRUG
ED50 VALUE ( u M )
LOCOMOTOR ACTIVITY
I.
GBR 13,069
.040 ~ .01
1817 ± 435 (I0) c
2.
GBR 13,098
.043 ± .01
1301 ±
3.
Amfonelic Acid
.17
± .09
2807 ± 248 (2)
4.
Mazindol
.28
± .02 a
1743 ± 352 (5)
5.
Win 35,428
.32
± .07 b
2569 ± 503 (5)
6.
Win 35,065-2
.74
± .3 b
2553 ± 52 (5)
7.
CDCI
3.8
± l.l
4C5 ± 90 (20)
8.
Cocaine
4.0
± l.O
1403 ± 773 (20)
9.
DMI
>>I0
133 ±
a) Reference I0 b) Reference II c) Dose used in mg/kg of compound in form listed in text
65 (20)
30 (20)
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Vol. 28, No. 17, 1981
400
>-
300
7 I0
200
0 o
I00
i
0
5O
60 TIME
i
0
I
i
30
r
60
(min)
FIGURE I
The e f f e c t of various drugs on amphetamine-induced a c t i v i t y in reseroinized mice. Data represent the mean a c t i v i t y for at least three experiments. All mice were aiven reserpine at 5 mg/kq. Three hours l a t e r ( f i r s t arrow) they were given H20 (curve I0) or the various uptake i n h i b i t o r s (numbers 1 to 9 and doses as in Table I ) . Fifteen minutes l a t e r the mice received 2.5 mg/kg of d-amphetamine s u l f a t e and a c t i v i t y was measured f o r one hour.
400
>p. a
p.. <[ 200 m,,
o '5
i
i
0
I
i
30
i
i
i
0
60 TIME
!
50
i
t
60
(rain)
FIGURE II The e f f e c t of amfonelic acid (AFA) and Win 35,428 on phenmetrazine (P) induced a c t i v i t y in reserpinized mice. Experiments are done as in Figure I. The reserpinized mice received H20, AFA or Win 35,428 at the f i r s t arrow and then 20 mQ/kg of phenmetrazine hydrochloride at the second arrow.
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Amphetamine at 2.5 mg/kg brought about increased motor a c t i v i t y in normal mice (mean of 1771 counts) as well as in reserpinized mice (Fiq. I , curve In). In contrast, V'ir 35,065-2 and Win 35,428, GBR 13,069 and GBR 13,098, mazindol, cocaine and amfonelic acid failed to increase a c t i v i t y in reserpinized mice in 15 min. (Fig. I) and at longer time periods (data not shown). However, these six druas were able to prevent the increased a c t i v i t y seen in reserpinized mice after amphetamine administration (Fig. I ) . Like these six drugs, DMI and CDCI failed to increase a c t i v i t y in reserpinized mice. But in contrast to the results obtained with the other drugs, DMI and CDCI had no inhibitory effects on the increases in a c t i v i t y brought about by amphetamine in reserpinized mice. This effect in preventing amphetamine-induced a c t i v i t y in reserpinized mice was clearly dependent upon the dose of the uptake inhibitor used. For example, as shown, GBR 13,069 at I0 mg/kg was able to almost completely i n h i b i t the amphetamine-induced increase in a c t i v i t y in the reserpinized mice (90% inhibition based on 1 hr. data). In contrast 1 mg/kg inhibited by only I~% while results with 5 mg/kg were intermediate (70% inhibition). Dose-response relationships were similarly observed for the other drugs that inhibited amphetamine-induced a c t i v i t y in reserpinized animals. Phenmetrazine at 20 mg/kg, increased a c t i v i t y in normal mice (mean of 1452 counts) and in reserpinized mice (Fiq. 2). Win 35,428 and amfonelic acid, given to the reserpinized mice prior to phenmetrazine (Fig. 2) prevented the increase in a c t i v i t y normally brought about by phenmetrazine. The other drugs were not tested aQainst phenmetrazine. DISCUSSION Indirectly acting CNS stimulants can be separated into two distinct grouns. One group consists of amphetamine-like agents and the other group consists of methylphenidate-like aQents. From his studies as well as from other in vitro studies from n~ny other laboratories, Ross (1,4) concluded that the amphetamine-like aqents ovid their CNS stimulant properties to the release of central DA and included d-and-l-amohetamine, ephedrine and phenmetrazine. He concluded that methylphenidate-like agents acted by virtue of their blockade of DA uptake and included besides methylphenidate, cocaine, Astra 2959, prolintane, amfonelic acid, mazindol and nomifensine. In spite of this and other information, there is s t i l l confusion over whether certain drugs are uptake inhibitors or releasing agents. Perhaps the best example of a drug for which confusion exists is amphetamine (2,3). The present study is an extension of that of Ross (1). The drugs used in the present study were selected for in vivo experiments for the following reasons: Two of the drugs, namely mazindol and cocaine, are commonly used as dopamine uptake inhibitors in vitro. Several of the others are relatively new experimental drugs that are also potent dopamine uptake inhibitors in vitro (see below). These include GBR 13,069 and GBR 13,098; amfonelic acid and Win 35,065-2 and Win 35,428. Moreover, GBR 13,069 and GBR 13,098 and CDCI are interesting in that they are among the few drugs presently known that are "specific DA uptake inhibitors". That is, they are better inhibitors of 3H-DA uptake into dopamine-containing brain areas than they are of 3Hnorepinephrine uptake into norepinephrine-containing brain areas (5,7). Win 35,065-2 and Win 35,428 are structural analogs of cocaine which are much more potent CNS stimulants than cocaine i t s e l f ( l l ) . DMI was chosen in that i t is an extremely weak inhibitor of 3H-DA uptake. In the present study we found several drugs (Nos. 1,2,3) to be potent uptake inhibitors for JH-DA in neostriatal slices (Table l ) . Previously published uptake values for mazindol, Win 35,065-2 and Win 35,428 (drugs 4,5,6)
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are presented for comparison. All six of these drugs in contrast were weak releasinq aqents for previously accumulated aH-DA. (For comparison to uptake results obtained by others with several of the same compounds, see references 1,4,5,6,7,8). In normal mice, cocaine as well as the six above-mentioned more potent inhibitors of 3H-DA uptake (drugs 1 to 6) caused large increases in locomotor a c t i v i t y (Table I ) . In contrast CDCl at 20 mg/kg and even at 50 mg/kg was without significant effect. Lower CDCI doses also did not increase a c t i v i t y . The results obtained with CDCl were somewhat unexpected; perhaps a sufficient quantity of CDCl to increase a c t i v i t y did not reach ~ r i t i c a l areas in the brain. All of the above compounds that were potent ~H-DA uptake inhibitors in v i t r o and increased a c t i v i t y in normal mice (thus excluding CDCl), did not increase a c t i v i t y in reserpinized mice (Fig. I ) . However, these same drugs given to reserpinized mice 15 min. prior to the dopamine releasing agent amphetamine, were able to prevent the increases in a c t i v i t y normally caused by amphetamine. CDCI and DMI in contrast failed to i n h i b i t amphetamine-induced increases in a c t i v i t y in reserpinized mice. In other experiments amfonelic acid and Win 35,428 also prevented phenmetrazine-induced increases in a c t i v i t y in reserpinized mice (Fig. 2). The failure of DMI to increase a c t i v i t y in normal mice is well known and the failure of DMI to prevent the actions of amphetamine in reserpinized mice predictable, since DMI is an extremely weak i n h i b i t o r of neostriatal 3H-DA uptake (Table I , see also 6). Again, as in non-reserpinized mice, the f a i l u r e of CDCl to i n h i b i t the actions of amphetamine in reserpinized mice is perhaps best explained by failure of a sufficient quantity of CDCI to reach the brain to be active. The data of the present study are consistent with the concepts set forth by Ross, (1,4,9) who also used mazindol and amfonelic acid. In the present study, all drugs (except CDCI) that were good inhibitors of 3H-DA uptake in neostriatal slices (but which were weak 3H-DA releasers in vitro) increased motor activity in normal mice and prevented amphetamine-induced increases in activity in reserpinized mice. All of these drugs are most likely methylohenidate-like and owe their behavioral effects to their capacity to act as DA uptake blockers in vivo. The present demonstration that a stimulant in normal mice (e.g. cocaine) can prevent stimulant action in a reserpinized animal is intriguing. Stated differently, under certain well-defined conditions, a stimulant can be an antagonist to another stimulant. These present data are also consistent with the concept that the potent inhibitors of 3H-DA uptake (except CDCI) also inhibit the transport (uptake) of amphetamine and phenmetrazine to their nermal intracellular site of action. Moreover, the findings suggest that the administration to a reserpinized animal of a drug suspected of being an uptake blocker or a releasing agent can help to distinguish which i f either of these actions might be important. And finally, hut not of least importance, the data indicate that the site of action of an uptake blocker and of a releasinq agent (i.e. the pool of DA on which they act) must be different. ACKNOWLEDGMENTS This work was supported by the Clinical Research Center for Parkinson's and Allied Diseases and USPHSGrants NS-05184 and AM-27328.
Vol. 28, No. 17, 1981
3H-dopamine Uptake and Locomotor Activity
REFERENCES I, 2.
3. 4. 5. 6. 7. 8.
9. lO. II.
S.B. ROSS, Life Sciences 24 159-168 (1979). R.E. HEIKKILA, H. ORLANSKYand G. COHEN, Biochem. Pharmacol 24 847-852 (1975). R.E. HEIKKILA, H. ORLANSKY, C. MYTILINEOUand G. COHEN, J. Pharmacol. Exp. Ther. 194 47-56 (1975). S.B. ROSS, ~Tc'£'a. Pharmacol. Toxicol. 41 392-396 (1977). W.F. HERBLIN, Neurochem Res. 2 lll-ll~--(1977). B.K. KOE, J. Pharmacol. Exp. Ther. 199 649-661 (1976). P. VAN DER ZEE, H.S. KOGERand W. HE-'~I~E,Abstr. 7th I n t l . Cong. Pharmacol. 591 (1978). P.A. SHORE, B.A. McMILLEN, H.H. MILLER, M.K. SANGHERA, R.S. KISER and D.C. GERMAN, In: Catecholamines: Basic and Clinical Frontiers E. Usdin, I.J. Kopin and J, Barchas eds., p. 722, Pergamon Press, New York, (1979). S.B. ROSS, J. Pharm. Pharmacol. 29 433-434 (1977). R.E. HEIKKILA, F.S. CABBAT and C.~MYTILINEOU, European J. Pharmacol. 45 329-333 (1977). ITT.E. HEIKKILA, F.S. CABBAT, L. MANZINOand R.C. DUVOISIN, J. Pharmacol. Exp. Ther. 211 189-194 (1979).
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Pergamon Press
DIFFERENTIAL EFFECTS OF SEVERAL DOPAMINE UPTAKE INHIBITORS AND RELEASING AGENTS ON LOCOMOTOR ACTIVITY IN NORMALAND IN RESERPINIZED ~ICE
Richard E. Heikkila Department of Neurology College of Medicine and Dentistry of New Jersey Rutgers Medical School University Heights Piscataway, New Jersey 08854 (Received in final form February 2, 1981)
SUMMARY Several drugs that were potent dopamine uptake i n h i b i t o r s in v i t r o (including GBR 13,069 and GBR 13,098, amfonelic acid, mazindol, Win 35,065 and Win 35,428) caused large increases in locomotor a c t i v i t y in normal mice but not in reserpinized mice. A d d i t i o n a l l y , these drugs given to reserpinized mice p r i o r to the dopamine releasinm a~ent d-amphetamine, were able to prevent the normal increase in a c t i v i t y caused by d-amphetamine. The data demonstrate markedly d i f f e r e n t in vivo effects of dopamine uptake i n h i ~ i t o r s and dopamine releasing agents. I t is well-known that drugs which increase dopaminergic a c t i v i t y d i r e c t l y , for example by dopamine receptor stimulation, as well as drugs which increase dopaminergic a c t i v i t y i n d i r e c t l y , for example by blockade of dopamine uptake or by f a c i l i t a t i o n of dopamine release, increase locomotor a c t i v i t y or stereotyped behavior in rodents. For example, i t was recently shown ( I ) that several rather potent dopamine uptake i n h i b i t o r s including methylphenidate mazindol, nomifensine and amfonelic acid increased locomotor a c t i v i t y in normal mice. However, these same agents did not increase a c t i v i t y in reserpinized mice. In contrast amphetamine, a drug thought by some to be an uptake i n h i b i t o r but by most to be a dopamine releasing agent (2,3), increased locomotor a c t i v i t y in both normal and reserpinized mice ( I ) . Furthermore, the four above mentioned dopamine uptake i n h i b i t o r s , given to reserpinized mice p r i o r to amphetamine, were able to prevent the increased a c t i v i t y normally brought about by amphetamine. I t was concluded that the amphetamine-like drugs owed t h e i r behavioral effect to dopamine release and the methylphenidatel i k e owed t h e i r behavioral effects to the blockade of uptake of synaptically released dopamine. In the present study we have given several drugs classified as dopamine uptake inhibitors from in vitro studies (including mazindol and amfonelic acid, which were done for comparative purposes) to both normal and reserpinized mice, and measured their effects on locomotor activity. We have also tested the effects of several of the drugs on amphetamine-induced increases in activity in reserpinized mice. The data will confirm and extend the concepts set forth by Ross, (1). 0024-3205/81/171867-07$02.00/0 Copyright (c) 1981 Pergamon Press Ltd.
1868
3H-dopamine Uptake and Locomotor Activity
Vol. 28, No. 17, 1981
MATERIALS AND METHODS The accumulation of 3H-do,amine into r a t neostriatal tissue slices was done with previously published standard methods (2,3). B r i e f l y , the neostriatum was dissected from male Sprague-Dawley rats (150 to 200 q) after decapitation. The tissue was sliced with a razor blade and then cross-chopped at 0.2 mm with a Mcllwain-Mickle tissue chopper. The slices ( I . 0 x 0.2 x 0.2 mm) were dispersed into 200 volumes of a modified Kreb's Ringer phosphate buffer at pH 7.a containing E.6 mM glucose, 1.3 mM EDTA, 1.7 mM ascorhic acid and 0.08 mM pargyline hydrochloride (3). Ten milligrams of tissue contained in 2 ml of the buffer was added to 8 ml of the buffer, and the samples equilibrated at 37oc for 5-10 min. Then 3H-dopamine (3H-DA, 12 Ci/mmole, New England Nuclear, Boston, Mass.) and the appropriate concent r a t i o n of the drug to be studied were added simultaneously. There were 4 concentrations used for each drug; these concentrations were chosen from preliminary experiments to qive a response ranging from 20% to 80% effect. The maximum concentration used was IO-~M. The samples ( t r i p l i c a t ~ t o quadruplicates) were mixed with shaking at 37oc for 15 min., the contents rapidly f i l t e r e d through 2.1 cm Whatman f i l t e r paper discs and rinsed with ice-cold saline. Radioactivity was measured by l i q u i d s c i n t i l l a t i o n spectrometry. In release studies, the 3H-DA was added and uptake was done as above. Then after the f i l t r a t i o n and saline rinse, the f i l t e r paper discs with the adhering tissue slices were placed in 30 ml beakers containing I0 ml of the buffer. The drugs at appropriate concentrations were then added, the samples equilibrated for 15 min., and then r a d i o a c t i v i t y remaining in the slices was measured after f i l t r a t i o n . GBR 13,0~9 { l - { 2 - { B i s (~-fluorophenyl) methoxy} ethyl} -~- (3-phenyl -2propenyl) piperazine dimethane sulfonate} and GBR 13,098 { I - { 2 {Bis (~fluorophenyl) methoxy} e t h y l } - 4 - {3- (4-fluorophenyl) propyl} piperazine dimethane sulfonate} were kindly supplied by Dr. W. Hespe (Gist-Brocades, Haarlem, The Netherlands); amfonelic acid ( 7 - b e n z y l - l - e t h y l - l , 4 - d i h y d r o - 4 - o x o 1,8- naphthyridine -3- carboxylic acid), Win 35,065-2 (Methyl(-)-3B-phenylImH, 5m H-tropane - 2B-carboxylate 1,5-naphthalene disulfonate), and Win 35,428 (Methyl (-) - 3B - (p-fluorophenyl) -ImH, 5m H-tropane-2~ -carboxylate 1,5 naphthalene disulfonate) were kindly supplied by Dr. Robert Clarke (Sterling-Winthrop Research I n s t i t u t e , Renssalaer, New York); mazindol (5Hydroxy-5-(41-chlorophenyl)-2,3-dihydro - 5H- imidazo (2, l-a) isoindole) was kindly supplied by Dr. W. Houlihan (Sandoz Pharmaceutics, E. Hanover, New Jersey); CDCI (N-cyclo-propylmethyl- I 0 , I I - dihydro - 5H-dibenzo- {a,d} cyclohepten -5-imine) was kindly supplied by Dr. W.F. Herblin (Dupont, Wilmington, Delaware.) The d-amphetamine sulfate was obtained from Smith, Kline and French (Philadelphia, Pa.), desmethylimipra~ine hydrochloride (DMI) was obtained from U.S.V. (Tuckahoe, New York), cocaine hydrochloride from Squibb (Princeton, New Jersey), phenmetrazine hydrochloride from the National Formulary (Washington, D.C.) and reserpine (Serpasil) from Ciba-Geigy (Summit, New Jersey). The mazindol and CDCI were dissolved in I00 ul of INHCI and 9.9 cc of d i s t i l l e d water, the amfonelic acid was dissolved in I00 ul of IN NaOH and 9.9 cc of d i s t i l l e d water, and all other drugs were dissolved in d i s t i l l e d water alone. For a c t i v i t y measurements, 4 male Swiss-Webster mice were put in each cage of a Varimex A c t i v i t y Chamber (Columbus I n s t . , Columbus, Ohio). The mice were allowed to acclimate to t h e i r surroundings for at least 2 hours. Some mice received reserpine at 5 mq/kg i . p . ; others received vehicle or no i n j e c t i o n . The mice then received i . p . injections of the drugs (0.I ml/lO g body weight) to be studied at appropriate times. A c t i v i t y was measured for successive 15 min. periods for I-2 hours p o s t - i n j e c t i o n . Each experiment was repeated
Vol. 28, No. 17, 1981
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several times. RESULTS ED50 values f o r several drugs as i n h i b i t o r s of 3H-DA uptake in v i t r o in n e o s t r i a t a l tissue s l i c e s are given in Table I. The f i r s t six drugs, with ED50 values of less than 1 uV a l l may be c l a s s i f i e d as r e l a t i v e l y potent inh i b i t o r s of 3H-DA uptake. CDCI and cocaine were less potent. All eight of these druQs were quite weak releasing aqents, with ED50 values f o r release of 3H-DA considerably higher than t h e i r respective EDSO values for blockade of 3H-DA uptake (data not shown; ED50 values enerally well above I0 uM). Drugs 1 to 8 thus caused a potent i n h i b i t i o n of ~H-DA uptake at concentrations at which they were quite weak in causing release. The drugs may thus be c l a s s i f i e d as true i n h i b i t o r s of uptake from this in v i t r o data. (For d i s cussion of these points, see references 2,3). Except CDCI and DMI all of the drugs listed in Table l ( i . e . numbers l through 6, and 8) caused large increases in locomotor a c t i v i t y in normal mice (Table l ) . The doses used ranged from 2 mg/kg to 20 mg/kg for the active drugs. These doses were chosen from preliminary screenin~ experiments to cause close to maximal a c t i v i t y . In separate experiments all drugs showed clear dose-response relationships. DMI and CDCI also caused no significant increases in a c t i v i t y at doses as high as 50 mg/kg or doses as low as l mg/kg. TABLE I
A comparison between the effects of drugs as uptake inhibitors for 3H-DA and their stimulatory effects on locomotor a c t i v i t y . Uptake data are given as mean ED50 values ± S.D. in uM (point of 50% inhibition) for at least three experiments. Locomotor a c t i v i t y data represent mean one hour a c t i v i t y ! S.D. for four mice for three to five experiments. Locomotor a c t i v i t y in vehicle treated controls (H20) was 560 ! 151. Similar values were obtained when the vehicle was dilute acid or base. NO.
DRUG
ED50 VALUE ( u M )
LOCOMOTOR ACTIVITY
I.
GBR 13,069
.040 ~ .01
1817 ± 435 (I0) c
2.
GBR 13,098
.043 ± .01
1301 ±
3.
Amfonelic Acid
.17
± .09
2807 ± 248 (2)
4.
Mazindol
.28
± .02 a
1743 ± 352 (5)
5.
Win 35,428
.32
± .07 b
2569 ± 503 (5)
6.
Win 35,065-2
.74
± .3 b
2553 ± 52 (5)
7.
CDCI
3.8
± l.l
4C5 ± 90 (20)
8.
Cocaine
4.0
± l.O
1403 ± 773 (20)
9.
DMI
>>I0
133 ±
a) Reference I0 b) Reference II c) Dose used in mg/kg of compound in form listed in text
65 (20)
30 (20)
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3H-dopamine Uptake and Locomotor Activity
Vol. 28, No. 17, 1981
400
>-
300
7 I0
200
0 o
I00
i
0
5O
60 TIME
i
0
I
i
30
r
60
(min)
FIGURE I
The e f f e c t of various drugs on amphetamine-induced a c t i v i t y in reseroinized mice. Data represent the mean a c t i v i t y for at least three experiments. All mice were aiven reserpine at 5 mg/kq. Three hours l a t e r ( f i r s t arrow) they were given H20 (curve I0) or the various uptake i n h i b i t o r s (numbers 1 to 9 and doses as in Table I ) . Fifteen minutes l a t e r the mice received 2.5 mg/kg of d-amphetamine s u l f a t e and a c t i v i t y was measured f o r one hour.
400
>p. a
p.. <[ 200 m,,
o '5
i
i
0
I
i
30
i
i
i
0
60 TIME
!
50
i
t
60
(rain)
FIGURE II The e f f e c t of amfonelic acid (AFA) and Win 35,428 on phenmetrazine (P) induced a c t i v i t y in reserpinized mice. Experiments are done as in Figure I. The reserpinized mice received H20, AFA or Win 35,428 at the f i r s t arrow and then 20 mQ/kg of phenmetrazine hydrochloride at the second arrow.
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3H-dopamine Uptake and Locomotor Activity
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Amphetamine at 2.5 mg/kg brought about increased motor a c t i v i t y in normal mice (mean of 1771 counts) as well as in reserpinized mice (Fiq. I , curve In). In contrast, V'ir 35,065-2 and Win 35,428, GBR 13,069 and GBR 13,098, mazindol, cocaine and amfonelic acid failed to increase a c t i v i t y in reserpinized mice in 15 min. (Fig. I) and at longer time periods (data not shown). However, these six druas were able to prevent the increased a c t i v i t y seen in reserpinized mice after amphetamine administration (Fig. I ) . Like these six drugs, DMI and CDCI failed to increase a c t i v i t y in reserpinized mice. But in contrast to the results obtained with the other drugs, DMI and CDCI had no inhibitory effects on the increases in a c t i v i t y brought about by amphetamine in reserpinized mice. This effect in preventing amphetamine-induced a c t i v i t y in reserpinized mice was clearly dependent upon the dose of the uptake inhibitor used. For example, as shown, GBR 13,069 at I0 mg/kg was able to almost completely i n h i b i t the amphetamine-induced increase in a c t i v i t y in the reserpinized mice (90% inhibition based on 1 hr. data). In contrast 1 mg/kg inhibited by only I~% while results with 5 mg/kg were intermediate (70% inhibition). Dose-response relationships were similarly observed for the other drugs that inhibited amphetamine-induced a c t i v i t y in reserpinized animals. Phenmetrazine at 20 mg/kg, increased a c t i v i t y in normal mice (mean of 1452 counts) and in reserpinized mice (Fiq. 2). Win 35,428 and amfonelic acid, given to the reserpinized mice prior to phenmetrazine (Fig. 2) prevented the increase in a c t i v i t y normally brought about by phenmetrazine. The other drugs were not tested aQainst phenmetrazine. DISCUSSION Indirectly acting CNS stimulants can be separated into two distinct grouns. One group consists of amphetamine-like agents and the other group consists of methylphenidate-like aQents. From his studies as well as from other in vitro studies from n~ny other laboratories, Ross (1,4) concluded that the amphetamine-like aqents ovid their CNS stimulant properties to the release of central DA and included d-and-l-amohetamine, ephedrine and phenmetrazine. He concluded that methylphenidate-like agents acted by virtue of their blockade of DA uptake and included besides methylphenidate, cocaine, Astra 2959, prolintane, amfonelic acid, mazindol and nomifensine. In spite of this and other information, there is s t i l l confusion over whether certain drugs are uptake inhibitors or releasing agents. Perhaps the best example of a drug for which confusion exists is amphetamine (2,3). The present study is an extension of that of Ross (1). The drugs used in the present study were selected for in vivo experiments for the following reasons: Two of the drugs, namely mazindol and cocaine, are commonly used as dopamine uptake inhibitors in vitro. Several of the others are relatively new experimental drugs that are also potent dopamine uptake inhibitors in vitro (see below). These include GBR 13,069 and GBR 13,098; amfonelic acid and Win 35,065-2 and Win 35,428. Moreover, GBR 13,069 and GBR 13,098 and CDCI are interesting in that they are among the few drugs presently known that are "specific DA uptake inhibitors". That is, they are better inhibitors of 3H-DA uptake into dopamine-containing brain areas than they are of 3Hnorepinephrine uptake into norepinephrine-containing brain areas (5,7). Win 35,065-2 and Win 35,428 are structural analogs of cocaine which are much more potent CNS stimulants than cocaine i t s e l f ( l l ) . DMI was chosen in that i t is an extremely weak inhibitor of 3H-DA uptake. In the present study we found several drugs (Nos. 1,2,3) to be potent uptake inhibitors for JH-DA in neostriatal slices (Table l ) . Previously published uptake values for mazindol, Win 35,065-2 and Win 35,428 (drugs 4,5,6)
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are presented for comparison. All six of these drugs in contrast were weak releasinq aqents for previously accumulated aH-DA. (For comparison to uptake results obtained by others with several of the same compounds, see references 1,4,5,6,7,8). In normal mice, cocaine as well as the six above-mentioned more potent inhibitors of 3H-DA uptake (drugs 1 to 6) caused large increases in locomotor a c t i v i t y (Table I ) . In contrast CDCl at 20 mg/kg and even at 50 mg/kg was without significant effect. Lower CDCI doses also did not increase a c t i v i t y . The results obtained with CDCl were somewhat unexpected; perhaps a sufficient quantity of CDCl to increase a c t i v i t y did not reach ~ r i t i c a l areas in the brain. All of the above compounds that were potent ~H-DA uptake inhibitors in v i t r o and increased a c t i v i t y in normal mice (thus excluding CDCl), did not increase a c t i v i t y in reserpinized mice (Fig. I ) . However, these same drugs given to reserpinized mice 15 min. prior to the dopamine releasing agent amphetamine, were able to prevent the increases in a c t i v i t y normally caused by amphetamine. CDCI and DMI in contrast failed to i n h i b i t amphetamine-induced increases in a c t i v i t y in reserpinized mice. In other experiments amfonelic acid and Win 35,428 also prevented phenmetrazine-induced increases in a c t i v i t y in reserpinized mice (Fig. 2). The failure of DMI to increase a c t i v i t y in normal mice is well known and the failure of DMI to prevent the actions of amphetamine in reserpinized mice predictable, since DMI is an extremely weak i n h i b i t o r of neostriatal 3H-DA uptake (Table I , see also 6). Again, as in non-reserpinized mice, the f a i l u r e of CDCl to i n h i b i t the actions of amphetamine in reserpinized mice is perhaps best explained by failure of a sufficient quantity of CDCI to reach the brain to be active. The data of the present study are consistent with the concepts set forth by Ross, (1,4,9) who also used mazindol and amfonelic acid. In the present study, all drugs (except CDCI) that were good inhibitors of 3H-DA uptake in neostriatal slices (but which were weak 3H-DA releasers in vitro) increased motor activity in normal mice and prevented amphetamine-induced increases in activity in reserpinized mice. All of these drugs are most likely methylohenidate-like and owe their behavioral effects to their capacity to act as DA uptake blockers in vivo. The present demonstration that a stimulant in normal mice (e.g. cocaine) can prevent stimulant action in a reserpinized animal is intriguing. Stated differently, under certain well-defined conditions, a stimulant can be an antagonist to another stimulant. These present data are also consistent with the concept that the potent inhibitors of 3H-DA uptake (except CDCI) also inhibit the transport (uptake) of amphetamine and phenmetrazine to their nermal intracellular site of action. Moreover, the findings suggest that the administration to a reserpinized animal of a drug suspected of being an uptake blocker or a releasing agent can help to distinguish which i f either of these actions might be important. And finally, hut not of least importance, the data indicate that the site of action of an uptake blocker and of a releasinq agent (i.e. the pool of DA on which they act) must be different. ACKNOWLEDGMENTS This work was supported by the Clinical Research Center for Parkinson's and Allied Diseases and USPHSGrants NS-05184 and AM-27328.
Vol. 28, No. 17, 1981
3H-dopamine Uptake and Locomotor Activity
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3. 4. 5. 6. 7. 8.
9. lO. II.
S.B. ROSS, Life Sciences 24 159-168 (1979). R.E. HEIKKILA, H. ORLANSKYand G. COHEN, Biochem. Pharmacol 24 847-852 (1975). R.E. HEIKKILA, H. ORLANSKY, C. MYTILINEOUand G. COHEN, J. Pharmacol. Exp. Ther. 194 47-56 (1975). S.B. ROSS, ~Tc'£'a. Pharmacol. Toxicol. 41 392-396 (1977). W.F. HERBLIN, Neurochem Res. 2 lll-ll~--(1977). B.K. KOE, J. Pharmacol. Exp. Ther. 199 649-661 (1976). P. VAN DER ZEE, H.S. KOGERand W. HE-'~I~E,Abstr. 7th I n t l . Cong. Pharmacol. 591 (1978). P.A. SHORE, B.A. McMILLEN, H.H. MILLER, M.K. SANGHERA, R.S. KISER and D.C. GERMAN, In: Catecholamines: Basic and Clinical Frontiers E. Usdin, I.J. Kopin and J, Barchas eds., p. 722, Pergamon Press, New York, (1979). S.B. ROSS, J. Pharm. Pharmacol. 29 433-434 (1977). R.E. HEIKKILA, F.S. CABBAT and C.~MYTILINEOU, European J. Pharmacol. 45 329-333 (1977). ITT.E. HEIKKILA, F.S. CABBAT, L. MANZINOand R.C. DUVOISIN, J. Pharmacol. Exp. Ther. 211 189-194 (1979).
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