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The effect of Δ9-tetrahydrocannabinol on the uptake and release of 14C-dopamine from crude striatal synaptosomal preparations
THE EFFECT OF A’-TETRAHYDROCANNABTNOL ON THE UPTAKE AND RELEASE OF 14C-DOPAMINE FROM CRUDE STRTATAL SYNAPTOSOMAL PREPARATIONS J. HOWESand P. OSGOOD Sheehan Institute and Sharps Associates (SISA), 767B Concord Avenue, Cambridge, Massachusetts 02138 (Acceptc)~ 6 April 1974) SummaryPA’-Tetrahydrocannabinol (A9-THC) and a water soluble ester derivative (compound I) caused a concentration-related decrease in the uptake of “‘C-dopamine into crude synaptosomal preparations derived from mouse striata. Both were less potent than amphetamine in this preparation. In the presence of ~O-‘M amphetamine the IC,, of A’-THC was unaffected. The IC,, is the concentration of drug in the medium which will inhibit the uptake of “C-dopamine into the synaptosomes by 50%. However in the presence of 3.0 x 10VhMA9-THC, the dose response curve to amphetamine was shifted to the right and the ICsO of amphetamine was increased. A’-Tetrahydrocannabinol and compound I increased the release of i4C-dopamine from preparations pre-incubated with “C-dopamine. The effect was small but significant. The effects of amphetamine and A9-THC combined were additive on this system. The mode of action of A9-THC with regard to the dopaminergic system of the striatum is discussed.
In previous communications we have demonstrated that agents such as amphetamine or pemoline, which are known to increase the availability of striatal dopamine, will antagonize many of the effects of the cannabinoids. Both compounds reverse cannabinoid induced catalepsy, ptosis, hypomobility and antinociception in rodents (HOWES. 1973a, b). One of the most important actions of amphetamine is its effect on striatal dopamine (COSTA. GROPETTI and NAIMZADA, 1972). Thus dopamine appears to be a prime candidate for consideration in the mode of action of A9-tetrahydrocannabinol (A9-THC). In view of these interactions, and the putative neurohumoural function of dopamine in the corpus striatum, we have used isolated striatal nerve ending particles (synaptosomes), to study changes in the uptake and release of 14C-dopamine. The purpose of this paper is to examine how A9-THC and a water soluble derivative (compound I) may alter the uptake and release of dopamine in preparations derived from the central nervous system and to try to account for the interactions reported with amphetamine and pemoline. METHODS
Crude preparations of isolated nerve ending particles (synaptosomes) were derived from the striata of male Charles-River mice (18-22 g) by the method described by FERRIS TANG and MAXWELL (1972), modified as follows: i4C-dopamine replaced the 3H-dopamine. The synaptosomes were prepared by homogenization of the striata in 032 M sucrose in 50 mM Tris buffer (pH 7.4) and centrifugation at 1000 g for 10 min. The supernatant was added to the incubation medium containing 2 x lo-‘M dopamine. Incubation was carried out at 37’C for 5 min in an atmosphere of 95% O2 + 5P:, CO,. Passive uptake or release of YI’ Ii ,I II
1109
1110
.I. HOWES and P. Oscoou
14C-dopamine was determined in preparations maintained at O-4°C in an ice bath. The uptake of 14C-dopamine into these preparations and the release from them was studied. Amphetamine and the water soluble compound I were dissolved in a small volume of the aqueous incubation medium. A’-Tetrahydrocannabinol was dissolved in ethanol and added to the medium in 10~1 volumes. Control preparations for A9-THC studies contained 10 ~1ethanol. The data are presented graphically as concentration-effect curves. The ICsO values were determined by replotting the data on log-probit paper and calculating the 95% confidence limits by the method of LITCHFIELD and WILCOXON (1949). The IC,, is the concentration of drug in the medium which will inhibit the uptake of 14C-dopamine into synaptosomes by 50%.
Compound
I
Ephedrine, L-DOPA and L-threo-3,4_dihydroxyphenylserine (L-DOPS) were obtained from Regis Chemical Co. Amphetamine was obtained from Sigma. Pargyline was a gift from Dr. N. Plotnikoff of Abbott Laboratories. A’-Tetrahydrocannabinol and the water soluble compound I were supplied by these laboratories. RESULTS
A9-Tetrahydrocannabinol, amphetamine and compound I caused a concentration related decrease in the uptake of 14C-dopamine into crude striatal synaptosomal preparations (Fig. 1). The I&, values were determined and are listed in Table 1. The value obtained for amphetamine agrees closely with recently published data (FERRISet al., 1972; 8.4 x lo-‘M using rat striata). In the presence of amphetamine (lo-‘M), a concentration which caused a decreased uptake of 14C-dopamine, the relative TC,, of A9-THC was unchanged (Fig. 2 and Table 1) whereas in the presence of an active concentration of A9-THC (3.0 x 10e6~)the effectiveness of amphetamine was reduced. The concentration-response curve for amphetamine was shifted to the right but calculation of the slope functions indicated that they were not parallel.
CONCENTRAT/ON
QF DRUG /N MEDlfJhf fmof NJ
Fig. 1. The effect of A9-THC, compound I and amphetamine on the uptake of ‘V-dopamine by mouse striatal synaptosomes. Preparations were incubated at 37’C for 5 min in an atmosphere of 95% 0, + 5’;” C02. 14C-Dopamine concentration was 2 x lo-‘M. Uptake is expressed as percentage of control.
A9-THC-synaptosomal
Table
dopamine
uptake
1111
I. Concentration of drugs required to reduce by 50% the 5-minute uptake of “C-dopamine 2 x IO-’ M solutions into crude synaptosomal preparations of mouse striatum
Drug A”-THC Compound 1 Amphetamine A9-THC in presence of lo-‘M amphetamine Amphetamine in presence of 3 x 10m6M A9-THC * Confidence
Number of experiments
IC,,
6 6 6 6
5.4 X IO-“M 1.3 X lo-‘M 4.6 x IO-‘M 4.7 X 10-hM
(24 1.2) (0.9-2.2) (2.3-9.2) (1.9-11.8)
6
2.1 X IOmhM
(1.4-5.3)
from
(95:;c.L.)*
limits for 95% probability
A9-Tetrahydrocannabinol and compound I caused a small but significant release of 14C-dopamine from synaptosomes pre-incubated with labelled material (Table 2). Amphetamine caused a marked effect alone and combinations of amphetamine and A”-THC were found to be additive.
Concentration
of drug in medium
(mol/l)
by mouse striatal synaptosomes Fig. 2. A: The effect of A9-THC on the uptake of ‘V-dopamine with lo-‘M amphetamine present in the incubation medium. B: The effect of amphetamine on the uptake of 14C-dopamine by mouse striatal synaptosomes with 3 x IO-‘M A9-THC present in the incubation medium. Preparations were incubated at 37’C for 5 min in an atmosphere of 95% 0, + 5% COz. ‘%Dopamine concentration was 2 x lo-‘M. Uptake is expressed as percentage of control. Table 2. Release of dopamine
from crude synaptosomal preparations of mouse striatum drugs which effectively inhibit uptake of this amine
Concentration A9-THC A9-THC Compound I Amphetamine A9-THC + Amphetamine A9-THC + Amphetamine
lo-‘M
10-6M 10-6M
'M lo-'M lo-'M lo-'M to-'M lo-
Number of experiments
by concentrations
Drug induced release expressed as I’:, k SE. - 1.1 +46 f4.3 +21.0 + 19.4
* f * + *
0.7 0.3* 0,9* 1.7t 2.1t
i-26.7
k 2.27
of
J. HOWESand P. OSGOOLI
1112
Table3.The efkct of ~tn~nt~dine pargfline plus L-DOPA, par&k plus L-DOPS and ephedrine on the motor depressant and antiIloci~ptive action of A"-THC ‘I<, Reversal
Pretreatment
Amantadine Pargyline + L-DOPA Pargyline + L-DOPS Ephedrine Control
ED,, A’-THC in hot plate procedure (mice)* _~ 117.9 (71.2..-208.4) 182(32~&1121~0)
mg/kg __-- “. 50 i.p. 40 p.0. 200 p.0. 40 p.0. 200 p.0, 200 i.p. -_
61.3 (27.1-l 18.0) > 200 54.0 (X2- 1065)
_
OF
decreased motor activity in mice
_~~~~~
_.
74.0 i 6+3 697 & 11.3 Motor activity was further decreased x9.4 * 55t
i P < 0.01. * For experimental details see (f%OWES, 1973a).
Table 3 shows that ephedrine, amantadine and pargyline plus L-DOPA were antagonistic to the motor depressant and antinociceptive actions of A’-THC. This is in agreement with our previously published reports (HOWES, 1973b). Pargyline with L-DOPS was not antagonistic. DISCUSSION
Delta-9-tetrahydrocannabinol produces a distinct syndrome in experimental animals. consisting of a state of catalepsy, accompanied by an increased reaction to sensory stimuli (DOMINO,1971). The biochemical mechanisms underlying these effects are not known but changes in central dopaminergic mechanjsms may be responsible. Many of the actions of amphetamine are attributed to the release of dopamine in the corpus striatum and several workers have described the in&actions of amphetamine with A’-THC (GARRIOTT.KING, FORNEYand HUGHES, 1967: HOWE& 1973a; ZITKO, HOWES,RAZDAN,DALZELL,DALZCLL, SHEEHAN,PARS, DEWEY and HARRIS, 1972; KLJ~ENAand BARRY, 1970; PIRC‘H,COHN, OSTERHOLM and BARRATT’, 1973). These workers have demonstrated that the actions of amphetamine may be potentiated or antagonized depending upon the dose of A”-THC used. WATERS and GLICK (1973) reported on the circling and rotary behaviour in rats induced by a derivative of A9-THC (Compound I). Circling behaviour in rats is seen after unilateral lesions of either the substantia nigra or the striatum in rats (U~~ERST~I~T and ARKUTHNOTT,1970; CHRISTIE and CROW, 1971; COSTALL, NAYL~R and OLLEY, 1972). The circling behaviour induced by amphetamine was potentiated when A9-THC was administered. The effect was dose related. In a previous communication (HOWES, 1973b), we have reported on antagonism between A’-THC and pemoline, using motor activity, catalepsy and antinociceptive activity as parameters. Other agents were also antagonistic (Table 3). I,Threo-3,4_dihydroxyphenylserine, an agent which is converted to norepinephrine directly without going via dopamine, was not antagonistic (Table 3). The agents which were found to be antagonistic to A9-THC all increase dopamine availability at the synapse (COSTA, rf al., 1972; BANERJEE and LIN, 1973; TAGLIAMONTE and TAGLIAMONTE. 1971; HENULEY, SNYDER.FARLEY and LA PINS. 1972; VON VOIGTLANDER and MOORE, 1973a, b). The fact that A9-THC interacts with all these agents is indicative of an effect on the dopaminergic system of the central nervous system. However, various authors have den~onstrated changes in central nervous system neurobumours after A”-THC or n~arijuana extracts.
A9-THC-synaptosomal
dopamine
uptake
1113
SCHILDKRAUT and EPHRON(1971) showed an accelerated rate of disappearance of 3H-norepinephrine from the brain of rats treated with 80mg/kg A9-THC. HOLTZMAN,LOVELL, JAFFEand FREEDMAN, (1964) have shown a slight decrease in brain norepinephrine which was not confirmed by other workers (WELCH,WELCH,MESSIHAand BERGER1971). FRIEDMANand GERSHON(1972) have demonstrated an alteration in rat brainstem dopamine with a concomittant increase in homovanillic acid. MAITRE, STAEHELINand BEIN (1970) have demonstrated an enhanced 3H-dopamine formation from 3H-tyrosine in brains of rats after treatment with A”-THC. However, changes in the blood levels of 3H-tyrosine caused by A”-THC during these experiments make interpretation of their data unclear. Increased levels of 5hydroxytryptamine (5HT) in the brain have been reported after A9-THC, as well as a decreased rate of turnover of 5-HT (SOFIA,DIXIT and BARRY,1971; WELCH et d., 1971). The work described in this communication was undertaken to determine whether A9THC affected the processes by which dopamine was taken up and released by synaptosoma1 preparations from the striatum. A”-Tetrahydrocannabinol and compound I caused a dose related inhibition of the uptake of 14C-dopamine into crude striatal synaptosomes at low concentration. Amphetamine was the most active compound tested and the value obtained agreed closely with a reported value (FERRISet al., 1972). When a fixed, active concentration of amphetamine was present in the medium, the relative concentration effect curve for A9-THC was unaltered; however, when a fixed concentration of A9-THC was used, the concentration effect curve for amphetamine was shifted to the right. A calculation of the slopes of these curves indicated that they were not parallel. Both A9-THC and amphetamine increased the release of 14C-dopamine from striatal synaptosomes. While the results do not satisfactorily explain the antagonism between A9-THC and amphetamine. they may help explain the reasons for the potentiation also observed with amphetamine by indicating an altered disposition of dopaminti. .4cl\llorl~lct/~~crrrc’rlrThis stud) was a part of a programme, Illinois.
supported
bq Abbott
Laboratories.
North
Chicago.
REFERENCES BANIKJEF, U. and LIP, G. S. (1973). On the mechanism of central action of amphetamine: the role of catcholamines. iVrurop/~clrrna~olu~~ 12: 9 I l-93 I CHRISTIE_J. E. and CROW. T. J. (197 I). Turning behavior as an index of the action of amphetamine and ephedrines on central dopamine-containing neurons. Br. J. Pharwnc. 43: 658-665. COSTA, E..GROPETTI. A. and NAIMZAI~A,M. (1972). Effects ofamphetamine on the turnover rate of brain catecholamines and motor activity. Br. J. Phwrmc. 44: 732-741, Cos-rALI_, B.. NAYLOK. R. J. and OLLEY. J. (1972). Catalepsy and circling behavior after intracerebral injections of neurolcptic. cholinergic and anticholinergic agents into the caudate putamen. globus pallidus and substantia nigra of rat brain. R'rurophu,rt~nco~~~~~ 1 I : 645 ~663. DOMINO. E. F. (1971). Ncuropsychopharmacologic studies of marijuana: some synthetic and natural THC derivatimes in animals and man. A~rr. N.Y. .4curl. Sci. 191: 166 191. FI.KRIS. R.. TAN(;. F. and MAXWELL. R. (1972). A comparison of the capacities of isomers of amphetamine, deoxypipradol and methylphenidate to inhibit the uptake of tritiated catecholamines into rat cerebral cortex slices. sqnaptosomal preparations of rat cerebral cortex, hypothalamus and striatum and into adrcnergic nerves of rabbit aorta. J. Phurmc~. c.xp. Thrr. 181: 407-416. FRII:I>MAPY. E. and GFIISHON, S. (1972). A*-Tetrahydrocannabinol (A’-THC): central dopamine (DA) metabolism and behavloral effects after acute and chronic treatment in rats. 5th /nte/~utional Co~qrrsb on Phar~lacoloy~. Abstract 439. GARRIOTT. J. C., KIX, L. J., FOKNF~. R. B. and HUGHES, F. W. (1967). Effects of some tetrahydrocannabinols on hexobarbital sleeping time and amphetamine induced hyperactivity in mice. L$ Sci. 6: ?I 19-2128.
1114
J. HOWESand P. OSGOOD
HENOLEY, E., SNYDER,S., FARLEY, J. and LAPIDUS, J. (1971). Stereoselectivity of catecholamine uptake by brain synaptosomes: studies with ephedrine, methyl phenidare and phenyl-2-piperidyl carbinol. J. Pharmuc. rxp. T&r. 183: 103-I 16. HOLTZMAN, D., LOVELL, R. A., JAFFE, J. H. and FREEDMAN, D. X. (1969). I-A9-Tetrahydrocannabinol: neurochemica1 and behavioral effects in the mouse. Sciencr, N.Y 163: 14641467. HOWES, J. F. (1973a). The effect of A3-tetrahydrocannabinol on amphetamine induced lethality in aggregated mice. Rex Conwn. chrm. Pathol. Phurmac. 6: 895-900. Howes, J. F. (1973b). Antagonism of the effects of A9-tetrahydrocannabinol by pemoline (Cylert). Rrs. Comm. them. Pathol. Pharmucol. 6: 901-908. K~JBENA, R. K. and BARRY, H. III (1970). Interactions of A’-tetrahydrocannabinols with barbiturates and methamphetamine. J. Pharmac. exp. Ther. 173: 94-100. LITCHFIELD. .I. T. and WILCOXON. F. (1949). A simolified method of evaluating dose-effect experiments. J. Pharmat. erp. Thu. 96: 99-113. MAITRE, L., STAEHELIN,M. and BAIN,H. (1970).Effect of an extract of cannabis and some cannabinols on catecholamine metabolism in rat heart and brain. Agents Actions 1: 136-143. PIRCH, J. H., COHN,R. A., OSTERHOLM, K. C. and BARRATT, E. S. (1973). Antagonism of amphetamine locomotor stimulation of single doses of marijuana extract administered orally. Neuropharmacology 12: 4X5-493. SCHILDKRAU~, J. and EPHRON, D. (1971). The effects of A9-tetrahydrocannabinol on the metabolism of norepinephrine in rat brain. Ps~chophuln~acologiu 20: 191k196. SOFIA. R. D.. DIXIT, B. N. and BARICY.H. (1971). The effects ofdelta-I-tetrahydrocannabinol on serotonin metabohsm in the rat brain. L$ Sci. 10: 425.-431. TAC;LIAMONTE,A. and TAGLIAMONTE,P. (1971). Stimulation of brain dopamine turnover by magnesium pemoline. Fedn. Proc. Frdn Am. Sots exp. Biol. Abstract 166. UNGERSTEUT, U. and ARBUTHNOTT, G. W. (1970). Quantitative recordings of rotational behaviour in rats after 6-hydroxydopamine lesions of the nigro-striatal dopamine system. Brain Rrs. 24: 485-493. VON VOIGTLANUER, P. F. and MOORE, K. E. (1973a). Turning behaviour in mice with unilateral 6-hydroxydopamine lesions in the striatum: effects of apomorphine, L-DOPA, amantadine, amphetamine and other psychomotor stimulants. Nruropharmacolog_v 12: 451-462. VON VOIGTLANDER, P. F. and MOORE, K. E. (I 973b). Involvement of nigrostriatal neurons in the i,l viva release of dopamine by amphetamine, amantadine and tyramine. J. Pharmac. rxp. Thu. 184: 542-552. WATERS. D. H. and GLICK, S. D. (1973). Assymetrical effect on delta 9-tetrahydrocannabinol (THC) on striatal dopamine and behaviour. Rrs. Comm. chehz. Puthol. Phurmac. 6: 57-63. WELCW. B. L.. WELCH. A. S.. MESSIHA.F. S. and BERGER, H. J. (1971). Rapid of adrenal aoineuhrine . depletion . 1 . and elevation of telencaphalic serotonin by (-)-trans-delta-9-tetrahydrocannabinol in mice. Rrs. Comm. chrm Patho/. Phurmac. 2: 382-390. ZI~KO. B.A.. H~WES. J. F., RAZDAN, R. K., DALZELL. B. C., DALZELL, H. C., SHEEHAN, J. C., PARS, H. C., DEWEY, W. L. and HARRIS, L. S. (1972). Water-soluble derivatives of A’-tetrahydrocannabinols. Science, N.Y 177: 442444.
In previous communications we have demonstrated that agents such as amphetamine or pemoline, which are known to increase the availability of striatal dopamine, will antagonize many of the effects of the cannabinoids. Both compounds reverse cannabinoid induced catalepsy, ptosis, hypomobility and antinociception in rodents (HOWES. 1973a, b). One of the most important actions of amphetamine is its effect on striatal dopamine (COSTA. GROPETTI and NAIMZADA, 1972). Thus dopamine appears to be a prime candidate for consideration in the mode of action of A9-tetrahydrocannabinol (A9-THC). In view of these interactions, and the putative neurohumoural function of dopamine in the corpus striatum, we have used isolated striatal nerve ending particles (synaptosomes), to study changes in the uptake and release of 14C-dopamine. The purpose of this paper is to examine how A9-THC and a water soluble derivative (compound I) may alter the uptake and release of dopamine in preparations derived from the central nervous system and to try to account for the interactions reported with amphetamine and pemoline. METHODS
Crude preparations of isolated nerve ending particles (synaptosomes) were derived from the striata of male Charles-River mice (18-22 g) by the method described by FERRIS TANG and MAXWELL (1972), modified as follows: i4C-dopamine replaced the 3H-dopamine. The synaptosomes were prepared by homogenization of the striata in 032 M sucrose in 50 mM Tris buffer (pH 7.4) and centrifugation at 1000 g for 10 min. The supernatant was added to the incubation medium containing 2 x lo-‘M dopamine. Incubation was carried out at 37’C for 5 min in an atmosphere of 95% O2 + 5P:, CO,. Passive uptake or release of YI’ Ii ,I II
1109
1110
.I. HOWES and P. Oscoou
14C-dopamine was determined in preparations maintained at O-4°C in an ice bath. The uptake of 14C-dopamine into these preparations and the release from them was studied. Amphetamine and the water soluble compound I were dissolved in a small volume of the aqueous incubation medium. A’-Tetrahydrocannabinol was dissolved in ethanol and added to the medium in 10~1 volumes. Control preparations for A9-THC studies contained 10 ~1ethanol. The data are presented graphically as concentration-effect curves. The ICsO values were determined by replotting the data on log-probit paper and calculating the 95% confidence limits by the method of LITCHFIELD and WILCOXON (1949). The IC,, is the concentration of drug in the medium which will inhibit the uptake of 14C-dopamine into synaptosomes by 50%.
Compound
I
Ephedrine, L-DOPA and L-threo-3,4_dihydroxyphenylserine (L-DOPS) were obtained from Regis Chemical Co. Amphetamine was obtained from Sigma. Pargyline was a gift from Dr. N. Plotnikoff of Abbott Laboratories. A’-Tetrahydrocannabinol and the water soluble compound I were supplied by these laboratories. RESULTS
A9-Tetrahydrocannabinol, amphetamine and compound I caused a concentration related decrease in the uptake of 14C-dopamine into crude striatal synaptosomal preparations (Fig. 1). The I&, values were determined and are listed in Table 1. The value obtained for amphetamine agrees closely with recently published data (FERRISet al., 1972; 8.4 x lo-‘M using rat striata). In the presence of amphetamine (lo-‘M), a concentration which caused a decreased uptake of 14C-dopamine, the relative TC,, of A9-THC was unchanged (Fig. 2 and Table 1) whereas in the presence of an active concentration of A9-THC (3.0 x 10e6~)the effectiveness of amphetamine was reduced. The concentration-response curve for amphetamine was shifted to the right but calculation of the slope functions indicated that they were not parallel.
CONCENTRAT/ON
QF DRUG /N MEDlfJhf fmof NJ
Fig. 1. The effect of A9-THC, compound I and amphetamine on the uptake of ‘V-dopamine by mouse striatal synaptosomes. Preparations were incubated at 37’C for 5 min in an atmosphere of 95% 0, + 5’;” C02. 14C-Dopamine concentration was 2 x lo-‘M. Uptake is expressed as percentage of control.
A9-THC-synaptosomal
Table
dopamine
uptake
1111
I. Concentration of drugs required to reduce by 50% the 5-minute uptake of “C-dopamine 2 x IO-’ M solutions into crude synaptosomal preparations of mouse striatum
Drug A”-THC Compound 1 Amphetamine A9-THC in presence of lo-‘M amphetamine Amphetamine in presence of 3 x 10m6M A9-THC * Confidence
Number of experiments
IC,,
6 6 6 6
5.4 X IO-“M 1.3 X lo-‘M 4.6 x IO-‘M 4.7 X 10-hM
(24 1.2) (0.9-2.2) (2.3-9.2) (1.9-11.8)
6
2.1 X IOmhM
(1.4-5.3)
from
(95:;c.L.)*
limits for 95% probability
A9-Tetrahydrocannabinol and compound I caused a small but significant release of 14C-dopamine from synaptosomes pre-incubated with labelled material (Table 2). Amphetamine caused a marked effect alone and combinations of amphetamine and A”-THC were found to be additive.
Concentration
of drug in medium
(mol/l)
by mouse striatal synaptosomes Fig. 2. A: The effect of A9-THC on the uptake of ‘V-dopamine with lo-‘M amphetamine present in the incubation medium. B: The effect of amphetamine on the uptake of 14C-dopamine by mouse striatal synaptosomes with 3 x IO-‘M A9-THC present in the incubation medium. Preparations were incubated at 37’C for 5 min in an atmosphere of 95% 0, + 5% COz. ‘%Dopamine concentration was 2 x lo-‘M. Uptake is expressed as percentage of control. Table 2. Release of dopamine
from crude synaptosomal preparations of mouse striatum drugs which effectively inhibit uptake of this amine
Concentration A9-THC A9-THC Compound I Amphetamine A9-THC + Amphetamine A9-THC + Amphetamine
lo-‘M
10-6M 10-6M
'M lo-'M lo-'M lo-'M to-'M lo-
Number of experiments
by concentrations
Drug induced release expressed as I’:, k SE. - 1.1 +46 f4.3 +21.0 + 19.4
* f * + *
0.7 0.3* 0,9* 1.7t 2.1t
i-26.7
k 2.27
of
J. HOWESand P. OSGOOLI
1112
Table3.The efkct of ~tn~nt~dine pargfline plus L-DOPA, par&k plus L-DOPS and ephedrine on the motor depressant and antiIloci~ptive action of A"-THC ‘I<, Reversal
Pretreatment
Amantadine Pargyline + L-DOPA Pargyline + L-DOPS Ephedrine Control
ED,, A’-THC in hot plate procedure (mice)* _~ 117.9 (71.2..-208.4) 182(32~&1121~0)
mg/kg __-- “. 50 i.p. 40 p.0. 200 p.0. 40 p.0. 200 p.0, 200 i.p. -_
61.3 (27.1-l 18.0) > 200 54.0 (X2- 1065)
_
OF
decreased motor activity in mice
_~~~~~
_.
74.0 i 6+3 697 & 11.3 Motor activity was further decreased x9.4 * 55t
i P < 0.01. * For experimental details see (f%OWES, 1973a).
Table 3 shows that ephedrine, amantadine and pargyline plus L-DOPA were antagonistic to the motor depressant and antinociceptive actions of A’-THC. This is in agreement with our previously published reports (HOWES, 1973b). Pargyline with L-DOPS was not antagonistic. DISCUSSION
Delta-9-tetrahydrocannabinol produces a distinct syndrome in experimental animals. consisting of a state of catalepsy, accompanied by an increased reaction to sensory stimuli (DOMINO,1971). The biochemical mechanisms underlying these effects are not known but changes in central dopaminergic mechanjsms may be responsible. Many of the actions of amphetamine are attributed to the release of dopamine in the corpus striatum and several workers have described the in&actions of amphetamine with A’-THC (GARRIOTT.KING, FORNEYand HUGHES, 1967: HOWE& 1973a; ZITKO, HOWES,RAZDAN,DALZELL,DALZCLL, SHEEHAN,PARS, DEWEY and HARRIS, 1972; KLJ~ENAand BARRY, 1970; PIRC‘H,COHN, OSTERHOLM and BARRATT’, 1973). These workers have demonstrated that the actions of amphetamine may be potentiated or antagonized depending upon the dose of A”-THC used. WATERS and GLICK (1973) reported on the circling and rotary behaviour in rats induced by a derivative of A9-THC (Compound I). Circling behaviour in rats is seen after unilateral lesions of either the substantia nigra or the striatum in rats (U~~ERST~I~T and ARKUTHNOTT,1970; CHRISTIE and CROW, 1971; COSTALL, NAYL~R and OLLEY, 1972). The circling behaviour induced by amphetamine was potentiated when A9-THC was administered. The effect was dose related. In a previous communication (HOWES, 1973b), we have reported on antagonism between A’-THC and pemoline, using motor activity, catalepsy and antinociceptive activity as parameters. Other agents were also antagonistic (Table 3). I,Threo-3,4_dihydroxyphenylserine, an agent which is converted to norepinephrine directly without going via dopamine, was not antagonistic (Table 3). The agents which were found to be antagonistic to A9-THC all increase dopamine availability at the synapse (COSTA, rf al., 1972; BANERJEE and LIN, 1973; TAGLIAMONTE and TAGLIAMONTE. 1971; HENULEY, SNYDER.FARLEY and LA PINS. 1972; VON VOIGTLANDER and MOORE, 1973a, b). The fact that A9-THC interacts with all these agents is indicative of an effect on the dopaminergic system of the central nervous system. However, various authors have den~onstrated changes in central nervous system neurobumours after A”-THC or n~arijuana extracts.
A9-THC-synaptosomal
dopamine
uptake
1113
SCHILDKRAUT and EPHRON(1971) showed an accelerated rate of disappearance of 3H-norepinephrine from the brain of rats treated with 80mg/kg A9-THC. HOLTZMAN,LOVELL, JAFFEand FREEDMAN, (1964) have shown a slight decrease in brain norepinephrine which was not confirmed by other workers (WELCH,WELCH,MESSIHAand BERGER1971). FRIEDMANand GERSHON(1972) have demonstrated an alteration in rat brainstem dopamine with a concomittant increase in homovanillic acid. MAITRE, STAEHELINand BEIN (1970) have demonstrated an enhanced 3H-dopamine formation from 3H-tyrosine in brains of rats after treatment with A”-THC. However, changes in the blood levels of 3H-tyrosine caused by A”-THC during these experiments make interpretation of their data unclear. Increased levels of 5hydroxytryptamine (5HT) in the brain have been reported after A9-THC, as well as a decreased rate of turnover of 5-HT (SOFIA,DIXIT and BARRY,1971; WELCH et d., 1971). The work described in this communication was undertaken to determine whether A9THC affected the processes by which dopamine was taken up and released by synaptosoma1 preparations from the striatum. A”-Tetrahydrocannabinol and compound I caused a dose related inhibition of the uptake of 14C-dopamine into crude striatal synaptosomes at low concentration. Amphetamine was the most active compound tested and the value obtained agreed closely with a reported value (FERRISet al., 1972). When a fixed, active concentration of amphetamine was present in the medium, the relative concentration effect curve for A9-THC was unaltered; however, when a fixed concentration of A9-THC was used, the concentration effect curve for amphetamine was shifted to the right. A calculation of the slopes of these curves indicated that they were not parallel. Both A9-THC and amphetamine increased the release of 14C-dopamine from striatal synaptosomes. While the results do not satisfactorily explain the antagonism between A9-THC and amphetamine. they may help explain the reasons for the potentiation also observed with amphetamine by indicating an altered disposition of dopaminti. .4cl\llorl~lct/~~crrrc’rlrThis stud) was a part of a programme, Illinois.
supported
bq Abbott
Laboratories.
North
Chicago.
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J. HOWESand P. OSGOOD
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