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The effect of phencyclidine on dopamine synthesis and metabolism in rat striatum
European Journal of Pharmacology, 65 (1980) 139--149 © Elsevier/North-Holland Biomedical Press
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THE EFFECT OF PHENCYCLIDINE ON DOPAMINE SYNTHESIS AND METABOLISM IN RAT STRIATUM JOHN D. DOHERTY, MILJANA SIMONOVIC, REBECCA SO and HERBERT Y. MELTZER
Department of Psychiatry and Physiological and Pharmacological Sciences, University of Chicago Pritzker School of Medicine, Chicago, Illinois 60637 and Illinois State Psychiatric Institute, 1601 W. Taylor Street, Chicago, Illinois 60612, U.S.A. Received 24 September 1979, revised MS received 11 January 1980, accepted 8 April 1980
J.D. DOHERTY, M. SIMONOVIC, R. SO and H.Y. MELTZER, The effect of phencyclidine on dopamine synthesis and metaolism in rat striatum, European J. Pharmacol. 65 {1980} 139--149. Previous behavioral and neurochemical studies indicate that phencyclidine (PCP), a potent psychotomimetic agent, interacts with central dopaminergic systems. We have examined the effects of PCP on the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) after the inhibition of L-aromatic amino acid decarboxylase and on the levels of dopamine (DA) metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum. PCP, in doses from 2.5 to 50 mg/kg, decreased the rate of striatal DOPA accumulation. PCP did not antagonize the increase in the rate of striatal DOPA formation caused by haloperidol, reserpine or ),-butyrolactone (GBL). When given alone, PCP decreased striatal levels of DOPAC and HVA, while it greatly potentiated the haloperidol-induced rise in striatal levels of these two metabolites. PCP is considerably less effective than d-amphetamine in promoting the release of 3H-DA from preloaded striatal slices in vitro. Our results are consistent with the interpretation that PCP potentiates the synaptic effects of endogenous DA. Its mechanism of action appears to be closely related to that of a category of drugs known as non-amphetamine stimulants, which, among others, includes methylphenidate, amfonelic acid and cocaine. Phencyclidine
Dopamine agonist
Reuptake blocker
1. Introduction
Phencyclidine (PCP) is a phenylcyclohexyl derivative with an unusual spectrum of pharmacologic actions. Its behavioral effects vary markedly, depending on the dose and species used, and range from stimulation in rodents to sedation in higher species (Chen et al., 1959). Subanesthetic doses of PCP may have profound psychotomimetic effects in humans. It sometimes induces behavioral changes which mimic the primary symptoms of schizoprenia in drug abusers or exacerbates symptoms in chronic schizophrenics (Luby et al., 1962; Meltzer et al., 1972). In rodents, low doses of PCP produce central stimulation characterized by increased locomotor activity and stereotyped behavior,
not unlike that caused by psychomotor stimulants such as amphetamine (Chen et al., 1959; Kanner et al., 1975a; Sturgeon et al., 1979). The effect of PCP on food-reinforced operant behavior is qualitatively similar to d-amphetamine (Wenger and Dews, 1976). PCP induces ipsilateral turning in rats with unilateral electrolytic or 6-hydroxydopamineinduced lesions in the nigro-striatal pathway, a finding consistent with an indirect rather than a direct DA agonist action (Fessler et al., 1979). The fact that inhibition of DA synthesis with ~-methylparatyrosine blocks this effect also suggests that PCP acts as an indirect DA agonist (Kanner et al., 1975b; Fessler et al., 1979). Numerous reports indicate that PCP has important effects on central dopaminergic,
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noradrenergic, cholinergic, serotonergic, GABA-ergic and endogenous opiates transmitter systems (Vincent et al., 1978; for review see Johnson, 1978). The mechanism underlying the characteristic psychotropic effects of PCP is not known. However, the possibility that behavioral stimulation seen in rats after PCP administration is related to its ability to enhance central dopaminergic transmission is of particular interest because of the possible role of DA in behavioral manifestations of psychomotor stimulants and schizophrenia (Von Voigtlander and Moore, 1973; Snyder et al., 1974; Meltzer and Stahl, 1976). In order to further elucidate the interaction of PCP with central dopaminergic systems, we have studied its effects on striatal DA synthesis and metabolism. A preliminary report of these findings has been presented at the 4th International Catecholamine Symposium (Simonovic et al., 1979).
2. Materi~l~ and methods
2.1. Animals Male, Sprague-Dawley rats, purchased from Sprague-Dawley, Inc., Madison, Wisconsin, weighing from 175-225 g were used throughout these studies. They were housed 6 per cage in a temperature (26°C) and humidity (60%) regulated room with a 12-h light/dark cycle. The animals had free access to Purina rat chow and water at all times.
2.2. Drugs d-Amphetamine sulphate was a gift from Smith, Kline and French, Philadelphia, Pa.; Ro4-4602 (seryl-trihydroxybenzylhydrazine) was kindly supplied by Dr. William Scott of Hoffmann-LaRoche Co., Nutley, N.J. and cocaine was obtained from the National Institute of Drug Abuse. The other drugs were
J.D. D O H E R T Y E T AL.
purchased: phencyclidine (Sernylan ®, BioCeutics Laboratories Inc., St. Joseph, Mo.); apomorphine HC1 (Mallinckrodt, St. Louis, Mo.); 7-butyrolactone (GBL) (Eastman Organic Chemicals Co., Rochester, N.Y.), haloperidol (Haldol ®, McNeil Laboratories, Fort Washington, Pa.); reserpine (Serpasil ®, CIBA Pharmaceutical Co., Summit, N.J.); pargyline HC1 (Abbott Laboratories, North Chicago, Ill.); L-3,4-dihydroxyphenylalanine (Sigma Chemical Co., St. Louis, Mo.). All injections were made intraperitoneally (i.p.) at time and dose schedules given in the Results section. Doses of basic compounds refer to salts. Control rats always received the same number of saline injections at corresponding time intervals.
2.3. Dopamine synthesis in vivo The rate of accumulation of striatal 3,4dihydroxyphenylalanine (DOPA) after decarboxylase inhibition has been shown to measure the rate of dopamine synthesis in this brain region (Carlsson et al., 1972). A rapidly acting inhibitor of aromatic amino acid decarboxylase, Ro4-4602 (800 mg/kg) was administered to rats 40 min before decapitation. It has been shown that following this dose of Ro44602, the accumulation of DOPA in the striatum is linear for at least 60 min (Walters and Roth, 1974). Immediately after decapitation, the brains were rapidly removed and corpora striata dissected on an ice-cold glass plate as described by Glowinski and Iversen (1966). The brain parts were frozen on dry ice and stored at --70°C until assayed. DOPA was isolated and measured fluorometrically by the technique of Kehr et al. (1972) with the modifications described by Walters and Roth (1974). The mean recovery of 300 ng of L-DOPA taken through the entire procedure was 61.2 + S.E.M. 2.2% (n = 50). Forty min after the administration of Ro4-4602, the mean striatal DOPAcontent was 2.15 + S.E.M. 0.11 t~g/g wet tissue (n = 40); all values were corrected for recovery.
PHENCYCLIDINE
2.4. Release o f tissue slices
AND
STRIATAL
DOPAMINE
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3. Results
3H-dopamine from striatal
3.1. The effect of PCP on the rate o f striatal DOPA formation after decarboxylase inhibition
The release of 3H
In agreement with previous studies (Roth et al., 1974; Kehr et al., 1975; Carlsson et al., 1977; Kehr et al., 1977), we have found that apomorphine inhibits, while d-amphetamine stimulates, the in vivo activity of striatal tyrosine hydroxylase as measured by the rate of DOPA accumulation after decarboxylase inhibition (fig. la). The dose-response curve for the effect of PCP on striatal tyrosine hydroxylase is presented in fig. l b . All doses of PCP decreased striatal DOPA accumulation, but the effect of 2.5 mg/kg did not reach statistical significance. In the dose range
2.5. Determination of DOPAC and HVA
Following drug treatment according to the schedules given in the Results section, rats were killed, their striata removed, homogenized in 1 N HC1 and frozen. The homogenate was shipped in dry ice for analysis by the electron capture gas chromatographic m e t h o d used in the laboratory of Dr. S. Wilk, Dept. of Pharmacology, Mt. Sinai School of Medicine, City University of New York (Wilk et al., 1975).
b
a
200
[] Saline • PCP [] Apomorphine [] d-Amphetamine
o Saline
• PCP I00
150
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~Q
o c o
8
0
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0
0 r-.
50
~-~ "'"'""
5
5
I
5
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2.5
1
I
I
5
I0
50
Fig. 1. The effect o f various drugs (a) and various doses of PCP (b) on the rate o f striatal DOPA accumulation following decarboxylase inhibition. Drugs were injected 45 min and Ro4-4602 (800 mg/kg) 40 rain before decapitation. The means for groups of four rats are given; vertical bars represent S.E.M. Significantly different from control (Tukey's HSD test): * P < 0.01, ** P < 0.05. Abscissa: dose (mg/kg).
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tested, the maximal decrease in the rate of striatal DOPA formation was attained with 10 mg/kg of PCP. Subsequent experiments were performed in an effort to define further the mechanism underlying the inhibitory effect of PCP on striatal tyrosine hydroxylase in vivo. Unlike apomorphine, a direct acting DA agonist (Ernst, 1967; And~n et al., 1967), which can reverse the increase in striatal DOPA accumulation due to haloperidol (Carlsson et al., 1977), PCP had no effect on haloperidol-induced activation of striatal tyrosine hydroxylase (fig. 2). These results suggest that the decrease in the activity of striatal tyrosine hydroxylase observed after the administration of PCP alone is probably not due to its ability to directly activate DA receptors. However, the high dose of haloperidol used may have masked the possible DA receptor agonist properties of PCP. We have therefore evaluated its effects on preand post-synaptic DA receptors in the absence of a DA receptor blocker. Reserpine, a drug which depletes granular DA stores (Shore, 1962), causes a profound increase in the activity of striatal tyrosine hydroxylase (Carlsson and Lindqvist, 1977), an effect which is antagonized by apomorphine in a dose
J.D. DOHERTY ET AL.
[]
SALINE
[]
~ERP~E
600
5OO
400
300
200
I00
5
5
Fig. 2. The effect of PCP on the rate of striatal DOPA formation in rats pretreated with haloperidol (1 mg/kg) or reserpine (5 mg/kg). Reserpine was administered 365 rain, haloperidol 50 rain, PCP 45 min and Ro4-4602 (800 mg/kg) 40 min before decapitation. The means for groups of four rats are given; vertical bars represent S.E.M. Significantly different from saline treated controls (Student's t-test); * P < 0.01. Ordinate: DOPA (% of control). Abscissa: drug, dose (mg/kg).
lethal. PCP can also potentiate the lethal effects of another anesthetic, pentobarbital (Chen et al., 1959). 3.2. The effect o f PCP on striatal levels o f D O P A C and H V A
PCP (10mg/kg) significantly decreased striatal levels of DOPAC (--28%) and HVA (--32%) (fig. 3). Haloperidol, as expected, increased striatal concentration of these two DA metabolites. When given in combination with haloperidol, PCP greatly enhanced the neuroleptic-induced rise in striatal content of DOPAC and HVA (fig. 3). 3.3. The effect o f PCP on 3H-DA release from striatal slices in vitro
In order to assess the DA releasing properties of PCP, we studied its effect on the release
PHENCYCLIDINE AND STRIATAL DOPAMINE
143
TABLE 1 The e f f e c t o f PCP on the rate o f striatal D O P A f o r m a t i o n in rats pretreated w i t h GBL. G B L w a s administered 5 0 rain, PCP 4 5 m i n and R o 4 - 4 6 0 2 ( 8 0 0 m g / k g ) 4 0 rain b e f o r e d e c a p i t a t i o n . Group
Drug 1
(mg/kg)
Drug 2
(mg/kg)
Striatal D O P A (% of c o n t r o l ) 1
Cornparison
P
(1) (2) (3) (4)
Saline Saline GBL GBL
--500 500
Saline PCP Saline PCP
-5 -5
100 50 231 251
1-2 1-3 3-4
<0.01
_+ 10 -+ 4 _+ 15 -+ 33
<0.01 n.s.
1 Mean for a group o f four rats _+ S.E.M. Significance o f difference assessed using S t u d e n t ' s t-test.
4
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-i
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~3 o
"1-
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-3
er
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2
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Fig. 3. T h e e f f e c t o f PCP ( 1 0 m g / k g ) alone or in c o m b i n a t i o n w i t h h a l o p e r i d o l (1 m g / k g ) o n striatal levels o f D O P A C and H V A as assayed b y the e l e c t r o n capture c h r o m a t o g r a p h y . Drugs were administered 4 5 m i n before d e c a p i t a t i o n . T h e means f o r groups o f four rats are given; vertical bars represent S.E.M. Statistical significance w a s assessed using S t u d e n t ' s t-test. [] S a l o n e ; [] PCP; ~ h a l o p e r i d o l ; m h a l o p e r i d o l + PCP. * P < 0 . 0 5 vs. saline. • * P < 0 . 0 0 1 vs. haloperidol.
TABLE 2 T h e e f f e c t o f PCP and o t h e r drugs on the release o f 3 H - d o p a m i n e f r o m striatal tissue slices. Drug
Concentration
% o f 3 H - d o p a m i n e remaining in slices
Control ( n o drug) PCP PCP PCP d-Amphetamine Cocaine
-5 2 1 1 1
100 58.0 71.0 72.2 33.4 85.0
× × × × ×
1 0 -4 1 0 -4 10 -~ 10-4 10- 4
_+ 0.2 (3) -+ 0.3 (3) + 6.3 (18) + 2.8 (9) + 0.004 (3)
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of 3H-DA from preloaded striatal slices, using the method of Heikkila et al. (1975a), which can differentiate release from the inhibition of uptake. PCP was found to be considerably less potent than d-amphetamine in promoting 3H-DA release, but slightly more potent than cocaine (table 2), indicating that in vitro PCP had a weak DA releasing action, at least in this model.
4. Discussion Considerable evidence supports the view that the activity of striatal tyrosine hydroxylase, and consequently the rate of DA synthesis, is under complex, receptor-mediated feedback control. Thus, a well-known DA receptor agonist, apomorphine, inhibits, while haloperidol, a potent DA receptor blocker, stimulates striatal DA synthesis (Kehr et al., 1975; Carlsson et al., 1977). We have tested the effect of PCP on striatal DA synthesis by measuring the rate of striatal DOPA accumulation after inhibition of L-aromatic amino acid decarboxylase, an index of the in vivo activity of striatal tyrosine hydroxylase (Carlsson et al., 1972). PCP produced a significant and apparently dose-related decrease in the rate of striatal DOPA formation. It is interesting to note that the maximal decrease in striatal DOPA levels was attained with 10 mg/kg of PCP, the dose which also produced maximal effect on turning behavior in rats with unilateral substantia nigra lesions (Finnegan et al., 1976). Our results are in good agreement with an earlier tracer study conducted in mice, which reported that PCP, at doses similar to those used here, decreased the accumulation of 14CDA when 14C-tyrosine, but not when 14CDOPA, was used as the precursor (Hitzemann et al., 1973). The possibility that PCP inhibits striatal DOPA formation by direct inhibition of tyrosine hydroxylase is unlikely because PCP actually stimulates tyrosine hydroxylase in vitro (Garey et al., 1977; Vickroy and
J.D. DOHERTY ET AL.
Johnson, 1979). This in vitro effect of PCP may be due to disinhibition of tyrosine hydroxylase following enhanced DA release from brain slices (Garey et al., 1977) or synaptosomes (Vickroy and Johnson, 1979). It is also unlikely that the decrease in striatal DOPA formation after PCP is due to reduced supply of tyrosine to the brain because PCP elevates brain and plasma levels of tyrosine (Tonge and Leonard, 1970). Further, PCP has no effect on the activity of brain monoamine oxidase (Tonge and Leonard, 1969). In addition to decreasing the rate of striatal DA synthesis, PCP also reduced striatal levels of DOPAC and HVA, the two principal DA metabolites. These effects of PCP are indistinguishable from those of apomorphine (Westerink and Korf, 1976; Carlsson et al., 1977). In the absence of any further information, this similarity with apomorphine would suggest that PCP acts as a DA receptor agonist. However, the inability of PCP to antagonize the increase in the rate of striatal DOPA formation caused by haloperidol, reserpine or GBL clearly demonstrates that the effect of PCP on striatal DA synthesis and metabolism could not be attributed to its ability to activate directly either pre- or post-synaptic DA receptors. This is further supported by the fact that apomorphine can antagonize (Lahti et al., 1972) while PCP potentiates (fig. 3) the rise in striatal DA metabolites caused by haloperidol. A direct DA agonist action of PCP is also inconsistent with its effect on turning behavior in rats with unilateral substantia nigra lesions (Fessler et al., 1979). PCP had no effect on in vitro binding of 3H-DA to synaptosomal membranes from rat striatum (Vincent et al., 1978). We have also found that PCP has a very limited ability to displace 3H-spiroperidol from rat striatal membranes (So and Meltzer, unpublished data). These findings further support the conclusion that PCP is devoid of DA receptor agonist properties. Our observation that reserpine, which depletes granular DA stores (Shore, 1962), or GBL, which blocks impulse flow in nigro-
PHENCYCLIDINE AND STRIATAL DOPAMINE striatal DA neurons (Walters et al., 1973) can both completely abolish the inhibitory effect of PCP on striatal tyrosine hydroxylase indicates that the reserpine-sensitive DA pool and uninterrupted impulse flow are necessary for t h e expression of this effect. PCP may inhibit tyrosine hydroxylase by enhancing the effect of synaptic DA on presynaptic DA receptors, which regulate DA synthesis, or on postsynaptic DA receptors, which regulate the activity of nigro-striatal DA neurons via the striato-nigral neuronal feedback loop (Carlsson et al., 1977). It is also possible that PCP releases DA from its storage sites within the granule during impulse flow and that increased intraneuronal levels of free DA inhibits tyrosine hydroxylase. Haloperidol, which is known to increase impulse flow in DA containing neurons, elevates striatal levels of deaminated DA metabolites, DOPAC and HVA. The ability of PCP to potentiate this effect of haloperidol (fig. 3) indicates that, at least during the conditions of increased impulse flow, PCP can enhance DA release. In contrast to its effect in haloperidol-treated rats. PCP decreased striatal levels of DOPAC and HVA when given alone (fig. 3). One possible explanation for this finding is that the decrease in striatal DOPAC and HVA is secondary to the inhibition of tyrosine hydroxylase by PCP. Alternatively, PCP could decrease striatal levels of these two DA metabolites by interfering with DA uptake in vivo. PCP is a potent, competitive inhibitor of DA uptake in vitro (Smith et al., 1975; Garey and Heath, 1976; Smith et al., 1977). Drugs which inhibit the high affinity uptake process may limit deaminated catabolite formation by reducing the amount of substrate available for intraneuronal monoamine oxidase (Azzaro et al., 1973; Rutledge, 1970). The latter possibility is consistent with the finding (Hitzemann et al., 1973) that, in the mouse, PCP increased the formation of 14C-3methoxytyramine (a methoxylated DA metabolite) from 14C-tyrosine, but did not significantly alter the accumulation of 14CDOPAC (a deaminated DA metabolite). Since
145 catechol-O-methyltransferase is believed to be an extraneuronal enzyme (Axelrod, 1966) their results suggest that PCP can increase synaptic DA levels without increasing the formation of its deaminated metabolites. As PCP is a competitive inhibitor of DA uptake, its ability to interfere with this process would be greatly reduced after haloperidol, because of increased concentration of DA in the synapse. Therefore, following haloperidol treatment the striatal levels of DOPAC and HVA would reflect better the ability of PCP to promote DA release. The prototype of the indirect DA agonist, d-amphetamine, appears to facilitate central dopaminergic transmission by promoting release of newly synthesized DA as well as by blocking DA uptake by presynaptic terminals (Glowinski et al., 1966; Carlsson et al., 1966; Besson et al., 1971; Chiueh and Moore, 1975). In spite of the similar behavioral effects in the rat, several lines of evidence indicate that PCP and d-amphetamine interact with central dopaminergic systems through different neurochemical mechanisms. For example, unlike PCP, d-amphetamine stimulates striatal tyrosine hydroxylase in vivo; it also reverses GBLinduced increase in the rate of striatal DOPA formation (Roth et al., 1974; Kehr et al., 1977). Further, d-amphetamine is a potent DA releasing agent in vitro (Heikkila et al., 1975a,b) while our results (table 2) show that PCP is much less effective in this respect. However, it is possible that PCP would have more potent effects on DA release in vitro with other methods of study. Recent behavioral studies performed in our laboratory indicate that the depletion of brain catecholamine stores with reserpine blocks the behavioral stimulation observed in rats after PCP (Fessler, Sturgeon and Meltzer, unpublished results); on the other hand, reserpine pretreatment enhances the behavioral effects of d-amphetamine (Scheel-Kri]ger, 1971). In addition to d-amphetamine, central dopaminergic transmission can also be enhanced by non-amphetamine stimulants, a
146
class of drugs which, among others, includes methylphenidate, amfonelic acid and cocaine. It has been proposed that these compounds facilitate dopaminergic transmission either by blocking the uptake of DA released by neuronal activity (Ross, 1977) or by making more DA available from so~alled storage pool DA for impulse-induced release (Shore, 1976a,b). The effect of non-amphetamine stimulants on striatal levels of DOPAC and HVA varies with each member of the group (Braestrup and Scheel-Krilger, 1976; Shore, 1976a,b), but compounds such as amfonelic acid and methylphenidate, can, like PCP, potentiate the rise in striatal levels of these two DA metabolites caused by neuroleptics (Shore, 1976b; Fuller and Snoddy, 1979), while amphetamine has the opposite effect (German et al., 1979). Again like PCP (Smith et al., 1977), drugs belonging to this class of psychomotor stimulants are potent inhibitors of DA uptake in vitro (Ferris et al., 1972; Hytell, 1978; Ross, 1979) with weak releasing action (Heikkila et al., 1975a; Hunt et al., 1974). Further, the behavioral effects of methylphenidate, as well as those of other nonamphetamine stimulants, are blocked by reserpine pretreatment (Scheel-Krfiger, 1971). We have also found (Simonovic and Meltzer, unpublished results) that methylphenidate and amfonelic acid decrease the rate of striatal DOPA formation. A cocaine-like mechanism of action has already been proposed for the circulatory effects of PCP (O'Donnell and Wanstall, 1968). The parallelism between the interaction of PCP and that of non-amphetamine stimulants with central dopaminergic systems suggests that they act through a related neurochemical mechanism. A fair degree of structural similarity between PCP and methylphenidate also favors such a possibility. Several studies have shown that PCP interacts with central cholinergic systems; PCP can block muscarinic receptors (Maayani et al., 1974) and its possesses a high affinity for the muscarinic binding sites in the brain (Vincent et al., 1978). It is well known that
J.D. DOHERTY ET AL.
cholinergic system has a profound effect on the activity and metabolism of striatal DA neurons. For example, anticholinergic drugs, such as atropine, decrease the release and turnover of DA in the striatum (And~n and B~dard, 1971; Bartholini and Pletscher, 1971). Although anticholinergic agents produce ipsilateral turning in rats with unilateral substantia nigra lesions (Pycock et al., 1978), an effect similar to that of PCP, it is clear that anticholinergic properties of PCP cannot provide a sufficient explanation for the observed effects of PCP on striatal DA metabolism. For example, the increase in central DA turnover caused by neuroleptics can be prevented by anticholinergic agents (O'Keefe et al., 1970; And~n, 1972), and the accumulation of striatal DOPA after decarboxylase inhibition was not modified by atropine (Javoy et al., 1975). PCP has the opposite effect on these two neurochemical parameters. While our results do not definitively identify the mechanism through which PCP interacts with dopaminergic neurons in the brain, the following conclusions can be drawn with some certainty. PCP is unable to activate directly either pre- or pos~synaptic DA receptors. It facilitates central dopaminergic transmission by altering DA release and/or blocking its uptake. Its mechanism of action differs from that of d-amphetamine and appears to be closely related to that of non-amphetamine stimulants. However, the possibility that the interaction of PCP with other neurotransmitter systems contributes to its dopaminergic effects requires further study.
Acknowledgements We wish to express our appreciation to Dr. Sherwin Wilk of the Mt. Sinai School of Medicine for determining the DOPAC and HVA levels and to Dr. William Scott of Hoffman La Roche for a generous supply of Ro4-4602. Supported in part by USPHS DA 02081 and MH 30,938 and by the Department of Mental Health, State of Illinois. HYM is recipient of RCSA HM 47,808. MS is a recipient of PSH MH 14,274.
PHFNCYCLIDINE AND STRIATAL DOPAMINE References And~n, N.-E., 1972, Dopamine turnover in the corpus striatum and the limbic system after treatment with neuroleptic and anti-acetyl-choline drugs, J. Pharm. Pharmacol. 24,905. And~n, N.-E. and P. B~dard, 1971, Influence of cholinergic mechanism on the formation and turnover of brain dopamine, J. Pharm. Pharmacol. 23,460. And~n, N.-E., A. Rubenson, K. F u x e and T. HSkfelt, 1967, Evidence for receptor stimulation by apomorphine, J. Pharm. Pharmacol. 1 4 , 6 2 7 . Axelrod, J., 1966, Methylation reactions in the formation and metabolism of catecholamines and other biogenic amines, Pharmacol. Rev. 18, 95. Azzaro, A.J., J.A. Gutrecht and D.J. Smith, 1973, Effect of diphenylhydantoin on the uptake and catabolism of L-[3H]-norepinephrine in vitro in rat cerebral cortex tissue, Biochem. Pharmacol. 22, 2719. Bartholini, G. and A. Pletscher, 1971, Atropineinduced changes of central dopamine turnover, Experientia 27, 1302. Besson, M.J., A. Ch~ramy, P. Feltz and J. Glowinski, 1971, Dopamine: spontaneous and drug-induced release from the caudate nucleus of the rat, Brain res. 3 2 , 4 0 7 . Braestrup, C. and J. Scheel-Kriiger, 1976, Methylphenidate-like effects of the new antidepressant drug nomifensine (HOE 984), European J. Pharmacol. 3 8 , 3 0 5 . Carlsson, A., J.M. Davis, W. Kehr, M. Lindqvist and C.V. Atack, 1972, Simultaneous measurement of tyrosine and t r y p t o p h a n hydroxylase activities in brain in vivo using an inhibitor of the aromatic amino acid decarboxylase, Naunyn-Schmiedeb. Arch. Pharmacol. 2 7 5 , 1 5 3 . Carlsson, A., K. Fuxe, B. Hamberger and M. Lindqvist, 1966, Biochemical and histochemical studies on the effects of imipramine-like drugs and (+)amphetamine on central and peripheral catecholamine neurons, Acta Physiol. Scand. 6 7 , 4 8 1 . Carlsson, A., W. Kehr and M. Lindqvist, 1977, Agonist-antagonist interaction of dopamine receptors in brain as reflected in the rates of tyrosine and t r y p t o p h a n hydroxylation, J. Neural. Trans. 40, 99. Carlsson, A. and M. Lindqvist, 1977, Effect of reserpine on monoamine synthesis and on apparent dopaminergic receptor sensitivity in rat brain, in: Neuropharmacology and Behavior, eds. B. Haber and M. Aprison (Plenum Press, New York) p. 89. Chen, G., C.R. Ensor, D. Russel and B. Bohner, 1959, The pharmacology o f 1-(1-phenylcyclohexyl) piperidine HC1, J. Pharmacol. Exp. Ther. 127, 241. Chiueh, C.C. and K.E. Moore, 1975, d-Amphetamine-
147 induced release of 'newly synthesized' and 'stored' dopamine from the caudate nucleus in vivo, J. Pharmacol. Exp. Ther. 1 9 2 , 6 4 2 . Ernst, A'M., 1967, Mode of action of apomorphine and dexarnphetamine on gnawing compulsion in the rats, Psychopharmacologia 10,316. Ferris, R.M., F.L.M. Tange and R.A. Maxwell, 1972, A comparison o f the capacities of isomers of amphetamine, deoxypipradrol and methylphenidate to inhibit the uptake of tritiated catecholamines into rat cerebral slices, synaptosomal preparations and rat cerebral cortex, hypothalamus, striatum into adrenergic nerves of rabbit aorta, J. Pharmacol. Exp. Ther. 1 8 1 , 4 0 7 . Fessler, R.G., R.D. Sturgeon and H.Y. Meltzer, 1979, Phencyclidine-induced ipsilateral rotation in rats with unilateral 6-hydroxydopamine-induced l c ~ n s of the substantia negra, Life Sci. 24, 1281. Finnegan, K.T., M.I. Kanner and H.Y. Meltzer, 1976, Phencyclidine-induced rotational behavior in rats with nigrostriatal lesions and its modulation by dopaminergic and cholinergic agents, Pharmaeol. Biochem. Behav. 5 , 6 5 1 . Fuller, R.W. and H.D. Shoddy, 1979, Synergistic elevation of brain 3,4-dihidroxyphenylacetic acid concentration by methylphenidated and spiperone in control but not reserpine-pretreated rats, J. Pharm. Pharmacol. 3 1 , 3 3 9 . Garey, R.E., C.W. Christensen, A.D. Howard and M.A. Spirtes, 1977, In vitro effects of phencyclidine on rat striatal tyrosine hydroxylase and adenylate eyclase, Nuerosci. Abs. 3 , 2 9 1 . Garey, R.E. and R.G. Heath, 1976, The effects of phencyclidine on the uptake o f 3H-catecholamines by rat striatal and hypothalamic synaptosomes, Life Sci. 18, 1105. German, D.C., H. Harden, M.K. Sanghera, D. Mann, R.S. Kiser, H.H. Miller and P.A. Shore, 1979, Dopaminergic neuronal responses to a nonamphetamine CNS stimulant, J. Neural Trans. 44, 39. Glowinski, J., J. Axelrod and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain: IV. Effects of drugs on the disposition and metabolism o f 3H-Dopamine, J. Pharmacol. Exp. Ther. 153, 30. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain: I. The disposition o f 3H-norepinephrine, 3H-dopamine, SH-DOPA in various regions of the rat brain, J. Neuroehem. 1 3 , 6 5 5 . Heikkila, R.E., H. Orlansky and G. Cohen, 1975a, Studies on the distinction between uptake inhibition and release of 3H-dopamine in rat brain tissue slices, Biochem. Pharmacol. 2 4 , 8 4 7 . Heikkila, R.E., H. Orlansky, C. Mytillineou and G. Cohen, 1975b, A m p h e t a m i n e : evaluation of dand 1-isomers as releasing agents and uptake inhibi-
148 tors for 3H-dopamine and 3H-norepinephrine in slices of rat neostriatum and cerebral cortex, J. Pharmacol. Exp. Ther. 194, 47. Hitzemann, R.J., H.H. Loh and E.F. Domino, 1973, Effect of phencyclidine on the accumulation of 14C-catecholamines formed from 14C-tyrosine, Arch. Int. Pharmacodyn. Ther. 202, 252. Hunt, P., M.H. Kannengiesser and J.P. Raynaud, 1974, Nomifensine: a new potent inhibitor of dopamine uptake into the synaptosomes from rat brain corpus striatum, J. Pharm. Pharmacol. 26, 370. Hyttel, J., 1978, Inhibition of 3H-dopamine accumulation in rat striatal synaptosomes by psychotropic drugs, Biochem. Pharmacol. 27, 1063. Javoy, F., Y. Agid and J. Glowinski, 1975, Oxotremorine- and atropine-induced changes in dopamine metabolism in the rat striatum, J. Pharm. Pharmacol. 2 7 , 6 7 7 . Johnson, K.M., 1978, Neurochemical pharmacology of phencyclidine, in: Phencyclidine Abuse: An Appraisal, eds. R.C. Petersen and R.C. Stillman, NIDA Research Monograph 21, p. 44. Kanner, M., H.Y. Meltzer and J.M. Davis, 1975a, Pharmacological aspects of the l o c o m o t o r stimulation produced by phencyclidine in the rat, Neurosci. Abstr. 1 , 2 3 7 . Kanner, M., K. Finnegan and H.Y. Meltzer, 1975b, Dopaminergic effects of phencyclidine in rats with nigro-striatal lesions, Psychopharmacol. Commun. 1,393. Kehr, W., A. Carisson, M. Lindqvist, 1972, A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain, Naunyn-Schmiedeb. Arch. Pharmacol. 2 7 4 , 2 7 3 . Kehr, W., A. Carlsson and M. Lindqvist, 1975, Biochemical aspects of dopamine agonists, Adv. Neurol. 9, 185. Kehr, W., W. Speckenbach and R. Zimmerman, 1977, Interaction of haloperidol and gamma-butyrolactone with (+)-amphetamine-induced changes in monoamine synthesis and metabolism in rat brain, J. Neural Trans. 40, 129. Lahti, R.A., B. McAllister and J. Wozniak, 1972, Apomorphine antagonism o f the elevation of homovanillic acid induced by antipsychotic drugs, Life Sci. 11,605. Luby, E.D., J.S. Gottlieb, G.A. Rosenbaum and E.F. Domino, 1962, Model psychoses and schizophrenia, Am. J. Pscyhiat. 119, 61. Maayani, S., H. Weinstein, N. Ben-Zvi, S. Cohen and M. Sokolovsky, 1974, Psychotomimetics as anticholinergic agents - - 1-cyclohexylpiperidine derivatives: Anticholinesterase activity and antagonistic activity to acetylcholine, Biochem. Pharmacol. 23, 1263. Meltzer, H.Y., P.S. Holzman, S.Z. Hassan and A. Guschwan, 1972, Effects o f phencyclidine and stress
J.D. DOHERTY ET AL. on plasma creatine phosphokinase (CPK) and aldolase activities in man, Psychopharmacologia 26, 44. Meltzer, H.Y. and S.M. Stahl, 1976, The dopamine hypothesis of schizophrenia: a review, Schiz. Bull. 2, 19. O'Donnell, S.R. and J.C. Wanstall, 1968, Actions of phencyclidine on the perfused rabbit ear, J. Pharm. Pharmacol. 20, 125. O'Keefe, R., D.F. Sharman and M. Vogt, 1970, Effect of drugs used in psychoses on cerebral dopamine metabolism, Br. J. Pharmacol. 38,287. Pycock, C., J. Milson, D. Tarsy and C.D. Marsden, 1978, The effect of manipulation of cholinergic mechanisms on turning behavior in mice with unilateral destruction of the nigro-neostriatal dopaminergic system, Neuropharmacology 17, 175. Ross, S.B., 1977, On the mode of action of central stimulatory agents, Acta Pharmacol. Toxicol. 41, 392. Ross, S.B., 1979, The central stimulatory action of inhibitors of. dopamine uptake, Life Sci. 24, 159. Roth, R.H., J.R. Walters and V.H. Morgenroth, III, 1974, Effects of alterations in impulse flow on transmitter metabolism in central dopaminergic neurons, in: Neuropsychopharmacology of Monoamines and their Regulatory Enzymes, ed. E. Usdin (Raven Press, New York) p. 369. Rutledge, C.O., 1970, The mechanism by which amphetamine inhibits oxidative deamination of norepinephrine in brain, J. Pharmacol. Exp. Ther. 171,188. Saner, A. and A. Pletscher, 1977, Increase of striatal dopamine turnover by drugs: Interference with granular storage or receptor blockade?, European J. Pharmacol. 4 2 , 1 5 5 . Scheel-Kriiger, J., 1971, Comparative studies of various amphetamine analogues demonstrating different interactions with the metabolism of the catecholamines in the brain, European J. Pharmacol. 14, 47. Shore, P.A., 1962, Release of serotonin and catecholamines by drugs, Pharmacol. Rev. 14,531. Shore, P.A., 1976a, On the role of storage granules in the functional utilization o f newly synthesized dopamine, J. Neural Trans. 39, 131. Shore, P.A., 1976b, Actions o f amfonelic acid and other non-amphetamine stimulants on the dopamine neuron, J. Pharm. Pharmacol. 28, 855. Simonovic, M., J. Doherty, R. So and H.Y. Meltzer, 1979, Phencyclidine inhibits the activity of striatal tyrosine hydroxylase in vivo, in: Catecholamines: Basic and Clinical Frontiers, eds. E. Usdin, I.J. Kopin and J. Barchas (Pergamon Press, New York) Vol. I, p. 710. Smith, R.C., H.Y. Meltzer, R.C. Arora and J.M. Davis, 1977, Effects of phencyclidine on 3H-serotonin
PHENCYCLIDINE AND STRIATAL DOPAMINE uptake in synaptosomal preparations from rat brain, Biochem. Pharmacol. 26, 1435. Smith, R.C., H.Y. Meltzer, H. Dekirmenjian and H. Davis, 1975, Effects of phencyclidine on biogenic amines in rat brain, Neurosci. Abstr. 1,468. Snyder, S.H., S.P. Banerjee, S.P. Yamamura and D. Greenberg, 1974, Drugs, neurotransmitters and schizophrenia, Science 184, 1243. Sturgeon, R.D., R.G. Fessler and H.Y. Meltzer, 1979, Behavioral rating scales for assessing phencyclidineinduced locomotor activity, stereotyped behavior and ataxia in rats, European J. Pharmacol. 59, 169. Tonge, S.R. and B.E. Leonard, 1969, The effects of some hallucinogenic drugs upon the metabolism of 5-hydroxytryptamine in the brain, Life Sci. 8, 805. Tonge, S.R. and B.E. Leonard, 1970, The effect of some hallucinogenic drugs on the amino acid precursors of brain monoamines, Life Sci. 9, 1327. Vickroy, T.W. and K.M. Johnson, 1979, Stimulation of synaptosomal tyrosine hydroxylase activity by phencyclidine, Neurosci. Abstr. 5,665. Vincent, J.P., D. Cavey, J.M. Kamenka, P. Geneste and M. Lazdunski, 1978, Interaction of phencyclidines with muscarinic and opiate receptors in the central nervous system, Brain Res. 152,176. Von Voigtlander, P.F. and K.E. Moore, 1973, Turning behavior in mice with unilateral 6-hydroxydopamine lesions in the striatum: Effects of apomorphine, 1-DOPA, amantadine, amphetamine and
149 other psychomotor stimulants, Neuropharmacology 12,451. Waiters, J.R. and R.H. Roth, 1974, Dopaminergic neurons: Drug-induced antagonism of the increase in tyrosine hydroxylase activity produced by cessation of impulse flow, J. Pharmacol. Exp. Ther. 191, 82. Walters, J.R. and R.H. Roth, 1976, Dopaminergic neurons: An in vivo system for measuring drug interaction with presynaptic receptors, NaunynSchmiedeb. Arch. Pharmacol. 296, 5. Waiters, J.R., R.H. Roth, G.K. Aghajanian, 1973, Dopaminergic neurons: Similar biochemical and histochemical effects of gamma-hydroxy-butyrate and acute lesion of the nigro-striatal pathway, J. Pharmacol. Exp. Ther. 186, 630. Wenger, G.R. and P.B. Dews, 1976, The effects of phencyclidine and ketamine, d-amphetamine and pentobarbital on schedule controlled behavior in the mouse, J. Pharmacol. Exp. Ther. 196, 616. Westerink, B.H.C. and J. Korf, 1976, Regional rat brain levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid: Concurrent fluorometric measurement and influence of drugs, Eur. J. Pharmacol. 38, 281. Wilk, S., E. Watson and M.D. Stanley, 1975, Differential sensitivity of two dopaminergic structures in rat brain to haloperidol and to clozaprine, J. Pharmacol. Exp. Ther. 195, 265.
139
THE EFFECT OF PHENCYCLIDINE ON DOPAMINE SYNTHESIS AND METABOLISM IN RAT STRIATUM JOHN D. DOHERTY, MILJANA SIMONOVIC, REBECCA SO and HERBERT Y. MELTZER
Department of Psychiatry and Physiological and Pharmacological Sciences, University of Chicago Pritzker School of Medicine, Chicago, Illinois 60637 and Illinois State Psychiatric Institute, 1601 W. Taylor Street, Chicago, Illinois 60612, U.S.A. Received 24 September 1979, revised MS received 11 January 1980, accepted 8 April 1980
J.D. DOHERTY, M. SIMONOVIC, R. SO and H.Y. MELTZER, The effect of phencyclidine on dopamine synthesis and metaolism in rat striatum, European J. Pharmacol. 65 {1980} 139--149. Previous behavioral and neurochemical studies indicate that phencyclidine (PCP), a potent psychotomimetic agent, interacts with central dopaminergic systems. We have examined the effects of PCP on the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) after the inhibition of L-aromatic amino acid decarboxylase and on the levels of dopamine (DA) metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum. PCP, in doses from 2.5 to 50 mg/kg, decreased the rate of striatal DOPA accumulation. PCP did not antagonize the increase in the rate of striatal DOPA formation caused by haloperidol, reserpine or ),-butyrolactone (GBL). When given alone, PCP decreased striatal levels of DOPAC and HVA, while it greatly potentiated the haloperidol-induced rise in striatal levels of these two metabolites. PCP is considerably less effective than d-amphetamine in promoting the release of 3H-DA from preloaded striatal slices in vitro. Our results are consistent with the interpretation that PCP potentiates the synaptic effects of endogenous DA. Its mechanism of action appears to be closely related to that of a category of drugs known as non-amphetamine stimulants, which, among others, includes methylphenidate, amfonelic acid and cocaine. Phencyclidine
Dopamine agonist
Reuptake blocker
1. Introduction
Phencyclidine (PCP) is a phenylcyclohexyl derivative with an unusual spectrum of pharmacologic actions. Its behavioral effects vary markedly, depending on the dose and species used, and range from stimulation in rodents to sedation in higher species (Chen et al., 1959). Subanesthetic doses of PCP may have profound psychotomimetic effects in humans. It sometimes induces behavioral changes which mimic the primary symptoms of schizoprenia in drug abusers or exacerbates symptoms in chronic schizophrenics (Luby et al., 1962; Meltzer et al., 1972). In rodents, low doses of PCP produce central stimulation characterized by increased locomotor activity and stereotyped behavior,
not unlike that caused by psychomotor stimulants such as amphetamine (Chen et al., 1959; Kanner et al., 1975a; Sturgeon et al., 1979). The effect of PCP on food-reinforced operant behavior is qualitatively similar to d-amphetamine (Wenger and Dews, 1976). PCP induces ipsilateral turning in rats with unilateral electrolytic or 6-hydroxydopamineinduced lesions in the nigro-striatal pathway, a finding consistent with an indirect rather than a direct DA agonist action (Fessler et al., 1979). The fact that inhibition of DA synthesis with ~-methylparatyrosine blocks this effect also suggests that PCP acts as an indirect DA agonist (Kanner et al., 1975b; Fessler et al., 1979). Numerous reports indicate that PCP has important effects on central dopaminergic,
140
noradrenergic, cholinergic, serotonergic, GABA-ergic and endogenous opiates transmitter systems (Vincent et al., 1978; for review see Johnson, 1978). The mechanism underlying the characteristic psychotropic effects of PCP is not known. However, the possibility that behavioral stimulation seen in rats after PCP administration is related to its ability to enhance central dopaminergic transmission is of particular interest because of the possible role of DA in behavioral manifestations of psychomotor stimulants and schizophrenia (Von Voigtlander and Moore, 1973; Snyder et al., 1974; Meltzer and Stahl, 1976). In order to further elucidate the interaction of PCP with central dopaminergic systems, we have studied its effects on striatal DA synthesis and metabolism. A preliminary report of these findings has been presented at the 4th International Catecholamine Symposium (Simonovic et al., 1979).
2. Materi~l~ and methods
2.1. Animals Male, Sprague-Dawley rats, purchased from Sprague-Dawley, Inc., Madison, Wisconsin, weighing from 175-225 g were used throughout these studies. They were housed 6 per cage in a temperature (26°C) and humidity (60%) regulated room with a 12-h light/dark cycle. The animals had free access to Purina rat chow and water at all times.
2.2. Drugs d-Amphetamine sulphate was a gift from Smith, Kline and French, Philadelphia, Pa.; Ro4-4602 (seryl-trihydroxybenzylhydrazine) was kindly supplied by Dr. William Scott of Hoffmann-LaRoche Co., Nutley, N.J. and cocaine was obtained from the National Institute of Drug Abuse. The other drugs were
J.D. D O H E R T Y E T AL.
purchased: phencyclidine (Sernylan ®, BioCeutics Laboratories Inc., St. Joseph, Mo.); apomorphine HC1 (Mallinckrodt, St. Louis, Mo.); 7-butyrolactone (GBL) (Eastman Organic Chemicals Co., Rochester, N.Y.), haloperidol (Haldol ®, McNeil Laboratories, Fort Washington, Pa.); reserpine (Serpasil ®, CIBA Pharmaceutical Co., Summit, N.J.); pargyline HC1 (Abbott Laboratories, North Chicago, Ill.); L-3,4-dihydroxyphenylalanine (Sigma Chemical Co., St. Louis, Mo.). All injections were made intraperitoneally (i.p.) at time and dose schedules given in the Results section. Doses of basic compounds refer to salts. Control rats always received the same number of saline injections at corresponding time intervals.
2.3. Dopamine synthesis in vivo The rate of accumulation of striatal 3,4dihydroxyphenylalanine (DOPA) after decarboxylase inhibition has been shown to measure the rate of dopamine synthesis in this brain region (Carlsson et al., 1972). A rapidly acting inhibitor of aromatic amino acid decarboxylase, Ro4-4602 (800 mg/kg) was administered to rats 40 min before decapitation. It has been shown that following this dose of Ro44602, the accumulation of DOPA in the striatum is linear for at least 60 min (Walters and Roth, 1974). Immediately after decapitation, the brains were rapidly removed and corpora striata dissected on an ice-cold glass plate as described by Glowinski and Iversen (1966). The brain parts were frozen on dry ice and stored at --70°C until assayed. DOPA was isolated and measured fluorometrically by the technique of Kehr et al. (1972) with the modifications described by Walters and Roth (1974). The mean recovery of 300 ng of L-DOPA taken through the entire procedure was 61.2 + S.E.M. 2.2% (n = 50). Forty min after the administration of Ro4-4602, the mean striatal DOPAcontent was 2.15 + S.E.M. 0.11 t~g/g wet tissue (n = 40); all values were corrected for recovery.
PHENCYCLIDINE
2.4. Release o f tissue slices
AND
STRIATAL
DOPAMINE
141
3. Results
3H-dopamine from striatal
3.1. The effect of PCP on the rate o f striatal DOPA formation after decarboxylase inhibition
The release of 3H
In agreement with previous studies (Roth et al., 1974; Kehr et al., 1975; Carlsson et al., 1977; Kehr et al., 1977), we have found that apomorphine inhibits, while d-amphetamine stimulates, the in vivo activity of striatal tyrosine hydroxylase as measured by the rate of DOPA accumulation after decarboxylase inhibition (fig. la). The dose-response curve for the effect of PCP on striatal tyrosine hydroxylase is presented in fig. l b . All doses of PCP decreased striatal DOPA accumulation, but the effect of 2.5 mg/kg did not reach statistical significance. In the dose range
2.5. Determination of DOPAC and HVA
Following drug treatment according to the schedules given in the Results section, rats were killed, their striata removed, homogenized in 1 N HC1 and frozen. The homogenate was shipped in dry ice for analysis by the electron capture gas chromatographic m e t h o d used in the laboratory of Dr. S. Wilk, Dept. of Pharmacology, Mt. Sinai School of Medicine, City University of New York (Wilk et al., 1975).
b
a
200
[] Saline • PCP [] Apomorphine [] d-Amphetamine
o Saline
• PCP I00
150
t
~Q
o c o
8
0
"6 < 50
~0c
0
0 r-.
50
~-~ "'"'""
5
5
I
5
J
[
2.5
1
I
I
5
I0
50
Fig. 1. The effect o f various drugs (a) and various doses of PCP (b) on the rate o f striatal DOPA accumulation following decarboxylase inhibition. Drugs were injected 45 min and Ro4-4602 (800 mg/kg) 40 rain before decapitation. The means for groups of four rats are given; vertical bars represent S.E.M. Significantly different from control (Tukey's HSD test): * P < 0.01, ** P < 0.05. Abscissa: dose (mg/kg).
142
tested, the maximal decrease in the rate of striatal DOPA formation was attained with 10 mg/kg of PCP. Subsequent experiments were performed in an effort to define further the mechanism underlying the inhibitory effect of PCP on striatal tyrosine hydroxylase in vivo. Unlike apomorphine, a direct acting DA agonist (Ernst, 1967; And~n et al., 1967), which can reverse the increase in striatal DOPA accumulation due to haloperidol (Carlsson et al., 1977), PCP had no effect on haloperidol-induced activation of striatal tyrosine hydroxylase (fig. 2). These results suggest that the decrease in the activity of striatal tyrosine hydroxylase observed after the administration of PCP alone is probably not due to its ability to directly activate DA receptors. However, the high dose of haloperidol used may have masked the possible DA receptor agonist properties of PCP. We have therefore evaluated its effects on preand post-synaptic DA receptors in the absence of a DA receptor blocker. Reserpine, a drug which depletes granular DA stores (Shore, 1962), causes a profound increase in the activity of striatal tyrosine hydroxylase (Carlsson and Lindqvist, 1977), an effect which is antagonized by apomorphine in a dose
J.D. DOHERTY ET AL.
[]
SALINE
[]
~ERP~E
600
5OO
400
300
200
I00
5
5
Fig. 2. The effect of PCP on the rate of striatal DOPA formation in rats pretreated with haloperidol (1 mg/kg) or reserpine (5 mg/kg). Reserpine was administered 365 rain, haloperidol 50 rain, PCP 45 min and Ro4-4602 (800 mg/kg) 40 min before decapitation. The means for groups of four rats are given; vertical bars represent S.E.M. Significantly different from saline treated controls (Student's t-test); * P < 0.01. Ordinate: DOPA (% of control). Abscissa: drug, dose (mg/kg).
lethal. PCP can also potentiate the lethal effects of another anesthetic, pentobarbital (Chen et al., 1959). 3.2. The effect o f PCP on striatal levels o f D O P A C and H V A
PCP (10mg/kg) significantly decreased striatal levels of DOPAC (--28%) and HVA (--32%) (fig. 3). Haloperidol, as expected, increased striatal concentration of these two DA metabolites. When given in combination with haloperidol, PCP greatly enhanced the neuroleptic-induced rise in striatal content of DOPAC and HVA (fig. 3). 3.3. The effect o f PCP on 3H-DA release from striatal slices in vitro
In order to assess the DA releasing properties of PCP, we studied its effect on the release
PHENCYCLIDINE AND STRIATAL DOPAMINE
143
TABLE 1 The e f f e c t o f PCP on the rate o f striatal D O P A f o r m a t i o n in rats pretreated w i t h GBL. G B L w a s administered 5 0 rain, PCP 4 5 m i n and R o 4 - 4 6 0 2 ( 8 0 0 m g / k g ) 4 0 rain b e f o r e d e c a p i t a t i o n . Group
Drug 1
(mg/kg)
Drug 2
(mg/kg)
Striatal D O P A (% of c o n t r o l ) 1
Cornparison
P
(1) (2) (3) (4)
Saline Saline GBL GBL
--500 500
Saline PCP Saline PCP
-5 -5
100 50 231 251
1-2 1-3 3-4
<0.01
_+ 10 -+ 4 _+ 15 -+ 33
<0.01 n.s.
1 Mean for a group o f four rats _+ S.E.M. Significance o f difference assessed using S t u d e n t ' s t-test.
4
4 o0
-i
"I
L}
~3 o
"1-
...l..: :.:.:.:.:
-3
er
..J
2
09
rr t-
I ....o....
......... ,,.,.,... .,.,.....
Fig. 3. T h e e f f e c t o f PCP ( 1 0 m g / k g ) alone or in c o m b i n a t i o n w i t h h a l o p e r i d o l (1 m g / k g ) o n striatal levels o f D O P A C and H V A as assayed b y the e l e c t r o n capture c h r o m a t o g r a p h y . Drugs were administered 4 5 m i n before d e c a p i t a t i o n . T h e means f o r groups o f four rats are given; vertical bars represent S.E.M. Statistical significance w a s assessed using S t u d e n t ' s t-test. [] S a l o n e ; [] PCP; ~ h a l o p e r i d o l ; m h a l o p e r i d o l + PCP. * P < 0 . 0 5 vs. saline. • * P < 0 . 0 0 1 vs. haloperidol.
TABLE 2 T h e e f f e c t o f PCP and o t h e r drugs on the release o f 3 H - d o p a m i n e f r o m striatal tissue slices. Drug
Concentration
% o f 3 H - d o p a m i n e remaining in slices
Control ( n o drug) PCP PCP PCP d-Amphetamine Cocaine
-5 2 1 1 1
100 58.0 71.0 72.2 33.4 85.0
× × × × ×
1 0 -4 1 0 -4 10 -~ 10-4 10- 4
_+ 0.2 (3) -+ 0.3 (3) + 6.3 (18) + 2.8 (9) + 0.004 (3)
144
of 3H-DA from preloaded striatal slices, using the method of Heikkila et al. (1975a), which can differentiate release from the inhibition of uptake. PCP was found to be considerably less potent than d-amphetamine in promoting 3H-DA release, but slightly more potent than cocaine (table 2), indicating that in vitro PCP had a weak DA releasing action, at least in this model.
4. Discussion Considerable evidence supports the view that the activity of striatal tyrosine hydroxylase, and consequently the rate of DA synthesis, is under complex, receptor-mediated feedback control. Thus, a well-known DA receptor agonist, apomorphine, inhibits, while haloperidol, a potent DA receptor blocker, stimulates striatal DA synthesis (Kehr et al., 1975; Carlsson et al., 1977). We have tested the effect of PCP on striatal DA synthesis by measuring the rate of striatal DOPA accumulation after inhibition of L-aromatic amino acid decarboxylase, an index of the in vivo activity of striatal tyrosine hydroxylase (Carlsson et al., 1972). PCP produced a significant and apparently dose-related decrease in the rate of striatal DOPA formation. It is interesting to note that the maximal decrease in striatal DOPA levels was attained with 10 mg/kg of PCP, the dose which also produced maximal effect on turning behavior in rats with unilateral substantia nigra lesions (Finnegan et al., 1976). Our results are in good agreement with an earlier tracer study conducted in mice, which reported that PCP, at doses similar to those used here, decreased the accumulation of 14CDA when 14C-tyrosine, but not when 14CDOPA, was used as the precursor (Hitzemann et al., 1973). The possibility that PCP inhibits striatal DOPA formation by direct inhibition of tyrosine hydroxylase is unlikely because PCP actually stimulates tyrosine hydroxylase in vitro (Garey et al., 1977; Vickroy and
J.D. DOHERTY ET AL.
Johnson, 1979). This in vitro effect of PCP may be due to disinhibition of tyrosine hydroxylase following enhanced DA release from brain slices (Garey et al., 1977) or synaptosomes (Vickroy and Johnson, 1979). It is also unlikely that the decrease in striatal DOPA formation after PCP is due to reduced supply of tyrosine to the brain because PCP elevates brain and plasma levels of tyrosine (Tonge and Leonard, 1970). Further, PCP has no effect on the activity of brain monoamine oxidase (Tonge and Leonard, 1969). In addition to decreasing the rate of striatal DA synthesis, PCP also reduced striatal levels of DOPAC and HVA, the two principal DA metabolites. These effects of PCP are indistinguishable from those of apomorphine (Westerink and Korf, 1976; Carlsson et al., 1977). In the absence of any further information, this similarity with apomorphine would suggest that PCP acts as a DA receptor agonist. However, the inability of PCP to antagonize the increase in the rate of striatal DOPA formation caused by haloperidol, reserpine or GBL clearly demonstrates that the effect of PCP on striatal DA synthesis and metabolism could not be attributed to its ability to activate directly either pre- or post-synaptic DA receptors. This is further supported by the fact that apomorphine can antagonize (Lahti et al., 1972) while PCP potentiates (fig. 3) the rise in striatal DA metabolites caused by haloperidol. A direct DA agonist action of PCP is also inconsistent with its effect on turning behavior in rats with unilateral substantia nigra lesions (Fessler et al., 1979). PCP had no effect on in vitro binding of 3H-DA to synaptosomal membranes from rat striatum (Vincent et al., 1978). We have also found that PCP has a very limited ability to displace 3H-spiroperidol from rat striatal membranes (So and Meltzer, unpublished data). These findings further support the conclusion that PCP is devoid of DA receptor agonist properties. Our observation that reserpine, which depletes granular DA stores (Shore, 1962), or GBL, which blocks impulse flow in nigro-
PHENCYCLIDINE AND STRIATAL DOPAMINE striatal DA neurons (Walters et al., 1973) can both completely abolish the inhibitory effect of PCP on striatal tyrosine hydroxylase indicates that the reserpine-sensitive DA pool and uninterrupted impulse flow are necessary for t h e expression of this effect. PCP may inhibit tyrosine hydroxylase by enhancing the effect of synaptic DA on presynaptic DA receptors, which regulate DA synthesis, or on postsynaptic DA receptors, which regulate the activity of nigro-striatal DA neurons via the striato-nigral neuronal feedback loop (Carlsson et al., 1977). It is also possible that PCP releases DA from its storage sites within the granule during impulse flow and that increased intraneuronal levels of free DA inhibits tyrosine hydroxylase. Haloperidol, which is known to increase impulse flow in DA containing neurons, elevates striatal levels of deaminated DA metabolites, DOPAC and HVA. The ability of PCP to potentiate this effect of haloperidol (fig. 3) indicates that, at least during the conditions of increased impulse flow, PCP can enhance DA release. In contrast to its effect in haloperidol-treated rats. PCP decreased striatal levels of DOPAC and HVA when given alone (fig. 3). One possible explanation for this finding is that the decrease in striatal DOPAC and HVA is secondary to the inhibition of tyrosine hydroxylase by PCP. Alternatively, PCP could decrease striatal levels of these two DA metabolites by interfering with DA uptake in vivo. PCP is a potent, competitive inhibitor of DA uptake in vitro (Smith et al., 1975; Garey and Heath, 1976; Smith et al., 1977). Drugs which inhibit the high affinity uptake process may limit deaminated catabolite formation by reducing the amount of substrate available for intraneuronal monoamine oxidase (Azzaro et al., 1973; Rutledge, 1970). The latter possibility is consistent with the finding (Hitzemann et al., 1973) that, in the mouse, PCP increased the formation of 14C-3methoxytyramine (a methoxylated DA metabolite) from 14C-tyrosine, but did not significantly alter the accumulation of 14CDOPAC (a deaminated DA metabolite). Since
145 catechol-O-methyltransferase is believed to be an extraneuronal enzyme (Axelrod, 1966) their results suggest that PCP can increase synaptic DA levels without increasing the formation of its deaminated metabolites. As PCP is a competitive inhibitor of DA uptake, its ability to interfere with this process would be greatly reduced after haloperidol, because of increased concentration of DA in the synapse. Therefore, following haloperidol treatment the striatal levels of DOPAC and HVA would reflect better the ability of PCP to promote DA release. The prototype of the indirect DA agonist, d-amphetamine, appears to facilitate central dopaminergic transmission by promoting release of newly synthesized DA as well as by blocking DA uptake by presynaptic terminals (Glowinski et al., 1966; Carlsson et al., 1966; Besson et al., 1971; Chiueh and Moore, 1975). In spite of the similar behavioral effects in the rat, several lines of evidence indicate that PCP and d-amphetamine interact with central dopaminergic systems through different neurochemical mechanisms. For example, unlike PCP, d-amphetamine stimulates striatal tyrosine hydroxylase in vivo; it also reverses GBLinduced increase in the rate of striatal DOPA formation (Roth et al., 1974; Kehr et al., 1977). Further, d-amphetamine is a potent DA releasing agent in vitro (Heikkila et al., 1975a,b) while our results (table 2) show that PCP is much less effective in this respect. However, it is possible that PCP would have more potent effects on DA release in vitro with other methods of study. Recent behavioral studies performed in our laboratory indicate that the depletion of brain catecholamine stores with reserpine blocks the behavioral stimulation observed in rats after PCP (Fessler, Sturgeon and Meltzer, unpublished results); on the other hand, reserpine pretreatment enhances the behavioral effects of d-amphetamine (Scheel-Kri]ger, 1971). In addition to d-amphetamine, central dopaminergic transmission can also be enhanced by non-amphetamine stimulants, a
146
class of drugs which, among others, includes methylphenidate, amfonelic acid and cocaine. It has been proposed that these compounds facilitate dopaminergic transmission either by blocking the uptake of DA released by neuronal activity (Ross, 1977) or by making more DA available from so~alled storage pool DA for impulse-induced release (Shore, 1976a,b). The effect of non-amphetamine stimulants on striatal levels of DOPAC and HVA varies with each member of the group (Braestrup and Scheel-Krilger, 1976; Shore, 1976a,b), but compounds such as amfonelic acid and methylphenidate, can, like PCP, potentiate the rise in striatal levels of these two DA metabolites caused by neuroleptics (Shore, 1976b; Fuller and Snoddy, 1979), while amphetamine has the opposite effect (German et al., 1979). Again like PCP (Smith et al., 1977), drugs belonging to this class of psychomotor stimulants are potent inhibitors of DA uptake in vitro (Ferris et al., 1972; Hytell, 1978; Ross, 1979) with weak releasing action (Heikkila et al., 1975a; Hunt et al., 1974). Further, the behavioral effects of methylphenidate, as well as those of other nonamphetamine stimulants, are blocked by reserpine pretreatment (Scheel-Krfiger, 1971). We have also found (Simonovic and Meltzer, unpublished results) that methylphenidate and amfonelic acid decrease the rate of striatal DOPA formation. A cocaine-like mechanism of action has already been proposed for the circulatory effects of PCP (O'Donnell and Wanstall, 1968). The parallelism between the interaction of PCP and that of non-amphetamine stimulants with central dopaminergic systems suggests that they act through a related neurochemical mechanism. A fair degree of structural similarity between PCP and methylphenidate also favors such a possibility. Several studies have shown that PCP interacts with central cholinergic systems; PCP can block muscarinic receptors (Maayani et al., 1974) and its possesses a high affinity for the muscarinic binding sites in the brain (Vincent et al., 1978). It is well known that
J.D. DOHERTY ET AL.
cholinergic system has a profound effect on the activity and metabolism of striatal DA neurons. For example, anticholinergic drugs, such as atropine, decrease the release and turnover of DA in the striatum (And~n and B~dard, 1971; Bartholini and Pletscher, 1971). Although anticholinergic agents produce ipsilateral turning in rats with unilateral substantia nigra lesions (Pycock et al., 1978), an effect similar to that of PCP, it is clear that anticholinergic properties of PCP cannot provide a sufficient explanation for the observed effects of PCP on striatal DA metabolism. For example, the increase in central DA turnover caused by neuroleptics can be prevented by anticholinergic agents (O'Keefe et al., 1970; And~n, 1972), and the accumulation of striatal DOPA after decarboxylase inhibition was not modified by atropine (Javoy et al., 1975). PCP has the opposite effect on these two neurochemical parameters. While our results do not definitively identify the mechanism through which PCP interacts with dopaminergic neurons in the brain, the following conclusions can be drawn with some certainty. PCP is unable to activate directly either pre- or pos~synaptic DA receptors. It facilitates central dopaminergic transmission by altering DA release and/or blocking its uptake. Its mechanism of action differs from that of d-amphetamine and appears to be closely related to that of non-amphetamine stimulants. However, the possibility that the interaction of PCP with other neurotransmitter systems contributes to its dopaminergic effects requires further study.
Acknowledgements We wish to express our appreciation to Dr. Sherwin Wilk of the Mt. Sinai School of Medicine for determining the DOPAC and HVA levels and to Dr. William Scott of Hoffman La Roche for a generous supply of Ro4-4602. Supported in part by USPHS DA 02081 and MH 30,938 and by the Department of Mental Health, State of Illinois. HYM is recipient of RCSA HM 47,808. MS is a recipient of PSH MH 14,274.
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