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In vitro effect of phencyclidine and other psychomotor stimulants in serotonin uptake in human platelets
Life Sciences, Vol. 27, pp. 1607-1613 Printed in the U.S.A.
Pergamon Press
IN VITRO EFFECTOF PHENCYCLIDINEAND OTHERPSYCHOMOTOR --S-TIMO-LANTS ON SEROTONINUPTAKE IN HUMANPLATELETS Ramesh C. Arora and Herbert Y. Meltzer I l l i n o i s State Psychiatric Institute, 1601W. Taylor St., Chicago, I l l i n o i s 60612 and the University of Chicago Pritzker School of Medicine Department of Psychiatry, Chicago, IL 60637 (Received in final form August 21, 1980)
Summary Phencyclidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and l-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (ICsQ, 2.5 ~M) was one-hundredth as potent as fluoxetine (ICs0, 22 nM) but ten times more potent than ketamine (ICs0, 25 pM) and d-amphetamine (ICso, 24~M) and three times more potent than l-amphetamine (ICs0, 80 pM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed. Phencyclidine [l-(Phencyclohexyl) piperidine, PCP] is one of the most widely abused drugs, despite i t s potent psychotomimetic effects (1). In doses from 2.5-15 mg/kg, i t produces locomotor activity, stereotypy and ataxia in rats that appear to be dopamine-dependent (2-4). PCP, in doses of 50 mg/kg, has been reported to induce behaviors in the rats thought to be consistent with the so-called "serotonin (5-HT) syndrome": e.g. forepaw treading, head weaving, r i g i d i t y , tremor, straub t a i l (5). These behaviors are antagonized by the putative 5-HT agonists, cinanserin and cyproheptadine, but not by the dopaminergic antagonists, haloperidol (5). Tonge and Leonard 6) reported that PCP increases 5-HT levels and decreases norepinephrine NE) levels in rat brain (7). These effects were found to be strain dependent (8). No significant effect of PCP on either brain monoamineoxidase (MAO) or aromatic amino acid decarboxylase has been reported (7). Tonge and Leonard also showed that PCP reduced the depletion of 5-HT in reserpineand p-chlorophenylalanine-treated rats and concluded that PCP had no effect on the binding of 3[ H ~5-HT to 5-HT receptors in rat brain homogenates (g). Previously, we (lO) and Garey and Heath (1"1) reported that PCP inhibits uptake of 5-HT and dopamine in crude rat brain sjn~aptosomal preparations. The dose needed for 50% inhibition of 5-HT uptake by PCP is 0.8 M. Concentrations of more than an order of magnitude higher are achieved in the rat brain after administration of high dose of PCP (12). I t could be argued, therefore, that 5-HT uptake inhibition could contribute to the functional effects of PCP, at least in higher doses. A number of similarities have also been shown between 5-HT uptake inhibition in brain synaptosomes and blood platelets (13). In the present study, we have studied the effect of PCP and ketamine, an analog of PCP, on 5-HT uptake In human blood platelets and compared its properties with three other psychomotor stimu-
0024-3205/80/431607-07502.00/0 Copyright (c) 1980 Pergamon Press Ltd
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PCP as Serotonin Uptake Inhibitor
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lants, d-amphetamine (d-Amp), l-amphetamlne (l-Amp) and methylphenidate (MP). Fluoxetine, a selective 5-HT uptake inhibitor in rat brain (147, was also studied for comparison purposes. Materials and Methods
Reagents: PCP was obtained from the National Institute of Drug Abuse. d- and l-Amp were g i f t s from Smith, Kline and French, Inc., Philadelphia, Pa. MP was purchased from Ciba Pharmaceutlcals Co., Summit, N.J. The radioactive (2-IWC) 5-HT binoxalate (sp. act. 47.2 mCi/mmole) was purchased from Amersham Searle Corp. A11 the other chemicals used were of analytical grade quality. Method: Blood (lO ml) was drawn in plastic syringes from normal volunteers and transferred into plastic tubes containing citrate-phosphate-dextrose (CPD) as anticoagulant (I.5 ml/lO ml of blood). Platele~-rich-plasma (PRP) was obtained by centrifugation at 600 x g for 2~ min. at 4". Platelet counts were made by means of a Coulter Thrombocounter after diluting lO ul PRP in 20 ml of counting solution (Coulter Electronics, Elk Grove Village, I l l i n o i s ) . Each count was done in t r i p l i c a t e and the average was taken. Uptake studies were done as follows. PRP (0.3 ml) was preincubated wlth 0.6 ml of KrebC) phosphate bufZer minus calcium chloride for lO min. After that, O.l ml of "~C-5-HT (4 x lO~M) was added and the mixture was incubated further for 4 min. The reaction was stopped by rapid cooling in ice. The platelets were isolated by centrifugation (10000 x g ) in a refrigerated centrifuge for 15 mlns. The pellet~as dissolved in Soluene-350 (0.5 ml) by incubating at 55°for 1 hour or at 37- overnight. The solubilized pellet was transferred into s c i n t i l l a t i o n vials with 5 ml of s c i n t i l l a t i o n solvent toluene,[l~, containing 500 ml absolute ethanol, 4 gm of 2.5 diphenyloxazole (PPO) and 50 mg of 1,4-bis-2(4-methyl-5-phenyloxazoly)-benzene .] Radioactivity was counted in a Nuclear Chicago S c i n t i l l a t i o n counter. A separate set of tubes were immediately cooled after addition of~C-5-HT to obtain a measure of passive diffusion and processed in identlcal fashion to the set of tubes previously described. Active uptake was determined by subtracting the dpm of the tubes immediately placed in ice baths from the dpm after incubation at 370. Inhibition Studies: Inhibition of 5-HT uptake into platelets was studied by preincubatlng PRP with various concentrations of the drugs in buffer for I0 min prior to the addition of I~C-5HT (4 x IO-?M). After the addition of 5-HT, the mixture was incubated further for 4 min and processed as described above. The degree of inhibition was calculated from the difference in dislntegrations per minute (dpm) from the samples with and without each drug. All determinations were done in duplicate and the average was taken. Nature of Inhibition: In order to determine the nature of the inhibition of platelet 5-HT uptake by PCP and other test drugs, PCP was incubated with concentrations of I~C-5-HT (2 x IO'?M to 1 x I0"6M). 5-HT uptake in the presence and absence of drug was plotted as a function of substrate concentration by the method of Lineweaver and Burke (15).
Vol. 27, No. 17, 1980
PCP as Serotonin Uptake Inhibitor
&
1609
m
!
!
i
D
!
!
f ~M,,J Fig. 1 Lineweaver and Burke plot for: A) PCP; B) Ketamine; C) Fluoxetine; D) MP. ( 0 ) human blood platelet; ( 0 ) human blood platelet + drug.
20
16
:E
I I
I 2
I 3
I 4
I 5
I
'-~-~ x ~M
Fig. 2 Lineweaver and Burke plot for d-Amp and l-Amp ( • ) human blood platelet (o )human blood platelet + l-Amp ( • ) human blood platelet + d-Amp.
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PCP as S e r o t o n l n Uptake I n h l b l t o r
Vol. 27, No. 17, 1980
Results The results of the inhibition studies are reported in Table 1 as ICs0 values (the concentration of the drug that produces 50% inhibition of 5-HT uptake by human blood platelets). TABLE 1 IC
Values for Inhibition of
Dru9 Fluoxetine PCP
C-SHTUptake in Human Platelets IC CX ± S.D.) pM 0.022 ± 0.008 2.5
± 0.22
Ketamine
25
± 3.5
d-amphetamine
24
± 2.6
l-amphetamine
80
± 8.6
400
± 2.5
Methylphenidate
PCP was the most potent of the psychomotor stimulants as an inhibitor of 5-HTuptake. The ICso of PCP was 2.5 ~M ± 0.22 i~. I t was ten times more potent than ketamine and d-Amp which had equivalent effects, d-Ampwas three times more potent as an uptake blocker than l-Amp. MP was found to be the least potent among the psychomotor stimulants tested in this study. Fluoxetine, a selective inhibitor of 5-HT uptake, was more potent than any of the CNS stimulants tested with an ICso of 0.022 p M ± 0.008 pM, 100 times more potent than PCP. A Lineweaver-Burke plot (Fig. 1) showed that PCP, ketamine, MP and fluoxetine inhibit 5-HT in a non-competitive manner. On the other hand, d-Amp and l Amp were found to be competitlve Inhibitors of platelet 5-HT uptake (Fig.
2). Discussion PCP, ketamlne, MP and fluoxetine, llke most trlcyclic antidepressants (16), inhibited S-HT uptake by platelets through a non-competitive mechanism. On the other hand, d- and l-Amp inhibited 5-HT uptake by blood platelets in a competitive manner. Fluoxetine was by far the most potent inhibitor of 5-HT uptake, followed by PCP, ketamine, d-Amp, l-Amp and MP. Similar results for the relative potency of d-and l-Amp have been reported earlier for inhibition of S-HT uptake in rat mldbraln (17). Because dopamine (DA) enters human platelets only by passive diffusion and not by active uptake (18), we did not investlgate the ability of PCP or other psychomotor stimulants to inhibit DA uptake into platelets. Although PCP inhibits 5-HT uptake by platelets in a non-competitive manner, we have previously shown that i t is a competitive inhibitor of S-HT accumulation by rat brain synaptosomes (lO). These differences maybe due, not only to the difference in organ specificity, i . e . , blood platelets vs central nervous system, but could also be due to species differences. The inhibition of 5-HT uptake into synaptosomes by PCP (ICsQ = 0.8 uM) (10) was three times greater than that for human platelets (I~o = 2.5~M). Theseresults indicate that
Vol. 27, No. 17, 1980
PCP as Serotonin Uptake Inhibitor
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the blood platelet has qualitative and quantitative limitations as a model for drug effects on brain 5-HT uptake. This is in accord with previous results of Smith et al (19) who found that the kinetics of 5-HT uptake by mouse platelets is different than those of mouse brain synaptosomes and concluded that 5-FFT uptake processes in platelets and brain may not be identical, at least under in v i t r o conditions. Ketamine, which is chemically similar to PCP, also inhibits platelet 5-FFT uptake, ICso= 25 ~M. This correlates with i t s lesser potency in man and laboratory animals in a variety of other effects. Ketamine, like PCP, also inhibits 5-HT uptak@ in a non-competitive manner. The non-competitive inhibition of 5-hFF uptake by PCP and ketamine may be due to an effect on Na~, K+ATPase a c t i v i t y , which has been shown to be an important component of the membrane transport processes (20). Ketamine has also been reported to i n h i b i t rat brain Na+, K+-ATPase a c t i v i t y in vivo (21). There is no data on the effect of PCP on Na+ , K+-ATPase. The apparent inhibition of 5-HT uptake by PCP or ketamine may contribute to some of the neurochemical effects of these drugs on brain 5-HTmetabolism. As previously mentioned, Tonge and Leonard (6) reported that a single dose of PCP (10 mg/kg) increased the 5-HT level in whole brain of Wistar rats with the peak effect occurring 10 min after administration. Sung et al. (22) also observed an increased level of 5-HT in rat brain after a single dose of ketamine (40 mg/i.v.). Increased rat brain 5-FFF and 5-hydroxyindoleacetic acid levels were also reported following ketamine administration (23,24). Inhibition of 5-HT uptake might produce these effects by inhibiting 5-){F neuronal a c t i v i t y through activation of a feedback loop (25). Inhibition of 5-HT uptake in brain by PCP may contribute to the serotonergic behavioral sJn~dromeobserved by Martin et al (5) in rats following high doses of PCP (50 mg/kg i . p . ) . However, we have found that the a b i l i t y of low doses (up to 10 mg/kg i . p . ) of PCP to increase locomotor a c t i v i t y , stereotypy and ataxia i n rats is not affected by putative 5-HT receptor blockers such as cinnanserin and cyproheptadine or by pretreatment with parachlorphenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, the rate limiting step in 5-HT s~thesis (26) (Sturgeon and Meltzer, unpublished data). At these low doses, the behavioral effects of PCP in rats are most l i k e l y primarily dopaminergic (2,3). The lack of effect of the 5-FFF antagonists and PCPA on PCP-induced stereotypy, locomotor a c t i v i t y and ataxia does not rule out the p o s s i b i l i t y that the CNS effects of PCP on 5-HT metabolism have other, possibly important, behavioral and physiological consequences in the rat, or that the CNS effects of PCP on 5-HT metabolism might be more important behaviorally in other species, including man. Recently, Sloviter et al (27) and Lees (28) have also reported that 5HT mediates some of the behavioral effects of high dose of Amp. The behavioral signs caused by high dose AMP (15 mg/kg or more) resemble those caused by 5-HT receptor stimulation (29). These behavioral effects increased when 5-~FF synthesis is enhanced by tryptophan administration and decreased when 5-HT synthesis is diminished (28). These effects are consistent with the uptake inhibition studies by PCP, d-Amp, l-Amp and fluoxetine. The relative potency of the CNS stimulants d-Amp, l-Amp and MP as inhibitors of 5-HT uptake by blood platelets correlates with their a b i l i t y to induce stereot)q~y and locomotor stimulation, e.g., d-Amp has been reported to be ten times as potent as l-Amp in enhancing locomotor actTvity in rats (30), twice as potent as the l-isomer in e l i c i t i n g stereotyped behavior in rats (31) and in activating pre-existing psychotic behaviors in humans (32). MP is weaker than d-Amp both with regard to induction of stereotyped behavior (33)
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PCP as Serotonin Uptake Inhibitor
Vol. 27, No. 17, 1980
and i n h i b i t i o n of 5-HT uptake in humanblood platelets. While this is conslstent with the p o s s i b i l i t y that i n h i b i t i o n of 5-HT uptake contributes to the behavioral effects of these agents, there is l i t t l e question that their effect on dopamine and norepinephrine are much more important to these behavloral effects of low doses of amphetamine and methylphenidate. We have found PCP is much more similar to MP in i t s effects on impulsedependent dopamine turnover in rat striatum than i t is to AMP (34). This would suggest that the effects of PCP on 5-HT and 5-HI~ levels are not mediated by an indirect effect on serotonergic neurons. The relevance, i f any, of the enhanced serotonergic a c t i v i t y produced by PCP in rats to the various behavioral syndromes produced by PCP in man w i l l require further study. Indole hallucinogens and some phenethylamine hallucinogens such as mescaline are 5-HT agonists (35). The a b i l i t y of PCP to potentiate serotonergic a c t i v i t y is weak compared to agents such as fluoxetine which are not hallucinogenic in man. This suggests that serotonergic effects may contribute to the psychotomimetic effects of PCP but that this is not a s u ffi c i e n t cause of psychomimetic effects. I t is possible that prolonged self-admlnistration of PCP by human PCP abusers may lead to incresed serotonergic a c t i v i t y which could contribute to some of the delayed behavioral effects of PCP. I t is posslble that enhanced 5-HT a c t i v i t y might contribute to a mood-elevating action of PCP. Acknowledgements This research was supported in part by USPHS MH 30,509, MH 30,938 and DA 02581. HYM is recipient of RCSA MH 47,808. We also wish to thank Dr. P. Muller for his helpful advice and critlcism. References I. 2. 3. 4.
5. 6. 7. 8. 9.
10. 11. 12. 13 14. 15, 16. 17. 18. 19.
E.D. LUBY, B. COHEN, G. ROSENBAUM,J.S. GOTTLIEBand R. KELLY, Arch. Neurol. Psychiatry. 81 363-369 (1969). M. KANNER,~ . FINNEGAN and H. Y. MELTZER, Psychopharmac. Communs. 114 393-401 (1979). ~'7~~. FESSLER, R.D. STURGEON, and H.Y. MELTZER, Life Sci. 24 1281-1288 (1979). R.D. STURGEON, R.G. FESSLER and H.Y. MELTZER, Eur. J. Pharmacology 59 169-179 (1980). J.R. MARTIN, M.H. BERMON, I. KREWSUNand S.F. SMALL, Life Sci. 24 169901704 (1979). S.R. TONGE and B.E. LEONARD, Life Sci. 8 805-814 (1969).
B.E. LEONARDand S.R. TONGE, Life Sci. ~815-825 (1969). S.R. TONGEand B.E. LEONARD, J. PHarm. ~harmacol. 23 711-712 (1971). J.P. VINCENT, D. CAVEY, J.M. KAMENKA, P. GENESTE~and M. LAZDUNSKI, R.C. Brain Res. 152 176-182 (1978). R.C. SMITH, H.Y. MELTZER, R.C. ARORA and J.M. DAVIS, Biochem. Pharmacco]. 26 1435-1439 (1977). ~ E . CAREYand R.G. HEATH, Life Sci. 18 1105-1110 (1976). S.H. JAMES and S.H. SCHNOLL, Clinica]'-Toxicol. 9 573-582 (1976). J,M. SNEDDON, Prog. Neurobiol. 1 151-198 (1973)T D,T. WONG, J.S. HORNG, F.P. BY~[ASTER, K.L. HAURSER and B.B. MOLLOY, Life Sci. 15 471-479 (1974). H. LII~I3"WEAVERand D. BURKE, J. Amer. Chem. Society. 56 658-666 (1934). G.L. FUR and A. UZAN, Biochem. Pharmacol. 26 497-503-'[1977). J.C. HOLMESand C.O. RUTLEDGE, Biochem. Ph~macol. 25 447-451 (1976). S.M. STAHL and H.Y. MELTZER, Exp. Neurol. 59 1-15 (~78). L.T. SMITH, D.R. HANSONand G.S. OMENN, Br~n Res. 146 400-403 (1978;.
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PCP as Serotonln Uptake Inhibitor
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20. H.H. KANNENGIESSIR, P. HUNT and J.P. TAYNAUD, Biochem. Pharmacol. 22 73-84 (1973). 21. H. KRISHNAN, R. SINGH and N.Z. BAQUER, 7TH International Congress of Pharmacology. Abstract No. 2825 (1978). 22. Y.F. SUNG, E.L. FREDERICKSON and S.G. HOLTZMAN, Anesthesiology. 39 478487 (1973). 23. G. BIGGIO, M.L. PORCEDOU, L. VARGIN, E. STEBANINI and P. ODESSA, Riv. Farm. Terap. 5 163-168 (1974). 24. H.P. KARl, P-~P. DAVIDSON, H.H. KOHL, M.M. KOCHHAR, Res. Commun. Chem. Patho]. Pharmacol. 20 475-488 (1978). 25. A. CARLSSONand M. I_-rNDQVIST, J. Neural Transmission, 43 73-91 (1978). 26. B.K. KOE and A. WEISSMAN, J. Pharmac. Exp. Ther. 154 4~-516 (1966). 27. R.S. SLOVITER, E.G. DRUST and J.D. CONNOR, J.-FITarmacol. Exp. Therap. 206 348-352 (1978). 28. ]T~.:J. LEES, J.C.R. FERNANDO and G. CURZON, Neuropharmacol. 18 153-158 29. 30. 31. 32. 33. 34.
(1979).
B.L. JACOBS, Psychopharmacologia, (Berl.) 39 81-86 (1974). K.M. TAYLOR and S.H. SNYDER, Sclence, 168 ~I-~[87-1489 (1970). K.M. TAYLOR and S.M. SNYDER, Brain Res. 28 295-309 (1971). D.S. JANOWSKY and J.M. DAVIS, Arch. Gen.~sychiatry. 33 304-308 (1976). I. SCHEEL-KRUGER, Eur. J. Pharmacol. 14 47-59 (1971). J.D. DOHERTY, M. SIMONOVIC, R. SO an--d H.Y. MELTZER, Eur. J. Pharmacol. (In press). 35. H.Y. MELTZER, R.G. FESSLER, M. SIMONOVIC and V.S. FANG, Psychopharmacology 56 255-259 (1978).
Pergamon Press
IN VITRO EFFECTOF PHENCYCLIDINEAND OTHERPSYCHOMOTOR --S-TIMO-LANTS ON SEROTONINUPTAKE IN HUMANPLATELETS Ramesh C. Arora and Herbert Y. Meltzer I l l i n o i s State Psychiatric Institute, 1601W. Taylor St., Chicago, I l l i n o i s 60612 and the University of Chicago Pritzker School of Medicine Department of Psychiatry, Chicago, IL 60637 (Received in final form August 21, 1980)
Summary Phencyclidine, ketamine and fluoxetine inhibited serotonin (5-HT) uptake in a non-competitive manner in human blood platelets whereas d- and l-amphetamine produced a competitive inhibition of 5-HT uptake. Phencyclidine (ICsQ, 2.5 ~M) was one-hundredth as potent as fluoxetine (ICs0, 22 nM) but ten times more potent than ketamine (ICs0, 25 pM) and d-amphetamine (ICso, 24~M) and three times more potent than l-amphetamine (ICs0, 80 pM) in inhibition of 5-HT uptake by human blood platelets. The possibility that inhibition of 5-HT may contribute to some of the proposed serotonergic effects of psychomotor stimulants is discussed. Phencyclidine [l-(Phencyclohexyl) piperidine, PCP] is one of the most widely abused drugs, despite i t s potent psychotomimetic effects (1). In doses from 2.5-15 mg/kg, i t produces locomotor activity, stereotypy and ataxia in rats that appear to be dopamine-dependent (2-4). PCP, in doses of 50 mg/kg, has been reported to induce behaviors in the rats thought to be consistent with the so-called "serotonin (5-HT) syndrome": e.g. forepaw treading, head weaving, r i g i d i t y , tremor, straub t a i l (5). These behaviors are antagonized by the putative 5-HT agonists, cinanserin and cyproheptadine, but not by the dopaminergic antagonists, haloperidol (5). Tonge and Leonard 6) reported that PCP increases 5-HT levels and decreases norepinephrine NE) levels in rat brain (7). These effects were found to be strain dependent (8). No significant effect of PCP on either brain monoamineoxidase (MAO) or aromatic amino acid decarboxylase has been reported (7). Tonge and Leonard also showed that PCP reduced the depletion of 5-HT in reserpineand p-chlorophenylalanine-treated rats and concluded that PCP had no effect on the binding of 3[ H ~5-HT to 5-HT receptors in rat brain homogenates (g). Previously, we (lO) and Garey and Heath (1"1) reported that PCP inhibits uptake of 5-HT and dopamine in crude rat brain sjn~aptosomal preparations. The dose needed for 50% inhibition of 5-HT uptake by PCP is 0.8 M. Concentrations of more than an order of magnitude higher are achieved in the rat brain after administration of high dose of PCP (12). I t could be argued, therefore, that 5-HT uptake inhibition could contribute to the functional effects of PCP, at least in higher doses. A number of similarities have also been shown between 5-HT uptake inhibition in brain synaptosomes and blood platelets (13). In the present study, we have studied the effect of PCP and ketamine, an analog of PCP, on 5-HT uptake In human blood platelets and compared its properties with three other psychomotor stimu-
0024-3205/80/431607-07502.00/0 Copyright (c) 1980 Pergamon Press Ltd
1608
PCP as Serotonin Uptake Inhibitor
Vol. 27, No. 17, 1980
lants, d-amphetamine (d-Amp), l-amphetamlne (l-Amp) and methylphenidate (MP). Fluoxetine, a selective 5-HT uptake inhibitor in rat brain (147, was also studied for comparison purposes. Materials and Methods
Reagents: PCP was obtained from the National Institute of Drug Abuse. d- and l-Amp were g i f t s from Smith, Kline and French, Inc., Philadelphia, Pa. MP was purchased from Ciba Pharmaceutlcals Co., Summit, N.J. The radioactive (2-IWC) 5-HT binoxalate (sp. act. 47.2 mCi/mmole) was purchased from Amersham Searle Corp. A11 the other chemicals used were of analytical grade quality. Method: Blood (lO ml) was drawn in plastic syringes from normal volunteers and transferred into plastic tubes containing citrate-phosphate-dextrose (CPD) as anticoagulant (I.5 ml/lO ml of blood). Platele~-rich-plasma (PRP) was obtained by centrifugation at 600 x g for 2~ min. at 4". Platelet counts were made by means of a Coulter Thrombocounter after diluting lO ul PRP in 20 ml of counting solution (Coulter Electronics, Elk Grove Village, I l l i n o i s ) . Each count was done in t r i p l i c a t e and the average was taken. Uptake studies were done as follows. PRP (0.3 ml) was preincubated wlth 0.6 ml of KrebC) phosphate bufZer minus calcium chloride for lO min. After that, O.l ml of "~C-5-HT (4 x lO~M) was added and the mixture was incubated further for 4 min. The reaction was stopped by rapid cooling in ice. The platelets were isolated by centrifugation (10000 x g ) in a refrigerated centrifuge for 15 mlns. The pellet~as dissolved in Soluene-350 (0.5 ml) by incubating at 55°for 1 hour or at 37- overnight. The solubilized pellet was transferred into s c i n t i l l a t i o n vials with 5 ml of s c i n t i l l a t i o n solvent toluene,[l~, containing 500 ml absolute ethanol, 4 gm of 2.5 diphenyloxazole (PPO) and 50 mg of 1,4-bis-2(4-methyl-5-phenyloxazoly)-benzene .] Radioactivity was counted in a Nuclear Chicago S c i n t i l l a t i o n counter. A separate set of tubes were immediately cooled after addition of~C-5-HT to obtain a measure of passive diffusion and processed in identlcal fashion to the set of tubes previously described. Active uptake was determined by subtracting the dpm of the tubes immediately placed in ice baths from the dpm after incubation at 370. Inhibition Studies: Inhibition of 5-HT uptake into platelets was studied by preincubatlng PRP with various concentrations of the drugs in buffer for I0 min prior to the addition of I~C-5HT (4 x IO-?M). After the addition of 5-HT, the mixture was incubated further for 4 min and processed as described above. The degree of inhibition was calculated from the difference in dislntegrations per minute (dpm) from the samples with and without each drug. All determinations were done in duplicate and the average was taken. Nature of Inhibition: In order to determine the nature of the inhibition of platelet 5-HT uptake by PCP and other test drugs, PCP was incubated with concentrations of I~C-5-HT (2 x IO'?M to 1 x I0"6M). 5-HT uptake in the presence and absence of drug was plotted as a function of substrate concentration by the method of Lineweaver and Burke (15).
Vol. 27, No. 17, 1980
PCP as Serotonin Uptake Inhibitor
&
1609
m
!
!
i
D
!
!
f ~M,,J Fig. 1 Lineweaver and Burke plot for: A) PCP; B) Ketamine; C) Fluoxetine; D) MP. ( 0 ) human blood platelet; ( 0 ) human blood platelet + drug.
20
16
:E
I I
I 2
I 3
I 4
I 5
I
'-~-~ x ~M
Fig. 2 Lineweaver and Burke plot for d-Amp and l-Amp ( • ) human blood platelet (o )human blood platelet + l-Amp ( • ) human blood platelet + d-Amp.
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PCP as S e r o t o n l n Uptake I n h l b l t o r
Vol. 27, No. 17, 1980
Results The results of the inhibition studies are reported in Table 1 as ICs0 values (the concentration of the drug that produces 50% inhibition of 5-HT uptake by human blood platelets). TABLE 1 IC
Values for Inhibition of
Dru9 Fluoxetine PCP
C-SHTUptake in Human Platelets IC CX ± S.D.) pM 0.022 ± 0.008 2.5
± 0.22
Ketamine
25
± 3.5
d-amphetamine
24
± 2.6
l-amphetamine
80
± 8.6
400
± 2.5
Methylphenidate
PCP was the most potent of the psychomotor stimulants as an inhibitor of 5-HTuptake. The ICso of PCP was 2.5 ~M ± 0.22 i~. I t was ten times more potent than ketamine and d-Amp which had equivalent effects, d-Ampwas three times more potent as an uptake blocker than l-Amp. MP was found to be the least potent among the psychomotor stimulants tested in this study. Fluoxetine, a selective inhibitor of 5-HT uptake, was more potent than any of the CNS stimulants tested with an ICso of 0.022 p M ± 0.008 pM, 100 times more potent than PCP. A Lineweaver-Burke plot (Fig. 1) showed that PCP, ketamine, MP and fluoxetine inhibit 5-HT in a non-competitive manner. On the other hand, d-Amp and l Amp were found to be competitlve Inhibitors of platelet 5-HT uptake (Fig.
2). Discussion PCP, ketamlne, MP and fluoxetine, llke most trlcyclic antidepressants (16), inhibited S-HT uptake by platelets through a non-competitive mechanism. On the other hand, d- and l-Amp inhibited 5-HT uptake by blood platelets in a competitive manner. Fluoxetine was by far the most potent inhibitor of 5-HT uptake, followed by PCP, ketamine, d-Amp, l-Amp and MP. Similar results for the relative potency of d-and l-Amp have been reported earlier for inhibition of S-HT uptake in rat mldbraln (17). Because dopamine (DA) enters human platelets only by passive diffusion and not by active uptake (18), we did not investlgate the ability of PCP or other psychomotor stimulants to inhibit DA uptake into platelets. Although PCP inhibits 5-HT uptake by platelets in a non-competitive manner, we have previously shown that i t is a competitive inhibitor of S-HT accumulation by rat brain synaptosomes (lO). These differences maybe due, not only to the difference in organ specificity, i . e . , blood platelets vs central nervous system, but could also be due to species differences. The inhibition of 5-HT uptake into synaptosomes by PCP (ICsQ = 0.8 uM) (10) was three times greater than that for human platelets (I~o = 2.5~M). Theseresults indicate that
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PCP as Serotonin Uptake Inhibitor
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the blood platelet has qualitative and quantitative limitations as a model for drug effects on brain 5-HT uptake. This is in accord with previous results of Smith et al (19) who found that the kinetics of 5-HT uptake by mouse platelets is different than those of mouse brain synaptosomes and concluded that 5-FFT uptake processes in platelets and brain may not be identical, at least under in v i t r o conditions. Ketamine, which is chemically similar to PCP, also inhibits platelet 5-FFT uptake, ICso= 25 ~M. This correlates with i t s lesser potency in man and laboratory animals in a variety of other effects. Ketamine, like PCP, also inhibits 5-HT uptak@ in a non-competitive manner. The non-competitive inhibition of 5-hFF uptake by PCP and ketamine may be due to an effect on Na~, K+ATPase a c t i v i t y , which has been shown to be an important component of the membrane transport processes (20). Ketamine has also been reported to i n h i b i t rat brain Na+, K+-ATPase a c t i v i t y in vivo (21). There is no data on the effect of PCP on Na+ , K+-ATPase. The apparent inhibition of 5-HT uptake by PCP or ketamine may contribute to some of the neurochemical effects of these drugs on brain 5-HTmetabolism. As previously mentioned, Tonge and Leonard (6) reported that a single dose of PCP (10 mg/kg) increased the 5-HT level in whole brain of Wistar rats with the peak effect occurring 10 min after administration. Sung et al. (22) also observed an increased level of 5-HT in rat brain after a single dose of ketamine (40 mg/i.v.). Increased rat brain 5-FFF and 5-hydroxyindoleacetic acid levels were also reported following ketamine administration (23,24). Inhibition of 5-HT uptake might produce these effects by inhibiting 5-){F neuronal a c t i v i t y through activation of a feedback loop (25). Inhibition of 5-HT uptake in brain by PCP may contribute to the serotonergic behavioral sJn~dromeobserved by Martin et al (5) in rats following high doses of PCP (50 mg/kg i . p . ) . However, we have found that the a b i l i t y of low doses (up to 10 mg/kg i . p . ) of PCP to increase locomotor a c t i v i t y , stereotypy and ataxia i n rats is not affected by putative 5-HT receptor blockers such as cinnanserin and cyproheptadine or by pretreatment with parachlorphenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, the rate limiting step in 5-HT s~thesis (26) (Sturgeon and Meltzer, unpublished data). At these low doses, the behavioral effects of PCP in rats are most l i k e l y primarily dopaminergic (2,3). The lack of effect of the 5-FFF antagonists and PCPA on PCP-induced stereotypy, locomotor a c t i v i t y and ataxia does not rule out the p o s s i b i l i t y that the CNS effects of PCP on 5-HT metabolism have other, possibly important, behavioral and physiological consequences in the rat, or that the CNS effects of PCP on 5-HT metabolism might be more important behaviorally in other species, including man. Recently, Sloviter et al (27) and Lees (28) have also reported that 5HT mediates some of the behavioral effects of high dose of Amp. The behavioral signs caused by high dose AMP (15 mg/kg or more) resemble those caused by 5-HT receptor stimulation (29). These behavioral effects increased when 5-~FF synthesis is enhanced by tryptophan administration and decreased when 5-HT synthesis is diminished (28). These effects are consistent with the uptake inhibition studies by PCP, d-Amp, l-Amp and fluoxetine. The relative potency of the CNS stimulants d-Amp, l-Amp and MP as inhibitors of 5-HT uptake by blood platelets correlates with their a b i l i t y to induce stereot)q~y and locomotor stimulation, e.g., d-Amp has been reported to be ten times as potent as l-Amp in enhancing locomotor actTvity in rats (30), twice as potent as the l-isomer in e l i c i t i n g stereotyped behavior in rats (31) and in activating pre-existing psychotic behaviors in humans (32). MP is weaker than d-Amp both with regard to induction of stereotyped behavior (33)
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Vol. 27, No. 17, 1980
and i n h i b i t i o n of 5-HT uptake in humanblood platelets. While this is conslstent with the p o s s i b i l i t y that i n h i b i t i o n of 5-HT uptake contributes to the behavioral effects of these agents, there is l i t t l e question that their effect on dopamine and norepinephrine are much more important to these behavloral effects of low doses of amphetamine and methylphenidate. We have found PCP is much more similar to MP in i t s effects on impulsedependent dopamine turnover in rat striatum than i t is to AMP (34). This would suggest that the effects of PCP on 5-HT and 5-HI~ levels are not mediated by an indirect effect on serotonergic neurons. The relevance, i f any, of the enhanced serotonergic a c t i v i t y produced by PCP in rats to the various behavioral syndromes produced by PCP in man w i l l require further study. Indole hallucinogens and some phenethylamine hallucinogens such as mescaline are 5-HT agonists (35). The a b i l i t y of PCP to potentiate serotonergic a c t i v i t y is weak compared to agents such as fluoxetine which are not hallucinogenic in man. This suggests that serotonergic effects may contribute to the psychotomimetic effects of PCP but that this is not a s u ffi c i e n t cause of psychomimetic effects. I t is possible that prolonged self-admlnistration of PCP by human PCP abusers may lead to incresed serotonergic a c t i v i t y which could contribute to some of the delayed behavioral effects of PCP. I t is posslble that enhanced 5-HT a c t i v i t y might contribute to a mood-elevating action of PCP. Acknowledgements This research was supported in part by USPHS MH 30,509, MH 30,938 and DA 02581. HYM is recipient of RCSA MH 47,808. We also wish to thank Dr. P. Muller for his helpful advice and critlcism. References I. 2. 3. 4.
5. 6. 7. 8. 9.
10. 11. 12. 13 14. 15, 16. 17. 18. 19.
E.D. LUBY, B. COHEN, G. ROSENBAUM,J.S. GOTTLIEBand R. KELLY, Arch. Neurol. Psychiatry. 81 363-369 (1969). M. KANNER,~ . FINNEGAN and H. Y. MELTZER, Psychopharmac. Communs. 114 393-401 (1979). ~'7~~. FESSLER, R.D. STURGEON, and H.Y. MELTZER, Life Sci. 24 1281-1288 (1979). R.D. STURGEON, R.G. FESSLER and H.Y. MELTZER, Eur. J. Pharmacology 59 169-179 (1980). J.R. MARTIN, M.H. BERMON, I. KREWSUNand S.F. SMALL, Life Sci. 24 169901704 (1979). S.R. TONGE and B.E. LEONARD, Life Sci. 8 805-814 (1969).
B.E. LEONARDand S.R. TONGE, Life Sci. ~815-825 (1969). S.R. TONGEand B.E. LEONARD, J. PHarm. ~harmacol. 23 711-712 (1971). J.P. VINCENT, D. CAVEY, J.M. KAMENKA, P. GENESTE~and M. LAZDUNSKI, R.C. Brain Res. 152 176-182 (1978). R.C. SMITH, H.Y. MELTZER, R.C. ARORA and J.M. DAVIS, Biochem. Pharmacco]. 26 1435-1439 (1977). ~ E . CAREYand R.G. HEATH, Life Sci. 18 1105-1110 (1976). S.H. JAMES and S.H. SCHNOLL, Clinica]'-Toxicol. 9 573-582 (1976). J,M. SNEDDON, Prog. Neurobiol. 1 151-198 (1973)T D,T. WONG, J.S. HORNG, F.P. BY~[ASTER, K.L. HAURSER and B.B. MOLLOY, Life Sci. 15 471-479 (1974). H. LII~I3"WEAVERand D. BURKE, J. Amer. Chem. Society. 56 658-666 (1934). G.L. FUR and A. UZAN, Biochem. Pharmacol. 26 497-503-'[1977). J.C. HOLMESand C.O. RUTLEDGE, Biochem. Ph~macol. 25 447-451 (1976). S.M. STAHL and H.Y. MELTZER, Exp. Neurol. 59 1-15 (~78). L.T. SMITH, D.R. HANSONand G.S. OMENN, Br~n Res. 146 400-403 (1978;.
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20. H.H. KANNENGIESSIR, P. HUNT and J.P. TAYNAUD, Biochem. Pharmacol. 22 73-84 (1973). 21. H. KRISHNAN, R. SINGH and N.Z. BAQUER, 7TH International Congress of Pharmacology. Abstract No. 2825 (1978). 22. Y.F. SUNG, E.L. FREDERICKSON and S.G. HOLTZMAN, Anesthesiology. 39 478487 (1973). 23. G. BIGGIO, M.L. PORCEDOU, L. VARGIN, E. STEBANINI and P. ODESSA, Riv. Farm. Terap. 5 163-168 (1974). 24. H.P. KARl, P-~P. DAVIDSON, H.H. KOHL, M.M. KOCHHAR, Res. Commun. Chem. Patho]. Pharmacol. 20 475-488 (1978). 25. A. CARLSSONand M. I_-rNDQVIST, J. Neural Transmission, 43 73-91 (1978). 26. B.K. KOE and A. WEISSMAN, J. Pharmac. Exp. Ther. 154 4~-516 (1966). 27. R.S. SLOVITER, E.G. DRUST and J.D. CONNOR, J.-FITarmacol. Exp. Therap. 206 348-352 (1978). 28. ]T~.:J. LEES, J.C.R. FERNANDO and G. CURZON, Neuropharmacol. 18 153-158 29. 30. 31. 32. 33. 34.
(1979).
B.L. JACOBS, Psychopharmacologia, (Berl.) 39 81-86 (1974). K.M. TAYLOR and S.H. SNYDER, Sclence, 168 ~I-~[87-1489 (1970). K.M. TAYLOR and S.M. SNYDER, Brain Res. 28 295-309 (1971). D.S. JANOWSKY and J.M. DAVIS, Arch. Gen.~sychiatry. 33 304-308 (1976). I. SCHEEL-KRUGER, Eur. J. Pharmacol. 14 47-59 (1971). J.D. DOHERTY, M. SIMONOVIC, R. SO an--d H.Y. MELTZER, Eur. J. Pharmacol. (In press). 35. H.Y. MELTZER, R.G. FESSLER, M. SIMONOVIC and V.S. FANG, Psychopharmacology 56 255-259 (1978).