Molecular Psychiatry (2005) 10, 877–883 & 2005 Nature Publishing Group All rights reserved 1359-4184/05 $30.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics P Seeman1,2,FKo1 and T Tallerico2 1Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada; 2Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state High of the dopamine D2 receptor, D2 , their dissociation constants (Ki) were obtained on [3H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high High affinity for D2 with a Ki of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a 3 Ki of 313 nM, as labeled by [ H]dizocilpine on rat striatal tissue. Ketamine also had a high High affinity for D2 with a Ki of 55 nM, an affinity higher than its 3100 nM Ki for the NMDA sites. High Dizocilpine had a Ki of 0.3 nM at D2 , but a Kd of 1.8 nM at the NMDA receptor. LSD had a Ki of 2nMatD2High. Because the psychotomimetics had higher potency at D2High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis. Molecular Psychiatry (2005) 10, 877–883. doi:10.1038/sj.mp.4001682; published online 26 April 2005 Keywords: dopamine receptor; phencyclidine; domperidone; ketamine; NMDA receptors; psychotomimetics While the clinical anti-dopaminergic actions of anti- pride at striatal D2 receptors, but has a dissociation psychotic drugs are compatible with the hyperdopa- constant of 1.5 nM at the D2High receptor when the mine hypothesis of psychosis and schizophrenia,1,2 link between dopamine D1 and D2 receptors is the psychoses caused by glutamate antagonists such blocked by the D1 antagonist SCH23390.10 The link as phencyclidine or ketamine suggest a hypo-gluta- between D1 and D2 receptors arises from several mate component in psychosis.3–5 However, phency- sources, including the colocalization of dopamine D1 clidine also lowers plasma prolactin6 and elicits and D2 receptors in at least 50% of the medium spiny rotation,7 suggesting a direct or indirect dopamine- neurons in the striatum, the cooperation and mutual mimetic action of phencyclidine. potentiation of D1 and D2 agonists on various Although phencyclidine and ketamine are not behaviors, and the biochemical conversion of D2 selective for glutamate NMDA receptors,8 the precise receptors from their functional high-affinity state, High affinities of these drugs for dopamine D2 receptors D2 , into their low-affinity state, D2Low, in the 10–13 need to be clarified in order to determine their presence of a D1 antagonist. This D1–D2 link has dopaminergic and non-dopaminergic components of been detected in vitro in human and rat striata,10,11 action. More specifically, although phencyclidine had and in tissue culture cells transfected with both D1 a dissociation constant of 37 000 nM at the D2 and D2,12 but has not yet been demonstrated in receptor in rat striatal homogenate,8 phencyclidine humans by means of positron emission tomography had a dissociation constant of 1.3 nM for the func- and [11C]raclopride. tional high-affinity site of the cloned D2 receptor, or However, in order to compare the potencies of the D2High receptor.9 psychotomimetics at dopamine D2 receptors with This apparent discrepancy may be resolved by the their potencies at NMDA receptors, it would be better recent finding that dopamine itself has a dissociation to avoid using SCH23390. This is because SCH23390 constant of 3000 nM when competing vs [3H]raclo- can also affect the potency of drugs at the NMDA receptor, especially since it is known that D1 and NMDA receptors interact.14–16 Correspondence: Dr P Seeman, MD, PhD, Departments of Although the blockade of D1 (by SCH23390) Pharmacology and Psychiatry, Medical Science Building, Room unmasks the functional D2High states,11 the addition 4344, University of Toronto, 1 King’s College Circle, Toronto, of SCH23390 is only needed when using [3H]raclo- Ontario, Canada M5S 1A8. E-mail: [email protected] 3 Received 23 November 2004; revised 4 March 2005; accepted 21 pride to measure D2 receptors. [ H]Domperidone, March 2005; published online 26 April 2005 however, is a selective ligand for D2 that readily Dopamine receptors, phencyclidine and ketamine P Seeman et al 878 detects D2High receptors without the addition of scintillant (Ready Solve, Beckman Co., CA, USA), SCH23390.17 and were monitored 6 h later for tritium in a Beckman Therefore, in order to obtain the potencies of L5000 scintillation spectrometer at 55% efficiency. phencyclidine, ketamine, dizocilpine and LSD at the The nonspecific binding of the [3H]ligand to the functional D2High receptor, the present study used dopamine D2 receptors was defined as that which [3H]domperidone to label D2 receptors. The results occurred in the presence of 10 mM S-sulpiride indicate that the potencies of the psychotomimetics (Ravizza, Milan). The dissociation constants (Kd)of were greater at D2High than at the NMDA receptor. [3H]domperidone for the dopamine D2 receptors (0.42 nM on the cloned D2 receptor and 0.47 nM on 17 Materials and methods the rat striatal tissue ) were obtained by the above method, using a range of 12 final concentrations of 3 We used the human cloned dopamine D2Long receptor 0.1–20 nM [ H]domperidone. The dissociation con- (in Chinese hamster ovary (CHO) cells18) . The cells stant for [3H]dizocilpine was 1.8 nM (rat striatum), were harvested by gently scraping the cells off the using 100 mM phencyclidine to define nonspecific bottom of the Petri dish, centrifuged, resuspended in binding. The Kd value was calculated by nonlinear phosphate-buffered saline (0.9% NaCl), recentrifuged regression analysis of the [3H]ligand saturation and the pellet frozen at À701. When used, the frozen curve.19 The competition data were analyzed as pellet was thawed, and the cells suspended at 200 mg previously described;20 the program provided two protein/ml. The suspension was homogenized for statistical criteria to judge whether a two-site fit was 5 s (Brinkmann Polytron, setting 5 (maximum on better than a one-site fit, or whether a three-site fit scale was 10)), without any further washing or was better than a two-site fit. centrifugation. The drug-induced incorporation of [35S]GTP-g-S Frozen rat brains were purchased (Pel-Freez, (1250 Ci/mmol; final concentration 0.2–0.3 nM) Rogers, AR, USA), partly thawed and the striata was carried out using the procedure previously removed. To prepare the tissues for the drug/[3H] described.8 ligand competition-type experiments, the striatal tissues were blotted and weighed; buffer was added Results (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2, with a final concentration Phencyclidine of 120 mM NaCl added later in the final incubation As noted above, both [3H]raclopride and [3H]domper- tubes) to yield 4 mg tissue per ml. The suspension idone selectively label D2 receptors, but [3H]domper- was homogenized with a glass-Teflon homogenizer idone is more sensitive than [3H]raclopride to the (10 up–down strokes of a piston rotating at 500 rpm). competitive action of dopamine and other drugs and The homogenate was not washed, centrifuged or readily allows the detection of D2High sites.17 There- preincubated because previous work found that these fore, using the more readily displaceable [3H]domper- procedures resulted in a loss of 30–60% of dopamine idone, phencyclidine had a dissociation constant at 19 High D2 receptors. D2 of 2.770.8 nM (n ¼ 9) (Figure 1, top) for the [3H]Dizocilpine (or [3H]( þ )MK801; 17 Ci/mmol; human cloned D2 receptor and 4.370.8 nM for the rat 3 nM final concentration in the incubation tubes) striatal tissue (Figure 1, middle). These Ki values were was purchased from PerkinElmer Life Sciences Inc. essentially unchanged when phencyclidine was (Boston, MA, USA). [3H]Domperidone was custom tested in the presence of 100 nM SCH23390 to synthesized as [phenyl-3H(N)]domperidone (42 Ci/ occlude other receptors such as dopamine D1 recep- mmol) by PerkinElmer Life Sciences Inc., and used tors and serotonin-2 receptors that may interact with at a final concentration of 1.2–1.8 nM. the D2 receptor.10 The competition-type experiment between a drug In contrast, the phencyclidine Ki for inhibiting and the [3H]ligand for binding at the receptors was the binding of [3H]dizocilpine to rat striatal homo- carried out as follows. Each incubation tube genate was 313748 nM (n ¼ 9) (Figure 1, bottom). (12 Â 75 mm, glass) received, in the following order, The addition of 2 mM glutamate did not change 0.5 ml buffer (with a final incubate concentration of the phencyclidine Ki values. either 10 mM or, more usually, 120 mM NaCl), 0.25 ml In order to determine the functional concentration [3H]ligand and 0.25 ml of tissue homogenate. The range for phencyclidine, the effect of phencyclidine tubes, containing a total volume of 1 ml, were on the incorporation of [35S]GTP-g-S was tested on incubated for 2 h at room temperature (201C), after the human cloned D2 receptors.