Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N-Methyl-d-Aspartate Receptor Ligands

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20-30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-D-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD = 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki = 31 nM) and the anticonvulsant MK-801 (Ki = 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Mode of binding of [3H]dibenzocycloalkenimine (MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its therapeutic implication

Binding of the labeled anticonvulsant drug [³H]dibenzocycloalkenimine (³H]MK-801) to the N-methyl-D-aspartate (NMDA) receptor and its dissociation from the receptor at 25°C are slow processes, both of which follow first order kinetics (t_1/270 and 180 min, respectively). Both reactions are markedly accelerated by glutamate and glycine (t_1/22-8 and 4 min, respectively), which allow bimolecular association kinetics of the labeled drug with the receptors whereas equilibrium binding of [³H]MK-801 (K_d 2–4 nM) is hardly affected by glutamate and glycine. The data suggest that MK-801 acts as a steric blocker of the NMDA receptor channel. The competitive antagonist D-(−)-2-amino-5-phosphovaleric acid (AP-5) freezes the receptor in a state which precludes either binding of [³H]MK-801 to the receptor channel or its dissociation from it. These findings have therapeutic implications.     

Pharmacological Characteristics and Distributions of σ and Phencyclidine Receptors in the Animal Kingdom

The phylogenetic distributions ofσ- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [³H]haloperidol and [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to σ- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [³H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [³H]N-[1-(2-thienyl)cyclohexyl]-3,4–piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that σ– and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.     

Drug-induced circling preference in rats

Drugs of abuse, such as phencyclidine (PCP), methamphetamine (METH), and cocaine (COC) are known to affect several behaviors in rats, such as motor activity, stereotypy, and circling. In this study, we evaluated whether these drugs produce circling preferences in the presence or absence of unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the caudate nucleus. Adult male CD rats were lesioned with 10 μg 6-OHDA/site. Animals were dosed with PCP (15 mg/kg, ip), its congener, (+) MK-801 (0.15 mg/kg, ip), METH (2 mg/kg, ip), COC (60 mg/kg, ip), or apomorphine (0.2 mg/kg, ip). circling preference was recorded in control and lesioned rats for 2 h before animals were sacrificed to determine monoamine levels by HPLC/EC. In control animals, administration of these drugs produced 60–70% left circling. In, lesioned animals, these drugs produced 78–90% ipsilateral (toward the lesion) circling, except apomorphine, which produced 60–80% contralateral (away from the lesion) circling. Dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations significantly decreased ipsilaterally in lesioned caudate nucleus (CN) and substantia nigra (SN). However, no significant changes were observed in nucleus accumbens (NA) and olfactory tubercles (OT). These data demonstrate that drugs of abuse like PCP, its congener (+) MK-801, METH, and COC produce a greater preference to turn toward the left than the right, a finding similar to that found in human psychosis. Since 6-OHDA lesions enhanced the circling bias and depleted DA and its metabolites DOPAC and HVA, it also suggests that the dopaminergic system may be involved in the circling behavior.     

Contrasting Neurochemical Interactions of Tiletamine, a Potent Phencyclidine (PCP) Receptor Ligand, with the N-Methyl-D-Aspartate-Coupled and -Uncoupled PCP Recognition Sites

Neurochemical interactions of tiletamine, a potent phencyclidine (PCP) receptor ligand, with the N-methyl-D-aspartate (NMDA)-coupled and -uncoupled PCP recognition sites were examined. Tiletamine potently displaced the binding of [3H]1-(2-thienyl)cyclohexylpiperidine with an IC50 of 79 nM without affecting sigma-, glycine, glutamate, kainate, quisqualate, or dopamine (DA) receptors. Like other PCP ligands acting via the NMDA-coupled PCP recognition sites, tiletamine decreased basal, harmaline-, and D-serine-mediated increases in cyclic cGMP levels and induced stereotypy and ataxia. Tiletamine was nearly five times more potent than PCP at inhibiting the binding of 3-hydroxy[3H]PCP to its high-affinity NMDA-uncoupled PCP recognition sites. However, following parenteral administration, dizocilpine maleate (MK-801), ketamine, PCP, dexoxadrol, and 1-(2-thienyl)cyclohexylpiperidine HCl, but not tiletamine, increased rat pyriform cortical DA metabolism and/or release, a response modulated by the NMDA-uncoupled PCP recognition sites. Pretreatment with tiletamine did not attenuate the MK-801-induced increases in rat pyriform cortical DA metabolism, a result suggesting that tiletamine is not a partial agonist of the NMDA-uncoupled PCP recognition sites in this region. However, following intracerebroventricular administration (100-500 micrograms/rat), tiletamine increased pyriform cortical DA metabolism with a bell-shaped dose-response curve. These data indicate a differential interaction of tiletamine with the NMDA-coupled and -uncoupled PCP recognition sites. The paradoxical effects of tiletamine suggest that tiletamine might activate receptor(s) or neuronal pathways of unknown pharmacology.     

Altered Dopamine Receptor and Dopamine Transporter Binding and Tyrosine Hydroxylase mRNA Expression Following Perinatal NMDA Receptor Blockade

This study examined how perinatal phencyclidine (PCP) treatment would affect dopamine D2 receptor and dopamine transporter (DAT) binding at different stages after treatment cessation. Female rat pups received injections of PCP (10 mg/kg, s.c.) or saline on postnatal day (PN)7, 9 and 11. D2 receptor and transporter binding was examined at four time-points (PN12, 18, 32 and 96) following injections. PCP treatment altered D2 receptor binding throughout development, with a final end-point of 22-33% decreased binding at adulthood in the nucleus accumbens and caudate putamen (P < 0.01), accompanied by a small but significant increase in DAT binding in the caudate putamen. Tyrosine hydroxylase mRNA expression was also significantly increased by 25% (P < 0.05) in the ventral tegmental area of adult rats, suggesting that this model may produce a long-term increase in dopamine output. This study demonstrates that early insult to the brain from NMDA receptor hypofunction alters the dopaminergic system at different stages of development.     

The role of antagonism of NMDA receptor-mediated neurotransmission and inhibition of the dopamine reuptake in the neuroendocrine effects of phencyclidine

Phencyclidine (PCP) activates the hypothalamo-pituitary-adrenal (HPA) axis and decreases plasma prolactin levels in the rat. PCP is a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, but it also inhibits the reuptake of dopamine, serotonin and norepinephrine. The purpose of the present study was to utilize the PCP analogue N-[1-(2-thienyl)cyclohexyl]piperidine; (TCP), the potent dopamine reuptake inhibitor N-[1-(2-benzo(b)thiophenyl) cyclohexyl]piperidine; (BTCP) and the nonselective monoamine reuptake inhibitor cocaine as pharmacologic probes in order to determine the roles of noncompetitive NMDA receptor blockade and inhibition of dopamine reuptake in the neuroendocrine effects of PCP. PCP, TCP and cocaine increased plasma levels of adrenocorticotropin and corticosterone, but BTCP had no effect. In contrast, PCP, BTCP and cocaine decreased plasma prolactin, but TCP produced no such effect. The data suggest that mechanisms besides inhibition of dopamine reuptake are involved in the effects of PCP on the HPA axis, and the PCP-induced decrease in plasma prolactin is not a consequence of inhibition of NMDA receptor-mediated neurotransmission.     

Phencyclidine and Analogues: Effects on Brain Protein Synthesis

Abstract: Phencyclidine and four analogues were tested for their capacity to inhibit total protein synthesis in a brain homogenate. At 1.0 m M they were all found to be potent inhibitors with values ranging from 36% to 96% inhibition. At this high concentration two of the analogues were equal to or more effective than the classic protein synthesis inhibitors cycloheximide and emetine. At lower concentrations the inhibition dropped off sharply to 18% at 1.0 × 10⁻⁴ M and 9% at 1.0 × 10⁻⁵ M for phencyclidine. If the inhibition observed in the brain homogenate occurs in vivo it may account for the high incidence of memory loss reported with phencyclidine use.     

The role of the hippocampo-prefrontal cortex system in phencyclidine-induced psychosis: A model for schizophrenia

Phencyclidine (PCP) is a psychotomimetic drug that induces schizophrenia-like symptoms in healthy individuals and exacerbates pre-existing symptoms in patients with schizophrenia. PCP also induces behavioral and cognitive abnormalities in non-human animals, and PCP-treated animals are considered a reliable pharmacological model of schizophrenia. However, the exact neural mechanisms by which PCP modulates behavior are not known. During the last decade several studies have indicated that disturbed activity of the prefrontal cortex (PFC) may be closely related to PCP-induced psychosis. Systemic administration of PCP produces long-lasting activation of medial PFC (mPFC) neurons in rats, almost in parallel with augmentation of locomotor activity and behavioral stereotypies. Later studies have showed that such PCP-induced behavioral abnormalities are ameliorated by prior administration of drugs that normalize or inhibit excess excitability of PFC neurons. Similar activation of mPFC neurons is not induced by systemic injection of a typical psychostimulant such as methamphetamine, even though behavioral hyperactivity is induced to almost the same level. This suggests that the neural circuits mediating PCP-induced psychosis are different to those mediating methamphetamine-induced psychosis. Locally applied PCP does not induce excitation of mPFC neurons, indicating that PCP-induced tonic excitation of mPFC neurons is mediated by inputs from regions outside the mPFC. This hypothesis is strongly supported by experimental results showing that local perfusion of PCP in the ventral hippocampus, which has dense fiber projections to the mPFC, induces tonic activation of mPFC neurons with accompanying augmentation of behavioral abnormalities. In this review we summarize current knowledge on the neural mechanisms underlying PCP-induced psychosis and highlight a possible involvement of the PFC and the hippocampus in PCP-induced psychosis.