Phencyclidine Receptor Ligand Research Articles

Role of N-methyl- D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity

Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl- D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [ 3H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca 2+] i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs.

Methylmercury increases N-methyl- d-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity

Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl- d-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to investigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [ 3H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25–1 μM). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25–5 μM). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 μM) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxicity. Intracellular calcium chelator BAPTA-AM (1 μM) partially attenuated the neurotoxicity effect of 1 μM MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs.

Quantitative Structure‐Activity Relationships of Phencyclidine Receptor Ligands. Part 2 [1]. Extension of the Scope of the DVDP Parameter

AbstractThe validity of a parameter, which represents the direction of the molecular dipole vector of phencyclidine (PCP) derivatives, was reviewed. This parameter, named DVDP, quantitatively predicted the receptor binding activity of various PCP analogues. DVDP also effectively explained the activity of remarkably different compounds such as the MK‐801 derivatives. For the comparison of the dipole direction, the arrangement of the molecules was investigated by superimposition. The molecules were superimposed on hypothetical receptor points that were assumed on the characteristic pharmacophores of centrally acting drugs. However, for the MK‐801 derivatives, the superimposition based on the steric overlapping volume and the electrostatic repulsion was dominant. The study confirmed the importance of the hydrocarbon portion of the PCP piperidine ring and suggested an opposite orientation of MK‐801 to the conventional 3‐point model.

MK801 induces immediate-early gene proteins and BDNF mRNA in rat cerebrocortical neurones.

Recent studies have shown that MK801, a potent phencyclidine receptor ligand, causes pathomorphological changes in rat cerebrocortical neurones. Here we report that doses of MK801 (1 and 5 mg kg-1) which have been shown to produce pathomorphological changes, induce the expression of immediate-early gene proteins (IEGPs) and brain-derived neurotrophic factor (BDNF) mRNA in rat cerebrocortical neurones. Blockade of central muscarinic receptors which has been shown to prevent MK801-induced pathomorphological changes in cerebrocortical neurones, also prevented MK801-induced expression of IEGPs and BDNF mRNA. The transiently increased expression of BDNF mRNA may be an acute compensatory response of these neurones to MK801-induced injury.

Characterization of [3H]desmethylimipramine binding in bovine adrenal medulla: interactions with sigma- and (or) phencyclidine-receptor ligands.

High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) sigma-receptor ligands, with the following order of potency: haloperidol > rimcazole > (-)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (-)-SKF-10047. The inhibition produced by sigma ligands was not attributed to stimulation of either sigma 1- or sigma 2-receptors, owing to inactivity of the selective sigma-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by sigma- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the sigma-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and sigma-ligands.

Dextromethorphan and phencyclidine receptor ligands: differential effects on K(+)- and NMDA-evoked increases in cytosolic free Ca2+ concentration.

The ability of dextromethorphan (DXM) and phencyclidine (PCP) receptor ligands to attenuate increases in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by N-methyl-D-aspartate (NMDA) and high extracellular [K+] was examined using the fluorescent dye Fura 2 in cultured rat hippocampal pyramidal neurons. The DXM receptor ligand caramiphen (40 microM) reduced K(+)-evoked rises in [Ca2+]i to a greater extent than NMDA-evoked rises; the reverse was true for the PCP receptor ligands ketamine (10-40 microM) and dextrorphan (10 microM). DXM itself, which has affinity for both DXM and PCP receptors, reduced both K(+)- and NMDA-evoked increases in [Ca2+]i in a concentration-dependent manner. The results suggest that DXM receptor ligands may at least in part exert their known anticonvulsant and neuroprotective effects by reducing Ca2+ influx through voltage-activated Ca2+ channels.

Binding affinity and antimuscarinic activity of σ and phencyclidine receptor ligands

The characterization of the σ receptor has been hampered by the lack of a functional bioassay system. Drugs that bind to σ receptors have been reported to inhibit carbachol-induced phosphatidylinositol turnover in rat brain; however, these drugs might directly affect muscarinic acetylcholine receptors. The purpose of the present study was to determine the affinity for muscarinic receptors and the antimuscarinic activity of σ and phencyclidine receptor ligands. All of the drugs tested inhibited the binding of [ 3H]N-methylscopolamine to guinea pig cerebral cortical membranes with K 1 values in the micromolar range and also inhibited carbachol-induced contractions in the guinea pig ileum. These results demonstrate that these compounds have substantial antimuscarinic activity which might limit the use of the inhibition of carbachol-induced phosphatidylinositol turnover as a functional assay system for studying σ ligands. Furthermore, this antimuscarinic activity must be considered when evaluating the effects of these compounds after in vivo administration.

Identification and characterization of a high-affinity glutamate-controlled TCP binding site in rat brain postsynaptic densities

The kinetic and equilibrium binding parameters of the phencyclidine receptor ligand [ 3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) to a postsynaptic density (PSD) subcellular fraction from rat brain were investigated. A single site was found, which was identified as the high-affinity TCP binding site by competition with dibenzocyloalkenimine (MK-801). In contrast, [ 3H]TCP binds to two sites on the plasma membrane fraction used as precursor for PSD; on both fractions, [ 3H]TCP binding responds to glutamate by an increase of the association rate, the dissociation constant and the number of sites being unchanged. In the PSD fraction [ 3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an antagonist specific for the N-methyl-D-aspartate (NMDA) site, bound to high- and low-affinity sites. These results ascertain the presence and identity of synaptic NMDA-gated ion channels, which are assumed in the current hypothesis about excitotoxicity, long-term potentiation and learning.

Non-competitive [formula omitted] antagonists are potent activators of ventral tegmental A 10 dopamine neurons

The response of ventral tegmental (VTA) A 10 dopamine neurons to a series of compounds covering the spectrum from high-affinity phencyclidine receptor ligands (MK-801, PCP) to high-affinity σ-receptor ligands ((+)-pentazocine, DTG) was measured using single-unit extracellular recording techniques in the rat. Dose-response comparisons revealed that MK-801 was 3, 6, 19 and 119 times more potent at activating A 10 neurons than PCP, (+)-SKF-10,047, ketamine and (+)-pentazocine, respectively. DTG (1,3-di- o-tolylguanidine), the most selective σ-ligand, and U50,488H, a κ-opiate, failed to produce any stimulation of firing. Also, pretreatment with haloperidol, a potent σ-receptor ligand, did not prevent MK-801-induced excitations. Thus, the activation of the A 10-mesolimbic-mesocortical dopamine pathways by PCP, PCP-like drugs and σ-psychotomimetics is mediated by the PCP receptor, not the haloperidol-sensitive σ-receptor, with potencies directly correlated to their activity as non-competitive N- methyl- d-aspartate (NMDA) antagonists.

Differential effects of σ and phencyclidine receptor ligands on learning

Several phencyclidine (PCP) and σ receptor ligands were examined for their effects on a single trial passive avoidance test in rats. Rats were administered the PCP receptor ligands (+)-5-methyl-10,11-dihydro-5Hdibenzo[1,d]cyclohepten-5,10-imine maleate (MK-801), PCP, ketamine or the σ receptor ligands (+)-N-allylnormetazocine ((+)-NANM), (+)-pentazocine, (+)-3-hydroxyphenyl)-N-n-propylpiperidine ((+)-3-PPP) or 1,3-Di(2-[5- 3H]tolyl)guanidine (DTG) subcutaneously prior to acquisition of the passive avoidance response, and tested 24 h later for retention. MK-801 (0.1–0.3 mg/kg), PCP (0.54–1.7 mg/kg), ketamine (10.0–17.2 mg/kg) and (+)-N-allylnormetazocine (5.4–10.0 mg/kg) produced significant memory deficits. (+)-Pentazocine (54 mg/kg) and (+)-PPP (30 mg/kg) also produced retention deficits, but at significantly higher doses. DTG (0.3–3.0 mg/kg s.c.) had no effect on retention. There was a positive correlation between production of retention deficits and the compounds' PCP receptor binding affinity. The results suggest that the σ receptor is not involved in learning the passive avoidance response.

Anticonvulsant actions of phencyclidine receptor ligands: Correlation with N-Methylaspartate Antagonism in vivo

1. 1. Drugs with phencyclidine (PCP)-like activity in behavioural discrimination and [ 3H]PCP binding studies share anticonvulsant properties. 2. 2. We have compared the rank order potency of a series of PCP-like compounds as N-methylaspartate (NMA) antagonists, determined from previously published studies from our laboratory, with their rank order anticonvulsant potencies as determined by two independent research groups in three different in vivo models of experimentally-induced epilepsy. 3. 3. Rank order potency for NMA antagonism correlated well with rank order anticonvulsant potency. Furthermore, the systemic doses required for an effective blockade of NMA-evoked excitations were, in most cases, similar to those which produced anticonvulsant activity. 4. 4. The results suggest that functional NMA antagonism may underlie the shared anticonvulsant properties of structurally dissimilar compounds with PCP-like activity.

MK-801 pretreatment enhances N-methyl- d-aspartate-mediated brain injury and increases brain N-methyl- d-aspartate recognition site binding in rats

Direct intracerebral administration of N-methyl- d-aspartate typically produces focal brain injury. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[ a,d]cyclohepten-5,10-immine maleate (MK-801), a non-competitive N-methyl- d-aspartate antagonist, can protect against N-methyl- d-aspartate-mediated brain injury when administered shortly before or after an intracerebral injection of N-methyl- d-aspartate. However, in this study we report that in perinatal rats if MK-801 (1 mg/kg) is administered intra-peritoneally 24 h prior to a unilateral intrastriatal N-methyl- d-aspartate injection, N-methyl- d-aspartate-mediated brain injury is paradoxically enhanced. The severity of resulting brain injury is 15–25% greater in groups that received MK-801 in comparison with saline-treated controls ( P < 0.001, linear regression analysis). In contrast, the severity of brain injury resulting from intrastriatal injection of the glutamate agonist quisqualate is not altered by a similar 24 h MK-801 pretreatment. Furthermore, the enhanced toxicity of N-methyl- d-aspartate produced by a 24 h pretreatment with MK-801 is completely blocked if a second dose of MK-801 is administered 15 min after the intrastriatal injection of N-methyl- d-aspartate. To determine if MK-801 produced alterations in glutamate receptor pharmacology co-incident with the enhanced toxicity of N-methyl- d-aspartate, in vitro quantitative autoradiography for excitory amino acid receptor subtypes was performed with [ 3H]glutamate and [ 3H] N-1-(2-thienyl)cyclohexyl-3,4-piperidine in seven-day-old rats killed 2 or 24 h after MK-801 (1 mg/kg) administration. A 2 h MK-801 pretreatment produced a 30–50% increase in [ 3H]glutamate binding at N-methyl- d-aspartate preferring recognition sites in all four brain regions examined (areas CA1 and CA3 of the hippocampus, corpus striatum, cingulate cortex) in comparison with saline-treated controls ( P <0.05, ANOVA). [ 3H] N-1-(2-Thienyl)cyclohexyl-3,4-piperidine binding to the phencyclidine site associated with the N-methyl- d-aspartate receptor was reduced by 60–80% in all brain regions examined ( P < 0.001). Quisqualate-sensitive [ 3H]glutamate binding was not altered by a 2 h MK-801 pretreatment. In animals that received a 24 h MK-801 pretreatment, N-methyl- d-aspartate-sensitive [ 3H]glutamate binding remained elevated (29% increase in CA1 and CA3, P <0.05), [ 3H] N-1-(2-thienyl)cyclohexyl-3,4-piperidine binding was still suppressed (40% reduction in CA1 and CA3, P < 0.05) and quisqualate-sensitive [ 3H]glutamate binding was reduced by 15–20% in area CA3 and in the corpus striatum ( P < 0.05, ANOVA). The results suggest that MK-801 induces rapid and persistent up-regulation of N-methyl- d-aspartate receptors and sensitizes the brain to N-methyl- d-aspartate-induced injury as the level of MK-801 in the brain declines. The observations provide new information about regulation of the N-methyl- d-aspartate receptor complex and may be relevant in the analysis of the neuroprotective properties of phencyclidine receptor ligands.

Interaction of [3H]MK-801 with multiple states of the N-methyl-D-aspartate receptor complex of rat brain.

N-Methyl-D-aspartate (N-Me-D-Asp) and phencyclidine receptors interactively mediate central nervous system processes including psychotomimetic effects of drugs as well as neurodegenerative, cognitive, and developmental events. To elucidate the mechanism of this interaction, effects of N-Me-D-Asp agonists and antagonists and of glycine-like agents upon binding of the radiolabeled phencyclidine receptor ligand [3H]MK-801 were determined in rat brain. Scatchard analysis revealed two discrete components of [3H]MK-801 binding after 4 hr of incubation. Incubation in the presence of L-glutamate led to an increase in apparent densities but not in affinities of both components of [3H]MK-801 binding as well as conversion of sites from apparent low to high affinity. Incubation in the presence of combined D-serine and L-glutamate led to an increase in the apparent density of high-affinity [3H]MK-801 binding compared with incubation in the presence of either L-glutamate or D-serine alone. These data support a model in which phencyclidine receptor ligands bind differentially to closed as well as open conformations of the N-Me-D-Asp receptor complex and in which glycine-like agents permit or factilitate agonist-induced conversion of N-Me-D-Asp receptors from closed to open conformations.

Differentiation of phencyclidine and sigma receptor types affecting the central inspiratory termination mechanism in cat

The effects of 1) the phencyclidine receptor ligand TCP, 2) sigma receptor ligands (+)3-PPP and DTG, and 3) N-methyl-D-aspartate receptor blockers MK-801 and dextrorphan were determined on a brainstem mechanism which controls the termination of the inspiratory phase of the breathing cycle. Inspiratory bursts were recorded from the phrenic nerve in decerebrate paralyzed cats ventilated by means of a phrenic driven servoventilator. The central mechanism which terminates inspiration was tested by witholding lung inflation, thus suppressing the contribution of the vagal feedback from the lungs to inspiratory termination. TCP increased the duration of test inspiration (tTi) by 17% at 0.03 mg/kg and by 14-fold (from 1.6 to 23 s) at 1 mg/kg. With dextrorphan, tTi was significantly increased at 3 mg/kg. In contrast, (+)3-PPP and DTG did not increase tTi at doses up to 10 mg/kg, although MK-801 (0.03 mg/kg), given after the sigma ligands, increased tTi by 59–90%. It is concluded that phencyclidine but not sigma receptor ligands block the central mechanism which terminates inspiration and that the likely site of action is the NMDA receptor complex.

Differences in results from in vivo and in vitro studies on the use-dependency of N-methylaspartate antagonism by MK-801 and other phencyclidine receptor ligands

We have used microelectrophoretic and intravenous administration of drugs to rat spinal cord neurones in vivo and bath application to rat cortical wedges in vitro to evaluate MK-801 and other phencyclidine (PCP) receptor ligands as N-methylaspartate (NMA) antagonists, paying particular regard to the possible use-dependent nature of their action. MK-801, 0.1–0.5 mg/kg, was a selective and long-lasting NMA antagonist. We were unable to demonstrate significant use-dependent onset of antagonism of NMA by any of the drugs in vivo. Recovery, however, for MK-801 was use-dependent. In vitro there was a gradation with MK-801 being very use-dependent, followed by (PCP), cyclazocine and ketamine, the last showing little or no use-dependence. Results of experiments modulating the in vitro environment suggest that a significant difference between the in vitro and in vivo systems was temperature. Raising the temperature of the wedge chamber from 23 to 33°C reduced the use-dependence of MK-801, and lowering the temperature to 13°C increased the use-dependence of PCP. The mechanism of action of PCP receptor ligands is discussed in the light of these results.

Quantitative autoradiographic localization of NMDA receptors in rat brain using [ 3H]CPP: comparison with [ 3H]TCP binding sites

The regional distribution of N-methyl-D-aspartate (NMDA) receptors in rat brain using the selective NMDA receptor ligand [ 3H]3-((±)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) has been quantitated by in vitro autoradiography. [ 3H]CCP binding was highest in the CA1 region of the hippocampus. Relatively high levels of binding were also observed in the cerebral cortex, while moderate binding was obtained in the thalamus, striatum and granule cell layer of the cerebellum. Concurrent studies examining the phencyclidine receptor ligand [ 3H]1-[1-(2-thienyl)cyclohexyl]-piperidine (TCP), revealed a similar pattern of binding that correlated well with the localization of [ 3H]CPP-labeled NMDA receptors (r = 0.88, P < 0.01).