dizocilpine-maleate and tenocyclidine

Prompted by our interest in neuroprotective agents with multiple mechanisms of action, we assessed the structure-activity relationship of a series of pentacycloundecylamine derivatives previously shown to have both L-type calcium channel blocking activity and N-methyl-d-aspartate receptor (NMDAR) antagonistic activity. We utilized a functional assay to measure NMDAR channel block using (45)Ca(2+) influx into synaptoneurosomes. The cage amine 8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6). 0(3,10).0(5,9)]undecane (NPG1-01) proved to be the most potent experimental compound with an IC(50) of 2.98microM, while 8-amino-pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane had the next most potent IC(50) of 4.06microM. Increasing the polycyclic cage size of NGP1-01 from a pentacycloundecane to a tridecane cage structure, but retaining the N-benzyl moiety decreased potency 10-fold, indicating a limitation on the volume of the cage that can be accommodated in the channel binding site. In the presence of NGP1-01, NMDA/glycine-induced maximal (45)Ca(2+) influx was attenuated by 34% with an insignificant effect on agonist potency. These results are consistent with uncompetitive antagonism for this group of compounds. Radioligand binding studies with [(3)H]MK-801 or [(3)H]TCP showed little or no displacement of these ligands by pentacycloundecylamines, suggesting that the latter compounds bind to a unique site in the NMDAR channel. The pentacycloundecylamines tested represent a novel group of NMDAR antagonists that have potential as therapeutic agents for neurodegenerative diseases including Parkinson's and Alzheimer's disease.

Topics: Amines; Animals; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Ion Channels; Male; Mice; Mice, Inbred ICR; Models, Molecular; Phencyclidine; Piperidines; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship; Synaptosomes; Thiophenes

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity (KD) of [3H]MK-801 for its binding site was 5.5 nM and the binding site density (Bmax) was 1.83 pmol/mg protein. Inhibition of [3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were -1. A series of eight non-competitive NMDA antagonists inhibited [3H]MK-801 binding with the following rank order of affinity (K(i), nM): MK-801 (5.5) > dexoxadrol (21.5) > or = TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated (P = 0.0002; r2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274-281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.

Topics: Animals; Binding, Competitive; Brain; Dextromethorphan; Dioxolanes; Dizocilpine Maleate; Dose-Response Relationship, Drug; Female; Ketamine; N-Methylaspartate; Phenazocine; Phencyclidine; Piperidines; Rats; Rats, Mutant Strains; Receptors, N-Methyl-D-Aspartate; Scintillation Counting; Tritium

Congeners of the potent dopamine (DA) re-uptake inhibitor 1-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine (BTCP) are unexpectedly able to bind in the rat cerebellum, although this structure is devoid of dopaminergic nerve endings. In line with previous studies the hypothesis that they bind to low affinity PCP sites labelled with [3H]TCP in the rat cerebellum, even though they do not bind to the high affinity PCP sites in the forebrain, was considered. Analogues of 1-[1-(2-thiophenyl)cyclohexyl]piperidine (TCP) and BTCP with a modified aromatic moiety and with O or S atoms substituted in the cyclohexyl ring were prepared and tested in competition experiments both in rat forebrain and cerebellum membranes labelled with [3H]TCP, and in rat striatum membranes labelled with [3H]BTCP. Results indicated that BTCP and congeners could bind to low affinity PCP sites labelled with [3H]TCP in the rat cerebellum with a decrease of the selectivity for the DA transporter. On the contrary, some TCP analogues displayed a very high selectivity for these low affinity sites; they might be important pharmacological tools to elucidate the nature and function at yet unknown of these sites.

Topics: Animals; Binding, Competitive; Cerebellum; Dizocilpine Maleate; Dopamine Agonists; Excitatory Amino Acid Antagonists; Kinetics; Male; Membranes; Neostriatum; Phencyclidine; Prosencephalon; Rats; Rats, Wistar; Receptors, Phencyclidine; Structure-Activity Relationship

Although the alkaloid ibogaine is a potent hallucinogenic agent some indications suggest that it may be useful for the treatment of opioid and cocaine addiction. The neurochemical mechanism(s) underlying ibogaine effects remain unclear. In the present study we investigated the interaction of ibogaine with the phencyclidine (PCP) site located in the ionophore of the N-methyl-D-aspartate (NMDA) receptor complex, with the NMDA receptor binding site, and with sigma binding sites. In well-washed membrane preparations of rat cortex and cerebellum, the PCP sites were labeled with [3H]MK-801 or [3H]1-[1(2-theinyl)-cyclohexyl]-piperidine ([3H]TCP), and the NMDA receptor with [3H]-CGP 39653. The sigma-1 and sigma-2 binding site in rat cortex and cerebellum were labeled with [3H]pentazocine and [3H]1,3-di-o-tolyl-guanidine ([3H]DTG), respectively. Results indicated that ibogaine interacts with high- and low-affinity PCP binding sites in the cortex: Ki(H) = 0.01-0.05 microM; Ki(L) = 2-4 microM, and only with low-affinity sites in the cerebellum: Ki = 2-4, microM. In contrast, ibogaine (> 100 microM) had no affinity for [3H]-CGP 39653 binding sites (cortex and cerebellum). The affinity of ibogaine for sigma-1 and -2 binding sites in cortex and cerebellum ranged from 1.5-3 microM. Since NMDA receptor antagonists (e.g., MK-801) are thought to attenuate opioid withdrawal symptoms and cocaine sensitization, it is possible that binding of ibogaine to the PCP sites contributes to its potential 'endabuse' properties. In turn, ibogaine interaction with sigma binding sites may be associated with its adverse effects.

Topics: Animals; Binding Sites; Brain; Dizocilpine Maleate; Ibogaine; Male; Phencyclidine; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Receptors, sigma

1. Electrically induced contractions of the epididymal portion of rat vas deferens were potentiated in concentration-dependent manner (0.1-30 microM) by different sigma and PCP receptor ligands (PCP, TCP, (+)-MK-801, dextromethorphan and (+)-3-PPP); dextrorphan did it in a minor extent. 2. Sigma and PCP receptor ligands also potentiated the effect of noradrenaline, inducing a reduction of the noradrenaline EC50 value in the rat vas deferens. The rank order of potencies was: PCP > TCP > (+)-3-PPP > (+)-MK-801 > dextrorphan > > > dextrometorphan. 3. In contrast, haloperidol (1 microM), a sigma receptor ligand, inhibited both the neurogenic and noradrenaline-induced responses in this tissue. 4. The effect of PCP and sigma receptor ligands on noradrenaline uptake was evaluated. All compounds tested, including haloperidol, inhibited the tritiated noradrenaline incorporation to the tissue. IC50 values were in the micromolar range, between 1.09 microM for dextrophan and 18 microM for dextrometorphan. 5. It is concluded that a direct interaction with the noradrenaline uptake system is involved in the potentiating effect of some sigma and PCP receptor ligands in the epididymal portion of rat vas deferens.

Topics: Adrenergic alpha-Agonists; Animals; Dextromethorphan; Dizocilpine Maleate; Dopamine Agonists; Electric Stimulation; Excitatory Amino Acid Antagonists; Ligands; Male; Muscle Contraction; Neuroprotective Agents; Norepinephrine; Phencyclidine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Phencyclidine; Receptors, sigma; Tritium; Vas Deferens

Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O2 and 95% N2 for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [3H]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK-801), on against in vivo hypoxia. The administration of MK-801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR.

Topics: Animals; Dentate Gyrus; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Hypoxia, Brain; Immunohistochemistry; Male; Neurons; Phencyclidine; Rats; Rats, Inbred F344; Receptors, N-Methyl-D-Aspartate; Tritium

Regulation of both the outgrowth and the survival of neurons involved in cognitive function can have a significant impact on the function of neural networks involved in memory and other cognitive processes. Results of this investigation demonstrated that 17beta-estradiol and the estrogenic steroids estrone, estriol, mestranol, and equilin induced significant increases in cortical nerve cell growth. Of the neurotrophic estrogenic steroids, equilin was most efficacious. We therefore conducted an extensive analysis of equilin-induced neurotrophism. Equilin induced highly significant increases in the growth of both the macro and micro features of cortical nerve cell morphology. The growth-promoting effects of equilin were present in both serum-containing and serum-free media, indicating that the growth-promoting effect of equilin is direct and not dependent upon factors present in serum. Analysis of the regional selectivity of equilin-induced neurotrophism in the cerebral cortex demonstrated that equilin significantly increased the growth of neurons from the frontal, temporal, and occipital regions, with neurons from the parietal region also influenced, though more modestly. We pursued the mechanism of equilin-induced neurotrophism and found that the growth-promoting effects of equilin were completely abolished in the presence of the glutamatergic NMDAreceptor antagonist AP5. Equilin is a major component of Premarin, the leading prescribed pharmaceutical for estrogen replacement therapy for postmenopausal women in the United States. Results of this investigation have the potential of influencing the application and design of therapeutic agents for the prevention of cognitive decline in estrogen deficient women and for the prevention of Alzheimer's disease in postmenopausal women, a group that comprises a large sector of the population, the size of which will continue to grow in the coming decades.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cells, Cultured; Cerebral Cortex; Cognition; Culture Media, Serum-Free; Dizocilpine Maleate; Equilin; Estradiol; Estrogen Replacement Therapy; Estrogens; Estrogens, Conjugated (USP); Excitatory Amino Acid Antagonists; Growth Substances; Microscopy, Video; Neurons; Phencyclidine; Rats; Receptors, N-Methyl-D-Aspartate

N-Methyl-D-aspartate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol-insensitive, but dizocilpine (MK-801)-sensitive, binding of N-[1-(2-[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration-dependent decrease in the maximal activation of [3H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by L-Glu when L-Glu was present at concentrations < 100 microM. However, ethanol (100 mM) inhibited completely the activation of [3H]TCP binding produced by high concentrations of L-Glu (200-400 microM). It also inhibited strongly the activation of [3H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has L-Glu-, Gly-, and [3H]TCP-binding sites, ethanol (100 mM) decreased significantly the maximal activation of [3H]TCP binding produced by either L-Glu or Gly. Activation constants (Kact) for L-Glu and Gly acting on the purified complex were 12 and 28 microM, respectively. Ethanol had no significant effect on the Kact of L-Glu but caused an increase in Kact of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both L-Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.

Topics: Animals; Brain Chemistry; Dizocilpine Maleate; Dopamine Antagonists; Ethanol; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Haloperidol; Illicit Drugs; Kinetics; Male; Membrane Proteins; N-Methylaspartate; Phencyclidine; Protein Binding; Proteins; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sensitivity and Specificity; Spermidine; Synaptic Membranes; Tritium

Various thienylcyclohexyl piperidine (TCP) derivatives were synthesized and evaluated as potential candidates for use as radiotracers for the in vivo study of the NMDA receptor ion-channel. Modification of the thienylcyclohexylpiperidine was accomplished by substituting its piperidine ring either with other cycloamine rings or N-alkyl-substituted amines, and these two classes of TCP derivatives were synthesized using two different methods. The compounds exhibited affinities ranging from 65 nM up to micromolar in competition assays for the receptor ion-channel labeled with 3H-(+)-MK-801 in rat brain homogenates. Radiosynthesis of 1-[1-(2-thienyl)-4-([18F]fluoro)-cyclohexyl]-1,2,5, 6-tetrahydropyridine was carried out by nucleophilic substitution reaction of 1-[1-(2-thienyl)-4-tosyloxycyclohexyl]-1,2,5,6-tetrahydropyridine with no carrier added 18F-, and the yield was approximately 5-10% (decay corrected) in specific activities of 500-1000 Ci/mmol after reverse-phase HPLC purification. The tracer showed good uptake in rat brains after i.v. injection (approx. 0.10% injected dose/g at 30 min. p.i.). However, the specific uptake in receptor-rich regions (striata, hippocampus, frontal cortex, and parietal cortex) improved only marginally with time compared to cerebellum. Three hours postinjection, parietal cortex showed a maximum ratio of 1.9. Preliminary PET experiment with this radiotracer in a rhesus monkey showed good uptake in the brain regions. However, little retention of the radiotracer was observed in the receptor-rich regions.

Topics: Animals; Brain; Calcium Channels; Dizocilpine Maleate; Fluorine Radioisotopes; Indicators and Reagents; Isotope Labeling; Macaca mulatta; Magnetic Resonance Spectroscopy; Male; Molecular Structure; Phencyclidine; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship; Tissue Distribution; Tomography, Emission-Computed

Metapramine, a pharmacological compound with antidepressant activity in humans, was tested for possible antiglutamatergic activity, in vitro. We investigated the effects of metapramine on the N-methyl-D-aspartic acid (NMDA) receptor complex, by determining whether this compound would interfere with the binding of [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes in the presence of either glycine NMDA, or both. Metapramine in the micromolar range inhibited the binding of [3H]TCP in the presence of both NMDA and glycine (IC50 = 1.4 +/- 0.2 microM). That very similar affinities were observed when either NMDA or glycine was present suggests that metapramine exerted a direct action at the PCP site. The affinity of metapramine for this site was about 25 and 350 times lower than that of PCP and MK-801, respectively. Metapramine inhibited the NMDA-evoked increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC50 = 13 microM). These results suggest that metapramine is a low-affinity antagonist of the NMDA receptor complex channel. This paper discusses the potential application of metapramine to the treatment of diseases linked to excessive stimulation of glutamatergic NMDA receptors.

Topics: Animals; Antidepressive Agents, Tricyclic; Binding Sites; Binding, Competitive; Cell Membrane; Cerebral Cortex; Cyclic GMP; Dibenzazepines; Dizocilpine Maleate; Glycine; In Vitro Techniques; Phencyclidine; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate

To investigate the modulatory effects of sigma ligands on the N-methyl-D-aspartate (NMDA) receptor-ion channel complex in vivo, we examined the intact cell binding of 3H-N-[1-(2-thienyl)cyclohexyl]piperidine (3H-TCP) to cultured neuronal cells prepared from fetal rat telencephalon. The 3H-TCP binding was saturable, reversible, and inhibited by a selective NMDA receptor antagonist, D-amino-5-phosphonovaleric acid. MII-limolar Mg2+ inhibited 3H-TCP binding both in the absence and presence of L-glutamate. 5-Methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate (MK801) inhibited 3H-TCP intact cell binding in a competitive manner, while haloperidol inhibited it in a noncompetitive manner. The effect of the test drugs to inhibit 3H-TCP intact cell binding was in the order of dextromethorphan, haloperidol > (+/-)MK 801 > (+)pentazocine > (-)pentazocine > DTG > PCP > (+)-N-allylnormetazocine [(+)SKF 10047] > (+)3-(3-hydroxyphenyl)-N- (1-propyl)piperidine [(+)3-PPP] > (-)SKF 10047 > (-)3-PPP. The IC50 values of the six sigma ligands for 3H-TCP binding were closely correlated with the Ki values of the corresponding drugs for DTG site 1 in the guinea pig brain reported by Rothman et al. (1991). These findings suggest that the sigma ligand indirectly modulates the NMDA receptor ion channel complex, presumably through sigma 1 sites in vivo as well as in vitro.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cells, Cultured; Dizocilpine Maleate; Glutamic Acid; Ion Channels; Ligands; Neurons; Phencyclidine; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, sigma

Quantitative receptor autoradiography was used to examine the regional binding characteristics of a diverse group of N-methyl-D-aspartate (NMDA)-receptor channel blockers that varied in potency 10(5)-fold. Full competition curves were generated in each of six brain regions for 11 different compounds. MK-801 was the most potent compound studied, with an IC50 of approximately 10 nM in the forebrain regions and 24 nM in the cerebellar granule cell layer (p < 0.05). The binding affinities of nine of the 11 compounds examined were significantly different in cerebellar granule cell layer than in forebrain regions. In addition, the apparent Hill slopes of five of the compounds were significantly different in cerebellum compared with forebrain. That the rank order of drug potencies in cerebellar diverges from that in forebrain suggests that cerebellar NMDA-receptor ion channels differ pharmacologically from those in forebrain. There was a general trend that drugs known to be well tolerated in humans (remacemide hydrochloride and its metabolites, amantadine, budipine, and memantine) had lower affinities than compounds with severe neurobehavioral or psychotomimetic effects. Moreover, all of the compounds known to be well tolerated in humans had a significantly higher affinity in the cerebellum than in forebrain regions, in contrast to MK-801, 1-[1-(2-thienyl)cyclohexyl]-piperidine hydrochloride (TCP), phencyclidine (PCP), and ketamine, which had lower affinities in cerebellum. Our results are consistent with the notion that low affinity (rapid kinetics) and, possibly, subunit specificity (as indicated by distinct regional pharmacologies) may be important determinants of the clinical tolerability of NMDA-receptor channel blockers.

Topics: Acetamides; Animals; Antiparkinson Agents; Autoradiography; Binding, Competitive; Brain; Dizocilpine Maleate; Ion Channels; Phencyclidine; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Tissue Distribution

Behavioral and in vitro receptor binding methods were used to evaluate and compare the effects of FR115427 ((+)-l-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride) with those of MK801, a non-competitive NMDA antagonist. FR115427 inhibited NMDA-induced convulsions in mice by intracerebroventrical(ICV) and systematic injection. FR115427 was found to be about ten times less potent than MK801. Furthermore, the inhibitory effect of FR115427 and MK801 on NMDA-induced convulsions was evaluated in time course studies in mice. MK801 exhibited a more sustained anticonvulsive activity than FR115427. In addition, PCP-like behaviors were examined in mice after ICV injection of these compounds. At the lowest dose FR115427 significantly increased locomotor activity, although the effect of this compound was about hundred times less potent than that of MK801. At higher dose a more complex pattern of behavior, e.g. head-movement and eventually ataxia was observed. In binding assays with rat brain membranes, FR115427 inhibited the binding of (3H)TCP (IC50 = 0.249 microM) and (3H)MK801 (IC50 = 0.312 microM) but did not inhibit the binding of (3H)CPP or (3H)glycine. These results suggest that FR115427 is a novel non-competitive NMDA antagonist that acts on a binding site located within the NMDA receptor associated ion channel.

Topics: Animals; Anticonvulsants; Behavior, Animal; Brain; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; In Vitro Techniques; Isoquinolines; Male; Membranes; Mice; Mice, Inbred ICR; Mice, Inbred Strains; Motor Activity; Phencyclidine; Piperazines; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Tetrahydroisoquinolines

NMDA channel blockers are potentially advantageous therapeutic agents for the treatment of ischemia and head trauma, which greatly elevate extracellular glutamate, because they should most effectively inhibit high levels of receptor activation. A novel high affinity TCP site ligand, WIN 63480, does not produce MK-801- or PCP-like behavioral activation at anti-ischemic doses. While WIN 63480, MK-801 and PCP were all observed to be effective blockers of open NMDA channels, WIN 63480 had much less access to closed NMDA channels. This difference may be due to the fact that WIN 63480 is hydrophilic (logD = -4.1) while MK-801 and PCP are lipophilic (logD = +1.8). In vivo, closed channel access may result in a non-competitive profile of antagonism for MK-801 and PCP compared to a more uncompetitive profile for WIN 63480. Release of glutamate, and depolarization, are likely to produce a high level of NMDA receptor activation in ischemic areas compared to normal tissue. Consequently, at anti-ischemic doses, WIN 63480 may produce less inhibition of physiological NMDA-mediated processes in neural systems involved in behavioral regulation than MK-801 or PCP, leading to an improved side effect profile.

Topics: Animals; Binding Sites; Cells, Cultured; Cerebral Cortex; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Kynurenic Acid; Membrane Potentials; Mice; Phencyclidine; Quinolizines; Receptors, N-Methyl-D-Aspartate; Transfection; Xenopus laevis

A series of novel N-methyl-D-aspartate antagonists acting at the phencyclidine site has been identified. Compound 2 has a Ki = 8 +/- 1 nM (vs [3H]thienylcyclidine, [3H]TCP) as a mixture of enantiomers. Resolution and further testing indicate that (-)-2, Ki = 4 +/- 0.7 nM, is a potent and selective TCP site ligand with neuroprotective activity in cultured neurons in the presence of excitotoxic concentrations of NMDA (IC50 = 26 nM). Compound (-)-2 is > 1000-fold selective for the TCP site vs a panel of receptor types including opiate, adrenergic, serotonergic, dopamine, adenosine, dihydropyridine, and benzodiazepine and displays increased selectivity for the activated (open) NMDA receptor-ion channel complex vs PCP and MK801 as measured by patch recordings in cultured, voltage-clamped neurons. Highly enhanced "open-channel" selectivity leads to tentative classification of these ligands as uncompetitive vs NMDA. Ligands with these characteristics may enable deconvolution of the pharmacologic effects associated with typical noncompetitive NMDA antagonists. We report here on the identification, synthesis, and activity of compounds of this structural class.

Topics: Animals; Binding Sites; Ion Channels; Male; Mice; N-Methylaspartate; Neuroprotective Agents; Phencyclidine; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship

Ibogaine is a putative anti-addictive drug with potential efficacy for the treatment of opiate, stimulant, and alcohol abuse. We now report ibogaine is a competitive inhibitor (Ki, 1.01 +/- 0.1 microM) of [3H]MK-801 binding to N-methyl-D-aspartate (NMDA) receptor coupled cation channels. Since MK-801 can attenuate the development of tolerance to morphine and alcohol as well as sensitization to stimulants in preclinical studies, the reported ability of ibogaine to modify drug-seeking behavior in man may be attributable to a blockade of NMDA receptor coupled cation channels.

Topics: Animals; Binding, Competitive; Dizocilpine Maleate; Ibogaine; In Vitro Techniques; Ion Channels; Ligands; Phencyclidine; Rats; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

Chronic treatment of adult rats with DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic carboxyethylester (CGP 39551) (30 mg/kg orally for 12 days) induced a significant increase, 72 h after the last dose, in the N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding in the hippocampal pyramidal layer (stratum oriens CA1, CA3: +51% on average; stratum radiatum CA1, CA3: +40% on average; stratum pyramidale CA1: +20%, CA3: +55%) and in the dentate gyrus (+43%) compared to vehicle-injected animals, as assessed by quantitative receptor autoradiography. Similar results were obtained using the NMDA receptor antagonist, [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653). Saturation experiments showed that the increase in [3H]CGP 39653 binding was due to the maximum number of receptors, without changes in affinity. The same regimen did not alter [3H]N-(1-[2-thienyl]-cyclohexyl)-3,4-piperidine (TCP) binding to the ion channel coupled to the receptor but prevented D-serine (5 microM)-induced enhancement of [3H]glutamate binding. NMDA (3-300 microM) enhanced [3H]noradrenaline release from hippocampal slices, and 7-Cl-kynurenic acid (5-100 microM) and (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclo-hepten-5,10-imine maleate (MK 801) (0.03-0.3 microM), antagonists at the glycine site and ion channel respectively, antagonized this effect to the same extent in CGP 39551-treated rats and controls. Chronic CGP 39551 did not affect the neurotoxic potency of quinolinic acid, a selective agonist at the NMDA receptor, injected in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; Animals; Autoradiography; Dizocilpine Maleate; Glutamic Acid; Hippocampus; Kynurenic Acid; Male; Norepinephrine; Phencyclidine; Quinolinic Acid; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

Binding of 1-[1-(2-[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or sigma recognition sites. In both tissues, [3H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [3H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [3H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [3H]TCP sites may be associated with sigma binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 nM [3H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [3H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [3H]TCP-labeled binding sites in the spinal cord may be associated with sigma but not the NMDA receptor-coupled PCP sites.

Topics: Animals; Binding Sites; Brain; Brain Chemistry; Dizocilpine Maleate; Glutamic Acid; Glycine; Haloperidol; Male; Mice; N-Methylaspartate; Phencyclidine; Spinal Cord; Tritium

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycl ohepten-5,10 - imine hydrochloride [(+)-MK801-NCS] was characterized for its ability to acrylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). The Kd values of [3H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18-24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [3H]TCP binding to site 2, but not to site 1. Although (+)-MK801-NCS decreased [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; ccyclohepten-5,10-imine maleate ([3H](+)-MK801) binding to site 1, it had no effect on [3H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [3H]TCP and [3H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.

Topics: Affinity Labels; Animals; Brain; Cell Membrane; Cerebral Ventricles; Dizocilpine Maleate; Illicit Drugs; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Phencyclidine; Receptors, Phencyclidine

[3H]TCP, an analog of the dissociative anesthetic phencyclidine (PCP), binds with high affinity to two sites in guinea pig brain membranes, one that is MK-801 sensitive and one that is not. The MK-801-sensitive site (PCP site 1) is associated with NMDA receptors, whereas the MK-801-insensitive site (PCP site 2) may be associated with biogenic amine transporters (BAT). Although several "BAT ligands" are known that bind selectively to PCP site 2 and not to PCP site 1 (such as indatraline), these compounds have low affinity for site 2 (Ki values > 1 microM). Here we demonstrate that the novel pyrrole RTI-4793-14 is a selective, high affinity ligand for PCP site 2. We determined the IC50 values of RTI-4793-14 and several reference compounds [PCP, (+)-MK801 and indatraline] for PCP site 1 (assayed with [3H](+)-MK801), PCP site 2 (assayed with [3H]TCP in the presence of 500 nM (+)-MK801) and a variety of BAT-related measures ([3H]CFT binding to the DA transporter, [3H]nisoxetine binding to the norepinephrine transporter, [3H]dopamine uptake, [3H]serotonin uptake). In addition, we determined the ability of RTI-4793-14 to block NMDA responses in cultured hippocampal neurons under voltage clamp. (+)-MK801 had high affinity for PCP site 1 (4.6 nM) and potently inhibited NMDA-induced responses, but was much less potent in the BAT-related measures (IC50 s > 10 microM). PCP had high affinity at PCP site 1 (IC50 = 92 nM) and PCP site 2 (IC50 = 117 nM), and was moderately potent in all BAT-related measures except [3H]nisoxetine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biogenic Monoamines; Brain; Dizocilpine Maleate; Electrophysiology; Guinea Pigs; In Vitro Techniques; Indenes; Ligands; Male; Microdialysis; Phencyclidine; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Phencyclidine; Synaptosomes

Human immunodeficiency virus (HIV-1) infection often results in central nervous system (CNS) dysfunction, yet the mechanism(s) of action for HIV-1 in the CNS are not fully understood. In the present study gp120, the HIV-1 envelope glycoprotein, was shown to selectively inhibit N-methyl-D-aspartate (NMDA) receptor function. In addition to inhibiting radioligand binding to rat NMDA receptors, gp120 inhibited NMDA-induced currents in Xenopus oocytes, attenuated NMDA-stimulated calcium flux and cytotoxicity in cultured cerebellar granule cells, and provided partial protection against NMDA-induced lethality in vivo. These findings suggest that NMDA receptor complex is a possible site of action of HIV-1 within the CNS.

Topics: Animals; Calcium Channel Blockers; Cells, Cultured; Dizocilpine Maleate; Electrophysiology; HIV Envelope Protein gp120; N-Methylaspartate; Oocytes; Phencyclidine; Pipecolic Acids; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Xenopus

Glutamate is the major excitatory neurotransmitter of the mammalian central nervous system. Two major classes of glutamate receptors have been reported. The actions of glutamate on its N-methyl-D-aspartate (NMDA)-type receptor may underlie developmental and adult plasticity as well as neurotoxicity. The NMDA-type of glutamate receptor in cat and monkey visual cortex was visualized by means of in vitro receptor autoradiography with the noncompetitive NMDA-receptor antagonist [3H]-MK-801. The kinetics, performed on tissue sections, revealed an apparently single, saturable site with an approximate dissociation constant (KD) of 18.5 nM in cat and 15.9 nM in monkey visual cortex. Autoradiography, performed on frontal sections of cat and monkey visual cortex, revealed a heterogeneous laminar distribution of NMDA receptors. Cat areas 17, 18, 19, and the lateral suprasylvian areas exhibited a similar NMDA-receptor distribution. In these areas, NMDA receptors were most prominent in layer II and the upper part of layer III. In monkey striate cortex, NMDA receptors were primarily concentrated in layers II, upper III, IVc, V, and VI. In monkey secondary visual cortex, [3H]-MK-801 labeling was most prominent in layers II, V, and VI; whereas in the temporal visual areas included in this study layer II displayed the heaviest receptor labeling. In neither cat nor monkey could we observe significant differences in NMDA-receptor distribution between different retinotopic subdivisions within a single visual area. Neither did we detect any periodic changes in NMDA-receptor distribution that would correspond to the compartments defined by cytochrome-oxidase in monkey V1 and V2.

Topics: Animals; Autoradiography; Binding, Competitive; Cats; Dizocilpine Maleate; Female; Freeze Drying; Glutamates; Glutamic Acid; Macaca fascicularis; Male; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Vision, Ocular; Visual Cortex

The addition of phosphatidylserine induced a significant interaction with radioligands widely used for labeling an ion channel associated with an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, such as [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) and [3H]N-[1-(2-thienyl)cyclohexyl]piperidine, in the absence of brain synaptic membranes irrespective of the addition of either L-glutamic acid or glycine when determined in the presence of the polyamine spermidine. The interaction with [3H]MK-801 increased in proportion to increasing concentrations of phosphatidylserine added at concentrations from 0.01 to 0.5 mg/ml. Similar interaction with [3H]MK-801 occurred for phosphatidylinositol to a lesser extent than for phosphatidylserine but not for either phosphatidylethanolamine or phosphatidylcholine at the similar concentration range. The interaction between phosphatidylserine and [3H]MK-801 was dependent partially on incubation temperature and on incubation time with reversible and saturable profiles. Among various natural and synthetic polyamines, both spermine and bis-(3-aminopropyl)amine in addition to spermidine were effective in markedly potentiating the interaction in a concentration-dependent manner at concentrations from 1 microM to 10 mM. However, the potentiation by spermidine was insensitive to antagonism by the respective antagonists for the NMDA and glycine domains at 0.1 mM. Moreover, the interaction between phosphatidylserine and [3H]MK-801 was sensitive to inhibition by several compounds with an antagonistic activity at calmodulin. The addition of phosphatidylserine completely concealed the potentiation by either glutamate alone or both glutamate and glycine of the binding of [3H]MK-801 in brain synaptic membranes with concomitant enhancement of the ability of spermidine to potentiate the binding, while phosphatidylserine failed to affect the affinities of the respective ligands for the NMDA and glycine domains. These results suggest that the acidic phospholipid phosphatidylserine may at least in part play crucial roles in mechanisms underlying the potentiation by polyamines of the binding of [3H]MK-801 to the open NMDA channel in rat brain.

Topics: Animals; Brain; Calmodulin; Dizocilpine Maleate; Glutamates; Glutamic Acid; Glycine; Illicit Drugs; Kinetics; Phencyclidine; Phosphatidylserines; Phospholipids; Polyamines; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship; Synaptic Membranes; Tritium

1. Homogenates of human infant and adult temporal cortex were used to measure [3H]-TCP and [3H]-MK-801 binding to the N-methyl-D-aspartate (NMDA)-coupled ion channel phencyclidine site. 2. Both [3H]-TCP and [3H]-MK-801 binding increased in infant cortex by > 100% between term and 26 weeks suggesting that the numbers of NMDA receptors increase during postnatal brain development. 3. [3H]-MK-801 binding was measured under non-equilibrium conditions in temporal cortex homogenates with the addition of 100 microM of L-glutamate plus a range of concentrations (0.05 microM-100 microM) of glycine. Glutamate and glycine increased [3H]-MK-801 binding by stimulating NMDA receptors and improving [3H]-MK-801 access to ion channel binding sites; maximum stimulation in adult and infant temporal cortex was achieved with 100 microM glutamate plus 5 microM glycine; a higher concentration of glycine (50 microM) reduced [3H]-MK-801 binding to below maximum. 4. The stimulation by 100 microM glutamate plus 5 microM glycine of [3H]-MK-801 binding in infant temporal cortex was affected by postnatal age. For example, although the stimulation of [3H]-MK-801 binding in 5-6 week infant cortex (236% of basal) was similar to adult cortex (230% of basal), in samples taken from infants aged 5-6 months glycine (plus glutamate) stimulation of [3H]-MK-801 binding (392% of basal) was substantially greater than that measured in adult temporal cortex. 5. The binding of [3H]-glycine to the glycine modulatory site associated with the NMDA receptor in infant cortex also increased with postnatal age by > 100% between term and 26 weeks. 6. It is concluded that NMDA receptors in infant cortex increase to levels greater than those in adult cortex during postnatal development. The results do not exclude the possibility that the transiently increased NMDA receptor-ion channel complex in infant cortex shows enhanced responses to agonists and modulators.

Topics: Adult; Aging; Cerebral Cortex; Dizocilpine Maleate; Female; Glutamates; Glutamic Acid; Glycine; Humans; In Vitro Techniques; Infant; Infant, Newborn; Male; Phencyclidine; Proteins; Receptors, N-Methyl-D-Aspartate; Stimulation, Chemical; Sudden Infant Death; Temporal Lobe

The non-psychotropic cannabinoid (+)-(3S,4S)-7-hydroxy-delta 6- tetrahydrocannabinol 1,1-dimethylheptyl (HU-211), a stereoselective inhibitor of the N-methyl-D-aspartate (NMDA) receptor, protects primary cultures of rat forebrain against NMDA receptor-mediated neurotoxicity. Cell mortality produced by exposure for 10 min to NMDA or glutamate was reduced to approximately 18 or 27%, respectively, by application of 50 microM HU-211 for 10-15 min during or after exposure of cultures to excitatory amino acid. This effect of HU-211 was dependent on its concentration (EC50 = 8.7 +/- 4 microM). HU-211 also reduced the toxicity induced by brief exposure (10 min) to kinase or quisqualate, though less effectively. HU-211 may therefore prove useful as a non-psychoactive drug that protects against neurotoxicity mediated by the NMDA receptor.

Topics: Animals; Calcium Channel Blockers; Cell Death; Cells, Cultured; Dizocilpine Maleate; Dronabinol; L-Lactate Dehydrogenase; Neurons; Phencyclidine; Prosencephalon; Rats; Receptors, N-Methyl-D-Aspartate

In rats trained to discriminate 0.04 mg/kg fentanyl from saline, phencyclidine (PCP) and the PCP-type drugs ketamine and (+/- )-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine produced effects that are usually referred to as partial generalization. Partial generalization could conceivably result from low efficacy actions at the receptor mediating the discriminative stimulus effects of the training drug. The PCP-type drugs produced maximum percentages of drug lever (DL) selection intermediate between those produced by the training conditions, but their curves relating dose to percentage of DL selection were not shallower than that of fentanyl. The PCP-type drugs decreased DL selection produced by the training dose of fentanyl, but there was no relationship between these antagonist effects and the DL selection produced by the PCP-type drugs when given alone. Naltrexone antagonized DL selection produced by fentanyl, but not that produced by the PCP-type drugs. The potency order of the PCP-type drugs to produce DL selection was in agreement with their relative affinities for PCP receptors, but not with those for morphine receptors. The intermediate levels of DL selection produced by the PCP-type drugs were associated with increased lever selection latencies and increased responding on the nonselected lever; this pattern of effects resembled the behavior of animals that had not yet acquired the discrimination. The results suggest that PCP-type drugs produce intermediate levels of drug-appropriate responding in fentanyl-trained rats through mechanisms involving not opioid receptors and partial generalization, but involving PCP receptors and performance deficits conceivably resulting from state dependency. Thus, the results stress the importance of a pharmacological and behavioral analysis of intermediate responding in drug discrimination to examine its validity as a measure of efficacy and of stimulus similarity.

Topics: Animals; Conditioning, Operant; Discrimination Learning; Dizocilpine Maleate; Fentanyl; Ketamine; Male; Phencyclidine; Rats; Rats, Wistar; Reinforcement, Psychology

Extracellular single-unit recordings were used to assess the effects of PCP and PCP-like drugs (MK-801 and TCP) on the burst firing of ventral tegmental A10 dopamine neurons in the rat. The effects of these noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists were compared to the potent and competitive NMDA antagonists CGS 19755 and (+/-)CPP, and to BTCP, a PCP-derivative possessing little affinity for the PCP binding site within the ion channel gated by NMDA. PCP, MK-801, and TCP produced dose-dependent increases in the firing rate, which were accompanied by increases in the amount of burst activity, the number of action potentials within a burst, and the conversion of nonbursty cells to bursty. However, the coefficient of variation, a measure of the regularity of firing, was not significantly altered. These predominately excitatory effects contrast with the inhibition of firing, decrease in bursting, and regularization of pattern produced by BTCP. CGS 197555 and (+/-)CPP failed to alter any of the measured parameters. Thus, the increase in firing rate and amount of burst activity of dopamine neurons produced by PCP and PCP-like drugs, and the resultant hyperdopaminergia within the mesolimbic-mesocortical regions, could underlie the psychotomimetic properties of these compounds. Moreover, this effect would not appear to be related to a loss of activity at the NMDA recognition site, as evidenced by the lack of effect of the competitive NMDA antagonists.

Topics: Action Potentials; Animals; Dizocilpine Maleate; Dopamine; Dose-Response Relationship, Drug; Illicit Drugs; Male; N-Methylaspartate; Neurons; Phencyclidine; Pipecolic Acids; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Stereotaxic Techniques; Tegmentum Mesencephali; Vibrissae

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.

Topics: Adrenal Medulla; Animals; Binding Sites; Cattle; Desipramine; Dextromethorphan; Dizocilpine Maleate; Phenazocine; Phencyclidine; Piperidines; Potassium Chloride; Receptors, Opioid, delta; Receptors, Phencyclidine; Tranquilizing Agents

ES-242-1 approximately 5 are novel microbial bioxanthracenes which do not contain nitrogen. The ES-242s inhibited the binding of [3H]TCP and [3H]CGS19755 to the N-methyl-D-aspartate (NMDA) receptor complex. They had no effect on the binding of the specific ligands for the non-NMDA receptor. The biochemical and pharmacological properties of ES-242-1 were fully examined since it is the most potent of the five compounds. ES-242-1 is highly specific for the NMDA receptor; it has no effect on other receptors. Kinetic analyses indicated that ES-242-1 inhibited the binding of [3H]TCP and [3H]CGS19755 in a competitive manner, respectively, suggesting that ES-242-1 interacts with both the transmitter recognition site and the channel domain. ES-242-1 selectively inhibited NMDA-induced Ca2+ influx in primary cultures of mouse hippocampal neurons. ES-242-1 also specifically blocked the increase in cyclic GMP accumulation induced by NMDA or L-glutamate in rat cerebellar slices. In a concentration range of 0.1-1.0 microM, ES-242-1 was as potent as MK-801 in preventing glutamate-induced neurotoxicity in primary cultures of mouse hippocampal neurons. These results show that ES-242-1 is a potent and specific antagonist for the NMDA receptor. The antagonistic properties of the ES-242s appear to be due to a novel mechanism of action at the NMDA receptor.

Topics: Animals; Binding, Competitive; Biological Transport; Calcium; Cells, Cultured; Cyclic GMP; Dizocilpine Maleate; Hippocampus; Ion Channels; Kinetics; Mice; N-Methylaspartate; Neurons; Neurotransmitter Agents; Phencyclidine; Pipecolic Acids; Pyrans; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes

A series of 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) analogs were synthesized with the aim of developing a potent ligand for the N-methyl-D-aspartate glutamate receptor subtype. The piperidine moiety of TCP was substituted at the nitrogen position with aliphatic chains of different length or with various polar groups. A correlation between the decrease in the potency of displacement of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-im ine maleate (MK-801) binding from bovine cortex membranes and the increase of length or polarity of the aliphatic chain was observed. Isonitrile, isothiocyanate and isoselenocyanate groups were substituted at position 4 of the thiophene ring, and the relative binding affinity and alkylating potencies of the derivatized compounds were studied. Among this set of compounds, the one carrying an isothiocyanate group at position 4 of the thiophene ring of the N-ethyl analog of TCP yielded the most efficient alkylating agent, demonstrated by its ability to irreversibly block up to 80% of the [3H]MK-801 binding sites. This affinity ligand did not significantly affect other ligand binding sites of the same N-methyl-D-aspartate receptor complex or of other receptor systems, further demonstrating its functional specificity as a potent alkylating probe for the TCP/MK-801 recognition site. Studies with a radiolabeled adduct of this isothiocyanate N-ethyl derivative, however, indicate that a substantial level of nonspecific covalent incorporation into the membranes occurs at concentrations as low as 10 nM, thereby obscuring any possibility of detecting a specifically labeled protein species.

Topics: Affinity Labels; Alkylation; Animals; Binding Sites; Binding, Competitive; Cattle; Dizocilpine Maleate; Female; Kinetics; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Structure-Activity Relationship; Thiocyanates; Thiophenes

A novel compound, ES-242-1, which binds to a site on N-methyl-D-aspartate (NMDA) receptor that is coupled to the channel domain, was isolated from the culture broth of a fungus, Verticillium sp. SPC-15898. ES-242-1 inhibited the [3H]thienyl cyclohexylpiperidine ([3H]TCP) binding to rat crude synaptic membrane fractions with an IC50 value of 116 nM, but did not inhibit the [3H]kainate binding to its receptor, which is another subtype of the excitatory amino acid receptor.

Topics: Animals; Bacteria; Binding, Competitive; Centrifugation; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Culture Techniques; Dizocilpine Maleate; Dose-Response Relationship, Drug; Fermentation; Kainic Acid; Ketamine; Mitosporic Fungi; Molecular Structure; Phencyclidine; Pyrans; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Soil Microbiology; Solubility; Synaptic Membranes

Several arylcyclohexylamines and opioid benzomorphans that bind to phencyclidine (PCP) receptors were studied for their effects on punished and unpunished responding maintained under fixed-interval schedules of food presentation. All of these drugs increased both punished and unpunished responding, although higher doses decreased responding. The order of potency for increasing punished responding was MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzoa(a,d)-cyclohepten-5,1 0-imine] greater than [1-[1-(2-thienyl)cyclohexyl]piperidine] = PCP greater than (+)-N-allylnormetazocine = (-)-N-allynormetazocine. There was a high correlation (0.95) between the relative potency of these drugs in increasing punished responding and their relative affinity for PCP receptors. Because some of these drugs also bind to sigma receptors, drugs with a high affinity for sigma receptors, such as haloperidol, BD 737 [1S,2R-(-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N- methyl-2-(1-pyrrolidinyl) cyclohexylamine] and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, were also studied for their effects on punished and unpunished responding. These drugs produced only rate-decreasing effects. The correlation between the relative potency of drugs in increasing punished responding and their relative affinity for sigma receptors was low (-0.19). These data suggest that the PCP receptor is involved in some drug-induced increases in punished responding.

Topics: Animals; Buspirone; Dizocilpine Maleate; Haloperidol; Learning; Male; Pentobarbital; Phenazocine; Phencyclidine; Punishment; Rats; Rats, Inbred Strains; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Phencyclidine; Receptors, sigma

Phencyclidine (PCP) has been found to affect neuroendocrine function by altering the release of the anterior pituitary hormones, adrenocorticotrophin, luteinizing hormone and prolactin. The purpose of this study was to examine the effect of PCP on release of the two pituitary hormones also derived from the adrenocorticotropin precursor, namely, alpha-melanocyte-stimulating hormone and beta-endorphin (beta-E), synthesized in the neurointermediate and anterior lobes of the pituitary. At behaviorally active doses, PCP administered i.c.v. increased plasma levels of immunoreactive beta-E (i beta-E) without affecting the concentration of immunoreactive alpha-melanocyte-stimulating hormone, suggesting that PCP increased the release of beta-E from only the anterior lobe of the pituitary. Dexamethasone pretreatment blocked the PCP-induced increase in i beta-E which indicated further the anterior lobe effects of PCP. MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate), a selective PCP ligand, at behaviorally active doses also increased the plasma concentration of i beta-E. The dose-response curves for induction of behavior was very different from that for increasing the concentration of i beta-E in plasma. The increase in release of i beta-E was stereoselective as (+)-(1-(1-phenylcyclohexyl)-3 methylpiperidine but not (-)-(1-(1-phenylcyclohexyl)-3 methylpiperidine increased release of i beta-E. The increase in plasma levels of beta-E was not due to an interaction with opioid receptors because naloxone did not block PCP-induced release of beta-E. In vitro, PCP also significantly increased release of i beta-E from anterior lobe of the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-MSH; Animals; Ataxia; Behavior, Animal; beta-Endorphin; Dexamethasone; Dizocilpine Maleate; Dose-Response Relationship, Drug; Male; Phencyclidine; Pituitary Gland, Anterior; Pituitary Gland, Posterior; Rats; Rats, Inbred Strains; Sympathomimetics

Phencyclidine (PCP) receptors have been solubilized from rat forebrain membranes with the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate. Specific binding of the potent PCP receptor ligands [3H]thienyl-phencyclidine (TCP) and [3H]MK-801 was restored by incorporating extracted membrane protein into lipid vesicles prepared from a total brain lipid extract. A nearly quantitative recovery of solubilized receptor activity was achieved; this was dependent upon both the concentration of detergent used during membrane solubilization and the concentration of added lipid used during the reconstitution. The single, saturable, binding site measured for both [3H]TCP and [3H]MK-801 in solubilized and reconstituted preparations exhibited properties similar to those of the high affinity PCP binding site labeled by these ligands in brain membranes. The ability of ligands selective for this site (MK-801, TCP, and dexoxadrol) to competitively displace specific [3H]TCP binding was retained after solubilization and reconstitution, although IC50 values measured for these ligands were shifted to higher concentrations. Levoxadrol and haloperidol were ineffective at displacing the radioligand binding in both membrane and vesicle preparations. The additive and dose-dependent ability of glutamate and glycine to enhance [3H]TCP binding to the solubilized/reconstituted receptor further suggests that a direct interaction with the N-methyl-D-aspartate receptor/ion channel complex has been preserved in the vesicle preparations. The photoaffinity labeling of two polypeptides (Mr 98,000 and 59,000) by azido-[3H]PCP was demonstrated in the vesicle preparations; this was largely prevented by competitive displacement of the radioligand with PCP before photolysis. These results establish both an essential lipid dependency and polypeptide composition for the high affinity, haloperidol-insensitive, PCP receptor in brain.

Topics: Affinity Labels; Animals; Binding Sites; Binding, Competitive; Cholic Acids; Dizocilpine Maleate; In Vitro Techniques; Male; Membranes; Molecular Weight; Phencyclidine; Photolysis; Prosencephalon; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Neurotransmitter; Receptors, Phencyclidine; Solubility

The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aged; Brain; Corpus Striatum; Dizocilpine Maleate; Female; Glutamates; Glutamic Acid; Glycine; Humans; Male; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Tritium

The inhibitory effects of spermidine, putrescine and magnesium on [3H]TCP binding in crude synaptosomes and well washed buffy coat membranes were studied. We report that the IC50 values for the drugs tested varied depending on the state of activation of the channel, being lowest when the channel is maximally activated and highest when the channel is least activated in the control buffy coat preparation. Comparison of the inhibitory characteristics of these 3 agents when activated by glutamate, glycine, and low concentrations of spermidine or magnesium strongly suggest that putrescine and magnesium share a common inhibitory mechanism, perhaps mediated by the voltage-dependent channel site for magnesium. Inhibition by spermidine, however, is clearly mediated by a distinct site, non-identical to the phencyclidine receptor. Glutamate appears to uncouple this inhibition in about one-half of the N-methyl-D-aspartate (NMDA) complexes, suggesting the possibility of distinct subpopulations of NMDA-operated ion channels. The potential physiological relevance of these findings is discussed.

Topics: Animals; Biogenic Polyamines; Dizocilpine Maleate; Erythrocyte Membrane; Glutamates; Glutamic Acid; Glycine; In Vitro Techniques; Magnesium; Phencyclidine; Putrescine; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Structure-Activity Relationship

Bioassay and spectrophotofluorometry were used to study the antagonistic effect of dextromethorphan (DM) on phencyclidine (PCP) vasoconstriction in rabbit ear artery. DM (5 mumols.L-1) antagonized enhancement of PCP, N-[1-(2-thienyl) cyclohexyl] piperidine (TCP) and dizocilpine maleate (MK-801) (5 mumols.L-1) on electrical stimulation-induced vasoconstriction by 86 +/- 18%, 84 +/- 17%, and 86 +/- 18%, respectively (n = 6, P less than 0.01), but had no obvious bioactivity itself at the same concentration. DM (1, 2.5, and 5 mumols.L-1) inhibited the PCP effect and reduced the maximal effect of PCP with pD2' = 5.3 +/- 0.3 (n = 4). The contents of norepinephrine (NE) in control, PCP, and DM + PCP groups were 5 +/- 6, 12 +/- 8, and 5 +/- 6 ng.ml-1, respectively (n = 9). PCP (10 mumols.L-1) increased the NE release (P less than 0.05) but DM (10 mumols.L-1) inhibited it (P less than 0.01). The results suggest DM may be a noncompetitive blockader for PCP receptors.

Topics: Animals; Biological Assay; Dextromethorphan; Dizocilpine Maleate; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Male; Norepinephrine; Phencyclidine; Rabbits; Vasoconstriction

Excessive activation of glutamate receptors, most notably the N-methyl-D-aspartate (NMDA) subtype, appears to be a crucial factor in the sequence of cellular events which lead to irreversible ischaemic damage to neurones. The ability of newly developed antagonists of the NMDA receptor to reduce ischaemic brain damage has been assessed in cat and rodent models of focal cerebral ischemia. Non-competitive NMDA receptor antagonists such as dizocilpine (CAS 77086-21-6) which act at a site within the receptor operated ion channel markedly reduce (by more than 50%) ischaemic brain damage when administered prior to the ischaemic episode or 2 h after the onset of ischaemia. Competitive NMDA receptor antagonists, such as D-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid, which act at the neurotransmitter recognition site are equally effective in reducing the ischaemic brain damage when administered prior to the onset of the ischaemic episode. The clinical utility of competitive and non-competitive NMDA receptor antagonists in man will, however, be determined not by their tremendous anti-ischaemic efficacy, but by their profile of adverse effects. Careful selection of the therapeutic target for NMDA antagonists will be necessary if beneficial effects are to be established in man.

Topics: Animals; Cats; Cerebral Arteries; Dextrorphan; Dizocilpine Maleate; Hematoma, Subdural; Ischemic Attack, Transient; Kynurenic Acid; Phencyclidine; Piperazines; Rats; Receptors, N-Methyl-D-Aspartate

Primary cultures of embryonic rat cerebral cortex were treated after 17 days in vitro for 10 min with a single dose of the non-competitive antagonists of N-methyl-D-aspartate (NMDA) receptor MK 801, TCP, and GK 11. They were then maintained in vitro for 31, 59, or 73 days, and then processed for the immunocytochemical detection of neuron-specific enolase (NSE). Immunoreactive cells were counted in treated and control cultures, and it was found that, except at 31 days, treated cultures contained far more NSE immunoreactive cells than controls. Moreover, this effect was dose-dependent, since with both TCP and GK 11 neuron survival was significantly higher with, respectively, 20 microM and 5 microM than with the lowest concentration of 2.5 microM. We tentatively conclude that spontaneous neuron death occurring in primary cultures in vitro is at least partly related to the NMDA-associated Ca++ channel, since the common property of the molecules we used is to block this channel. The relevance of this mechanism of cell death in vitro to neuronal death in vivo is discussed.

Topics: Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Cyclohexanes; Cyclohexenes; Dizocilpine Maleate; Embryo, Mammalian; Female; N-Methylaspartate; Neurons; Phencyclidine; Piperidines; Pregnancy; Rats; Rats, Inbred Strains

Topics: Animals; Dizocilpine Maleate; Glutamates; Glutamic Acid; Guinea Pigs; In Vitro Techniques; Kinetics; Membranes; Molecular Conformation; Phencyclidine; Piperazines; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine

Recently, the presence of two high affinity binding sites for phencyclidine were described in guinea pig brain, with one site coupled to the glutamate excitatory amino acid receptor, specifically activated by N-methyl-D-aspartate (NMDA) (site 1) and the other site associated with the dopamine (DA) reuptake carrier (site 2). Phencyclidine and its analogs, as well as the benzomorphan opiates, are known to interact with binding sites for phencyclidine. In this study, the equilibrium dissociation constants (Kd) of these compounds for the two binding sites for phencyclidine were determined. Phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), an analog of PCP, were essentially non-selective between the two sites and also were the two drugs of the group observed to have the highest affinity for site 2. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine [(+)MK801] was the most selective agent for site 1, while none of the drugs tested showed selectivity for site 2. In humans, phencyclidine produces psychotomimetic effects, while (+)MK801 has been reported to produce minimal, if any, psychotomimetic effects, at doses sufficient to reduce seizures. These clinical observations, in conjunction with the present biochemical binding data, suggest that (+)MK801 may serve as a "marker" for site 1 and that the psychotomimetic effects of phencyclidine might be mediated by site 2.

Topics: Analgesics; Animals; Benzomorphans; Brain; Dioxolanes; Dizocilpine Maleate; Guinea Pigs; In Vitro Techniques; Kinetics; Ligands; Narcotics; Phencyclidine; Piperidines; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine; Structure-Activity Relationship

The phencyclidine analogue [3H](1-[2-thienyl]cyclohexyl)piperidine (3H-TCP) binds to the ion channel associated with the N-methyl-D-aspartate receptor channel complex. In vitro autoradiography indicates that the distribution of 3H-TCP binding in brain closely parallels that of [3H]glutamate binding to the N-methyl-D-aspartate receptor. In nine 7-day-old rats, an acute focal hypoxic-ischemic insult produced by unilateral carotid artery ligation and subsequent exposure to 8% oxygen acutely reduced 3H-TCP binding ipsilateral to the ligation by 30% in the CA1, by 27% in the CA3, by 26% in the dentate gyrus, and by 17% in the striatum compared with values from the contralateral hemisphere. In 10 littermates that received 1 mg/kg of the neuroprotective noncompetitive N-methyl-D-aspartate antagonist MK-801 immediately before hypoxic exposure, the regional distribution of 3H-TCP binding in hypoxic-ischemic brain was relatively preserved and there were no interhemispheric asymmetries in 3H-TCP binding densities. In addition, in three unoperated rats decapitated 24 hours after MK-801 treatment, 3H-TCP binding was reduced by 15-35%; similar bilateral suppression of 3H-TCP binding was detected in MK-801-treated ligates. Our data indicate that 3H-TCP autoradiography can be used to assay the efficacy of neuroprotective agents in this experimental model of perinatal stroke.

Topics: Animals; Animals, Newborn; Aspartic Acid; Autoradiography; Binding Sites; Brain; Brain Ischemia; Dibenzocycloheptenes; Dizocilpine Maleate; Glutamates; Glutamic Acid; Hypoxia; N-Methylaspartate; Phencyclidine; Rats; Rats, Inbred Strains; Tritium

At concentrations greater than or equal to 100 microM, phencyclidine (PCP), N-(1-(2-thienyl)-cyclohexyl)piperidine (TCP), and MK-801 induced [3H]dopamine release from dissociated cell cultures of rat mesencephalon. This release was Ca2+ independent and tetrodotoxin insensitive. Tetrodotoxin (2 microM) itself had no effect on spontaneous release of [3H]dopamine. [3H]Dopamine release was induced by 1,3-di(2-tolyl)guanidine, a sigma ligand, and by 4-aminopyridine (1-3 mM), a K+ channel blocker. No stereoselectivity was observed for [3H]dopamine release evoked by the dioxadrol enantiomers, dexoxadrol, and levoxadrol, or by enantiomers of N-allylnormetazocine (SKF 10,047). The selective dopamine uptake inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909) did not affect spontaneous or TCP-evoked [3H]dopamine release. Together, these data suggest that the dopamine-releasing effects of PCP-like compounds on the mesencephalic cells were not mediated by actions at the PCP receptor or sigma binding site, Ca2+, or Na+ channels, or at the high affinity dopamine uptake site. It remains conceivable that blocking actions of PCP-like compounds at voltage-regulated K+ channels may at least partly explain the response. These results are discussed in comparison with findings in intact brain.

Topics: 2-Amino-5-phosphonovalerate; Animals; Anticonvulsants; Binding Sites; Cells, Cultured; Dioxolanes; Dizocilpine Maleate; Dopamine; Dose-Response Relationship, Drug; Guanidines; Mesencephalon; N-Methylaspartate; Neurotransmitter Uptake Inhibitors; Phencyclidine; Piperazines; Piperidines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine; Sigma Factor; Tritium

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 dibenzocycloalkenimine (MK-801). In contrast, [3H]TCP binds to two sites on the plasma membrane fraction used as a 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.

Topics: Animals; Brain; Cell Membrane; Dizocilpine Maleate; Glutamates; Glutamic Acid; In Vitro Techniques; Kinetics; Phencyclidine; Piperazines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine; Subcellular Fractions; Synapses; Synaptosomes

A series of eight C5-substituted analogues of (+-)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (1) have been prepared by the directed lithiation-alkylation (and acylation) of its (+-)-N-tert-butylformamidinyl derivative 2 followed by formamidine solvolysis. An additional 10 analogues were prepared by elaboration of the C5-ethyl ester derivative. Analogues possessing large (e.g. propyl and larger) lipophilic substituents displace [3H]-1-(1-thienylcyclohexyl)piperidine [( 3H]TCP) from the high-affinity phencyclidine (PCP) binding site in rat brain homogenates only at high concentrations (Ki greater than 1000 nM); however, the presence of a polar amino functionality (e.g. 2-aminoethyl) offsets this effect (Ki = 20 nM). Thus, the boundary condition for lipophilic substituents larger than ethyl appears to be polar in nature. Interaction of the 11 relatively small (MR less than 14) C5-substituted analogues of 1 with the high-affinity PCP binding site associated with the N-methyl-D-aspartate (NMDA) receptor is best described by the equation log (1/Ki) = -5.83F + 0.64 pi + 7.41 (r = 0.90).

Topics: Animals; Binding Sites; Dibenzocycloheptenes; Dizocilpine Maleate; In Vitro Techniques; Ligands; Phencyclidine; Rats; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine; Regression Analysis; Structure-Activity Relationship

Tonic-clonic convulsions of mutant quaking mice were antagonized by the intracerebroventricular injection of N-methyl-D-aspartate receptor antagonists. The competitive antagonists, CPP (3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) and CGS 19755 (cis-4-(phosphonomethyl)-2-piperidine carboxylic acid), exerted a partial anticonvulsant action, with ED50S of 0.115 and 0.076 nmol, respectively. The non-competitive antagonists, TCP (1-(1-(2-thienyl)cyclohexyl)piperidine) and MK-801 [+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine), provided full protection, with ED50s of 4.49 and 2.67 nmol, respectively. The competitive antagonists elicited a marked ataxia whereas the non-competitive antagonists did not have side-effects. These results might reflect the involvement of glutamatergic neurotransmission in the convulsions of the quaking mutants.

Topics: Animals; Anticonvulsants; Dibenzocycloheptenes; Dizocilpine Maleate; Epilepsy; Injections, Intravenous; Male; Mice; Mice, Quaking; Phencyclidine; Physical Stimulation; Piperazines; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

The mouse neuroblastoma-Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptor-like sites. We report here that polypeptide components of NCB-20 cell PCP sites were identified with the photolabile PCP derivative [3H]N-[1-(3-azidophenyl)cyclohexyl]piperidine ([3H]AZ-PCP). The pharmacological selectivity of [3H]AZ-PCP binding (under reversible conditions) was similar to that observed for [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to NCB-20 cell membranes. Inhibition of [3H]TCP binding by AZ-PCP, dexoxadrol or MK-801 was biphasic, suggesting the presence of two types of PCP sites on NCB-20 cells. Photolysis of NCB-20 cell membranes pre-equilibrated with [3H]AZ-PCP, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), revealed the presence of 5 major labeled bands (Mr 90,000, 68,000, 49,000, 40,000 and 33,000), a pattern similar to that observed for rat brain membranes. MK-801 and D-2-amino-5-phosphonovaleric acid (D-(-)-AP5) selectively inhibited the labeling of Mr 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The Mr 68,000 and 90,000 components appear to correspond to a high-affinity site, which comprises approximately 20% of total [3H]TCP sites in these cells, and exhibits the pharmacology expected for the PCP receptor of the N-methyl-D-aspartate (NMDA)-gated channel.

Topics: Animals; Azides; Binding, Competitive; Cell Line; Cricetinae; Dibenzocycloheptenes; Dizocilpine Maleate; Mice; Molecular Weight; Neuroblastoma; Phencyclidine; Photochemistry; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine

Two high affinity phencyclidine (PCP) binding sites, labelled by [3H] 1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP), have been identified in guinea pig brain, with one site coupled to the N-methyl-D-aspartate (NMDA) receptor (site 1) and the other site associated with the dopamine reuptake carrier complex (site 2). In this study, PCP enhanced the dissociation of [3H]TCP from PCP site 1 and site 2, while (+)-MK801 only enhanced dissociation of [3H]TCP from PCP site 1. Although additional studies will be required to determine the exact mechanism(s) of these effects, these data demonstrate that the interactions of PCP with both site 1 and site 2 are more complex than previously appreciated.

Topics: Allosteric Regulation; Allosteric Site; Animals; Binding, Competitive; Brain; Cell Membrane; Dizocilpine Maleate; Guinea Pigs; Kinetics; Ligands; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine

Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. This compound is assumed to be a non-competitive antagonist for the N-methyl-D-aspartate(NMDA)-sensitive subclass of central excitatory amino acid receptors. Binding was quite low but detectable in Triton-treated membranes irrespective of the incubation temperature, and the temperature-dependent portion of the binding was greatly reduced in these Triton-treated membranes. However, binding was drastically potentiated by the inclusion of L-glutamate and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists for the NMDA-sensitive subclass also potentiated binding, with agonists for the other subclasses being ineffective. Glycine at a concentration above 10 nM was not only effective as a stimulant of potentiated binding by glutamate, but was also active in enhancing binding in the absence of added glutamate. Glycine increased both the association and dissociation rates without significantly affecting the dissociation constant. Pharmacological profiles of binding in Triton-treated membranes were not significantly different from those in untreated membranes, except for that of haloperidol. Haloperidol is proposed to be highly selective for brain sigma-receptors on the basis of a potent inhibition of sigma-receptor binding. The inhibitory potency of this sigma-ligand was markedly attenuated in the presence of both glutamate and glycine in Triton-treated membranes, as compared with that in untreated membranes. These results suggest that [3H]TCP binding in Triton-treated membranes is a useful biochemical tool to evaluate predominantly the activated state of ion channels associated with the NMDA-sensitive receptors in terms of freedom from the confounding effects of endogenous amino acids.

Topics: Animals; Binding, Competitive; Brain; Detergents; Dizocilpine Maleate; Kinetics; Octoxynol; Phencyclidine; Polyethylene Glycols; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes; Tritium

We have used a variety of selective radioligands to identify and localize sigma- and phencyclidine (PCP)-binding sites in rat endocrine organs. [3H]Haloperidol-labeled sigma-receptors were identified in membrane homogenates of rat pituitary, adrenal, testis, and ovary which had kinetic and pharmacological characteristics similar to those of the well characterized sigma-receptors in rat cerebellum. The highest density of sigma-receptors was present in the ovary, with progressively lower densities present in the testis, pituitary, adrenal, and cerebellum, respectively. In autoradiographic studies, sigma-receptors [labeled with d-3-(3-hydroxyphenyl)N-(1-propyl-2,3-[3H]piperidine or [3H]1,3-di-(2-tolyl)guanidine] were discretely localized within the endocrine tissues. In the pituitary, the highest density of sigma-receptors was found in the anterior lobe. In the adrenal, sigma-receptors were localized primarily in the cortex. In the testis, sigma-receptors were present in highest concentrations in the ductuli efferentes and ductus epididymis; lower densities of binding sites were present in the seminiferous tubules, and no binding was seen in the interstitial tissue. In the ovary, sigma-receptors were localized in high density in the maturing follicles, and lower densities were present in resting follicles. After hypophysectomy, there were relative increases in the densities of sigma receptors in the remaining tissue in the adrenal gland and testis. In contrast, hypophysectomy resulted in a marked depletion of sigma-binding sites in the ovary. The data from hypophysectomized rats indicate that the highest densities of sigma-receptors in the ovary are localized to (LH-dependent) maturing follicles, while sigma-binding sites in adrenal and testis are localized to cells that are not dependent on trophic maintenance by the pituitary. In contrast, high affinity PCP receptors were not detected in pituitary, adrenal, testis, or ovary either by homogenate binding studies with 3,4-[3H]N-[1-(2-thienyl)cyclohexyl]piperidine or in vitro autoradiography using 3,4-[3H]N-[1-(2-thienyl)cyclohexyl]piperidine and d-[3H]5-methyl-10,11-dihydro-5H-dibenzo-[a,d] + cyclohepten-5,10-imine. In summary, the data suggest that the reported endocrine effects of PCP and the prototypic sigma-receptor agonist N-allylnormetazocine are probably mediated either through direct action on sigma-receptors in the pituitary and/or target endocrine organs or by actions on sigma- and/or PCP receptors

Topics: Adrenal Glands; Animals; Autoradiography; Brain Chemistry; Cell Membrane; Dibenzocycloheptenes; Dizocilpine Maleate; Female; Haloperidol; Hypophysectomy; Male; Ovary; Phencyclidine; Piperidines; Pituitary Gland; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, sigma; Testis; Tissue Distribution

The selective non-competitive NMDA receptor antagonist, MK-801, potently blocked convulsions induced in the mouse by N-methyl-DL-aspartic acid (NMDLA) with an i.v. ED50 dose of 0.2 mg/kg. Similar doses of MK-801 were also effective in blocking seizures induced by pentylenetetrazol (PTZ), electroshock and by sound in audiogenic seizure-prone animals. Other less selective non-competitive NMDA receptor antagonists including phencyclidine (PCP), thienylcyclohexylpiperidine (TCP), (+)-N-allylnormetazocine [+)-NANM, (+)-SKF 10,047) and ketamine also blocked NMDLA-induced seizures with a rank order of potency of MK-801 greater than PCP greater than TCP = (+)-NANM greater than ketamine. The competitive NMDA receptor antagonist, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) blocked NMDLA-induced seizures with an ED50 of 4.5 mg/kg, 22- and 560-fold more potently than the competitive antagonists, 2-DL-amino-7-phosphonoheptanoic acid (2-APH) and 2-DL-amino-5-phosphonovaleric acid (2-APV), respectively. MK-801 was the most potent of the non-competitive antagonists to induce a motor syndrome including head weaving, body rolling, increased locomotion and ataxia, characteristic of the behavioural response to PCP in the mouse. The syndrome was also present following injection of the competitive NMDA receptor antagonists, although they were generally less potent (probably a reflection of poor brain penetration) and less efficacious than the non-competitive antagonists. For all compounds except CPP, the anticonvulsant ED50 dose was close to the minimum effective dose to induce motor stimulation: CPP was 5- to 10-fold more potent as an anticonvulsant.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anticonvulsants; Aspartic Acid; Behavior, Animal; Binding, Competitive; Dibenzocycloheptenes; Discrimination Learning; Dizocilpine Maleate; Dose-Response Relationship, Drug; Ketamine; Male; Motor Activity; N-Methylaspartate; Phenazocine; Phencyclidine; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Seizures

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 withholding 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.

Topics: Animals; Brain Stem; Cats; Decerebrate State; Dextrorphan; Dibenzocycloheptenes; Dizocilpine Maleate; Guanidines; Phencyclidine; Phrenic Nerve; Piperidines; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Phencyclidine; Receptors, sigma; Respiration

Numerous studies have now demonstrated that a binding site for the psychotomimetic drug phencyclidine (PCP) exists within the receptor channel complex for the excitatory amino acid neurotransmitter glutamate, specifically the glutamate receptor selectively activated by N-methyl-D-aspartate (NMDA). Several lines of evidence support the hypothesis that all PCP receptors in rat brain are associated with the NMDA receptor complex. In the present study, we reexamine this hypothesis. We report that the PCP analog [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) labels two high affinity binding sites in membranes prepared from guinea pig brain site 1 (Kd = 14.1 nM, Bmax = 631 fmol/mg of protein) and site 2 (Kd = 46.5 nM, Bmax = 829 fmol/mg of protein). (+)-5-Methyl-10 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate bound to site 1 with high affinity (Kl = 3.2 nM) and to site 2 with low affinity (Kl = 5208 nM). The order of potency of drugs for inhibiting [3H]TCP binding to site 1 correlated with their ED50 values for inhibition of NMDA-mediated responses reported in the literature, whereas the order of potency of drugs for inhibiting [3H]TCP binding to site 2 correlated with their ED50 values for inhibition of [3H]dopamine reuptake reported in the literature. Kinetic experiments demonstrated that glutamate, 2-amino-7-phosphonoheptanoic acid, and Mg2+ modulated [3H]TCP binding to site 1 but not site 2. Preincubation of guinea pig striatal membranes with varying concentrations of the high affinity dopamine reuptake inhibitors N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3- phenylpropyl]piperazine caused a wash-resistant inhibition of [3H]TCP binding to site 2 but not site 1. Taken collectively, these data demonstrate the existence of a high affinity PCP binding site associated with the dopamine reuptake carrier and raise the possibility that the therapeutic and psychotomimetic effects of PCP in humans are separable and mediated via different binding sites.

Topics: Acetylcholine; Animals; Binding Sites; Brain; Dibenzocycloheptenes; Dizocilpine Maleate; Dopamine; Guinea Pigs; In Vitro Techniques; Kinetics; Magnesium; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine

Topics: Animals; Binding, Competitive; Brain; Carrier Proteins; Culture Techniques; Dibenzocycloheptenes; Dizocilpine Maleate; Dopamine; Dopamine Plasma Membrane Transport Proteins; Guinea Pigs; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Phencyclidine; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Receptors, Phencyclidine

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.

Topics: Animals; Anticonvulsants; Brain; Cations, Divalent; Cell Membrane; Cerebral Cortex; Dibenzocycloheptenes; Dizocilpine Maleate; Kinetics; Male; Phenazocine; Phencyclidine; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Tissue Distribution; Tritium

Brief exposure of cortical or hippocampal cell cultures to glutamate (500 microM) resulted in a progressive neuronal necrosis which is maximal 18-24 h later. Pretreatment of the cultures by a phencyclidine derivative: thienylphencyclidine (TCP), or the TCP precursor: GK86, or MK801 protected against glutamate toxicity. Non-competitive antagonists of N-methyl-D-aspartate receptors appear as potent tools for the in vitro protection of neuronal cells against excitatory amino acid toxicity.

Topics: Animals; Cells, Cultured; Cerebral Cortex; Dibenzocycloheptenes; Dizocilpine Maleate; Glutamates; Glutamic Acid; Hippocampus; Neurotoxins; Phencyclidine; Prodrugs; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter

MK-801 was found to be more potent than phencyclidine (PCP) as an inhibitor of N-methyl-D-aspartate-induced [3H]norepinephrine (NE) release and [3H]TCP binding in the hippocampus. On the other hand, MK-801 was slightly less potent than PCP to enhance kainate-stimulated [3H]NE release and to inhibit hippocampal [3H]NE uptake. Further, MK-801 was strikingly less potent than PCP as an inhibitor of striatal synaptosomal [3H]dopamine uptake. These data are discussed with reference to the therapeutic potential of MK-801.

Topics: Animals; Aspartic Acid; Catecholamines; Dibenzocycloheptenes; Dizocilpine Maleate; Hippocampus; In Vitro Techniques; N-Methylaspartate; Norepinephrine; Phencyclidine; Rats

Topics: Animals; Brain Chemistry; Convulsants; Dibenzocycloheptenes; Dizocilpine Maleate; Phencyclidine; Rats; Receptors, Neurotransmitter; Receptors, Phencyclidine