Adjunctive high-dose glycine in the treatment of schizophrenia.

Glycine is an agonist at brain N-methyl-D-aspartate receptors and crosses the blood-brain barrier following high-dose oral administration. In a previous study, significant improvements in negative and cognitive symptoms were observed in a group of 21 schizophrenic patients receiving high-dose glycine in addition to antipsychotic treatment. This study evaluated the degree to which symptom improvements might be related to alterations in antipsychotic drug levels in an additional group of 12 subjects. Glycine treatment was associated with an 8-fold increase in serum glycine levels, similar to that observed previously. A significant 34% reduction in negative symptoms was observed during glycine treatment. Serum antipsychotic levels were not significantly altered. Significant clinical effects were observed despite the fact that the majority of subjects were receiving atypical antipsychotics (clozapine or olanzapine). As in earlier studies, improvement persisted following glycine discontinuation.

Phencyclidine, excitatory amino acids, psychiatry and drug abuse: Historical perspectives on clinical-laboratory interactions.

Some 40 years ago phencyclidine (PCP) was developed as the prototype of a proposed new class of 'dissociative' general anesthetics, so called because it induced a marked dissociation from the environment without complete loss of consciousness. In the earliest clinical trials of PCP anesthesia, it was observed that as many as half the subjects experienced severe psychotic reactions during and beyond emergence. This striking clinical observation at once marked the failure of PCP as a suitable general anesthetic, and the beginning of a remarkable new era in basic and clinical neuroscience which can serve as an example of the interaction between clinical observation and basic science. At once, clinical researchers turned their focus upon the characterization of the PCP-induced psychosis, and recognized striking similarities between PCP-induced symptoms and signs and both the negative and positive symptoms of schizophrenia, proposing the PCP psychosis as a new model of that illness. Several years later PCP suddenly emerged as a major drug of abuse, with the result that emergency rooms and psychiatric inpatient units were observing and treating large numbers of these patients, in many of whom a diagnosis of schizophrenia could not be ruled out until toxicological analyses were performed. This natural experiment yielded a huge amount of additional data, and contributed a strong public-health based impetus to research into the nature and treatment of PCP intoxication.

Amelioration of negative symptoms in schizophrenia by glycine.

Phencyclidine induces a psychotomimetic state by blocking neurotransmission at N-methyl-D-aspartic acid (NMDA) receptors. In a double-blind, placebo-controlled fashion, 14 medicated patients with chronic schizophrenia were treated with glycine, a potentiator of NMDA-receptor-mediated neurotransmission. Significant improvement in negative symptoms occurred in the group given glycine but not in the group given placebo, suggesting that potentiation of NMDA-receptor-mediated neurotransmission may represent an effective treatment for neuroleptic-resistant negative symptoms in schizophrenia.

Activation-related and activation-independent effects of polyamines on phencyclidine receptor binding within the N-methyl-D-aspartate receptor complex.

The phencyclidine (PCP) receptor is located within the N-methyl-D-aspartate (NMDA) receptor-gated ion channel. The functional state of the NMDA receptor complex thus influences parameters of radioligand binding to the PCP receptor, and PCP receptor ligands can serve as in vitro probes for elucidation of NMDA receptor activation mechanisms. PCP receptor binding is stimulated by NMDA receptor agonists such as L-glutamate and also by distinct classes of modulatory agents such as glycine-like amino acids and polyamines such as spermidine (SPD). The present study utilizes a kinetic approach permitting differentiation of PCP receptor binding within closed and activated conformations of the NMDA receptor complex. The results demonstrate that SPD increases radioligand binding to the PCP receptor through two distinct mechanisms. First, SPD, like glycine, increases the percentage of time that NMDA channels remain in the open state in the presence of L-glutamate, consistent with a role as a positive allosteric modulator of NMDA receptor activation. Second, unlike glycine, SPD increases the affinity of the PCP receptor for its ligands. The latter effect does not appear to reflect increased NMDA receptor activation. SPD does not induce glycine-like alteration of the EC50 value for stimulation of PCP receptor binding by L-glutamate, suggesting that the effects of SPD cannot be attributed solely to augmentation of glycine binding. These findings demonstrate first that total specific PCP receptor binding cannot, of itself, be used as an index of NMDA receptor activation and second, glycine and polyamines differ in the mechanisms by which they potentiate PCP receptor binding.

High efficiency reconstitution of a phencyclidine/MK-801 receptor binding site solubilized from rat forebrain membranes.

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.

Recent advances in the phencyclidine model of schizophrenia.

OBJECTIVE: Phencyclidine (PCP, "angel dust") induces a psychotomimetic state that closely resembles schizophrenia. As opposed to amphetamine-induced psychosis, PCP-induced psychosis incorporates both positive (e.g., hallucinations, paranoia) and negative (e.g., emotional withdrawal, motor retardation) schizophrenic symptoms. PCP-induced psychosis also uniquely incorporates the formal thought disorder and neuropsychological deficits associated with schizophrenia. The purpose of the present paper is to review recent advances in the study of the molecular mechanisms of PCP action and to describe their implications for the understanding of schizophrenic pathophysiology. METHOD: Twenty-five papers were identified that described the clinical dose and serum and CSF levels at which PCP induces its psychotomimetic effects. The dose range of PCP-induced effects were compared to the dose range at which PCP interacts with specific molecular targets and affects neurotransmission. RESULTS: It was found that PCP-induced psychotomimetic effects are associated with submicromolar serum concentrations of PCP. At these concentrations PCP interacts selectively with a specific binding site (PCP receptor) that is associated with the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. Occupation of its receptor by PCP induces noncompetitive inhibition of NMDA receptor-mediated neurotransmission. Other NMDA antagonists such as the dissociative anesthetic ketamine induce PCP-like neurobehavioral effects in proportion to their potency in binding to the PCP receptor and inducing NMDA receptor inhibition. CONCLUSIONS: These findings suggest that endogenous dysfunction of NMDA receptor-mediated neurotransmission might contribute to the pathogenesis of schizophrenia. The relative implications of the PCP and amphetamine models of schizophrenia are discussed in relationship to the diagnosis and etiology of schizophrenia.

Polyamine effects upon N-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists.

Polyamines such as spermidine potentiate activation of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [3H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and without D-(-)-2-amino-5-phosphonovaleric acid (D(-)-AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [3H]MK-801 binding with an EC50 value of 13.3 microM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 microM (n = 10, S.E.M. = 24.66, range = 25-250 microM). Glutamate (10 microM) decreased the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding to 3.4 microM. Glycine (10 microM) did not significantly alter either maximum spermidine-induced [3H]MK-801 binding or the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding. Incubation in the presence of the specific glutamate antagonist D(-)AP-5 attenuated [3H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [3H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding while D(-)AP-5 was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic mechanisms of glycine requirement for N-methyl-D-aspartate channel activation.

Glycine potentiates N-methyl-D-aspartate (NMDA) receptor-mediated responses via its interaction with a strychnine-insensitive glycine recognition site. We have previously shown that the potent glycine receptor antagonist 7-chlorokynurenic acid (7Cl-KYN) dose-dependently inhibits [3H]MK-801 binding to the PCP receptor and that this effect is reversed by glycine. [3H]MK-801 binding to the PCP receptor within the NMDA receptor-gated ion channel is a measure of channel activation. Association of PCP receptor ligands is biexponential with the fast component of binding serving as a marker of activated NMDA channels. In the present study we utilize 7Cl-KYN as a probe of the kinetic mechanism of the glycine effect upon NMDA receptor functioning. In the presence of L-glutamate, incubation with 7Cl-KYN completely abolished the fast component of [3H]MK-801 association in 4 out of 5 experiments. In the fifth experiment where the fast component was detected, it accounted for less than half of that seen in the presence of L-glutamate alone. 7Cl-KYN-induced inhibition of the fast component of [3H]MK-801 association was reversed by the addition of glycine. Since the fast component represents ligand binding to the PCP receptor via the open NMDA channel, selective reduction of this component by 7Cl-KYN indicates that glycine receptor antagonists reduce the probability of channel opening, and also that the selective reduction in the component of [3H]MK-801 binding that manifests fast kinetics can serve as a marker for glycine antagonists.

Rat brain N-methyl-D-aspartate receptors require multiple molecules of agonist for activation.

N-Methyl-D-aspartate (NMDA) receptors mediate important physiological and pathological processes, including long term potentiation and neuronal excitotoxicity. Elucidation of mechanisms underlying NMDA receptor functioning will promote understanding of the molecular bases of NMDA receptor-mediated processes. The localization of the phencyclidine (PCP) receptor within the ionophore of the NMDA receptor-gated ion channel permits the binding of PCP receptor ligands to serve as a functional marker of channel activation. We have previously demonstrated that the highly selective PCP receptor ligand [3H]MK-801 displays multiexponential kinetics of association, indicating that the NMDA receptor functions according to a multistate model. Using the fast component of [3H]MK-801 binding to PCP receptors as a marker for activated NMDA channels, we demonstrate here a Hill coefficient of 2 for activation of NMDA channels by L-glutamate. A multistate model of NMDA receptor functioning analogous to the model known to account for the functioning of nicotinic cholinergic and gamma-aminobutyric acidA receptors fits well to our experimental data, supporting the concept that the NMDA receptor is properly classified in the Class 1 superfamily of ligand-gated channels.

Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line.

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.

The role of excitatory amino acids in neuropsychiatric illness.

Over the past several years, research has demonstrated that the excitatory amino acids serve as the major excitatory neurotransmitters in cerebral cortex and hippocampus. Neurons that contain excitatory amino acids play crucial roles in neuropsychological functioning, learning, and memory. In addition, these neurons exert descending control over subcortical structures that is behaviorally antagonistic to the effects mediated by the ascending dopaminergic system. Disturbances of excitatory amino acid systems may contribute to the pathogenesis of schizophrenia and to other neuropsychiatric syndromes associated with delirium or dementia.

Glycine reverses 7-chlorokynurenic acid-induced inhibition of [3H]MK-801 binding.

7-Chlorokynurenic acid (7-Cl KYNA) has been reported to attenuate N-methyl-D-aspartate (NMDA) receptor functioning by a potent and selective inhibitory action mediated at the strychnine-insensitive glycine recognition site of the NMDA complex. Here we report that 7-Cl KYNA dose-dependently inhibits [3H]MK-801 binding to the PCP receptor, and that this effect is reversed by addition of glycine. Since [3H]MK-801 binding is a measure of channel activation, our results are consistent with the hypotheses that 7-Cl KYNA exerts its NMDA receptor antagonism by acting at the glycine site, and that activation of the glycine site is required for NMDA channel activity to occur.

Biexponential kinetics of [3H]MK-801 binding: evidence for access to closed and open N-methyl-D-aspartate receptor channels.

The phencyclidine (PCP) receptor is a site within the ion channel gated by the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. In the present study, kinetics of association and dissociation of the specific PCP receptor ligand [3H]MK-801 were determined in order to elucidate the mechanism of functioning of the NMDA receptor complex. Two distinct components of [3H]MK-801 association with apparent t1/2 values of approximately 10 min and 3 hr were resolved. Incubation with the NMDA receptor agonist L-glutamate increased the total steady state binding of [3H]MK-801 and increased the relative percentage of [3H]MK-801 binding that manifested fast rather than slow kinetics, without altering the observed rate constant of either the fast or slow component of association. The competitive NMDA receptor antagonist D(-)-2-amino-5-phosphonovaleric acid decreased total steady state binding of [3H]MK-801. These data support a model in which [3H]MK-801 can gain access to its binding site via two distinct paths, a fast hydrophilic path associated with a conformation of the NMDA receptor in which the channel is open and a slow hydrophobic path independent of the open channel. In the presence of L-glutamate, incubation with glycine increased the relative percentage of [3H]MK-801 binding that manifested fast rather than slow kinetics. The Hill coefficient for stimulation of specific [3H]MK-801 binding by L-glutamate was significantly greater than unity in either the absence or presence of glycine. Our data support a model of NMDA receptor functioning in which two molecules of agonist are required to convert the receptor complex to a conformation that is in equilibrium with the open conformation and in which glycine regulates the percentage of NMDA receptor complexes bound to two molecules of agonist that convert to the open configuration.

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

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

Characterization of opioid, sigma, and phencyclidine receptors in the neuroblastoma-brain hybrid cell line NCB-20.

Opioid, sigma, and phencyclidine (PCP) receptors were characterized in the mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20. Quantitative receptor assays under equilibrium binding conditions with highly specific radioligands demonstrated the presence of delta, but not mu or kappa, opioid receptors on NCB-20 cell membranes. NCB-20 cells were shown to possess two distinct sites specific for sigma opioids and PCP derivatives. One site was labeled by (+)-[3H]N-allylnormetazocine [(+)-[3H]SKF-10,047] (Kd = 69 nM; Bmax = 4100 fmol/mg of protein). The rank order of potency of drugs at this site was (+)-3-(3-hydroxy-phenyl)-N-(1-propyl)piperidine [(+)-3-PPP] greater than haloperidol greater than (+)-SKF-10,047 greater than (+/-)-ethylketocyclazocine greater than (+/-)-bremazocine greater than N-[1-(2-thienyl) cyclohexyl]piperidine (TCP) greater than dexoxadrol. This site is similar in its ligand selectivity to the haloperidol-sensitive sigma receptor of rat brain. The other site was labeled by the potent phencyclidine derivative [3H]TCP (Kd = 335 nM; Bmax = 9300 fmol/mg of protein). This density is equivalent to approximately 60,000 sites/cell. The rank order of potency of drugs at this site was TCP greater than (+)-3-PPP greater than PCP greater than dexoxadrol greater than haloperidol greater than cyclazocine greater than levoxadrol greater than (+)-SKF-10,047; mu and delta ligands were inactive. This site is similar to the rat brain PCP receptor. The NCB-20 cell line is the only cultured cell line that has been demonstrated to have PCP receptors.