Joseph B. Monahan

d-Cycloserine: A ligand for the N-methyl-d-aspartate coupled glycine receptor has partial agonist characteristics

We have previously shown that D-cycloserine displaces [3H]glycine binding to a recognition site with properties consistent with an N-methyl-D-aspartate (NMDA) receptor modulatory site. Additionally, D-cycloserine positively modulates the NMDA receptor as evidenced by its dose-dependent enhancement of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the NMDA receptor-coupled ionophore. Further evaluation of this compound indicates that the maximal stimulation of [3H]TCP binding induced by D-cycloserine is lower than that produced by other compounds acting at the NMDA receptor associated glycine modulatory site (glycine and D-serine). Moreover, the stimulation of [3H]TCP binding induced by D-cycloserine in the presence of various fixed concentrations of glycine results in a family of dose-response curves which asymptotically converge to 40-50% of the maximal stimulation induced by glycine alone. These results are consistent with D-cycloserine acting as a partial agonist of the NMDA receptor via its interaction with the coupled glycine modulatory site.

D-cycloserine, a positive modulator of the N-methyl-D-aspartate receptor, enhances performance of learning tasks in rats.

Glycine has recently been shown to positively modulate the N-methyl-D-aspartate (NMDA) subclass of acidic amino acid receptors which are important in neural pathways involved in learning and memory. We report that d-cycloserine (DCS), an antimycobacterial agent known to cross the blood-brain barrier, binds with high affinity to this glycine modulatory site, functions as a positive modulator, and facilitates performance of learning tasks in rats. In addition, DCS appears to be a potent cognitive enhancer at doses lower than those required for antibacterial activity. Based on these data, we propose that modulation of NMDA receptors via glycinergic mechanisms may be a means of influencing cognitive processes.

Identification and Characterization of an N‐Methyl‐D‐Aspartate‐Specific L‐[3H]Glutamate Recognition Site in Synaptic Plasma Membranes

Abstract: Conditions have been developed for an L‐[3H]glu‐tamate binding assay in which 85‐95% of the specific binding is to a site that corresponds to the N‐methyl‐D‐aspartate subclass of acidic amino acid receptors. Incubation of synaptic plasma membranes with L‐[3H]glutamate in 50 mM Tris/acetate, pH 7.4, for 2‐20 min at 2°C results in binding with pharmacological characteristics of the electrophysio‐logically defined N‐methyl‐D‐aspartate receptor. The fraction of glutamate binding to this subclass of receptors, relative to the total, decreases with both increased time and temperature. This binding is reversible, is concentrated in the synaptic plasma membrane fraction, has a pH optimum of 7.0‐7.4, and is linear with respect to tissue protein concentration. The binding is unaffected by 1 mM concentrations of the anions sulfate, chloride, bromide, thiocyanate, phosphate, acetate, nitrate, or carbonate and the monovalent cations potassium or ammonium. However sodium and the divalent cations copper, cobalt, zinc, cadmium, and manganese decrease binding to this N‐methyl‐D‐aspartate site.

Sigma receptors modulate both A9 and A10 dopaminergic neurons in the rat brain: functional interaction with NMDA receptors

The sigma receptor ligands, (+)-pentazocine and (+)-SKF 10,047, were found to increase dopamine metabolism (DOPAC, HVA) and release (3-MT) in both the striatum and olfactory tubercle of the rat, in a dose-dependent manner, after central as well as peripheral administration. The effect of (+)-SKF 10,047 was stereospecific. The increase in dopamine metabolism was not blocked by naloxone pretreatment, excluding an action via opioid receptors. More interestingly, this modulation was blocked by pretreatment with the NMDA receptor antagonist, CPP. Neither sigma ligand exhibited any affinity for D1 or D2 dopamine receptors or for NMDA, PCP or NMDA-associated glycine receptors. Sigma receptors thus appear to modulate dopaminergic function in both A9 and A10 projections. This modulation appears to involve a functional interaction with NMDA receptors or an NMDA-utilizing synapse downstream to neurons modulated by sigma receptors.

A Review of the In Vitro and In Vivo Neurochemical Characterization of the NMDA/PCP/Glycine/Ion Channel Receptor Macrocomplex*

ConclusionsCurrent neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.

Glycine modulation of the phencyclidine binding site in mammalian brain.

Neurophysiological studies have shown that glycine potentiates the NMDA response in cultured neurons by a strychnine-insensitive mechanism. Autoradiographic data have demonstrated a correspondence between strychnine-insensitive [3H]glycine binding sites and NMDA-sensitive [3H]glutamate binding sites. Here we report that in synaptic plasma membranes from rat brain, the binding of a PCP analog, [3H]TCP, was enhanced more than 5-fold by 1 microM glycine. This glycine stimulation of binding of [3H]TCP was blocked by the competitive NMDA-receptor antagonist, D-AP7. These data provide support for the hypothesis that a unique amino acid recognition site is associated with the proposed NMDA/PCP receptor complex in brain.

N-Methyl-D-Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site

Abstract: In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1‐[1‐(2‐thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine‐insensitive [3H]glycine binding are altered in the presence of N‐methyl‐D‐aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis‐4‐phosphonomethyl‐2‐piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L‐glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 γM vs. 0.129 γM). Similar decreases in affinity are observed for the glycine site agonists, D‐serine and 1‐aminocyclopropane‐1‐carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1‐hydroxy‐3‐amino‐2‐pyrrolidone and 1‐aminocyclobutane‐1‐carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L‐glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

cis-2,4-methanoglutamate is a potent and selective N-methyl-D-aspartate receptor agonist

Cis- and trans-2,4-methanoglutamate were compared with L-glutamate as acidic amino acid ligands. Cis-2,4-methanoglutamate had a Ki of 0.052 microM against N-methyl-D-aspartate (NMDA)-specific L-[3H]glutamate binding compared with 0.050 microM for L-glutamate. Cis-2,4-methanoglutamate exhibited no significant affinity against [3H]kainate or [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate ([3H]AMPA) binding. Trans-2,4-methanoglutamate had no significant affinity for any of these sites. Cis-2,4-methanoglutamate increased [3H]N-1[2-thienyl]cyclohexyl-3,4-piperidine [( 3H]TCP) binding with EC50 of 0.35 +/- 0.14 microM. It produced an inward current in rat brain mRNA-injected Xenopus oocytes which was blocked by the NMDA antagonist, D-2-amino-7-phosphonoheptanoate (D-AP7). Cis-2,4-methanoglutamate (EC50 = 15.9 microM) was 100-fold more potent than L-glutamate (EC50 = 1,584 microM) in reducing the excitatory postsynaptic potential in CA1 of hippocampal slices. Cis-2,4-methanoglutamate is the most potent, selective NMDA agonist known.

Evidence for a functional coupling of the NMDA and glycine recognition sites in synaptic plasma membranes

Activation of the N-methyl-D-aspartate (NMDA) receptor complex is subject to modulation via interactions at a coupled [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM). We examined the effect of the potent and specific glycine site antagonists, 1-hydroxy-3-amino-2-pyrrolidone (HA-966) and 1-aminocyclobutane-1-carboxylate (ACBC), on the NMDA recognition site. These glycine analogs were found to significantly stimulate the binding of the competitive NMDA antagonist, [3H]3-(2-carboxypiperazin-4-y1)propyl-1-phosphonate ([3H]CPP) in a dose-dependent fashion, whereas both compounds inhibited NMDA-specific L-[3H]glutamate (agonist) binding. Additionally, both glycine antagonists reduced the binding of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to SPM, a functional assessment of activation of the NMDA receptor-channel complex. The glycine site agonists, glycine and serine reversed these effects in a dose-dependent manner, with the serine reversal being stereospecific for D-serine. The relative potencies of these compounds in reversing the glycine antagonist effects on the NMDA recognition site corresponded with their ability to competitively displace strychnine-insensitive [3H]glycine binding. These results provide evidence for a functional coupling between the glycine and NMDA recognition sites and further, may provide a mechanism by which compounds interacting at the glycine recognition site may modulate NMDA receptor activity.

[3H]MK-801 binding in Alzheimer's disease

The density of [3H]MK-801 binding sites was studied in homogenates prepared from different cortical regions of postmortem brains of Alzheimer patients and age matched controls. Highest number of binding sites for this noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor was in temporal pole (Brodmann area A-38) in both groups. There were no consistent differences between patients and controls in either binding density or affinity constant in any of the areas studied. Involvement of glutamatergic neurotransmission in Alzheimers disease does not seem to be at the level of the MK-801 recognition site on the NMDA receptor complex.