Geoffrey Neil Woodruff

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

Abstract: 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 homogenous 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 > cortex > olfactory bulb = striatum > medulla‐pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]HIMK‐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 σ 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.

Light microscopic autoradiographic localisation of [3H] glycine and [3H] strychnine binding sites in rat brain

Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive.

The neuroprotective action of dizocilpine (MK‐801) in the rat middle cerebral artery occlusion model of focal ischaemia

1 An acute model of focal ischaemia, which involves permanent occlusion of the middle cerebral artery of the rat with 4 h survival, was used to find the minimum effective plasma concentration of dizocilpine (MK‐801) and to determine its dose‐effect relationship. 2 MK‐801 was administered at the time of occlusion and was given as an i.v. bolus followed by an infusion for 4 h to maintain a steady state plasma concentration of the drug throughout the study. MK‐801 was given at 3 dose levels; 0.04 mg kg−1 i.v. bolus + 0.6 μg kg−1 min−1 infusion; 0.12 mg kg−1 i.v. bolus + 1.8 μg kg−1 min−1 infusion; 0.4 mg kg−1 i.v. bolus + 6 μg kg−1 min−1 infusion, which gave mean plasma levels over the 4h of 8.0 ng ml−1, 18.9 ng ml−1 and 113.2 ng ml−1 respectively. 3 MK‐801 at 8.0 ng ml−1 gave 10% reduction in the volume of ischaemic brain damage in the cerebral cortex which just reached significance. The middle dose of MK‐801 (18.9 ng ml−1) gave a highly significant reduction in the volume of ischaemic brain damage in the cerebral cortex and hemisphere, volumes of ischaemic tissue being reduced by 60% and 50% compared to saline‐treated animals, respectively. The highest plasma concentration of MK‐801 (113.2 ng ml−1) resulted in a 35% reduction in the volume of hemispheric damage and a 40% reduction in the volume of cortical damage, which were significant. 4 The reduction in the amount of protection afforded by the highest dose of MK‐801 may be due to the hypotensive effect of this dose. There was no protection against the volume of damage in the caudate nucleus for any of the doses of MK‐801 tested. 5 Therefore the minimum effective plasma concentration of MK‐801 was 8.0 ng ml−1, although the greatest protection was seen with a plasma level of 18.9 ng ml−1. This correlates well with the concentration of MK‐801 required to block N‐methyl‐d‐aspartate (NMDA) receptors and prevent NMDA receptor mediated neurotoxicity in vitro.

The neuroprotective actions of kynurenic acid and MK-801 in gerbils are synergistic and not related to hypothermia

The broad spectrum excitatory amino acid antagonist, kynurenic acid was evaluated in a transient forebrain ischaemia model in gerbils. When administered i.p., 15 min prior to a 5 min period of ischaemia, a dose-related neuroprotective effect was seen with 800 mg/kg of kynurenic acid showing very good protection. A combination of kynurenic acid (200 or 400 mg/kg) and MK-801 (0.1 mg/kg) gave a synergistic neuroprotective effect. Neither kynurenic acid (200 or 400 mg/kg) nor MK-801 (0.3 mg/kg) was neuroprotective when administered by itself 30 min post-ischaemically, but when co-administered significant protection of the CA1 pyramidal neurones of the hippocampus was seen. In addition, we examined the effect of maintaining core body temperature on the neuroprotective action of MK-801 and kynurenic acid following the suggestion that it was a hypothermic effect of MK-801 which resulted in neuroprotection in gerbils. When the body temperature of the gerbils was maintained at 37 degrees C for a period of 24 h it did not affect the neuroprotective action of MK-801 (0.1 or 10 mg/kg) or kynurenic acid (200 mg/kg).

Antagonism of responses to excitatory amino acids on rat cortical neurones by the spider toxin, argiotoxin636

1 Several low molecular weight spider toxins have recently been shown to block potently glutama‐tergic neuromuscular transmission at the invertebrate neuromuscular junction. The aim of the present investigation was to evaluate the effects of one such toxin, argiotoxin636, on excitatory amino acid receptor‐mediated responses in mammalian neurones. 2 Membrane currents were recorded from rat cortical neurones after 2–6 weeks in cell culture, by the whole‐cell variant of the patch‐clamp technique. N‐methyl‐D‐aspartate (NMDA) and kainate were used as selective agonists for their respective receptor subtypes. α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) was used as a selective agonist for the quisqualate receptor subtype. 3 Responses to these agonists were characterised with respect to their concentration and voltage‐dependence. Argiotoxin636 (3–30 μm) was found to attenuate markedly responses to NMDA in an agonist‐ and voltage‐dependent manner. Thus, argiotoxin636 progressively reduced successive responses to NMDA when membrane potentials were voltage clamped between −40 mV to −100 mV. The more negative the membrane potential the more rapid the development of the block of inward current. 4 The antagonism of NMDA‐induced currents by argiotoxin636 could be reversed by clamping the membrane at positive potentials (+ 20 to + 60 mV) and reapplying NMDA. 5 Responses to AMPA and kainate were less affected by argiotoxin636, with an antagonist action only becoming evident at a concentration of 100 μm 6 These results suggest that argiotoxin636 is an open‐channel blocker of the NMDA activated ion‐channel in mammalian neurones. Furthermore, our results indicate at least a 30 fold selectivity for NMDA over the quisqualate‐ and kainate‐activated ion‐channels.

In vivo interaction of a polyamine with the NMDA receptor

We have investigated the ability of spermidine to modulate N-methyl-DL-aspartic acid (NMDLA)-induced tonic seizures in mice. Here we present evidence that the polyamine site may not normally be fully activated and that interactions at this site may have important consequences in the whole animal

The affinities, potencies and efficacies of some benzodiazepine-receptor agonists, antagonists and inverse-agonists at rat hippocampal GABAA-receptors

1 The abilities of some benzodiazepine‐receptor agonists, antagonists and inverse agonists to modulate the inhibitory potency of the γ‐aminobutyric acid (GABA)A‐receptor agonist, isoguvacine, on the CA1 population spike recorded from slices of rat hippocampus, were determined. 2 Concentration‐response curves were constructed of the extent to which the benzodiazepine‐receptor ligands shifted the isoguvacine concentration‐response curve to the left or right. These were compared to their displacement curves of [3H]‐Ro15‐1788 binding to rat hippocampal membranes under near physiological assay conditions. 3 The above comparisons suggest that the effect on the potency of isoguvacine produced by the benzodiazepine‐receptor agonists, diazepam and flunitrazepam, and the partial agonists, Ro16‐6028 and Ro17‐1812, closely parallels their degree of benzodiazepine‐receptor occupancy. Thus, the partial agonists, Ro16‐6028 and Ro17‐1812, were unable to produce as large a maximum response as the full agonists, diazepam and flunitrazepam. 4 The maximum effects produced by diazepam, flunitrazepam, Ro16‐6028, Ro17‐1812, the antagonist, propyl‐β‐carboline‐3‐carboxylate, and the inverse agonist, methyl‐6, 7‐dimethyl‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM), on the potency of isoguvacine in the hippocampal slice corresponded to the change in their affinities produced by the addition of GABA in the radioligand binding studies (GABA‐shift). This suggests that the changes in affinity of benzodiazepine‐receptor ligands produced by GABAA‐receptor activation reflects their ability to modify GABAA‐receptor function. 5 The benzodiazepine‐receptor antagonists, Ro15‐1788 and CGS 8216, had apparent agonist and inverse agonist effects, respectively, on the potency of isoguvacine. These effects occurred at concentrations above those required for saturation of the benzodiazepine‐receptor, as labelled by [3H]‐Ro15‐1788, and were not in agreement with the absence of any effect of GABAA‐receptor stimulation in the GABA‐shift experiments. This indicates that these events are not mediated by an action at the classical benzodiazepine‐receptor site. 6 It is concluded that hippocampal GABAA‐receptor function can be allosterically modulated in a manner consistent with the agonist/inverse‐agonist model of benzodiazepine‐receptor activation, and that compounds exist with varying efficacies throughout this range.

Similar binding of 3H-ADTN and 3H-apomorphine to calf brain dopamine receptors

The binding of 3H(+/-)-ADTN (of high specific activity; 7.6 Ci/mmole) to homogenates of calf striatum was investigated. The dissociation constant (KD) for the specific, saturable binding of 3H-(+/-)-ADTN was 1 nM and the density of specific sites was 100 fmoles/mg protein. The IC50 values (nM concentrations inhibiting specific binding by 50%) were 0.9 for (+/-)-N-propyl-norapomorphine, 3.0 for dopamine, 7 for (--)-adrenaline, 60 for (--)-noradrenaline and 4000 for isoproterenol, a series of potencies compatible with properties for a dopaminergic site. The (+)-enantiomer of ADTN was 10 times more potent than (--)-ADTN in competing for 3H-(+/-)-ADTN, while the (--)-enantiomer of 5-OH-dipropyl-ATN was 40 times more potent than the (+)-isomer. The IC50 values for various agonists against 3H-(+/-)-ADTN were similar to those against 3H-apomorphine or 3H-dopamine in the calf striatum. A comparison of these 3H-(+/-)-ADTN data to those for 3H-spiperone suggests that the two 3H-ligands label different receptor sites.

The inhibitory effect of somatostatin peptides on the rat anococcygeus muscle in vitro

1 Electrically evoked contractions of the rat anococcygeus muscle were inhibited in a concentration‐dependent manner by somatostatin‐14 (SS14), −28 (SS28) and two synthetic hexapeptide analogues: L‐363,301 and L‐363,586 , with pIC50 values of 7.41, 7.38, 7.07 and 8.34, respectively. 2 The inhibitory effects of SS14 were dependent on stimulation frequency and external calcium ion concentration. Calcium behaved as a non‐competitive antagonist of SS14, it reduced the maximal inhibitory effect of the peptide and at a concentration of 5.08 mm it significantly affected the pIC50 value. 3 SS14 (3 times 10−7 m) did not affect the tonic actions of bath‐applied noradrenaline in the absence of field stimulation. 4 The effects of SS14 persisted in naloxone (10−5 m) and were, therefore, not due to an action at opiate receptors. Furthermore, experiments involving the lyophilisation of bath contents, showed no evidence to support an indirect mechanism involving the release of an endogenous inhibitory substance. 5 High concentrations (10−5 m) of SS14 or L‐363,301 inhibited the relaxation response evoked by electrical stimulation of guanethidine (3 times 10−4 m)‐treated preparations. 6 These results are consistent with similar actions of SS14 on other smooth muscle preparations and are presumed to reflect a presynaptic inhibition of transmitter release by a direct action on somatostatin receptors. The antagonistic effect of calcium on this response is discussed with reference to a possible role in receptor desensitization.

The characterization of [3H]sulpiride binding sites in pig striatal membranes

Pig striatal membranes have [3H]sulpiride-binding sites similar to those identified in rat striatal membranes. The pharmacological profile indicates that this binding is to dopamine receptors. Agonist displacement of [3H]sulpiride binding in pig striatal membranes is subject to guanine nucleotide regulation. This effect is mimicked by heat treatment. N-ethyl maleamide (20 microM) and dithioerythritol (3 mM) decrease agonist affinity for the [3H]sulpiride-binding site in pig striatal membranes without significantly affecting maximal displacement.