John A. Kemp

Ionotropic and metabotropic glutamate receptor structure and pharmacology

Rationalel-Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and mediates its actions via activation of both ionotropic and metabotropic receptor families. The development of selective ligands, including competitive agonists and antagonists and positive and negative allosteric modulators, has enabled investigation of the functional roles of glutamate receptor family members.ObjectiveIn this review we describe the subunit structure and composition of the ionotropic and metabotropic glutamate receptors and discuss their pharmacology, particularly with respect to selective tools useful for investigation of their function in the CNS.ResultsA large number of ligands are now available that are selective either for glutamate receptor subfamilies or for particular receptor subtypes. Such ligands have enabled considerable advances in the elucidation of the physiological and pathophysiological roles of receptor family members. Furthermore, efficacy in animal models of neurological and psychiatric disorders has supported the progression of several glutamatergic ligands into clinical studies. These include ionotropic glutamate receptor antagonists, which have entered clinical trials for disorders including epilepsy and ischaemic stroke, α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor positive allosteric modulators which are under evaluation as cognitive enhancers, and metabotropic glutamate receptor 2 (mGluR2) agonists which are undergoing clinical evaluation as anxiolytics. Furthermore, preclinical studies have illustrated therapeutic potential for ligands selective for other receptor subtypes in various disorders. These include mGluR1 antagonists in pain, mGluR5 antagonists in anxiety, pain and drug abuse and mGluR5 positive allosteric modulators in schizophrenia.ConclusionsSelective pharmacological tools have enabled the study of glutamate receptors. However, pharmacological coverage of the family is incomplete and considerable scope remains for the development of novel ligands, particularly those with in vivo utility, and for the their use together with existing tools for the further investigation of the roles of receptor family members in CNS function and as potentially novel therapeutics.

Non-competitive antagonists of excitatory amino acid receptors

Abstract Non-competitive antagonists have become important tools for investigating the basic mechanisms of NMDA receptor function. Such compounds (e.g. MK-801, PCP) are thought to act at the level of the NMDA receptor-associated ion channel and many show a marked use-dependence in their antagonist properties. Little information is available concerning selective non-competitive antagonists of quisqualate or kainate receptors. A number of structurally diverse compounds act as non-competitive antagonists of glutamate receptors at invertebrate neuromuscular junctions, although it remains to be determined whether these compounds have effects at excitatory amino acid receptors in the mammalian CNS. The ability of the non-competitive NMDA receptor antagonists to penetrate the brain following systemic administration, and the use-dependency of their antagonism may confer distinct therapeutic advantages in the treatment of neurological disorders where an overstimulation of NMDA receptors is thought to occur (such as in epilepsy or cerebral ischaemia).

The glycine site of the NMDA receptor - five years on

The glycine site on the NMDA receptor complex has generated an enormous amount of interest since it was first described five years ago. In this review by John Kemp and Paul Leeson the structure-activity relationships of agonists, partial agonists and antagonists acting at the glycine site are reviewed, along with what has been learned from studies with these compounds about the role of this site in physiological and pathological processes. Studies with prototype antagonists, and low-efficacy partial agonists that penetrate the brain, show that these compounds possess anticonvulsant and neuroprotective properties but lack some of the side-effects of other types of NMDA antagonists.

NMDA receptor pathways as drug targets

Since the mid 1980s, there has been a great deal of enthusiasm within both academia and industry about the therapeutic potential of drugs targeting the NMDA subtype of glutamate receptors. That early promise is just beginning to translate into approvable drugs. Here we review the reasons for this slow progress and critically assess the future prospects for drugs that act on NMDA receptor pathways, including potential treatments for some major disorders such as stroke and Alzheimers disease, for which effective therapies are still lacking.

Cholinergic modulation of the functional organization of the cat visual cortex

The cortex receives a cholinergic input which is considered to be involved in mediating the effects of arousal. The experiments reported here have examined the nature of the cholinergic influence on the neuronal organization of the cat visual cortex. Out of 83 cells studied, 92% exhibited a modification in their visual response properties during the iontophoretic application of ACh. These comprised 61% in which responses were facilitated and 31% in which responses were depressed. The facilitatory effects were associated with a striking increase in stimulus specific responses without any concomitant loss in the selectivity. This comment applied equally to orientation and direction selectivity. It is argued that the facilitatory action of ACh on stimulus specific responses is consistent with a modulation of potassium conductance and most probably the conductance associated with a voltage dependent channel. We found no evidence to support the view that the facilitatory action involved disinhibition; the action of bicuculline, which blocks inhibitory influences in the visual cortex, was quite distinct to that of ACh. The facilitatory and depressive effects of ACh did not show any correlation with the simple-complex classification of cells or any other obvious parameter of receptive field organization, but there was a correlation with cortical lamination. Cells facilitated by ACh were found in all cortical laminae, but those depressed by ACh were found in laminae III and IV.

A re-evaluation of the mechanisms underlying simple cell orientation selectivity

Following from evidence supporting GABA as a putative inhibitory transmitter in the visual cortex, we have iontophoretically applied the GABA antagonist N-methyl bicuculline (Nmb) to simple cells in order to block the inhibitory inputs acting on them. We found that under these conditions previously sharply-tuned simple cells responded equally to all orientations. Moreover receptive field dimensions, judged by the response to stimuli at the optimal and orthogonal orientations, equated best with that expected from a single dLGN cell input. It seems thus, that asymmetries in the excitatory input are not a significant factor in the generation of simple cell orientation selectivity. The asymmetry underlying orientation selectivity rather originates from the operation of an intracortical inhibitory mechanism.

Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site.

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.

A novel allosteric modulatory site on the GABAA receptor β subunit

Abstract Cloning of cDNAs that code for GABA A receptor subunits has revealed multiple receptor populations constructed from different subunit combinations. On native rat and cloned human GABA A receptors, the anticonvulsant compound loreclezole strongly potentiated GABA-mediated chloride currents. Using different combinations of human GABA A receptor subunits expressed in Xenopus oocytes and transfected 293 cells, loreclezole was highly selective for receptors containing the β2 or β3 subunit over those containing the β1 subunit. Loreclezole was demonstrated to act at a site distinct from the benzodiazepine, barbiturate, and steroid sites with a unique subunit dependence. These results describe a previously unidentified modulatory site on the GABA A receptor β subunit that allows pharmacological discrimination of different GABA A receptor subpopulations in the brain and provides a new target for putative anticonvulsant/anxiolytic drugs.

Preferential co-assembly of recombinant NMDA receptors composed of three different subunits

cDNAs ENCODING NMDA receptor subunits have recently been cloned and been shown to have different distributions in the CNS. However, no studies on the possible in vivo combinations or subunit stoichiometry have yet been carried out. By combining human NR1 with rat NR2A and NR2C we have studied the pharmacological properties of three possible NMDA receptor subtypes; NR1 + NR2A, NR1 + NR2C and NR1 + NR2A + NR2C. By performing glycine concentration-response curves and comparing EC50s, it was possible to show that the NR1 + NR2A + NR2C receptor preferentially co-assembled when all three subunit cDNAs were present. This receptor had an affinity for glycine intermediate between that of NR1 + NR2A and NR1 + NR2C, but a similar Hill coefficient. Thus, two different NR2 subunits can combine in the same receptor, conferring unique pharmacological properties, suggesting that it is likely that two or more NR2 subunits co-assemble together in the same NMDA receptor complex.

The cholinergic influence on the function of the cat dorsal lateral geniculate nucleus (dLGN).

The functional influence of the cholinergic input to cat dLGN has been examined by assessing the action of iontophoretically applied acetylcholine (ACh) on the visual responses of cells in layers A and A1. Iontophoretically applied pulses of ACh exerted a strong excitatory action on all 113 cells studied within these layers. In the presence of a sustained application of ACh, the excitatory responses to an optimal stimulus such as a spot of light located within the receptive field centre were greatly facilitated, but at the same time stimulus-specific inhibitory influences were also enhanced. The action of ACh on the stimulus-specific inhibitory influences had the consequence that the responses to non-optimal stimuli were not facilitated to the same extent as those to optimal stimuli and in some cases even diminished. The stimulus-specific inhibitory effects seen in the presence of ACh were very powerful and frequently resulted in complete suppression of the elevated background discharge. We suggest that the ACh directly excites both the relay cells and the Golgi type II inhibitory interneurones within the dLGN. The facilitation of the stimulus-specific inhibition may follow from a direct action on the presynaptic dendrites of the Golgi type II cells which arborize within the dendritic field of the relay cell. Supplementary observations on cells in the perigeniculate nucleus confirm previous findings showing that ACh has an inhibitory effect on these cells. We suggest a tripartite action for the cholinergic influence on the dLGN, involving direct facilitation of relay cells, enhancement of stimulus-specific inhibition via the Golgi type II cells, and disinhibition of the non-specific inhibitory influence form the perigeniculate nucleus.