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6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [3H]NBQX binding to rat brain. The association rate of [3H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0 degree C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 mM unlabelled L-glutamate. [3H]NBQX bound to a single class of sites with KD and Bmax values of 47 nM and 2.6 pmol mg-1 of protein, respectively. The rank order of inhibition of [3H]NBQX binding by AMPA receptor ligands was NBQX > > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > or = (S)-5-fluorowillardiine > or = AMPA > > L-glutamate. The chaotrope KSCN had no effect on the IC50 value of unlabelled NBQX displacement of [3H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [3H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [3H]NBQX to coronal sections showed a distribution compatible with that of [3H]AMPA binding. These data indicate that [3H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.

The effects of exogenous phospholipase A2 on the binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) to rat cortical membranes in the presence of the chaotrope potassium thiocyanate were assessed. Pretreatment of membranes with secretory phospholipase A2 (sPLA2) elicited a concentration-dependent decrease in specific [3H]AMPA binding due mainly to a decrease in affinity (KD). This observation, together with protease inhibitor and western blot evidence, suggest that the sPLA2 effect is not due to proteolysis. The sPLA2-evoked decrease was temperature and calcium dependent. Inclusion of the specific inhibitor oleoyloxyethyl phosphocholine or preincubation of the enzyme with reducing agents to degrade its secondary structure significantly reduced the sPLA2 inhibition. These results suggest that the effects of sPLA2 arise from an enzymatic action rather than a competitive interaction at the AMPA binding site. However, arachidonic acid, a major metabolite of sPLA2 action, did not cause a similar decrease in the affinity of [3H]AMPA binding. In contrast to the effects on [3H]AMPA binding, sPLA2 caused an increase in [3H]CNQX binding, which is in accordance with the functionality of the AMPA receptor complex. These results suggest that sPLA2 may play a role in the physiological and pathophysiological regulation of AMPA receptors.

Recent reports have suggested that a major proportion of [3H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [3H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTP gamma S resulted in an approximately twofold decrease in the affinity of [3H]kainate binding and a 50% reduction in the apparent Bmax values in both Mg2+/Na+ and Mg2+/Na(+)-free buffer when assayed at 0 degrees C. The pharmacology of [3H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known metabotropic glutamate receptors, with neither 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) nor ibotenic acid being effective competitors. The molecular mass of the [3H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP gamma S also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[3H]cyano-7-nitroquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [3H]kainate and 6-[3H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.

alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) binds to 1-day-old chick telencephalon membranes with KD and Bmax values of 138 nM and 2.56 pmol/mg of protein, respectively. High-energy radiation bombardment of intact frozen telencephalon resulted in a biphasic inactivation curve for [3H]AMPA binding. At a 5.8-Mrad radiation dose, the affinity of [3H]AMPA binding was increased (54 nM), but there was no apparent alteration in the Bmax value (2.76 pmol/mg of protein). We attribute this phenomenon to the inactivation of a high molecular weight modulatory protein that down-regulates the affinity of [3H]AMPA binding. The estimated molecular masses of the AMPA binding site and of the modulatory component were 59 and 108 kDa, respectively. Solubilisation with n-octyl-beta-glucopyranoside resulted in an increase in the Bmax (4.7 pmol/mg of protein) with no pronounced alteration in the affinity (109 nM) of [3H]AMPA binding. However, the solubilisation-induced increase in Bmax did not occur in telencephalon irradiated before solubilisation. In contrast, the increase in affinity induced by radiation treatment was still detected in solubilised extracts. These results suggest that the number and affinity of [3H]AMPA sites in chick telencephalon are closely regulated and that the modulatory systems involved are affected by both irradiation and solubilisation.

[3H]Kainate bound to chick cerebellar membranes with a KD of 0.6 microM and with an exceptionally high Bmax of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [3H]kainate was reduced (KD = 2.7 microM), but the Bmax was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate greater than kainate greater than 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) greater than glutamate. Binding sites for alpha-[3H]amino-3- hydroxy-5-methylisoxazolepropionate ([3H]AMPA) were much less abundant, with KD and Bmax values in membranes of 86 nM and 1 pmol/mg of protein, respectively. The affinity of [3H]AMPA binding was also reduced on solubilisation (KD = 465 nM), but there was an increase in the Bmax (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [3H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [3H]kainate, domoate was markedly less potent than kainate at displacing [3H]AMPA. These results suggest that [3H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [3H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non-N-methyl-D-aspartate receptors.

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]Kainate binding to membrane homogenates and detergent extracts prepared from Xenopus central nervous system was evaluated in 50 mM Tris-citrate buffer, pH 7.0. In membrane fragment preparations, [3H]kainate bound with a KD of 54.4 nM to a large number of sites (Bmax = 27.8 pmol/mg of protein). Up to 80% of the total number of membrane-bound binding sites were solubilised using the nonionic detergent n-octyl-beta-D-glucopyranoside. Values for the KD of [3H]kainate for solubilised binding sites were 46.0 nM and 53.6 nM derived from equilibrium and kinetic binding experiments, respectively. Competitive binding studies revealed that a variety of ligands had similar Ki values in both membranes and solubilised extracts, with domoate and kainate being the most potent inhibitors of [3H]kainate binding. The dissociation rate of [3H]kainate from solubilised binding sites was 0.022 min-1. The binding component migrated in sucrose density gradients in a single 8.6S peak. These results demonstrate that the kainate receptor in Xenopus central nervous system, although similar to the [3H]kainate binding site from goldfish brain, differs in a number of important respects. In particular, the slower dissociation rate and higher affinity of [3H]kainate suggest that Xenopus provides the most convenient model system yet investigated for biochemical analysis of kainate receptors.