Janine M. Barnes

Cyclothiazide unmasks AMPA‐evoked stimulation of [3H]‐L‐glutamate release from rat hippocampal synaptosomes

The effect of α‐amino‐3‐hydroxy‐5‐methylisoxazolepropionate (AMPA) on Ca2+‐sensitive, tetrodotoxin (TTX)‐insensitive K+‐stimulated [3H]‐L‐glutamate release from rat hippocampal synaptosomes was determined. AMPA in the presence, but not in the absence of cyclothiazide, a drug which blocks AMPA receptor desensitization, elicited a dose‐dependent increase in K+‐stimulated [3H]‐L‐glutamate release but had no effect on basal release. The AMPA/cyclothiazide stimulation was blocked by CNQX and by GYKI 52466, an antagonist at the cyclothiazide site. These results indicate that AMPA receptors are present on presynaptic terminals and suggest that they may play a role in the regulation of neurotransmitter release.

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— 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 GTPγS 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°c. The pharmacology of [3H]kainate binding is similar to that of well‐characterised ionotropic kainate receptors but unlike that of known me‐tabotropic glutamate receptors, with neither 1S,3R‐amino‐1,3‐cyclopentanedicarboxylic acid (1S,3R‐ACPD) nor ibo‐tenic 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‐γS also inhibited the binding of the non‐NMDA receptor‐selective antagonist 6‐[3H]cyano‐7‐ni‐troquinoxaline‐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.

Autoradiographic distribution of [3H]-(S)-zacopride-labelled 5-HT3 receptors in human brain

Autoradiographic binding studies using the 5-HT3 (5-hydroxytryptamine3) receptor radioligand, [3H]-(S)-zacopride (0.5 nM), identified a heterogeneous distribution of specific binding sites (defined by granisetron, 1 microM) throughout the human brain. Highest radiolabelled 5-HT3 receptor densities were detected in discrete nuclei within the brainstem (nucleus tractus solitarius, area postrema, spinal trigeminal nerve nucleus; 50-200 fmol/mg tissue equivalent) with more modest levels of expression in the forebrain (e.g. hippocampus, nucleus accumbens, putamen, caudate; 4-17 fmol/mg tissue equivalent). Within the hippocampal formation, radiolabelled 5-HT3 receptors were differentially distributed with highest levels in the granule cell layer of the dentate gyrus. Saturation studies with [3H]-(S)-zacopride (0.05-16 nM; non-specific binding defined by granisetron, 10 microM) binding to homogenates of human putamen indicated that [3H]-(S)-zacopride (0.05-16 nM; non-specific binding defined by granisetron, 10 microM) binding to homogenates of human putamen indicated that [3H]-(S)-zacopride labelled an apparently homogenous population of binding sites (Bmax = 72 + 7 fmol mg-1 protein, pKd = 8.69 +/- 0.09, Hill coefficient = 0.99 +/- 0.06, mean +/- SEM, n = 4). The pharmacological profile of [3H]-(S)-zacopride binding to homogenates of putamen indicated the selective labelling of the human variant of the 5-HT3 receptor. The marked differences, however, in the pharmacology (e.g. low affinity for D-tubocurarine) and relative distribution (e.g. presence of 5-HT3 receptors in the human extrapyramidal system) of 5-HT3 receptors in the human forebrain when compared with other species further necessitates caution in predicting clinical responses based on data generated in animal models of disease.

Behavioural pharmacology of 5-HT3 receptor ligands

Extensive studies have ascribed a role for the central 5-HT3 receptor in the modulation of behaviour. Much of the work stems from the actions of potent and selective 5-HT3 receptor antagonists; these agents reduce mesolimbic dopamine initiated hyperactivity, release suppressed behaviour, reduce the reinforcing properties and withdrawal symptoms of drugs of abuse, enhance cognitive performance and modulate appetite. This article reviews the preclinical and clinical evidence implicating the 5-HT3 receptor in these indications and discusses the potential neurochemical mechanisms underlying the behavioural changes.

Identification and characterisation of angiotensin II receptor subtypes in human brain

Autoradiographic and homogenate binding studies using the radioligand, [125I]angiotensin II, identified a heterogeneous distribution of specific binding sites (defined by angiotensin II, 1.0 microM) throughout the human forebrain. Highest AT receptor densities were detected in the paraventricular nucleus, median eminence, substantia nigra, putamen and caudate nucleus (2.4, 1.2, 1.0, 0.30 and 0.24 fmol/mg tissue equivalent, respectively). The AT1 receptor antagonist, losartan (1.0 microM) competed for the majority of the specific binding. [125I]Angiotensin II-specific binding (although not consistently above non-specific binding levels) was also detected in various other brain regions (e.g. amygdala, entorhinal cortex, frontal cortex, hippocampus, inferior colliculus, nucleus accumbens, parietal cortex, periaquaductal grey, superior colliculus, striate cortex, temporal cortex, thalamus). In the presence of losartan (1.0 microM), angiotensin II, saralasin, losartan and PD123177 competed for [125I]angiotensin II binding to membranes prepared from the cerebellum or substantia nigra with a rank order of affinity; angiotensin II = saralasin > PD123177 > losartan. In the presence of PD123177 (1.0 microM), the rank order of affinity of losartan and PD123177 was reversed. These studies indicate the presence of both AT1 and AT2 receptor subtypes within various regions of the human forebrain.

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1 The pharmacological properties of 5‐HT3 receptor recognition sites labelled with [3H]‐(S)‐zacopride, [3H]‐LY278,584, [3H]‐granisetron and [3H]‐GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5‐HT3 receptor complex. 2 In rat entorhinal cortex homogenates, [3H]‐(S)‐zacopride, [3H]‐LY278,584, [3H]‐granisetron and [3H]‐GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 μm) with high affinity (Bmax = 75 ± 5, 53 ± 5, 92 ± 6 and 79 ± 6 fmol mg−1 protein, respectively; pKd = 9.41 ± 0.04, 8.69 ± 0.14, 8.81 ± 0.06 and 10.14 ± 0.04 for [3H]‐(S)‐zacopride, [3H]‐LY278,584, [3H]‐granisetron and [3H]‐GR67330, respectively, n = 3–8). 3 Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38–8.51, granisetron 8.62–8.03), whilst the agonists, 5‐hydroxytryptamine (5‐HT), phenylbiguanide (PBG) and 2‐methyl‐5‐HT competed at sub‐micromolar concentrations (pIC50; 5‐HT 7.16–6.42, PBG 7.52–6.40, 2‐methyl‐5‐HT 7.38–6.09). 4 Competition curves generated with increasing concentrations of quipazine, PBG, 5‐HT and 2‐methyl‐5‐HT displayed Hill coefficients greater than unity when the 5‐HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]‐LY278,584, [3H]‐granisetron and [3H]‐GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]‐(S)‐zacopride. Competition for the binding of [3H]‐(S)‐zacopride, [3H]‐LY278,584, [3H]‐granisetron and [3H]‐GR67330 by granisetron generated Hill coefficients around unity. 5 The nature of the interaction of competing compounds (quipazine, granisetron, PBG, 5‐HT, 2‐methyl‐5‐HT) for the [3H]‐(S)‐zacopride binding site in the rat entorhinal cortex preparation was not altered by the removal of the Krebs ions or the addition of the monoamine oxidase inhibitor, pargyline, to the HEPES/Krebs buffer. 6 In conclusion, the present studies provide further evidence towards the presence of cooperativity within the 5‐HT3 receptor macromolecule and indicate that either [3H]‐(S)‐zacopride labels a different site on the receptor complex from [3H]‐LY278,584, [3H]‐granisetron or [3H]‐GR67330, or it binds in such a manner as to prevent the conformatory change in the receptor protein responsible for the cooperative binding of agonists (and quipazine).

Identification of angiotensin II receptor subtypes in human brain.

The present study assessed the binding characteristics of [125I]angiotensin II to slices of human cerebellum adhered to glass slides using quantitative receptor autoradiography. Specific [125I]angiotensin II binding, defined by the inclusion of unlabelled angiotensin II (1.0 microM), was detected in the molecular layer of the cerebellum (0.09 +/- 0.02 fmol/mg tissue equivalent, mean +/- s.e.m., n = 3). The angiotensin II-2 receptor subtype selective ligand, PD123177, competed for approximately 65% of the specific binding in the molecular layer whilst the remainder of the specific binding was displaced by the angiotensin II-1 receptor subtype selective ligand, DuP753. It is concluded that angiotensin II receptor subtypes exist in human brain tissue and provide potential therapeutic sites of action.

Differential binding characteristics of agonists at 5-HT3 receptor recognition sites in NG108-15 neuroblastoma-glioma cells labelled by [3H]-S-zacopride and [3H]granisetron

The pharmacological characteristics of 5-HT3 receptor (5-hydroxytryptamine3 receptor) recognition sites labelled with [3H]-(S)-zacopride and [3H]granisetron in membranes prepared from NG108-15 neuroblastoma-glioma cells were directly compared to investigate further differences in the binding characteristics of these two radioligands. Competition curves generated with increasing concentrations of 5-HT3 receptor ligands emphasized the pharmacological similarity of the two recognition sites labelled by [3H]-(S)-zacopride and [3H]granisetron. However, analysis of the nature of the competition curves indicated that 5-HT3 receptor agonists (5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, phenylbiguanide) and quipazine generated Hill coefficients greater than unity when the 5-HT3 receptor recognition sites were labelled with [3H]granisetron whilst these competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for either [3H]-(S)-zacopride or [3H]granisetron binding by the 5-HT3 receptor antagonists granisetron and ondansetron generated Hill coefficients around unity. Furthermore, addition of unlabelled (S)-zacopride (1.0 nM) failed to alter the nature by which quipazine competed for the [3H]granisetron-labelled 5-HT3 receptor recognition site. Consistent with 5-HT3 receptors radiolabelled in rat cortical membranes, the present studies indicate that [3H]-(S)-zacopride may label a different site on the 5-HT3-receptor complex compared to [3H]granisetron.

Quantitative analysis of the distributions of glutamatergic ligand binding sites in goldfish brain

Goldfish brain is a widely used model system for the study of the mechanisms involved in neuronal regeneration and synaptic plasticity. Because of the proposed role of glutamate receptors in these processes we have investigated the anatomical localisations of [3H]AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate), [3H]kainate, [3H]CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and [3H]L-glutamate binding sites in horizontal and sagittal sections. Binding sites for [3H]L-glutamate were the most widespread and both NMDA (N-methyl-D-aspartate) and non-NMDA sensitive components were detected. The density of [3H]kainate binding was very high in the cerebellum compared to other regions and in comparison with the other radioligands used. Conversely, relatively low amounts of [3H]AMPA binding were present with the telencephalon being the most densely labelled structure. [3H]CNQX binding was most densely localised in the tectum with the cerebellum also possessing high binding. In addition, there was a small population of [3H]CNQX binding sites located in the telencephalon and lobus vagi that appeared insensitive to AMPA and kainate.

Distribution of S(-)-zacopride-insensitive [125I]R(+)-zacopride binding sites in the rat brain and peripheral tissues.

Increasing evidence indicates that the 5-HT3 receptor antagonist R(+)-zacopride labels an additional site in brain tissue that is not sensitive to 5-HT (non-5-HT R(+)-zacopride site, R(+)-site). Since the levels of R(+)-sites in the brain are relatively low, the present studies explored the use of [125I]R(+)-zacopride to label the R(+)-site; the incorporation of an [125I] atom considerably increasing the specific activity of the radioligand relative to [3H]R(+)-zacopride that has been utilised previously. Competition experiments with [125I]R(+)-zacopride (1.0 nM) binding to rat whole brain homogenates, in the presence of the 5-HT3 receptor antagonist granisetron (1.0 microM), identified that R(+)-zacopride and prazosin bound to two sites (pIC50: 7.59 and 5.28, respectively, for R(+)-zacopride; 6.75 and 4.42, respectively, for prazosin) whereas S(-)-zacopride and mianserin possessed relatively low affinity (pIC50: 4.37 and 3.80, respectively) while (-)sulpiride and 5-HT failed to compete for [125I]R(+)-zacopride binding at concentrations up to 10 microM. Autoradiographic radioligand binding studies using [125I]R(+)-zacopride (0.5 nM) identified a heterogeneous distribution of specific binding sites (defined by unlabelled R(+)-zacopride, 1.0 microM) throughout the rat brain. In the presence of a saturating concentration of granisetron (1.0 microM), highest levels of specific [125I]R(+)-zacopride, binding sites (defined by R(+)-zacopride, 1.0 microM; R(+)-site), were detected in the olfactory tubercle, thalamus, corpus callosum, colliculus, dorsal and median raphe nucleus, spinal cord and the pons (8.0-13.0 fmol/mg). Moderate densities of R(+)-sites were located in the striatum, nucleus accumbens, substantia nigra, ventral tegmental area, globus pallidus, septal nuclei, frontal cortex and cerebellum (2.0-7.9 fmol/mg). In the hippocampus, amygdala and cortical areas. R(+)-site levels were low but detectable (0.1-1.9 fmol/mg). [125I]R(+)-zacopride labelled R(+)-sites were also detected in some rat peripheral tissues, for instance kidney cortex, adrenal gland and liver (2.4-6.8 fmol/mg). The present results indicate that specific non-5-HT [125I]R(+)-zacopride sites are heterogeneously distributed throughout the rat brain and are expressed in various peripheral tissues.