Identification of a novel nicotinic binding site in mouse brain using [(125)I]-epibatidine.

[(125)I]-Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high affinity. In this study, [(125)I]-epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates. Binding of [(125)I]-epibatidine was saturable and apparently monophasic in each brain region (K:(D:)=71+/-12 pM mean+/-s.e.mean across regions) but inhibition of [(125)I]-epibatidine binding (200 pM) by A85380, cytisine and (-)-nicotine was biphasic, indicating the presence of multiple binding sites. The sites with lower agonist affinity comprised 30.0+/-2.2, 58.6+/-0.1 and 48.7+/-3.3% of specific [(125)I]-epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively. The affinity difference between A85380-sensitive and -resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to differentiate agonist-sensitive and -resistant sites. The pharmacological profiles of the A85380-resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and five antagonists. The profiles were indistinguishable across regions, implying that A85380-resistant [(125)I]-epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype. The pharmacological profile of the A85380-resistant sites is very different from that previously reported for high affinity (-)-[(3)H]-nicotine-, [(125)I]-alpha-bungarotoxin-, or [(125)I]-alpha-conotoxin MII-binding sites, suggesting that they represent a novel nAChR population in mouse brain.

125I-alpha-conotoxin MII identifies a novel nicotinic acetylcholine receptor population in mouse brain.

alpha-Conotoxin MII (CtxMII), a peptide toxin from the venom of the predatory cone snail Conus magus, displays an unusual nicotinic pharmacology. Specific binding of a radioiodinated derivative ((125)I-alpha-CtxMII) was identified in brain region homogenates and tissue sections. Quantitative autoradiography indicated that (125)I-alpha-CtxMII binding sites have an unique pharmacological profile and distribution in mouse brain, being largely confined to the superficial layers of the superior colliculus, nigrostriatal pathway, optic tract, olivary pretectal, and mediolateral and dorsolateral geniculate nuclei. Expression of alpha-CtxMII binding sites in the nigrostriatal pathway, combined with evidence for alpha-CtxMII-sensitivity of nicotine-induced [(3)H]dopamine release in rodent striatal preparations indicates that (125)I-alpha-CtxMII binding nicotinic acetylcholine receptors are likely to be physiologically important. Unlabeled alpha-CtxMII potently (K(i) < 3 nM) competed for a subset of [(3)H]epibatidine binding sites in mouse brain homogenates, but weakly (IC(50) > 10 microM) interacted with (125)I-alpha-bungarotoxin and (-)-[(3)H]nicotine binding sites, confirming this compound's novel nicotinic pharmacology. Quantitative autoradiography revealed that alpha-CtxMII binds with high affinity at a subset of [(3)H]epibatidine binding sites with relatively low cytisine affinity ("cytisine-resistant" sites), resolving [(3)H]epibatidine binding into three different populations, each probably corresponding to a receptor subtype. The majority population seems to correspond to that which binds nicotine and cytisine with high affinity ("cytisine-sensitive" sites). Comparison of the cytisine-resistant population's distribution with that of alpha3 subunit mRNA expression suggests that the fractions both more and less sensitive to alpha-CtxMII probably contain the alpha3 subunit, perhaps in combination with different beta subunits.

Pharmacological and null mutation approaches reveal nicotinic receptor diversity.

We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [125I]alpha-Bungarotoxin-, (-)-[3H]nicotine-, and [3H]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [3H]dopamine, [3H]gamma-aminobutyric acid ([3H]GABA), and 86Rb(+) efflux techniques. Additionally, the effects of beta2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [125I]alpha-bungarotoxin-binding nicotinic acetylcholine receptors; beta2-subunit-dependent and -independent high-affinity (-)-[3H]nicotine-binding sites; beta2-dependent and -independent cytisine-resistant [3H]epibatidine-binding sites; and a beta2-dependent low-affinity [3H]epibatidine binding site. Comparative pharmacology suggested that [3H]GABA and dihydro-beta-erythroidine (DHbetaE)-sensitive 86Rb(+) efflux are mediated by the same (probably alpha4beta2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [3H]dopamine and DHbetaE-resistant 86Rb(+) efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was beta2 dependent. The majority of beta2-independent [3H]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors.

UB-165: a novel nicotinic agonist with subtype selectivity implicates the alpha4beta2* subtype in the modulation of dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain.

[3H]-Methyllycaconitine ([3H]-MLA) is a new radioligand with selectivity for alpha7-type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679-690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well-established alpha7-ligand [125I]-alpha-bungarotoxin. However, a small population of [3H]-MLA binding sites was apparently insensitive to alpha-bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]-MLA and [125I]-alpha-bungarotoxin binding sites. [3H]-MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nM and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nM), was completely inhibited by (-)-nicotine (Ki = 1.62 microM), whereas alpha-bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nM). The distributions of [125I]-alpha-bungarotoxin and [3H]-MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]-MLA in the absence or presence of unlabelled MLA or alpha-bungarotoxin provided no evidence for any alpha-bungarotoxin-resistant binding. The results are discussed in terms of binding sites that are inaccessible to alpha-bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]-MLA for characterization of alpha7-type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]-alpha-bungarotoxin.

Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit.

Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit.

Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition.

Chronic nicotine up-regulates the number of high affinity nicotinic acetylcholine receptors (nAChRs) in mammalian brain. Here, we studied up-regulation of the nAChR composed of alpha4 and beta2 subunits in the M10 cell line by using [3H]epibatidine to measure nAChR in cells in situ and in membrane preparations. Cultures were exposed to drugs for 2 days before assay. All agonists up-regulated [3H]epibatidine binding sites with EC50 values typically 10-100-fold higher than their respective Ki values from competition binding assays. Maximum up-regulation ranged from 40% to 250% above control values. Maximally effective concentrations of the less efficacious agonists methylcarbamylcholine or (+/-)-epibatidine together with nicotine resulted in less up-regulation than that produced by nicotine alone, showing that they are partial up-regulatory agonists. The antagonists dihydro-beta-erythroidine, methyllycaconitine, d-tubocurarine, hexamethonium, decamethonium, and mecamylamine either failed to up-regulate [3H]epibatidine binding sites or up-regulated mildly at high concentrations. When tested at non-up-regulating concentrations, only d-tubocurarine significantly inhibited agonist-induced up-regulation; this inhibition seemed to be noncompetitive. Comparison of [3H]epibatidine displacement in intact M10 cells and membrane preparations by membrane-impermeant ligands indicated that 85% of [3H]epibatidine binding sites are intracellular. On chronic treatment with agonist, the proportion of surface receptors did not change significantly, indicating that most up-regulated [3H]epibatidine binding sites are internal. However, up-regulation is mediated at the cell surface because the impermeant ligand tetramethylammonium was as efficacious as nicotine in eliciting up-regulation, and methylcarbamylcholine (i.e., impermeant but with low efficacy) blocked nicotine induced up-regulation. Thus, agonists elicit up-regulation (mainly of intracellular receptors) by interacting with cell surface nAChRs that are not compatible with either an active or high affinity desensitized conformation.

Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats.

1. Isoarecolone was approximately 250 times less potent than nicotine as an inhibitor of [3H]-nicotine binding to rat brain membranes. Isoarecolone failed to inhibit the binding of the nicotinic ligand [125I]-alpha-bungarotoxin or of the muscarinic ligand [3H]-QNB. 2. Nicotine (0.01-30 microM) evoked the release of [3H]-dopamine from striatal and frontal cortex synaptosomes, with EC50 values of approximately 0.5 microM in each case. This release was largely mecamylamine-sensitive. 3. Isoarecolone (1-200 microM) evoked predominantly mecamylamine-sensitive dopamine release from both striatal and cortical synaptosomes, with a potency at least 20 times less than that of nicotine. The maximum effect of isoarecolone was less than that of nicotine, particularly in the frontal cortex preparation. 4. In control rats treated chronically with saline, neither nicotine nor isoarecolone had clear effects on locomotor activity at the doses tested. Chronic treatment with nicotine clearly sensitized rats to the locomotor activating effect of isoarecolone was seen at a dose about 40 times larger than that of nicotine. 5. The low potency and efficacy of isoarecolone in facilitating sensitized locomotor activity resembled its lower potency and efficacy, compared with nicotine, in evoking dopamine release in vitro. The agonist profile of the nicotinic receptor population mediating dopamine release may determine the pharmacological characteristics of consequent locomotor behaviour.