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S Wonnacott

Publications and source records attributed to S Wonnacott.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of a nicotinic acetylcholine receptor from the insect Manduca sexta.

Manduca sexta is a nicotine-insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose alpha subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [125I]alpha-bungarotoxin bound specifically to both larval and adult membranes, with Kd values of 7.6 and 6.5 nM and Bmax values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [1251]alpha-bungarotoxin binding was similar in both tissues. In particular, nicotine (Ki values: 1.6 microM and 2 microM for larvae and adults, respectively) competed with an affinity similar to that found for nicotine-sensitive insects. No alpha-bungarotoxin-insensitive binding sites labelled by [3H]epibatidine could be detected. Using the alpha-like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other alpha-subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an alpha-like subunit capable of binding alpha-bungarotoxin, and it retains all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta is the result of a nicotinic receptor with diminished nicotine binding.

Aging↗
PubMed1998

Differential inhibition by alpha-conotoxin-MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices.

The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, alpha-conotoxin-MII has been reported to inhibit potently and selectively the rat alpha3beta2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (+/-)-anatoxin-a-evoked release of [3H]dopamine from rat striatal synaptosomes and slices. Alpha-conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of alpha3beta2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 +/- 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nM alpha-conotoxin-MII. On perfused striatal synaptosomes and slices, alpha-conotoxin-MII dose-dependently inhibited [3H]dopamine release evoked by 1 microM (+/-)-anatoxin-a with IC50 values of 24.3 +/- 2.9 and 17.3 +/- 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by alpha-conotoxin-MII (112 nM) was 44.9 +/- 5.4% for synaptosomes and 25.0 +/- 4.1% for slices, compared with an inhibition by 10 microM mecamylamine of 77.9 +/- 3.7 and 88.0 +/- 2.1%, respectively. These results suggest the presence of presynaptic alpha3beta2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (+/-)-anatoxin-a-evoked [3H]dopamine release by alpha-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an alpha-conotoxin-MII-insensitive nAChR.

Animals↗
PubMed1998

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.

Binding Sites↗
PubMed1997

Parallel increases in [alpha-125I]bungarotoxin binding and alpha 7 nicotinic subunit immunoreactivity during the development of rat hippocampal neurons in culture.

Previous studies have shown that hippocampal neurons cultured at high density express alpha-bungarotoxin binding sites and have alpha 7 nicotinic acetylcholine receptor subunit immunoreactivity [Barrantes, G.E., Rogers, A.T., Lindstrom, J. and Wonnacott, S., Brain Res., 672 (1995) 228-236]. We now examine both of these parameters in well-characterized hippocampal neurons cultured at sufficiently low densities to resolve individual neurons and their processes. The specific binding of [alpha-125I]bungarotoxin is first detectable after 3 days in culture and increases during the next 12 days in culture, reaching a maximum of approximately 30,000 binding sites per cell. This is accompanied, over the same timecourse, by an increase in immunoreactivity for two antibodies that specifically bind to the alpha 7 subunit. Both cell bodies and processes were labelled by 9 days in culture. The timecourse of alpha 7-type nicotinic receptor expression resembles that previously described for synapse formation in hippocampal cultures.

Animals↗
PubMed1997

Presynaptic nicotinic ACh receptors.

Nicotinic ACh (nACh) receptors in the CNS are composed of a diverse array of subunits and have a range of pharmacological properties. However, despite the fact that they are ligand-gated cation channels, their physiological functions have not been determined. This has led to increased interest in presynaptic nACh receptors that act to modulate the release of transmitter from presynaptic terminals.

Animals↗
PubMed1997

Presynaptic nicotinic modulation of dopamine release in the three ascending pathways studied by in vivo microdialysis: comparison of naive and chronic nicotine-treated rats.

The modulation of dopamine release by presynaptic nicotinic receptors in vitro is well established, but the significance of this effect in vivo is unclear. We have characterised the effect of nicotine, locally applied via a microdialysis probe, on dopamine release from the terminal regions of three ascending dopaminergic pathways in conscious, freely moving rats. Nicotine caused a dose-dependent increase in dopamine release in the striatum, the nucleus accumbens, and, to a lesser extent, the frontal cortex. Metabolite levels were unaltered by any concentration of nicotine. Prior administration of mecamylamine via the probe abolished the nicotine-evoked increase in dopamine release, confirming the mediation of nicotinic receptors. The dose dependence of mecamylamine-sensitive, nicotine-evoked dopamine release was similar in all three brain regions. However, 10(-5) M tetrodotoxin totally blocked nicotine-stimulated dopamine release in the striatum and the accumbens but not the cortex. Daily subcutaneous injections of nicotine (0.4 mg kg-1 for 7 days) increased the response to a subsequent local application of nicotine in the striatum, and a similar trend was found in the other brain areas. The same daily dose of nicotine given as a continuous infusion had no effect, whereas infusion of 4 mg kg-1 day-1 increased the response to a subsequent nicotine challenge. The localisation and regulation of nicotinic receptors in the terminal fields of dopaminergic pathways are discussed.

Animals↗
PubMed1997

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties.

Accumulating preclinical and clinical evidence data suggests that compounds that selectively activate neuronal nicotinic acetylcholine receptor (nAChR) subtypes may have therapeutic utility for the treatment of several neurological disorders. In the present study, the in vitro pharmacological properties of the novel cholinergic channel modulator ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine], are described. In radioligand binding studies, ABT-089 was shown to display selectivity toward the high-affinity (-)-cytisine binding site present on the alpha4beta2 nAChR subtype (Ki = 16 nM) relative to the [125I]alpha-bungarotoxin binding site present on the alpha7 (Ki > or = 10,000 nM) and alpha1beta1deltagamma (Ki > 1000 nM) nAChR subtypes. In cation flux and channel current studies, ABT-089 displayed a more complex profile than (-)-nicotine having agonist, partial agonist and inhibitory activities depending on the nAChR subtype with which it interacts. ABT-089 differentially stimulated neurotransmitter release. The compound displayed a similar potency and efficacy to (-)-nicotine to facilitate ACh release (ABT-089, EC50 = 3 microM; (-)-nicotine, EC50 = 1 microM), but was markedly less potent and less efficacious than (-)-nicotine to stimulate dopamine release (ABT-089, EC50 = 1.1 microM; (-)-nicotine, EC50 = 0.04 microM). Additionally, ABT-089 was neuroprotective against the excitotoxic insults elicited by exposure to glutamate in both rat cortical cell cultures (EC50 = 10 +/- 3 microM) and differentiated human IMR32 cells (EC50 = 3 +/- 2 microM). The differential full agonist/partial agonist profile of ABT-089, as compared with (-)-nicotine and ABT-418, illustrates the complexity of nAChR activation and the potential to target responses at subclasses of the neuronal and peripheral receptors.

Acetylcholine↗
PubMed1996

Nudicauline and elatine as potent norditerpenoid ligands at rat neuronal alpha-bungarotoxin binding sites: importance of the 2-(methylsuccinimido)benzoyl moiety for neuronal nicotinic acetylcholine receptor binding.

Methyllycaconitine (MLA, 1) is a novel, potent probe for mammalian and insect nicotinic acetylcholine receptors (nAChR) and displays remarkable selectivity toward neuronal [125I]-alpha-bungarotoxin (alpha BgTX) binding sites that correspond to alpha 7-type nAChR in mammalian brain. We have shown that, among a number of selected norditerpenoid alkaloids, elatine (2) and nudicauline (3) are equipotent with, or better than, MLA (1) in binding to brain [125I]-alpha BgTX binding sites, with IC50 values of 6.1, 1.7, and 7.6 nM, respectively. The 2-((S)-methylsuccinimido)benzoyl moiety of these ligands is crucial for high-affinity binding, whereas structural modifications to the norditerpenoid core of the ligand can be tolerated without loss of activity or selectivity. In addition to MLA (1), elatine (2), and nudicauline (3), we have examined lycoctonine (4), inuline (6), lappaconitine (7), N-desacetyllappaconitine (8), delsoline (10), delcorine (11), deltaline (12), condelphine (13), and karacoline (14). This study therefore extends the range of norditerpenoids, other than MLA, which can be used to probe this important class of nAChR. All 12 alkaloids were assessed for activity at [3H]nicotine binding sites which are considered to represent alpha 4 beta 2 nAChR. Furthermore, the 1H and 13C NMR spectroscopic data of MLA and elatine have been critically compared.

Aconitine↗
PubMed1996

Relationship between up-regulation of nicotine binding sites in rat brain and delayed cognitive enhancement observed after chronic or acute nicotinic receptor stimulation.

(-)-Nicotine tartrate (2 mg/kg), and a nicotinic agonist, RJR 2403 (1.4 mg/kg), and antagonist, mecamylamine (1 mg/kg), were administered to separate groups of rats SC twice daily for 10 days. Two other groups received the same doses of nicotine or RJR 2403 for 1 day followed by saline for 9 days. Twenty-four hours after the final injection, the rats were compared to a 10-day saline-injected group on acquisition of a hidden platform position in the Morris water maze (20 trials, 30-min inter-trial interval). The rats were killed 48 h after the last drug injection and frontal, entorhinal and posterior cingulate cortex and dorsal and ventral hippocampus assayed for [3H]-nicotine binding density. Chronic nicotine significantly increased the number of frontal and entorhinal cortical and dorsal hippocampal, but not posterior cingulate cortical or ventral hippocampal, nicotinic receptors, and improved rate of learning. Chronic mecamylamine and RJR 2403 also significantly increased the number of nicotinic receptors in frontal cortex, though not other regions, but retarded rate of learning. Nicotine given for 1 day 11 days earlier marginally increased nicotinic receptors in entorhinal cortex (but not other regions) and significantly increased rate of learning, though significantly less than 10-day nicotine. Entorhinal cortical and dorsal hippocampal nicotinic receptor numbers were positively associated with rate of learning but not performance at asymptote. Thus cognitive enhancement after chronic nicotine is in part a delayed consequence of nicotine administration 11 days earlier, and may reflect regional changes in nicotinic receptor up-regulation.

Animals↗
PubMed1996

Pharmacological characterization of a nicotinic autoreceptor in rat hippocampal synaptosomes.

The modulation of [3H]ACh release by nicotinic compounds was studied in superfused rat hippocampal synaptosomes loaded with [3H]choline, (-)-Nicotine (0.1-10 microM) evoked a dose-dependent increase in [3H]ACh release; higher concentrations were less effective. Nicotine-evoked release was Ca(2+)-dependent, and blocked by the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, and pempidine. The alpha 7-selective antagonist methyllycaconitine did not inhibit nicotine-evoked release when tested at 1 microM, although at 10 microM some attenuation of the response was observed. Six agonists tested were equally efficacious in stimulating [3H]ACh release, as judged by the maximum responses, and gave the following EC50 values: (+/-)-epibatidine 0.12 microM; (+)-anatoxin-a 0.14 microM; (-)-nicotine 0.99 microM; (-)-cytisine 1.06 microM; ABT-418 2.6 microM; isoarecolone 43 microM. Each agonist generated a "bell-shaped" dose response curve, suggesting desensitisation at higher concentrations. This is supported by analysis of repetitive stimulation with (-)-nicotine and (-)-cytisine: S2/S1 ratios declined sharply with increasing concentration, whereas subsequent KC1-evoked release remained constant. These results are discussed in terms of possible nicotinic receptor subtypes that might be present on hippocampal nerve terminals.

Acetylcholine↗
PubMed1996

Tetrodotoxin-sensitivity of nicotine-evoked dopamine release from rat striatum.

Recent observations from synaptosome preparations have questioned the tetrodotoxin (TTX) insensitivity of nicotine-evoked release in the striatum, a characteristic previously considered diagnostic of presynaptically located nicotinic acetylcholine receptors (nAChRs). Therefore, we have undertaken a comparison of nicotine-evoked dopamine release in the presence of TTX from the rat striatum in vitro, using synaptosomes and brain slices, and in vivo, using microdialysis. In P2 and Percoll-purified synaptosome preparations, 1.5 microM TTX partially inhibited nicotine-evoked [3H]dopamine release by 54% and 37%, respectively, whereas in more intact preparations (brain slices and microdialysis) TTX completely inhibited mecamylamine-sensitive nicotine-stimulated dopamine release. These results suggest that caution should be exercised in the interpretation of TTX sensitivity of nicotine-evoked responses with regard to the location of nAChRs.

Animals↗
PubMed1996

Voltage-sensitive Ca2+ channels involved in nicotinic receptor-mediated [3H]dopamine release from rat striatal synaptosomes.

The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [3H]dopamine release from striatal synaptosomes, and this action is both Na+ and Ca2+ dependent and is blocked by Cd2+. This suggests that stimulation of presynaptic nicotinic receptors results in Na+ influx and local depolarisation that activates voltage-sensitive Ca2+ channels, which in turn provide the Ca2+ for exocytosis. Here we have investigated the subtypes of Ca2+ channels implicated in this mechanism. [3H]-Dopamine release evoked by anatoxin-a (1 microM) was partially blocked by 20 microM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a 86Rb+ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca2+ channel blocker omega-conotoxin GVIA (1 microM) blocked anatoxin-a-evoked [3H]dopamine release by 60% but had no significant effect on 86Rb+ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker omega-agatoxin IVA (90 nM) also inhibited KCl-evoked release by approximately 30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker omega-conotoxin MVIIC (1 microM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [3H]dopamine release by activation of N-type Ca2+ channels. In contrast, KCl-evoked [3H]dopamine release appears to involve both N-type and P-type channels.

Animals↗
PubMed1995

A nicotinic acetylcholine receptor subunit from insect brain forms a non-desensitising homo-oligomeric nicotinic acetylcholine receptor when expressed in Xenopus oocytes.

The locust alpha-like nicotinic receptor subunit alpha L1 was expressed in Xenopus oocytes. Small but reproducible currents were elicited by application of high concentrations of nicotine, demonstrating that alpha L1 is capable of forming homo-oligomeric channels. Nicotine-evoked currents were blocked by alpha-bungarotoxin and methyllycaconitine. Comparison with chick alpha 7 receptors showed that the two receptors differ with respect to nicotine sensitivity and time course of evoked currents. Nicotine dose-response curves gave EC50 values of 24 and 830 microM for alpha 7 and alpha L1 respectively. Whereas alpha 7 responses showed characteristic fast onset and rapid desensitization within 3 s, alpha L1 currents displayed a slow onset and showed no tendency to desensitize during 45 s of agonist application. Thus alpha L1 is a novel nicotine subunit for the further exploration of structure-function relationships of ligand-gated ion channels. The question of the subunit composition of native insect receptors remains open.

Animals↗
PubMed1995

Nicotine increases intracellular calcium in rat hippocampal neurons via voltage-gated calcium channels.

The effect of nicotinic receptor activation on intracellular calcium concentrations ([Ca2+]i) was quantitated in populations of cultured hippocampal neurons loaded with Fura-2. Nicotine (50 microM) and cytisine (50 microM) increased [Ca2+]i by 100%. This response was abolished in the presence of the nicotinic antagonist methyllycaconitine (MLA) whereas KCl-evoked increases in [Ca2+]i were insensitive to MLA. Glial cultures were unaffected by nicotine, although they did respond to glutamate with increased [Ca2+]i. In hippocampal neurons, responses to nicotinic agonists and KCl were dependent on the presence of extracellular Ca2+ and were similarly sensitive (85% inhibition) to CdCl2. These results are consistent with the presence of functional nicotinic receptors on hippocampal neurons. The receptors appear to elevate [Ca2+]i by promoting the influx of extracellular Ca2+ through voltage-gated calcium channels.

Aconitine↗
PubMed1995

Anatoxin-a is a potent agonist of the nicotinic acetylcholine receptor of bovine adrenal chromaffin cells.

(+)-Anatoxin-a is a neurotoxic alkaloid produced by the cyanobacterium Anabaena flos-aquae. In this study synthetic (+/-)-anatoxin-a was tested on isolated bovine adrenal chromaffin cells to determine its ability to evoke secretion of endogenous catecholamines through neuronal-type nicotinic receptor activation. Anatoxin-a was found to act as a potent agonist of the secretory response of chromaffin cells with an EC50 of 1-2 microM, compared with an EC50 of 4-5 microM for nicotine. The cells responded to anatoxin-a and nicotine with bell-shaped concentration-response curves consistent with desensitisation at concentrations of anatoxin-a greater than 5 microM and of nicotine greater than 20 microM. The secretion of catecholamines stimulated by anatoxin-a was completely inhibited in a non-competitive manner by the nicotinic antagonist mecamylamine with an IC50 of 0.4-0.5 microM. In the presence of depolarising concentrations of K+ (15 or 50 mM), anatoxin-a increased the secretion of catecholamines in a concentration-dependent manner up to the same maximum as that achieved by anatoxin-a alone. It is concluded that anatoxin-a acts as a potent and selective nicotinic agonist, capable of evoking secretion of endogenous catecholamines from chromaffin cells via their neuronal-type nicotinic receptor.

Animals↗