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S Wonnacott

Publications and source records attributed to S Wonnacott.

At least 55 records · Page 3Linked to original sources
PubMed1995

Conversion of the sodium channel activator aconitine into a potent alpha 7-selective nicotinic ligand.

Methyllycaconitine (MLA) is a competitive antagonist of nicotinic acetylcholine receptors, with a remarkable preference for neuronal [125I]alpha Bgt binding sites. We have begun to investigate the structural basis of its potency and subtype selectivity. MLA is a substituted norditerpenoid alkaloid linked to a 2-(methylsuccinimido)benzoyl moiety. Hydrolysis of the ester bond in MLA to produce lycoctonine diminished affinity for rat brain [125I]alpha Bgt binding sites 2500-fold and abolished affinity for [3H]nicotine and muscle [125I]alpha Bgt binding sites. The voltage-gated Na+ channel activator aconitine, also a norditerpenoid alkaloid, but with significant structural differences from lycoctonine, displayed comparable weak or absent nicotinic activity. Addition of a 2-(methylsuccinimido)benzoyl sidechain to O-demethylated aconitine, to mimic MLA, abolished Na+ channel activation and conferred nanomolar affinity for brain [125I]alpha Bgt binding sites, comparable to that of MLA. We propose that the ester-linked 2-(methylsuccinimido)benzoyl group is necessary for nicotinic potency, but alpha 7 selectivity resides in the norditerpenoid core of the molecule.

Aconitine↗
PubMed1995

alpha-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with alpha 7 subunits and up-regulation by chronic nicotine treatment.

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [3H]nicotine or [3H]cytisine to live cells in situ was detected although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [3H]QNB were present at a density of 0.75 pmol/mg protein. [125I]alpha-Bungarotoxin (alpha Bgt) bound to hippocampal cultures with a Bmax of 128 fmol/mg protein and a Kd of 0.6 nM; cortical cultures expressed five times fewer [125I]alpha-Bgt binding sites. Fluorescence cytochemistry with rhodamine-alpha-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 x 10(4) and 2 x 10(4) binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat alpha 7 nicotinic receptor subunit) and rhodamine-alpha-Bgt gave coincident labelling patterns, supporting the correlation between the alpha 7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 microM nicotine for 14 days elicited a 40% increase in the numbers of [125I]alpha-Bgt binding sites, mimicking the up-regulation observed in in vivo studies. Primary cultures offer a useful in vitro system for investigating the expression and regulation of brain alpha-Bgt-sensitive receptors.

Animals↗
PubMed1995

Sensitivity of rat frontal cortical neurones to nicotine is increased by chronic administration of nicotine and by lesions of the nucleus basalis magnocellularis: comparison with numbers of [3H]nicotine binding sites.

The effects of chronic nicotine treatment and of unilateral AMPA lesion of the nucleus basalis magnocellularis (nbm) on the sensitivity of frontal cortical neurones to iontophoretically applied nicotine were studied. Chronic nicotine treatment increased the number of [3H]nicotine binding sites from 2.9 to 3.9 pmol g-1 wet weight, and increased the proportion of cortical neurones responding to nicotine from 32.3% to 60.0%. After unilateral nbm lesions, the densities of AChE-positive fibers and [3H]nicotine binding sites were reduced by approximately 97% and 55%, respectively, and the proportion of neurones responding to nicotine increased from 32.3% to 53.8%. The two treatments, chronic nicotine administration and nbm lesion, also increased the size of individual neuronal responses, prolonged their duration, and shortened the response latency. Responses to glutamate were unaffected by either procedures. The results show that the increase in [3H]nicotine binding produced by chronic nicotine administration is associated with an increased response to iontophoretically applied nicotine, suggesting that the receptor upregulation induced by the chronic treatment were functional. Less easily explained is the association between increased sensitivity of frontal cortical neurons to nicotine after nbm lesion with a decreased receptor density. It is suggested that a substantial proportion of nicotinic receptors are located presynaptically, and that their loss after lesion concealed an upregulation at postsynaptic sites.

Animals↗
PubMed1995

Anatoxin-a-evoked [3H]dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. Using rat striatal synaptosomes loaded with [3H]dopamine, we have characterized the action of the selective nicotinic agonist, (+/-)anatoxin-a, with respect to [3H]dopamine release, in order to explore the mechanisms coupling nicotinic receptor activation to exocytosis. Anatoxin-a evoked [3H]dopamine release in a concentration-dependent and mecamylamine-sensitive manner, EC50 = 0.11 microM. The maximum [3H]dopamine release elicited by anatoxin-a was only 20% of the maximum elicited by KCl depolarization; there was no additivity between anatoxin-a and sub-maximal concentrations of KCl. Both agents stimulated Ca(2+)-dependent release that was equally sensitive to inhibition by 200 microM Cd2+. This result suggests that anatoxin-a-stimulated exocytosis is mediated by Ca2+ influx via voltage-sensitive Ca2+ channels, with little contribution from Ca2+ entering directly through the nicotinic receptor channel. This view is supported by the abolition of anatoxin-a-evoked [3H]dopamine release in Na(+)-depleted medium. A partial (40%) inhibition by tetrodotoxin was observed. These data suggest that activation of presynaptic nicotinic acetylcholine receptors by anatoxin-a results in an influx of Na+, producing sufficient local depolarization to open voltage-sensitive Ca2+ and Na+ channels. The latter may then amplify the response, activating further Ca2+ channels. The particular voltage-sensitive Ca2+ channels involved remain to be determined.

Animals↗
PubMed1995

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.

Amphetamine↗
PubMed1994

Interaction of p-aminophenyldichloroarsine, an arsenical with specificity for vicinal cysteines, with [3H]cytisine binding sites in rat brain membranes.

The arsenical compound p-aminophenyldichloroarsine (APA) is selective for spatially close thiols with which it forms a stable complex. The alpha subunits of nicotinic acetylcholine receptors are defined by the presence of a pair of adjacent cysteines close to the agonist binding site. Here the interaction of APA with [3H]cytisine binding sites, which correspond to the major subtype of nicotinic receptors in rat brain has been examined. Incubation of brain membranes with 10 microM APA abolished [3H]cytisine binding. The action of APA was dependent on prior reduction of sulphydryls with dithiothreitol. APA effects could not be reversed by oxidizing agents but could be reversed by the antiarsenical reagent 2,3-dimercapto-1-propane sulphonic acid. Under the conditions used, the concentration of APA producing a half-maximal decrease in binding was 130 nM. The loss of [3H]cytisine binding was due to a decrease in the number of binding sites (Bmax) with no effect on affinity for the radioligand (Kd). Nicotinic ligands failed to protect against the reduction and arsenylation of neuronal receptor sites. These observations are consistent with the potent interaction of APA with this neuronal nicotinic receptor.

Alkaloids↗
PubMed1993

Agonist pharmacology of the neuronal alpha 7 nicotinic receptor expressed in Xenopus oocytes.

The potencies and efficacies of seven agonists at chick alpha 7 nicotinic receptors expressed in Xenopus oocytes were determined by whole cell recording. (+)-Anatoxin-a was the most potent agonist (EC50 = 0.58 microM) and acetylcholine was the least potent (EC50 = 320 microM). The rank order of agonist potencies was: (+)-anatoxin-a >> cytisine > (-)-nicotine > (+)-nicotine > DMPP > 1-acetyl-4-methylpiperazine methiodide > acetylcholine. DMPP evoked only very small currents: comparison of maximally effective agonist concentrations showed that DMPP was only one-fifth as efficacious as other agonists. Previously published IC50 values for rat brain [125I]alpha-bungarotoxin sites show a similar agonist profile, and the identity of homo-oligomeric alpha 7 receptors with native alpha-bungarotoxin-sensitive neuronal nicotinic receptors is discussed.

Acetylcholine↗
PubMed1993

Upregulation of nicotinic receptors following continuous infusion of nicotine is brain-region-specific.

Rats receiving 4 mg nicotine/kg/day via implanted minipumps sustained plasma nicotine concentrations of 40 ng/ml throughout two weeks of nicotine infusion. Numbers of brain [3H]nicotine binding sites were increased by about 50% in cortex and hippocampus whereas numbers of [3H]nicotine binding sites in striatum were unaffected by nicotine treatment at either of the timepoints examined (7, 14 days). Cortical [125I] alpha-bungarotoxin and [3H]QNB binding sites were also unchanged. The regional selectivity of nicotinic receptor modulation may reflect the low dose of nicotine used and the mode of administration. The changes observed may be pertinent to the continuous administration of nicotine in man, via transdermal nicotine patches.

Animals↗
PubMed1993

Behavioural and ligand-binding studies in rats with 1-acetyl-4-methylpiperazine, a novel nicotinic agonist.

The novel nicotinic agonist 1-acetyl-4-methylpiperazine (AMP) has been studied in ligand-binding and behavioural studies. AMP methiodide potently inhibited [3H]-(-)-nicotine and [125I]-alpha-bungarotoxin binding to P2 membranes from rat brain and [125I]-alpha-bungarotoxin binding to rat skeletal muscles. AMP HCl also inhibited nicotinic binding, but it was 100 times less potent than AMP methiodide. In behavioural studies, AMP HCl reduced locomotor activity of experimentally naive rats and mecamylamine blocked this effect. In rats receiving (-)-nicotine chronically, AMP HCl did not increase locomotor activity consistently or to the same extent as (-)-nicotine. In rats trained to discriminate (-)-nicotine from saline in a two-bar operant conditioning procedure with food reinforcement, there was generalization to AMP HCl, but only at doses that reduced the overall rate of responding. The potency and effectiveness of AMP relative to (-)-nicotine varied across the different behavioural procedures. The results suggest that the pharmacodynamic action of AMP differs from that of (-)-nicotine and that it usefully extends the range of agonists that can be used as probes for central nicotinic mechanisms.

Animals↗
PubMed1992

Homoanatoxin: a potent analogue of anatoxin-A.

The natural toxin anatoxin-a (AnTx) is a potent nicotinic agonist that is valuable for the study of nicotinic receptors. We have synthesized 2-(propan-1-oxo-1-yl)-9-azabicyclo[4.2.1]non-2-ene, the homologue of AnTx in which the side-chain is extended by one methylene unit from a methyl to an ethyl ketone. This chemistry would allow the generation of a tritiated product and the homologue, designated homoanatoxin (HomoAnTx), has been characterized here with that aim in mind. In competition binding assays at neuronal nicotinic ligand binding sites characterized by [3H]nicotine and [125I]-alpha bungarotoxin, HomoAnTx retained the same potency as the parent molecule, with Ki values of 7.5 nM and 1.1 microM, respectively. In contrast, it showed little inhibition of muscarinic binding defined by [3H]-quinuclidinyl benzilate. HomoAnTx is a potent nicotinic agonist in frog muscle contracture assays, having four times the potency of carbamylcholine and one tenth of the activity of AnTx itself. The N-methylated version of HomoAnTx was more than two orders of magnitude weaker in both functional and binding assays. The successful synthesis of HomoAnTx with retention of high nicotinic potency offers a route for the generation of novel, potent radiolabelled nicotinic ligands.

Animals↗
PubMed1992

Characterization of nicotinic receptor-mediated [3H]dopamine release from synaptosomes prepared from mouse striatum.

This study establishes that presynaptic nicotinic receptors modulate dopamine release in the mouse striatum. Nicotinic agonists elicit a dose-dependent increase in the release of [3H]dopamine from synaptosomes prepared from mouse striatum. At low concentrations, this release is Ca2+ dependent, whereas at higher concentrations Ca(2+)-independent, mecamylamine-insensitive release was also observed. The Ca(2+)-dependent nicotine-evoked release was not blocked by alpha-bungarotoxin but was effectively blocked by neuronal bungarotoxin as well as several other nicotinic receptor antagonists. The relationship between potency for stimulation of release for agonists and potency for inhibition of release for antagonists was compared to the affinity of these compounds for the [3H]nicotine binding site. The overall correlation between release and binding potency was not high, but the drugs may be classified into separate groups, each of which has a high correlation with binding. This finding suggests either that more than one nicotinic receptor regulates dopamine release or that not all agonists interact with the same receptor in an identical fashion.

Animals↗
PubMed1992

Blockade of nicotinic currents in hippocampal neurons defines methyllycaconitine as a potent and specific receptor antagonist.

Methyllycaconitine, a toxin isolated from the seeds of Delphinium brownii, inhibited acetylcholine- and anatoxin-induced whole-cell currents in cultured fetal rat hippocampal neurons, at picomolar concentrations. This antagonism was specific, concentration dependent, reversible, and voltage independent. Furthermore, methyllycaconitine inhibited 125I-alpha-bungarotoxin binding to adult rat hippocampal membranes, protected against the alpha-bungarotoxin-induced pseudoirreversible blockade of nicotinic currents, and shifted the concentration-response curve of acetylcholine to the right in fetal rat hippocampal neurons, suggesting a possible competitive mode of action for this toxin. Remarkably low concentrations of methyllycaconitine (1-1000 fM) decreased the frequency of anatoxin-induced single-channel openings, with no detectable decrease in the mean channel open time. These actions of methyllycaconitine commend this neurotoxin for the characterization of the alpha-bungarotoxin-sensitive subclass of neuronal nicotinic receptors, which has hitherto eluded functional demonstration.

Aconitine↗
PubMed1991

The relevance of receptor binding studies to tobacco research.

The initial event in the process by which nicotine acts on the nervous system is its interaction with specific receptor molecules in the neuronal membrane. This interaction can be characterized in radioligand binding assays, using [3H]nicotine. While this approach has generated a detailed description of recognition sites for nicotine in the brain, caution is necessary in correlating these binding proteins with physiologically relevant sites of action of nicotine. Of particular interest is the disparity between effective nicotine concentrations in binding and functional assays, which may reflect receptor desensitization and could be pertinent to the development of dependence. Receptor heterogeneity, reflecting subtypes of nicotine receptor having different radioligand binding specificities and differing properties, introduces a new dimension in the consideration of nicotine's actions. Thus, receptor binding assays have an important role to play, in conjunction with other approaches, in unravelling the complex mechanisms that can lead to nicotine dependence.

Animals↗