Purification of L-[3H] nicotine eliminates low affinity binding

doi:10.1016/0024-3205(90)90095-9

Life Sciences

Volume 46, Issue 13, 1990, Pages 935-943

https://doi.org/10.1016/0024-3205(90)90095-9 Get rights and content

Abstract

Some studies of L-[³H] nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicates that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-[³H] nicotine eliminates the low affinity site.

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Cited by (41)

86Rb+ efflux mediated by α4β 2-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization
2010, Biochemical Pharmacology
Citation Excerpt :
Association kinetics was measured using two concentrations of each ligand, one near the KD and a second higher concentration. [ 3H]Nicotine was purified as described previously [21]. The algorithms in Sigma Plot were used for all curve fitting.
The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration–effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine-stimulated ⁸⁶Rb⁺ efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC₅₀ values for each agonist for desensitization of ⁸⁶Rb⁺ efflux both with high (EC₅₀ ≈ 3 μM) and low (EC₅₀ ≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective K_D values for receptor binding. Even though the two components of α4β2*-nAChR-mediated ⁸⁶Rb⁺ efflux from mouse brain differ markedly in EC₅₀ values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/h and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high-sensitivity and low-sensitivity acetylcholine-stimulated ⁸⁶Rb⁺ efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal ⁸⁶Rb⁺ efflux from thalamus, IC₅₀ values in both brain regions were unaffected by chronic nicotine treatment.
The effect of nicotine and haloperidol co-treatment on nicotinic receptor levels in the rat brain
2001, Molecular Brain Research
Citation Excerpt :
Single point saturation assays for high affinity nicotinic receptor binding were performed as previously described [6,28,35]. Briefly, re-purified [3H]-nicotine (N-methyl-[3H]-nicotine, specific activity 82 Ci/mmol, Amersham Corp., Arlington Heights, IL) [41] or [3H]-epibatidine (specific activity 52 Ci/mmol, Amersham Corp., Arlington Heights, IL) were used to determine high affinity nicotinic receptor binding in striatum, hippocampus and thalamus. All incubations were performed in 96-well microtiter plates in a final volume of 200 μl using 75–100 μg of homogenized protein in the presence of either 10 nM [3H]-nicotine or 500 pM [3H]-epibatidine.
Genetic and biological data have suggested a role for the neuronal nicotinic acetylcholine receptors in the neuropathophysiology of schizophrenia. Studies in human postmortem brain demonstrate dose-dependent increases in nicotinic receptor binding in normal smokers. We found this upregulation to be reduced in schizophrenic smokers, many of whom had taken typical neuroleptics during their lifetime. The present study examined the hypothesis that typical antipsychotic drug treatment might modulate nicotinic receptor upregulation in a rat model. Nicotine, administered alone or in combination with haloperidol, increased both high and low affinity neuronal nicotinic receptors in a region specific manner. Haloperidol had no generalized effect on basal levels of nicotinic receptor binding or nicotine induced upregulation of nicotinic receptors. However, haloperidol attenuated high affinity nicotinic receptor upregulation in thalamus and low affinity receptor upregulation in hippocampus. These results suggest that haloperidol is not likely to affect nicotinic receptor regulation by smoking in most brain regions.
Abnormal regulation of high affinity nicotinic receptors in subjects with schizophrenia
2000, Neuropsychopharmacology
Previous studies have suggested that an abnormality in neuronal nicotinic acetylcholine receptor expression or function may be involved in the neuropathophysiology of schizophrenia. [³H]-nicotine and [³H]-epibatidine binding were compared in postmortem brain from control and schizophrenic subjects with varying smoking histories. In control subjects, increased receptor binding was seen in hippocampus, cortex, and caudate with increasing tobacco use. In contrast, schizophrenic smokers had reduced nicotinic receptor levels in these brain regions compared to control smokers. Chronic haloperidol and nicotine treatment, in the rat, was used to assess neuroleptic effects on receptor up-regulation by nicotine. A significant increase in cortical nicotinic receptors was seen in both nicotine treated as well as haloperidol and nicotine co-treated animals, suggesting that the abnormal regulation of high affinity neuronal nicotinic receptors in schizophrenics following nicotine use was not related to chronic neuroleptic treatment.
Up-regulation of nicotinic acetylcholine receptors following chronic exposure of rats to mainstream cigarette smoke or α4β2 receptors to nicotine
1995, Biochemical Pharmacology
Smokers are reported to have a higher density of central nicotinic acetylcholine receptors (nAChRs) that non-smokers at autopsy. Whether this increased receptor density is a response to smoking or a result of genetic variability is not known. While sub-chronic treatment of rats and mice with nicotine results in upregulation of central nAChRs, changes in receptor density in response to cigarette smoke have not been studied previously. In this study, male Sprague-Dawley rats were exposed nose-only for 13 weeks to mainstream cigarette smoke followed by assessment of [³H]nicotine binding in five brain regions of smoke- and sham-exposed animals. In smoke-exposed animals, there was a significant increase in nAChR density in the cortex, striatum, and cerebellum (35, 25, and 31% increases, respectively), while there was no significant change in receptor density in the thalamus and hippocampus. Smoke exposure did not alter markedly the affinity of the receptor for nicotine in these brain regions. Furthermore, up-regulation of nAChRs did not alter the biphasic binding properties by which nicotine binds to its receptor. There were no changes in the association (fast phase) or isomerization (slow phase) rate constants, and the percent contribution of slow and fast phase binding to nAChRs was not altered in the up-regulated receptor population compared with control. Similar results were observed following chronic nicotine exposure of cultured cortical cells from fetal rat brain or cells transfected with the α4β2 nAChR subtype. These results show that the up-regulation following smoke exposure in the rat is phenomenologically similar to that observed in vitro. These data provide preliminary evidence for a relationship between cigarette smoking and nAChR up-regulation in vivo and suggest that similar mechanisms of upregulation may underlie chronic smoke exposure of live animals and nicotine exposure of artificially expressed α4β2 receptors in vitro.
α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment
1995, Brain Research
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]cyttine 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]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.
Localization of nicotinic cholinergic receptors in rat brain: Autoradiographic studies with [3H]cytisine
1994, Neuroscience
There is a great deal of interest in the role of nicotinic acetylcholine receptors in the central nervous system, although their function is not well understood at present. Currently, central nicotinic receptors can be classified broadly as either α-bungarotoxin binding sites with low affinity for acetylcholine agonists, or as high-affinity agonist binding sites with low affinity for a-bungarotoxin. Neuronal nicotinic receptors with a high affinity for agonists are distributed widely in the central nervous system. Evidence from molecular biology and electrophysiology suggests that multiple nicotinic receptor types exist in the brain. In this study we have used the agonist [³H]cytisine as a ligand for autoradiography to generate a detailed quantitative map of the high-affinity agonist binding nicotinic receptor in the rat brain. Optimized binding conditions, characterization of the kinetic and equilibrium binding properties, and demonstration of the nicotinic pharmacology of this binding site in tissue sections confirm the usefulness of [³H]cytisine as a ligand for nicotinic receptor autoradiography. [³H]Cytisine autoradiography provides excellent anatomic resolution with very low non-specific binding. This property has allowed us to describe variations in receptor density within subnuclei and gradients of receptor density in larger brain regions. Data from several studies suggest that the predominant high-affinity agonist binding nicotinic receptor in the central nervous system is composed of the α4 and β2 subunits. The data in the current study are consistent with the suggestion that [³H]cytisine labels only the α4β2 nicotinic receptor with high affinity, offering the possibility of localizing a specific nicotinic receptor subtype in the central nervous system.
In summary, we characterize the optimum experimental conditions for the use of [³H]cytisine in tissue section autoradiography. [³H]Cytisine proves to be an excellent marker for nicotinic cholinergic receptors with a very high affinity and very low background. We provide a detailed quantitative characterization of nicotinic receptor density in the rat central nervous system and we find there are significant variations and gradients in receptor density within specific brain regions, including subrogions previously thought to be homogeneous.

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Life Sciences

Abstract

References (12)

J. Bio. Chem.

FEBS LETTERS

Eur J. Pharmacol.

Biochem. Pharmacol.

Human Toxicol.

Mol. Pharmacol.

Cited by (41)

<sup>86</sup>Rb<sup>+</sup> efflux mediated by α4β 2-nicotinic acetylcholine receptors with high and low-sensitivity to stimulation by acetylcholine display similar agonist-induced desensitization

The effect of nicotine and haloperidol co-treatment on nicotinic receptor levels in the rat brain

Abnormal regulation of high affinity nicotinic receptors in subjects with schizophrenia

Up-regulation of nicotinic acetylcholine receptors following chronic exposure of rats to mainstream cigarette smoke or α4β2 receptors to nicotine

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

Localization of nicotinic cholinergic receptors in rat brain: Autoradiographic studies with [<sup>3</sup>H]cytisine