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Re-evaluation of nicotinic acetylcholine receptors in rat brain by a tissue-segment binding assay.

Wang MH, Yoshiki H, Anisuzzaman AS, Uwada J, Nishimune A, Lee KS, Taniguchi T, Muramatsu I - Front Pharmacol (2011)

Bottom Line: The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum).The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2).The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui Fukui, Japan.

ABSTRACT
Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [(3)H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [(3)H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [(3)H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pK(i) = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pK(i) = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [(3)H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

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Effects of chronic administration of nicotine for 3 weeks. The tissue segments or homogenates of the cerebral cortex and cerebellum in nicotine-treated and untreated rats were incubated with [3H]-epibatidine for 28 h (segments) or 5 h (homogenates) at 4°C. The total density of [3H]-epibatidine binding sites was estimated from saturation experiments in the cortex and from 2 nM [3H]-epibatidine binding in the cerebellum. From the competition curves for nicotine, high-affinity (open column), and low-affinity sites (hatched column) for nicotine were calculated in the cerebral cortex. The binding sites in the cerebellum were not characterized by nicotine and were represented as black columns. Control, rats not treated with nicotine; nicotine, nicotine-treated rats. Mean ± SEM of four experiments. a: Significantly different from those of the control rats.
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Figure 4: Effects of chronic administration of nicotine for 3 weeks. The tissue segments or homogenates of the cerebral cortex and cerebellum in nicotine-treated and untreated rats were incubated with [3H]-epibatidine for 28 h (segments) or 5 h (homogenates) at 4°C. The total density of [3H]-epibatidine binding sites was estimated from saturation experiments in the cortex and from 2 nM [3H]-epibatidine binding in the cerebellum. From the competition curves for nicotine, high-affinity (open column), and low-affinity sites (hatched column) for nicotine were calculated in the cerebral cortex. The binding sites in the cerebellum were not characterized by nicotine and were represented as black columns. Control, rats not treated with nicotine; nicotine, nicotine-treated rats. Mean ± SEM of four experiments. a: Significantly different from those of the control rats.

Mentions: Chronic administration of nicotine is known to cause an increase in nAChRs in the brain, which is called upregulation. Because the number of [3H]-epibatidine binding sites was underestimated in homogenates as mentioned above, the nicotine-induced upregulation was re-evaluated by the tissue-segment binding approach. Administration of nicotine for 3 weeks increased the binding density of [3H]-epibatidine with a slight decrease in affinity in the rat cortex segments (Table 1). A similar tendency was observed in the cortex homogenates, but the density and affinity were also significantly different from the values estimated in the cortex segments of chronically nicotine-treated rats (Table 1). The binding affinities for nicotine in nicotine-treated rat cortex (pKihigh and pKilow = 7.8 ± 0.2 and 6.3 ± 0.2 in the segments and pKi = 8.1 ± 0.1 in the homogenates) were not significantly different from those in rats not treated with nicotine (Table 2). However, the proportion of nicotine-high-affinity sites in the cortex segments increased from 72% in control rats to 86% in nicotine-treated rats (Figure 4). On the other hand, no upregulation was caused by chronic nicotine administration in the cerebellum.


Re-evaluation of nicotinic acetylcholine receptors in rat brain by a tissue-segment binding assay.

Wang MH, Yoshiki H, Anisuzzaman AS, Uwada J, Nishimune A, Lee KS, Taniguchi T, Muramatsu I - Front Pharmacol (2011)

Effects of chronic administration of nicotine for 3 weeks. The tissue segments or homogenates of the cerebral cortex and cerebellum in nicotine-treated and untreated rats were incubated with [3H]-epibatidine for 28 h (segments) or 5 h (homogenates) at 4°C. The total density of [3H]-epibatidine binding sites was estimated from saturation experiments in the cortex and from 2 nM [3H]-epibatidine binding in the cerebellum. From the competition curves for nicotine, high-affinity (open column), and low-affinity sites (hatched column) for nicotine were calculated in the cerebral cortex. The binding sites in the cerebellum were not characterized by nicotine and were represented as black columns. Control, rats not treated with nicotine; nicotine, nicotine-treated rats. Mean ± SEM of four experiments. a: Significantly different from those of the control rats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198036&req=5

Figure 4: Effects of chronic administration of nicotine for 3 weeks. The tissue segments or homogenates of the cerebral cortex and cerebellum in nicotine-treated and untreated rats were incubated with [3H]-epibatidine for 28 h (segments) or 5 h (homogenates) at 4°C. The total density of [3H]-epibatidine binding sites was estimated from saturation experiments in the cortex and from 2 nM [3H]-epibatidine binding in the cerebellum. From the competition curves for nicotine, high-affinity (open column), and low-affinity sites (hatched column) for nicotine were calculated in the cerebral cortex. The binding sites in the cerebellum were not characterized by nicotine and were represented as black columns. Control, rats not treated with nicotine; nicotine, nicotine-treated rats. Mean ± SEM of four experiments. a: Significantly different from those of the control rats.
Mentions: Chronic administration of nicotine is known to cause an increase in nAChRs in the brain, which is called upregulation. Because the number of [3H]-epibatidine binding sites was underestimated in homogenates as mentioned above, the nicotine-induced upregulation was re-evaluated by the tissue-segment binding approach. Administration of nicotine for 3 weeks increased the binding density of [3H]-epibatidine with a slight decrease in affinity in the rat cortex segments (Table 1). A similar tendency was observed in the cortex homogenates, but the density and affinity were also significantly different from the values estimated in the cortex segments of chronically nicotine-treated rats (Table 1). The binding affinities for nicotine in nicotine-treated rat cortex (pKihigh and pKilow = 7.8 ± 0.2 and 6.3 ± 0.2 in the segments and pKi = 8.1 ± 0.1 in the homogenates) were not significantly different from those in rats not treated with nicotine (Table 2). However, the proportion of nicotine-high-affinity sites in the cortex segments increased from 72% in control rats to 86% in nicotine-treated rats (Figure 4). On the other hand, no upregulation was caused by chronic nicotine administration in the cerebellum.

Bottom Line: The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum).The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2).The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

View Article: PubMed Central - PubMed

Affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui Fukui, Japan.

ABSTRACT
Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [(3)H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [(3)H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [(3)H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pK(i) = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pK(i) = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [(3)H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

No MeSH data available.


Related in: MedlinePlus