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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.

No MeSH data available.


Related in: MedlinePlus

Competition curves for various dugs at [3H]-epibatidine binding sites in intact segments and homogenates of rat cerebral cortex (A,B) and rat cerebellum (C,D). Intact segments (A,C) were incubated with 400 pM [3H]-epibatidine for 28 h at 4°C. Homogenates were incubated with 200 pM [(B): cortex] or 300 pM [(D): cerebellum] [3H]-epibatidine for 5 h at 4°C. Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.
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Figure 3: Competition curves for various dugs at [3H]-epibatidine binding sites in intact segments and homogenates of rat cerebral cortex (A,B) and rat cerebellum (C,D). Intact segments (A,C) were incubated with 400 pM [3H]-epibatidine for 28 h at 4°C. Homogenates were incubated with 200 pM [(B): cortex] or 300 pM [(D): cerebellum] [3H]-epibatidine for 5 h at 4°C. Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.

Mentions: Because the binding capacity and dissociation constant for [3H]-epibatidine were significantly different between tissue segments and homogenates, the pharmacological profiles of [3H]-epibatidine binding sites in both preparations of rat cortex were compared in competition binding experiments using various drugs. Nicotine, dihydro-β-erythroidine, and cytisine showed shallow competition curves in the segments of cortex and approximately 70% of the specific binding sites were identified as high-affinity sites for these ligands (Figure 3A). In the homogenates, dihydro-β-erythroidine and cytisine also showed shallow curves, but the competition curve for nicotine better fitted a one-site model with a slightly higher pKi value than that in the segments (Figure 3B and Table 2). Hexamethonium at concentrations higher than 10 μM were competitive, but mecamylamine (1 mM) and α-bungarotoxin (1–30 nM) failed to compete in the [3H]-epibatidine binding assay with both segment and homogenate preparations (Table 2). In general, the estimated ligand affinities were higher in the homogenates compared with the segments.


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)

Competition curves for various dugs at [3H]-epibatidine binding sites in intact segments and homogenates of rat cerebral cortex (A,B) and rat cerebellum (C,D). Intact segments (A,C) were incubated with 400 pM [3H]-epibatidine for 28 h at 4°C. Homogenates were incubated with 200 pM [(B): cortex] or 300 pM [(D): cerebellum] [3H]-epibatidine for 5 h at 4°C. Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Competition curves for various dugs at [3H]-epibatidine binding sites in intact segments and homogenates of rat cerebral cortex (A,B) and rat cerebellum (C,D). Intact segments (A,C) were incubated with 400 pM [3H]-epibatidine for 28 h at 4°C. Homogenates were incubated with 200 pM [(B): cortex] or 300 pM [(D): cerebellum] [3H]-epibatidine for 5 h at 4°C. Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.
Mentions: Because the binding capacity and dissociation constant for [3H]-epibatidine were significantly different between tissue segments and homogenates, the pharmacological profiles of [3H]-epibatidine binding sites in both preparations of rat cortex were compared in competition binding experiments using various drugs. Nicotine, dihydro-β-erythroidine, and cytisine showed shallow competition curves in the segments of cortex and approximately 70% of the specific binding sites were identified as high-affinity sites for these ligands (Figure 3A). In the homogenates, dihydro-β-erythroidine and cytisine also showed shallow curves, but the competition curve for nicotine better fitted a one-site model with a slightly higher pKi value than that in the segments (Figure 3B and Table 2). Hexamethonium at concentrations higher than 10 μM were competitive, but mecamylamine (1 mM) and α-bungarotoxin (1–30 nM) failed to compete in the [3H]-epibatidine binding assay with both segment and homogenate preparations (Table 2). In general, the estimated ligand affinities were higher in the homogenates compared with the segments.

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