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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

Time course and saturation isotherm of [3H]-epibatidine binding in the intact segments (A,C) and homogenates (B,D) of rat cerebellum. Time course (A,B): The intact segments were incubated with 1000 pM [3H]-epibatidine at 4°C for 1–24 h and the homogenates were incubated with 500 pM [3H]-epibatidine at 4°C for 15 min to 5 h. Saturation binding (C,D): The intact segments and homogenates were incubated with [3H]-epibatidine (up to 2000 pM) at 4°C for 28 h (segments) or 5 h (homogenates). Specific binding (open circles) was determined by subtracting the amount bound in the presence of 100 μM nicotine (asterisks) from total binding (closed squares). Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.
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Figure 2: Time course and saturation isotherm of [3H]-epibatidine binding in the intact segments (A,C) and homogenates (B,D) of rat cerebellum. Time course (A,B): The intact segments were incubated with 1000 pM [3H]-epibatidine at 4°C for 1–24 h and the homogenates were incubated with 500 pM [3H]-epibatidine at 4°C for 15 min to 5 h. Saturation binding (C,D): The intact segments and homogenates were incubated with [3H]-epibatidine (up to 2000 pM) at 4°C for 28 h (segments) or 5 h (homogenates). Specific binding (open circles) was determined by subtracting the amount bound in the presence of 100 μM nicotine (asterisks) from total binding (closed squares). Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.

Mentions: The representative time courses of [3H]-epibatidine binding to the intact segments (Figure 1A) and homogenate preparations (Figure 1B) of rat cerebral cortex and to the intact segments (Figure 2A) and homogenate preparations (Figure 2B) of rat cerebellum are shown. At 4°C, the binding to the segments was extremely slow compared with that in the homogenates; therefore, different incubation periods were applied in the segment binding assay (26–30 h) and the homogenate binding assay (5 h). In the saturation experiments, the specific binding of [3H]-epibatidine to both the intact segments (Figure 1C) and homogenate preparations (Figure 1D) of rat cortex was saturable at the concentrations tested and fitted a single-site model. However, the slopes of the saturation curves were apparently different; the slope was steeper for the homogenates than for the segments. Thus, the dissociation constant (Kd) for [3H]-epibatidine was estimated to be four times higher in the segment binding assay than in the homogenate binding assay (Table 1). Furthermore, the maximum binding capacity (Bmax) of [3H]-epibatidine was also approximately three times higher in the segments than in the homogenates of rat cortex (Table 1). Differences in the time course, the density, and the dissociation constant for [3H]-epibatidine binding were also observed between the segments and homogenates of rat cerebellum (Figure 2 and Table 1). The affinity for [3H]-QNB (a muscarinic receptor ligand that was used as control) was also lower in the cortex segments than in the homogenates, but the binding capacity was not significantly different between the segments and the homogenates (Table 1).


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)

Time course and saturation isotherm of [3H]-epibatidine binding in the intact segments (A,C) and homogenates (B,D) of rat cerebellum. Time course (A,B): The intact segments were incubated with 1000 pM [3H]-epibatidine at 4°C for 1–24 h and the homogenates were incubated with 500 pM [3H]-epibatidine at 4°C for 15 min to 5 h. Saturation binding (C,D): The intact segments and homogenates were incubated with [3H]-epibatidine (up to 2000 pM) at 4°C for 28 h (segments) or 5 h (homogenates). Specific binding (open circles) was determined by subtracting the amount bound in the presence of 100 μM nicotine (asterisks) from total binding (closed squares). 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 2: Time course and saturation isotherm of [3H]-epibatidine binding in the intact segments (A,C) and homogenates (B,D) of rat cerebellum. Time course (A,B): The intact segments were incubated with 1000 pM [3H]-epibatidine at 4°C for 1–24 h and the homogenates were incubated with 500 pM [3H]-epibatidine at 4°C for 15 min to 5 h. Saturation binding (C,D): The intact segments and homogenates were incubated with [3H]-epibatidine (up to 2000 pM) at 4°C for 28 h (segments) or 5 h (homogenates). Specific binding (open circles) was determined by subtracting the amount bound in the presence of 100 μM nicotine (asterisks) from total binding (closed squares). Each point represents the mean of duplicate determinations. Each figure represents four to five experiments.
Mentions: The representative time courses of [3H]-epibatidine binding to the intact segments (Figure 1A) and homogenate preparations (Figure 1B) of rat cerebral cortex and to the intact segments (Figure 2A) and homogenate preparations (Figure 2B) of rat cerebellum are shown. At 4°C, the binding to the segments was extremely slow compared with that in the homogenates; therefore, different incubation periods were applied in the segment binding assay (26–30 h) and the homogenate binding assay (5 h). In the saturation experiments, the specific binding of [3H]-epibatidine to both the intact segments (Figure 1C) and homogenate preparations (Figure 1D) of rat cortex was saturable at the concentrations tested and fitted a single-site model. However, the slopes of the saturation curves were apparently different; the slope was steeper for the homogenates than for the segments. Thus, the dissociation constant (Kd) for [3H]-epibatidine was estimated to be four times higher in the segment binding assay than in the homogenate binding assay (Table 1). Furthermore, the maximum binding capacity (Bmax) of [3H]-epibatidine was also approximately three times higher in the segments than in the homogenates of rat cortex (Table 1). Differences in the time course, the density, and the dissociation constant for [3H]-epibatidine binding were also observed between the segments and homogenates of rat cerebellum (Figure 2 and Table 1). The affinity for [3H]-QNB (a muscarinic receptor ligand that was used as control) was also lower in the cortex segments than in the homogenates, but the binding capacity was not significantly different between the segments and the homogenates (Table 1).

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