Re-Evaluation of Nicotinic Acetylcholine Receptors in Rat Brain by a Tissue-Segment Binding Assay

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. [3H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [3H]-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 [3H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pKi = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pKi = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [3H]-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.

Regional quantification of muscarinic acetylcholine receptors and β‐adrenoceptors in human airways

“…In particular, the decrease in binding was greater for [ 3 H]‐NMS‐binding (more than 10% reduction) than for [ 3 H]‐CGP‐12,177‐binding sites (20–50% reduction) in the three regions. This preliminary result implies that many airway samples should be pooled in the case of homogenate binding, and that homogenization causes a large loss in yield of airway tissue receptors, similar to other tissues and radioligands (Tanaka et al., 2004; Wang et al., 2011).…”

“…Regardless of the presence or absence of cartilage, the airway segments were directly incubated with radioligands in physiological solution. As epithelial and muscle layers were not scraped from the cartilage, and in contrast to a marked reduction of [ 3 H]‐NMS and [ 3 H]‐CGP‐12,177 binding in homogenates (Figure 2), the present segment binding assay is less prone to loss of receptors and to modification of the natural environment/conformation of the receptors (Wang et al., 2011). However, the airway segments used in the present study were heterogeneous tissues that included various types of cells, as shown in Figure 1.…”

Cholinergic Receptor Binding Profile of Hypericum perforatum L. and its Active Constituents

“…The 6 mL of filter count scintillation cocktail (Perkin-Elmer Life Science, Boston, MA) was added to each vial and radioactivity was counted by Tri-Carb Liquid Scintillation Counter (Perkin-Elmer Life Science, Boston, MA). The 10G 5 M concentration of nicotine bitartrate was used for non-specific binding 20 . Muscarinic and nicotinic receptor binding inhibition data were presented as B/B 0 (%) (transformed from the cpm counts of the tested compounds) and means of logarithmic concentrations±standard error of the mean of four to six experiments conducted in duplicate.…”