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.
Highlights
Nicotinic acetylcholine receptors in the nervous system are members of a family of ligand-gated ion channels, pentametric in structure, with at least 11 different subunits (α2–α10 and β2– β4) characterized (Lindstrom et al, 1996; Alexander et al, 2009)
In general, α4β2 and α7 are the predominant subtypes expressed in the central nervous system (CNS), whereas the α3β4 subtype is the predominant subtype expressed in the autonomic ganglia (Lindstrom, 2000; Alexander et al, 2009)
The Nicotinic acetylcholine receptors (nAChRs) in the CNS are physiologically relevant in a wide range of neuronal functions, and may be involved in pathological states such as Alzheimer’s and Parkinson’s diseases (Grady et al, 2007; Quik et al, 2007; Drever et al, 2010)
Summary
Nicotinic acetylcholine receptors (nAChRs) in the nervous system are members of a family of ligand-gated ion channels, pentametric in structure, with at least 11 different subunits (α2–α10 and β2– β4) characterized (Lindstrom et al, 1996; Alexander et al, 2009). In most studies of nAChRs and other receptors in the CNS, brain tissue was homogenized, and the resulting membrane or particulate preparations have been exclusively used in the radioligand binding assay (Bylund and Toews, 1993; Houghtling et al, 1995; Yang et al, 1998; Marubio et al, 1999; Whiteaker et al, 2000; Schneider and Michel, 2010) Such a grind-and-bind approach has been successfully used to identify receptor pharmacology for more than two decades, tissue homogenization may cause alterations in receptor environments, resulting in different properties from the inherent profile in some receptors (HiraizumiHiraoka et al, 2004; Anisuzzaman et al, 2008, 2011; Morishima et al, 2008). The effects on nAChRs of chronic nicotine administration were examined by the tissue-segment binding assay
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