Assessment of Novel Muscarinic Acetylcholine Receptors in Rat Cerebral Cortex by a Tissue Segment Binding Method

Kung-Shing Lee; Atsushi Nishimune; Hatsumi Yoshiki; Abu Syed Md Anisuzzaman; Fumiko Suzuki; Mao-Hsien Wang; Juei-Tang Cheng; Ikunobu Muramatsu

doi:10.1254/jphs.10016FP

Full Papers

Assessment of Novel Muscarinic Acetylcholine Receptors in Rat Cerebral Cortex by a Tissue Segment Binding Method

Kung-Shing Lee, Atsushi Nishimune, Hatsumi Yoshiki, Abu Syed Md Anisuzzaman, Fumiko Suzuki, Mao-Hsien Wang, Juei-Tang Cheng, Ikunobu Muramatsu

Author information

Kung-Shing Lee
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Department of Surgery, Kaohsiung Municipal Hsiao-Kang Hospital, and Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Taiwan
Atsushi Nishimune
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Organization for Life Science Advancement Programs, University of Fukui, Japan
Hatsumi Yoshiki
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Abu Syed Md Anisuzzaman
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Fumiko Suzuki
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Organization for Life Science Advancement Programs, University of Fukui, Japan
Mao-Hsien Wang
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Juei-Tang Cheng
Department of Pharmacology, College of Medicine, National Cheng Kung University, Taiwan
Ikunobu Muramatsu
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Japan
Organization for Life Science Advancement Programs, University of Fukui, Japan

Keywords: muscarinic acetylcholine receptor, intracellular receptor, quinuclidinyl benzilate, rat cerebral cortex, tissue segment binding assay

JOURNAL FREE ACCESS

2010 Volume 112 Issue 4 Pages 444-451

DOI https://doi.org/10.1254/jphs.10016FP

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Abstract

Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [³H]-Quinuclidinyl benzilate (QNB, a hydrophobic ligand) specifically bound to mAChRs in the cortex segments. The total mAChRs level was approximately 2,000 fmol/mg protein, which was estimated after incubation for 120 min at 37°C or for 8 h at 4°C. These mAChRs were a mixture of high- and low-affinity sites for N-methylscopolamine (NMS) in a 70:30 ratio. In contrast, only a single high-affinity site for NMS was detected following incubation for 30 min at 37°C, whose abundance was about 70% of that of the total mAChRs. Atropine showed a single affinity for mAChRs under all conditions. These indicate that mAChRs are constitutively expressed not only on plasma membrane sites but also at intracellular sites in rat cerebral cortex and that the receptors at both sites have different affinities for NMS. Acetylcholine completely inhibited [³H]-QNB binding to both mAChRs without any change in the subcellular distribution, suggesting the possibility that acetylcholine can access, and bind to, both mAChRs in intact tissue. Two different affinity states for acetylcholine were detected only in plasma membrane mAChRs at 37°C. The present study demonstrates a unique subcellular distribution, and distinct pharmacological profiles, of mAChRs in rat cerebral cortex.

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