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

Abstract

Abstract. Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [3 H]-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 [ 3 H]-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.

Intracellular distribution of functional M1‐muscarinic acetylcholine receptors in N1E‐115 neuroblastoma cells

“…The main finding of this study was that, as in cerebral cortex and rabbit cornea cells (Lind and Cavanagh 1993;Lee et al 2010), the N1E-115 neuroblastoma cells constitutively possess both intracellular and plasma membrane-localized populations of M 1 -mAChRs. Furthermore, our data clearly indicate that the mAChRs at both sites are independently activated by carbachol or the endogenous ligand, acetylcholine.…”

“…The main finding of this study was that, as in cerebral cortex and rabbit cornea cells (Lind and Cavanagh 1993; Lee et al. 2010), the N1E‐115 neuroblastoma cells constitutively possess both intracellular and plasma membrane‐localized populations of M 1 ‐mAChRs.…”

“…Recently, we identified intracellular M 1 ‐mAChRs in rat cortex and hippocampus (Lee et al. 2010 and unpublished observations).…”

“…Apart from the multiple intracellular mechanisms whereby the M 1 -mAChR activates ERK1/2, it is uniformly presumed that the initial signal is generated by a cell surface receptor. In our recent work with rat cerebral tissue segments, as opposed to membrane receptor preparations, we have obtained evidence that there are two 'pools' of M 1 -mAChR in intact tissues, one localized on plasma membrane that can interact with the cell-impermeable 'hydrophilic' antagonist, N-methyl-scopolamine (NMS) and the other, intracellularly localized, that interacts with the cell-permeable 'hydrophobic' antagonist, qunuclidinyl benzilate (QNB) (Lee et al 2010). However, it was unclear whether the intracellular M 1 -mAChR is actually functional or non-functional as a backup for the plasma membrane localized receptors.…”

Military Incapacitating Agent Bz (3-Quinuclidinyl Benzilate) - Past, Present and Future

“…Nowadays, BZ is commonly used for identification of M receptors via several laboratory techniques, mainly radio-ligand binding assay [e.g. 2,3], and, due to its psychotomimetic properties, it has also been used to generate a cognitive deficit in animal models of neurodegenerative disorders, such as Alzheimer's disease [4][5][6].…”