A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents

BACKGROUND: Cholinergic neurons utilize choline (Ch) to synthetize acetylcholine (ACh) and contain a high-affinity Ch transporter, Ch acetyltransferase (ChAT), ACh receptors, and acetylcholinesterase (AChE). As the depletion or malfunction of each component of the cholinergic system has been reported in patients with dementia, many studies have sought to evaluate whether treatment candidates affect each of the cholinergic components. The associated changes in the cholinergic components may be reflected by intra- or extra-cellular ACh levels, with an increase in extracellular ACh levels occurring following AChE inhibition. We hypothesized that increases in intracellular ACh levels can be more sensitively detected than those in extracellular ACh levels, thereby capturing subtle effects in the cholinergic components other than AChE. The objective of this study was to test this hypothesis. METHODS: We developed an in vitro model to measure both extracellular and intracellular ACh levels using the human cholinergic neuroblastoma cell line, LA-N-2, which have been reported to express Ch transporter, ChAT, muscarinic ACh receptor (mAChR), and AChE. With this model, we evaluated several drug compounds and food constituents reported to improve cholinergic function through various mechanisms. In addition, we conducted western blotting to identify the subtype of mAChR that is expressed on the cell line. RESULTS: Our cell-based assay system was capable of detecting increases in extracellular ACh levels induced by an AChE inhibitor at relatively high doses, as well as increases in intracellular ACh levels following the administration of lower AChE-inhibitor doses and an mAChR agonist. Moreover, increases in intracellular ACh levels were observed even after treatment with food constituents that have different mechanisms of action, such as Ch provision and ChAT activation. In addition, we revealed that LA-N-2 cells expressed mAChR M2. CONCLUSION: The findings support our hypothesis and indicate that the developed assay model can broadly screen compounds from drugs to food ingredients, with varying strengths and mechanisms of action, to develop treatments for ACh-relevant phenomena, including dementia and aging-related cognitive decline.