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

Abstract

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.

Oral Administration of Egg- and Soy-Derived Lysophosphatidylcholine Mitigated Acetylcholine Depletion in the Brain of Scopolamine-Treated Rats

“…In our previous study, a cholinergic neuroblastoma cell line was used to demonstrate that LPC increased intracellular ACh levels [ 23 ], prompting the investigation of the cholinergic activity of LPC in vivo. To investigate the functional benefits of a wide variety of LPC species, different LPC sources, i.e., egg, soy, and rapeseed, were used for the experiments.…”

“…Furthermore, it is intriguing to know that the meta-analyses on the patients with Alzheimer’s disease showed significantly lower levels of Ch-containing lipids in the brain and PC in blood [ 37 ]. Considering that LPC treatment elevated ACh levels in cholinergic neurons [ 23 ], brain LPC availability may also be related to ACh synthesis in addition to brain Ch and PC, which suggests the additional benefit of LPC supplementation. It is clear that brain LPC levels after LPC administration should be quantified to investigate this possibility.…”

“…Sample pretreatment and measurement of ACh and Ch levels were performed as described previously [ 23 ]. The divided brain samples were sonicated in perchloric acid and centrifuged (15,000× g , 15 min, 4 °C).…”