Investigating the Alterations in Muscarinic M1 Receptor Signalling Caused by Prolonged Agonist Activation

F Calvert1, KA Bennett1, AJH Brown1, R Prihandoko2. 1Heptares Therapeutics, Welwyn Garden City, UK, 2Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK

Background: Agonists of the muscarinic M1 receptor have potential in the treatment of Alzheimer’s disease. However, G-protein coupled receptor signalling is highly regulated and prolonged agonist exposure may lead to receptor desensitisation, internalisation and down-regulation. Here we describe development of a signalling assay (IPone) to detect alterations in M1-mediated signalling in response to prolonged agonist (acetylcholine) exposure.

Methods: CHO cells stably expressing M1 receptors were maintained in MEME (α-modification) supplemented with 10 % FBS, 1 % penicillin/streptomycin and 1 % G-418. To determine levels of inositol phosphates produced in response to acetylcholine an IP-One HTRF® assay kit (Cisbio assays) was used as per manufacturer’s instructions. Cells were seeded (10,000-35,000 cells/well) in 96 well half-area plates, 24 h later cells were stimulated with agonist (5-125 minutes; 37 ºC) in IPone stimulation buffer before assay was terminated and read following manufacturer’s instructions.

For assessment of prolonged agonist exposure (‘pre-stimulation experiments’) cells were seeded (35,000 cells/well) in 96 well half-area plates. After 4 hours media was removed and replaced with 1 % serum media supplemented with 10 µM donepezil and, where appropriate, cells were pre-stimulated with an EC100 concentration of acetylcholine (~1 µM). After 24 hours cells were washed three times with 1 % serum media and challenged with varying concentrations of acetylcholine in IPone stimulation buffer (1 h; 37 °C), 100 µM ionomycin was used as a control.

Results: A response to acetylcholine was measured in CHO M1 cells (pEC50 ± S.E.M = 7.31 ±0.18) but not in parental CHOs. A cell seeding density of 35,000 cells/well and 1 h incubation time with acetylcholine was determined to give the optimal signal window. The washout protocol for pre-stimulation compounds was optimised to three washes (no residual agonist response observed).

Pre-stimulation with ~1 µM acetylcholine in the presence of 10 µM donepezil appeared to increase the ACh pEC50 shift (n=2, pEC50 shift ± S.E.M = -0.014 ± 0.044 without donepezil and 0.52 ± 0.0095with donepezil).

A significant change in the response to 0.31 µM, 0.10 µM, 0.03 µM acetylcholine was seen in cells pre-stimulated with 1 µM acetylcholine; (unpaired two tailed t test; p<0.05 n=3, p<0.01 n=4, p<0.01 n=3 respectively). Responses to 100 µM ionomycin were unaltered in pre-stimulated cells.

Conclusion: We have optimised an IPone assay that can detect significant alterations in the acetylcholine response after pre-stimulation with an EC80 concentration of acetylcholine for 24 hours. The addition of donepezil to the pre-stimulation media appeared to cause a greater reduction in the challenge response in comparison to cells pre-stimulated in the absence of donepezil; suggesting acetylcholine may be broken down by endogenous acetylcholine esterases in CHO cells. Ionomycin responses were similar in both pre-stimulated and control cells suggesting that the decreased response to acetylcholine observed in pre-stimulated cells is not due to depleted inositol phosphate levels or cell death. We aim to test a range of compounds (differing efficacy/affinity profiles) in this assay to observe how, in this system, these affect signalling of the receptor.