Probenecid

Chinese hamster ovary (CHO) cells stably expressing human FFAR1 with different receptor mRNA expression levels (clones #104 and #2) [32 (link)] were cultured with minimum essential medium-alpha (FUJIFILM Wako) containing 10% dialyzed fetal bovine serum (FBS, GE Healthcare Life Sciences, Buckinghamshire, UK), 10 mM HEPES (Thermo Fisher Scientific, Waltham, MA), 100 IU/mL penicillin, and 100 μg/mL streptomycin in 5% CO₂ at 37°C. Cells (1 x 10⁴ cells /well) were seeded in 384 well plates and then incubated overnight. After removing of the medium, cells were incubated in 30 μL of loading buffer (Hanks' Balanced Salt Solution containing 20 mM HEPES, 0.1% fatty acid-free BSA (FUJIFILM Wako), 0.08% Pluronic F127 (#CSK-01F, Dojindo Kumamoto, Japan), 2.5 mmol/L Probenecid (#CSK-03F, Dojindo) and 2.5 μg/mL Fluo4 (#F311, Dojindo)) for 60 min in 5% CO₂ at 37°C. Test compounds at various concentrations were added into the cells and increase of the intracellular Ca²⁺ concentration was monitored by the FLIPR Tetra system (Molecular Devices, Tokyo, Japan) for 180 sec. EC₅₀ was calculated by data analysis using a 4-parameter logistic equation in Graphpad Prism 7 software.

Pure hiPSC-CM spheroids on day 5, after they started beating, were analyzed after replacing the medium with fresh medium and performing incubation at 37°C for 30 min. We exposed the spheroids to several compounds at different concentrations for 30 min and recorded the findings with a BZX-710. The beating rates were calculated based on moving images. We also detected the onset of the changes in beating after the administration of COA-Cl. We continuously observed the beating spheroids before and after the administration of COA-Cl using fluorescent calcium imaging. The spheroids were incubated for 1 h at 37°C in loading buffer containing 10 μM fluo4 acetoxymethyl ester (Dojindo, Kumamoto, Japan) and detergents (0.02% Pluronic F-127, and 1.25 mmol/l Probenecid) (Dojindo). The samples were washed with recording buffer (Dojindo) for 20 min at 37°C, and calcium transients were recorded using fluorescence imaging with an excitation wavelength of 488 nm. The videos were converted to a series of TIFF format pictures, and the relative fluorescence (in relative fluorescence units [RFU]) of the spheroids was measured using the BZ-X Analyzer software program. The measured RFU was graphed to determine the beating profiles of RFU with the Excel software program.

The culture medium of the mpkCCD cells or dissection solution of mouse kidneys was replaced with loading buffer containing 5 μg ml⁻¹ Fluo4-AM (Dojindo), 1.25 mmol l⁻¹ probenecid (Dojindo), and 0.04% Pluronic F-127 (Dojindo). After incubation for 1 h at 37 °C, loading buffer was replaced with recording medium containing 1.25 mmol l⁻¹ probenecid. The mpkCCD cells and isolated CCD of mouse kidneys were then placed under confocal laser scanning microscopy. Wnt5a (500 ng ml⁻¹) was added, and calcium responses were monitored by the LSM510 Meta. Fluorescence intensities of Fluo4 were quantified from six regions of interest using Zen 2009 software.

Chinese hamster ovary (CHO)‐K1 cells expressing M₁R–M₅R were plated on a 96‐well black, clear bottom plate at 30 000 cells/well and incubated at 37°C in an atmosphere of 5% CO₂ for 1 day. On the day of the assay, cells were incubated with calcium dye buffer (Hanks’ Balanced Salt Solution (HBSS) containing 20 mmol/L HEPES, 0.1% fatty acid‐free bovine serum albumin (BSA), 0.08% pluronic F127 (Dojindo Laboratories), 2.5 μg/mL Fluo‐4 (Dojindo Laboratories), and 1.25 mmol/L probenecid (Dojindo Laboratories)) for 30 minutes at 37°C in an atmosphere of 5% CO₂ and then incubated for 30 minutes at room temperature. To measure Ca²⁺ mobilization using CellLux (PerkinElmer), cells were stimulated with T‐495 or MK‐7622 (0.01‐1000 nmol/L for PAM activity; 0.3‐10 000 nmol/L for agonist activity) in assay buffer (HBSS containing 20 mmol/L HEPES and 0.1% fatty acid‐free BSA) with or without an EC₂₀ concentration of ACh. The inflection point (IP) and EC₅₀ values were calculated using the following equation by GraphPad Prism 5 software (GraphPad Software Inc): $Y=\text{Bottom}+\frac{(\text{Top}-\text{Bottom})}{1+{10}^{(\left(\text{Log IP}\text{orEC}50-X\right)\times \text{HillSlope}))}}$
where X and Y are the log concentration of a compound and the percentage of Ca²⁺ response, respectively, and Top and Bottom are the upper and lower plateaus, respectively.

Ca²⁺ influx assay using primary neurons was performed as previously described^{21 (link)}. After 5 days of culture, the cells plated on poly-D-lysine coated 96-well plates (Corning Incorporated) at 2 × 10⁴ or 5 × 10⁴ cells/well were used for experiments with PF-04958242 or TAK-653, respectively. Fluorescent calcium indicator dye solution (Calcium4 assay Kit, Dojindo, Kumamoto, Japan) in Ca²⁺ reaction buffer (DMEM, HEPES and BSA) containing Probenecid (Dojindo) was added and incubated for 60 min in 5% CO₂ at 37 °C. After washing once, the relative increases in intracellular Ca²⁺ levels stimulated by compounds in the presence and absence of AMPA were monitored. Details are described in the Supplementary Information.