| Assay Method Information | |
| | Measurement of Apelin Receptor Agonist Activity |
| Description: | The apelin receptor agonist activity of the compound represented by formula (I) or (II) of the present invention was measured by cell-based assay for determining the amount of intracellular cAMP.CHO cells (DiscoveRx Corp.) stably expressing the human apelin receptor were inoculated to wells of a microtiter plate and cultured overnight at 37° C. under conditions of 5% CO2.Each test compound and Forskolin were added to the wells at the same time. Specifically, the test compound dissolved in DMSO was diluted with a Forskolin-containing buffer [DMEM/F-12, HEPES, no phenol red (#11039, GIBCO)] to prepare a mixed solution containing the test compound at a final concentration of 0 to 10 μM (common ratio: 3, serial dilution: 11), Forskolin at a final concentration of 10 μM, and DMSO at a final concentration of 0.3% v/v, which was then added to the wells (N=4).After 30 minutes, the cells were lysed, and HitHunter cAMP XS+ Assay #90-0075L (DiscoveRx Corp.) was added to the cell lysate. The mixture was left overnight, and the resulting chemiluminescence signals were then detected using Analyst GT (Molecular Devices LLC.) to determine the amount of cAMP.The amount of cAMP determined by the addition of a positive control [Pyr1]-apelin-13 (final concentration: 0.300 μM) and Forskolin (final concentration: 10 μM) at the same time was used as an index to define 100% efficacy. The amount of cAMP determined by the addition of Forskolin (final concentration: 10 μM) alone was used as an index to define 0% efficacy.A dose response curve was prepared from the final concentration of the test compound and the determined amount of cAMP using Sigmaplot 12.0. |
| Affinity data for this assay | |
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