A high-throughput glow-type aequorin assay for measuring receptor-mediatedchanges in intracellular calcium levels

A glow-type aequorin luminescence assay for measuring receptor-mediated sti mulation of intracellular calcium levels is described and characterized. Th e human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryo nic kidney cells with apoaequorin was used to characterize the system and s howed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist, Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values tha t were stable over this time period. Testing a range of 5-hydroxytryptamine (2A) receptor agonists gave the expected rank order of potency for this rec eptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine (2A) receptor antagonists, generating affinity values that directly correla ted with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologicall y relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer. ( C) 2000 Academic Press.