A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels.
A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed 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 that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.
George
S E
SE
Merck, Sharp & Dohme, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, United Kingdom.
Schaeffer
M T
MT
Cully
D
D
Beer
M S
MS
McAllister
G
G
D000331:Aequorin / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002623:Chemistry Techniques, Analytical / Q000379:methods; D015870:Gene Expression; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D014162:Transfection
D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D017366:Serotonin Receptor Agonists; D000331:Aequorin; D002118:Calcium