Symposium 1997: Abstract for J Stables et al

Jenny Stables, Steve Rees, Elaine Murrison, Melanie Lee
Receptor Systems Unit, GlaxoWellcome Research and Development,
Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK

The jellyfish photoprotein apoaequorin, in the presence of the cofactor coelenterazine and O₂, emits a characteristic luminescence which is dependent on Ca²⁺ concentration. The apoaequorin cDNA has been expressed in mammalian cells and used to detect agonist activation at a number of Gaq/11 coupled receptors (Sheu et al. 1993, Button and Brownstein 1993, Hedley et al. 1996).

In this study, CHO cells were stably transfected with apoaequorin and either the human oxytocin receptor or the human V1a vasopressin receptor. Using these cell lines, a 96-well format luminescent assay was developed. A series of standard agonists induced large concentration-related increases in luminescence, in general with an expected rank order of potency. The EC₅₀ estimates obtained at the oxytocin receptor were as follows: oxytocin= 23±6nM, vasopressin=31±18nM, desmopressin=418±163nM, Thr⁴Gly⁷oxytocin=17±4nM. The EC₅₀ estimates obtained at the vasopressin receptor were as follows: oxytocin=1.9±0.5µM, Vasopressin=28±8nM, Thr⁴Gly⁷oxytocin>10µM.

The oxytocin receptor antagonist L-371,257 inhibited oxytocin-induced luminescence at the oxytocin receptor with a pKB of 9, while it had no significant effect on vasopressin-induced luminescence in the V1a receptor expressing cell line. In conclusion, we confirm that co-expression of apoaequorin and receptor of interest offers a rapid and sensitive read-out of agonist activation of Gaq/11 coupled receptors, which is suitable for medium throughput of compounds in a 96-well format.

Y Sheu, L Kricka, D Pritchett. Anal Biochem 1993; 209:343-7.
D Button, M Brownstein. Cell Calcium 1993; 14:663-71.
L Hedley, S Phagoo, I.James. Anal Biochem 1996; 236:270-4.