( with a rapid flash of light. The light-...)Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)

with a rapid flash of light. The light-emitting reaction was essentially complete within 10 seconds. The amount of light emitted increased with increasing UTP concentration. In addition, the higher the UTP concentration, the faster the peak emission was reached.

Figure 3 compares the UTP dose response curves for CHOMQ6 cells incubated with either native coelenterazine or coelenterazine isoform h, plotting fluorescence counts collected for a 10 second period versus concentration of UTP. Cells preincubated with coelenterazine isoform h produced 3 to 4 times the luminescence quantum yield compared to the cells preincubated with native coelenterazine upon UTP activation. Furthermore, the EC50 of UTP was determined to be 0.1uM in cells incubated with coelenterazine isoform h compare to 0.3 uM in cells incubated with native coelenterazine.

SUMMARY
These studies demonstrate that the Lmax can be used to determine the GPCR-mediated pharmacological response of cells expressing apo-aequorin. Apoaequorin can be used as a calcium sensor, allowing researchers to determine the initiation of calcium flux within cells upon receptor activation. The lack of reagent toxicity and ease of use of aequorin-expressing cells may provide a useful alternative method to calcium-sensitive dye-loading procedures for determining receptor activation. The Lmax offers high assay sensitivity and automated reagent injection in a 96-well format, as well as SOFTmax PRO for Lmax, a powerful and convenient instrument control, data analysis and presentation package. With the use of apo-aequorin expressing cells, the Lmax can be a useful GCPR pharmacology tool and may also be useful for medium throughput antagonist screening.

REFERENCES
1. J. Chem. Soc. Chem. Comm. 21, 1566 (1986)

2. Meth. Enzymol. 57, 271 (1978)

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