( Jinfang Liao M.D. Ph.D. and Evel...)Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)

Jinfang Liao, M.D., Ph.D. and Evelyn McGown, Ph.D.
Molecular Devices Corporation, 8/00

INTRODUCTION
The photoprotein aequorin, a bioluminescent protein from the coelentratejellyfish Aequorea victorea, has proven to be highly sensitive to the trace amount of Ca⁺⁺ in cells. The aequorin complex which contains the 22,000 MW apo-aequorinprotein, molecular oxygen, and the luminophore coelenterazine^1,2 emits blue light (469 nm) upon binding calcium ions (Figure 1). The binding of Ca⁺⁺ to aequorin induces a conformational change resulting in the oxidation ofcoelenterazine through an intramolecular reaction. This bioluminescent processpresents an advantage over Ca⁺⁺-sensitive fluorescent dyes by being easilytargeted to specific cells and to subcellular compartments with appropriateregulatory elements and peptide signals³. Moreover, the aequorin complex doesnot require light excitation, thus eliminating autofluorescence, photobleachingand biological degradation problems. Its high affinity for Ca⁺⁺ (Kd = 10 M) makes aequorin a good sensor in the biological range of intracellular Ca⁺⁺ concentrations. The quick flash luminescence that is produced by aequorin canbe measured easily with the Lmax microplate luminometer equipped withautomated reagent injectors.

In a related previous application note⁴, we have described how to use Molecular Devices Lmax microplate luminometer to measure luciferase activity. Here, wedemonstrate how to use Lmax to detect receptor-activated Ca⁺⁺ signals in cellsstably expressing apo-aequorin. In these cell-based assays, we were able toconstruct agonist concentration-response curves for the activation of a Gqprotein-coupled purinergic receptor expressed endogenously in CHO cells. Thus,the Lmax microplate luminometer can be used to study GPCR pharmacology.Furthermore, with a constant agonist concentration in the injector reservoir, theLmax microplate luminometer could potentially be used to run high-thoughputluminescent assays for GPCR antagonist screening.

PRINCIPLE OF ASSAY
CHO cells expressing the apo-aequorin protein were tested in the L-max using uridine triphosphate (UTP) to activate the endogenous P2Y purinergic receptor. This receptor is known to be coupled to the Gq pathway, causing an increase in intracellular calcium. In order to form the aequorin complex, the cells were first loaded with the coenzyme coelenterazine. There are many different isoforms of this coenzyme available, for these studies. We chose to use the native coelenterazine and the coelenterazine(h) isoform.

MATERIALS
1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)

2. CHOMQ6 cell line stably expressing apo-aequorin, kindly provided by Jenny Stables (GlaxoWellcome Research, U.K.)

3. Coelenterazine, both native and h iso forms from Molecular Probes, Inc.; http://www.probes.com

4. UTP, Calbiochem Cat. No. 6701; Tel: 1-800-492-1110

5. Hams F-12 Cat. No. 9058, Fetal Bovine Serum Cat. No. 3000, Glutamine Pen-Strep Solution Cat. No. 9316, G418 sulfate #98529, and 1 M HEPES buffer solution Cat. # 9319 were obtained from Irvine Scientific Tel. 1-800-437-5706.

6. 10X Hanks Balanced Salt Solution, GIBCO BRL Cat# 14065-056; Tel 800-8286686

7. Clear-bottomed white 96-well microplates. CorningCostar Cat. No. 3903. Tel: 18004921110.

8. The native coelenterazine and h isoform loading solutions (2.5 M) were prepared by dilution in Hanks Balanced salt solution + 20 mM HEPES (H&H) with 1 mg/ml BSA.

PREPARATION OF CELLS
1. The stable CHOMQ6 cell line expressing apo-aequorin was maintained in F12 growth medium with 10% fetal bovine serum, 1% Glutamine Pen-Strep and 400 g/mL G418.

2. The day before the experiment, cells in growth medium as above were seeded in 96-well microplates at a density of 60,000 cells/well and incubated overnight in a 37C, 5% CO₂ incubator.

3. Cells were loaded with coelenterazine by aspirating the growth medium from the wells and replacing it with 100 L coelenterazine loading solution/well. They were then incubated at 37C with 5% CO₂ for an additional 2 hours.

ASSAY PROCEDURE
1. A dilution series of UTP was prepared in H&H with 1% BSA concentrations ranging from 0.2 M to 200 M, with the diluent (0 M UTP) as the blank. All were placed in 15-mL conical tubes.

2. The plate containing the coelenterazine-loaded cells (100 uL/well) was placed into the drawer of the Lmax.

3. The SOFTmax PRO for Lmax was set up to inject 100 L of UTP reagent through the M injector and to begin reading immediately and take 20 consecutive 1-second reads on selected replicate wells.

4. Starting with the blank (0 M UTP in diluent) the M injection system was sequentially filled with increasing concentrations of UTP solutions by pumping 1.5 mL through it and then the selected replicate target wells for each concentration were read. The UTP-containing tubes were placed in a small rack on top of the Lmax so that the injector inlet tubing could easily be moved from one tube to the next higher concentration after each set of measurements was complete.

REACTION PROFILES AND DOSE/RESPONSE RESULTS
The reaction profiles of coelenterazine(h)-loaded cells to 0.1 - 3 uM UTP (final concentrations) are shown in Figure 2. When CHO cells expressing apo-aequorin protein (CHOMQ6 cells) were incubated with 2.5 uM coelenterazine(h), then exposed to UTP (a potent purinergic agonist), they responded 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)

3. Meth. Enzymol. 305, 479 (2000

4. MDC MAXline App. Note #38, August 2000.

ACKNOWLEDGMENT
The authors wish to thank Jenny Stables (GlaxoWellcome Research, U.K.) for providing aequorin-expressing CHO cells and coelenterazine loading procedures.

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