Study of GPCR pharmacology using the DiscoveRx HitHunter™ cAMP HS assay on the FLUOstar OPTIMA

Julie M.-N. Rainard, Stewart E. Mireylees and Mark G. Darlison
School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK

The assay directly measures the activity of G-protein coupled receptors (GPCRs), coupled to either Gi or Gs proteins
The assay is sensitive and can detect low levels of cAMP (ideal for cell-lines expressing endogenous receptors)
The assay was performed on the FLUOstar OPTIMA, a foreruner of the FLUOstar Omega
The multidetection reader accepts different assay formats from 6- to 1536-well plates (suitable for academic environments and high-throughput screening; HTS)

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Introduction
G-protein coupled receptors (GPCRs) are cell surface receptors, which represent the most predominant drug targets. Following stimulation of these receptors, intracellular signalling pathways are activated and this leads to a decrease (coupling to a Gi protein) or increase (via Gs or Gq proteins) in the production of intracellular second messengers. The common way of determining the activity of compounds is by measuring the cellular formation of second messengers such as cAMP and calcium.
DiscoveRx assays offer a non-radioactive alternative for the detection of a decrease or increase in second messenger production in cells. They can be used with both cell-lines that express native receptors and cells transfected with a GPCR of interest, and can be employed in conjunction with high-throughput screening (HTS).
The HitHunter™ cAMP High Sensitivity (HS) assay is able to measure low cAMP levels and is, therefore, particularly suitable for cell-lines that endogenously express receptors at a level much lower than in transfected cells overexpressing a cloned GPCR.
HitHunter™ cAMP assays are in vitro-based competitive immunoassays that rely on enzyme fragment complementation technology (EFC, Fig. 1).

Fig. 1: The HitHunter™ cAMP assay principle

Free cAMP molecules from cell lysates compete for antibody binding with a labelled enzyme donor (ED)-cAMP conjugate, which contains a small peptide fragment of ß-galactosidase. In the absence of free cAMP, the ED-cAMP conjugates are captured by the cAMP-specific antibody and are unavailable for complementation with the enzyme acceptor (EA), resulting in a low signal. In the presence of free cAMP, antibody sites are occupied, allowing the ED-cAMP conjugate to complement with EA, forming an active ß-galactosidase enzyme; substrate hydrolysis by this enzyme produces a chemiluminescent signal. The signal generated is in direct proportion to the amount of free cAMP bound by the antibody (1). Any luminescence reader used with the HitHunter™ cAMP HS assay has to be sensitive enough to detect small changes in cAMP levels. The BMG LABTECH FLUOstar OPTIMA microplate reader has this required sensitivity.
The purpose of this Application Note is to explain how to set up the FLUOstar OPTIMA, for use with the HitHunter™ cAMP HS assay, to detect changes in cAMP levels following agonist stimulation of cells in the absence and presence of receptor antagonists. In this study, we used the rat PC12 phaeochromocytoma cell-line which endogenously expresses adenosine A2A and A2B receptors (2). Both of these couple to Gs proteins, which promotes an increase in intracellular cAMP levels upon receptor stimulation. Here, we have utilised compounds selective for the A2A receptor.

Materials and Methods
All materials were purchased from the manufacturers stated.

HitHunter™ cAMP HS kit reagents (DiscoveRx):

Lysis buffer and antibody mixture
- ED reagent
EA reagent and CL substrate mixture
cAMP standard (0.25mM)

Other reagents and materials:

Rat PC12 phaeochromocytoma cell-line (ATCC)
Dulbecco’s Phosphate Buffered Saline (PBS ; Cambrex)
Microplate, low volume, white with clear bottom, tissue culture treated, sterile, 96-well (Corning)
Agonist (CGS21680; Tocris)
Antagonist (ZM241385; Tocris)
IBMX (Sigma; optional)

A full description of the use of the HitHunter™ cAMP assay is included with the kit.
Low volume 96-well microplates were used. These allow the user to reduce the cost of each experiment by half, by using the volumes of reagents for a 384-well format.
To produce the standard curve, the cAMP standard provided in the kit was diluted 1 in 25 to prepare the highest working concentration, which was then used to prepare 1 in 3 serial dilutions in PBS giving a range of concentrations from 2.7 × 10^-6M to 4.6 × 10^-11M cAMP in a final assay volume of 55 µL. PBS alone was used as the control. PBS was also used, with PC12 cells, to measure the cAMP produced by constitutive receptor activity (basal activity).

Assay protocol (low volume 96-well plate):
PC12 cells were seeded 48 hours prior to the experiment at a density of 20,000 cells per well.

Add cAMP standard dilutions (15 µL) to empty wells of the microplate
Remove media from the cells and resuspend them in PBS containing 500µM IBMX (10 µL)
Add antagonist to the cells (5 µL)
Incubate at 37°C for 15 min
Add agonist to the cells (5 µL)
Incubate at 37°C for 30 min
Add 10 µL of Lysis buffer and antibody mixture to each well
Incubate at room temperature for 60 min
Add 10 µL of ED reagent to each well
10. Incubate at room temperature for 60 min
Add 20 µL of EA reagent and CL substrate mixture to each well
Incubate at room temperature for at least 60 min
Chemiluminescence is then read on the FLUOstar OPTIMA 4 hours after addition of the last reagent.

FLUOstar OPTIMA settings:
Basic parameters for luminescence plate mode detection are listed below, and shown in Fig. 2:

Read Mode:	Plate Mode
Positioning delay:	0.2 sec
No. of kinetic windows:	1
No. of multichromatics:	1
Emission filter:	lens
Gain:	3000
Measurement interval time:	1 sec

Fig. 2: Screenshot of the settings window from the FLUOstar OPTIMA multimode reader for the HitHunter™ cAMP HS assay

Results and Discussion
The HitHunter™ cAMP HS assay from DiscoveRx was used in 96-well format, and data from standard and agonist curves were obtained from the FLUOstar OPTIMA (BMG LABTECH) in luminescence mode.
Reagents were added according to the manufacturer’s protocol, and chemiluminescence was read 4 hours after the addition of the last reagent.
Data was evaluated using Microsoft Excel™ in conjunction with the FLUOstar OPTIMA Excel™ evaluation package and the software package GraphPad Prism.

Fig. 3: cAMP standard curve for the HitHunter™ cAMP HS assay (standards were measured in triplicate)

Fig. 3 illustrates the cAMP standard curve obtained using luminescence detection in a 96-well format. The curve shows a dose-dependent increase with a good signal-to-background noise (S/B) value of 11.
Dose-response curves for the selective A2A receptor agonist CGS21680 either alone or in the presence of the A2A receptor selective antagonist ZM241385 were generated (Fig. 4).

Fig. 4: Dose-response curves for CGS21680 in the presence or absence of ZM241385. pEC50 values were calculated, using GraphPad Prism software, from three individual experiments, each performed in triplicate.

A rightward shift of the agonist dose-response curve, and a decrease in the maximal response, was observed in the presence of 10^-7M ZM241385. This shows that ZM241385 non-competitively antagonised (by 4- to 5-fold) the agonist-induced increase in intracellular cAMP levels.

Conclusion
The HitHunter™ cAMP HS assay is particularly suitable for detecting small changes in cAMP levels such as those seen in, for example, the rat PC12 phaeochromocytoma cell-line, which endogenously expresses GPCRs.
Both agonist and antagonist data can be generated, using the HitHunter™ cAMP HS assay, in conjunction with the FLUOstar OPTIMA microplate reader.
The FLUOstar OPTIMA, which can also be used for absorbance and fluorescence detection, was used here in luminescence mode. It offers user-friendly software for both protocol set up and data analysis. It is very amenable for use in an academic environment (e.g. 96-well format) but can also be employed in all formats up to 1536-well plates for HTS of library compounds.

References

Eglen, R. M. (2002). Enzyme fragment complementation: a flexible high throughput screening assay technology. ASSAY and Drug Development Technologies 1, 97-104.
Florio, C., Frausin, F., Vertua, R., Gaion, R. M. (1999). Amplification of cyclic AMP response to forskolin in pheochromocytoma PC12 cells through adenosine A2A purinoceptors. Journal of Pharmacology and Experimental Therapeutics 290, 817-824.

The views expressed herein are those of the authors and do not necessarily represent those of Nottingham Trent University.

First published in Intl. Labmate (2006) Vol. XXXI, Is. VI.