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Comparison of human recombinant adenosine A2B receptor function assessed by Fluo-3-AM fluorometry and
H Patel1, R H P Porter, A M Palmer
1Department of Neuroinflammation, Faculty of Medicine at Imperial College, Charing Cross Hospital, Fulham Palace Road, London W6 8RF.
British Journal of Pharmacology
|February 25, 2003
Summary
This study demonstrates that the fluorometric imaging plate reader (FLIPR) assay, using a specific cell line, is a valuable high-throughput method for assessing adenosine A(2B) receptor function, showing higher potency and efficacy than microphysiometry.
Area of Science:
- Pharmacology
- Cellular Biology
- Assay Development
Background:
- Adenosine A(2B) receptors play roles in various physiological processes.
- Accurate assessment of A(2B) receptor function is crucial for drug discovery.
- Existing methods may have limitations in throughput or sensitivity.
Purpose of the Study:
- To establish the utility of a fluorometric imaging plate reader (FLIPR) assay for human adenosine A(2B) receptor function.
- To characterize the receptor pharmacology using FLIPR and compare it to microphysiometry.
- To evaluate a novel high-throughput screening method.
Main Methods:
- Utilized a fluorometric imaging plate reader (FLIPR) assay with a Ca(2+)-sensitive dye (Fluo-3-AM).
- Employed a Chinese Hamster Ovary (CHO-K1) cell line stably expressing human A(2B) receptor and G(alpha16) protein.
- Compared results with microphysiometry using a Human Embryonic Kidney (HEK-293) cell line expressing human A(2B) receptor.
Main Results:
- FLIPR assay demonstrated concentration-dependent increases in intracellular calcium upon A(2B) receptor activation.
- Rank order of agonist potency was consistent across assays (NECA > N(6)-Benzyl NECA > adenosine > R-PIA > CPA > S-PIA > CHA > CGS 21680).
- FLIPR assay showed higher absolute potency and efficacy compared to microphysiometry; ZM-241385 was the most potent antagonist (pA(2) = 8.0).
Conclusions:
- The FLIPR assay, coupled with a specific transfected cell line, provides a useful, high-throughput method for assessing A(2B) receptor function.
- The FLIPR assay offers a sensitive and efficient alternative to microphysiometry for receptor pharmacology studies.
- This method facilitates the screening of A(2B) receptor agonists and antagonists.