Kinetic measurement of cAMP accumulation in human primary cells using the promega GloSensor™ cAMP technology

Elizabeth Rosethorne, Steven Charlton. Novartis Institues for Biomedical Research, Receptor Biology, RH12 5AB, Horsham, W. Sussex, United Kingdom.

Traditional high throughput methods for measuring cAMP generation utilize interactions between labelled antibodies and cAMP probes which, although accurate in determining the amount of cAMP generated within a cell, are not able to determine the rate at which that cAMP is generated. Alternative methods, such as [3H]-adenine incorporation and sequential Dowex/alumina chromatography allows reaction termination and accurate measurement of early time points, however this method relies on the use of radioactive probes and generally has low throughput. The GloSensor™ cAMP (Promega) technology utilizes a genetically modified form of firefly luciferase that contains a cAMP-binding motif. Upon cAMP binding, a conformational change leads to increased light output that can be measured in real-time using an injection luminescence reader. We have evaluated the ability of this new technology to measure increases in cAMP in real-time to allow determination of efficacy, potency and rate of intracellular signalling in human primary cells.

Human bronchial smooth muscle cells (hBSMc’s) were transiently transfected with the GloSensor™ cAMP vector using Lipofectamine2000. After 48 hours, cells were harvested and incubated with a luminescent substrate for 2 hours prior to agonist treatment. Cells were stimulated with a range of β2 adrenoceptor agonists (at EC80) and luminescence read on the FDSS7000 (Hamamatsu, Japan) over a 25 min incubation period. Results for efficacy and rate of cAMP generation are shown in the table below, data are expressed as mean ± s.e. mean (n ≥ 3).

There was a good correlation between GloSensor cAMP assay and traditional methods for both the rate (r2 = 0.988) of cAMP generation, when compared to [3H]-adenine incorporation, and the efficacy of the different agonist (r2 = 0.855) in the AlphaScreen assay. In conclusion, we have utilized the Promega GloSensor™ kit to measure the rate and extent of cAMP accumulation in primary human cells, and compared this to conventional antibody and radio methods.

Rosethorne EM, Turner RJ, Fairhurst RA, Charlton SJ (2010) Efficacy is a contributing factor to the clinical onset of bronchodilation of inhaled β2 adrenoceptor agonists. Naunyn-Schmiedeberg’s Archives of Pharmacology 382, 255-263