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High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels

2003; Elsevier BV; Volume: 478; Issue: 1 Linguagem: Inglês

10.1016/j.ejphar.2003.08.036

1879-0712

Rainer K. Reinscheid, Joon Kim, Joanne Zeng, Olivier Civelli,

Phosphodiesterase function and regulation

Cyclic adenosine-monophosphate (cAMP) is one of the major second messenger molecules transmitting extracellular stimuli into short- and long-term changes of intracellular homeostasis. Measurements of cellular cAMP levels are often used to quantify and characterize signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. We have developed a technology to monitor real-time changes of cAMP levels in living cells. This method uses a modified cyclic nucleotide-gated (CNG) Ca(2+) channel which is opened by intracellular cAMP. Thus, changes in cAMP levels are translated into changes in free Ca(2+) which can easily be measured using fluorimetric imaging technologies compatible with high-throughput screening formats. The new assay method was used to characterize the pharmacology of various endogenously and heterologously expressed G protein-coupled receptors and allows for the simultaneous study of G(s), G(i) and G(q)-linked receptors in the same cell population.