High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels.
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.
Reinscheid
Rainer K
RK
Department of Pharmacology, University of California Irvine, 101 Theory Dr, Irvine, CA 92612, USA.
Kim
Joon
J
Zeng
Joanne
J
Civelli
Olivier
O
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000819:agonists / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000819:agonists / Q000378:metabolism*; D009655:Octopamine / Q000494:pharmacology; D051381:Rats; D013997:Time Factors
D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D009655:Octopamine; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D004837:Epinephrine