Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging.
The spatial and temporal dynamics of two intracellular second messengers, cAMP and Ca2+, were simultaneously monitored in living cells by digital fluorescence ratio imaging using FlCRhR, a single-excitation dual-emission cAMP indicator, and fura-2, a dual-excitation single-emission Ca2+ probe. In single C6-2B glioma cells, isoproterenol- or forskolin-evoked cAMP accumulation (measured in vivo as an increased FlCRhR emission ratio) was reduced when cytosolic free Ca2+ concentration was elevated before, simultaneously with, or after cAMP activation. However, in REF-52 fibroblasts, Ca2+ neither prevented nor reduced forskolin-stimulated cAMP production. These results provide novel in vivo evidence for the Ca2+ modulation of the cAMP transduction pathway in C6-2B cells. The simultaneous microscopic measurement of cAMP and Ca2+ kinetics in single cells makes it now possible to study the regulatory interactions between these second messengers at the cellular and even the subcellular level.
DeBernardi
M A
MA
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Brooker
G
G
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008856:Microscopy, Fluorescence; D051381:Rats; D015290:Second Messenger Systems; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D000242:Cyclic AMP; D002118:Calcium; D016257:Fura-2