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Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca(2+)-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer-dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin-sensitive Galpha(i) and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.

Original publication




Journal article


J Cell Biol

Publication Date





303 - 312


Acetylcysteine, Biosensing Techniques, Calcium, Calcium Signaling, Cell Line, Cell Membrane, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Erythromycin, Fluorescence Resonance Energy Transfer, GTP-Binding Protein alpha Subunits, Gi-Go, Humans, Pertussis Toxin, Receptors, Calcium-Sensing, Sensitivity and Specificity, Signal Transduction