Termination of cAMP signals by Ca(2+) and Gα(i) via extracellular Ca(2+) sensors: a link to intracellular Ca(2+) oscillations

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 Gα(i) and the CaR-stimulated increase in intracellular [Ca(2+)]. In parallel measurements with fura-2, CaR activation elicited robust Ca(2+) oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca(2+) 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 Ca(2+) 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 Ca(2+) spiking stimulated by Ca(2+)-mobilizing agonists under physiological conditions.