Termination of cAMP signals by Ca²⁺ and Gα_i via extracellular Ca²⁺ sensors : a link to intracellular Ca²⁺ oscillations

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca²⁺-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 [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 Ca²⁺-mobilizing agonists under physiological conditions.