Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations

Abstract

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.

Figure 1.

Changes in the 480/535 nm FRET emission ratio in HEK CaR cells expressing cAMP sensors in response to cAMP-elevating agonists. (A) Cells expressing the PKA-based sensor (R-CFP + C-YFP) were stimulated with 100 nM isoproterenol (ISO), 100 nM PGE₂, and 100 μM forskolin. (B) Ratio images corresponding to the time points (a–f) indicated in the trace in A. The probe was excluded from the nuclear compartment, although in this nonconfocal image, a signal emanating from above and below the nucleus gives the appearance of a nuclear ratio change. (C) Response of cells expressing Epac-based sensor to 5 nM PGE₂ and 100 μM forskolin.

Figure 2.

Stimulation of HEK CaR cells with CaR agonists prevents or reverses PGE₂-induced cAMP formation as measured by the 480/535 nm emission ratio of PKA sensors. (A) Response to 100 nM PGE₂ is prevented by 5 μM of the specific synthetic CaR modulator NPS-R-476. (B) Another CaR agonist, spermine (used at 1 mM throughout this study), largely inhibited the ratio change elicited by a supramaximal dose (3 μM) of PGE₂; comparison with response to 100 μM forskolin. (C) Similarly, spermine or 3 mM Ca²⁺ completely blocked response to a lower dose (100 nM) of PGE₂. (D) Acute addition of spermine during stimulation with 100 nM PGE₂ reverses the ratio elevation. (E) Experiments using the low-affinity cAMP indicator R230K show smooth nonoscillatory decline in FRET ratio during acute spermine (1 mM) treatment (bold trace); the thinner line represents control recording. (F) Comparison with the action of the Ca²⁺-mobilizing agonist 100 μM carbachol. Experiments using the low-affinity cAMP indicator R230K show a significantly reduced action on the FRET ratio during acute carbachol treatment compared with spermine treatment.

Figure 3.

Stimulation of cAMP-generating pathways alters CaR-mediated intracellular Ca ²⁺ oscillations as measured by fura-2 in HEK CaR cells. (A) Spermine-stimulated Ca²⁺ oscillations (1 mM spermine) are significantly enhanced in frequency and amplitude by acute addition of PGE₂. (B) PGE₂ pretreatment converts the oscillatory spiking pattern into a pattern of a large spike followed by several rapid oscillations that fuse into a sustained plateau. (C) Consistent spiking pattern after three consecutive control stimulations with spermine. (D) Summary of oscillation frequency data (peaks per minute ± SEM) corresponding to experimental protocols shown in A–C. The left bar represents the first spermine stimulation; the middle, gray bar represents the second stimulation; and the right bar represents the third stimulation. ***, P < 0.0001.

Figure 4.

Cyclic AMP accumulation is sensitive to [Ca ²⁺ ] _i . The 480/535 nm emission ratio of the cAMP/FRET probe. (A) HEK WT cells. An artificial pulse of intracellular Ca²⁺ was generated by pretreating cells with thapsigargin in Ca²⁺-free solutions and then re-adding 5 mM Ca²⁺ at the time point indicated during the 100-nM PGE₂ response (left). The corresponding time course for the [Ca²⁺]_i increase as measured in separate experiments by fura-2 is shown in the right panel. (B) HEK CaR cells. (left) Persistent elevation of [Ca²⁺]_i using 10 μM ionomycin in the presence of extracellular Ca²⁺ yields a persistent increase in [Ca²⁺]_i (not depicted) that inhibits both PGE₂- and forskolin-induced increases in the cAMP/FRET ratio. (right) Transient increase [Ca²⁺]_i generated by ionomycin treatment in Ca²⁺-free solutions (not depicted) does not prevent PGE₂- or forskolin-induced increases in the cAMP/FRET ratio.

Figure 5.

CaR inhibits cAMP production through PTX-sensitive Gα_i. Measurements of the 480/535 nm emission ratio of the PKA-based sensor were performed in thapsigargin-pretreated HEK CaR cells maintained in Ca²⁺-free solutions (to eliminate contributions from Ca²⁺ signaling). (A) Control experiment showing two successive responses to 100 nM PGE₂ followed by maximal response to 100 μM forskolin. (B) 1 mM spermine prevents PGE₂-induced ratio elevation in the absence of Ca²⁺ signaling. (C) Pretreatment with PTX rescues the PGE₂ response, in spite of the presence of spermine.

Figure 6.

Profile of the spermine-induced decline in cAMP after PTX pretreatment as measured by PKA- and Epac-based sensors. (A) In PTX-pretreated HEK CaR cells (100 ng/ml × 16 h), spermine added during the peak of PGE₂ stimulation causes a smooth decline in the 480/535 nm ratio, even though [Ca²⁺]_i, as measured by fura-2, initially maintains an oscillatory pattern under these conditions (not depicted). (B) A similar smooth decline in the 480/535 nm emission ratio is observed when spermine is added during the peak of the response to 5 nM PGE₂ as measured using the Epac sensor. (C) As in B, but in PTX-pretreated cells. (D) Concurrent fura-2 and FRET ratio measured in fura-2–loaded HEK CaR cells transfected with the Epac sensor shows persistence of spermine-stimulated Ca²⁺ oscillations in Epac-expressing cells, although there is significant optical contamination of the FRET channel by the fura-2 signal.

Figure 7.

PKA dissociation is immune to high-frequency Ca ²⁺ oscillations. Parallel measurements of [Ca²⁺]_i using fura-2 and cAMP using the low-affinity R230K version of the PKA/FRET probe in HEK WT cells. (A) Effect of high-frequency artificial Ca²⁺ pulses during peak of PGE₂ response in cells preincubated in thapsigargin/Ca²⁺-free solutions. (B) Control experiment in digitonin-permeabilized WT cells alternately superfused with 50 and 30 μM cAMP for 1 min each shows the response time of the probe to rapid fluctuations in [cAMP]. (C) Low-frequency, long-duration Ca²⁺ pulses generate complex staircase patterns of cAMP in HEK WT cells.