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

Gerbino A, Ruder WC, Curci S, Pozzan T, Zaccolo M, Hofer AM - J. Cell Biol. (2005)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Veterans' Affairs Boston Healthcare System, West Roxbury, MA 02132, USA.

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.

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Cyclic AMP accumulation is sensitive to [Ca2+]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.
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fig4: Cyclic AMP accumulation is sensitive to [Ca2+]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.

Mentions: We first examined the actions of a large, single, “artificial” pulse of [Ca2+]i on cAMP formation in HEK 293 WT cells, which do not express CaR. Before the start of the experiment, WT cells were treated with the irreversible sarco/endoplasmic reticulum Ca2+-ATPase pump inhibitor thapsigargin to deplete intracellular Ca2+ stores. Cells were initially maintained in Ca2+-free solution. Under these conditions, it is expected that readmission of Ca2+ to the bath will provoke a large entry of Ca2+ into the cell because of influx through capacitative Ca2+ entry pathways (Berridge et al., 2000). Parallel experiments with fura-2 showed that addition of 5 mM Ca2+ resulted in a large increase in [Ca2+]i (Fig. 4 A, right), comparable in amplitude with the initial peak of the agonist-evoked Ca2+ spike (n = 110 cells in four experiments). In measurements using the PKA-FRET indicator under the same conditions, 5 mM Ca2+ administered during the peak of PGE2-stimulated cAMP production (100 nM PGE2) completely and reversibly decreased the ratio increase (Fig. 4 A; n = 28 cells in six experiments), although with a small delay.


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

Gerbino A, Ruder WC, Curci S, Pozzan T, Zaccolo M, Hofer AM - J. Cell Biol. (2005)

Cyclic AMP accumulation is sensitive to [Ca2+]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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171199&req=5

fig4: Cyclic AMP accumulation is sensitive to [Ca2+]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.
Mentions: We first examined the actions of a large, single, “artificial” pulse of [Ca2+]i on cAMP formation in HEK 293 WT cells, which do not express CaR. Before the start of the experiment, WT cells were treated with the irreversible sarco/endoplasmic reticulum Ca2+-ATPase pump inhibitor thapsigargin to deplete intracellular Ca2+ stores. Cells were initially maintained in Ca2+-free solution. Under these conditions, it is expected that readmission of Ca2+ to the bath will provoke a large entry of Ca2+ into the cell because of influx through capacitative Ca2+ entry pathways (Berridge et al., 2000). Parallel experiments with fura-2 showed that addition of 5 mM Ca2+ resulted in a large increase in [Ca2+]i (Fig. 4 A, right), comparable in amplitude with the initial peak of the agonist-evoked Ca2+ spike (n = 110 cells in four experiments). In measurements using the PKA-FRET indicator under the same conditions, 5 mM Ca2+ administered during the peak of PGE2-stimulated cAMP production (100 nM PGE2) completely and reversibly decreased the ratio increase (Fig. 4 A; n = 28 cells in six experiments), although with a small delay.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Veterans' Affairs Boston Healthcare System, West Roxbury, MA 02132, USA.

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.

Show MeSH
Related in: MedlinePlus