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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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CaR inhibits cAMP production through PTX-sensitive Gαi. 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.
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fig5: CaR inhibits cAMP production through PTX-sensitive Gαi. 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.

Mentions: We used the specific blocker of Gαi, PTX, to determine whether CaR expressed in HEK cells could also inhibit cAMP production through this pathway. Previous attempts to characterize this aspect of CaR signaling in HEK CaR cells using conventional radioimmune assays for cAMP have been only partially successful (Chang et al., 1998). During these experiments we eliminated the Ca2+ signaling component by pretreating the cells with thapsigargin in Ca2+-free solutions. Control experiments (Fig. 5 A) demonstrate that store-depleted HEK CaR cells maintained in Ca2+-free solutions were still able to repeatedly respond to PGE2 and forskolin (n = 37/39 cells in six experiments). Somewhat surprisingly, this response was entirely abolished by spermine (Fig. 5 B; n = 28/29 cells in four experiments). This implies that activation of CaR is capable of regulating cAMP signaling through a pathway entirely independent of Ca2+. However, the response to PGE2 was recovered in the presence of spermine when Gαi, the link between CaR and AC inactivation, was inhibited by PTX (n = 19 cells in five experiments; Fig. 5 C).


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)

CaR inhibits cAMP production through PTX-sensitive Gαi. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171199&req=5

fig5: CaR inhibits cAMP production through PTX-sensitive Gαi. 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.
Mentions: We used the specific blocker of Gαi, PTX, to determine whether CaR expressed in HEK cells could also inhibit cAMP production through this pathway. Previous attempts to characterize this aspect of CaR signaling in HEK CaR cells using conventional radioimmune assays for cAMP have been only partially successful (Chang et al., 1998). During these experiments we eliminated the Ca2+ signaling component by pretreating the cells with thapsigargin in Ca2+-free solutions. Control experiments (Fig. 5 A) demonstrate that store-depleted HEK CaR cells maintained in Ca2+-free solutions were still able to repeatedly respond to PGE2 and forskolin (n = 37/39 cells in six experiments). Somewhat surprisingly, this response was entirely abolished by spermine (Fig. 5 B; n = 28/29 cells in four experiments). This implies that activation of CaR is capable of regulating cAMP signaling through a pathway entirely independent of Ca2+. However, the response to PGE2 was recovered in the presence of spermine when Gαi, the link between CaR and AC inactivation, was inhibited by PTX (n = 19 cells in five experiments; Fig. 5 C).

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