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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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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.
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fig2: 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.

Mentions: We next took advantage of the extraordinary sensitivity of these cAMP indicators to examine how activation of CaR influenced cAMP signaling. Fig. 2 A shows that the elevation of the emission ratio induced by 100 nM PGE2 was completely abolished by brief preexposure to NPS-R-467, a specific synthetic allosteric activator of CaR (n = 38 cells in six experiments; Nemeth, 2004). This calcimimetic by itself did not have any effect on cAMP levels, as measured by FRET. The data in Fig. 2 B further support this result. In the presence of the CaR agonist spermine, an extremely high dose (3 μM) of PGE2 produced only a very small transient increase in the ratio, followed by a small undershoot, showing that CaR is extremely effective in inhibiting cAMP production. The cAMP signal was partially recovered after spermine washout. Prestimulation of CaR with spermine or 3 mM Ca2+ was able to completely abolish the ratio increase when a lower dose (100 nM) of PGE2 was applied (Fig. 2 C; n = 16/19 cells in four experiments for spermine, and n = 15/15 cells in three experiments for 3 mM Ca2+). Thus, preexposure to a variety of CaR agonists effectively prevented the increase in cAMP production.


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)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: We next took advantage of the extraordinary sensitivity of these cAMP indicators to examine how activation of CaR influenced cAMP signaling. Fig. 2 A shows that the elevation of the emission ratio induced by 100 nM PGE2 was completely abolished by brief preexposure to NPS-R-467, a specific synthetic allosteric activator of CaR (n = 38 cells in six experiments; Nemeth, 2004). This calcimimetic by itself did not have any effect on cAMP levels, as measured by FRET. The data in Fig. 2 B further support this result. In the presence of the CaR agonist spermine, an extremely high dose (3 μM) of PGE2 produced only a very small transient increase in the ratio, followed by a small undershoot, showing that CaR is extremely effective in inhibiting cAMP production. The cAMP signal was partially recovered after spermine washout. Prestimulation of CaR with spermine or 3 mM Ca2+ was able to completely abolish the ratio increase when a lower dose (100 nM) of PGE2 was applied (Fig. 2 C; n = 16/19 cells in four experiments for spermine, and n = 15/15 cells in three experiments for 3 mM Ca2+). Thus, preexposure to a variety of CaR agonists effectively prevented the increase in cAMP production.

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