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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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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.
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fig6: 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.

Mentions: Fig. 6 A shows that in the absence of Gαi-mediated inhibition (PTX pretreatment), spermine still caused a smooth monotonic reduction in the FRET ratio (compare with control in Fig. 2 E), and this occurred with a substantial delay (n = 20 cells in four experiments). We expected to see fluctuations or stepwise decrements in cAMP because parallel experiments in fura-2–loaded cells revealed initial persistence of Ca2+ oscillations (similar to those observed in Fig. 3; not depicted) after PTX treatment, and the data of Fig. 5 showed cAMP accumulation to be very sensitive to intracellular Ca2+. We considered the possibility that the PKA-based sensors we were using had not been fast enough to resolve rapid fluctuations in cAMP because a relatively complex binding reaction involving four cAMP molecules and the dissociation of four PKA subunits is required to see a change in FRET. However, a similar profile was observed in experiments using the fast monomeric Epac-based sensor described on the previous page. As shown in Fig. 6 B, spermine addition during stimulation of cells with a low dose (5 nM) of PGE2 caused a smooth reversible decline in the FRET ratio. Fig. 6 C illustrates the powerful inhibitory effect of the PTX-sensitive component of CaR stimulation on cAMP signaling. After PTX pretreatment, the action of spermine-induced Ca2+ signals can be examined in isolation. A smooth, slow decline in the FRET ratio that occurred with a notable delay was observed upon spermine addition. Concurrent measurements of the 340:380 nm fura-2 excitation ratio and 480/535 nm FRET emission ratio in fura-2–loaded HEK CaR cells expressing the Epac sensor confirmed that the presence of Epac did not alter Ca2+ oscillations (Fig. 6 D). However, we noted significant “bleed through” of the fura-2 signal into the FRET channels (see Materials and methods for details), giving rise to apparent oscillations in the FRET ratio that were never observed in cells not loaded with fura-2 (n = 9 cells in five experiments).


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)

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.
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Related In: Results  -  Collection

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fig6: 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.
Mentions: Fig. 6 A shows that in the absence of Gαi-mediated inhibition (PTX pretreatment), spermine still caused a smooth monotonic reduction in the FRET ratio (compare with control in Fig. 2 E), and this occurred with a substantial delay (n = 20 cells in four experiments). We expected to see fluctuations or stepwise decrements in cAMP because parallel experiments in fura-2–loaded cells revealed initial persistence of Ca2+ oscillations (similar to those observed in Fig. 3; not depicted) after PTX treatment, and the data of Fig. 5 showed cAMP accumulation to be very sensitive to intracellular Ca2+. We considered the possibility that the PKA-based sensors we were using had not been fast enough to resolve rapid fluctuations in cAMP because a relatively complex binding reaction involving four cAMP molecules and the dissociation of four PKA subunits is required to see a change in FRET. However, a similar profile was observed in experiments using the fast monomeric Epac-based sensor described on the previous page. As shown in Fig. 6 B, spermine addition during stimulation of cells with a low dose (5 nM) of PGE2 caused a smooth reversible decline in the FRET ratio. Fig. 6 C illustrates the powerful inhibitory effect of the PTX-sensitive component of CaR stimulation on cAMP signaling. After PTX pretreatment, the action of spermine-induced Ca2+ signals can be examined in isolation. A smooth, slow decline in the FRET ratio that occurred with a notable delay was observed upon spermine addition. Concurrent measurements of the 340:380 nm fura-2 excitation ratio and 480/535 nm FRET emission ratio in fura-2–loaded HEK CaR cells expressing the Epac sensor confirmed that the presence of Epac did not alter Ca2+ oscillations (Fig. 6 D). However, we noted significant “bleed through” of the fura-2 signal into the FRET channels (see Materials and methods for details), giving rise to apparent oscillations in the FRET ratio that were never observed in cells not loaded with fura-2 (n = 9 cells in five experiments).

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