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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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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.
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fig1: 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.

Mentions: Several different types of fluorescent cAMP sensors have been described previously (Adams et al., 1991; Zaccolo et al., 2000; Zaccolo and Pozzan, 2002; DiPilato et al., 2004; Mongillo et al., 2004; Ponsioen et al., 2004; Landa et al., 2005). Here we used recently developed FRET sensors based on PKA (Zaccolo and Pozzan, 2002) and Epac (Ponsioen et al., 2004) to continuously monitor cAMP levels for extended periods of time in single HEK CaR cells. Fig. 1 A shows that stimulation of endogenous β-adrenoceptors using 100 nM isoproterenol produced rapid and reversible elevation in the FRET ratio. Repeated stimulation of prostanoid receptors (EP2/EP4) with 100 nM of the inflammatory mediator PGE2 yielded largely reproducible responses in the same cell. This agonist dose generally caused ratio responses that were smaller than the saturating activation elicited by a supramaximal dose (100 μM) of forskolin. This stimulation protocol was therefore used for much of the remainder of the study (typical of results from 32 cells in five experiments). Fig. 1 B shows pseudocolor images of the 480/535 nm FRET emission ratio of the PKA-based sensor, corresponding to the plot shown in Fig. 1 A. CFP- and YFP-labeled PKA subunits were distributed throughout the cytoplasm in transfected cells but were excluded from the nuclear compartment (not depicted). Significantly, because of the high sensitivity of these FRET-based techniques, we were able to detect ratio changes in response to low nanomolar concentrations of cAMP-generating agonists (e.g., 6.25 nM VIP or 10 nM isoproterenol; not depicted). One drawback, in fact, of the high sensitivity of these probes is that it is possible to saturate the indicators when GFP-tagged PKA subunits are dissociated to the maximal extent, even though intracellular cAMP continues to rise. This issue is discussed further on the following page. We also used a second type of cAMP probe based on Epac that exhibits a faster response time (Ponsioen et al., 2004). As illustrated in Fig. 1 C, reversible ratio changes after stimulation with 5 nM PGE2 were readily discriminated in HEK CaR cells using this sensor.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171199&req=5

fig1: 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.
Mentions: Several different types of fluorescent cAMP sensors have been described previously (Adams et al., 1991; Zaccolo et al., 2000; Zaccolo and Pozzan, 2002; DiPilato et al., 2004; Mongillo et al., 2004; Ponsioen et al., 2004; Landa et al., 2005). Here we used recently developed FRET sensors based on PKA (Zaccolo and Pozzan, 2002) and Epac (Ponsioen et al., 2004) to continuously monitor cAMP levels for extended periods of time in single HEK CaR cells. Fig. 1 A shows that stimulation of endogenous β-adrenoceptors using 100 nM isoproterenol produced rapid and reversible elevation in the FRET ratio. Repeated stimulation of prostanoid receptors (EP2/EP4) with 100 nM of the inflammatory mediator PGE2 yielded largely reproducible responses in the same cell. This agonist dose generally caused ratio responses that were smaller than the saturating activation elicited by a supramaximal dose (100 μM) of forskolin. This stimulation protocol was therefore used for much of the remainder of the study (typical of results from 32 cells in five experiments). Fig. 1 B shows pseudocolor images of the 480/535 nm FRET emission ratio of the PKA-based sensor, corresponding to the plot shown in Fig. 1 A. CFP- and YFP-labeled PKA subunits were distributed throughout the cytoplasm in transfected cells but were excluded from the nuclear compartment (not depicted). Significantly, because of the high sensitivity of these FRET-based techniques, we were able to detect ratio changes in response to low nanomolar concentrations of cAMP-generating agonists (e.g., 6.25 nM VIP or 10 nM isoproterenol; not depicted). One drawback, in fact, of the high sensitivity of these probes is that it is possible to saturate the indicators when GFP-tagged PKA subunits are dissociated to the maximal extent, even though intracellular cAMP continues to rise. This issue is discussed further on the following page. We also used a second type of cAMP probe based on Epac that exhibits a faster response time (Ponsioen et al., 2004). As illustrated in Fig. 1 C, reversible ratio changes after stimulation with 5 nM PGE2 were readily discriminated in HEK CaR cells using this sensor.

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