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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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PKA dissociation is immune to high-frequency Ca2+ oscillations. Parallel measurements of [Ca2+]i using fura-2 and cAMP using the low-affinity R230K version of the PKA/FRET probe in HEK WT cells. (A) Effect of high-frequency artificial Ca2+ pulses during peak of PGE2 response in cells preincubated in thapsigargin/Ca2+-free solutions. (B) Control experiment in digitonin-permeabilized WT cells alternately superfused with 50 and 30 μM cAMP for 1 min each shows the response time of the probe to rapid fluctuations in [cAMP]. (C) Low-frequency, long-duration Ca2+ pulses generate complex staircase patterns of cAMP in HEK WT cells.
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fig7: PKA dissociation is immune to high-frequency Ca2+ oscillations. Parallel measurements of [Ca2+]i using fura-2 and cAMP using the low-affinity R230K version of the PKA/FRET probe in HEK WT cells. (A) Effect of high-frequency artificial Ca2+ pulses during peak of PGE2 response in cells preincubated in thapsigargin/Ca2+-free solutions. (B) Control experiment in digitonin-permeabilized WT cells alternately superfused with 50 and 30 μM cAMP for 1 min each shows the response time of the probe to rapid fluctuations in [cAMP]. (C) Low-frequency, long-duration Ca2+ pulses generate complex staircase patterns of cAMP in HEK WT cells.

Mentions: We generated repetitive artificial Ca2+ spikes in HEK WT cells using a pattern that mimicked spermine-stimulated Ca2+ oscillations in HEK CaR cells. WT cells were pretreated with thapsigargin in Ca2+-free solutions, and intermittent pulses of Ca2+ (1 mM) were applied during PGE2-stimulated cAMP accumulation. As shown in Fig. 7 A, with a frequency of one Ca2+ spike (lasting 1 min) every 2 min, no effect could be discerned on the FRET ratio (n = 8 cells in three experiments). Fig. 7 B shows that the low-affinity PKA-based indicator should, in principle, be able to respond to alterations in [cAMP] on the minute time scale. In these experiments, cells were permeabilized with digitonin in an intracellular-like buffer. Cells were then alternately superfused for 1-min pulses with solutions containing 30 or 50 μM cAMP (n = 9 cells in four experiments). These seemingly high concentrations of cAMP were used because in situ calibration of the low-affinity probe revealed this to be the range of [cAMP] levels achieved during stimulation with 500 nM PGE2 (not depicted; n = 12 cells in five experiments). In contrast to the results of Fig. 7 A, when the frequency and duration of the Ca2+ pulses were altered (one 3-min-long spike every 6 min), distinctive stepwise reductions in Ca2+ were evident (Fig. 7 C; n = 22 cells in five experiments). It was consistently observed that >1 min was required after admission of Ca2+ in the superfusion before the FRET ratio began to decline (during the first Ca2+ pulse, the ratio started to decrease only after 1.63 ± 0.60 min, for the second Ca2+ pulse, after 1.40 ± 0.66 min, and for the third pulse, after 1.22 ± 0.70 min; n = 22 cells in five experiments), explaining why the pattern of rapid Ca2+ spiking in Fig. 7 A failed to elicit any effect. These results indicate that in this model system the cAMP machinery is immune to rapid Ca2+ spiking but does respond to large static increases in Ca2+ (as sensed by PKA and Epac), which sets cAMP at a new, reduced level.


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)

PKA dissociation is immune to high-frequency Ca2+ oscillations. Parallel measurements of [Ca2+]i using fura-2 and cAMP using the low-affinity R230K version of the PKA/FRET probe in HEK WT cells. (A) Effect of high-frequency artificial Ca2+ pulses during peak of PGE2 response in cells preincubated in thapsigargin/Ca2+-free solutions. (B) Control experiment in digitonin-permeabilized WT cells alternately superfused with 50 and 30 μM cAMP for 1 min each shows the response time of the probe to rapid fluctuations in [cAMP]. (C) Low-frequency, long-duration Ca2+ pulses generate complex staircase patterns of cAMP in HEK WT cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171199&req=5

fig7: PKA dissociation is immune to high-frequency Ca2+ oscillations. Parallel measurements of [Ca2+]i using fura-2 and cAMP using the low-affinity R230K version of the PKA/FRET probe in HEK WT cells. (A) Effect of high-frequency artificial Ca2+ pulses during peak of PGE2 response in cells preincubated in thapsigargin/Ca2+-free solutions. (B) Control experiment in digitonin-permeabilized WT cells alternately superfused with 50 and 30 μM cAMP for 1 min each shows the response time of the probe to rapid fluctuations in [cAMP]. (C) Low-frequency, long-duration Ca2+ pulses generate complex staircase patterns of cAMP in HEK WT cells.
Mentions: We generated repetitive artificial Ca2+ spikes in HEK WT cells using a pattern that mimicked spermine-stimulated Ca2+ oscillations in HEK CaR cells. WT cells were pretreated with thapsigargin in Ca2+-free solutions, and intermittent pulses of Ca2+ (1 mM) were applied during PGE2-stimulated cAMP accumulation. As shown in Fig. 7 A, with a frequency of one Ca2+ spike (lasting 1 min) every 2 min, no effect could be discerned on the FRET ratio (n = 8 cells in three experiments). Fig. 7 B shows that the low-affinity PKA-based indicator should, in principle, be able to respond to alterations in [cAMP] on the minute time scale. In these experiments, cells were permeabilized with digitonin in an intracellular-like buffer. Cells were then alternately superfused for 1-min pulses with solutions containing 30 or 50 μM cAMP (n = 9 cells in four experiments). These seemingly high concentrations of cAMP were used because in situ calibration of the low-affinity probe revealed this to be the range of [cAMP] levels achieved during stimulation with 500 nM PGE2 (not depicted; n = 12 cells in five experiments). In contrast to the results of Fig. 7 A, when the frequency and duration of the Ca2+ pulses were altered (one 3-min-long spike every 6 min), distinctive stepwise reductions in Ca2+ were evident (Fig. 7 C; n = 22 cells in five experiments). It was consistently observed that >1 min was required after admission of Ca2+ in the superfusion before the FRET ratio began to decline (during the first Ca2+ pulse, the ratio started to decrease only after 1.63 ± 0.60 min, for the second Ca2+ pulse, after 1.40 ± 0.66 min, and for the third pulse, after 1.22 ± 0.70 min; n = 22 cells in five experiments), explaining why the pattern of rapid Ca2+ spiking in Fig. 7 A failed to elicit any effect. These results indicate that in this model system the cAMP machinery is immune to rapid Ca2+ spiking but does respond to large static increases in Ca2+ (as sensed by PKA and Epac), which sets cAMP at a new, reduced level.

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