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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 cAMP-generating pathways alters CaR-mediated intracellular Ca2+ oscillations as measured by fura-2 in HEK CaR cells. (A) Spermine-stimulated Ca2+ oscillations (1 mM spermine) are significantly enhanced in frequency and amplitude by acute addition of PGE2. (B) PGE2 pretreatment converts the oscillatory spiking pattern into a pattern of a large spike followed by several rapid oscillations that fuse into a sustained plateau. (C) Consistent spiking pattern after three consecutive control stimulations with spermine. (D) Summary of oscillation frequency data (peaks per minute ± SEM) corresponding to experimental protocols shown in A–C. The left bar represents the first spermine stimulation; the middle, gray bar represents the second stimulation; and the right bar represents the third stimulation. ***, P < 0.0001.
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fig3: Stimulation of cAMP-generating pathways alters CaR-mediated intracellular Ca2+ oscillations as measured by fura-2 in HEK CaR cells. (A) Spermine-stimulated Ca2+ oscillations (1 mM spermine) are significantly enhanced in frequency and amplitude by acute addition of PGE2. (B) PGE2 pretreatment converts the oscillatory spiking pattern into a pattern of a large spike followed by several rapid oscillations that fuse into a sustained plateau. (C) Consistent spiking pattern after three consecutive control stimulations with spermine. (D) Summary of oscillation frequency data (peaks per minute ± SEM) corresponding to experimental protocols shown in A–C. The left bar represents the first spermine stimulation; the middle, gray bar represents the second stimulation; and the right bar represents the third stimulation. ***, P < 0.0001.

Mentions: Parallel experiments were performed with fura-2–loaded HEK CaR cells to examine whether PGE2 had any effect on the pattern of intracellular Ca2+ signaling. CaR stimulation elicits robust and long-lasting oscillations in [Ca2+]i (Breitwieser and Gama, 2001; Young and Rozengurt, 2002; De Luisi and Hofer, 2003). As shown in Fig. 3 A, stimulation with spermine alone gave rise to Ca2+ oscillations with a frequency of 1.78 ± 0.03 per minute (n = 122 cells in four experiments). This frequency was dramatically increased after acute PGE2 addition (2.27 ± 0.06; P < 0.0001), until the spikes fused into an elevated plateau after ∼2 min (1.85 ± 0.08 min). A third exposure to spermine after PGE2 washout elicited oscillations that were faster than the control (2.41 ± 0.05; P < 0.0001, compared with the first stimulation), indicating that the effect of PGE2 was somewhat prolonged. The amplitude of the spikes was also significantly affected by PGE2, with the overall effect being an increased delivery of Ca2+ to the cytoplasm. As shown in Fig. 3 B, a 3-min preexposure to PGE2 (when [cAMP] is typically near its maximum; see Fig. 2 D) dramatically changed the profile of the spermine response. We observed a few initial rapid spikes, which eventually fused into a prolonged plateau after 2.2 ± 0.13 min (n = 118 cells in four experiments). A third challenge with spermine yielded a persistent effect on oscillation frequency compared with the first control stimulation (2.03 ± 0.03 spikes per minute in control vs. 2.66 ± 0.04 for third stimulation; P < 0.0001). Similar results were obtained when carbachol was used in place of spermine (not depicted; n = 90 cells in four experiments). Although the carbachol-stimulated Ca2+ oscillations under control conditions were typically much less robust than those observed during spermine stimulation, the pattern of initial increase in oscillation frequency that fused into a sustained plateau was still dependably observed. Fig. 3 C shows that the frequency of oscillations recorded during three sequential control stimulations with spermine were very consistent (n = 87 cells in four experiments; no statistical difference). The data on oscillation frequency are summarized in Fig. 3 D.


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 cAMP-generating pathways alters CaR-mediated intracellular Ca2+ oscillations as measured by fura-2 in HEK CaR cells. (A) Spermine-stimulated Ca2+ oscillations (1 mM spermine) are significantly enhanced in frequency and amplitude by acute addition of PGE2. (B) PGE2 pretreatment converts the oscillatory spiking pattern into a pattern of a large spike followed by several rapid oscillations that fuse into a sustained plateau. (C) Consistent spiking pattern after three consecutive control stimulations with spermine. (D) Summary of oscillation frequency data (peaks per minute ± SEM) corresponding to experimental protocols shown in A–C. The left bar represents the first spermine stimulation; the middle, gray bar represents the second stimulation; and the right bar represents the third stimulation. ***, P < 0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171199&req=5

fig3: Stimulation of cAMP-generating pathways alters CaR-mediated intracellular Ca2+ oscillations as measured by fura-2 in HEK CaR cells. (A) Spermine-stimulated Ca2+ oscillations (1 mM spermine) are significantly enhanced in frequency and amplitude by acute addition of PGE2. (B) PGE2 pretreatment converts the oscillatory spiking pattern into a pattern of a large spike followed by several rapid oscillations that fuse into a sustained plateau. (C) Consistent spiking pattern after three consecutive control stimulations with spermine. (D) Summary of oscillation frequency data (peaks per minute ± SEM) corresponding to experimental protocols shown in A–C. The left bar represents the first spermine stimulation; the middle, gray bar represents the second stimulation; and the right bar represents the third stimulation. ***, P < 0.0001.
Mentions: Parallel experiments were performed with fura-2–loaded HEK CaR cells to examine whether PGE2 had any effect on the pattern of intracellular Ca2+ signaling. CaR stimulation elicits robust and long-lasting oscillations in [Ca2+]i (Breitwieser and Gama, 2001; Young and Rozengurt, 2002; De Luisi and Hofer, 2003). As shown in Fig. 3 A, stimulation with spermine alone gave rise to Ca2+ oscillations with a frequency of 1.78 ± 0.03 per minute (n = 122 cells in four experiments). This frequency was dramatically increased after acute PGE2 addition (2.27 ± 0.06; P < 0.0001), until the spikes fused into an elevated plateau after ∼2 min (1.85 ± 0.08 min). A third exposure to spermine after PGE2 washout elicited oscillations that were faster than the control (2.41 ± 0.05; P < 0.0001, compared with the first stimulation), indicating that the effect of PGE2 was somewhat prolonged. The amplitude of the spikes was also significantly affected by PGE2, with the overall effect being an increased delivery of Ca2+ to the cytoplasm. As shown in Fig. 3 B, a 3-min preexposure to PGE2 (when [cAMP] is typically near its maximum; see Fig. 2 D) dramatically changed the profile of the spermine response. We observed a few initial rapid spikes, which eventually fused into a prolonged plateau after 2.2 ± 0.13 min (n = 118 cells in four experiments). A third challenge with spermine yielded a persistent effect on oscillation frequency compared with the first control stimulation (2.03 ± 0.03 spikes per minute in control vs. 2.66 ± 0.04 for third stimulation; P < 0.0001). Similar results were obtained when carbachol was used in place of spermine (not depicted; n = 90 cells in four experiments). Although the carbachol-stimulated Ca2+ oscillations under control conditions were typically much less robust than those observed during spermine stimulation, the pattern of initial increase in oscillation frequency that fused into a sustained plateau was still dependably observed. Fig. 3 C shows that the frequency of oscillations recorded during three sequential control stimulations with spermine were very consistent (n = 87 cells in four experiments; no statistical difference). The data on oscillation frequency are summarized in Fig. 3 D.

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