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Published 24 October 2005. doi:10.1083/jcb.200507054
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 171, Number 2, 303-312
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Article

 Termination of cAMP signals by Ca2+ and G{alpha}i via extracellular Ca2+ sensors : a link to intracellular Ca2+ oscillations



Andrea Gerbino1,2, Warren C. Ruder1, Silvana Curci1,2, Tullio Pozzan3, Manuela Zaccolo4, and Aldebaran M. Hofer1,2

1 Veterans' Affairs Boston Healthcare System, West Roxbury, MA 02132
2 Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115
3 Department of Biomedical Sciences, Venetian Institute of Molecular Medicine, University of Padua, 35129 Padua, Italy
4 Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, 35129 Padua, Italy

Correspondence to Aldebaran M. Hofer: ahofer{at}rics.bwh.harvard.edu

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca2+-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 G{alpha}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 Ca2+-mobilizing agonists under physiological conditions.

A. Gerbino's present address is Dipartimento di Fisiologia Generale ed Ambientale, Universita' di Bari, 70126 Bari, Italy.

Abbreviations used in this paper: AC, adenylyl cyclase; [Ca2+]i, intracellular calcium concentration; CaR, extracellular Ca2+-sensing receptor; Epac, exchange protein activated by cAMP; FRET, fluorescence resonance energy transfer; GPCR, G protein–coupled receptor; HEK, human embryonic kidney; InsP3, inositol 1,4,5-trisphosphate; PDE, phosphodiesterase; PGE2, prostaglandin E2; PKA, protein kinase A; PTX, pertussis toxin; VIP, vasoactive intestinal peptide; WT, wild-type.


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