Termination of cAMP signals by Ca2+ and G(alpha)i via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations.
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.
Gerbino
Andrea
A
Veterans' Affairs Boston Healthcare System, West Roxbury, MA 02132, USA.
Ruder
Warren C
WC
Curci
Silvana
S
Pozzan
Tullio
T
Zaccolo
Manuela
M
Hofer
Aldebaran M
AM
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004917:Erythromycin / Q000031:analogs & derivatives / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D037342:Pertussis Toxin / Q000494:pharmacology; D044169:Receptors, Calcium-Sensing / Q000187:drug effects / Q000502:physiology*; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D044169:Receptors, Calcium-Sensing; D004917:Erythromycin; C057513:erythromycin propionate-N-acetylcysteinate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium; D000111:Acetylcysteine