Termination of cAMP signals by Ca2+ and Gαi via extracellular Ca2+ sensors

Abstract

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 Gαi 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.

Transcriptional Regulation of the Claudin-16 Gene by Mg²⁺Availability

“…HEK293 cells stably transfected with the human CaSR (HEK293 -CaSR) (provided by Dr. A. Hofer, Harvard Medical School, Boston, MA) [18]) were grown and maintained in DMEM/F-12 supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 5IU/ml penicillin and 5 µg/ml streptomycin at 37°C in a humidified atmosphere of 95% air-5% CO 2 .…”

Allosteric Modulation of the Calcium-sensing Receptor by γ-Glutamyl Peptides

“…8). These results suggest that the double mutant has little or no effect on modulator binding but instead impairs the coupling between Ca o on intracellular cAMP levels in HEK-CaR cells that were transfected with the cAMP reporter CFPnd-EPac1-cpVenus since the CaR is known to suppress cAMP levels in a G i and Ca 2ϩ i -dependent manner (27). cAMP levels were first elevated by exposure to 5 M forskolin and the cells were then exposed to one or more CaR activators in the continuing presence of forskolin.…”

“…S1). Of the compounds tested, S-methylglutathione exhibited the highest potency activation of Ca i -dependent mechanisms have been identified (27). In the current study, we transiently expressed a FRET-based cAMP sensor, CFPnd-EPac1-cpVenus (33) in CaR-expressing HEK-293 cells to permit detection of changes in intracellular cAMP levels in real-time.…”

Simultaneous Optical Measurements of Cytosolic Ca 2+ and cAMP in Single Cells

“…The intracellular signaling pathways mediated by these two second messengers are thought to be interconnected (1)(2)(3), and resolving the spatial and temporal interrelationships of Ca 2+ and cAMP signaling requires simultaneous measurement of both molecules. FlCRhR, a bimolecular recombinant Förster or fluorescence resonance energy transfer (FRET)-based cAMP indicator, has been used together with the Ca 2+ indicator Fura-2 or with patch-clamp recordings of Ca 2+ current to concurrently measure cAMP and Ca 2+ responses in single cells (4,5).…”