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First published online 14 February 2006
doi: 10.1242/jcs.02812

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Ca²⁺ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP

Department of Pharmacology, Tennis Court Road, University of Cambridge, CB2 1PD, UK

View larger version (35K): [in a new window]   Fig. 1. CCE-induced stimulation of AC8 activity detected with the fluorescent Epac-1 probe. (Ai) Averaged and calibrated fura-2 measurements show Tg-induced (100 nM) emptying of intracellular Ca2+ stores and induction of increasing degrees of CCE upon addition of 0.2, 1 and 2 mM Ca2+. (Aii) Parallel changes in cAMP levels in three separate cells that express the Epac-1 biosensor during CCE with 1 mM Ca2+. A decrease in YFP:CFP emission-ratio represents an increase in cAMP. 10 nM PGE1 was added at 60 seconds. (Aiii) After Tg-induced Ca2+-store depletion a Ca2+ dose-dependent rise in [cAMP] was seen due to increasing degrees of CCE. (Bi-iii) Parallel experiments to A, but 500 µM carbachol (CCh) was added instead of Tg, to deplete the Ca2+ stores before inducing CCE. Data are plotted as mean ± s.e.m., n values range from 22 to 40 cells. *P<0.01, **P<0.001.

View larger version (31K): [in a new window]   Fig. 2. Temporal limitations of CCE-evoked synchronous Ca2+- and cAMP-oscillations. All panels show overlays of single-cell calibrated fura-2 (black trace) and Epac-1 signals (coloured traces) during imposed Ca2+ changes. Cells were pre-treated with Tg (100 nM) in Ca2+-free saline to deplete Ca2+ stores, and PGE1 (10 nM) was added to stimulate basal AC8 activity. Real-time cAMP and Ca2+ changes were obtained at various frequencies and durations. (A) Periodic CCE-evoked Ca2+ entry (2 minutes), and washes in Ca2+-free saline (2 minutes). (B) Ca2+ entry (1 minute), and washes (1 minute) in Ca2+-free saline. (C) Ca2+ entry (20 seconds), and washes (40 seconds) in Ca2+-free saline. (D) Ca2+ entry (10 seconds), and washes (20 seconds) in Ca2+-free saline.

View larger version (45K): [in a new window]   Fig. 3. cAMP oscillations depend on changes in cytosolic [Ca2+] and PDE activity. (A) Two neighbouring Epac-1 probe-expressing cells, showing synchronous cAMP oscillations imposed by Ca2+ changes due to Tg-induced CCE. (Ba) YFP emission image, showing cytosolic distribution of the fluorescent Epac-1 probe in AC8-expressing HEK293 cells. (Bb-i) Pseudocolour images of the FRET emission ratio at various time points indicated in A, showing oscillating cAMP levels in the imaged cells. Bar, 20 µm. (C) Comparison of on-off-rates of Ca2+- and cAMP-transients presented as t1/2. Data are shown as mean ± s.e.m. (n=12); P values were calculated using Student's t-test. (D) Effect of 100 µM IBMX on cAMP- and Ca2+-oscillations.

View larger version (26K): [in a new window]   Fig. 4. Recovery from Ca2+-evoked cAMP changes is PDE4-dependent. (A) Averaged and calibrated fura-2 signal from 50 HEK293 cells that had been pre-treated with Tg (100 nM) at 60 seconds, and were subsequently exposed to periodic 2-minute-long CCE events followed by 2-minute-long washes (after each Ca2+ rise) in Ca2+-free saline. (B-E) Effects of selective PDE inhibitors on cAMP recovery. Epac-1 FRET ratios from parallel experiments with 10 nM PGE1 and (B) without phosphodiesterase inhibitors, (C) with 10 µM MMX, (D) with 10 µM cilostamide, (E) with 10 µM rolipram. (F) Bar graph shows the data presented in B-E plotted as mean ± s.e.m. (n values range from 9 to 23 individual cells). In addition, cAMP recovery following treatment with the broad-spectrum phosphodiesterase inhibitor IBMX (100 µM) is shown. *P<0.05 and **P<0.001 compared with control recoveries.

View larger version (31K): [in a new window]   Fig. 5. Comparison of Ca2+- and cAMP-interplay in the presence of different AC8 activators. Parallel experiments of cytosolic Ca2+ changes (black trace) and Epac-1 FRET ratios (coloured traces) after Tg-induced store-depletion at 60 seconds and subsequent imposed 2-minute-long Ca2+ oscillations. (A) Modest cAMP oscillations in a single cell in response to Ca2+ changes without adenylyl cyclase activator. (B-D) CCE-evoked cAMP oscillations following addition of (B) 10 nM PGE1 at 310 seconds, (C) 10 nM isoprenaline at 310 seconds, (D) 10 nM forskolin at 310 seconds.

View larger version (17K): [in a new window]   Fig. 6. Agonist-induced oscillations in levels of Ca2+ and cAMP. (A) Single-cell cytosolic Ca2+ changes in response to 10 µM CCh showing either (i) sustained Ca2+ increase or (ii) regular Ca2+ oscillations. (B) Parallel experiments measuring cAMP signals in individual Epac-1-probe- and AC8-expressing cells with and without external Ca2+. 10 nM PGE1 was present throughout the period of CCh application.