Ca2+ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP.
The spatial and temporal complexity of Ca2+ signalling is central to the regulation of a diverse range of cellular processes. The decoding of dynamic Ca2+ signals is, in part, mediated by the ability of Ca2+ to regulate other second messengers, including cyclic AMP (cAMP). A number of kinetic models (including our own) predict that interdependent Ca2+ and cAMP oscillations can be generated. A previous study in Xenopus neurons illustrated prolonged, low-frequency cAMP oscillations during bursts of Ca2+ transients. However, the detection of more dynamic Ca2+ driven changes in cAMP has, until recently, been limited by the availability of suitable cAMP probes with high temporal resolution. We have used a newly developed FRET-based cAMP indicator comprised of the cAMP binding domain of Epac-1 to examine interplay between Ca2+ and cAMP dynamics. This probe was recently used in excitable cells to reveal an inverse relationship between cAMP and Ca2+ oscillations as a consequence of Ca2+-dependent activation of phosphodiesterase 1 (PDE1). Here, we have used human embryonic kidney (HEK293) cells expressing the type 8 adenylyl cyclase (AC8) to examine whether dynamic Ca2+ changes can mediate phasic cAMP oscillations as a consequence of Ca2+-stimulated AC activity. During artificial or agonist-induced Ca2+ oscillations we detected fast, periodic changes in cAMP that depended upon Ca2+ stimulation of AC8 with subsequent PKA-mediated phosphodiesterase 4 (PDE4) activity. Carbachol (10 microM) evoked cAMP transients with a peak frequency of approximately 3 minute(-1), demonstrating phasic oscillations in cAMP and Ca2+ in response to physiological stimuli. Furthermore, by imposing a range of Ca2+-oscillation frequencies, we demonstrate that AC8 acts as a low-pass filter for high-frequency Ca2+ events, enhancing the regulatory options available to this signalling pathway.
Department of Pharmacology, Tennis Court Road, University of Cambridge, CB2 1PD, UK.
Debbie
D
Willoughby
Dermot M F
DM
Cooper
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000527:Alprostadil / Q000494:pharmacology; D001683:Biological Clocks / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D013997:Time Factors
D015220:Calcium Channels; D005456:Fluorescent Dyes; D005576:Colforsin; D002217:Carbachol; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; D000527:Alprostadil; D007545:Isoproterenol; D002118:Calcium