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

Abstract

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 μM) evoked cAMP transients with a peak frequency of ∼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.