Abstract
Cyclic adenosine monophosphate (cAMP) serves as an intracellular second messenger to transduce the effects of the stimulation of Gs-coupled receptors. Recent studies indicated that cAMP can be extruded by the multidrug resistance associated-protein 4 (MRP4) from many cell types including cardiac myocytes (CM). The present study investigated whether the cAMP extruded from CM acts as a signalling molecule. We employed a fluorescence resonance energy transfer (FRET)-based sensor (Epac2-cAMPs) to directly assay for the formation of cAMP in CM and fibroblasts (CF). We found cAMP to be extruded from neonatal and adult CM upon β-adrenergic receptor (βAR) stimulation (1µM isoproterenol, ISO), an effect that was largely sensitive to MRP inhibition. Extracellular cAMP blocked the βAR-mediated increase in intracellular cAMP in CM. This negative feedback was reversed by a non-specific adenosine receptor antagonist (DPSPX) and an A1 adenosine receptor specific antagonist (DPCPX) but not by an A2 adenosine receptor specific antagonist (ZM-241385) (FRET ratio: 12.6±0.6 after ISO treatment vs. 4.5±0.8 after ISO+ecto-cAMP, 12.6±1.3 after ISO+ecto-cAMP+DPSPX, p<0.001). In contrast, application of extracellular cAMP onto CF induced an increase in intracellular cAMP levels while A2 adenosine receptor specific antagonist blocked this effect (FRET ratio: 8.1±1.1 after ecto-cAMP treatment vs 0.07±0.3 after ecto-cAMP+ZM, p<0.001). To determine if extracellular cAMP released from CM is sufficient to stimulate cAMP formation in CF, we stimulated CF isolated from β1β2-AR-deficient mice with conditioned medium (coM) from ISO or control-treated CM. Exposure of βAR-deficient CF to coM from ISO-treated CMs yielded a significant increase in intracellular cAMP compared to control-coM. DPSPX again markedly attenuated the elevation of cAMP in response to ISO-coM (FRET ratio: control-coM: 3.1±0.6, ISO-coM: 7±1.2, ISO-coM+DPSPX: 2.2±1.3, p<0.05). In conclusion, this study provides evidence that βAR stimulation of CM induces an inside-out signaling through secreted cAMP. cAMP is exported from CM, metabolized to adenosine and in an autocrine and paracrine manner stimulates adenosine receptors on CM (A1 receptors) and CF (A2a,2b receptors).
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