10.6084/m9.figshare.6031892.v1 Pleli T. Pleli T. Mondorf A. Mondorf A. Ferreiros N. Ferreiros N. Thomas D. Thomas D. Dvorak K. Dvorak K. Biondi R.M. Biondi R.M. Heringdorf D.M. Heringdorf D.M. Zeuzem S. Zeuzem S. Geisslinger G. Geisslinger G. Zimmermann H. Zimmermann H. Waidmann O. Waidmann O. Piiper A. Piiper A. Supplementary Material for: Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors Karger Publishers 2018 Adenosine receptors cAMP Adenylyl cyclase eNPP2 MRP4 PKA 2018-03-27 07:11:35 Dataset https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Activation_of_Adenylyl_Cyclase_Causes_Stimulation_of_Adenosine_Receptors/6031892 <b><i>Background/Aims:</i></b> Signaling of G<sub>s</sub> protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. <b><i>Methods:</i></b> Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. <b><i>Results:</i></b> Here we show that the activation of GsPCRs and the GsPCRs-independent activation of G<sub>s</sub> proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A<sub>2A</sub> or A<sub>2B</sub> adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A<sub>2A</sub> adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A<sub>2B</sub> adenosine receptors. <b><i>Conclusion:</i></b> Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.