T., Pleli A., Mondorf N., Ferreiros D., Thomas K., Dvorak R.M., Biondi D.M., Heringdorf S., Zeuzem G., Geisslinger H., Zimmermann O., Waidmann A., Piiper Supplementary Material for: Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors <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. Adenosine receptors;cAMP;Adenylyl cyclase;eNPP2;MRP4;PKA 2018-03-27
    https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Activation_of_Adenylyl_Cyclase_Causes_Stimulation_of_Adenosine_Receptors/6031892
10.6084/m9.figshare.6031892.v1