Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

<bold><italic>Background/Aims:</italic></bold> Signaling of G_s 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. <bold><italic>Methods:</italic></bold> 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. <bold><italic>Results:</italic></bold> Here we show that the activation of GsPCRs and the GsPCRs-independent activation of G_s proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A_2A or A_2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A_2A 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_2B adenosine receptors. <bold><italic>Conclusion:</italic></bold> Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors. [ABSTRACT FROM AUTHOR]