A cell-based luciferase-dependent assay for the quantitative determination of free extracellular adenosine with paracrine signaling activity.

Abstract Extracellular adenosine exerts powerful paracrine effects on immune cells. Thus, adenosine signaling has to be strictly regulated. This is achieved by its rapid internalization or enzymatic degradation. Consequently, free adenosine is extremely difficult to measure in cell culture systems and may escape from detection by time-consuming endpoint measurements like high-performance liquid chromatography (HPLC). Therefore, we have now developed a highly sensitive assay which enables the quantification of biologically relevant extracellular adenosine via the activation of an ectopically expressed Adenosine 2a-receptor (ADORA2A) in HEK-293 reporter cells. Binding of the short-lived nucleoside to this receptor induces a cAMP-dependent signal which can be detected via a cAMP-responsive luciferase construct. Tests with exogenously added adenosine confirmed that the resulting luminescence signals correlate with the respective adenosine levels and thus allow quantitative measurements in a range from 20 nM to 80 μM free extracellular adenosine. Inhibition of adenosine uptake by dipyridamole further increased the sensitivity of the assay. We further validated our approach by quantifying the adenosine levels that are generated by regulatory T cells via ectonucleotidase-mediated cleavage of ATP. As expected, values returned to baseline when ADORA2A was inhibited. This confirmed that this new cell-based reporter assay constitutes a biologically relevant, technically easy, versatile, scalable and cost-effective approach that allows the non-radioactive quantification of adenosine as a signaling intermediate.