Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A_2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A_2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC₅₀ value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gα_q family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gα_q proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gα_q protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gα_q-protein-binding radio-tracers as novel diagnostics.