Comparative pharmacology of adrenergic α2C receptors coupled to Ca2+ signaling through different Gα proteins

Adrenergic α1, α2 and β receptors are members of the G-protein-coupled receptor families (GPCRs) mediating physiological responses to adrenaline (epinephrine) and noradrenaline (norepinephrine). Since GPCRs are major targets for potential therapeutic agents, development of robust, reliable and cost effective functional screening methods for these receptors is in the focus of pharmacological research. For this reason, the aim of the present study was to develop an intracellular calcium assay for investigating the pharmacology of the α2C type of adrenergic receptors (α2C-AR). Although activation of α2C-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Gαqi5 protein, containing the five carboxyl-terminal amino acids from Gi, or promiscuosus Gα16 protein can divert receptor signaling to the Gq pathway generating Ca2+ release from intracellular stores.In order to assess the functional potency of α2-AR agonists and antagonists, we established a fluorometric Ca2+ assay using cell lines stably and constitutively co-expressing α2C-AR and Gαqi5 or Gα16 proteins (Gαqi5/α2C and Gα16/α2C). As part of the pharmacological characterization, we measured the changes in cytoplasmic Ca2+ levels due to activation of the chimeric Gαqi5 or Gα16 coupled recombinant α2C receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine, oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of α2-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in α2C-AR expressing cells were also measured. These results confirmed that the Gαqi5/α2C and Gα16/α2C recombinant systems can be useful for modelling the native Gi-coupled system. Our results indicate that a plate-reader based fluorometric Ca2+ assay may be suitable in high-throughput screening for α2C-AR ligands as well.