Comparative pharmacology of adrenergic α 2C receptors coupled to Ca 2+ signaling through different Gα proteins

Abstract

Adrenergic α 1, α 2 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 α 2C type of adrenergic receptors (α 2C-AR). Although activation of α 2C-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Gα qi5 protein, containing the five carboxyl-terminal amino acids from G i, or promiscuosus Gα 16 protein can divert receptor signaling to the G q pathway generating Ca 2+ release from intracellular stores. In order to assess the functional potency of α 2-AR agonists and antagonists, we established a fluorometric Ca 2+ assay using cell lines stably and constitutively co-expressing α 2C-AR and Gα qi5 or Gα 16 proteins (Gα qi5/α 2C and Gα 16/α 2C). As part of the pharmacological characterization, we measured the changes in cytoplasmic Ca 2+ levels due to activation of the chimeric Gα qi5 or Gα 16 coupled recombinant α 2C receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine, oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of α 2-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in α 2C-AR expressing cells were also measured. These results confirmed that the Gα qi5/α 2C and Gα 16/α 2C recombinant systems can be useful for modelling the native G i-coupled system. Our results indicate that a plate-reader based fluorometric Ca 2+ assay may be suitable in high-throughput screening for α 2C-AR ligands as well.