Comparative pharmacology of adrenergic alpha(2C) receptors coupled to Ca(2+) signaling through different Galpha proteins.

Adrenergic alpha(1), alpha(2) and beta 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 alpha(2C) type of adrenergic receptors (alpha(2C)-AR). Although activation of alpha(2C)-AR is not linked to calcium mobilization, co-expression of these receptors with the chimeric Galpha(qi5) protein, containing the five carboxyl-terminal amino acids from G(i), or promiscuosus Galpha(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 alpha(2)-AR agonists and antagonists, we established a fluorometric Ca(2+) assay using cell lines stably and constitutively co-expressing alpha(2C)-AR and Galpha(qi5) or Galpha(16) proteins (Galpha(qi5)/alpha(2C) and Galpha(16)/alpha(2C)). As part of the pharmacological characterization, we measured the changes in cytoplasmic Ca(2+) levels due to activation of the chimeric Galpha(qi5) or Galpha(16) coupled recombinant alpha(2C) receptors as a function of increasing concentration of several agonists (noradrenaline, brimonidine, oxymetazoline, clonidine, moxonidine) and antagonists (MK912, yohimbine). The binding affinities of alpha(2)-AR agonist and antagonists and the inhibition of the forskolin-stimulated cAMP accumulation in alpha(2C)-AR expressing cells were also measured. These results confirmed that the Galpha(qi5)/alpha(2C) and Galpha(16)/alpha(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 alpha(2C)-AR ligands as well.