Comparative pharmacology of adrenergic alpha sub(2C) receptors coupled to Ca super(2+) signaling through different G alpha proteins

• Published in: Neurochemistry international Vol. 55; no. 7; pp. 467 - 475
• Main Authors: Kurko, Dalma, Bekes, Zsofia, Gere, Aniko, Baki, Andrea, Boros, Andras, Kolok, Sandor, Bugovics, Gyula, Nagy, Jozsef, Szombathelyi, Zsolt, Ignacz-Szendrei, Gyoergyi
• Format: Journal Article
• Language: English
• Published: 01.12.2009
• ISSN: 0197-0186
• DOI: 10.1016/j.neuint.2009.04.015

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