Pharmacological characterization of human recombinant melatonin mt1 and MT2 receptors

We have pharmacologically characterized recombinant human mt1 and MT2 receptors, stably expressed in Chinese hamster ovary cells (CHO‐mt1 and CHO‐MT2), by measurement of [3H]‐melatonin binding and forskolin‐stimulated cyclic AMP (cAMP) production. [3H]‐melatonin bound to mt1 and MT2 receptors with pKD values of 9.89 and 9.56 and Bmax values of 1.20 and 0.82 pmol mg−1 protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt1 and MT2 receptors, a number of putative antagonists had substantially higher affinities for MT2 receptors, including luzindole (11 fold), GR128107 (23 fold) and 4‐P‐PDOT (61 fold). In both CHO‐mt1 and CHO‐MT2 cells, melatonin inhibited forskolin‐stimulated accumulation of cyclic AMP in a concentration‐dependent manner (pIC50 9.53 and 9.74, respectively) causing 83 and 64% inhibition of cyclic AMP production at 100 nM, respectively. The potencies of a range of melatonin receptor agonists were determined. At MT2 receptors, melatonin, 2‐iodomelatonin and 6‐chloromelatonin were essentially equipotent, whilst at the mt1 receptor these agonists gave the rank order of potency of 2‐iodomelatonin>melatonin>6‐chloromelatonin. In both CHO‐mt1 and CHO‐MT2 cells, melatonin‐induced inhibition of forskolin‐stimulated cyclic AMP production was antagonized in a concentration‐dependent manner by the melatonin receptor antagonist luzindole, with pA2 values of 5.75 and 7.64, respectively. Melatonin‐mediated responses were abolished by pre‐treatment of cells with pertussis toxin, consistent with activation of Gi/Go G‐proteins. This is the first report of the use of [3H]‐melatonin for the characterization of recombinant mt1 and MT2 receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.