Pharmacological characterization of human recombinant melatonin mt1 and MT2 receptors

1 We have pharmacologically characterized recombinant human mt(1) and MT2 receptors, stably expressed in Chinese hamster ovary cells (CHO-mt(1) and CHO-MT2), by measurement of [H-3]melatonin binding and forskolin-stimulated cyclic AMP (cAMP) production. 2 [H-3]-melatonin bound to mt, and MT2 receptors with pK(D) values of 9.89 and 9.56 and B-max values of 1.20 and 0.82 pmol mg(-1) protein, respectively. Whilst most melatonin receptor agonists had similar affinities for mt(1) 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). 3 In both CHO-mt(1) and CHO-MT2 cells, melatonin inhibited forskolin-stimulated accumulation of cyclic AMP in a concentration-dependent manner (pIC(50) 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 mt, receptor these agonists gave the rank order of potency of 2-iodomelatonin > melatonin > 6-chloromelatonin. 4 In both CHO-mt(1) 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 pA(2) 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 G(i)/G(o) G-proteins. 5 This is the first report of the use of [H-3]-melatonin for the characterization of recombinant mt, and MT2 receptors. Our results demonstrate that these receptor subtypes have distinct pharmacological profiles.