Pharmacological characterisation of melatonin mt, receptor-mediated stimulation of [35S]-GTPγS binding

The activation of G-proteins by melatonin mt(1) receptors was studied by measuring [S-35]-guanosine-5'-(3-thiotriphosphate) ([S-35]-GTP gamma S) binding to membranes prepared from Chinese hamster ovary (CHO) cells stably expressing human mt(1) receptors. Melatonin stimulated [S-35]-GTP gamma S binding in a concentration-dependent manner (pEC(50), 8.77 +/- 0.02). The optimal (212 +/- 4%) increase over basal levels of binding (basal = 100%) was observed following incubation of membranes (12.5 mu g protein/well) for 120 min at 30 degrees with [S-35]-GTP gamma S (0.1 nM), in the presence of GDP (10 mu M), NaCl (100 mM), and MgCl2 (10 mM). Melatonin analogues stimulated [S-35]-GTP gamma S binding with a rank order (2-iodomelatonin > melatonin = S20098 > GR196429 > 6-chloromelatonin = 6-hydroxymelatonin much greater than N-acetylserotonin greater than or equal to GR135531 = mt(1) luzindole = 5-HT = 0), which was identical to their affinities for the high affinity state of the receptor (correlation coefficient 0.94). All agonists evoked similar maximum increases in [S-35]-GTP gamma S binding. EC50 values were 14- to 63-fold lower than binding affinities. The melatonin receptor antagonist luzindole (0.1-10 mu M) evoked a parallel rightward shift in the melatonin concentration-response curve, with a pK(B) Of 7.19 +/- 0.13, which is similar to its affinity in radioligand binding studies for human mt(1) receptors. Stimulation of [S-35]-GTP gamma S binding was abolished by pretreatment of cells with pertussis toxin (18 hr, 100 ng/mL) prior to preparation of membranes. Melatonin was without effect in CHO cells which lacked the mt(1) receptor. Thus, melatonin and melatonin analogues stimulate [S-35]-GTP gamma S binding with a profile which is consistent with binding to mt(1) receptors causing activation of G(i)/G(o) G-proteins. (C) 1998 Elsevier Science Inc.