Pharmacological characterisation of melatonin mt 1 receptor-mediated stimulation of [ 35S]-GTPγs binding

Abstract

The activation of G-proteins by melatonin mt 1 receptors was studied by measuring [ 35S]-guanosine-5′-(3-thiotriphosphate) ([ 35S]-GTPγS) binding to membranes prepared from Chinese hamster ovary (CHO) cells stably expressing human mt 1 receptors. Melatonin stimulated [ 35S]-GTPγS 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 μg protein/well) for 120 min at 30° with [ 35S]-GTPγS (0.1 nM), in the presence of GDP (10 μM), NaCl (100 mM), and MgCl 2 (10 mM). Melatonin analogues stimulated [ 35S]-GTPγS binding with a rank order (2-iodomelatonin > melatonin = S20098 > GR196429 > 6-chloromelatonin = 6-hydroxymelatonin ≫ N-acetylserotonin ≥ 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 [ 35S]-GTPγS binding. EC 50 values were 14- to 63-fold lower than binding affinities. The melatonin receptor antagonist luzindole (0.1–10 μM) 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 [ 35S]-GTPγS 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 [ 35S]-GTPγS binding with a profile which is consistent with binding to mt 1 receptors causing activation of G i/G o G-proteins.