Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor

Abstract

We have identified subtype selective agonists, partial agonists and antagonists, which distinguish the human recombinant Mel1a and Mel1b melatonin receptors expressed in COS-7 cells. Melatonin receptor agonists showed higher affinity for competition of 2-[125I]-iodomelatonin binding for the Mel1b than the Mel1a melatonin receptor. The dissociation constants (Ki) of 16 agonists determined on the recombinant human Mel1a and Mel1b melatonin receptor subtypes showed a significant correlation (r2 = 0.85, slope = 0.97, P < 0.0001, n = 16). However, six agonists showed 10 to 60 fold higher affinity for the Mel1b melatonin receptor as indicated by the affinity selectivity ratios (Mel1a/Mel1b) [8-methoxy-2-acetamidotetraline (11); S20098 (14); 8-methoxy-2-propionamidotetraline (20); 6, 7 di-chloro-2-methylmelatonin (21); 6-chloromelatonin (57); 6-methoxymelatonin (59)]. Dissociation constants for competition of 11 partial agonists and antagonist for 2-[125I]-iodomelatonin binding were between 15.5 (luzindole, pKi: 7.7) to 362 (4-phenyl-2-chloroacetamidotetraline, pKi: 9.1) fold higher for the Mel1b than for the Mel1a melatonin receptor. The lack of correlation between the pKi values (r2 = 0.23, P > 0.1, n = 11) strongly suggest that the two human melatonin receptor subtypes can be distinguished pharmacologically. The partial agonist: 5-methoxyluzindole (pKi: 9.6) and the competitive melatonin receptor antagonists: GR128107 (pKi: 9.6), 4-phenyl-2-chloroacetamidotetraline (pKi: 9.1), 4-phenyl-2-acetamidotetraline (pKi: 8.9) and 4-phenyl-2-propionamidotetraline (pKi: 8.8) are selective Mel1b melatonin receptor analogues as their affinity selectivity ratios (Mel1a/Mel1b) are bigger than 100. We conclude that the 40% overall amino acid difference in the sequence of the human recombinant Mel1a and Mel1b melatonin receptors is reflected in distinct pharmacological profiles for the subtypes. We compared the pharmacological profile of the presynaptic ML1 melatonin heteroreceptor of rabbit retina mediating inhibition of the calcium-dependent release of dopamine to that of the recombinant Mel1a and Mel1b melatonin receptors. Melatonin inhibited [3H]dopamine release by 50% (1C50) at 20 pM with a maximal inhibitory effect (80%) at 1 nM. The partial agonists, i.e., N-acetyltryptamine (1C50 5.6, maximal inhibition 55%) and 5-methoxyluzindole (1C50: 1.3, maximal inhibition 40%) showed various degrees of efficacy while none of the competitive melatonin receptor antagonists did inhibit [3H]dopamine release on their own. The potency (1C50) of full melatonin receptor agonists significantly correlated with their affinity to compete for 2-[125I]-iodomelatonin binding to either the Mel1a (r2 = 0.76, slope = 0.77, P < 0.0001, n = 17) or Mel1b (r2 = 0.63, slope = 0.75, P < 0.001, n = 17) human melatonin receptors. By contrast, the apparent dissociation constants (KB) for partial agonists and antagonists to antagonize the inhibition of [3H]dopamine release mediated by activation of the ML1 heteroreceptor by melatonin, significantly correlated with...

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

“…In agreement with published data (20), GTP␥ 35 S binding experiments classified S20928 and luzindole as neutral antagonists, 4P-PDOT as a weak inverse agonist, and melatonin and S20098 as full agonists (Fig. 5b).…”

“…As shown in Fig. 5a, melatonin stimulation promoted a dosedependant increase in BRET for the MT2 homodimer with an EC 50 value of 1 nM, consistent with the affinity of melatonin for this receptor (20). In contrast, no change in constitutive BRET was observed for the MT1R homo-dimer.…”

The mt1 Melatonin Receptor and RORβ Receptor Are Co-localized in Specific TSH-immunoreactive Cells in the Pars Tuberalis of the Rat Pituitary

“…Two high-affinity receptors for melatonin have thus far been cloned in rodents: the mt1 (Mel 1a ) and the MT2 (Mel 1b ) receptor subtypes (Reppert et al 1994(Reppert et al ,1995Roca et al 1996) which belong to the seven-transmembrane domain, G-protein-coupled receptor family (Dubocovich et al 1999). However, the mt1 receptor appears to be the highly dominant subtype in the PT because targeted disruption of its gene in mice leads to undetectable levels of 2-iodomelatonin binding (Liu et al 1997), whereas cells transfected with the MT2 receptor gene bind this ligand (Dubocovich et al 1997). Furthermore, the MT2 receptor subtype is naturally nonfunctional in the golden hamster, which is a highly photoperiodic species .…”

Leucocyte Infiltration in Lymphoid Organs and Peritoneal Cavity upon Immunization: Dependence on Circadian Rhythmicity and Melatonin 24-H Profile

“…The melatonin analogue 4P-PDOT was described as a high-affinity and selective antagonist of MT2 melatonin receptor. It has >300-fold higher affinity for the MT2 vs MTl (30). Our experiments revealed that the action of 4P-PDOT in vivo was similar to that under constant lighting.…”