Melatonin (5-methoxy-N-acetyltryptamine), dubbed the hormone of darkness, is released following a circadian rhythm with high levels at night. It provides circadian and seasonal timing cues through activation of G protein-coupled receptors (GPCRs) in target tissues (1). The discovery of selective melatonin receptor ligands and the creation of mice with targeted disruption of melatonin receptor genes are valuable tools to investigate the localization and functional roles of the receptors in native systems. Here we describe the pharmacological characteristics of melatonin receptor ligands and their various efficacies (agonist, antagonist, or inverse agonist), which can vary depending on tissue and cellular milieu. We also review melatonin-mediated responses through activation of melatonin receptors (MT1, MT2, and MT3) highlighting their involvement in modulation of CNS, hypothalamic-hypophyseal-gonadal axis, cardiovascular, and immune functions. For example, activation of the MT1 melatonin receptor inhibits neuronal firing rate in the suprachiasmatic nucleus (SCN) and prolactin secretion from the pars tuberalis and induces vasoconstriction. Activation of the MT2 melatonin receptor phase shifts circadian rhythms generated within the SCN, inhibits dopamine release in the retina, induces vasodilation, enhances splenocyte proliferation and inhibits leukocyte rolling in the microvasculature. Activation of the MT3 melatonin receptor reduces intraocular pressure and inhibits leukotriene B4-induced leukocyte adhesion. We conclude that an accurate characterization of melatonin receptors mediating specific functions in native tissues can only be made using receptor specific ligands, with the understanding that receptor ligands may change efficacy in both native tissues and heterologous expression systems.

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...

This study demonstrates the involvement of the MT2 (Mel1b) melatonin receptor in mediating phase advances of circadian activity rhythms by melatonin. In situ hybridization histochemistry with digoxigenin-labeled oligonucleotide probes revealed for the first time the expression of mt1 and MT2 melatonin receptor mRNA within the suprachiasmatic nucleus of the C3H/HeN mouse. Melatonin (0.9 to 30 microg/mouse, s.c.) administration during 3 days at the end of the subjective day (CT 10) to C3H/HeN mice kept in constant dark phase advanced circadian rhythms of wheel running activity in a dose-dependent manner [EC50=0.72 microg/mouse; 0.98+/-0.08 h (n=15) maximal advance at 9 microg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P-ADOT and 4P-PDOT (90 microg/mouse, s.c.) nor luzindole (300 microg/mouse, s.c.), which shows 25-fold higher affinity for the MT2 than the mt1 subtype, affected the phase of circadian activity rhythms when given alone at CT 10. All three antagonists, however, shifted to the right the dose-response curve to melatonin, as they significantly reduced the phase shifting effects of 0.9 and 3 microg melatonin. This is the first study to demonstrate that melatonin phase advances circadian rhythms by activation of a membrane-bound melatonin receptor and strongly suggests that this effect is mediated through the MT2 melatonin receptor subtype within the circadian timing system. We conclude that the MT2 melatonin receptor subtype is a novel therapeutic target for the development of subtype-selective analogs for the treatment of circadian sleep and mood-related disorders.

In rat caudal artery, contraction to melatonin results primarily from activation of MT 1 melatonin receptors; however, the role of MT 2 melatonin receptors in vascular responses is controversial. We examined and compared the expression and function of MT 2 receptors with that of MT 1 receptors in male rat caudal artery. MT 1 and MT 2 melatonin receptor mRNA was amplified by reverse transcription-polymerase chain reaction from caudal arteries of three rat strains (i.e., Fisher, Sprague-Dawley, and Wistar). Antisense (but not sense) 33 P-labeled oligonucleotide probes specific for MT 1 or MT 2 receptor mRNA hybridized to smooth muscle, as well as intimal and adventitial layers, of caudal artery. In male Fisher rat caudal artery denuded of endothelium, melatonin was 10 times more potent than 6-chloromelatonin to potentiate contraction to phenylephrine, suggesting activation of smooth muscle MT 1 melatonin receptors. The MT 1 /MT 2 competitive melatonin receptor antagonist luzindole (3 M), blocked melatonin-mediated contraction (0.1-100 nM) with an affinity constant (K B value of 157 nM) similar to that for the human MT 1 receptor. However, at melatonin concentrations above 100 nM, luzindole potentiated the contractile response, suggesting blockade of MT 2 receptors mediating vasorelaxation and/or an inverse agonist effect at MT 1 constitutively active receptors. The involvement of MT 2 receptors in vasorelaxation is supported by the finding that the competitive antagonists 4-phenyl 2-acetamidotetraline and 4-phenyl-2-propionamidotetraline, at MT 2 -selective concentrations (10 nM), significantly enhanced contractile responses to all melatonin concentrations tested (0.1 nM-10 M). We conclude that MT 2 melatonin receptors expressed in vascular smooth muscle mediate vasodilation in contrast to vascular MT 1 receptors mediating vasoconstriction.Accumulating evidence indicates that the hormone melatonin regulates vascular tone; however, the nature of the response remains controversial. Both vasoconstrictor and vasodilator responses have been reported; however, data supporting the presence of melatonin receptors is found in some, but not all, vascular beds (Mahle et al., 1997). In isolated rat caudal arteries, nanomolar concentrations of melatonin potentiate contraction induced by either endogenous or exogenous vasoconstrictors (Viswanathan et al

This study investigated the receptor mechanism(s) by which the hormone melatonin directly affects ovarian function. Expression of MT 1 and MT 2 melatonin receptor mRNA was detected in the rat ovaries both by reverse transcriptase-polymerase chain reaction and in situ hybridization with digoxigenin-labeled oligoprobes. Exposure of granulosa cells in culture to 17␤-estradiol seems to alter the state of melatonin receptor coupling. Indeed, the efficacy of 4P-PDOT on forskolin-stimulated cAMP formation was reversed from an MT 2 partial agonist in vehicle-treated cells to that of an MT 1 inverse agonist in 17␤-estradiol (0.1 M)-treated granulosa cells. We conclude that MT 1 and MT 2 melatonin receptors expressed in antral follicles and corpus luteum may affect steroidogenesis through cAMPmediated signaling. These results underscore the implications of the levels of ovarian estrogen when melatonin receptor ligands are used as therapeutic agents.