Mammalian Melatonin Receptors

2.1. Melatonin Receptor Nomenclature and Classification

This review will use the nomenclature and classification for melatonin receptors adopted by the International Union of Pharmacology (23) in 1998 (Figure 1). Accordingly, the melatonin receptors are referred to with the letters MT (MelaTonin). This nomenclature uses lower case "mt" followed by its number to describe receptors for which only the molecular structure is known (e.g., mti former MeL) (8). Melatonin receptors with a well defined functional pharmacology in a native tissue, as well as known molecular structure are referred in upper case followed by a number subscript (e.g. MT2 former MeU) (8,24-25). Upper case in italics (MT) followed by the corresponding number is reserved for receptors pharmacologically characterized in native tissues for which the molecular structure is not known (e.g., MT3 former ML2) (14).

2.2. Melatonin Receptor Subtypes

The original classification of melatonin receptors differentiated the ML1 and ML2 subtypes based on kinetics and pharmacological characteristics (12) (Figure 1). High (30-300 pM) affinity (MLi) 2-[125I]-iodomelatonin binding sites found in mammalian retina, suprachiasmatic nucleus, and pars tuberalis, show a pharmacology profile (2-iodomelatonin > melatonin >> N-acetylserotonin) characterized by a low affinity for the precursor of melatonin, N-acetylserotonin. This pharmacological

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Figure 1. Nomenclature for mammalian melatonin receptors. The nomenclature described here was adopted by the Nomenclature Committee of International Union of Pharmacology in 1998. For further details please see reference 25. Luzindole: MLT: melatonin (N-acetyl-5-methoxytryptamine); NA 5-HT N-acetyl-5-hydroxytryptamine; 5-MCA-NAT 5-methoxy-carbonylamino- N-acetyltryptamine; 4-P-PDOT 4-phenyl 2-propionamidotetraline; 4-P-ADOT: 4-phenyl 2-acetamidotetraline.

Figure 1. Nomenclature for mammalian melatonin receptors. The nomenclature described here was adopted by the Nomenclature Committee of International Union of Pharmacology in 1998. For further details please see reference 25. Luzindole: MLT: melatonin (N-acetyl-5-methoxytryptamine); NA 5-HT N-acetyl-5-hydroxytryptamine; 5-MCA-NAT 5-methoxy-carbonylamino- N-acetyltryptamine; 4-P-PDOT 4-phenyl 2-propionamidotetraline; 4-P-ADOT: 4-phenyl 2-acetamidotetraline.

profile corresponds closely to that of the functional melatonin receptor of rabbit retina (14).

Cloning studies revealed two mammalian melatonin receptors (Melia and MeU now termed mtl and MT2) encoding 2-[125I]-iodomelatonin binding sites showing the general pharmacology of the high affinity (ML1) melatonin receptor. These two melatonin receptors were defined as unique subtypes on the basis of their molecular structure and chromosomal localization (8,26). The human melatonin receptor subtypes show 60% homology at the amino acid level and distinct pharmacological profiles of partial agonists and antagonists (24). The use of cell lines expressing the human mti and MT2 melatonin receptors has led to the discovery of subtype selective analogues (24) (Figure 1).

Activation of high affinity melatonin receptors in both neuronal and nonneuronal native tissues and transfected cell lines inhibits cAMP formation through a pertussis toxin sensitive inhibitory G proteins (17,27-28). However, coupling of the high affinity melatonin receptors to modulation of cGMP formation (29), synthesis of diacylglycerol and release of arachidonic acid, changes in calcium influx (30) and potassium conductances (31) were also reported. Melatonin receptor mediated vasoconstriction appears to involve closing of calcium-activated potassium channels (BKca) (32). In cell lines expressing the h mt1 and h MT2 melatonin receptors, melatonin inhibits forskolin stimulated cAMP accumulation (27-28). In this preparation a parallel signal transduction mechanism has been proposed for mt1 receptors whereby GPy subunits may activate phospholipase CP sabsequent to PGF2 a stimulation (27).

The putative MT3 melatonin receptor (former ML2 binds 2-[125I]-iodomelatonin with nanomolar affinity and shows a pharmacological profile (2-iodomelatonin > melatonin = N-acetylserotonin) distinct from that of the high affinity site where melatonin has higher affinity than N-acetylserotonin (12,33-34) (Figure 1). 2-[125I]-Iodomelatonin and the novel MT3 subtype selective radioligand 2-[125I]-5-MCA-NAT bind with low nanomolar affinity (Kd: 0.9-10nM) and show fast kinetics of association and dissociation to membranes of hamster brain, kidney and testes, to mouse brain and to RPMI 1846 melanoma cells (34-35). Melatonin receptors with pharmacology and kinetics similar to those in hamster brain have been described in brown fat (36). Activation of the MT3 receptor appears to signal through increases in phosphoinositide turnover (37). In guinea pig colon melatonin induced contraction through activation of a receptor with a pharmacological profile comparable to that of the MT3 receptor (38). In summary, MT3 melatonin binding sites show distinct pharmacological profiles and distribution than high affinity melatonin receptors (i.e., mt1 and MT2).

2.3. Subtype Selective Melatonin Analogues

The structural molecular differences between the human recombinant mt1 and MT2 melatonin receptors are reflected in distinct pharmacological profiles of melatonin analogues to compete for either 3H-melatonin or 2-[125I]-iodomelatonin binding (24,39). A number of synthetic melatonin analogues (e.g., S20098, 6-chloromelatonin, GR 196429) that mimic the effect of melatonin in functional responses (e.g., inhibition of dopamine release) do show small differences in affinities for the mammalian subtypes (24-25). For example 6-chloromelatonin competes with 57 times higher affinity for 2-[125I]-iodomelatonin binding to the MT2 than the mt1 human melatonin receptors expressed in CHO cells.

Subtype selective melatonin receptor antagonists are essential to identify melatonin receptor subtypes in native tissues. In this review we describe the specificity and selectivity of three competitive melatonin receptor antagonists, luzindole, 4P-ADOT and 4P-PDOT that are used to identify melatonin receptor subtypes in native tissues (14,24) (Figure 2). These antagonists show melatonin receptor specificity as they did not compete for binding of forty-nine radioligands to receptors, channels, transporters and second messengers and various degree of selectivity for the h MT2 melatonin receptor subtype (24-25). Figure 2 shows the chemical structures of luzindole, 4P-ADOT and 4P-PDOT and their affinity constants to compete for 2-[125I]-iodomelatonin binding to the recombinant h mt1 and h MT2 melatonin receptors stably expressed in CHO cells. Luzindole (Ki = 7.3 ± 2.0 nM, n = 4), 4P-ADOT (Ki = 0.4 ± 0.02 nM, n = 3) and 4P-PDOT (Ki = 0.41 ± 0.04 nM, n = 3) showed higher affinity for competition with 2-[125I]-iodomelatonin binding to the h MT2 melatonin receptor. The calculated affinity ratios (KiMT2/Kimt1) showed that luzindole has 25 fold higher affinity for the h MT2 than the h mt1 melatonin receptor subtype, while 4P-ADOT and 4P-PDOT are 951 and 1560 fold MT2 subtype selective (Figure 2).

2.4. Melatonin Receptor Subtype Function

Specific functions of melatonin believed to be mediated through activation of melatonin receptors include: inhibition of dopamine release in retina (9-11,13), acute inhibition of electrical activity (40-41), phase shifts of circadian rhythms in the suprachiasmatic nucleus slice (42-43) and potentiation by melatonin of endogenous and exogenous norepinephrine mediated vasoconstriction (22,32). This hypothesis is based on the observation that melatonin responses were blocked by either the melatonin receptor antagonists, luzindole or S20928. The use of subtype selective melatonin analogues is essential to identify functional melatonin receptor subtypes in

MELATONIN (0.6) LUZINDOLE (25)

OQ-Y

Figure 2. Chemical structures of melatonin and competitive melatonin receptor antagonists. Melatonin, luzindole (2-benzyl-N-acetyltryptamine), 4P-ADOT and 4P-PDOT competed for 2-[125I]-iodomelatonin binding to the human mt1 and MT2 melatonin receptor subtypes expressed in CHO cells (24-25).The number in parenthesis shows the affinity ratios (Ki m,1/ Ki MT2) with represent the fold differences in affinity for the subtypes. The higher the number the higher the affinity for the MT2 subtype. Analogues with affinity ratios of equal or higher than 100 are considered subtype selective (25).

native tissues. Although targeted gene disruption or the use of antisense provide evidence for the function of a given receptor protein, the presence of a receptor subtype is better characterized using subtype selective competitive melatonin receptor agonists and antagonists. Here, we describe the use of subtype selective MT2 melatonin receptor antagonists to characterize functional melatonin receptor subtypes in mammals.

2.4.1. Retina Receptors. In rabbit retina melatonin at picomolar concentrations inhibits the calcium dependent release of 3H-dopamine through activation of a presynaptic heteroreceptor. The release of 3H-dopamine is antagonized by luzindole (Kb = 20 nM) as well as by the MT2 subtype selective melatonin receptor antagonists, 4P-ADOT and 4P-PDOT (Kb = 1.6 nM and 0.3 nM, respectively) (24). The excellent correlation found between the affinity of a number of melatonin receptor antagonists to block the inhibition of dopamine release by melatonin (KB) with the affinity (Ki values) of these analogues to compete for 2-[125I]-iodomelatonin binding to the MT2 recombinant melatonin receptor strongly supports the classification of the presynaptic melatonin heteroreceptor of rabbit retina as an MT2 subtype (24).

2.4.2. Melatonin Receptors in the Circadian Timing System. The mti melatonin receptor was initially suggested to mediate circadian functions in mammals due to the high levels of mRNA expression within the mammalian SCN, which correlated with the specific 2-[125I]-iodomelatonin binding (8). However, recently Liu et al. (41) reported that targeted disruption of the mt1 melatonin receptor in the C57BL/6 mouse blocks the melatonin-induced inhibition of neuronal firing in SCN slices without impairing the melatonin-mediated phase shifts of circadian firing rhythms. This phase shifting effect of melatonin is pertussis toxin sensitive, suggesting the involvement of a G-protein coupled receptor (41-42).

In order to assess the melatonin receptor subtype mediating phase shifts of cir-cadian rhythms in mammals we investigated the effect of subtype selective melatonin receptor antagonists on the wheel running activity in the C3H/HeN mouse. We used the C3H/HeN mouse in these studies because it produces melatonin in the pineal gland (44) and in this strain melatonin phase-shifts circadian activity rhythms with periods of sensitivity identical to those found in humans (45-46).

The suprachiasmatic nucleus of the C3H/HeN mouse shows high density of 2-[125I]-iodomelatonin binding sites (18) as well as expression of both the mti and MT2 melatonin receptor mRNA (detected by in situ hybridization histochemistry with digoxigenin labeled oligonucleotide probes) (25). However, the selective melatonin receptor antagonist, 4P-ADOT did not compete for 2-[125I]-iodomelatonin binding to the mouse SCN. These findings suggest that either the density of the MT2 melatonin receptor protein in the C3H/HeN mouse SCN is below the limits of detection or 2-[125I]-iodomelatonin is not able to recognize the native MT2 melatonin receptor protein in brain frozen sections. It is noteworthy that undetectable levels of 2-[125I]-iodomelatonin binding were also reported in the SCN of the mti knockout C57BL/6 mouse (41).

In C3H/HeN mice, as in humans, melatonin administration for three consecutive days phase advances circadian activity rhythms when given at the end of the subjective day (CT 10) (18,45-46). Treatment with vehicle followed by saline administration for three consecutive days at CT 10 did not affect the rhythm of wheel running activity (45). Melatonin administration at CT 10 induced advances in the phase of the cir-cadian activity rhythms in a dose dependent manner (0.3 to 30 (g/mouse) (Figure 3). The dose of melatonin inducing a half-maximal phase advance (EC50) was 0.72(g/ mouse with a maximal advance of 0.98 ± 0.08 h (n = 15) at 9(g/mouse. The selective MT2 melatonin receptor antagonists, 4P-ADOT and 4P-PDOT (90 (g/mouse, sc) did not affect the phase of circadian activity rhythms when given alone at CT 10 (Figure 3). Both 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 (g melatonin (25) (Figure 3). The melatonin receptor antagonist luzindole, which shows 25 fold higher affinity for the MT2 than the mt1 melatonin receptor also antagonized the phase advance by melatonin at CT 10 (25). Together this study suggests that activation of the MT2 melatonin receptor subtype within the circadian timing system mediates the phase advances of circadian activity rhythms.

2.5. Summary

The MT2 melatonin receptor appears to be emerging as the subtype involved in mediating important physiological functions by melatonin in the visual, circadian and vascular systems. Pharmacological studies using subtype selective antagonists have demonstrated that the melatonin receptor involved in mediating inhibition of dopamine release from rabbit retina is the MT2 subtype (24). In the mammalian SCN, activation of G-protein coupled receptors by melatonin appear to mediate two distinct functional responses, i.e., acute inhibition of neuronal firing through the mt1 subtype and time-dependent phase shifts of circadian rhythms through the MT2 subtype (40-41). The dual effect of melatonin on phenylephrine-mediated vasoconstriction in rat caudal artery appears to be mediated by activation of two distinct receptor

What Melatonin Antagonist

Figure 3. The MT2 selective melatonin receptor antagonists blocked the melatonin-induced phase advances of circadian rhythms in the C3H/HeN mouse. Circadian wheel running activity rhythms were recorded from C3H/HeN mice held in constant dark. Mice received two treatments a day on three consecutive days. Each day the first treatment was at CT 10 and the second treatment 10min later.The ordinate represents the phase advance of circadian activity rhythms at CT 10. C3H/HeN mice were first treated with vehicle and ten minutes later with saline or melatonin (0.9-30^g/mouse). Mice treated with 4P-ADOT (90 ^g/mouse) or 4P-PDOT (90 ^g /mouse) did not show change in phase, however, they antagonized the phase advance induced by various doses of melatonin (0.9-30^g/mouse).The effect of the antagonists for each dose of melatonin was assessed by one way ANOVA (p < 0.01 for 0.9^g; p < 0.001 for 3^g and p < 0.05 for 9^g) (Data from reference 5).

Figure 3. The MT2 selective melatonin receptor antagonists blocked the melatonin-induced phase advances of circadian rhythms in the C3H/HeN mouse. Circadian wheel running activity rhythms were recorded from C3H/HeN mice held in constant dark. Mice received two treatments a day on three consecutive days. Each day the first treatment was at CT 10 and the second treatment 10min later.The ordinate represents the phase advance of circadian activity rhythms at CT 10. C3H/HeN mice were first treated with vehicle and ten minutes later with saline or melatonin (0.9-30^g/mouse). Mice treated with 4P-ADOT (90 ^g/mouse) or 4P-PDOT (90 ^g /mouse) did not show change in phase, however, they antagonized the phase advance induced by various doses of melatonin (0.9-30^g/mouse).The effect of the antagonists for each dose of melatonin was assessed by one way ANOVA (p < 0.01 for 0.9^g; p < 0.001 for 3^g and p < 0.05 for 9^g) (Data from reference 5).

subtypes, i.e., vasoconstriction by the mtj subtype and vasodilation by the MT2 subtype (47).

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