Margarita L. Dubocovich

Functional MT1 and MT2 melatonin receptors in mammals

Melatonin, dubbed the hormone of darkness, is known to regulate a wide variety of physiological processes in mammals. This review describes well-defined functional responses mediated through activation of high-affinity MT1 and MT2 proteinteoupled receptors viewed as potential targets for drug discovery. MT1 melatonin receptors modulate neuronal firing, arterial vasoconstriction, cell proliferation in cancer cells, and reproductive and metabolic functions. Ativation of MT2 melatonin receptors phase shift circadian rhythms of neuronal firing in the suprachiasmatic nucleus, inhibit dopamine release in retina, induce vasodilation and inhibition of leukocyte rolling in arterial beds, and enhance immune responses. The melatonin-mediated responses elicited by activation of MT1 and MT2 native melatonin receptors are dependent on circadian time, duration and mode of exposure to endogenous or exogenous melatonin, and functional receptor sensitivity. Together, these studies underscore the importance of carefully linking each melatonin receptor type to specific functional responses in target tissues to facilitate the design and development of novel therapeutic agent.

Melatonin receptors: are there multiple subtypes?

There is now evidence for more than one site of action for the hormone melatonin (N-acetyl-5-methoxy-tryptamine). Recent pharmacological and molecular advances are providing the tools to address the characterization of melatonin receptor subtypes. The development of novel melatonin receptor agonists and antagonists, high-affinity radioligands, quantitative bioassays, and the recent cloning of melatonin receptors are furthering our understanding of native and recombinant melatonin receptors. In this article, Margarita Dubocovich discusses the properties of melatonin receptors, and the basis for their classification into at least two subtypes, the ML1 and ML2.

Molecular pharmacology, regulation and function of mammalian melatonin receptors.

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.

Melatonin-Depleted Blood from Premenopausal Women Exposed to Light at Night Stimulates Growth of Human Breast Cancer Xenografts in Nude Rats

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 microW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 microW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.

Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms

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 μg/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 μg/mouse; 0.98 ± 0.08 h (n = 15) maximal advance at 9 μg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P‐ADOT and 4P‐PDOT (90 μ/mouse, s.c.) nor luzindole (300 μg/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 mg 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.—Dubocovich, M. L., Yun, K., Al‐Ghoul, W. M., Benloucif, S., Masana, M. I. Selective MT2 melatonin receptor antagonists block melatonin‐mediated phase advances of circadian rhythms. FASEB J. 12, 1211–1220 (1998)

International Union of Basic and Clinical Pharmacology. LXXV. Nomenclature, Classification, and Pharmacology of G Protein-Coupled Melatonin Receptors

The hormone melatonin (5-methoxy-N-acetyltryptamine) is synthesized primarily in the pineal gland and retina, and in several peripheral tissues and organs. In the circulation, the concentration of melatonin follows a circadian rhythm, with high levels at night providing timing cues to target tissues endowed with melatonin receptors. Melatonin receptors receive and translate melatonins message to influence daily and seasonal rhythms of physiology and behavior. The melatonin message is translated through activation of two G protein-coupled receptors, MT1 and MT2, that are potential therapeutic targets in disorders ranging from insomnia and circadian sleep disorders to depression, cardiovascular diseases, and cancer. This review summarizes the steps taken since melatonins discovery by Aaron Lerner in 1958 to functionally characterize, clone, and localize receptors in mammalian tissues. The pharmacological and molecular properties of the receptors are described as well as current efforts to discover and develop ligands for treatment of a number of illnesses, including sleep disorders, depression, and cancer.

Pharmacology and function of melatonin receptors.

The hormone melatonin is secreted primarily from the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone, through an action in the brain, appears to be involved in the regulation of various neural and endocrine processes that are cued by the daily change in photoperiod. This article reviews the pharmacological characteristics and function of melatonin receptors in the central nervous system, and the role of melatonin in mediating physiological functions in mammals. Melatonin and melatonin agonists, at picomolar concentrations, inhibit the release of dopamine from retina through activation of a site that is pharmacologically different from a serotonin receptor. These inhibitory effects are antagonized by the novel melatonin receptor antagonist luzindole (N‐0774), which suggests that melatonin activates a presynaptic melatonin receptoir. In chicken and rabbit retina, the pharmacological characteristics of the presynaptic melatonin receptor and the site labeled by 2‐[125I]iodomelatonin are identical. It is proposed that 2‐[125I]iodomelatonin binding sites (e.g., chicken brain) that possess the pharmacological characteristics of the retinal melatonin receptor site (order of affinities: 2‐iodomelatonin > 6‐chloromelatonin ≥ melatonin ≥ 6,7‐di‐chloro‐2‐methylmelatonin > 6‐hydroxymelatonin ≥ 6‐methoxymelatonin > N‐acetyltryptamine ≥ luzindole > N‐acetyl‐5‐hydroxytryptamine > 5‐methoxytryptamine >>>> 5‐hydroxytryptamine) be classified as ML‐1 (melatonin 1). The 2‐[125I]iodomelatonin binding site of hamster brain membranes possesses different binding and pharmacological characteristics from the retinal melatonin receptor site and should be classified as ML‐2. In summary, the recent advances in the pharmacological characterization of melatonin receptors in the central nervous system will further stimulate the search for potent and selective melatonin receptor agonists and antagonists, and should aid in our understanding of the mechanism of action of melatonin in mammalian brain.— Dubocovigh, M. L. Pharmacology and function of melatonin receptors. FASEB J. 2: 2765‐2773; 1988.

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% (IC50) at 20 pM with a maximal inhibitory effect (80%) at 1 nM. The partial agonists, i.e., N-acetyltryptamine (IC50: 5.6, maximal inhibition 55%) and 5-methoxyluzindole (IC50: 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 (IC50) 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 the affinity constants (Ki) for 2-[125I]-iodomelatonin binding determined on the Mel1b (r2 = 0.77, slope = 0.55, P<0.001; n = 11) but not the Mel1a (r2 = 0.27, P<0.1, n = 11) subtype. Together these results demonstrate that the pharmacological profile of the human recombinant Mel1b melatonin receptor is similar to that of the functional presynaptic melatonin heteroreceptor of rabbit retina, which we referred as an ML1B subtype. We conclude that the selective Mel1b melatonin partial agonists and antagonists described here can be used to identify melatonin receptor subtypes in native tissues and to search for subtype selective analogues with therapeutic potential.

The Impact of School Daily Schedule on Adolescent Sleep

Objectives. This study was initiated to examine the impact of starting school on adolescent sleep, to compare weekday and weekend sleep times, and to attempt to normalize the timing of the circadian sleep/wake cycle by administering bright light in the morning. This was a collaborative project involving high school students and their parents, as well as high school and university faculty members, for the purpose of contributing information to the scientific community while educating students about research processes and their own sleep/wake cycles and patterns. Methods. Sixty incoming high school seniors kept sleep/wake diaries beginning in August and continuing through 2 weeks after the start of school in September. Sleep diaries were also kept for 1 month in November and 1 month in February. Early-morning light treatments were given to 19 students in the last 2 weeks of November and the last 2 weeks of February. Neuropsychologic performance was measured with computer-administered tests. Paper-and-pencil tests were used for assessment of mood and vigor. A testing period consisted of 2 consecutive days at the beginning and end of November and at the beginning and end of February. Tests were given 3 times per day, ie, in the morning before school (6:30–8:00 am), during midday lunch periods (11:30 am to 1:00 pm), and in the afternoon (3:00–4:30 pm), on each of the test days. Results. Adolescents lost as much as 120 minutes of sleep per night during the week after the start of school, and weekend sleep time was also significantly longer (∼30 minutes) than that seen before the start of school (August). No significant differences were found between weekday sleep in the summer and weekend sleep during the school year. Early-morning light treatments did not modify total minutes of sleep per night, mood, or computer-administered vigilance test results. All students performed better in the afternoon than in the morning. Students in early morning classes reported being wearier, being less alert, and having to expend greater effort. Conclusions. The results of this study demonstrated that current high school start times contribute to sleep deprivation among adolescents. Consistent with a delay in circadian sleep phase, students performed better later in the day than in the early morning. However, exposure to bright light in the morning did not change the sleep/wake cycle or improve daytime performance during weekdays. Both short-term and long-term strategies that address the epidemic of sleep deprivation among adolescents will be necessary to improve health and maximize school performance.

Melatonin mediates two distinct responses in vascular smooth muscle

The pineal hormone melatonin was found to produce two distinct contractile responses in vascular smooth muscle. In isolated rat caudal artery segments, denuded of endothelium, melatonin (10(-10)-10(-7) M) potentiated phenylephrine-induced contractions in a concentration-dependent manner. At higher melatonin concentrations (10(-7)-10(-5) M), however, the potentiating effect was attenuated. In the presence of the melatonin MT2 receptor antagonist, 4-phenyl-2-acetamidotetraline (4P-ADOT), the attenuated constrictor responses were selectively enhanced. These results are consistent with the hypothesis that melatonin activates two receptor subtypes in vascular smooth muscle; MT2 receptors may induce relaxation, while a second receptor subtype mediates vasoconstriction.